Contains Nonbinding Recommendations 
 
Utilizing Animal Studies to Evaluate 
Organ Preservation Devices 
______________________________________________________________________________ 
Guidance for Industry and  
Food and Drug Administration Staff 
 
Document issued on May 8, 2019. 
 
The draft of this document was issued on September 15, 2017. 
 
 
For questions about this document, contact DHT3A: Division of Renal, Gastrointestinal, Obesity, 
and Transplant Devices at 301-796-7030. 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Devices and Radiological Health 
 
  Contains Nonbinding Recommendations 
 
Preface 
 
Public Comment 
 
You may submit electronic comments and suggestions at any time for Agency consideration to 
https://www.regulations.gov. Submit written comments to the Dockets Management Staff, Food 
and Drug Administration, 5630 Fishers Lane, Room 1061, (HFA-305), Rockville, MD 20852. 
Identify all comments with the docket number FDA-2017-D-4886. Comments may not be acted 
upon by the Agency until the document is next revised or updated. 
 
Additional Copies 
 
Additional copies are available from the Internet. You may also send an e-mail request to 
CDRH-Guidance@fda.hhs.gov to receive a copy of the guidance. Please use the document 
number 1500083 to identify the guidance you are requesting. 
 
  Contains Nonbinding Recommendations 
 
Table of Contents 
 
I.  Introduction ........................................................................................................................... 1 
II.  Scope ..................................................................................................................................... 2 
III.  Definitions ............................................................................................................................. 3 
IV.  Overview and General Study Design Considerations ........................................................... 3 
A. Procedure Duration .......................................................................................................... 5 
B. Contamination .................................................................................................................. 5 
C. Transportability ................................................................................................................ 5 
V.  Reperfusion Models .............................................................................................................. 5 
A. Ex Vivo Models ................................................................................................................ 6 
B. In Vivo Models ................................................................................................................. 7 
C. Conclusion ........................................................................................................................ 8 
 
  Contains Nonbinding Recommendations 
 
Utilizing Animal Studies to Evaluate 
Organ Preservation Devices  
 
Guidance for Industry and  
Food and Drug Administration Staff  
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or 
Agency) on this topic. It does not establish any rights for any person and is not binding on 
FDA or the public. You can use an alternative approach if it satisfies the requirements of the 
applicable statutes and regulations. To discuss an alternative approach, contact the FDA staff 
or Office responsible for this guidance as listed on the title page.  
I.  Introduction 
While the national transplant waiting list continues to grow, donation and transplant rates remain 
stagnant. The shortage of organs available for transplants has propelled a new wave of 
innovation in organ preservation technologies. These technologies are evaluated in animal 
models to demonstrate that they are suitable for clinical experience.  
 
The intent of this guidance is to provide recommendations regarding best practices for utilizing 
animal studies for the evaluation of organ preservation devices. For information regarding Good 
Laboratory Practice (GLP) requirements that may apply to such studies, you should refer to 21 
CFR Part 58 Good Laboratory Practice for Nonclinical Laboratory Studies. FDA recommends 
balancing the ethical principles of The Three Rs (replacement, reduction and refinement)1 as 
well as regulatory least burdensome principles, with the goal of using the minimum number of 
animals necessary to generate data to demonstrate device safety. You should consider the best 
practices for the development, conduct and presentation of these animal studies while 
incorporating modern animal care and use strategies. 
 
FDA recognizes that best practices for conducting animal studies to evaluate organ preservation 
devices are evolving with the rapid advancements in such technologies. This guidance is not 
intended to be comprehensive or prescriptive. Instead, it aims to highlight FDAs initial thoughts 
on how animal transplant models can be utilized to evaluate organ preservation technologies, 
with careful considerations of regulatory least burdensome principles. While FDA expects that at 
this time, most of these animal studies will be initially submitted to support investigational 
device exemption (IDE) applications, and may also be used to support premarket approval 
                                                           
1 Russell WMS, Burch, RL. The Principles of Humane Experimental Technique. London: Methuen & Co.; 1959. 
Special edition published by Universities Federation for Animal Welfare, 1992. Available online at: 
http://altweb.jhsph.edu/pubs/books/humane_exp/het-toc. 
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applications (PMA), premarket notifications (510(k)), humanitarian device exemption (HDE) 
applications, or De Novo classification requests.  
 
FDA encourages members of industry to submit a Pre-Submission to obtain feedback for specific 
animal study protocols to evaluate organ preservation devices. For more information on Pre-
Submissions, you should refer to Requests for Feedback on Medical Device Submissions: The 
Pre-Submission Program and Meetings with Food and Drug Administration Staff.2 
 
FDA's guidance documents, including this guidance, do not establish legally enforceable 
responsibilities. Instead, guidances describe FDAs current thinking on a topic and should be 
viewed only as recommendations, unless specific regulatory or statutory requirements are cited. 
The use of the word should in FDA guidance means that something is suggested or 
recommended, but not required. In this document, the terms you and your refer to members 
of industry, also known as sponsors or submitters. The terms we, us, and our refer to 
FDA. 
 
II.  Scope 
The recommendations in this guidance document are applicable to devices intended to preserve 
human vascularized organs via machine perfusion (hypothermic or normothermic) from the time 
of organ procurement until transplant. The Health Resources and Services Administration 
(HRSA), not FDA, oversees the donation and transplantation of human organs.  
 
Many of the devices to which this guidance applies are currently under product development and 
are not yet classified. This guidance document is also applicable, but is not limited to, devices 
regulated under: 
 
  Product code QBA (Normothermic machine perfusion system for the preservation of 
standard criteria donor lungs prior to transplantation); 
  Product code PHO (Normothermic preservation system for transplantation of initially 
unacceptable donor lungs); and 
  21 CFR 876.5880 with the product code KDN (System, Perfusion, Kidney). 
 
The recommendations in this guidance document do not apply to devices intended to preserve 
organs via cold static storage, including those associated with product codes KDK, PIN, KDL, 
and MSB that are regulated as class II devices under 21 CFR 876.5880 (Isolated kidney 
perfusion and transport system and accessories). In addition, human cells, tissues, or cellular or 
tissue-based products (HCT/Ps) regulated under 21 CFR 1271.3(d)(1) and sections 351 and 361 
of the Public Health Service Act, and the devices utilized to preserve and transport these 
products, are also outside the scope of this guidance document. 
 
                                                           
2 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/requests-feedback-and-meetings-
medical-device-submissions-q-submission-program   
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III.  Definitions 
 
For purposes of this guidance document, the following definitions apply: 
 
Cold ischemia time: The amount of time that an organ is cold (4C) and not receiving 
adequate blood supply.  
 
Cold static storage: The current standard method to preserve most organs. Organs are 
submerged in a preservation solution in a closed container that maintains the temperature 
at 4C. 
 
Extended criteria organs: Donor organs that are suboptimal for transplant (e.g., 
donation after cardiac death (DCD) donor organs). The criteria may differ depending on 
organ type.  
 
Ischemia reperfusion injury: Inflammation and oxidative damage to the tissue caused 
by the restoration of blood supply after a period of ischemia. 
 
Machine perfusion: A dynamic method to preserve organs, utilizing a device with a 
pump that drives the movement of a perfusate. Devices performing machine perfusion of 
organs may also contain oxygenators, heat exchangers, sensors, disposable circuits, and 
computer units for processing and displaying hemodynamic and metabolic data. Machine 
perfusion can be performed at various temperatures, e.g., 4C (hypothermic), 37C 
(normothermic).  
 
Perfusate: The solution that is pumped through the donor organ. 
 
Reperfusion: The restoration of blood supply to an organ.  
 
Warm ischemia time: The amount of time that an organ is at body temperature or room 
temperature and not receiving adequate blood supply.  
IV.  Overview and General Study Design Considerations
 
FDA recommends a risk-based approach for developing animal study protocols for evaluating 
organ preservation devices. In order to determine the specific risks to be evaluated in an animal 
study, you should consider the risks inherent to the proposed indications for use and other known 
risks of your device identified through literature review, bench testing, exploratory animal 
studies, and, where appropriate, perfusion studies using human organs from consented donors for 
research. For example, compared to cold static storage, machine perfusion may subject the 
organs to additional risks of injuries due to organ manipulation and contamination of the 
perfusion circuit. As another example, a device indicated to preserve extended criteria organs 
may also subject the organs to different risks than that indicated to preserve standard criteria 
organs.  
 
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After determining the specific risks and their corresponding failure modes, you should develop a 
protocol with focused objectives and a priori acceptance criteria. When appropriate, FDA 
recommends including the scientific rationales for the chosen acceptance criteria. In addition, 
FDA recommends that you provide a rationale for the selection of a particular animal model for 
your study, with careful considerations of anatomical, physiological, and immunological 
similarities and differences between the animal model and humans. 
 
A typical experimental setup for such animal studies will consist of three phases: organ 
procurement, organ preservation, and organ reperfusion (see Figure 1 below).  
 
 
Figure 1. The Three Phases of a Typical Animal Study for Evaluating Organ Preservation 
Devices. 
To begin the safety assessment of the organ preservation device, selected organs are procured 
from appropriate animal model donors. Then, these organs are preserved using either an 
experimental method (e.g., machine perfusion) or a control method (e.g., cold static storage). 
Organs from both groups are reperfused in either an in vivo or ex vivo model, to evaluate 
reperfusion injury. Due to its complexity, the reperfusion phase will be discussed in detail in 
Section V. In the section below, our recommendations focus on general study design 
considerations. 
 
 
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A.  Procedure Duration 
Procedure duration has a significant effect on the outcome of transplant studies. You should 
carefully consider the following recommendations regarding the duration of the experimental 
procedures: 
 
  Procurement Phase: FDA recommends specifying warm ischemia time and cold 
ischemia time as part of the animal organ procurement protocol. The ischemia time 
should reflect the indications for use of the device. For instance, when evaluating a 
device indicated to preserve organs from non-heart-beating donors, you should extend the 
period of warm ischemia by leaving the organ in situ after inducing cardiac arrest in the 
animal. 
 
  Preservation Phase: Prior to initiating preservation, the time to successfully cannulate 
and connect the organ to the device should be evaluated based on a priori acceptance 
criteria. The total preservation time should take into consideration the expected maximum 
transportation time that is consistent with the indications for use of the device. 
Preservation time may vary based on organ type. 
 
  Reperfusion Phase: At the end of the preservation phase, the organ should be cold-
flushed per standard protocol and exposed to a realistic preparation period prior to the 
start of ex vivo reperfusion or in vivo transplant. The duration of reperfusion will be 
discussed in detail in Section V. 
B.  Contamination 
Compared to cold static storage, machine perfusion has a higher risk of contamination due to the 
increased complexity of the perfusion circuit and manipulation of the organ. Therefore, FDA 
recommends performing bacterial cultures on perfusate samples taken at the end of a perfusion 
session.  
C.  Transportability 
If your organ preservation device is transportable, your animal protocol should assess whether 
the device and the organ can withstand the turbulence during transport (e.g., being driven in an 
ambulance). Normal handling, such as tilting the device, during transport may jeopardize the 
organ support system or cause transient changes in the perfusion parameters. FDA recommends 
developing and evaluating strategies that mitigate the risk of organ injury from mechanical 
trauma. For instance, if you plan to administer a vasodilator to regulate the spikes in 
hemodynamic parameters (e.g., vascular pressure) during transport, you should evaluate whether 
the amount of vasodilator administered achieves the intended effect. 
V.  Reperfusion Models 
 
After an organ undergoes preservation, the clinical concern centers on the severity of the 
reperfusion injury. There are generally two models to assess reperfusion injury: an in vivo model 
in which the organ is transplanted into a recipient animal and an ex vivo model in which the 
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organ is reperfused in an isolated setup. In order to establish a more focused animal study 
protocol, it is important to discuss the advantages and limitations of each model, in the context of 
recent technological advancements in organ preservation technologies. 
A. Ex Vivo Models 
The development of new organ preservation technologies (e.g., normothermic machine 
perfusion) has unlocked the potential to monitor and assess organs ex vivo prior to 
transplantation. Compared to the more traditional ex vivo models (e.g., Langendorff heart 
model), ex vivo models utilizing these new technologies are capable of continuously collecting 
more detailed hemodynamic, metabolic, and functional data under more relevant physiological 
conditions. In addition, compared to their in vivo counterparts, these ex vivo models typically 
offer a more controlled study environment with fewer potential confounders (i.e., non-device 
related factors that may affect the interpretation of study outcomes). Nevertheless, ex vivo 
models have two important limitations: 
 
  The evaluation of ischemia reperfusion injury attributed to interactions between the 
coagulation and inflammatory cascades is hindered by 1) the use of anticoagulants (e.g., 
heparin) in the blood-based perfusates and 2) the lack of whole-body immune response. 
 
  The association between organ viability and the hemodynamic, metabolic, and functional 
data collected in an ex vivo model has not yet been well-established. While the perfusate 
can be sampled during ex vivo reperfusion to measure levels of biomarkers for organ 
injury and function, some of these biomarkers are considered exploratory and are not 
well-accepted as surrogates for organ viability post-transplant.  
 
While some of these limitations are inherent to the ex vivo model, other limitations can be 
mitigated through improved study design. FDA has the following recommendations for study 
designs in an ex vivo model: 
 
  Control group: Due to the limitations discussed above, an ex vivo model cannot 
determine the absolute extent of ischemia reperfusion injury. Therefore, we recommend 
including a control group (e.g., cold static storage) in the study, so that the relative effects 
of the injury can be evaluated. 
 
  Near-physiological conditions: In order to simulate in vivo conditions, ex vivo 
reperfusion should be performed under near-physiological conditions (e.g., temperature, 
pressure, flow, oxygenation). The performance of critical device components (e.g., 
pumps, sensors, oxygenators) should be validated using exploratory animal studies or 
studies using human organs not suitable for transplant. 
 
  Perfusate and its additives: If your device utilizes a blood-based perfusate, FDA 
recommends the use of whole blood. If you plan to supplement your perfusate with 
additives (e.g., sodium bicarbonate, vasodilators) through bolus or continuous infusions, 
FDA recommends establishing pre-specified conditions for administering these additives 
to minimize bias. 
 
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  Reperfusion duration: You should specify the duration of ex vivo reperfusion to allow 
adequate assessment of organ function and viability. For instance, if you plan to assess 
the ability of a liver to synthesize a coagulation factor post-preservation, you should 
consider the half-life of the coagulation factor when specifying the duration of 
reperfusion. 
 
  Biomarkers: Ischemia reperfusion injury may affect several distinct structures and 
functions of a single organ; therefore, FDA recommends evaluating a panel of 
biomarkers (e.g., molecular, functional, imaging) targeted to assess both organ injury and 
organ function. Due to the aforementioned limitations of the ex vivo model, special 
consideration should be given to the evaluation of biomarkers for endothelial cell injury 
and activation of the inflammatory cascades.  
 
  Edema: FDA recommends weighing organs before and after reperfusion to assess the 
risk of machine perfusion-related edema. Machine perfusion parameters such as 
preservation duration, perfusate composition, temperature, pressure, and flow can 
contribute to edema, which in turn can adversely affect organ function. Extended 
hypothermic machine perfusion of the heart, for instance, is known to induce myocardial 
edema,3,4 which is directly associated with increased ventricular stiffness and diastolic 
dysfunction. 
 
  Histopathology: You should collect tissue biopsies from multiple representative regions 
of the organ before and after reperfusion. FDA recommends that a qualified independent 
pathologist evaluate the histopathology, with a focus on the integrity of endothelial cells 
using appropriate stains (e.g., CD31 immunohistochemistry stains for assessing 
sinusoidal endothelial cell integrity in the liver).  
B.  In Vivo Models 
After an organ undergoes preservation, transplanting the organ in a survival model offers the 
most direct method for evaluating the preservation technology. Compared to ex vivo models, in 
vivo models rely on the most clinically relevant endpointgraft survival, instead of biomarkers 
for organ injury and function. In addition, in vivo models allow for the whole-body immune 
response and the complex interplay between the coagulation and inflammatory cascades, so you 
can evaluate the full extent of ischemia reperfusion injury. Despite these advantages, in vivo 
models introduce many non-device related variables, which may affect transplant outcomes and 
hinder meaningful interpretation of data. To address these challenges, FDA recommends that you 
carefully consider the following: 
  Confounders in the organ recipient: FDA recommends that you collect baseline 
hemodynamic profiles in organ recipients and provide immunosuppressants to limit the 
effects of hemodynamic instability and immunologic heterogeneity, respectively, on 
transplant outcome. 
                                                           
3 Van Caenegem O., et al., Hypothermic continuous machine perfusion improves metabolic preservation and 
functional recovery in heart grafts. Transpl Int (2015) 28(2):224-231.  
4 Collins MJ, et al., Preserving and evaluating hearts with ex vivo machine perfusion: an avenue to improve early 
graft performance and expand the donor pool. Eur J Cardiothorac Surg (2008) 34(2):318-325.  
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  Confounders in the transplant procedure: The animal studies should be conducted in a 
highly controlled facility by qualified personnel with extensive experience in surgical 
transplants and post-operative care. Standard procedures, including antibiotic and 
immunosuppressant regimens and post-operative monitoring and care, should be applied 
to both the experimental and control groups. In order to reduce the risk of confounders 
such as rejections, FDA recommends a follow up period no longer than one-week post-
transplant, with endpoints that evaluate early injury patterns.  
C.  Conclusion 
In the field of organ preservation, the outlook and utility of ex vivo and in vivo models will 
evolve with continued innovation in technology and our improved understanding of basic 
science. On one hand, as machine perfusion more closely mimics physiologic conditions and 
more biomarkers are accepted as surrogates for organ injury and function, the data collected in ex 
vivo models are expected to become increasingly predictive of transplant outcomes and 
subsequently reduce the number of animals used in the studies. On the other hand, in vivo models 
have the potential to utilize genetically-engineered animals with specific immunologic 
deficiencies or ischemic tolerance in order to simulate clinical scenarios.  
 
While FDA understands that the choice of the model may be restricted by many factors including 
utilizing animals and other available resources, your study should primarily be based on the 
study objectives and the risks of the device. For example, ex vivo models may be sufficient to 
support, for example, a device modification or protocol modification of a previously approved 
IDE application. In vivo models may be necessary to support an IDE application for a first-of-its-
kind device or a perfusion solution with multiple novel components. Finally, the two models 
should not be regarded as mutually exclusive; the in vivo models can be used to verify findings 
from ex vivo models. Recognizing that each scenario is unique and that our understanding of 
these devices continues to evolve, FDA recommends that sponsors submit a Pre-Submission to 
obtain feedback on proposed animal studies. 
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