CARDIAC ORGANOGENESIS: 3D BIOSCAFFOLDS,
BIOENERGETICS AND REGENERATIONby
Alice S. Ferng

Copyright © Alice S. Ferng 2015
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF GRADUATE INTERDISCIPLINARY PROGRAMSIn Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
WITH A MAJOR IN PHYSIOLOGICAL SCIENCES
In the Graduate College
THE UNIVERSITY OF ARIZONA
2015

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THE UNIVERSITY OF ARIZONAGRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation
prepared by Alice S. Ferng, titled “Cardiac Organogenesis: 3D Bioscaffolds, Bioenergetics andRegeneration” and recommend that it be accepted as fulfilling the dissertation requirement for
the Degree of Doctor of Philosophy.
___________________________________________________________ Date: 11/19/2015
Janis Burt___________________________________________________________ Date: 11/19/2015
Mark D. Pagel___________________________________________________________ Date: 11/19/2015
Paul McDonagh___________________________________________________________ Date: 11/19/2015
John Konhilas

Final approval and acceptance of this dissertation is contingent upon the candidate’s submissionof the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommendthat it be accepted as fulfilling the dissertation requirement.
________________________________________________ Date: 11/19/2015
Dissertation Director: Zain I. Khalpey

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STATEMENT BY AUTHORThis dissertation has been submitted in partial fulfillment of the requirements for an advanced
degree at the University of Arizona and is deposited in the University Library to be madeavailable to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that an
accurate acknowledgement of the source is made. Requests for permission for extended
quotation from or reproduction of this manuscript in whole or in part may be granted by the
copyright holder.
SIGNED: Alice Ferng

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ACKNOWLEDGEMENTS
I would like to acknowledge the members of both my Comprehensive Exam Committee andDissertation Committee for their help, guidance, and support through this PhD – Dr. Zain
Khalpey (mentor), Dr. Janis Burt, Dr. Marty Pagel, Dr. Paul McDonagh, Dr. John Konhilas, andDr. Torsten Falk.
In particular, I want to thank Dr. Khalpey for his amazing personal and professional support overthese years, for the opportunity to pursue a PhD in his lab, and for giving me his full trust andfaith in growing into the physician-scientist, innovator, and engineer I wanted to be.
I would like to thank Dr. Pagel for taking the time to mentor me and show me the ropes tomagnetic resonance imaging, and taking the time to offer advice and edit my manuscripts.
I would like to thank Holly Lopez for all of the moral and administrative support that got me
through my graduate studies in the Department of Physiological Sciences.
I would like to thank the many mentors I have had through my academic career who have been
there for me, taught me through example how to pay-it-forward, encouraged me to pursue mydreams, and challenged me to keep striving for excellence.
I would also like to thank all of the members of the Khalpey lab and our collaborators for their
ongoing assistance, camaraderie, and friendship.
I would like to thank my absolutely wonderful friends and family who have been my source ofstrength, sanity, and reason. Thank you for always being there for me and looking out for me.
Finally, I would like to acknowledge the Technology and Research Initiative Fund (TRIF)
Imaging Fellowship and other sources of funding that made my PhD possible.

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DEDICATION

To my mother and father –you are everything to me,
the reason I am who I am today,
and the true embodiment of truthfulness, compassion, and tolerance;

To my beloved brother and sister, and my dearest friends –you are my world and guiding light;

To those who dare to be their true selves –never give up.

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TABLE OF CONTENTS
LIST OF FIGURES ...................................................................................................................... 8
LIST OF ACRONYMS ................................................................................................................ 9
ABSTRACT .................................................................................................................................. 10
PREFACE: DISSERTATION LAYOUT & RATIONALE..................................................... 12
CHAPTER 1 .................................................................................................................................. 16
BACKGROUND & INTRODUCTION
1.1 Cardiac Organogenesis ............................................................................................................ 17
1.2 Cellular Bioenergetics .............................................................................................................. 18
1.3 Tissue and Organ Regeneration ............................................................................................... 20
CHAPTER 2 .................................................................................................................................. 24
PRESENT STUDY 1: PRE-CLINICAL EX-VIVO HUMAN RECELLULARIZATION OF
ACELLULAR PORCINE HEARTS
2.1 Introduction ............................................................................................................................. 25
2.2 Methods .................................................................................................................................... 27
2.3 Results ...................................................................................................................................... 31
2.4 Discussion ................................................................................................................................ 35
2.4 Future Directions...................................................................................................................... 37
CHAPTER 3 .................................................................................................................................. 39
PRESENT STUDY 2: CARDIAC REPAIR AND REGENERATION: RESEEDING POTENTIAL OF
CLINICALLY USED COMMERCIAL MATRICES
3.1 Introduction ............................................................................................................................. 40
3.2 Methods .................................................................................................................................... 42
3.3 Results ...................................................................................................................................... 44
3.4 Discussion ................................................................................................................................ 48
REFERENCES ............................................................................................................................. 50
APPENDIX A ................................................................................................................................ 55
ACELLULAR PORCINE HEART MATRICES: WHOLE ORGAN PHYSIOLOGICDECELLULARIZATION WITH 3D-BIOSCAFFOLD & VASCULAR PRESERVATION
Abstract .......................................................................................................................................... 56
Introduction .................................................................................................................................... 56
Methods .......................................................................................................................................... 59
Results ............................................................................................................................................ 65
Discussion ...................................................................................................................................... 75
References ...................................................................................................................................... 79

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APPENDIX B ................................................................................................................................ 82
HUMAN ADIPOSE-DERIVED STEM/STROMAL CELLS: COMPARATIVE ORGAN SPECIFIC
MITOCHONDRIAL BIOENERGY PROFILES
Abstract .......................................................................................................................................... 83
Introduction .................................................................................................................................... 83
Methods .......................................................................................................................................... 85
Results ............................................................................................................................................ 88
Discussion ...................................................................................................................................... 91
References ...................................................................................................................................... 95
APPENDIX C .............................................................................................................................. 100
CARDIAC ORGAN PRESERVATION SOLUTIONS: MITOCHONDRIAL SUSPENDEDANIMATION
Abstract ........................................................................................................................................ 101
Introduction .................................................................................................................................. 101
Methods ........................................................................................................................................ 105
Results .......................................................................................................................................... 107
Discussion .................................................................................................................................... 111
References .................................................................................................................................... 115
APPENDIX D.............................................................................................................................. 117
CARDIAC REGENERATION IN THE HUMAN LEFT VENTRICLE FOLLOWING CORMATRIXIMPLANT
Abstract ........................................................................................................................................ 118
Case Presentation ......................................................................................................................... 118
Discussion .................................................................................................................................... 119
References .................................................................................................................................... 123
APPENDIX E .............................................................................................................................. 124
TRANSLATION OF FIRST NORTH AMERICAN 50CC AND 70CC TOTAL ARTIFICIAL HEART
VIRTUAL & CLINICAL IMPLANTATIONS: UTILITY OF 3D COMPUTED TOMOGRAPHY TO
TEST FIT DEVICES
Abstract ........................................................................................................................................ 125
Introduction .................................................................................................................................. 126
Case History ................................................................................................................................. 128
Methods ........................................................................................................................................ 130
Results .......................................................................................................................................... 131
Comments .................................................................................................................................... 133
References .................................................................................................................................... 138
APPENDIX F: FUTURE DIRECTIONS .................................................................................. 141
Summary ...................................................................................................................................... 142
Future Directions.......................................................................................................................... 143
APPENDIX G.............................................................................................................................. 146
First in Man: Sternal Reconstruction with Autologous Stem Cells

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LIST OF FIGURES
Figure 1.1. Mitochondrial respiration and glycolytic function profiles generated on a Seahorse
Bioscience XFe96 Extracellular Flux Analyzer ........................................................................... 19
Figure 2.1. Recellularization of acellular matrix .......................................................................... 32
Figure 2.2. Cell count following recellularization ........................................................................ 33
Figure 2.3. Langendorff Bioreactor Schematic............................................................................. 33
Figure 2.4. Angiogram of whole acellularized heart .................................................................... 34
Figure 2.5. Langendorff recellularized LAD ................................................................................ 34
Figure 3.1. Seeding of CorMatrix with Human Amniotic Stem Cells ................................................. 45
Figure 3.2. Seeding of Amniotic Membrane with Human Amniotic Stem Cells .................................. 45
Figure 3.3. Scanning Electron Microscopy of CorMatrix and Amniotic Membrane............................. 46
Figure 3.4. Transmission Electron Microscopy of CorMatrix and Amniotic Membrane ...................... 46
Figure 3.5. Treatment of seeded CorMatrix with OLFM-1 and TGF-β1 ............................................. 47

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LIST OF ACRONYMS
2-DG
AMP
AmSCs
ASCs
ECM
EMT
ETC
HUVECs
LAD
LV
MV
OCT
OLFM1
OPTN
SEM
SIS
SRTR
TEM
TGF

2-Deoxy-D-glucose
Adenosine monophosphate
Amniotic stem cells
Adipose-derived stromal/stem cells
Extracellular matrix
Epithelial-to-mesenchymal transition
Electron transport chain
Human umbilical vein endothelial cells
Left anterior descending coronary arteryLeft ventricle
Mitral valve
Optimal cutting temperatureOlfactomedin-1
Organ Procurement and Transplantation
Network
Scanning electron microscopy
Small intestinal submucosa
Scientific Registry of Transplant
Recipients
Transmission electron microscopy
Transforming growth factor

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ABSTRACT
Each year the Organ Procurement and Transplantation Network (OPTN) reports anincrease in patients requiring an organ transplant without an increase in available donor organs,leading to a transplant gap that continues to widen. Over 70% of donor hearts are deemed
unsuitable for transplantation each year, and a large number of these organs (~50%) arediscarded due to poor organ function, decreased ejection fraction, disease, or cardiac arrest(Scientific Registry of Transplant Recipients (SRTR) Annual Data Report 2011).
We therefore set out to improve knowledge in the field of cardiac transplantation in termsof organogenesis, bioenergetics, and regeneration. The main goal through tissue bioengineering
is to regenerate and salvage discarded hearts through organogenesis, or to lengthen the totalorgan preservation time such that organs would not be thrown away while a recipient was
waiting to be found. Our first hypothesis was that an optimized acellular extracellular matrix
scaffold would allow for cell adherence, growth and proliferation, and could potentially begrown into a clinically transplantable organ. To achieve these goals, an optimized protocol wasdeveloped for the total acellularization of a whole porcine heart, leaving behind a 3D bioscaffold.
We showed that acellularized matrices could be successfully seeded using endothelial cells foracellular vasculature and stem cells for other acellular tissues, both as a 2D matrix and within aconstantly perfused 3D Langendorff setup bioreactor.
In order to best understand cell-cell and cell-matrix interactions, cellular bioenergetics
were evaluated. We hypothesized that the bioenergetic demand of the type and anatomical originof stem cells would affect the regeneration potential dependent on intrinsic metabolic demand.
We therefore showed a differential of the bioenergetic profiles of human adipose-derived stem
cells isolated from various adipose depots, concluding that the physiological microenvironment

11that supports stem cells in specific anatomic locations can regulate how stem cells participate intissue regeneration, maintenance and repair, and also will vary based on donor-differences.
During organ transplantation, organ preservation solutions are created for use at specificconditions, such as on ice or at room temperature. We hypothesized that hypothermia would
slow down cellular metabolism, and that solutions containing a higher content of antioxidants
and other protective substrates against ischemic reperfusion injury would create the best organ
storage conditions. We tested three organ preservation solutions against control media andnormal saline at 4 and 21 degrees C, for 4 to 8 hours, investigating the bioenergetics of organ
preservation solution effects on cardiac cells. By simulating clinical conditions, we were able todetermine that one of our solutions was ideal and had protective effects for cells for up to 8 hours
at 4 degrees C.
Finally, we believed that studying existing cardiac patches and optimizing cardiac matrix
design would lead to improved cardiac physiological function and would aid in healing andrepair during cardiac surgery. Following a clinical case report showing new cardiac tissue growth
after implantation of an acellular porcine extracellular matrix, we devised a proof-of-concept
study to show that clinical matrices could be easily cultured in vitro. We successfully seeded
these clinical matrices using human amniotic stem cells, a commonly used cell type forregeneration and repair after surgery. Our preliminary studies suggest that preconditioned
matrices can be potentially used clinically for greater efficacy and tissue regeneration.

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PREFACE: DISSERTATION LAYOUT & RATIONALE
The purpose of this dissertation was: 1) to better understand cardiac organogenesis, tissue
regeneration and engineering, as well as the underlying bioenergetics, cell-cell and cell-matrix
interactions involved when using cardiac and stem cells, and 2) to utilize our knowledge in basic
sciences in a way such that our research could be translated from bench-to-bedside.
Chapter 1 therefore begins by recognizing the shortage of donor organs in what is
termed the “transplant gap,” and lays out the relevant background necessary to integrate andweave the basic sciences together with the clinical sciences.
Chapter 2 is an ongoing study. The data presented is exciting proof-of-concept work thathas shown that we are able to recellularize the acellular matrices created through our whole heart
decellularization process that was optimized (Appendix A). We have additionally shown that weare able to recellularize part of the vasculature matrix in an ex vivo circuit using a Langendorff
setup as a bioreactor. Experiments are currently being conducted to further this work, and will beultimately added to this chapter, which has been written for submission once data has beencollected in a manuscript titled “Pre-Clinical Ex-Vivo Human Recellularization of Acellular
Porcine Hearts.”
Chapter 3 is another ongoing study. This preliminary proof-of-concept work that will be
developed into a manuscript once experiments have been completed is titled “Cardiac Repair
and Regeneration: Reseeding Potential of Clinically Used Commercial Matrices.” This study
involves taking cardiac patch matrices currently used in the operating room and showing thatthese matrices can be seeded similarly to acellular cardiac tissues.
Because we are a translational research lab, this was a project derived from the operating
room to help augment our understanding of the matrices and stem cells being used in our13patients. Amniotic stem cells are often injected by surgeons to aid in cardiac repair andregeneration, and extracellular matrix also offers these benefits, and more. We wanted to showthat amniotic stem cells could adhere to CorMatrix extracellular matrix and amniotic membrane,
and then additionally show that the epithelial-to-mesenchymal transition could be induced once
cells had adhered to these matrices. The clinical counterpart of this project is presented inAppendix D, where I was in the operating room and involved in the laboratory components ofthis clinical case, helped analyze and assemble the data, and helped with writing.
Appendices A-E is a series of projects and manuscripts that were written by me, where I
performed the majority of experiments, did the data analysis, and created the figures. These
manuscripts are in submission.
Appendix A is a manuscript titled: “Acellular Porcine Heart Matrices: Whole Organ
Physiologic Decellularization with 3D-Bioscaffold & Vascular Preservation” that summarizes
the conclusions gleaned after many overnight organ experiments (>125 organs, pig and human)performed over 3 years in an effort to understand the protocols and methods published byprominent scientists in the field of cardiac organogenesis—that is, to “de-cellularize” andremove organs of all cells, blood, and other constituents to effectively leave behind the shell,
which is the 3D bioscaffold of the heart. In this process, we cycled through different apparatuses
and decellularization setups, adapting and rebuilding our system, testing temperatures, flow
rates, and perfusion pressures, as well as testing many other published decellularization methods,
such as freeze/thaw cycles, sonication, and so on. In the end, we were able to find an optimized
method for complete whole porcine heart decellularization that preserved organ architecture, as
well as tissue elasticity.
Left with a 3D bioscaffold, or essentially a cardiac extracellular matrix, the next large

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step was then to “re-cellularize” the acellular cardiac extracellular matrix with stem cells;
however, this quickly became a daunting task, especially given the lack of success inrecellularizing whole organs of larger animals and creating functional organs that could betransplanted. This is in part because the thickness of the heart walls in porcine and human hearts
is much greater than in rodent hearts, which means that it may not be possible to properly
recellularize each layer of the heart wall without a deeper understanding of cells and matrix
interactions. However, we will try to tackle this issue anyway. The recellularization project ispresented in Chapter 2.
Appendix B then follows as a manuscript titled: “Human Adipose-Derived Stem/Stromal
Cells: Comparative Organ Specific Mitochondrial Bioenergy Profiles” where we have taken
adipose tissue from different anatomical locations and studied their baseline energy profiles. In
order to better understand how our human cell populations could best be used forrecellularization, we had to first understand how these cells were different, what the ideal sourceof adipose-derived stem/stromal cells was, and under what conditions they were healthy andthriving. As a proof-of-concept that adipose-derived stem/stromal cells isolated from the stromal
vascular fraction could be used in organ/tissue regeneration, our lab isolated autologous patient
stem cells from the stromal vascular fraction using sonication in the clinical operating room, andused these isolated cells as a point-of-care treatment within 2 hours of extraction. Ultimately,
there was full sternal reconstruction, which is shown in a case report as Appendix G: “First in
Man: Sternal Reconstruction with Autologous Stem Cells.”
Appendix C is a manuscript titled “Cardiac Organ Preservation Solutions:
Mitochondrial Suspended Animation” to mimic in vitro conditions on cardiac cells and test theboundaries of metabolic cellular function by replicating a state of “suspended animation” that is15

achieved when transporting a donor organ to the recipient. There are times at which the organ
may be at room temperature (roughly 21 degrees C) briefly or on ice (at 4 degrees C). For thismanuscript, we studied some of the popularly used organ preservation solutions used fortransplant organs: Somahlution, Perfadex and Celsior. We created a situation where cells were
unsupported for 4 or 8 hours, and studied their ability to recover at 37 degrees C, in other words,
at physiological body temperature with the proper nutrients. From the knowledge we gained
here, we decided to investigate cell-to-cell interactions in co-culture.
Appendix D is a case report titled “Cardiac Regeneration in the Human Left Ventricle
Following Cormatrix Implant” describing how following CorMatrix implant in a patient, cardiac
cells were able to grow over the patch and ultimately regenerate some of the damaged tissue. We
were able to process some samples at the lab and use some histology and immunohistochemistry
to show growth of new cardiomyocytes.
Appendix E is a manuscript titled: “Translation of First North American 50cc and 70cc
Total Artificial Heart Virtual & Clinical Implantations: Utility of 3D Computed Tomography to
Test Fit Devices” that shows that we are able to take patient chest CT and MRI scans, anddetermine which size of total artificial heart could be implanted in a patient. This provides aclinical proof-of-concept, and relevant case series for 3D modeling will be useful in the lab tomodel patient pathologies and create a customized cardiac patch for tissue regeneration through3D bioprinting.
Appendix F includes a final summary and future directions where I discuss the next steps
to take, and an ongoing 3D bioprinting project not previously described in this dissertation.
Appendix G is a published case report titled “First in Man: Sternal Reconstruction withAutologous Stem Cells.”

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CHAPTER 1
BACKGROUND & INTRODUCTION

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1.1 Cardiac Organogenesis
In brief, the ultimate goal of organogenesis is to grow a clinically transplantable organ. Many
methods exist to first remove all cell and potentially immunogenic components of a biologicalorgan through decellularization, and then reintroduce stem and terminal cell types to thedecellularized organ through a process known as recellularization.
Broadly, the process of decellularization involves: lysis of cell membranes; separation of thecellular components from the extracellular matrix (ECM); and removal of cytoplasmic, nuclear
and cell debris. The optimal decellularized biological matrix is one that is free of donor cellmaterial and highly preserves the critical ECM. Much of the groundwork for whole organ
decellularization has been proven and shown to be a potential option in rodent models (Ott et al.,2008); however, nearly a decade later, this same work has failed to be translated fully into larger
animal models and humans. This implies that there is much work to be done in this field, in orderto create viable and transplantable organs.
The end-goal of decellularization is to produce a bioartificial scaffold that resembles thethree-dimensional structure of the heart while maintaining an ECM structure sufficient forcellular adhesion and growth, and with mechanical properties that resemble that of native hearttissue. It is ideal to produce a scaffold with both minimal “fraying” of ECM fibers and structures
and also a total volume and size similar to the initial state of each respective heart used. The
scaffold must also have similar tensile and biaxial strain characteristics as does a normal healthy
heart. The ECM is the direct environment for cells during recellularization, and it consists of acomplex network of proteins, proteoglycans, and glycosaminoglycans. Far from being an inert
scaffold, the ECM is increasingly being recognized as having an important role in cell signaling,differentiation, and tissue homeostasis. Additionally, it is through the ECM that cells perceive

18

signals such as shear stress and tensile forces through membrane receptors known as integrins
and mechanosensitive ion channels (Fernandes, Moroni, van Blitterswijk, & de Boer, 2009).
The final engineered heart muscle must have the structural integrity to be able to develop
systolic force. It must be sufficiently compliant to withstand physiological diastolic loads, andform an electromechanical syncytium. Hence, it is important to preserve the structural and matrix
components of the heart, which consists of collagen, elastin, proteoglycans, glycosaminoglycans,fibronectin, and laminin, among other components. Ideal detergents for decellularization arethose that result in maximally preserved bioscaffold architecture, which can be determinedthrough various histology and imaging techniques, mass spectrometry, and dynamic materials
analyses.

1.2 Cellular Bioenergetics
Evaluation and understanding of cellular bioenergetics is an effective and quick methodof identifying which substrates and nutrients are most preferred by different cell types, assessing
cell-cell interactions, cell-matrix interactions, and detecting cellular dysfunction. Bioenergetics isa field of biochemistry that involves studying energy flux through living systems and focuses on
different cellular processes, such as mitochondrial respiration or glycolysis, that lead to the
production and utilization of energy (e.g., ATP molecules).
Using the XFe96 Seahorse Bioscience extracellular flux analyzer, mitochondrial
respiration and glycolytic capacity can be measured for homogenous and heterogenous cell
populations. A mitochondrial stress test can be performed by sequentially injecting drug
inhibitors in the media of cells. These drugs include: oligomycin (ATP synthase inhibitor), FCCP(an uncoupler that acts as electron transport chain (ETC) accelerator), antimycin A (binds to19cytochrome C reductase and inhibits complex III of ETC), and rotenone (inhibits complex I and
II of the ETC).
Oligomycin A will significantly reduce electron flow through the electron transport
chain, although electron flow is not completely ceased due to proton leak. FCCP is a compoundthat carries protons across the inner mitochondrial membrane and dissipates the membranepotential that drives ATP synthesis. In order to maintain the membrane potential, themitochondria need to increase the flow of electrons and thus oxygen consumption. Antimycin A
inhibits complex III of the electron transport chain, which stops all mitochondrial respiration;
this allows us to subtract the non-mitochondrial sources of oxygen consumption and calculate themitochondrial-specific component of respiration. Rotenone blocks NADH dehydrogenase andhas a similar ETC inhibitory function to antimycin A, as the two drugs are used together.
Injection of these drugs while measuring oxygen consumption rate over time results in a
mitochondrial respiration profile as seen in Figure 1.1.

Figure 1.1. Mitochondrial respiration and glycolytic function profiles generated on a Seahorse
Bioscience XFe96 Extracellular Flux Analyzer. OCR=oxygen consumption rate; ECAR=extracellularacidification rate.

20

Parameters that can be calculated regarding mitochondrial function are: basal respiration,
ATP turnover, proton leak, and maximal respiration, or spare respiratory capacity (Figure 1.1A).
By looking at mitochondrial respiration, it is also possible to investigate the substrates andgrowth factors that cell populations prefer for expansion or differentiation (if coupled with othertechniques including flow cytometry and qPCR). Examining the metabolic demands of various
progenitor/stem cell and primary cell types cultured homogenously and in co-cultures allow for
an understanding of what nutrients, substrates, growth factors, and other factors are required
and/or preferred in the microenvironment of each cell type. Specifically looking at purine andpyrimidines through the use of high-pressure liquid chromatography (HPLC) would offer
another snapshot of nucleotide metabolism in various environments.
A glycolytic function profile can be generated by injecting glucose, oligomycin, and 2Deoxy-D-glucose (2-DG; a glycolysis inhibitor). This allows for the calculation of glycolysis,
which is the extracellular acidification rate due to the utilization of exogenously provided
glucose, glycolytic capacity, and glycolytic reserve.

1.3 Tissue and Organ Regeneration
Following complete decellularization of the whole porcine heart, the next step is torepopulate the acellular organ with populations of terminal and stem cells. Recellularization, or
re-seeding, of the decellularized bioscaffold can be a very complicated process due to thestructure of the heart of larger animal models or humans. Specifically, the thickness of the
cardiac walls is greater than that of a rodent model, and the density of cells and tissues is also
varied within the same heart. The ventricular wall will not have the same cellular composition as
the mitral valve, and the vasculature and nerve conduction systems also have different and21

specific cell populations. Each of these components must be approached in a different way that
ultimately requires specific placement and homing of cells into the correct anatomical locations.
Cardiac structure and cellular composition must be appropriately considered during
recellularization.
Layers of the heart wall can be divided up into 3 major tissue layers: the endocardium,myocardium, and pericardium. The innermost endocardium layer of the heart is a thin, smoothlayer of epithelial tissue that lines the inner surface of the heart’s chambers and valves. The
epithelial layer consists of simple squamous cells that are in direct contact with the blood that is
pumped by the heart. Next, the thickest layer of the heart is the myocardium, which is an
involuntary striated muscle layer of the heart and is made up of myocytes (cardiac muscle cells).
Finally, the pericardium is a double-walled sac that contains the heart and the roots of the great
vessels. Serous fluid produced by epicardium cells fills the space between the two layers toprotect the heart from external movements by reducing friction from muscle movements andproviding lubrication.
The two layers of the pericardial sac are the outermost fibrous pericardium and the innerserous pericardium. The fibrous pericardium is primarily composed of connective tissue, which
provides a tough layer that protects the heart from being overstretched and also acts as an anchor.
Internally, the double-layered serous pericardial sac is a transparent membrane that consists of aparietal pericardium fused to the fibrous pericardium, and a visceral pericardium (e.g.,
epicardium) that covers the surface of the heart, which is comprised of mesothelial simple
squamous epithelial cells. Innervating nerves and coronary arteries run through the epicardial
layer of the heart. Finally, the endothelium and pericardium are thinner layers that consist
primarily of simple squamous epithelial cells. The myocardium contains the most diverse group22of cells in the heart due to its function as the muscle layer, and the cells contained are thus
involved in contraction, regeneration, repair, and maintenance of the myocardium.
The major cell types that make up the heart are cardiac fibroblasts, myocytes, endothelial
cells, and vascular smooth muscle cells. Adult stem cells involved in the tissue replacement,
regeneration, and repair process include bone marrow cells, haemopoietic stem cells, endothelial
progenitor cells, mesenchymal stem cells, and resident cardiac stem cells. Adult murine
myocardium contains the following cell types and numbers during cardiac development: 56%
myocytes, 27% fibroblasts, 7% endothelial cells, and 10% vascular smooth muscle cells(Banerjee, Fuseler, Price, Borg, & Baudino, 2007), whereas, human myocardium is composed of
2 to 3 billion cardiomyocytes, which is roughly 75% by volume, and about ~25-30% by number(Cohen, Ruvinov, & Sapir, 2012). These differences, along with the anatomical differences
between a rodent vs. large animal vs. human, account for some of the difficulties in translating
previously successful recellularization studies from the rodent to a pig or human heart.
Heart tissue is mainly composed of fibroblasts (about two-thirds of the total cell number)and endothelial cells (Cohen et al., 2012). Unlike skeletal muscle cells, cardiomyocytes arecontrolled by the autonomic (involuntary) rather than the somatic (voluntary) nervous system.
Therefore, cardiomyocytes can generate their own excitatory impulses to function as a biologicalpacemaker; however, during whole organ organogenesis, an external pacemaker is likely
necessary to jumpstart contraction. External pacemakers are often the final step in cardiac
organogenesis, and are included as part of the Langendorff setup.
The Langendorff setup was developed by Oscar Langendorff in 1895 to study andunderstand the fundamental physiology of the heart: its contractile function, coronary blood flow
regulation, and cardiac metabolism. This is a setup that can be used for isolated retrograde

23

perfused hearts in an ex vivo application, but which for the purposes of this dissertation has been
repurposed as a bioreactor. The Langendorff setup allows for the control of temperature,
perfusion rates, and gas exchange through a membrane oxygenator that is necessary to grow awhole heart through retrograde perfusion through the aorta.

24

CHAPTER 2
PRESENT STUDY 1: PRE-CLINICAL EX-VIVO HUMAN RECELLULARIZATION OF
ACELLULAR PORCINE HEARTSManuscript (to be submitted): Ferng AS, Bajaj N, Iwanski J, Nunez M, Runyan R, Khalpey Z.
Pre-Clinical Ex-Vivo Human Recellularization of Acellular Porcine Hearts.

25

2.1 Introduction
Biological scaffold materials composed of extracellular matrix(ECM) are a viableoption for cardiac repair and regeneration. Current commercially used surgical meshes andcardiac patches are comprised of ECM harvested from allogeneic or xenogeneic tissues. Thesematrices help provide structural support, facilitate wound healing, and enhance tissue repair bypositively effecting cell mitogenesis, chemotaxis, (Agrawal, Kelly, Tottey, & Daly, 2011a;
Agrawal et al., 2011b; Reing et al., 2009) and differentiation, (Agrawal et al., 2011b; Crapo etal., 2012), They also contribute to the modulation of the host innate immune response, (Badylak,
Valentin, Ravindra, McCabe, & Stewart-Akers, 2008; Brown et al., 2012a; Brown, Ratner,
Goodman, Amar, & Badylak, 2012b) and therefore can be used effectively for repairing different
tissues in multiple organ systems. While it has not yet been proven whether tissue specificity ofsurgical meshes is a requirement for reconstruction of complex tissues, studies have shown that
ECM does affect cellular migration, proliferation and differentiation.
ECM bioscaffolds can be created in a variety of methods, including partial or wholeorgan decellularization, or acellularization, lyophilization and processing of biological matrices
into a clinically functional material, or via de novo synthesis of ECM using artificial materials.
However, the method of ECM scaffold preparation has been shown to affect downstream
remodeling and repair, thus affecting clinical efficacy. Most often, commercial ECMs are
generated from several tissue sources, including small intestinal submucosa (SIS), urinary
bladder, or pericardium. For the SIS, the mucosa, serosa, and muscular layers are removed toreveal a strong collagenous matrix that is then disinfected and sterilized for clinical use.
While porcine ECM scaffolds made of SIS have been shown to promote myocardial
tissue repair without the usual scar tissue formation via increased vascularization, success of this26

ECM scaffold depends on the sufficient removal of immunogenic components. These acellularscaffolds have been shown to allow for cellular adhesion, differentiation, and growth in animal(Badylak, Obermiller, Geddes, & Matheny, 2003) and human models (M. Stelly & Stelly, 2013;
Yanagawa, Rao, Yau, & Cusimano, 2013).
Stem cell differentiation is a very complicated process, and controlling the outcome ofco-cultured cells and heterogenous cell populations can be very difficult. However, recent studies
have indicated that tissue-specific and organ-specific ECMs can promote location appropriate
differentiation of progenitor cells (Petersen et al., 2010) into a site-appropriate phenotype(Sellaro, Ravindra, & Stolz, 2007). Therefore, decellularization to produce the ECM of eachtissue type or organ represents a logical method of manufacturing scaffolds for tissue repair andregeneration. Over the past decade, many scientists have done experiments involving wholeorgan decellularization (Crapo, Gilbert, & Badylak, 2011; Freytes, Godier-Furnemont, Duan,O’Neill, & Vunjak-Novakovic, 2014; Gilpin & Ott, 2015; Guyette et al., 2014; Ott et al., 2008;
Song & Ott, 2011; Sullivan et al., 2012; Tapias & Ott, 2014; Wainwright et al., 2010), by using
various techniques to create bioscaffolds or synthetically-created scaffolds for the purpose ofclinical transplantation. Alternatively, acellular organs or tissues have also been repurposed fortheir ECM, which can be reprocessed into another form, such as a powder to be used as an
injectable hydrogel (Johnson & Christman, 2013; Seif-Naraghi, Horn, Schup-Magoffin, &Christman, 2012; Young, Ibrahim, Hu, & Christman, 2011).
This study design involved the use of a whole organ acellularization method for porcine
hearts developed by our lab. Experimental studies were carried out to examine cell growth andproliferation over time as a proof-of-concept to show that our ECM scaffold could allow for celladherence and growth. Recellularization, or re-seeding, of an acellularized bioscaffold is a very

27complicated process due to the structure of the heart in larger animal models and humans.
Specifically, the thickness of the cardiac walls are greater than that of a rodent model, and thedensity of cells and tissues is variable within the same heart. The ventricular wall will not havethe same cellular composition as the mitral valve. The vasculature and nerve conduction systems
will also have differing but specific cell populations. Each of these components must be
approached in a different way so that ultimately specific placement and homing of cells into thecorrect anatomical location can be achieved. Cardiac structure and cellular composition must be
appropriately considered during recellularization. Therefore, we used endothelial cells to reseed
vasculature and stem cells to reseed the left ventricle and mitral valve.
In addition to seeding two-dimensional acellular ECM scaffolds, a pressure constantLangendorff perfusion circuit was assembled to generate the necessary force for pushing
nutrients from the perfusate through the thick walls of a whole acellularized heart (SkrzypiecSpring, Grotthus, Szeląg, & Schulz, 2007). The basic principle of the Langendorff circuit is tomaintain cardiac activity by perfusing the heart via the coronary arteries. Using an aortic cannula
inserted into the ascending aorta, distal to the left and right coronary ostia, perfusate can bedelivered to the heart in a retrograde manner, providing adequate nutrients to the main vascularsystems (Bell, Mocanu, & Yellon, 2011). Using this device, the ideal bioreactor system can beused for recellularization of the acellular three-dimensional heart scaffold.

2.2 Methods
Acellularization of Whole Porcine Hearts
Adult porcine Yorkshire hearts (n=9) around 130±10 kg were obtained at the Food
Product and Safety Laboratory of the University of Arizona. Hearts were processed immediately

28

following procurement, by excising the heart at the great vessels, aorta, and adjacent mediastinal
attachments. The aorta was cannulated with a 3/8” attachment connected to perfusion tubing.
Acellularization was achieved through constant perfusion at 90 mmHg, not exceeding 120
mmHg during fluctuations caused by different solution viscosities and the loss of cellular andblood content over time. 3% SDS was perfused for 36 hours, followed by 24 hours in 3% Triton
X-100, with multiple subsequent and intermittent washes with 1X PBS and diH2O.

Cell Culture
Commercially bought Human Umbilical Vein Endothelial Cells (HUVECs) weremaintained in the recommended EMG2 media (Lonza Group, Ltd.). Adipose-derived
stem/stromal cells (ASCs) were isolated from thymic adipose tissue that was designated as
surgical discard. In brief, stem cell isolation followed a published protocol involving mincing of
the fat, digestion of the fat in 0.1% wt/vol type I collagenase (Worthington Biochemical
Products, catalog # LS004197) at 37oC for 45 to 60 minutes, then a series of centrifugation andfiltration steps to isolate the ASCs (Estes, Diekman, Gimble, & Guilak, 2010). Expansion media
for ASCs included 10% FBS (Seradigm, Product # 1400-500), 1% L-glutamine (Corning, Lot #25005289), and 1% Antibiotic-Antimicotic (Sigma Aldrich, catalog # A5955) in 1X DMEM
with 4.5g/L-glucose & L–glutamine (Corning, Lot # 10017257).

Cryosectioning of Acellular Tissues
After a complete decellularization of porcine hearts, small sections up to 2 cm indiameter were taken from the left ventricle (LV), mitral valve (MV), and left anterior descending
coronary artery (LAD). The ECM samples were stored in 90% (v/v) ethanol for a minimum of29

30 minutes and washed vigorously with 1X DMEM with 4.5g/L-glucose & L–glutamine(Corning, Lot # 10017257). Samples were then rinsed and frozen gradually via intermittent
exposures to liquid nitrogen a few seconds at a time in optimal cutting temperature (OCT)compound using customized tissue molds. The OCT blocks were stored overnight at -80oC
before cryosectioning them. Sectioned tissues were 5-7 µm in thickness and collected on square
cover slips.

Reseeding of Acellularized ECM Heart Scaffolds
LAD sections were reseeded at 100,000 cells/cm2 with HUVECs. MV and LV sections
were reseeded at 100,000 cells/cm2 with ASCs. Metal rings were sterilized and placed on top of
sectioned ECM scaffolds, and media with the respective cells was added inside the inner ring.
Each sample was kept in its own well within a 6-well plate, and cultured at 37oC with 95% O2,
5% CO2 for 4 hours and 24 hours to monitor cell adherence, survival, and growth. ECM
scaffolds were also incubated in media without cells as a control. After the incubation period,
reseeded matrices were fixed with 4% paraformaldehyde for 5 minutes and stained with DAPI
mount before imaging on a confocal microscope.

Reseeding of LAD in Whole Acellularized Heart Scaffold
Using a Langendorff preparation as a bioreactor (Figure 2.4), a whole acellularized
porcine heart was cannulated at the ascending aorta using a 3/8” cannula. Perfusion solution
consisted of 1X DMEM with 4.5g/L-glucose & L–glutamine (Corning, Lot # 10017257)
supplemented with 20% FBS (Seradigm, Product # 1400-500) and 10% Antibiotic-Antimicotic(Sigma Aldrich, catalog # A5955) was delivered to the heart in a retrograde manner via the aortic

30

cannula. The chamber was kept at 37oC and a membrane oxygenator was used to keep the gas
content of the perfusion solution at 95% O2 and 5% CO2. A manometer was used to measure thepressure, and perfusion was delivered at a constant pressure of 50 mmHg. 4 million HUVECs
were injected via a sterile 32G needle into the LAD of the acellularized heart as the heart was
being perfused at 50 mmHg. Fresh perfusion solution was provided daily for a total of 5 days.

Cell Count Analysis
After fluorescence images were taken on a confocal microscope using a DAPI filter, they
were saved in 8-bit RGB jpeg files, cropped to a 1:1 ratio. Three images for each tissue type andtime point were selected at random for analysis. Image threshold and cell counts were performedin ImageJ (Schneider, Rasband, Eliceiri, 2012). Data was collected and the standard error was
calculated and plotted in GraphPad Prism 6 (La Jolla, CA).

Angiography
A Toshiba mobile C-arm (Surginix SXT-2000A) system was used to image acellularized
porcine hearts. Perfusion of the coronary sinus was imaged using a 14Fr Edwards LifesciencesTM
Retrograde Cardioplegia Catheter (Edward Lifesciences Services GmbH, Germany) with Isovue300 Iopamidol Injection 61% for contrast media (Bracco Diagnostics Inc., BIPSO GmbH,Germany). Subsequent fluoroscopy images were taken of the left and right main coronaries using
a 2.1 mm right angle Coronary Artery Perfusion Cannula with a self-inflating 5.0 mm balloon(Vitalcor, Westmont Illinois). A 25 G PrecisionGlide needle (Becton Dickinson & CO, Franklin
Lakes NJ) was used for epicardial injections into the apex of the left ventricle. Digitalangiography videos were obtained for each injection route, and a snapshot was taken from the31video showing left coronary artery patency.

2.3 Results
Cellular adherence was seen within 4 hours for all tissue types. ASCs were successfully
seeded onto the left ventricle (Figure 2.1A-C) and mitral valve (Figure 2.1D-F), while HUVECs
were successfully seeded onto the LAD (Figure 2.1G-I). A subsequent cell count using ImageJ
was performed to analyze whether there was increased cell proliferation/adherence over time.
Measured at 4 and 24 hours, there was an increase in the total number of cells in each area withcellular adhesion at 4 hours (Figure 2.2).
Angiograms were taken of the whole acellularized porcine heart prior to reseeding to
identify patency of vasculature. Seen in Figure 2.4, the left coronary artery of the acellular heart
was patent and therefore suitable for recellularization. Branches of the left coronary artery canalso be clearly visualized, including the LAD, left circumflex artery, and diagonal arteries. The
acellular heart was subsequently connected to a Langendorff bioreactor (Figure 2.3). Following 5days of perfusion at constant pressure, the whole acellularized heart was removed from theLangendorf and the LAD portion of the heart was excised and fixed for H&E staining. Cellularadherence of HUVECs was seen in LAD sections (Figure 2.5).

32

Figure 2.1. Recellularization of acellular matrix. Adipose-derived stem cells were seeded at adensity of 100,000 cells/cm2 on acellularized left ventricular (B,C) or mitral valve tissue (E,F),
and maintained in culture for 4 hours and 24 hours, respectively. The tissue prior torecellularization is shown in A and D. Endothelial cells were seeded at a density of 100,000
cells/cm2 on acellularized left anterior descending artery (H,I) and maintained in culture for 4
hours and 24 hours, respectively. The tissue prior to recellularization is shown in G. Images weretaken at 10x magnification.

33

Figure 2.2. Cell count following recellularization. In each respective tissue, the cell count on
the reseeded acellular matrix was higher at 24 hours than 4 hours, showing celladhesion/proliferation. LV=left ventricle; LAD=left anterior descending artery; MV=mitralvalve. Scale bars are represented as SD, with an n=3 for each group and time point.

Figure 2.3. Langendorff Bioreactor Schematic. Perfusate from the reservoir chamber was
pulled into the roller pump and pushed through the membrane oxygenator into the ascending
aorta. Retrograde flow delivered fluid into the right and left coronaries, which ultimately drained
into the right atrium. Effluent exited the heart via the pulmonary artery and was removed fromthe reservoir daily during perfusate change-out through the waste port. Perfusion was kept at aconstant pressure of 50 mmHg, temperature was maintained at 37oC, and perfusate flowing into
the aorta was kept at 95% O2 and 5% CO2.

34

Figure 2.4. Angiogram of whole acellularized heart. Vasculature of the whole acellularized
porcine heart is seen to be patent with vascular integrity. The left coronary artery (white arrow)and its branches (e.g. left anterior descending artery and left circumflex artery) are clearlyvisualized in this angiogram.

Figure 2.5. Langendorff recellularized LAD. A whole acellularized porcine heart was perfusedover 5 days in a Langendorff setup as a bioreactor. 4 million HUVECs were injected into the left
anterior descending artery (LAD) right as it branched from the left coronary artery. The LAD of
the acellularized heart was successfully recellularized with HUVECs at 5 days (black arrows

35

point to nuclei of HUVECs). Some endothelial cells have also lined the surface of the artery wall(hollow white arrows). Tissue is stained with H&E. 10x objective magnification.
2.4 Discussion
Following whole organ acellularization of a large animal heart model, the next challenge
is in the recellularization of the resultant 3-D ECM bioscaffold. Since the thickness of the
myocardium in porcine hearts is much greater than that of rodents and the density of cells andtissues is highly variable it is an extremely complicated process to properly recellularize eachmyocardial layer. Without sufficient recellularization of the heart wall and its many layers, it ismuch more challenging to elicit functional contractions as was accomplished in the muchthinner, acellular rodent hearts (Ott & Taylor, 2008). It is even more difficult to properly injectcells deep within the heart muscle. This is why a bioreactor model may be required to simulate
physiological stress/strain forces experienced during diastole and systole.
A properly functioning vasculature is essential to heart function and cell viability. By
providing a renewable source of proliferating, functional cardiomyocytes and contributing to thedevelopment of a network of blood vessels, healthy vasculature provides support and nourishes
newly formed cardiomyocytes and the surrounding myocardium. Thus, the first issue we faced
was to overcome successful cell-adherence and re-seeding of the vasculature with endothelial
cells onto acellular porcine artery matrix. To check for blood vessel patency, an angiogram was
performed for the acellularized heart, and the vessels were shown to be fully intact (Figure 2.4).
The left coronary artery could be traced as it branches into the left anterior descending artery, left
circumflex artery, and other associated branches. Subsequently, the LAD portion of the acellular
heart was excised and processed into 2D frozen section slices of acellular matrix forrecellularization with HUVECs. After just 4 hours, cell adherence is seen, and after 24 hours in36

culture, there is increased cell adhesion/proliferation (Figure 2.2).
Next, since the myocardium cannot be regenerated by recellularization withcardiomyocytes as these are terminal cells that cannot divide or replicate, stem cells must be usedon many acellular heart surfaces. These stem cells must eventually differentiate into
cardiomyocytes and they must be seeded onto contractile regions of the heart in order for thewhole acellular heart to become functional. Recellularization was performed in this manner
within the acellularized left ventricular tissue and mitral valve (Figure 2.1). Cell adherence by 4hours and proliferation by 24 hours was seen (Figure 2.2). After discovering that our cell types
could adhere to the acellular tissue, we tried recellularization of the acellular heart scaffold in abioreactor Langendorff setup (Figure 2.3). A membrane oxygenator was used to ensure the heart
was perfused with media containing 95% O2 and 5% CO2 at a low constant pressure of 50
mmHg, and at 37oC. The LAD was injected with 4 million endothelial cells, and after 5 days,
there was successful cellular adhesion on the tissue (Figure 2.5).
Utilizing the Langendorff setup as a bioreactor demonstrated that the LAD, one of themain arteries of the heart, was successfully recellularized as shown by cell adherence. Thisshows potential that cells would adhere similarly to other vessels in the heart such as the rightcoronary artery, circumflex artery and the posterior descending artery. Since images obtained
from the angiogram depicted patent blood vessels, it appears possible to re-seed other vascularsystems within the heart. While it would have been interesting to observe cell proliferation overtime, taking samples of the LAD multiple times would have disrupted this proof-of-concept
experiment.
From this study, we therefore propose that recellularized acellular ECM scaffolds can beused instead as a surgical mesh device. Since the major cell types that make up the heart are37

cardiac fibroblasts, myocytes, endothelial cells, and vascular smooth muscle cells, we wanted todemonstrate that appropriate cell types could be reseeded onto our acellular matrix. In addition,
we chose human cell lines to seed onto the acellular matrix to show that current cell types being
administered during cardiac surgery can be used with acellular porcine ECM for recellularizationof different areas of the heart.

2.5 Future Directions
Since our initial data suggests that cells can properly adhere to vessels, valves and tissue
within the porcine heart, it would follow that there is potential to recellularize the different
myocardial chambers. However, future experiments will involve washing the 2D matrices after 4hours of cell adhesion such that proliferation rates can be monitored. Recellularization of the
myocardial chambers would be performed using the Langendorff setup in order to ensure properdelivery of cells to the inner muscular layer of the heart. By maintaining adequate perfusionpressures stem cells could have the opportunity to penetrate myocardial tissue and adhere to thenascent acellular myocardium. Despite the possibility of increased cell adhesion to theendomyocardial tissue, further adherence onto the intramyocardial tissue may be more difficult
to achieve due to the thick nature of the musculature. This is important as the necessity forproper cellular homing, chemotaxis and growth is dependent on the specific placement of stem
cells within different structures of the heart. Even though a bioreactor can encourage cells to
migrate into the deeper layers of heart tissue, guided recellularization will likely be necessary
and more effective than simple stem cell injections. Creating functionally contracting ventricles
in thick acellular ventricular tissue will take a combination of clever and elegant engineering
methods as well as advanced cellular differentiation techniques to make this a clinical reality.

38

Therefore, other current experiments include enhancing cellular invasion through theaddition of olfactomedin-1, which modulates the epithelial-to-mesenchymal transition (EMT),
and TGF-beta 1, which is a stimulus of the EMT. Additional EMT markers will be identified andincluded in future data sets. Similarly, recellularization of the whole heart in the Langendorff
setup will be attempted to repopulate the endocardial surface with stem cells. Stains will include
endothelial markers such as Sca-1 and VEGF.

39

CHAPTER 3
PRESENT STUDY 2: CARDIAC REPAIR AND REGENERATION: RESEEDING POTENTIAL OF
CLINICALLY USED COMMERCIAL MATRICES

Manuscript (to be submitted): Ferng AS, Bajaj N, Nunez M, Runyan R, Khalpey Z. Cardiac
Repair and Regeneration: Reseeding Potential of Clinically Used Commercial Matrices.

40

Introduction
Extracellular matrix (ECM)-containing biological scaffolds are commonly used inreconstruction surgical applications and for functional tissue repair. The ECM serves as not only
a structural scaffold, but is a matrix with a composition that is influenced by the secreted
products of resident cells of each specific tissue and organ. It is known to affect cellularmigration, proliferation and differentiation, and is therefore in a dynamic state of reciprocity withsurrounding cells and its microenvironment.
Creation of ECM bioscaffolds can involve tissue or organ decellularization,lyophilization and processing of biological matrices into a clinically functional material, or denovo synthesis using artificial materials. Many marketed biological scaffold materials are
regulated as surgical mesh devices that are composed of ECM harvested from allogeneic or
xenogeneic tissue sources, such as porcine small intestinal submucosa (CorMatrix®) or humanamniotic membrane (PalinGen KardiaMembrane). During cardiac surgery, surgical meshes andECM are often placed onto the epicardial surface of the heart following wound closure. It isthought that the matrix offers structural support and also facilitates wound healing and tissue
repair by positively effecting cell mitogenesis and chemotaxis (Agrawal, Kelly, Tottey, & Daly,
2011a; Agrawal et al., 2011b; Reing et al., 2009), differentiation (Agrawal et al., 2011b; Crapo etal., 2012), and modulation of the host innate immune response (Badylak, Valentin, Ravindra,
McCabe, & Stewart-Akers, 2008; Brown et al., 2012a; Brown, Ratner, Goodman, Amar, &Badylak, 2012b).
In order for ECM to modulate cellular responses, it is important to understand the 3Dultrastructure, surface ligand landscape and composition of the ECM of the particular material. It
has been shown that there is an improvement in cardiac function using small intestine ECM that41

is associated with the recruitment of c-kit cells, myofibroblasts, and macrophages postmyocardial infarction (Zhao, Puskas, Xu, & Wang, 2010). Given the benefits, cardiac surgeons
will use both a surgical ECM or mesh in addition to stem cells to facilitate cardiac repair andregeneration.
There are many clinical trials in place involving the use of stem cells in tissue repair,including amniotic stem cell injections both locally and systemically. This is due to the fact thatamniotic fluid isolated from placental tissue contains a heterogeneous population of cells
including multipotent amniotic stem cells (AmSCs) that are mesenchymal in nature, a very small
subset of embryonic stem cells, amniotic extracellular matrix (ECM), and multiple growth
factors that have demonstrated wound healing and antimicrobial effects in animal models (Yanget al., 2013). AmSCs have been shown to display immunomodulation properties similar to that ofbone marrow derived stem cells, however, multipotent AmSCs possess greater stem cellproliferative capacities (Yang et al., 2013; Yoon et al., 2010).
Since our lab uses amniotic membrane and CorMatrix during cardiac repair in theoperating room and in the laboratory, we were interested whether the human amniotic stem cells
we used for stem cell therapy could be seeded onto these surgical meshes, and if these cells could
interact with endogenous cells to ultimately grow new tissue at the repair site. In addition toseeding with human amniotic stem cells and studying cell adherence, olfactomedin-1 (OLFM1),
also known as Neolin-1, has been identified as an extracellular matrix protein expressed in thedeveloping heart that is involved in mediating cell invasion during the epithelial-mesenchymaltransition (EMT) in chick embryonic hearts (Lencinas et al., 2013). EMT is a cellular processthat mediates transition from two-dimensional cellular sheets to a three-dimensional structure,
which is possible because mesenchymal cells have the ability, which true epithelia do not, to42

invade and migrate through the ECM (Hay, 1995). EMT is a multistep process that can beinitiated through a variety of signals including growth factors, inflammatory signals andextracellular proteases with the goal of inducting or stabilizing transcriptional repressors (e.g.,
SNAI1 or ZEB1) that will lead to loss of cell-cell adhesion and initiate the cascade of processes
leading to EMT (Nieto, 2011). In the avian heart, EMT is mediated by activities of transforming
growth factor (TGF) β2 that acts as a stimulus to start the cascade that leads to cell invasion withthe upregulation of EMT markers (Lencinas et al., 2013). As a result, our study investigates
whether AmSCs can adhere to clinical matrices, and whether EMT can be induced in an in vitro
invasion assay.

Methods
Seeding of Clinical ECM Scaffolds
ECM scaffolds often used in surgical repair, PalinGen amniotic membrane allograft(Amnio Technology, LLC) and CorMatrix® ECM (CorMatrix Cardiovascular, Inc.), were cut
into 1 cm2 pieces for reseeding. Scaffolds were weighted down with 1-1.5 cm2 metal rings in 6
well plates to prevent curling of the ECM from the edges, to minimize the amount of media
needed, and to keep cells on ECM.
Human amniotic stem cells were purchased (AllCells, Alameda, CA) and cultured in
Celprogen media (Celprogen, Torrance, CA) under standard cell culture conditions before
reseeding on the membrane patches. AmSCs were seeded at 100,000 cells/cm2 per membrane.
Seeded ECM was incubated at 37oC, 95% O2 with 5% CO2 for 4 hours. Control matrices without
cells were incubated with media under the same conditions. After the incubation period,
scaffolds were additionally treated by adding 5 mM of AMP (adenosine monophosphate) in each43

well for 5 minutes for future analysis, and cell-matrix supernatants were frozen for HPLC
analysis. The reseeded ECM was fixed with 4% paraformaldehyde for 5 minutes and stainedwith DAPI mount.

Treatment of Commercial ECM Scaffolds with OLFM1 and TGF-β1
ECM scaffolds were similarly prepared as the 4 hour experiments, where 1 cm2 pieces of
the commercial matrices were cut for reseeding. Scaffolds were weighted down with 1-1.5 cm2
metal rings in 6 well plates to prevent curling of the ECM from the edges. AmSCs were seeded
at 100,000 cells/cm2 per membrane, and cultured for 72 hours with 200 ng/ml OLFM1-V1, 2
ng/mL TGF-β1, or a combination of OLFM1 and TGF-β1. Reseeded ECM then was fixed with
4% paraformaldehyde for 5 minutes and stained with DAPI mount.

Scanning Electron Microscopy
5-mm3 pieces of CorMatrix and amniotic membrane were cut for processing and analysis.
Since these specimens come in a dehydrated form, no further processing was needed. Samples
were mounted on aluminum stubs with carbon double-sided tape and sputter-coated with a 5-nmthin layer of gold (Pelco SC4, Ted Pella, Inc., Redding, CA) to improve conduction andresolution.

Transmission Electron Microscopy
5-mm3 specimens of CorMatrix and amniotic membrane were cut for processing andanalysis. Since samples are dehydrated, no additional sectioning or preparatory work done fornormal tissue sections was needed. Sections were stained with 2% lead citrate and viewed in an

44

FEI Tecnai Spirit electron microscope operated at 100 kV. 8 bit TIFF images were collected via
an AMT 4 megapixel camera.

Results
Successful amniotic stem cell adherence was seen on CorMatrix (Figure 3.1) after 4hours in cell culture. Similarly, amniotic stem cells adhered to amniotic membrane after 4 hours(Figure 3.2). To image surface topography of the matrices, scanning electron microscopy was
performed and the collagenous structure of both Cormatrix and amniotic membrane can be seen
(Figure 3.3). Using transmission electron microscopy, the collagen structure of CorMatrix can beseen, but there is no obvious cellular content visible (Figure 3.4A). On the other hand, theamniotic cell monolayer of amniotic membrane can be clearly visualized, with the nucleus andcell boundaries seen in a monolayer adjacent to the supporting structural membrane (Figure
3.4B).
Addition of OLFM1 alone did not result in increased cell adherence, while addition of
TGF-β1 resulted in increased cell adherence, and addition of combined OLFM1 and TGF-β1
resulted in even more cell adherence than in cells treated with TGF-β1 alone (Figure 3.5).

45

Figure 3.1. Seeding of CorMatrix with Human Amniotic Stem Cells. Amniotic stem cells were seeded
at a density of 100,000 cells/cm2 on CorMatrix (white arrows; C) and maintained in culture for 4 hours.
The matrix prior to seeding is shown in A and B. Images were taken at 10x objective magnification.

Figure 3.2. Seeding of Amniotic Membrane with Human Amniotic Stem Cells. Amniotic stem cells
were seeded at a density of 100,000 cells/cm2 on amniotic membrane (C) and maintained in culture for 4
hours. The matrix prior to seeding is shown in A and B. Images were taken at 10x objective
magnification.

46

Figure 3.3. Scanning Electron Microscopy of CorMatrix and Amniotic Membrane. Images of
CorMatrix and amniotic membrane were taken at 500x, 5,000x, and 10,000 magnification (A-C and D-F,
respectively).

Figure 3.4. Transmission Electron Microscopy of CorMatrix and Amniotic Membrane. The
structural collagen of CorMatrix can be seen in A, with no obvious cellular content. Amniotic membrane
is lined by an amniotic cell monolayer, where the nucleus and cell boundaries can be visualized next tothe supporting membrane (B).

47

Figure 3.5. Treatment of seeded CorMatrix with OLFM-1 and TGF-β1. Amniotic stem cells wereseeded at 100,000 cells/cm2 on CorMatrix, and cultured for 72 hours with 200ng/ml OLFM1-V1,
2ng/mL TGF-β1, or a combination of OLFM1 and TGF-β1.

48

Discussion
ECM of the porcine small intestine consists of a strong ECM that contains collagens,proteoglycans, glycosaminoglycans, glycoproteins such as fibronectin, and growth factors. Thisbiochemically rich and diverse ECM of the submucosa makes CorMatrix an ideal surface forreseeding. As patches are not typically preconditioned in the operating room prior to use, theidea of this proof-of-concept experiment was to seed these clinical patches without additionalsurface adhesion proteins or preconditioning. Since amniotic stem cells are used for cardiac
regeneration during surgery in the form of intramural or intramuscular injections, we wereinterested in their ability to adhere to clinically used ECM scaffolds.
After the addition of amniotic stem cells for just 4 hours at 37oC, cell adherence can beseen on CorMatrix (Figure 1). The thickness of CorMatrix is 0.60 ± 0.15 mm in thickness. Dueto the thickness, it seemed unlikely that amniotic stem cells would be able to adhere within 4hours, but cells can be clearly visualized when compared to control ECM, without the addition ofcells.
In contrast, amniotic membrane is the innermost layer of the placenta, and it ishistologically a multilayer membrane approximately 0.02 to 0.5 mm thick (Meller, Pauklin, &Thomasen, 2011). The amniotic membrane that was seeded was between 5-10 µm thick, whichsuggested an easier surface for cells to adhere. After 4 hours, amniotic stem cells are seen tohave adhered evenly throughout the amniotic membrane (Figure 2). Both CorMatrix andamniotic membrane have similar appearing structural surfaces as seen on SEM (Figure 3).
Amniotic membrane is processed with an amniotic cell monolayer that is visualized with TEM,
which could potentially affect cell adherence and engraftment onto the membrane (Figure 4).
TGF-β1 is a necessary stimulus to induce the epithelial-to-mesenchymal response that is

49

in part mediated by OLFM1. As expected, addition of OLFM1 alone did not result in increased
cell adherence, while addition of TGF-β1 resulted in increased cell adherence (Figure 5).
However, addition of combined OLFM1 and TGF-β1 resulted in even more cell adherence thanin cells treated with TGF-β1 alone (Figure 5) since there was a presumed cellular response from
AmSCs to first the TGF-β1, and then to the OLFM1 that aided in additional cell adherence andinvasion onto the CorMatix. Similar experiments are being conducted on amniotic membraneseeded with AmSCs. If cell invasion is also seen on amniotic membrane, then pre-seeded andpre-conditioned commercial patches with a combination of OLFM1 and TGF-β1 could offer apotential future surgical intervention during cardiac repair. Alternatively, AmSCs injected duringstem cell therapy may be more beneficial during a local injection when coupled with OLFM1
and TGF-β1 to aid in increased cellular invasion and adherence.

50

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55

APPENDIX AACELLULAR PORCINE HEART MATRICES: WHOLE ORGAN PHYSIOLOGICDECELLULARIZATION WITH 3D-BIOSCAFFOLD & VASCULAR PRESERVATION

Manuscript (in submission): Ferng AS, Connell AM, Marsh KM, Qu N, Bajaj N, Palomares D,
Tran PL, Iwanski J, Medina AO, Louis AV, Schipper D, Lotun K, Lagrand D, Johnson K,
Khalpey Z. Acellular Porcine Heart Matrices: Whole Organ Physiologic Decellularization with
3D-Bioscaffold & Vascular Preservation. Tissue Engineering Part A

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Abstract
Regenerative medicine has been increasingly studied due to the growing donor organ
shortage, particularly decellularization-recellularization methods via whole-organ tissue
engineering. Though numerous decellularization protocols exist, the ideal decellularization
protocol for optimal recellularization is unclear. This study was performed to optimize existingheart decellularization protocols and compare current methods using the detergents SDS (sodium
dodecyl sulfate), Triton X-100, OGP (octyl β-D-glucopyranoside), and CHAPS (3-[(3cholamidopropyl) dimethylammonio]-1-propanesulfonate) through retrograde aortic perfusion
via aortic cannulation of a whole porcine heart. The goal of decellularization is to preserve
extracellular matrix integrity and architecture, which was analyzed in this study throughhistology, microscopy, DNA analysis, hydroxyproline content analysis, materials analysis andangiography. Effective decellularization was determined by analyzing the tissue organization,geometry, and biological properties of the resultant extracellular matrix scaffold. Using theseparameters, optimal decellularization was achieved between 90 and 120 mmHg pressure with 3%
SDS as a detergent.

Introduction
While heart transplantation is currently the definitive treatment for end-stage heart
failure, the massive organ shortage has led to increased regenerative medicine and whole-organ
tissue engineering research (1-3). One approach to tissue engineering is the decellularizationrecellularization method (4,5). This method has been successful in regenerating skin, bladders,bone, vessels and upper airways; however, the same level of success has been much moredifficult to achieve in organs with functional units, such as the heart (6,7). The vascularization,

57

high metabolic demand and the low regenerative potential of hearts all contribute tobioengineering difficulties (6,7). However, using a perfusion decellularization method withsubsequent recellularization, the first bioengineered whole rat heart was reported in 2008 (8).
The perfusion decellularization method involved cannulation of the ascending aorta withretrograde aortic perfusion using detergents (8). More recently, an alternate decellularization
approach using serial perfusion and agitation of hypotonic solution has been used in porcine
hearts (9), though the retrograde aortic perfusion method is more standardized (10).
Regardless of the delivery method, the end-goal of decellularization is to produce abioartificial scaffold that resembles the three-dimensional structure and mechanical properties ofnative heart tissue, while maintaining structure of the extracellular matrix (ECM) sufficient forcellular adhesion and growth. The ECM is the direct environment for cells during
recellularization, and it consists of a complex network of proteins, proteoglycans andglycosaminoglycans. Far from being an inert scaffold, the role of ECM has been increasingly
recognized in cell signaling, differentiation and tissue homeostasis. Additionally, it is through the
ECM that cells perceive signals such as shear stress and tensile forces through membrane
receptors known as integrins and mechanosensitive ion channels (11). Decellularized heart ECM
would ideally have minimal fiber “fraying,” as well as a total volume and size similar to theinitial state of each respective heart used. The scaffold must also have similar tensile and biaxial
strain characteristics as a normal healthy heart. Currently, numerous decellularization protocols
exist in an attempt to reach these goals.
One of the variables that affects these quantitative outcomes is pressure control during
the decellularization process. The study of this topic is limited. It has been shown that
automating pressure during decellularization with digital pressure sensors improves whole heart

58

decellularization (12), however the effects of pressures have not been studied despite thevariations in pressure between protocols. The same holds true for total decellularization time.
Few studies focus on the differences in detergent exposure times within non-toxic ranges, andmost studies simply try to limit total exposure time (13). However, the latter goal of limiting
exposure time may be sufficient for producing a useable scaffold matrix. Instead, the maindifferentiating factor between protocols has been the detergents or other decellularization agentsused. The detergents used in the first successful rat heart decellularization were Triton-X andsodium dodecyl sulfate (SDS) (8). The authors perfused heparinized PBS with adenosine for 15
minutes, 1% SDS for 12 hours, and 1% Triton X-100 in a retrograde fashion for 30 minutes witheach step followed by rinsing with deionized water (8). At a larger scale using porcine hearts via
retrograde aortic perfusion, successive perfusates of 0.02% trypsin/0.05% EDTA, 3% Triton X100, and 4% deoxycholate with PBS rinses between reagents also resulted in successful
decellularization (14). Other porcine heart decellularization protocols implemented similar
methods(14,15),

with

a

few

additional

detergents

such

as

CHAPS

(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) (16) used in heart valves and otherorgans (17,18). All detergents have the potential to inflict damage on the ECM, including
damaging the collagen and glycosaminoglycans and disrupting the ultrastructure (18). In
particular, CHAPS retained ECM and mechanical elasticity following lung decellularization
fairly robustly, which made this a detergent of interest. Another novel decellularization agent,
OGP (octyl β-D-glucopyranoside), recently showed promising results compared to otherdecellularization agents in this regard, and with less cytotoxicity to porcine pericardium seen inother solutions (19). This solution has not yet been used in whole heart decellularization, norhave tissues exposed to OGP been tested in recellularization.

59

In the present study, OGP is used along another nonionic detergent (Triton-X 100), an
ionic detergent (SDS), and a zwitterionic detergent (CHAPS) to decellularize whole porcine
hearts. The present study observes the effects of these detergents at varying pressures todetermine the minimum retrograde perfusion pressure necessary to perform a complete
decellularization. Furthermore, our study aims to investigate the mechanical differences between
the decellularized matrices following various detergent treatments, and to ultimately optimize themethodology used for whole heart decellularization.

Materials and Methods

Harvest and surgical preparation of porcine hearts
Adult porcine hearts were obtained from healthy Yorkshire and Hampshire pigs at the
Food Product and Safety Laboratory of the University of Arizona and processed immediately
following procurement. At the time of collection, each whole animal weighed 130 +/- 10 kg.
Procured porcine hearts weighed from 350 to 500 g.
The heart was surgically excised at the great vessels and aorta from adjacent mediastinal
attachments. Cannulation of the aorta was performed by using a cable tie to secure the aorta to a
3/8” cannula connected to the perfusion tubing. A total of 34 hearts were procured, and 7 hearts
were excluded from the study due to errors in procurement, mechanical or computer errors.
Therefore a total of 27 porcine hearts were included in the present study.

Bioreactor Apparatus
The decellularization system used was a custom-built apparatus situated inside a laminarflow hood throughout the entire procedure (Figure 1). Peristaltic pumps were used to exchange

60

solutions within the decellularization apparatus throughout the experiment. Digital pressuregauges and an analog manometer were used to determine the individual perfusion rates necessary
to maintain continuous perfusion pressures of 70-80, 90, 120 or 140 mmHg per experimental
condition.

	
  Figure 1. Apparatus and setup used for porcine heart decellularization.

Whole heart decellularization
The decellularization duration was determined initially by gross examination of the heart,and followed up by more specific analysis and testing that allowed determination of a time pointthat resulted in full decellularization of the heart. Retrograde aortic perfusion of the solutions

61

took advantage of coronary perfusion while keeping pressure constant throughout the procedure
with flow rates adjusted to remain around 90 mmHg, but under physiological pressures ≤120mmHg. The aorta of porcine hearts was cannulated and kept in the customized chamber
described above at room temperature within a cell culture hood for the entire duration of theprocedure. All solutions were introduced via retrograde aortic perfusion through a series ofpumps and carboys containing autoclaved, sterile-filtered solutions. Constant pressure wasmaintained at 70-80, 90, 120 or 140 mmHg for exploratory experiments, with ideal flow rates
remaining around 90 mmHg.
The decellularization solutions used were: 3%, 5%, and 10% SDS, CHAPS, and 1%
OGP, and 3% Triton X-100. All solutions were autoclaved or filtered. Decellularization with
SDS involved use of Triton X-100 to aid in removal of residual SDS, and subsequent PBS to
remove Triton X-100. Following decellularization, the heart was washed with diH2O and 1X
PBS. The optimal decellularization using SDS is as follows: (1) 45 mins: heparin rinse with
10,000 U/L, (2) repeat step 1, (3) 10 mins: diH2O rinse, (4) 12 hours: 3% SDS, (5) 10 minutes:
diH2O rinse, (6) 24 hours: fresh 3% SDS, (7) 10 minutes: diH2O rinse, (8) 24 hours: 3% Triton
X-100, (9) 10 mins: 1X PBS rinse, (10) repeat step 9 twice more, (11) 24 hours: 1X PBS, (12) 24hours 1X PBS. This leads to a total decellularization time of approximately 110 hours.
Decellularization with CHAPS and OGP was performed as a single detergent experiment, withthe same amount of time in detergent as SDS/Triton X-100 experiments. Decellularization withCHAPS and OGP was suboptimal.

62

Histological Assessment
Each decellularized and native heart was biopsied at the right and left atria, right and left
ventricle, and right and left auricles for morphological analysis. The samples were fixed
overnight at room temperature with 10% neutral-buffered formalin, embedded in paraffin, andsectioned into 10-micron thick adjacent sections. Hematoxylin and eosin (H&E) staining wasused to evaluate the presence of nuclear material by standard light microscopy on a Leica
microscope. Masson’s trichome stain was used also to evaluate the collagen in the decellularizedheart ECM.

Transmission electron microscopy (TEM)
From both decellularized and native hearts, 5-mm3 specimens were cut for processing andanalysis. Samples were fixed in 2.5 % glutaraldehyde in PIPES buffer (pH 7.4) overnight.
Samples were washed three times for 10 minutes with PIPES, fixed in 1% osmium tetroxide andthen followed by two washes in DI water. Samples were stained in a block with 2% uranyl
acetate and dehydrated through a graded ethanol series (50%, 70%, 90%, and 100%). Following
infiltration with Spurr’s Resin, the blocks were polymerized at 60°C overnight. 70 nm sections
were cut on a Leica EMUC6 ultra microtome onto 150 mesh copper grids. Sections were stainedwith 2% lead citrate and viewed in an FEI Tecnai Spirit electron microscope operated at 100 kV.
8 bit TIFF images were collected via an AMT 4 megapixel camera.

Scanning electron microscopy (SEM)
From both decellularized and native hearts, 5-mm3 specimens were cut for processing andanalysis, using a standardized protocol. The specimens were first fixed with 2.5% glutaraldehyde

63

in PBS (pH 7.4) at 4°C overnight and washed with DI water. Samples were then dehydrated in a
graded ethanol series (50%, 70%, 90%, and 100%) v/v in DI water at 10-minute intervals foreach concentration. Following this, specimens were freeze-dried with liquid CO2 using a criticalpoint drying apparatus (Polaron model 3100, Energy Beam Sciences, East Granby, CT). Samples
were mounted on aluminum stubs with carbon double-sided tape and sputter-coated with a 5-nmthin layer of gold (Pelco SC4, Ted Pella, Inc., Redding, CA) to provide surficial conduction.

DNA quantification
Approximately 100 mg of native and decellularized porcine hearts were incubated with
400 µl cell lysis buffer and 8 µl Proteinase K (Viagen, 20 mg/ml) overnight in a 55oC water bath.
Samples were then placed in 90oC for 10 minutes to ensure inactivation of Proteinase K. The
digest was vortexed for 1 minute and centrifuged at 13,000 rpm for 10 minutes. The supernatant
was transferred to a new tube and the DNA was precipitated with isopropanol and centrifuged topellet the DNA. The DNA pellet was rinsed with 70% ethanol, dried, and re-suspended innuclease-free water. DNA was quantified using a Thermo Scientific NanoDropTM 1000
spectrophotometer. The DNA concentration of each chamber of the heart was measured, alongwith the left and right auricles. Tissue samples were separated into the epicardial and endocardial
layers. DNA analysis was performed for each sample, and in this case, 3 separate experiments
using the same protocol was performed, and the DNA concentration results were averaged.

Mechanical testing of cardiac tissue
The dynamic mechanical behavior of decellularized epicardial left ventricular cardiac
tissue was measured with a Perkin-Elmer Pyris Diamond dynamic mechanical analyzer (DMA)64

through sinusoidal oscillation of rectangular specimens in tension. The tissue was cut into
rectangular bars of width 10 mm, thickness 1 mm, and length ~20 mm. The rate of oscillation
was 250 µm/min, with a duration of 20 minutes and an endpoint of 5000 µm, or until the sample
slipped from the harness. Prior to each run, the DMA was calibrated by adjusting the force readby the DMA with a 50 g standard weight. Characterization was performed in 1X PBS at 37°C, at
a pH of 7.4. Strain was calculated as the % change in length (m), which was plotted against thecorresponding stress (kPa). Young’s modulus for elasticity was determined from the initial,
linear section of the plotted traces.

Assessment of Collagen Content
A hydroxyproline assay kit (Sigma Aldrich, MAK008-1KT) was used to assess thecontent of collagen in cardiac tissue samples. The manufacturer’s protocol was used for theassay, and absorbance readings were taken at an excitation wavelength of 560 nm.

Angiogram of decellularized heart
Decellularized hearts were imaged using a Toshiba mobile C-arm (Surginix SXT-2000A)
system. Images were obtained in the cranial, caudal, RAO and LAO planes. Perfusion of thecoronary sinus was imaged using a 14Fr Edwards LifesciencesTM Retrograde Cardioplegia
Catheter (Edward Lifesciences Services GmbH, Germany) with Isovue-300 (Iopamidol Injection
61%, molecular weight: 777.08 g/mol) for contrast media (Bracco Diagnostics Inc., BIPSO
GmbH, Germany). Subsequent fluoroscopy images were taken of the left and right main
coronaries using a 2.1 mm right angle Coronary Artery Perfusion Cannula with a self-inflating
5.0 mm balloon (Vitalcor, Westmont Illinois). A 25 G PrecisionGlide Needle (Becton Dickinson

65& CO, Franklin Lakes NJ) was used for epicardial injections into the apex of the left ventricle.
Digital angiography videos were obtained for each injection route.

Results
Decellularization of whole porcine heart
The ultimate objective of organ decellularization is to remove all of the cellular material
without adversely affecting the composition, biologic activity, or structural integrity of theremaining three-dimensional extracellular matrix. In this study, decellularization conditions were
controlled using a customized bioreactor system. 3% SDS with subsequent Triton X-100 resulted
in a successful decellularized heart. Observing the endocardium grossly at the surface (Figure 2)
and with a sagittal cut (Figure 2B), decellularization appeared successful. As expected, eachheart also had a decreased weight after decellularization (pre-decellularization weight: 431 ± 54
g; post-decellularization weight: 320 ± 70 g; average ± SD). The results from the exploratory
experiments performed at constant pressures of 70-80, 90, 120 or 140 mmHg, separately, aresummarized in Table 1.
By gross visualization and microscopically, CHAPS and OGP treated hearts were notsuccessfully decellularized (data not shown). Cellular components remained even after
lengthening the usual 5-day method to 21 days at a constant pressure of 120 mmHg. A higher
perfusion pressure of 140 mmHg produced similar results.

66

	
  
	
  
	
  
	
  

	
  
Figure 2. Gross images of the native control porcine heart (A,C) and decellularized porcine heart (B,D)
before and after experiment. C and D are cut in the sagittal plane.

67

Experimental
Condition	  Decellularization Method Used	
  Result Summary	  70-80 mmHg	
  

SDS + Triton X-100	
  Incomplete: Pressures were not highenough for complete decellularization
after 5 days.	
  90 mmHg	  

SDS + Triton X-100	
  Complete: This was the ideal pressure touse, and resulted in complete
decellularization.	
  120 mmHg	  

SDS + Triton X-100; CHAPS; OGP	  Complete: While the results were withinreason using SDS, the matrix at 90mmHg more closely resembled native
cardiac tissue. CHAPS and OGP showed
an incomplete decellularization after 5days, and even waiting 21 days total
using fresh solutions, decellularization
was still incomplete.	
  140 mmHg	  

SDS + Triton X-100; CHAPS; OGP	  Complete: Even though fulldecellularization was seen in SDS hearts,
this pressure was too high, and resulted
in matrix that had wide spacing. At thispressure, CHAPS and OGP did not resultin a fully decellularized heart.	
  Table 1. Experimental conditions for heart decellularization. All experiments listed were performed atconstant pressure.

68

Histological assessment
H&E staining revealed no visible basophilic staining representative of cellular nuclear
material in the left ventricle after 5 days using the SDS decellularization protocol (Figure 3B). In
contrast, cell nuclei were visible in the left ventricle of the native control heart (Figure 3A), as
evidenced by the presence of basophilic staining. The tissue morphology remained largely intact
following decellularization, as native and decellularized cardiac histology can be directlycompared, with cell ghosting seen in the decellularized heart where cardiomyocytes are present
in native cardiac tissue (Figure 3B). Cardiac architecture after trichrome collagen staining ofdecellularized samples (Figure 3D) was representative of native hearts (Figure 3C).

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM)
SEM analysis (Figure 4) indicated the maintenance of architecture after intact porcine
heart 5-day decellularization. The epicardial left ventricular wall of the porcine heart in thenative control (Figure 4A) was comparable to the epicardial wall of decellularized heart tissue(Figure 4B). This shows that there was architectural preservation following the 5-day
decellularization process. The endocardial surface of the decellularized heart (Figure 4D)
indicated a topographic variance and intact ECM fibers without the presence of cells (shown byarrows in Figure 4C), which showed that ECM components, particularly collagen, were
preserved.
TEM imaging indicated that there were no nuclei at the completion of the 5-day
decellularization process (Figure 5B). However, tissue basement membrane was intact andarchitectural integrity was largely maintained while cellular material has been predominantly
removed since collagen bundles were still present and clearly recognizable on TEM (Figure 5B).

69

In the native heart tissue, cellular components such as the mitochondria were clearly visualized(Figure 5A).

	
  Figure 4. Scanning Electron Microscopy (SEM) images of native and decellularized porcine heart.
Surfaces of the porcine heart were similar between the native and decellularized tissues (A vs. B), and theremoval of cells can be seen between C (white arrows) and D. 	
  

70

	
  
	
  
	
  
	
  

	
  
Figure 5. Transmission Electron Microscopy (TEM) images of native (A) and decellularized (B)porcine heart. There was an absence of cellular structures, such as mitochondria, from the decellularized
cardiac tissue. However, collagen bundles were preserved in the decellularized cardiac tissue.	
  

71

	
  
	
  
	
  

	
  
Figure 3. Histological images of the native and decellularized heart. Under standard H&E staining,
nuclei (denoted by arrows) in the native heart (A) are absent following decellularization (B). Masson’s
trichrome stain demonstrated preservation of collagen in the decellularized heart tissue (D) whencompared to the native heart (C). Images taken at 4x magnification.

72

DNA quantification
DNA quantification analysis from native and decellularized hearts demonstrated asignificant decrease in the amount of DNA present in decellularized heart tissue compared tonative heart tissue (Figure 6). Using a NanoDrop spectrophotometer, it was determined that over
90% of DNA was removed in decellularized tissue from each chamber of the heart via a constantperfusion pressure of 120 mmHg, prior to the use of nucleases for recellularization.

Figure 6. DNA analyses of each chamber of the heart following decellularization. RA=right atrium;
RV=right ventricle; LA=left atrium; LV=left ventricle; epi=epicardium; endo=endocardium; R=right,
L=left.	
  73

Cardiac tissue mechanics
The epicardial layer of the left ventricle from each respective experiment was stretched
via sinusoidal oscillation at 37oC until the tissue tore apart or slipped out of the machine.
Young’s modulus of elasticity was then calculated from the slope of the initial linear portion ofeach data plot collected. The native heart demonstrated the largest modulus of elasticity whencompared with the decellularized heart. Hearts decellularized with 3% SDS exhibited an elastic
modulus closest to the native heart, whereas decellularization with CHAPS and OGP resulted invery low elasticity values.

Experimental
Condition
Native Heart
Decell with 3% SDS
Decell with 5% SDS
Decell with 10% SDS
Decell with CHAPS
Decell with OGPYoung’s Modulus(kPa)
331.79320.44205.8
27.8294.237
0.6187

Table 2. Dynamic Materials Analysis of Cardiac Tissue. The epicardial layer of the left ventricle wasstretched via sinusoidal oscillation at 37oC. Young’s modulus of elasticity was calculated, and values are
representative from one sample in each group due to limited tissue availability.

74

Assessment of Collagen Content
The collagen content of the hearts decellularized with SDS was similar to the collagencontent found in the native heart (Figure 7). OGP and CHAPS had significantly less collagencontent than the native heart.

	
  Figure 7. Hydroxyproline Assay for Collagen Content. The collagen content of the hearts
decellularized with SDS was similar to the native heart. OGP and CHAPS had significantly less collagencontent than the native heart. n=3 for each group; * = p<0.05; error bars expressed as SEM.

75

Angiogram of decellularized heart
The results of the angiogram through the right coronary artery, left coronary artery,coronary sinus, as well as the associated branches and circumflex arteries can be seen in Figure
8, showing patency of the vasculature.

	
  

	
  
Figure 8. Angiogram of Decellularized Heart. The angiogram shows patency and vascular integrity ofthe vessels through the right coronary artery (A), left coronary artery (B), and coronary sinus (C).

Discussion
Despite many years of research on the topic of decellularization, a multitude ofdifficulties remain. The replicability of decellularized bio-scaffolds using existing methods
remains inconsistent, calling for a decellularization standard (20). In an attempt to quantitatively
standardize decellularization results, Crapo et al. reviewed general parameters with an emphasison minimizing residual DNA. Though decellularization techniques have thus far been unable toremove 100% of cell material in larger animal model and human hearts, these parameters all

76

focus on limiting nucleic material since residual DNA is directly correlated to adverse hostreactions and may contribute to cytocompatibility issues upon reintroduction of cells (20,21).
Prior to the use of nucleases to remove DNA, we were interested in the effectiveness ofretrograde perfusion through the aorta and wanted to identify which regions had higher residual
DNA content. In line with the established parameters, the decellularized porcine hearts in thepresent experiment had <50 ng DNA per mg ECM dry weight from each of the 4 heart chambers(Figure 6) and lacked visible nuclear material in tissue sections stained with H&E (Figure 3).
The final DNA content in the endocardium and epicardium of the right ventricle was greater thanthat of the left ventricle, whereas the right and left atria had comparable DNA concentrations.
The differences in the DNA content of the ventricles might be explained in part by the use ofretrograde aortic perfusion, defined this way due to the retrograde flow through the aortic
cannula. Perfusion in this manner inevitably causes differences in perfusion pressures acrossregions of the heart, with left heart pressures greater than the right. This therefore results invariation of decellularization across the heart chambers and vasculature.
Another general complication in the field of decellularization is “scaling up” current
methods to human-sized organs. Much of the existing research involves the use of rodent organs,which are orders of magnitude smaller than a porcine heart. The larger overall size and increased
heart wall thickness pose a challenge to achieving full decellularization of porcine at low
pressures. Using a pressure of 70 mmHg resulted in incomplete decellularizations, whereas apressure of 140 mmHg disturbed the ECM integrity (Table 1). After testing various perfusionpressures between 70-140 mmHg, it was determined that a minimum perfusion pressure >80
mmHg but <120 mmHg was optimal for achieving complete decellularization through a whole
porcine heart. Maintaining a truly constant perfusion pressure was not possible due to the fact

77

that flow rates change depending on the amount of material removed from the native heart, as
well as the viscosity of the fluids both newly introduced and over time. We used a digitalpressure sensor (PendoTECH, Princeton, NJ) set to adjust the flow rate to create a constantperfusion pressure, which was checked by a manual manometer. Due to the flux ofviscoelasticity of the heart ECM and remaining blood/cellular contents over the duration of
decellularization, there is a ±10-20 mmHg variability in perfusion pressure across experiments.
Our proposed decellularization method results a complete decellularization determined byhistology, microscopy, and DNA analysis (Figures 3-6). Moreover, a complete decellularization
can be grossly determined by a translucent-white tissue appearance (Figure 2), as opposed tomaintaining native tissue coloring. Finally, an angiogram performed on a heart decellularizedwith 3% SDS showed that the vasculature was intact and patent through the right coronary
artery, left coronary artery, coronary sinus, circumflex arteries, and associated branches (Figure
8).
Beyond the DNA, histological and microscopic analyses, as well as the gross appearance
and intact vasculature, the final engineered heart tissue must have the structural integrity todevelop systolic force following recellularization. It must be sufficiently compliant to withstandphysiological diastolic loads, and form an electromechanical syncytium. Hence, it is important topreserve the structural and matrix components of the heart, consisting of collagen, elastin,
proteoglycans, glycosaminoglycans, fibronectin, and laminin, among other components. A
hydroxyproline assay was done to analyze collagen content on decellularization experiments
performed with 10% SDS, 5% SDS, 3% SDS, OGP, and CHAPS (Figure 7). The collagencontent of the hearts decellularized with SDS was not significantly different than native heart.
OGP and CHAPS had significantly less collagen content than the native heart.

78

SDS hearts had similar positive findings in measurements of tissue elasticity, evaluatedby recording the maximum force necessary to tear the outside layer of the heart. With systolic
function in mind, dynamic materials analysis was performed on left ventricular epicardial tissue
and the elasticity modulus was calculated for decellularization experiments performed with 10%
SDS, 5% SDS, 3% SDS, OGP and CHAPS (Table 2). Native cardiac tissue had the highest
elastic modulus, followed by 3% SDS, 5% SDS, 10% SDS, CHAPS, and OGP, respectively.
While we tested a series of detergent concentrations, it was determined that 3% SDS was theminimum concentration required to perform a complete decellularization within our 5-day
experiment. Greater amounts of detergents had a degenerative effect on the ECM, demonstrated
by the lower elasticity values. CHAPS and OGP were continued for a total of 21 days, including
PBS and water washes. The longer experiment time may explain, in part, its lower elastic
modulus.
In summary, this study aimed to build upon existing heart decellularization protocols tofurther optimize and standardize current methods. Perfusion pressures were added as anadditional experimental variable and mechanical tissue properties as an additional endpoint. To
optimize reseeding potential of a decellularized bioscaffold, we propose using retrograde aortic
perfusion via aortic cannulation, between 90 and 120 mmHg pressure, and 3% SDS as adetergent for optimal decellularization.

79

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Tapias LF, Ott HC. Decellularized scaffolds as a platform for bioengineered organs. Curr Opin
Organ Transplant. 19(2), 145, 2014.

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Faulk DM, Johnson SA, Zhang L, Badylak SF. Role of the extracellular matrix in whole organ
engineering. J. Cell. Physiol. 229(8), 984, 2014.

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Yagi H, Soto-Gutierrez A, Kitagawa Y. Whole-organ re-engineering: a regenerative medicine
approach to digestive organ replacement. Surg. Today. 43(6), 587, 2013.

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Song JJ, Ott HC. Organ engineering based on decellularized matrix scaffolds. Trends Mol Med.
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Murphy SV, Atala A. Organ engineering--combining stem cells, biomaterials, and bioreactors to
produce bioengineered organs for transplantation. Bioessays. 35(3), 163, 2013.

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Orlando G, Baptista P, Birchall M, De Coppi P, Farney A, Guimaraes-Souza NK, et al.
Regenerative medicine as applied to solid organ transplantation: current status and futurechallenges. Transpl Int. 24(3), 223, 2011.

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Tee R, Lokmic Z, Morrison WA, Dilley RJ. Strategies in cardiac tissue engineering. ANZ J Surg.
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Ott HC, Matthiesen TS, Goh S-K, Black LD, Kren SM, Netoff TI, et al. Perfusion-decellularized
matrix: using nature's platform to engineer a bioartificial heart. Nat Med. 14(2), 213, 2008.

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Methe K, Bäckdahl H, Johansson BR, Nayakawde N, Dellgren G, Sumitran-Holgersson S. An
alternative approach to decellularize whole porcine heart. Biores Open Access. 3(6), 327, 2014.

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Guyette JP, Gilpin SE, Charest JM, Tapias LF, Ren X, Ott HC. Perfusion decellularization ofwhole organs. Nat Protoc. 9(6), 1451, 2014.

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Fernandes H, Moroni L, van Blitterswijk C, de Boer J. Extracellular matrix and tissue engineering
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Momtahan N, Poornejad N, Struk JA, Castleton AA, Herrod BJ, Vance BR, et al. Automation ofPressure Control Improves Whole Porcine Heart Decellularization. Tissue Eng Part C Methods.
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Friedrich LH, Jungebluth P, Sjoqvist S, Lundin V, Haag JC, Lemon G, et al. Preservation of aortic
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Wainwright JM, Czajka CA, Patel UB, Freytes DO, Tobita K, Gilbert TW, et al. Preparation ofcardiac extracellular matrix from an intact porcine heart. Tissue Eng Part C Methods. 16(3), 525,
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Weymann A, Patil NP, Sabashnikov A, Jungebluth P, Korkmaz S, Li S, et al. Bioartificial Heart:
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Petersen TH, Calle EA, Colehour MB, Niklason LE. Matrix Composition and Mechanics ofDecellularized Lung Scaffolds. Cells Tissues Organs. 195(3), 222, 2012.

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Remlinger NT, Wearden PD, Gilbert TW. Procedure for Decellularization of Porcine Heart by
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Booth C, Korossis SA, Wilcox HE, Watterson KG, Kearney JN, Fisher J, et al. Tissue engineeringof cardiac valve prostheses I: Development and histological characterization of an acellular
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Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization
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82

APPENDIX B
HUMAN ADIPOSE-DERIVED STEM/STROMAL CELLS: COMPARATIVE ORGAN SPECIFIC
MITOCHONDRIAL BIOENERGY PROFILES

Manuscript (in submission): Ferng AS, Marsh KM, Fleming JM, Schipper D, Bajaj N, Connell
AM, Pilikian T, Johnson K, Runyan R, Black S, Szivek JA, Khalpey Z. Acellular Porcine Heart
Matrices: Human Adipose-Derived Stem/Stromal Cells: Comparative Organ Specific
Mitochondrial Bioenergy Profiles. Stem Cells

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Abstract
Adipose-derived stromal/stem cells (ASCs) isolated from the stromal vascular fraction(SVF) are a source of mesenchymal stem cells that have been shown to be beneficial in many
regenerative medicine applications. ASCs are an attractive source of stem cells, in particular due
to the lack of immunogenicity. This study examines differences between mitochondrial
bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial,
thymic, knee, shoulder and abdomen. Through an investigation of mitochondrial respiration andglycolysis, it was demonstrated that bioenergetic profiles do not significant differ by region due
to depot-dependent and donor-dependent variability. Thus, although the physiological function,
microenvironment and anatomical harvest site may directly affect the characteristics of ASCs
isolated from different organ regions, the ultimate utility of ASCs is independent of the
anatomical harvest site.

Introduction
Adipose-derived stromal/stem cells (ASCs) isolated from the stromal vascular fraction(SVF) have long been considered an abundant and ideal source of stem cells for tissue
regeneration and stem cell research. The SVF is the densest component of the lipoaspirate
obtained from liposuction of excess adipose tissue. SVF isolates typically contain many
components also found in adipose tissue including adipocytes, fibroblasts, preadipocytes, tissue
resident macrophages, and vascular constituents [1-3]. Most importantly, both adipose tissue and
SVF provide a source of mesenchymal stem cells (MSCs) that do not elicit an immunologicalresponse, particularly if autologous ASCs are used during point-of-care applications [4,5]. Of thethree main sources of MSCs currently used for human studies and therapies— bone marrow,

84

umbilical cord blood, and adipose tissue— ASCs show important differences in regard to harvestand cell yield, despite similarities to bone-marrow derived MSCs in morphology and phenotype.
Therefore, ASCs represent an attractive, abundant and readily available cell type for regenerative
medicine. ASCs have been successfully used in tissue or wound regeneration such as sternal
reconstruction [6], cosmetic reconstruction [7], or chondrogenesis [8,9]. A major reason ASCs
have shown great promise is that ASCs from the SVF have 50-74% of the matrix-forming abilityof fibroblasts, which can be helpful in wound healing [10].
There are a variety of methods of ASC isolation that include mechanical, chemical, or
non-enzymatic techniques. However, collagenase-based isolation methods yield the highest levelof cell recovery. Mechanical methods have reported cell yields ranging from 6,250 to 25,000
ASCs/mL of adipose tissue, whereas collagenase methods yield 100,000 to 500,000 viable
cells/mL [2,11-13]. While the use of collagenase-isolated ASCs in patient-specific applications
may cause local allergic reaction, infection, evidence of local tissue destruction, and otherpossible injection-related complications, clinical studies in 164 patients have shown little or nocomplications related to collagenase isolations [13].
ASCs all share similar characteristics despite isolation protocol or anatomic location [14],and it has recently it has been recognized that adipocytes demonstrate significant intrinsic
inflammatory properties [15]. Since adipose tissue is a mediator of inflammation and innate
immunity, its anatomic location plays a defining role in its specific metabolic functions [15,16].
The bioenergetic demand of cells from various locations is therefore expected to vary, in part due
to individual physiologic organ functions. Furthermore, the physiological microenvironment that
supports stem cells in specific anatomic locations can regulate how stem cells participate intissue regeneration, maintenance and repair, and may also be donor-dependent [17]. We

85

therefore hypothesized that bioenergetic demand of adipose tissue will be correlated to intrinsic
metabolic function and dependent on anatomical location. In this study, we compare thebioenergetic profiles of freshly isolated adipose tissue of the SVF obtained from the abdomen(subcutaneous), thymus, pericardium, knee (peri-patellar), and shoulder of healthy humanpatients.

Methods

Primary Culture of Human Adipose-Derived Stem Cells
ASCs were isolated from freshly excised human subcutaneous adipose tissue following
adapted methods described by Estes et al. [9]. All samples were obtained as surgical discards
with approval from the University of Arizona Internal Review Board from the adipose tissue
surrounding the following regions: abdominal (subcutaneous), knee (peri-patellar), shoulder,
pericardium, and thymus. In brief, adipose tissue was minced and washed extensively with 1X
PBS + 1% Antibiotic-Antimycotic (Thermo Scientific, Waltham, MA), and then incubated at
37oC for 60 minutes in 2 mg/ml of type I collagenase (Worthington Biochemical, Lakewood, NJ,
USA). Enzyme activity was neutralized with Dulbecco’s modified Eagle’s medium-low glucose(DMEM, Gibco, Grand Island, NY, USA), containing 10% FBS, and cells were centrifuged at
1200 rpm for 10 minutes to remove adipocytes and filtered through a 100 µm nylon mesh toremove debris, and incubated in a culture flask at 37oC at standard cell culture conditions of 95%
O2 and 5% CO2. Following incubation for 3 days, the cells were washed with 1X PBS to remove
non-adherent cells. ASCs were isolated from adipose of 12 donors, with an n=4 for each tissue
type.

86

Bioenergetic ProfilingStress tests were completed using the Seahorse Bioscience XFe96 Flux analyzer
according to the manufacturer’s instructions to measure oxygen consumption (mitochondrial
stress test) and extracellular acidification (glycolysis stress test). The reagents used for the
mitochondrial stress tests were optimized for maximal effect for ASCs isolated from each periorgan region, and all chemicals were obtained from Seahorse Biosciences or Sigma Aldrich.
Cells were seeded into 96-well plates at their previously optimized density and allowed to adhere
overnight. Prior to running the mitochondrial or glycolysis stress assays, cells were first washedwith assay media containing appropriate bioenergetic substrates, and incubated at 37oC in a nonCO2 environment 60 minutes prior to the Seahorse assay in order to allow the cells to become
equilibrated with the assay medium. The XFe96 sensor cartridge was pre-hydrated overnight atroom temperature prior to assay with calibrant solution.
Oxygen consumption rates (OCR) were measured in the presence of oxidative
phosphorylation (OXPHOS) driving substrates. After 3 basal measurements, 3 measurements
each were taken after the subsequent addition of oligomycin, FCCP (carbonyl cyanide-4trifluoromethoxyphenylhydrazone), and rotenone/antimycin A combination. These injecteddrugs block ATP synthase, uncouple the oxygen consumption from ATP synthesis, and block
mitochondrial complexes I and III, respectively. For the glycolysis stress test, drugs weresimilarly optimized for each cell type and injected in the following order: glucose, oligomycin,
and 2-DG (2-deoxy-D-glucose; glycolysis inhibitor).

87

Mitochondrial Stress Test Calculations
The basal respiration was recorded as the basal OCR minus non-mitochondrial OCR.
Following inhibition of ATP synthase by oligomycin, ATP production was calculated bysubtracting the proton leak from basal respiration. Proton leak was defined as remaining basal
respiration not coupled to ATP production. After injection of the uncoupler FCCP, maximal
respiration was calculated by subtracting the non-mitochondrial respiration from the highest
OCR value obtained. The respiratory control ratio (RCR) was then calculated by dividing
maximal respiration (state 3u) by proton leak (state 4o). Coupling efficiency was calculated as apercentage by dividing ATP production by basal respiration and multiplying the final value by
100.

Glycolysis Stress Test Calculations
Glycolysis was defined as the extracellular acidification rate (ECAR) due to theutilization of exogenously provided glucose. After addition of glucose, glycolysis was calculated
by subtracting non-glycolytic acidification (NGA) ECAR with the highest ECAR value, where
NGA was extracellular acidification not due to utilization of exogenous glucose. Following
injection of oligomycin, glycolytic capacity was calculated as the basal ECAR subtracted fromthe highest ECAR used to meet cellular demands after ATP synthase is inhibited. Glycolytic
reserve is calculated as a percentage by dividing glycolytic capacity with glycolysis, andmultiplying by 100.

88

Statistical Analysis
A one-way ANOVA post hoc analysis was conducted to compare for significancebetween each parameter for ASCs isolated from different adipose tissue organ locations. Where
there was significance found in the ANOVA test for a measured parameter (alpha = 0.05), a
Tukey’s HSD test was performed to identify pairwise significance. Statistics were performed in
R, and graphs were created in GraphPad Prism 6 (La Jolla, CA).

Results
Basal respiration of shoulder-region ASCs had the highest OCR when compared to theother groups: pericardial, thymic and abdominal (Figure 1A). OCR associated with ATP
production was similar across all groups with no significance (Figure 1B). Proton leak OCR was
the highest in shoulder ASCs, with significance when compared to abdominal ASCs, whereas
there were no significant differences between the other groups (Figure 1C). Maximal respiration
OCR was similar in all groups (Figure 1D). Although the respiratory control ratio (RCR) was
highest in abdominal ASC there were no significant differences between the groups (Figure 1E).
Similarly, there were no significant differences in coupling efficiency (CE) between the groups(Figure 1F).
There were no significant differences in glycolytic ECAR between the groups (Figure2A). The glycolytic capacity was significantly higher in shoulder ASCs than thymic andpericardial ASCs (Figure 2B). There was no were no significant differences in glycolytic reserve
between the groups (Figure 2C).

89

Although non-mitochondrial respiration was highest in knee ASCs, there were no
significant differences between the groups (Figure 3A). ECAR for non-glycolytic acidification
was significantly lower in abdominal ASCs compared to knee and shoulder ASCs (Figure 3B).

Figure 1. Mitochondrial Stress Test Calculations. Significant oxygen consumption rates (OCR) wereidentified in shoulder ASCs, where OCR values were higher than other ASC groups under basal
respiration and proton leak. Graphs are presented as mean ± SEM, with n=4 for all organ specific adipose
tissue types.

90

Figure 2. Glycolysis Stress Test Calculations. Significant extracellular acidification rates (ECAR) wereidentified for shoulder ASCs under glycolytic capacity, where the ECAR value was highest in this group.
Graphs are presented as mean ± SEM, with n=4 for all organ specific adipose tissue types.

Figure 3. Non-Mitochondrial Respiration and Non-Glycolytic Acidification Calculations. Abdominal
ASCs had the lowest ECAR rates under non-glycolytic acidification. OCR: oxygen consumption rate;
ECAR: extracellular acidification rate. Graphs are presented as mean ± SEM, with n=4 for all organ
specific adipose tissue types.

91

Discussion
The emerging field of regenerative medicine has identified adipose tissue as an abundantsource of stromal/stem cells for tissue engineering applications. Due to its mesodermal origin,
ASCs can differentiate into adipose lineage cells [30], osteogenic cells [3], chondrogenic cells[31] and myogenic cells [32], making ASCs ideal for tissue repair and regeneration.
Interestingly, ASCs isolated from subcutaneous tissue have been shown to demonstrate enhanced
adipogenic differentiation capacity relative to ASCs derived from other areas [18]. Extrapolating
from

this

concept,

wecomparedthe bioenergetic

profilesof adipose-

derived stromal/stem cells isolated from five different subcutaneous adipose sites. Though 12
patients were included in this study, it is likely that the general trends reported here will change
between patient populations depending on the patient’s age, state of health, genetics,
environment, gender, race, ethnicity, etc. Indeed in both humans [18] and animals [17,19] there
is adipose depot-dependent and donor-dependent variability that alters cell yield, viability,
immunophenotype, doubling time, differentiation capacities and metabolic differences.
Overall, shoulder ASCs had the highest basal respiration, proton leak and glycolytic
capacity. To discuss these parameters, basal respiration is defined as the oxygen consumed that isused to meet cellular ATP demand. This means that basal respiration is strongly controlled by
ATP turnover, and partly by substrate oxidation and proton leak [20]. Proton leak refers to theremaining basal respiration not coupled to ATP production. Glycolytic capacity refers to the partof ECAR used to meet cellular demands when ATP synthesis has been inhibited by oligomycin.
Conceptually, isolated mitochondria can reach the respiratory state 4o when ATP
synthase is inhibited by oligomycin, stopping proton re-entry through the synthase and slowing
respiration. Respiration is then strongly controlled by proton leak kinetics and partially by

92

substrate oxidation in state 4o, making respiration responsive to dysfunction caused byuncoupling, relatively insensitive to changes in substrate oxidation, and completely insensitive tochanges in ATP turnover [20,22]. State 4, which is similar to state 4o but with a contributing
factor of ATP recycling, is sensitive to changes in both proton leakiness and ATP turnover [21].
In these hypothetical states where the mitochondria are isolated, a large increase in proton leakwould indicate that mitochondria are severely damaged due to uncoupling. However, proton leakis not just an artifact of isolated mitochondria; it is also observed in intact cells. Indeed, proton
leak can account for a large percentage of oxygen consumption in resting cells [19,21,23],
though it should be noted that the proton leak data in the current experiment is unavoidably
elevated since the addition of oligomycin inhibits phosphorylation and subsequently increases
proton leak. In vivo, standard metabolic rate can vary between different major oxygenconsuming tissues. Cardiac tissue can have a low proton leak, whereas liver and skeletal muscle
will have moderate and high proton leaks, respectively [19,24,25]. Therefore, it might be
expected that ASCs isolated from an area near skeletal muscle would have a higher proton leakvalue, since the ASCs within the shoulder adipose tissue contribute to healing and repair ofshoulder muscles. Indeed in a rotator cuff model using ASCs, the adipose tissue and increased
fatty infiltration of the muscle resulted in better healing and regeneration [26]. Moreover, ASCs
derived from rotator cuff regions have greater myogenic potential than bone marrow derived
mesenchymal stem cells [27,28]. Since cellular ATP demand is also greater in skeletal muscle, it
is logical for shoulder ASCs to have a higher basal respiration. In line with this logic,
subcutaneous abdominal ASCs are isolated from a region that does not require fast cell turnoverand cells that support an increased bioenergetic state, unlike ASCs from omental adipose tissue[18,29]. In the present experiment, the ECAR of abdominal ASCs was found to be lowest of all93groups under non-glycolytic acidification. Moreover, the higher glycolytic capacity observed inshoulder ASCs can be explained by the higher ECAR necessary to meet the cellular demands
associated with increased basal respiration and proton leak.
In our studies we also estimated the coupling efficiency (CE) and the respiratory control
ratio (RCR), of the isolated ASCs. CE is calculated as the proportion of mitochondrial
respiratory rate used to drive ATP synthesis (i.e., perfectly coupled OXPHOS will have a
CE=100%, while a pure state 4 respiration will have CE=0%) [21]. RCR is calculated as a ratio
between the cellular proton leak and their maximal capacity. Therefore, a change in either one ofthese 2 parameters will alter RCR, offering a solid estimation of overall bioenergetic
dysfunction. Since CE and RCR are calculated as ratios, both measures are useful internal
controls. However, the CE and RCR did not significantly differ between ASCs from eachlocation in this study. The fact that we observed no significant differences in CE and RCR
between groups could be explained by biomechanics and organ function. Due to the constant, but
normal, wear and tear of pressures imposed upon the knee joints during ambulation, the cells inthat peri-organ region require a faster rate of healing, proliferation and regeneration. Similarly,
the function of human heart requires the peri-organ milieu to support quick cell turnover,proliferation and regeneration. Furthermore, due to the high oxygen demand of cardiac cells
required to sustain diastolic and systolic contractions, it is logical that pericardial ASCs would
display a maximal respiration state compared to ASCs from skeletal muscle regions (e.g.,shoulder).
In summary, our data suggest that ASC utility is bioenergetically independent ofanatomical adipose tissue harvest site. However, it is still likely that bioenergetic differences willvary depending on the individual “stem cell niche”, which describes the physiological94

microenvironment that supports stem cells in specific anatomic locations and regulates how stem
cells participate in tissue generation, maintenance and repair [17]. Therefore, while subcutaneous
ASC utility may be overall independent of the anatomical harvest site [34], the bioenergetic
profile of ASCs from adipose tissue of various peri-organ regions will be reflected by its
respective physiological functions, niche and microenvironment.

95

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100

APPENDIX CCARDIAC ORGAN PRESERVATION SOLUTIONS: MITOCHONDRIAL SUSPENDEDANIMATION

Manuscript (in submission): Ferng AS, Schipper D, Bajaj N, Connell AM, Marsh KM, Knapp
S, Johnson K, Khalpey Z. Cardiac Organ Preservation Solutions: Mitochondrial Suspended
Animation. Journal of Regenerative Medicine & Tissue Engineering

101

Abstract
Heart transplantation remains the gold standard for end-stage heart failure patients, withcurrent ex vivo organ storage times limited to 4 to 6 hours before critical tissue damage occurs.
The debate between the benefits of warm versus cold storage continues, with warm organ
preservation methods being more expensive and complicated. Therefore, we sought to focus on
cold storage preservations solutions and to mimic a transplant setting at clinically relatabletemperatures of 4o and 21oC. Experimental plates seeded with cardiac myoblasts acted as thedonor heart and were kept in suspended animation for 4 to 8 hours at 4o and 21oC, in Celsior,Pefadex, or Somah storage solutions. Cells were then reanimated for 1 hour at 37oC with regularcell culture media to stimulate the transplant scenario. Cellular bioenergetics were measured
immediately thereafter to examine biochemical differences between organ preservation solutions
and their effectiveness on preserving metabolic and mitochondrial function. The results
confirmed published literature, suggesting that Somah is bioenergetically preferred over Celsior
and Perfadex when used at 4oC, and its beneficial effects can last from 4 hours up to 8 hours.

Introduction
The gold standard for patients suffering from end-stage heart failure remains hearttransplantation. With the number of patients requiring transplant growing and the available
organs remaining constant, there is a need for improved donor organ and graft preservation
methods to extend the life of donor organs [1]. Conserving organ viability outside of an organism
can be accomplished by warm or cold storage, with or without perfusion. Currently, the standard
approach in heart preservation is cold static storage, often supplemented by the use ofpreservation solutions [2]. These approaches lead to an ex vivo storage time of about 4-6 hours

102

before critical tissue damage occurs [3]. Preservation of organs for longer than 4-6 hours results
in increased ROS levels, ATP depletion, Na+/K+ ATPase alterations, mitochondrial disturbances,
accumulation of xanthine oxidase, and dysregulation of Ca2+ homeostasis that will negatively
effect cellular viability [2]. In an attempt to avoid these negative effects, the cold storage
approach exists. Though warm preservation might result in injury from non-controlled warm
ischemic periods [2], is less researched and could be a more effective and sophisticated storage
method [4]. Regardless of storage temperature during perfused or static storage, preservation
solutions are commonly used to decrease ischemic reperfusion injury (IRI) from reactive oxygen
species (ROS), prevent intra- and extracellular swelling, and minimize energy usage by lowering
metabolic demand.
Cellular metabolic demand decreases as much as 12-fold under hypothermic conditionsof 4oC [5], but metabolic processes persist and will continue to result in cellular damage. Duringstorage and upon reperfusion, IRI can be attenuated by using storage solutions such as Universityof Wisconsin (UW) solution [6]. However, UW contains high molecular weight compounds suchas hydroxyethyl starch that resulted in a highly viscous solution that is linked to organ
dysfunction. Newer alternatives including Celsior, Perfadex, and Somah have since been createdto allow for reliable organ preservation up to 6 hours by providing immunosuppressant andantioxidant properties that minimize IRI [6]. The differences between Somah, Celsior, andPerfadex are summarized in Table 1. Properties of these three preservation solutions are fairly
similar, however, Somah contains many additional protective and unique substrates, such as
adenosine, insulin, and abscorbic acid, among others [7]. Under normal physiological conditions,the heart utilizes ATP as energy for the Na+/K+ ATPase, both of which are required for sustained
myocardial contractility. During ischemia, ATP levels decrease while intracellular H+ increases

103

due to the shift from aerobic to anaerobic respiration via glycolysis and lactate production.
ATPase pumps that maintain homeostasis become dysfunctional as the synthesis of ATP slows.
Disruption of these ATPase pumps in the mitochondria can be measured through examining
extracellular flux and mitochondrial respiration. As previously mentioned, both the cold storage
method and utilization of storage solutions in clinical practice attempt to address these concerns
and limit damage to the donor organ.
In the present study, we sought to compare the biochemical differences between organ
preservation solutions and their effectiveness on preserving metabolic and mitochondrial
function. Our protocol involves keeping cells in suspended animation for 4 to 8 hours at 4o or
21oC, then reanimating the cells over 1 hour at 37oC with regular media, and immediately
recording the bioenergetics thereafter. This study allows a glimpse of mitochondrial function
under clinically relatable conditions, investigates mitochondrial safeguarding, and examines the
bioenergetic state of cells after exposure to Celsior, Perfadex, and Somah storage solutions.

104

	
  
Table	
   1.	
   All	   units	
   expressed	
   in	
   mmol/L	
   unless	
   otherwise	
   indicated.	
   Abbreviations:	
   IC,	
   intracellular;	
   EX,	
  extracellular;	
   Und,	
   undetermined;	
   LactoB,	
   lactobionate;	
   HES,	
   hydroxyethyl	
   starch;	
   Phos,	
   phosphate;	
   Bicarb,	
  bicarbonate;	
  GSH,	
  glutathione.	
  Source	
  1:	
  Reference	  [6];	
  2:	
  Reference	  [7].

105

Methods

Microplate Coating
Cell-Tak was purchased from Corning (Product #354240). Cell-Tak was prepared in a
2:1 ratio to 1 M NaOH. The wells of a 96-well Seahorse microplate (Seahorse Bioscience, North
Billerica, MA) were coated with 40 µl Cell-Tak solution and the plate was allowed to dry atroom temperature for 20 minutes. Following the drying period, wells were aspirated and washedtwice with 1x DMEM. The coated microplates were stored at 4oC and used within 72 hours.

Preservation Solution Treatments
Rat embryonic myoblasts, H9C2s (ATCC® CRL-1446™), were maintained at standard
cell culture conditions in DMEM medium enriched with 10% FBS (Seradigm, Product # 1400500), 5% L-glutamine (Corning, Lot # 25005289), and 1% Antibiotic-Antimicotic Solution(Sigma Aldrich, catalog # A5955).
H9C2s were seeded in the previously described Cell-Tak coated 96-well Seahorse
microplates at 25,000 cells/well. The seeded microplates were incubated for 48 hours at 37oC
before introducing experimental conditions to the cells. Each well was washed with 1X PBS(phosphate buffered saline) prior to the addition of 200 µl of experimental solution or control
media. Experimental groups included Celsior (Sanofi, Bridgewater, NJ), Perfadex (XVIVO
Scientific Animation, Wethersfield, CT), Somah (Somahlution, Jupiter, FL) solutions, 1X PBS,
and standard H9C2 media. The 10 experimental groups were named 4CEL, 21CEL, 4PER,
21PER, 4SOM, 21SOM, 4PBS, 21PBS, 4MED and 21MED to indicate the temperature
condition and storage solution of each group: CEL=Celsior, PER=Perfadex, SOM=Somah, and106

MED=media. The microplates were then exposed to either 4oC or 21oC for 4-hour and 8-hourtime points. Each treatment group consisted of 12 wells and each microplate included all of theexperimental groups. Each experimental condition was repeated twice using freshly madesolutions and a separately started H9C2 cell line. After each experiment, supernatant from eachgroup was collected and snap-frozen for High Performance Liquid Chromatography (HPLC)
analysis.

Mitochondrial Stress Test
Immediately after administering the experimental treatments, mitochondrial respiration of
H9C2 cells was assessed using XFe96 Extracellular Flux Analyzer (Seahorse Biosciences, North
Billerica, MA). Oxygen consumption rates (OCR) were measured in the presence of oxidative
phosphorylation (OXPHOS) driving substrates. After 3 basal measurements, 3 measurements
each were taken after the addition of oligomycin, FCCP, and rotenone/antimycin A combination.
These injected drugs block ATP synthase, uncouple the oxygen consumption from ATP
synthesis, and block mitochondrial complexes I and III, respectively. Results were then used to
calculate the respiratory control ratio (RCR) and coupling efficiency (CE), which were theprimary outcome measurements along with basal respiration. RCR was calculated by thefollowing equation: proton leak ÷ maximal respiration; and CE by the following equation:[(basal respiration – proton leak) ÷ (basal respiration)] * 100.

Statistical analysis
The data were analyzed as a split-plot design. A plate was considered a "whole plot" withtreatments: time, temperature, and the time-temperature interaction. Each solution was

107

considered the split-plot treatment with individual wells used as the experimental unit for asolution. Where there was a statistically significant (alpha = 0.05) effect of the solution, Tukey's
HSD was used to compare all pairs of solutions for differences in mean response. Where there
was a statistically significant interaction effect involving a solution, a slice was conducted to test
the significance of drug at each level of time, temperature, and/or time-temperature combination
and all pairs of solutions were tested for differences in mean response at each level of the time
and/or temperature, again, using Tukey's HSD multiple comparison procedure. Initial analysis
suggested non-constant variance among treatment combinations. Therefore, a weighted analysis
was conducted using the inverse variance for each solution treatment-plate combination as theweight. All analyses were conducted using SAS PROC MIXED.

Results
Tukey's HSD was used for pairwise comparisons of solutions for differences in mean
response under each respective time and temperature condition. Only organ preservation solution
pairwise comparisons with at least a significance of alpha=0.05 are mentioned in the figurelegends for each experimental condition (Figures 1-4). A side-by-side comparison of allconditions and groups are shown in Figure 5.

4 Hour Experiments
Comparisons of different solutions are presented in Figure 1 and 2, showing experiments
conducted at 4C and 21C, respectively. H9C2 cells in 4SOM and 4PER both showed a higher
mean basal oxygen consumption rate (OCR) than cells treated with either 4CEL, without

108

significant differences between 4SOM and 4PER. H9C2 cells in 21SOM solution showed ahigher mean basal OCR than H9C2s in 21CEL or 21PER.
At the 4 hour time point, 4SOM and 21SOM treated cells had a higher OCR than cells in
4MED and 21MED, respectively. 4SOM also had a higher coupling efficiency (CE) than 4CEL
and 4PER, and 21SOM showed a higher CE than 21PER at 4 hours. 4SOM showed a higher
respiratory control rate (RCR) than both 4CEL and 4PER.
21MED showed decreased CE in comparison to 21SOM. 21MED had higher RCR values
than all other solutions.	
  8 Hour Experiments
Comparisons of different solutions are presented in Figure 3 and 4, showing experiments
conducted at 4C and 21C respectively. After 8 hours, H9C2s in 4SOM had a higher basal OCR
than cells in 4PER and 4CEL. The significant elevation in basal OCR seen in 21SOM during the
4 hour experiment did not persist after 8 hours.
4SOM had a higher CE than 4CEL; 21SOM had a higher CE than 21CEL and 21PER.
4SOM also showed this same RCR trend above 4CEL and 4PER. As seen in the 4 hour
experiments, 21MED had a lower CE than 21SOM. Finally, 21MED had a higher RCR than theother solutions, in accordance with the trend seen during 4 hour experiments.

109

Figure 2. Experimental Conditions at 4 hours in 4oC. For basal respiration (A), coupling efficiency (B) andrespiratory control ratio (C), there is significance between the Somah-Celsior, and Somah-Perfadex pairwise
comparisons. Error bars shown are SEM.

Figure 3. Experimental Conditions at 4 hours in 21oC. For basal respiration (A), there is significance between the
Somah-Celsior and Somah-Perfadex pairwise comparisons. For coupling efficiency (B), there is significancebetween the Somah-Perfadex pairwise comparison. For respiratory control ratio (C), there is no significancebetween the pairwise comparison of the organ preservation solutions (Somah, Celsior and Perfadex). Error bars
shown are SEM.

110

Figure 4. Experimental Conditions at 8 hours in 4oC. For basal respiration (A), there is significance between the
Somah-Celsior and Somah-Perfadex pairwise comparisons. For coupling efficiency (B) and respiratory control ratio(C), there is significance between the Somah-Celsior, and Somah-Perfadex pairwise comparisons. Error bars shown
are SEM.

Figure 5. Experimental Conditions at 8 hours in 21oC. For basal respiration (A), there is no significance between
the pairwise comparisons of the organ preservation solutions. For coupling efficiency (B), there is significancebetween the Somah-Celsior, and Somah-Perfadex pairwise comparisons. For respiratory control ratio (C), there is no
pairwise significance between the organ preservation solutions. Error bars shown are SEM.

111

Discussion
The goal of this study was to evaluate cellular bioenergetics following a protocol thatsimulated a clinical transplantation scenario. To mimic a transplant setting, the experimental
plate seeded with cardiac myoblasts acted as the donor heart. The cells were submerged in
Somah, Celsior, or Perfadex preservation solution for 4 and 8 hours either at room temperature(21oC) or “on ice” (4oC). Normal saline (phosphate buffered saline; PBS) with no substrates waschosen as the negative control, whereas normal H9C2 media acted as the positive control since
this media is supplemented with the ideal growth substrates for this cell type at 37oC.
It was hypothesized that the ideal preservation solution could create a state of suspended
animation, in which cellular function and metabolism could be preserved for an extended periodof time with the least amount of resultant metabolic dysfunction. Altered cellular metabolism is amore sensitive indicator of stress than some of the traditional end-point markers that may taketime to accumulate and detect (e.g., DNA and protein analyses). Additionally, the role ofmitochondria in ischemic reperfusion injury that occurs during cardiac transplant is becoming
increasingly evident. Safeguarding mitochondrial bioenergetics could play a key role in
improving transplant outcomes. Thus, to examine this endpoint, extracellular flux analyses wereused to compare mitochondrial respiration of H9C2s after each treatment condition. Targeting
various complexes and pathways in the mitochondria allowed us to identify any changes insubstrate metabolism and mitochondrial (dys)function. Basal respiration was analyzed as abaseline bioenergetic measurement for each experimental condition. The basal respiration isoften controlled by high ATP turnover, and in part dictated by substrate oxidation and proton
leak [8]. This measure is therefore altered in response to ATP demand. Coupling efficiency andrespiratory control ratios were analyzed since both parameters are ratios, and are therefore useful112

internal controls that are unaffected by differences in cell number. Additionally, changes in CE
and RCR are good indicators of cellular and mitochondrial dysfunction.
Overall, Somah had a greater OCR than that of both treatment and control groups. Thisfinding was statistically significant after 4 hours at room temperature compared to Celsior andPerfadex. Compared to the control media, the OCR of Somah-treated H9C2s (4SOM and21SOM) were increased compared to the control media (Figures 1 and 2) at both at 4o and 21oC
after 4 hours. By 8 hours, the media and the preservation solution conditions become moresimilar and the significant trend diminishes. 4SOM had an increased OCR value compared to
4PER and 4CEL experimental groups, as well as 4MED and 4PBS controls. Also at the 8-hourtime point, 21PBS had a significantly lower OCR than the other treatment groups, while 21SOM,
21CEL and 21PBS have similar OCR values. As expected, PBS was overall the least-ideal
solution since it has the lowest OCR.
The basal respiratory OCR of Somah-treated cells was greatest and allowed cells tomaintain metabolism at a more consistent OCR through the 4-hour time point (Figures 1 and 3).
In comparison, media, Celsior, and Perfadex took up to 8 hours to return to a higher OCR at
21oC (Figures 2 and 4). These same differences are even more pronounced at 4oC, where 4CEL
is found to have a lower OCR than both 4PER and 4SOM.
As previously mentioned, coupling efficiency (CE) is defined as the proportion of
mitochondrial respiratory rate used to drive ATP synthesis (e.g., perfectly coupled OXPHOS hasa coupling efficiency of 100%) [9]. CE is calculated as the change in basal respiration rate withthe addition of oligomycin, and is thus presented as the fraction of basal mitochondrial oxygen
consumption used for ATP synthesis. Since it takes the basal respiration into account, the CE
also varies with ATP demand and is most sensitive to changes in proton conductance. Since CE

113

is a ratio of two rates, it is valuable as an internally normalized value and also a good indicator of
mitochondrial dysfunction. Overall, both Somah and media treated cells have a high CE (90%),
which shows that even though maximal respiration may be damaged (see RCR), the normal ATP
turnover when compared to the leak of protons over the membrane has not changed. After both 4
and 8 hours at 4oC, the CE of H9C2s treated with either Somah or media was significantly higher
than all other solutions (Figures 1 and 3) and Celsior and Perfadex were not significantly
different at either time point. The shorter time point of 4 hours led to a higher CE in Somah over
the other solutions. 4SOM is comparable to the media control condition, while at 21SOM the CE
was significantly lower than compared to Celsior and Perfadex, yet the CE of media treated cells
remained significantly higher than the other groups. This might be due to an increased proton
leak or a decrease in ATP production. If caused by a rise in proton leak, there might be anincreased production of superoxide anions and other ROS that cause tissue or organ damage. In
previous research, Somah has shown to function best at 21oC [7]. This is an interesting link to
our significantly lowered CE result and should be the subject of future research. However, given
the similarities between the Somah and media treatments in the hypothermic conditions of thisexperiment, Somah is considered preferred at 4oC, while media is favored at a warmer
temperature.
The respiratory control ratio (RCR) is calculated as a ratio of the uncoupled rate of
mitochondrial respiration to the rate with ATP synthase inhibited by oligomycin. The RCR issensitive to changes in substrate oxidation and proton leak, which makes it sensitive overall topotential dysfunction [8]. One major benefit of the RCR as a measurement is that it is internallynormalized, similar to CE. At 4 hours, 4SOM results in a higher RCR than H9C2s treated witheither 4CEL or 4PER, but has a similar RCR to media. Similar to the CE results, this change is114

attributable to either an increase in proton leak or a decrease in maximal ATP turnover, or a
combination of both. Unlike the CE results, the RCR of Somah-treated cells remains
significantly higher than all of the other solutions over the entire 8 hours. 21MED also have ahigh RCR over both 4 and 8 hours, with significance over all other treatment groups. As
expected by 8 hours, 21PBS has the lowest RCR of all groups. The experimental group mostsimilar to the media-treated cells was the Somah-treated cells. While Somah best maintained ahigh RCR under hypothermic conditions, media maintained a high RCR at warmer room
temperature conditions.
These results suggest Somah solution sustains a higher and more consistent OCR whencompared to both Celsior and Perfadex 4oC, and these effects can last from 4 hours up to 8 hours,
which is additionally confirmed in literature [7]. Given these results, Somah offers benefits overthe solutions at 4oC and should be a target in future organ preservation solution research.

115

References[1]

Colvin-Adams M, Smith JM, Heubner BM, Skeans MA, Edwards LB, Waller CD, et al.
OPTN/SRTR 2013 Annual Data Report: heart. Am J Transplant 2015;15 Suppl 2:1–28.
doi:10.1111/ajt.13199.

[2]

Guibert EE, Petrenko AY, Balaban CL, Somov AY, Rodriguez JV, Fuller BJ. Organ
Preservation: Current Concepts and New Strategies for the Next Decade. Transfus Med
Hemother 2011;38:125–42. doi:10.1159/000327033.

[3]

Bourge RC, Naftel DC, Costanzo-Nordin MR, Kirklin JK, Young JB, Kubo SH, et al.
Pretransplantation risk factors for death after heart transplantation: a multiinstitutional
study. The Transplant Cardiologists Research Database Group. J Heart Lung Transplant
1993;12:549–62.

[4]

Talaei F. Limiting the side effects of organ preservation. Global Journal of Surgery 2013;
1:31-36. doi:10.12691/js-1-3-6.

[5]Belzer FO, Southard JH. Principles of solid-organ preservation by cold storage.
Transplantation 1988;45(4):673-6.

[6]

Latchana N, Peck JR, Whitson B, Black SM. Preservation solutions for cardiac andpulmonary donor grafts: a review of the current literature. J Thorac Dis 2014;6:1143–9.
doi:10.3978/j.issn.2072-1439.2014.05.14.

[7]

Thatte HS, Rousou L, Hussaini BE, Lu X-G, Treanor PR, Khuri SF. Development andevaluation of a novel solution, Somah, for the procurement and preservation of beating
and nonbeating donor hearts for transplantation. Circulation 2009;120:1704–13.
doi:10.1161/CIRCULATIONAHA.108.808907.

116

[8]

Brand MD, Nicholls DG. Assessing mitochondrial dysfunction in cells. Biochem J
2011;435:297–312. doi:10.1042/BJ20110162.

[9]

Divakaruni AS, Brand MD. The regulation and physiology of mitochondrial proton leak.
Physiology 2011;26:192–205. doi:10.1152/physiol.00046.2010.

117

APPENDIX DCARDIAC REGENERATION IN THE HUMAN LEFT VENTRICLE FOLLOWING CORMATRIXIMPLANT

Manuscript (in submission): Ferng AS, Connell AM, Johnson K, Nunez M, Braunhut B, Lick S,
Desai A, Kazui T, Runyan R, Khalpey Z. Cardiac Regeneration in the Human Left Ventricle
Following Cormatrix Implant. Journal of Heart and Lung Transplantation

118

Abstract
CorMatrix is an organic extracellular matrix derived from porcine small intestine
submucosa used for pericardial closure and cardiac tissue repair. During HeartMate II LVAD
explant for infection, CorMatrix was used to repair the left ventricular apex and aorta. Threemonths later, a HeartWare H-VAD was implanted for recurrent heart failure. Excised apical
CorMatrix samples showed cardiac tissue remodeling with viable cardiomyoblasts similar tonative myocardium. Excised CorMatrix from the aorta showed organization of collagen andelastin similar to native aortic tissue.

Case Presentation
A 62-year old Caucasian male with a history of hypertension, type II diabetes mellitus,
atrial fibrillation with automatic implantable cardioverter defibrillator (AICD), New York Heart
Association (NYHA) class IV congestive heart failure underwent HeartMate II left ventricularassist device (LVAD) implantation for ischemic cardiomyopathy in 2011. Due to a persistentventricular assist device (VAD) pocket infection, his HeartMate II VAD was explanted in
November 2014. All foreign material was removed, and CorMatrix was used to reconstruct theleft ventricular apical defect and the aortic graft defect. He was discharged off inotropes 3 days
post-operatively, but ultimately failed medical therapy and was re-admitted for worsening heart
failure on inotropes 3 months after explant. He underwent HeartWare H-VAD implant withexcision of the apical and aortic CorMatrix patches, and the device inflow cannula and outflow
grafts attached to native tissue at these sites, respectively. The patient made a slow but steadyrecovery after his second LVAD, and was discharged to go home.

119

Discussion
CorMatrix is an organic extracellular matrix (ECM) derived from porcine small intestine
submucosa that when implanted provides an interim bioscaffold onto which a patient’s cells will
migrate and integrate to repair damaged tissues. CorMatrix has been used to repair cardiac
structures such as the pericardium, tricuspid valve, aortic root, or ventricular tissue [1-4]. The
ECM of various tissue types has structural and functional roles depending upon theircomposition, but all ECM share these common factors: structural proteins (collagen and elastin),adhesion glycoproteins (fibronectin and laminin), glycosaminoglycans and proteoglycans(hyaluronan,

heparinsulfate,heparin,dermatansulfate),andmatricellular

proteins(thrombospondin, osteopondin, and tenascin). The idea is that a patient’s cells will utilize the
CorMatrix bioscaffold and lay down collagen to eventually replace the foreign ECM withpermanent tissue repair. As the CorMatrix patch is largely acellular, it does not encapsulate when
surgically implanted, but has been shown to remodel over time to leave behind healthy tissue.
Furthermore, as an acellular bioscaffold, the patch does not activate the human complement
cascade, thus avoiding acute rejection responses.
To date, CorMatrix has been implanted in over 50,000 patients worldwide in a number ofcardiac closure applications [1]. In our patient, we had the opportunity for CorMatrix
explantation after 3 months in vivo, allowing us to examine the histopathological fate of the
CorMatrix following implantation. CorMatrix samples were specifically taken from the aorta(where the previous Heartmate II outflow graft was reconstructed) and the left ventricular
CorMatrix reconstruction (where the inflow cannula was previously removed) and stained forcells and structure.

120

From our histology, there is cardiac tissue growing on the CorMatrix apical material, as
there are cardiac striations seen in Figure 2 are similar native cardiac tissue, and the hematoxylin
and eosin staining further show mature cardiomyocytes found within the same plane as the
CorMatrix in a background of dense fibrotic stroma (Figure 2A) that is likely part of theresorption of CorMatrix following the resolution of the initial inflammatory response that occurs
following CorMatrix implantation [5]. Furthermore, cardiomyocytes can be visualized in Figure
2B (black arrows), which shows that the CorMatrix material was incorporated as part of the heart
naturally over time. Biopsies of the aorta CorMatrix shows a pattern of growth of elastin andcollagen similar to that found in normal aorta histology (Figure 3). To investigate cardiac
regeneration and the growth of new cardiomyoblasts, HOP was identified as a gene involved in
the developing heart. HOP is highly expressed in the development heart where its expression isdependent on cardiac-restricted homeodomain protein Nkx2.5, and it acts as an antagonist ofserum response factor, which regulates the opposing processes of proliferation and myogenesis[6]. CorMatrix sections from the apex, but not from the aorta, stained positive for anti-HOPX,
therefore showing there was growth of cardiomyoblasts involved in regenerating cardiac tissue(Figure 4).
This case report describes human evidence of left ventricle and aortic regeneration
following the CorMatrix ECM reconstruction after a 3-month period of implantation. These
results found support the hypothesis that CorMatrix plays an important role in neocardiac
remodeling and growth. In another similar case report, it was shown that CorMatrix is involved
in remodeling of cardiac tissue into neo-pericardium [2]. Therefore, placement of CorMatrix andits surrounding microenvironment appears to determine the phenotypic properties of the tissueduring repair and regeneration.

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Figure 1. Intraoperative pictures of where CorMatrix was isolated from the rim of theHeartMate II. After the patient had his HeartMate II left ventricular assist device explanted,
CorMatrix was placed onto the heart for closure of defects in the left ventricular apex and aorta.
After 3 months, the patient required a HeartWare LVAD implantation as destination therapy, andduring this procedure CorMatrix biopsies were taken for analysis. White arrows denote where
the CorMatrix patch is visible. CorMatrix biopsies were specifically taken from the aorta and therim of the device (D).

Figure 2. CorMatrix isolated 3 months post-op from the rim of the HeartMate II near theleft ventricle. Hematoxylin & Eosin stained sections show mature cardiac myocytes in a
background of dense fibrotic stroma (A; 100x). The myocytes have conspicuous cross striations
and frequent vacuolar cytoplasmic change. No coagulative necrosis or wavy fibers are seen;
however, hypereosinophilic fibers are apparent on the right. Myocyte nuclei are readily apparent
throughout and are normal in size to slightly enlarged. In the area of hypereosinophilic change,
the myocytes nuclei are hyperchromatic. Blood vessels are regularly distributed within theinterstitium. On higher power (B; 200x), the myocyte cross striations are readily apparent, as is
the cytoplasmic vacuolization. Cardiomyocytes are present within a dense fibrotic stroma, shownby black arrows. Blood vessels are regularly distributed and appear normal.

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Figure 3. CorMatrix isolated 3 months post-op from the aorta. Routine stained sections taken
at 100x magnification of CorMatrix isolated from the aorta shows a tissue growth pattern ofconsistent with native aortic tissue. H&E staining shows cells in the same plane as the explanted
CorMatrix (A). The VVG elastic tissue stain (B) and trichrome stain (C) shows uniform patternsof elastic fibers and collagen in the media.

Figure 4. CorMatrix isolated 3 months post-op from the aorta. Hoechst staining (blue) wasused to stain for DNA of fixed cells, and anti-HOPX stained (red) shows cardiomyocytes of theregenerating heart.

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References[1]

Boyd WD, Tyberg JV, Cox JL. A review of the current status of pericardial closure
followingcardiacsurgery.

ExpertRev

CardiovascTher2012;10:1109–18.

doi:10.1586/erc.12.87.
[2]Stelly M, Stelly TC. Histology of CorMatrix bioscaffold 5 years after pericardial closure.
Ann Thorac Surg 2013;96:e127–9. doi:10.1016/j.athoracsur.2013.06.114.

[3]

Suendermann SH, Biefer HRC, Emmert MY, Falk V. Use of Extracellular Matrix
Materials in Patients with Endocarditis. Thorac Cardiovasc Surg 2014;62:76–9.
doi:10.1055/s-0032-1328920.

[4]

Brinster DR, Patel JA. The use of CorMatrix extracellular matrix for aortic root
enlargement. J Cardiothorac Surg 2014;9. doi:10.1186/s13019-014-0178-5.

[5]

Zaidi AH, Nathan M, Emani S, Baird C, del Nido PJ, Gauvreau K, et al. Preliminary
experience with porcine intestinal submucosa (CorMatrix) for valve reconstruction incongenital heart disease: Histologic evaluation of explanted valves. J Thorac Cardiovasc
Surg 2014;148:2216–25. doi:10.1016/j.jtcvs.2014.02.081.

[6]

Chen F, Kook H, Milewski R, Gitler AD, Lu MM, Li J, et al. Hop is an unusual
homeobox gene that modulates cardiac development. Cell 2002;110:713–23.

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APPENDIX E
TRANSLATION OF FIRST NORTH AMERICAN 50CC AND 70CC TOTAL ARTIFICIAL HEART
VIRTUAL & CLINICAL IMPLANTATIONS: UTILITY OF 3D COMPUTED TOMOGRAPHY TO
TEST FIT DEVICES

Manuscript (in submission): Ferng AS, Oliva I, Jokerst C, Avery R, Connell AM, Trans PL,
Smith RG, Kazui T, Khalpey Z. Translation of First North American 50cc and 70cc Total
Artificial Heart Virtual & Clinical Implantations: Utility of 3D Computed Tomography to Test
Fit Devices. Artificial Organs

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Abstract
Background:
Since the creation of SynCardia’s 50cc Total Artificial Hearts (TAHs), patients with irreversible
biventricular failure now have two sizing options. Herein, we present a case series of 3 patients
who have undergone successful 50cc and 70cc TAH implantation with complete closure of thechest cavity utilizing preoperative “virtual implantation” of different sized devices for surgicalplanning.
Methods and Results: Computed tomography (CT) images were used for preoperative planningprior to TAH implantation. 3D reconstructions of preoperative chest CT images were generated
and both 50cc and 70cc TAH were virtually implanted into patients’ thoracic cavities. During thesimulation, the TAHs were projected over the native hearts in a similar position of the actual
implantation, and the relationship between the devices and the atria, ventricles, chest wall, anddiaphragm were assessed.
The 3D reconstructed images and virtual modeling were used to simulate and determine for eachpatient if the 50cc or 70cc TAH would have a higher likelihood of successfully implantation
without complications. Subsequently, all 3 patients received clinical implants of the properly
sized total artificial heart based on virtual modeling, and their chest cavities were fully closed.
Conclusions: This virtual implantation increases our confidence that the selected TAH will better
fit within the thoracic cavity allowing for improved surgical outcome. Clinical implantation of
the TAHs showed that our virtual modeling was an effective method for determining the correct
fit and sizing of 50 and 70cc TAHs.

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Introduction
Heart failure is the final common pathway of all cardiac diseases, accounting for nearly
250,000 deaths per year1. With shortages in available donor hearts, mechanical circulatory
support systems have emerged as life-saving augmentation of heart failure for bridge-to-decision,bridge-to-recovery, and bridge-to-transplantation or destination therapy2–4. Patients withirreversible biventricular failure have two options: biventricular assist device (BIVAD) or
temporary TAH. However, the BIVAD system can be complex, involving multiple controllers
and batteries; and still dependent upon native ventricles (i.e. pharmaceutical supports) and proper
valvular function. On contrary, the TAH system replaces both ventricles, all valves and has only
one controller; negating any common problems routinely seen in BIVAD patients5. In fact,patients with the TAH system have a higher survival to transplant rate6,7 and lesser incidence ofneurological events8 than patients with BIVAD support.
Since the implantation of the Liotta TAH by Dr. Cooley in 1969 followed by Jarvik-7 (apredecessor of SynCardia 70cc TAH) by Dr. DeVries in 19829, the Food and Drug
Administration (FDA) has approved the 70cc TAH (Syncardia Systems Inc, Tucson, AZ, USA)in 2004 as bridge-to-transplant, and again in 2014 for destination therapy study. It is currently
the only FDA approved and CE marked TAH with more than 1,300 patients worldwide andcommands a 79% bridge-to-transplant rate6,7,10. However, implantation of these TAHs hasremained limited to patients with a larger chest cavity, or who have adequate intrathoracic space
to accommodate this device. Patient selection criteria includes having a body-surface area (BSA)of 1.7-2.5 m2, or a distance greater or equal to 10 cm from the anterior vertebral body to innertable of the sternum at the 10th thoracic vertebra. Now with the birth of the 50cc TAH, under an127investigational device exemption study, adolescents and smaller adults with irreversible
biventricular failure will have a second chance at life.
Preoperative TAH sizing was less of an issue previously when there was only one sizing
option available with the 70cc TAH since the patients with smaller chest cavities simply did nothave another option. SynCardia Systems Inc. has postulated that a 50cc TAH would fit smaller
statured patients at BSA 1.2-1.7 m2 or a distance of 7 cm or greater between the anteriorvertebral and the 10th thoracic vertebra. However, these selection metrics have never been fully
characterized. Examining the fit of both 50cc and 70cc TAHs using three-dimensional (3D)reconstructions of pre-operative computed tomography (CT) images for preoperative “virtual
implantation” would help surgeons select the proper TAH; eliminating any chance of placing a
TAH into a space that cannot accommodate it, resulting in inability to close the chest. In fact,
Moore RA et al 2015 have found that 2 patients outside of the proposed sizing criteria would fit a
50cc TAH11. However, the in silico study lacked clinical implantation of the 50cc TAH as
validation. Furthermore, there is a limitation with CT images, specifically its static nature that
does not take into account the pliability of the surrounding tissues (i.e. the mobility of thediaphragm), which allows for surgical maneuvers that can improve fit (Figure 1). Herein, we
report a series of 3 cases involving the use of 3D-CT virtual implantation to fit the first North
American implantation of a 50cc TAH. Subsequently, 50cc and 70cc TAHs were placed into two
additional patients.

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Case History

Case 1 - The first 50cc TAH implanted in North America
Patient was a 53-year-old female (BSA 2.1m2) status post orthotopic heart transplant 25 years
ago with chronic rejection, severe allograft vasculopathy, left ventricular dysfunction, acute on
chronic heart failure with moderately depressed left ventricular systolic function on chronic
milrinone support. She presented to the emergency department (ED) with severe abdominal pain,
nausea, vomiting, and diarrhea with bright red blood per rectum for two days. She had multiple
comorbidities including persistent sinus tachycardia, pulmonary embolism, non-ST segment
myocardial infarction, chronic kidney disease, and chronic pancreatitis. She denied chest pain or
shortness of breath. In the ED, she was found to be in metabolic acidosis with elevated lactate
and became hypotensive to a systolic blood pressure of 80 mmHg. Her milrinone infusion was
discontinued and replaced with dopamine and dobutamine. The patient was then transferred tothe cardiac intensive care unit (ICU), and her workup revealed elevated cardiac enzymes andcorresponding EKG changes. While in the ICU, the patient underwent pulseless electricalactivity (PEA) for which she was resuscitated and intubated. She experienced a second PEA
while in the cardiac catheterization lab. An intra-aortic balloon pump was placed in the left
femoral artery and despite additional vasoactive medications she continued to show diminishedpressures. She had high filling pressures, low cardiac output, and a diffusely hypocontractile
heart. She was taken emergently for bilateral extracorporeal biventricular assist device (BIVAD)
implantation of Jogstra RotaFlow blood pump with Thoratec CentriMag cannulas as bridge todecision. She was cardiac tamponade overnight and required multiple mediastinal exploration.
One week later, we decannulated the BIVAD and implanted a 70cc TAH after partial restoration

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of multiple organ failure. However, when we tried to close the sternum upon implantation of the
70 cc TAH, this resulted in kinking of an abnormally enlarged vena cavae; thus restricting fillvolume and blood flow. Consequently, Mersilene tapes were used around the lower left rib cage
to draw the TAH leftward, but this still compromised flow when closing, so the sternum was left
open. The sternal edges were coated with thrombin Gelfoam using Tisseel glue, and then a thickGore-Tex patch was placed over the open sternum with an Ioban over the patch. The patient was
taken back to the operating room later the same day to treat post-op bleeding. Following
placement of the 70cc TAH, the patient received mediastinal washouts on post-operative day(POD) 4, 7, and 10. On POD 10, the patient also received wound VAC placement withvancomycin. At the point, the chest remained not fully closed, and 3D virtual modeling was used
to determine if a 50cc TAH would allow for chest closure. Furthermore, we used an MRI scan of
the patient obtained prior to device implantation to generate dynamic modeling, which helped thesurgeon to determine that with partial resection of the diaphragm, a 50cc TAH could successfully
be implanted. We then exercised the “one time emergency clause of compassionate use” toreplace the 70cc TAH with a 50cc TAH with partial removal of a left rib to successfully close thechest on POD 40. Unfortunately, she suffered from gastric perforation; prompting family towithdraw care despite stable cardiopulmonary support on POD 81.

Case 2
Patient was a 22-year-old male with NYHA stage IV systolic heart failure due to a hereditary
titin mutation non-ischemic cardiomyopathy with an ejection fraction of 15%. He underwent
CentriMag (Thoratec Corp, Pleasanton, CA, USA) BIVAD support using PROTEK Duo
cannulas (CardiacAssist Inc, Pittsburgh, PA, USA). He was extubated and ambulated on POD 2

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and inotropes weaned. On POD 10, the CentriMag BIVAD was switched to HVAD (HeartWare
Inc, Framingham, MA, USA) and CentriMag as bridge to transplant. On POD 34, devices wereexplanted for the placement of a 50cc TAH. Preoperative virtual implantation of the 70cc TAH
revealed a tight fit (despite the fact that the patient did not have a small chest cavity, BSA 1.9m2) whereas the 50cc TAH appeared to fit perfectly. In the operating room the 50cc TAH wentin and the chest was closed without trouble. He is currently awaiting heart transplant.

Case 3
Patient was a 22-year-old female with a history of Marfan's syndrome, non-ischemic
cardiomyopathy, ascending arch replacement, aortic valve replacement, and type B aortic
dissection who presented with decompensated heart failure. Her automatic implantablecardioverter defibrillator (AICD) was explanted and a redo sternotomy was performed following
by placement of a 70cc TAH. Interestingly, this patient (BSA 1.5 m2) had a relatively small chest
cavity with a pectus excavatum; however, she had a large heart and virtual implantation of the
70cc TAH demonstrated a good fit. In the operating room the 70cc TAH went in and the chest
was closed without trouble. She is currently awaiting heart transplant.

Methods
There are several software programs on the market that allow the user to superimpose CTdata sets. Our methodology is to load CT scans of potential TAH patients into Amide's a Medical
Image Data Examiner (AMIDE), along with CT scans of both 50 and 70cc TAHs. AMIDE is an
open-source software tool for displaying and analyzing multimodality volumetric medical
images that runs on multiple operating systems. Of note, each component of the TAHs (right and131left) was scanned separately. The SynCardia TAH has 2 halves that are not fused, but rather
Velcro together, which allows for greater flexibility. The 2 halves of the TAH can be fully
disarticulated and placed above, below, or beside each other as needed (Figure 1).
Once the patient’s CT scan is loaded into the system we first insert the left component
into the space occupied by the left ventricle. The mitral annulus is relatively fixed in position
within the chest and serves as the anchor point for our virtual TAH implantation. The location of
the pumping chamber itself and of the aortic graft is less important as there is flexibility inpositioning of these devices. The left component is placed as posteriorly as possible to allow
maximum space anteriorly for the right component.
The right component is then virtually implanted using the tricuspid annulus as the anchor
point. Sometimes the components may need to be translated or rotated relative to one another toensure best possible fit. As long as the mitral and tricuspid valves are at or near (1-2 cm distal)their respective annulus the location of the chamber and outflow grafts can be manipulated toensure best possible fit. After the TAH is manipulated into place we examine the relationshipbetween the right component and the anterior chest wall to predict if there will be any difficultywith chest closure.

Results
Ultimately, if the TAH can fit in the space remaining once the ventricles are resected,
then there should be no issues with chest closure. If the TAH is larger than the space left behind
after the ventricles are removed, then there is a chance that the chest will not close or that closure
will result in compression of the IVC or pulmonary veins12. While the size of the chest cavity isimportant, the size of the heart is of equal or more importance. If the patient has a very large

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heart, left ventricle end diastolic diameter (LVEDD) > 6 cm, it is usually straightforward to placea TAH regardless of the size of the thoracic cavity. The volume left after resection of theventricles is large enough to accommodate the TAH. Patients with average-sized hearts, as well
as average-sized thoracic cavities, pose the greatest challenge.
Herein, we have used preoperative 3D CT imaging to perform prospective “virtual
implantation” of 50 and 70cc TAHs in patients as described above. The first patient in this caseseries had a BSA of 2.1 m2 and T10 distance of 11.4 cm. Both measurements indicated that a
70cc TAH should fit and allow for post-op chest closure. Unfortunately, the latter was not thecase as she had a relatively average-sized heart with LVEED of ~5 cm and an abnormallyenlarged inferior vena cava. Two weeks following placement of the 70cc TAH, to allow for
closure of the sternum and waiting for swelling to subside, we explanted the intracorporeal 70cc
TAH, put the patient on central cardiopulmonary bypass, and implanted a new 50cc TAH, which
resulted in successful chest closure (Figure 1). This is the first reported case of a successful
implantation of a smaller-sized 50cc TAH in a patient that was achieved with 3D virtual
implantation (Figure 2A,B). Retrospective “virtual implantation” of the 70cc TAH in this patientdemonstrated that the 70cc TAH was not going to fit. Had the virtual implantation been doneprospectively instead of retrospectively for this first case a 50cc TAH might have been placed
initially saving the patient unnecessary operations.
Following the first successful 50cc TAH implantation, similar 3D virtual modeling wasused for two successive patients. We were able to assess TAH fit preoperatively and thus
resulted in a successful execution of TAH implantation and chest closure. In patient 2, virtual
implantation modeling showed that 70cc TAH would have a tight fit despite a BSA of 1.9 m2;
whereas 50cc TAH would fit quite easily. After FDA approval for IDE study, HVAD and the133

CentriMag devices were removed and replaced with a 50cc TAH for bridge-to-transplant based
on RVAD wean failure in spite of extensive fluid removal during the BIVAD support (Figure
2C,D). In patient 3, virtual implantation showed the 70cc TAH would fit despite her relatively
small thoracic cavity (BSA 1.5 m2). She had a larger-sized heart with an LVEDD of > 7 cm.
Hence, an automatic implantable cardioverter defibrillator was explanted and a 70cc TAH was
implanted with successful chest closure (Figure 2E,F).
To the best of our knowledge, this is the first time a 50cc TAH was successfully
implanted in North America. This is also the first full report of utilization of 3D reconstructed
images and virtual modeling for the implantation of 50cc and 70cc TAHs coupling both 3D chest
CT with MRI pre-operatively that was subsequently used in patients. As reported, all 3 patients
received clinical implants of the properly sized total artificial heart without complications.

Comments
3D reconstruction of CT images is a technique that is widely used to assess volume ofdevices and organs. As part of the preoperative planning, some centers create a 3D model of thedevice to be implanted that is virtually placed into reconstructed CT images of the patient’s chestin order to assess if the device will fit within the patient’s thoracic cage. This assessment is madebased on CT images acquired of the thoracic cavity. These images are acquired while the patientis holding their breath at maximum inspiratory effort. The act of breathing is dynamic andinvolves several muscles. CT images are acquired at a single time point during the respiratory
cycle, limiting our ability to assess the physiologic changes that happen within the thoracic
cavity with normal respiration.

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Magnetic resonance imaging (MRI) is a relatively new and rapidly evolving imaging
modality that can allow improved evaluation of the thoracic cavity during respiration to better
assess these physiologic changes that CT scans cannot detect. We have discovered thatpreoperative planning based on dynamic chest MRI acquisition can better depict volumetricchanges in the thoracic cage and detect physiologic adjustments of the respiratory muscles.
Moreover, assessments based on static CT images also do not take into consideration small
surgical procedures that can be done to expand the volumetric capacity of the thoracic cavity,
such as diaphragmatic plication, which was used in this the first patient case to allow the 50cc
TAH to fit into the cavity and allow for closure (Figure 2A,B).
Although MRI has a lot of potential for preoperative device sizing, preoperative virtual
implantation of a device into a chest CT is a relatively quick and easy way to assess device fit
prior to surgery. A CT is comparatively cheaper, fast, and widely available. Many patients
undergoing evaluation for TAH placement are too sick to get an MRI or may havecontraindications to MRI such a being pacer-dependent. In these patients checking device fit
preoperatively with CT is feasible. Metrics such as body surface area (BSA) >1.7 m2, LVEDD>6 cm, and intrathoracic antero-posterior dimension at the level of the T10 vertebral body (T10)of >10 cm have been used as cut-offs to determine whether the patient’s thoracic cavity canaccommodate a 70cc TAH12,13.
Preoperative “virtual implantation” of TAHs into a patient’s chest CT appears to be ahelpful method for determining preoperative “fit” of TAH. This is especially important now
since the 50cc TAH has been added to the armamentarium, and there are currently no guidelines
for minimum thoracic cavity size for the 50cc TAH. As stressed before, limitations of thismethodology is the reliance on static CT imaging. Previous techniques to improve fitting of the135

TAH within the chest involves a 3D reconstruction of the chest CT coupled with a 3D renderingof the 50cc and 70cc TAH11. However, these fitting techniques were part of a study that was a
retrospective analysis of the 70cc TAH for the virtual implantation of a 50cc TAH. Our paper
documents the first time a 50cc TAH was implanted successfully in North America after preoperative virtual fitting. Additionally, the thoracic cavity is dynamic and changes in size andconfiguration with respiration. The chest CT also fails to take into account the fact that there are
surgical maneuvers and manipulations that can help improve fit of TAHs, which was highlighted
by our paper following surgical implantation. Patient 3 (Figure 2E,F) is an example of how
surgical maneuvers could be used to create more space in the chest such that a device thatshowed a tight virtual fit could still be implanted. MRI has potential for improved preoperative
sizing since it is a more dynamic imaging technique. Therefore, a combination of multiple
imagining modalities, including CT and MRI, can be used to aid a cardiothoracic surgeon inpredicting through “virtual implantation” whether or not there will be difficulty with chest
closure following TAH placement.

Acknowledgements
The authors would like to acknowledge the patient and his family.

Consent
Written informed consent was obtained from the patient for publication of this case report andany accompanying images. A copy of the written consent is available for review by the Editorin-Chief.

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Figure 1. Atypical TAH positioning. There is atypical positioning of the TAH components (*)
on a frontal chest X-ray (A), with the right component being placed below the left component. In
contrast, an X-ray from a different, larger patient (B) shows the more conventional side-by-side
relationship between the components of the TAH (*).

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Figure 2. Virtual implantation of a 70 and 50 cc TAHs into Patient 1. The 70 cc TAH (A)depicted in red shows an impossible fit, whereas the superimposed 50 cc TAH (B) shown ingreen demonstrates potential fit into patient 1. Neither device fits perfectly due to the patient’s
left ventricle being only mildly enlarged and normal size of the right ventricle; however,successful implantation of the 50 cc device was possible after surgical manipulation of thesurrounding organs and diaphragm. Virtual implantation of a 70 and 50 cc TAHs into Patient
2. Part of the 70cc TAH (C) projects over the anterior chest wall and beyond the lateral wall of
the LV posteriorly (arrows), indicating extensive surgical maneuvering is required for successful
implantation. The 50 cc device (D) projects entirely within the ventricular cavities. From thevirtual modeling, the decision was made to place the 50 cc TAH, which was successfully
implanted into the patient without complication. Virtual implantation of a 70 and 50 cc TAH
into Patient 3. The patient has pectus excavatum with anteroposterior chest cavity diameter of
9.6 cm at T10 level, indicating a low probability of successful implantation of a 70cc TAH. On
virtual implantation of the 70cc device (E), the RV component impinges upon the anterior chestwall (arrow) but the LV is dilated, providing extra space posteriorly (arrow head) that allows thesurgeon to maneuver for an appropriate fitting. This predicts a high likelihood of successful
implantation, despite the small thoracic cavity. Virtual implantation of a 50cc TAH showsadequate space for successful implantation (F). The decision was made to place the 70 cc device,
which was successfully implanted without complication; this highlights the limitation of static
CT images, which do not take in consideration the dynamics of the diaphragm, pliability ofsurrounding tissues, and effect of surgical maneuvers. We recommend factoring inapproximately 1 cm of extra space when performing virtual TAH implantation to account forsuch factors.

138

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APPENDIX FSUMMARY & FUTURE DIRECTIONS142

SUMMARY
The goal of this dissertation was to contribute to the field of cardiac transplantation by
studying the fields of organogenesis, cellular energetics, and tissue and organ regeneration. We
have presented an optimized whole heart acellularization technique not previously found inliterature that results in a 3D bioscaffold. We next took this bioscaffold and successfully showedthat human endothelial and stem cells could not only adhere to our acellular matrix, but also
proliferate over time. Moreover, we took clinically used surgical matrices and showed that thesematrices could be similarly seeded with human stem cells, perhaps offering an alternate
treatment intervention. We complemented this study with a clinical case study investigating the
growth of new cardiac tissue onto CorMatrix, a clinically used matrix.
We also studied through bioenergetics the differential profile of adipose-derived
stem/stromal cells isolated from the stromal vascular fraction of adipose tissue to better
understand the profile of cells we have translated from the bench-side into the operating room,
which is published as a case report. Our hypothesis that the origin of adipose tissue affected stem
cell metabolism was confirmed, which gives us the option to choose a certain type of adipose
tissue over another depending on the application.
To study organ storage conditions, we compared the bioenergetics of three organ
preservation solutions: Celsior, Somah and Perfadex. Through clinically relatable times andconditions, we showed that Somah performed best at hypothermic conditions for up to 8 hours.
Given the 150+ organ preservation solutions on the market being used by different active hearttransplant hospitals through the United Network of Organ Sharing (UNOS), it is difficult tochoose the ideal solution. Therefore, we wanted to take a few common solutions used in heartlung transplantation and provide a clear picture of preservation solution effects on mitochondrial

143

respiration and cell metabolism. Knowing these differences can allow scientists in the field tostart investigating more of the antioxidants and other substrates (e.g., adenosine and insulin) thatmay provide additional protective cellular effects that other solutions may not offer.
Finally, we used a Langendorff setup to create a bioreactor for a whole heart bioscaffold.
We showed that we could successfully recellularize part of the vasculature of our scaffold while
it was under constant perfusion for up to 5 days. This was the first step in the direction ofpreferentially delivering various stem and terminal cell types to the whole heart bioscaffold inorder to one day grow a clinically transplantable heart. Understanding various aspects oforganogenesis and organ preservation offers knowledge that will one day allow us to groworgans, and to extend organ viability in preservation solutions from hours to potentially days.

FUTURE DIRECTIONSIn addition to the present and future experiments described for recellularization andseeding of commercial patches in the discussion section of Chapters 2 and 3, there is one otherrelevant project I would like to describe. One of the biggest issues when recellularizing matrices,or a whole organ, is cell placement, especially considering multiple layers of myocardium. 3D
bioprinting can provide an alternative option for tissue engineering applications and heartorganogenesis. For this reason, I have built several 3D bioprinters over the course of my PhD
with the goal of bioprinting customized stem cells and tissues. The first model was an inkjet
printer hacked to print cells with a drop-down Z-axis. However, the spray of the cells using thismodel could not be effectively controlled, so a different type of 3D printer was needed.
The current version of the printer I built, where I assembled, designed, remodeled andengineered all parts that I needed, was done by building off of open source RepRap and144

Fab@Home Model 1 3D printer designs online. This 3D bioprinter utilizes a stepper motordriven syringe dual-syringe system that can additionally be retrofitted with a pneumaticallycontrolled extruder. The 3D bioprinter is driven by a Rambo 1.3Vb driver board with a LCD
microcontroller that runs off of an SD card that provides for axes control (X, Y, and Z planes)
and syringe dispension control. I am currently in the process of retrofitting this bioprinter withpneumatic and temperature-controlled dispensing systems to allow for better control of thebioprinting of cells, and investigating various biomaterials for development of a scaffold andprintable bioink. One of the approaches involves utilizing the acellular porcine matrix andprocessing this matrix into a printable hydrogel that can be printed in tandem with bioink.
Currently, researchers have attempted to create an effective cell therapy delivery vehicle
by seeding commercial patches with fibroblasts and cardiomyocytes; however, these cardiac
patches are either only a single cell-layer thick on both sides, or the patch itself is too thick to beeffectively seeded. Bioprinting can potentially overcome this problem and offer additional celldelivery vehicles. This work can potentially be coupled with clinically used matrices to create
alternative cardiac patches, or even repair parts of damaged organs by printing the necessary
tissues and/or cell populations onto the damaged surface in the correct locations and spatialconformations.
We have been involved in 3D modeling of heart valves and the whole heart to determine
the ideal 3D structures to print with using biomaterials. The ideal clinical imaging methods forthis will also be determined for the generation of the best type of imaging data, after cardiac
gating, to use for the clearest 3D printing resolution (CT vs. MRI vs. 2D echocardiography vs.
3D echocardiography). This imaging can also help in determining what pathologies exist, andwhat they look like such that 3D bioprinting can be used to help with tissue regeneration.

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Various biomaterials to be used both separately and together for the scaffold structure and cellsuspensions will be tested. Scaffold structure can be tested via dynamic materials analysis. Cell
viability can be accessed either by cell staining or by live/dead assays. Culturing of thesematerials can be performed in the various bioreactor systems mentioned (e.g., Langendorff
setup). It will be very exciting to see what unfolds over the next decade in this field.

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APPENDIX GFIRST IN MAN: STERNAL RECONSTRUCTION WITH AUTOLOGOUS STEM CELLS

Manuscript (published): Khalpey Z, Marsh KM, Ferng A, Riaz IB, Hemphill C, Johnson K,
Oliva I, Friedman M. First in Man: Sternal Reconstruction with Autologous Stem Cells. ASAIO
J. 2015 Sep-Oct;61(5):e31-2. doi: 10.1097/MAT.0000000000000236.

ASAIO Journal 2015

Case Reports

First in Man: Sternal Reconstruction with Autologous Stem Cells
Zain Khalpey,* Katherine M. Marsh,* Alice Ferng,* Irbaz Bin Riaz,† Courtney Hemphill,* Kitsie Johnson,*
Isabel Oliva,‡ and Mark Friedman§

Sternal nonunion is associated with high morbidity and treatedusing rigid plate and screw fixation. This is the first reported
example of successful sternal reconstruction using adiposederived stromal vascular fraction (SVF) stem cells in addition
to traditional techniques. Mesenchymal stem cells, one component of the SVF, play an important role in bone healing andwere therefore used to promote remedial processes in a patientwith sternal nonunion. A 3D printed model of the patient’s sternum was used for preoperative planning of the plating. Intraoperatively, SVF was isolated using ultrasonic cavitation andpreviously planned sternal plating was completed. A total of
300 million cells were delivered via both local injection andintravenously before chest closure. The patient’s pain dramatically decreased, commensurate with healed areas of nonunionby 3 months and maintained at 6 months postoperatively,
supported by three-dimensional computed tomography imaging. Utilizing autologous stem cells from the SVF in conjunction with existing plating techniques may provide an optimal
platform to stabilize the sternum and promote bone healing,although additional study is recommended. ASAIO Journal
2015; 61:e31–e32.

the past decade. Their potential as progenitor/stem cells has beenshown to be equivalent to the properties of pluripotent stem cells
or even advantageous to using other populations of stem cells dueto their low immunogenicity.2 These cells can be readily isolated
via noninvasive lipoaspiration from subcutaneous fat at a muchhigher yield than other sources, and has since emerged as an alternative tissue source for use in regenerative medicine. These adipose-tissue SVF cells, or adipose-derived stem cells (ASCs), havebeen used in studies that include cartilage and bone regeneration,wound healing, articular cartilage defects, and tissue regeneration.
Because SVF cells can be used without expansion and cell-culture
treatment, the stem cells undergo minimal manipulation before
clinical use. Though in vitro expansion also shows that human
MSCs do not seem to undergo malignant transformation, SVF cells
are even more safe and efficacious due to the minimal manipulation.2 Other studies show further evidence that there are various components of crude SVF that act synergistically with ASCs,
which may be more clinically beneficial than ASCs alone.2 Adipose-derived stem cells are also more immunosuppressive thanbone marrow-derived stem cells, another common source used in
preclinical and clinical studies.2 There are many current, ongoingclinical trials involving the use of autologous adipose tissue SVF
cells for various disease etiologies including osteoarthritis, spinal
cord injury, multiple sclerosis, and acute myocardial infarction.2,4Although there is always a possibility of side effects from the delivery of stem cells, minimal side effects are expected from SVF MSCs
compared with other cell types, because MSCs have been shown
to modulate T-cell-mediated immunological responses.5 Methodsof delivery include both intravenous injection and local injection,
both of which have been shown to cause cell homing to the site ofischemia or injury.5–7Here, we outline the first reported case of successful sternal
reconstruction with autologous stem cells from the SVF. Utilizing this accessible heterogeneous mixture of cells in conjunction with existing plating techniques may provide an optimal
platform to stabilize the sternum and promote bone healing.

Key Words: sternal reconstruction, autologous stem cells,
human adipose-derived stem cells, hADSCs, adipose-derived
stromal vascular stem cells, ASCS

ternal nonunion, although rare, is associated with high morbidity.1 Various fixation techniques have been described with theultimate goal of rigid fixation and bony union to promote bonehealing.1 Natural bone healing involves many processes, including stem cell involvement. Research has highlighted the importantrole of mesenchymal stem cells (MSCs) in bone repair, which has
lead to the exploration of additional therapeutic options.2 Emerging treatments include the use of adipose-derived stromal vascular
fraction (SVF) cells, which contain multi-potent CD31+, CD34+,
CD44+, CD90+, CD73+, and CD105+ cells characteristic ofMSCs, in addition to exhibiting mesodermal capacity.3 Stromal
vascular fraction cells have been studied extensively in vitro overCase Report
A 65 year old male presented with disabling pleuritic chest
pain due to chronic sternal nonunion with bone loss after coronary artery bypass grafting. This began following an episode ofsevere coughing secondary to upper respiratory tract infection.
Three-dimensional computed tomography of the chest (3D CT)
showed a comminuted lower sternal fracture and nondisplacedhealing fractures of the left fifth and right fifth and sixth ribs.
Although the wired upper portion of the sternum remained intact,
the lower sternum had fractured in three places and distracted
into four principal pieces greater than 5 cm apart (Figure 1A). Aftermedical therapy proved unsuccessful, a sternal reconstruction
and unilateral pectoralis flap reconstruction was recommended.
Given the extensive sternal nonunion, we constructed a preoperative 3D printed image of the patient’s sternum augmented with

From the *Division of Cardiothoracic Surgery, Department of Surgery, †Department of Internal Medicine, ‡Department of Radiology,
and §Division of Cardiology, Department of Internal Medicine, Banner
University Medical Center, Tucson, Arizona.
Submitted for consideration August 2014; accepted for publicationin revised form December 2014.
Disclosure: The authors have no conflicts of interest to report.
Supported by The University of Arizona Medical Center and Collegeof Medicine, Department of Surgery.
Correspondence: Zain Khalpey, 1501 N. Campbell Ave, Rm 4302A,
P.O. Box 245071 Tucson, AZ 85724-5071. Email: zkhalpey@surgery.
arizona.edu.
Copyright © 2015 by the American Society for Artificial Internal
Organs
DOI: 10.1097/MAT.0000000000000236

e31

Copyright © American Society of Artificial Internal Organs. Unauthorized reproduction of this article is prohibited.

e32

KHALPEY et al.

Figure 1. Three-dimensional computed tomography image. Fracture sites and regions of sternal nonunion are indicated by arrows
preoperatively (A), and healed fractures are indicated by arrows at 3 months (B) and 9 months postoperatively (C).

biomaterials and mock-prepared the joints and the plating system (Synthes, DePuy, West Chester, PA). This was completed toboth plan and practice the procedure in three dimensions before
surgery. Although in vitro stem cell injection was not completed
during the planning phase, after completion of the plating, we
identified potential locations for local stem cell injection.
Intraoperatively, we performed sternal plating with bone putty
as planned preoperatively, and harvested autologous adiposederived SVF stem cells with ultrasonic cavitation (IntelliCell Biosciences, New York, NY). A sterile section of the operating room(OR) was used for the SVF isolation procedure, which included
a centrifuge, flow cytometer, sonicator probe, and the necessary accessories. Before the plating procedure, lipoaspirate wasobtained via standard liposuction from the patient’s abdomen.
The SVF isolation procedure was immediately implemented
using the lipoaspirate and completed 1 hour later, by the time
the surgeon had completed the sternal reconstruction. This
allowed for prompt injection of the freshly isolated, autologous
SVF cells: 100 million via localized injection and 200 million
via intravenous injection. The SVF was a heterogeneous population of cells and extracellular matrix that can be used clinicallywithout expansion,2 and was mostly composed of CD34+ cellswith approximately 88% cell viability. Finally, a unilateral pectoralis flap reconstruction was completed before closure.
Serial 3D CT imaging demonstrated fracture healing andclosure of areas of nonunion 3 and 6 months postoperatively
compared with preoperative imaging (Figure 1B, C). Since surgery, the patient has established regular follow up at our outpatient facility. His sternum is stabilized and he now reports
minimal to no pain, with normal exercise tolerance. This caseis the first reported example in man of successful sternal reconstruction with adipose-derived SVF.
Discussion
Medial sternontomies are the gold standard for many cardiothoracic surgical procedures. Sternal nonunion occurs inapproximately 0.5–3% of all patients with standard wire fixation, with greater risk in patients who are on prolonged ventilation, female, older age, or overweight.8 Treatment options
have included repeat circumferential peristernal rewiring, steel
banding, polymer tapes, and absorbable sutures, but currently
rigid plate and screw fixation is most widely utilized.1
Despite improvements in fixation technologies and allogeneic bone matrices, there is no guarantee that healing will
occur. As mentioned earlier, human ASC transplantation has
shown promise in current orthopedic-related clinical studies,including osteoarthritis and spinal cord injury.2,4 In addition,
the usefulness of ASCs in various wound and bone healing
applications has been studied in multiple animal models withevidence that they promote the type of accelerated regeneration we have seen in our patient.2 Due to the ability to easily acquire both autologous stem cells (adipose-derived andstromal vascular progenitor cells) and native micromatrix fromliposuction-derived adipose tissue in the operating room, ourmethod of SVF isolation and application make this an attractive
therapeutic option. The engraftment of the SVF and generationof stable bone highlights the importance of cell–matrix combination therapy options, rather than stem cell therapy alone.
This has implications for cardiac and orthopedic regeneration.
Acknowledgment
The authors acknowledge IntelliCell BioSciences and Dr. Steven Victor, MD for implementation of the SVF isolation protocol using ultrasonic cavitation. The authors thank Synthes who helped 3D print thediseased sternum for preoperative surgical planning.

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Copyright © American Society of Artificial Internal Organs. Unauthorized reproduction of this article is prohibited.