A Pilot Study: Evaluating the Safety and Feasibility of Using Autologous Adipose-Derived Stromal Vascular Fraction (SVF) for the Treatment of Aerodigestive & Enterocutaneous Fistulae in Adults
Phase: PHASE1
Conditions: Tracheoesopharyngeal Fistula, Bronchoesophageal Fistula, Tracheoesophageal Fistula
Sponsor: Mayo Clinic

Adipose-Derived SVF for Aero-digestive Fistulae 
 
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A PILOT STUDY: EVALUATING THE SAFETY AND 
FEASIBILITY OF USING AUTOLOGOUS ADIPOSE-DERIVED 
STROMAL VASCULAR FRACTION (SVF) FOR THE 
TREATMENT OF AERO-DIGESTIVE FISTULAE IN ADULTS 
 
 
Principal Investigator: 
Timothy A. Woodward, M.D. 
Mayo Clinic Florida 
4500 San Pablo Road 
Jacksonville, Florida 32224 
 
Study Product: 
Uncultured, autologous, adipose-derived stromal vascular 
fraction (SVF) 
Protocol Number: (IRBe) 
17-003774 
IND Number: 
17796 
 
 
Version 1.0: 
[10/20/2017] 
Version 1.1: 
[07/23/2018] Tracked Changes 
Version 2.0 
[07/25/2018] Changes Accepted 
Version 2.1 
[9/17/2018] Tracked Changes 
Version 3.0 
[9/18/2018] Changes Accepted 
 
 
 

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Table of Contents 
STUDY SUMMARY ...................................................................................................................................... 5 
1 
INTRODUCTION ................................................................................................................................. 6 
1.1 
SPECIFIC AIMS ................................................................................................................................. 6 
1.2 
BACKGROUND.................................................................................................................................. 6 
Stem Cell Therapy ................................................................................................................................... 6 
Pathology and impact of aerodigestive fistulas .......................................................................................... 7 
1.3 
INVESTIGATIONAL AGENT ................................................................................................................. 9 
1.4 
CLINICAL DATA TO DATE ................................................................................................................ 10 
1.5 
DOSE RATIONALE AND RISK/BENEFITS ............................................................................................. 12 
2 
STUDY OBJECTIVES ........................................................................................................................ 13 
3 
STUDY DESIGN ................................................................................................................................. 13 
3.1 
GENERAL DESIGN ........................................................................................................................... 13 
3.2 
PRIMARY STUDY ENDPOINTS ........................................................................................................... 16 
3.3 
SECONDARY STUDY ENDPOINTS ...................................................................................................... 16 
3.4 
PRIMARY SAFETY ENDPOINTS.......................................................................................................... 16 
4 
SUBJECT SELECTION ENROLLMENT AND WITHDRAWAL ...................................................... 16 
4.1 
INCLUSION CRITERIA ...................................................................................................................... 16 
4.2 
EXCLUSION CRITERIA ..................................................................................................................... 17 
4.3 
SUBJECT RECRUITMENT, ENROLLMENT AND SCREENING .................................................................... 17 
4.4 
EARLY WITHDRAWAL OF SUBJECTS ................................................................................................. 18 
4.4.1 
When and How to Withdraw Subjects ...................................................................................... 18 
4.4.2 
Data Collection and Follow-up for Withdrawn Subjects ............................................................ 19 
5 
STUDY DRUG .................................................................................................................................... 19 
5.1 
PREPARATION AND ADMINISTRATION OF STUDY DRUG ...................................................................... 19 
5.2 
TREATMENT REGIMEN .................................................................................................................... 19 
5.3 
ISOLATION OF STROMAL VASCULAR FRACTION (SVF) USING KISO: ................................................... 20 
5.4 
SUBJECT COMPLIANCE AND PRIOR THERAPY ..................................................................................... 21 
5.5 
PACKAGING ................................................................................................................................... 21 
5.6 
BLINDING OF STUDY ....................................................................................................................... 21 
5.7 
RECEIVING, STORAGE, DISPENSING AND RETURN .............................................................................. 21 
5.7.1 
Receipt of Drug Supplies ........................................................................................................ 21 
5.7.2 
Storage ................................................................................................................................. 22 
5.7.3 
Dispensing of Study Drug ....................................................................................................... 22 
5.7.4 
Return or Destruction of Study Drug ....................................................................................... 22 
6 
STUDY PROCEDURES ...................................................................................................................... 22 
6.1 
VISIT 1 .......................................................................................................................................... 22 
6.2 
VISIT 2 .......................................................................................................................................... 22 
6.3 
VISIT 3 .......................................................................................................................................... 23 
6.4 
FOLLOW-UP VISITS ......................................................................................................................... 23 
6.5 
STUDY VISIT TABLE ....................................................................................................................... 24 
7 
STATISTICAL PLAN ......................................................................................................................... 24 
7.1 
SAMPLE SIZE DETERMINATION ........................................................................................................ 24 
7.2 
STATISTICAL METHODS .................................................................................................................. 25 
8 
SAFETY AND ADVERSE EVENTS ................................................................................................... 25 

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8.1 
DEFINITIONS .................................................................................................................................. 25 
8.2 
RECORDING OF ADVERSE EVENTS .................................................................................................... 26 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS ....................................... 26 
8.3.1 
Sponsor-Investigator reporting: notifying the Mayo IRB ........................................................... 27 
8.3.2 
Sponsor-Investigator reporting: Notifying the FDA .................................................................. 27 
8.4 
STOPPING RULES ............................................................................................................................ 28 
8.5 
MEDICAL (CLINICAL) MONITORING ................................................................................................. 28 
8.5.1 
Internal Data and Safety Monitoring Board ............................................................................. 28 
9 
DATA HANDLING AND RECORD KEEPING .................................................................................. 28 
9.1 
CONFIDENTIALITY .......................................................................................................................... 28 
9.2 
SOURCE DOCUMENTS ..................................................................................................................... 29 
9.3 
CASE REPORT FORMS ..................................................................................................................... 29 
9.4 
RECORDS RETENTION ..................................................................................................................... 29 
10 
STUDY MONITORING, AUDITING, AND INSPECTING ................................................................ 30 
10.1 
STUDY MONITORING PLAN .............................................................................................................. 30 
10.2 
AUDITING AND INSPECTING ............................................................................................................. 30 
11 
ETHICAL CONSIDERATIONS ......................................................................................................... 30 
12 
STUDY FINANCES ............................................................................................................................ 31 
12.1 
FUNDING SOURCE .......................................................................................................................... 31 
12.2 
CONFLICT OF INTEREST ................................................................................................................... 31 
13 
PUBLICATION PLAN ....................................................................................................................... 31 
14 
REFERENCES ................................................................................................................................... 31 
 
 
 

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 LIST OF ABBREVIATIONS 
 
AE 
Adverse Event/Adverse Experience 
AMSC 
Adipose-derived Mesenchymal Stem Cells 
RNP 
Registered Nurse Practitioner 
CAD 
Coronary Artery Disease 
CFR 
Code of Federal Regulations 
CLD 
Chronic Liver Disease 
CRD 
Chronic Renal Disease 
CRF 
Case Report Form 
DSMB 
Data and Safety Monitoring Board 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
HTN 
Hypertension 
IB 
Investigator’s Brochure 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
MNCs 
Mononuclear Cells 
MSCs 
MADF 
Mesenchymal Stem Cells 
Malignant Aerodigestive Fistula 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PVD 
RTEF 
Peripheral Vascular Disease 
Recurrent Tracheoesophageal Fistula 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
SVF 
Standard Operating Procedure 
Stromal Vascular Fraction 
WOC RN 
Wound Ostomy Continence Registered Nurse 
 
 
 

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Study Summary 
Title 
A Pilot Study: Evaluating the Safety and Feasibility of Using 
Autologous Adipose-Derived Stromal Vascular Fraction  (SVF) for the 
Treatment of Aerodigestive Fistulae in Adults 
 
Running Title 
Adipose Derived SVF for Aero-digestive Fistulae 
Protocol Number 
17-003774 
Phase 
Pilot Study 
Methodology 
Pilot of a small number of patients to test the safety and feasibility of 
autologous SVF administration for the treatment of aerodigestive 
fistulae. 
Overall Study 
Duration 
5 years  
Subject Participation  Patients with tracheoesophageal, bronchoesophageal, and 
tracheopharyngeal fistulae. 
Single or Multi-Site  
Single site – Mayo Clinic in Florida 
Objectives 
Determine safety and feasibility of using institutionally prepared 
autologous, uncultured SVF on patients with aerodigestive fistulae 
secondary to malignancy, trauma or surgery. 
Number of Subjects 
10 patients. 
Diagnosis and Main 
Inclusion/Exclusion 
Criteria 
Patients older than 18 years old with aerodigestive fistulae. Either 
recurrent or de novo fistulae measuring between 2 - 15 mm Exclusion 
criteria: patients with severe malnutrition BMI <16 (which would 
interfere with the lipoaspiration procedure)  
Study Product, Dose, 
Route, Regimen 
Study Product: Uncultured, autologous, adipose-derived stromal 
vascular fraction(SVF) 
Two-thirds of the obtained SVF will be injected into the submucosa of 
the de-epithelialized fistula and one-third will be suspended without 
expansion in a fibrin sealant containing 5 x106 mononuclear cells per 
ml. The fibrin glue will be endoscopically injected inside the fistulous 
tract.  
Duration of 
Administration 
Permanently. Single SVF administration or until further management is 
warranted by either endoscopic or surgical intervention. 
Statistical 
Methodology 
There will be descriptive statistics for the pilot of 10 cases. 
 
 
 
 
 
 
 

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1 Introduction 
This document is a protocol for a human research study. This study will be carried out in 
accordance with the applicable regulations and Mayo Clinic research policies and procedures.  
 
1.1 
Specific Aims 
The primary aim of this pilot study is to evaluate the feasibility, time, cost, safety, limitations, 
and efficacy of the use of institutionally processed SVF for management and closure of aero-
digestive fistulae. This pilot study would help identify design issues and the potential success of 
fistulae closure by the means of autologous SVF administration before a full-scale trial is 
performed.  
 
A secondary aim is the description of aero-digestive fistulae characteristics such as size, 
etiology, recurrence, localization, and the association of these factors with the outcome after SVF 
administration. The SVF quantification, characterization and differentiation in vitro will be 
described. 
 
This process will help identify the type of fistulae that are susceptible to closure with human cell 
therapy. 
 
1.2 
Background 
 
Stem Cell Therapy 
Mesenchymal stem cells (MSCs) are multipotent cells capable of developing into several types 
of connective tissue such as muscle, fat, cartilage and bone. The use of MSCs for tissue 
regeneration is promising and the field of bioengineering has developed a variety of scaffolds for 
stem cell administration. Different biological sources and processing options are available. For 
instance, adipose-derived stem cells (AMSCs) can be isolated from human adipose tissue by the 
means of needle biopsy, excision or lipoaspiration. Subsequently, they can be transplanted to the 
same individual (autologous) or to a different individual (allogenic), with or without cell 
expansion and with or without the use of a scaffold. The scaffold may be either cellular or 
acellular. These variations may have an impact in the outcome of therapy and need to be 
evaluated for each case. 
 
Adipose-derived stem cells have recently emerged as a more viable source for clinical 
applications, compared to bone-marrow mesenchymal stromal cells (BM-MSCs) because of their 
abundance and easy access. They have been used for the treatment of refractory and complex 
perianal fistulas in Crohn’s disease as well as for the treatment of pressure ulcers and 
rectovaginal fistulas with promising outcomes. The stromal vascular fraction (SVF), isolated 
when fresh lipoaspirates are enzymatically digested with a collagenase, contains a heterogeneous 
cellular and extracellular milieu.   SVF is an aqueous fraction, consisting of endothelial cells and 
their precursors, macrophages, smooth muscle cells, lymphocytes, pericytes, pre-adipocytes and 
actual AMSCs.  Past studies have shown that such tissues contain progenitor cells that can 
release multiple angiogenic growth factors and cytokines including vascular endothelial growth 
factor (VEGF), hepatocyte growth factor (HGF) and chemokine stromal cell-derived factor-1 
(SDF-1).1 
 

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Pathology and impact of aerodigestive fistulas 
An aero-digestive fistula refers to an abnormal communication between the respiratory and 
digestive tracts. These may be secondary to trauma, malignancy, surgery, and congenital 
malformations. Some examples of aero-digestive fistulae include: tracheo-esophageal, tracheo-
pharyngeal, and broncho-esophageal fistulae. The esophagus has an immediate contact with the 
membranous portion of the trachea and the left bronchus. This predisposes to the formation of 
fistulous tracts between these structures.2 Esophagorespiratory fistulae are uncommon. They 
usually form a communication between the esophagus and the trachea and less frequently, they 
enter a bronchus or the pleural cavity. They may also terminate in the mediastinum, pericardium 
or skin.3 
 
The congenital type occurs with or without esophageal atresia or stenosis and usually 
communicates with the trachea. Esophagorespiratory fistulae in adults may be congenital. The 
etiology of the acquired fistulae, in their probable order of frequency, is as follows: 1. Carcinoma 
of the esophagus. 2. Trauma due to ingested foreign bodies, caustics, instrumental dilatation, and 
crushing injuries of the thorax. 3. Infections of the esophagus, trachea, bronchi, pleura, or 
mediastinum, due chiefly to tuberculosis and syphilis. Pathogenic organisms, fungi, and 
nonspecific infections may also be the cause. 4. Unknown etiology. 5. Esophageal traction 
diverticula. 6. Broncholithiasis. 7. Aneurysm. 8. Esophagomalacia. 3 
The symptoms and signs depend on the etiology, primary pathologic changes, and site of the 
fistula. Difficulty in swallowing of liquids is usually the initial symptom. Dysphagia increases, 
and later there are strangling, cough, and often dyspnea during meals. Swallowing in the dorsal 
recumbent position may minimize these symptoms. Sputum mixed with food is often raised in 
increasing amounts. It may contain purulent material and blood. Substernal or referred pain to 
the shoulders may be present. Food intake may be reduced to a minimum, with resulting loss of 
weight. A pathognomonical triad has been proposed (Saegesser et al., 1968): paroxysmal cough 
after liquid ingestion, episodical abdominal distension and recurrent bronchopulmonary 
infections .2 Complications include chronic cough, recurring pneumonia, bronchiectasis, 
pulmonary abscess and gangrene, mediastinal abscess, fatal hemorrhage, and death if the trachea 
is suddenly blocked. 3 
 
Most esophagorespiratory fistulas are single; double or more complicated tracts have been 
recorded in cancer and after lye ingestion. The fistulous opening is located in the middle third of 
the esophagus in 60% of the cases and in the upper third in 25% of the cases. The remainder is 
located in the lower third or cervical esophagus. 2 
The pathological basis of malignant aerodigestive fistula (MADF) formation is the spreading of 
the esophageal cancer into the airways or lungs, or the propagation of pulmonary and mediastinal 
tumors into the esophagus. Primary malignancies that are most often causative are esophageal, 
lung and thyroid cancers, as well as, lymphoma.4 Presumably, it occurs more frequently than the 
5-10% incidence noted in literature, especially at the end stage of the malignant disease. Its early 
diagnosis and treatment is extremely important, because sealing the fistula can improve the 
survival and the quality of life of the patient.5 
 
 

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Current treatment for aerodigestive fistulas 
Treatment varies according to the primary disease, complications, condition of the patient, and 
extent of the pathologic changes. There is rarely spontaneous closure and healing in view of the 
recurrent infections due to passage of food and saliva into the respiratory tract. Surgical therapy 
often comprises division of the fistulous tract and resection of irreversibly damaged lung tissue. 
Treatment of MADFs raises difficulties, especially when healing is compromised by prior 
irradiation. Endoluminal approaches have proven useful for these patients, especially when at 
time of diagnosis, the patient’s performance and nutritional status precludes aggressive surgical 
therapy. Palliative measures have evolved over the years and have included surgical (i.e. enteric 
bypasses or surgical exclusion of the fistula), esophageal endoprothesis, esophageal intubation, 
application of self-expandable metallic stents and supportive medical therapy, with or without 
enterostomies.4 
 
Impact of recurrent fistulae 
Despite the relative success of surgical repair, recurrent tracheoesophageal fistula (RTEF) has 
been reported in up to 20% of patients. Unfortunately, open procedures on RTEF have been met 
with high levels of postoperative morbidity, mortality, and recurrence. This has led investigators 
to explore less invasive techniques in the surgical repair of RTEF. Endoscopic approaches using 
a ventilating bronchoscope were first introduced in the German literature in the mid-1970s. Since 
then, various endoscopic techniques in the repair of RTEF have been described and 
unfortunately, several attempts may be required before complete closure is achieved, which can 
result in repeated and potentially unnecessary, exposure to anesthetic risk.6 
 
In a review of literature by Gresham et. al., a total of 15 articles were identified in the literature 
from 1975 to 2007 describing endoscopic techniques for the repair of RTEF. Various, but 
similar, techniques to endoscopic repair have been performed. These can be categorized into 3 
approaches: de-epithelialization of the fistula, application of tissue adhesives, or a combination 
of the two. Fibrin glue and Histoacryl have been applied alone or in combination with de-
epithelialization techniques to promote fibrin and scar tissue formation. The greatest chance for 
success, both as a first attempt and overall, was achieved in cases where a combined approach of 
de-epithelialization and placement of tissue adhesive was performed, 67.7% and 93.3%, 
respectively. Authors repeated therapy as needed to achieve successful closure. A collection of 
the entire population of reported cases, including all 3 approaches, illustrated a success rate of 
only 48.6% after one attempt at repair. Although techniques and likely surgical experience 
varied, the overall success rate including all techniques and despite the number of repairs was 
81.1%.6 
 
This data demonstrates the current need of novel therapies in order to enhance early fistulae 
closure and decrease the number of interventions before fistula healing is achieved.  
 
 
 

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Cell Therapy in the management of fistulas 
In fistulising Crohn’s disease, local injection of stem cells is considered to be beneficial with 
sustained efficacy based on the fact that AMSCs have the ability to inhibit surrounding 
inflammation by suppressing proliferation of activated lymphocytes and have some regenerative 
potential for replacement of the defective fistula tract. 7 Multiple trials demonstrate that AMSC 
treatment for patients with Crohn’s fistulae (perianal8, rectovaginal9 and enterocutaneous10) was 
well tolerated with favorable therapeutic outcomes.  
 
Autologous AMSC administration has  been used in vivo for the closure of recurrent 
bronchopleural fistulae. Wigle et al reported the case of a recurrent bronchopleural fistula which 
after 30 bronchoscopic and surgical interventions, was finally closed by the means of a surgically 
implanted autologous adipose stem cell-seeded matrix.11 Garcia-Arranz et al described a 
successful bronchoscopic intervention for the closure of recurrent bronchopleural fistulae in two 
patients using autologous, non-expanded AMSCs endoscopically injected into the fistulous de-
epithelialized area.12  Similarly, Garcia-Olmo et al treated a tracheomediastinal fistula with 
bronchoscopic injection of AMSCs suspended in fibrin glue with successful fistula re-
epithelialization and closure.13It is hopeful that a heterogeneous population of stromal cells 
containing AMSCs would be similarly beneficial in RTEF. 
 
 
Study Population 
The population to be studied includes adults between 18 and 90 years old with tracheo-
esophageal, tracheo-pharyngeal and broncho-esophageal fistulae measuring up to 1.5 cm. De 
novo and recurrent fistulas will be included in the inpatient and outpatient setting. Subjects will 
be identified in the inpatient or outpatient setting during consults and/or appointments with the 
Gastroenterology and/or Thoracic Surgery Departments. When a potential patient is identified, a 
research nurse (RN) consult will be initiated in order to identify if the patient meets all the 
inclusion criteria for the study. Baseline values, fistula assessment and a picture of the fistula 
will be obtained. Approximately twenty to thirty patients with aerodigestive fistulae are seen in 
this healthcare center per year across the departments of gastroenterology, thoracic surgery and 
otorhinolaryngology.  In this study our goal is to have 10 subjects complete all study procedures 
up to at least the 3 month follow-up visit. 
 
1.3 
Investigational Agent 
Adult stem cells have been found to be available in several tissues and organs such as skin, brain, 
liver, and bone marrow. The ability to self-renew and differentiate in multiple cell phenotypes is 
well established.  Several research studies have used adult bone marrow derived stem cells but 
unfortunately, small numbers of stem cells are generated from bone marrow in the absence of 
culture expansion, and the amount of stem cells also decreases with age. More recently, adipose 
tissue has been used to isolate stem cells through a process of filtration, collagenase digestion, 
and centrifugation. It is more easily obtained and is found to differentiate into cells of the 
mesenchymal lineage (eg, bone, fat, cartilage, or muscle). Adipose-derived MSCs have a 
potential for angiogenesis and vasculogenesis and are able to produce cytokines in the pathologic 
tissue. For these reasons, they are of interest in the process of wound healing.1 

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More specifically, Mesenchymal stem cells (MSCs) are multipotent or able to differentiate into 
multiple tissue types. MSCs can give rise to bone, cartilage, fat, tendons, and ligaments. MSCs 
express the cell surface markers CD73, CD90, CD105, SCF R, STRO-1, but lack CD34, CD45, 
CD14, CD11b, CD19, CD79a, and HLA-DR (Kim et al., 2014). In addition to differentiating 
into different types of tissue to accelerate healing, MSCs secrete many different cytokines and 
growth factors that may aid in wound healing. These factors include: VEGF-α, G-CSF, GM-
CSF, IGF-1, IL-2, IL-6, IL-7, IL-11, IL-11, LIF, bFGF, MCP-1 and SDF-1, among other factors 
(Blaber et al., 2012).  
 
The stromal vascular fraction (SVF), isolated from fresh lipoaspirates and enzymatically digested 
results in an aqueous fraction of heterogeneous cells proteins and growth factors consisting of 
endothelial cells and their precursors, macrophages, smooth muscle cells, lymphocytes, 
pericytes, pre-adipocytes and actual AMSCs.  The ultimate goal is to create a shorter healing 
time for the patient so immediate, same day, autologous application is a benefit. There is less 
manipulation of the cells in the uncultured state, possibly increasing the safety to the patient. 
This will also be an autologous application, making the risk of infection or rejection very low. 
 
1.4 
Clinical Data to Date 
Most data to date involve the use of AMSCs. ADSC were systematically evaluated for their 
ability to rebuild volume in depressed scars following the subcutaneous injection of ADSC 
which differentiated towards adipocytes. This trial of 36 patients was completed in 2007 but has 
not, to our knowledge, yet been documented in any peer-reviewed international journal. Only 
one report relates to a field in which ADSCs have been evaluated for their reconstructive 
properties based on their classic mesenchymal differentiation potentials, specifically in the field 
of bone or cartilage reconstruction when the osteogenic or chondrogenic potentials of ADSC are 
well-established and widely investigated. In this case report with 36 months of follow-up, the 
defect was successfully reconstructed with a microvascular flap using betatricalcium phosphate, 
autologous ADSCs and bone morphogenetic protein-2 to trigger their osteogenesis. Although 
this result is encouraging, a case report cannot give prove a general effect and no conclusions can 
be definitively drawn until phase I and II trials have been conducted.  
 
 
 
 

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Most other ADSC trials concern fistula complications that result from tissue degeneration 
following an uncontrolled inflammatory process. In a noteworthy case report, Garcia-Olmo et al 
found that expanded ADSCs are more efficient than the freshly-prepared conterpart in treating 
Crohn’s disease. The trials on fistula indicate that ADSCs are very efficient in controlling 
inflammation and improving the healing process. Garcia-Olmo et al published the conclusions of 
randomized phase I and II trials in which they compared injection of ADSC into rectal mucosa 
with fibrin glue to fibrin glue alone on 25 patients with complex perianal fistulas associated or 
not with Crohn’s disease.  
 
Following a first dose of 20 million ADSCs, a second dose of 40 million was administered 8 
weeks later in cases where there was no initial healing. Patients were considered healed when a 
total epithelialization of the external opening was evident after 8 wk. Seventy one percent of 
patients treated with ADSC and fibrin glue displayed fistula healing compared to 16% observed 
in patients treated with fibrin glue alone (P < 0.001). This positive effect on healing is all the 
more remarkable as it is otherwise poorly documented, even in rodent models. Two further 
clinical trials have focused on Graft versus Host disease, an application which is not surprising 
given the efficiency of MSCs at the clinical level and the similarity in immunomodulation 
properties between MSCs and ADSCs. Multiple sclerosis can be also included in the field of the 
modulation of inflammation/immune response. In a rodent model, it was demonstrated that 
intravenously injected ADSCs can home to the lymph nodes and brain and that they act by 
suppressing the autoimmune response in early phases of disease as well as by inducing local 
neuroregeneration by endogenous progenitors in animals with established disease. The strong 
immunosuppressive effects of ADSC reported by various independent groups naturally led to 
investigation the effect of ADSC on MS.  
 
Based on the positive and well-documented positive effects of intravenous administration of 
MSCs, positive effects of ADSCs are also expected in this field. Only two trials have 
investigated the effect of ADSCs on chronic critical limb ischemia, one after intra-muscular 
injections, the second with intravenous injections in diabetic patients. A further planned trial also 
intends to use allogenic ADSCs in the context of fistula. This trial is important as it could open 
up the field of ADSC-associated regenerative medicine. 14 
 
 
 

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Table from Casteilla et al. 14 
 
 
1.5 
Dose Rationale and Risk/Benefits 
From the point of view of safety and adverse-side effects, two key issues are the possibility of 
undesirable differentiation and the possibility of interaction between ADSCs and resident cancer 
cells. Concerning the first point, undesirable calcifications have been observed after the 
administration of bone marrow stem cells in the heart after infarction. More recently, cysts and 
microcalcifications were detected in 4 out of 70 patients after breast reconstruction using 
lipoaspirate associated with crude SVF. To our knowledge, no other cases of undesirable 
differentiation have been described, suggesting either that such events are rare or have not been 
fully and systematically evaluated.14 
No definitive conclusion can be reached on the possible interaction between ADSCs and cancer 
cells as contradictory reports have been published. Immunosuppressive capacity and modulation 
of inflammation are shared with ADSCs, which seem very efficient both in vitro and in different 
in vivo situations. This immunosuppressive effect associated with the angiogenic properties of 
ADSC raises questions about the interactions between these cells and cancer cells. Donnerberg et 
al. concluded that ADSCs could trigger the growth of tumors from active cancer cells but not 

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from dormant cells.14 Another systematic analysis of the issue found that malignancy occurred 
only in studies involving participants with ongoing or previous malignancies; no de novo 
malignancies were observed.15 
 
In a meta-analysis of clinical trials that examined the use MSCs to evaluate their safety, the 
review did not detect an association between acute toxicity related to the infusion of MSCs, 
organ system complications, infection, death or malignancy. There was a significant association 
between MSCs and transient fever however no long term sequelae developed.15 Risk is assumed 
to be very small for this study because of a focal injection and smaller dose versus systemic 
injection.  
 
2 Study Objectives 
Primary Objective 
To assess the safety and tolerability of a single dose of autologous stromal vascular fraction 
(SVF) applied endoscopically into tracheoesophageal fistulae. 
 
Secondary Objective 
To assess the efficacy of stromal vascular fraction’s (SVF) ability to improve the success of 
surgical repair and prevent recurrence of tracheoesophageal fistula (RTEF).  
 
 
3 Study Design 
 
3.1 General Design 
This is an open label pilot study to assess the safety and feasibility of harvesting and applying 
autologous adipose derived SVF  and the efficacy of the SVF for closure of aero-digestive 
fistulas. Both the researcher and the participant in this research study will know the treatment the 
participant is receiving. No control group will be required as every participant will receive the 
same therapy. Subjects will be identified in the inpatient or outpatient setting during consults 
and/or appointments with Gastroenterology and/or Thoracic Surgery Departments. When a 
potential patient is identified, a research coordinator nurse (RN) consult will be initiated in order 
to identify if the patient meets all the inclusion/exclusion criteria for the study.  
After signing of informed consent and successful screening, baseline values, fistula 
measurements and a radiographical assessment of the fistula will be obtained from the subject.  
The adipose tissue lipoaspiration and subsequent endoscopy will be scheduled. . The adipose 
tissue harvest will be performed in the Regenerative Medicine Therapy Suites by qualified 
providers. The SVF will be manufactured on site in the Regenerative Medicine Therapy Suites 
and injected into the thrombin portion of the TISSEEL syringe and labeled for the individual 
patient. Then the final product will be taken to the gastrointestinal/bronchoscopic laboratory for 
endoscopic administration.  
 
 

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Placement of an Alimaxx or Wallflex stent covering the fistulous tract after SVF administration 
procedure will be considered in order to prevent mechanical recanalization of the fistula. The 
patient will be allowed to eat low residue meals 8 hours after the procedure. The status of the 
fistulae will be assessed with contrast studies (e.g. barium swallow) or other imaging studies 
such as CT scan or MRI, immediately after the procedure. Other radiographic studies can be 
performed within 24 hours post procedure. The patient and fistula status will be reassessed with 
imaging studies after 1 month. If a stent was placed, the latter will be removed during this visit.  
 
Follow up visits will take place every 3 months for the first year and then yearly for five years. 
Imaging studies, such as barium swallow or CT scan to assess fistula closure will be performed 
on every visit as clinically indicated. Evaluations will consist of an assessment of fistula healing 
including fistula characteristics and measurements. Clinical signs and symptoms such as cough, 
dysphagia, dyspnea, abdominal distention, sputum mixed with food, wheezing and lower 
respiratory tract infections will be documented. 
 
The nurse will also examine the lipoaspiration site for any signs of bleeding and/or infection. The 
patient’s temperature will be obtained because of the potential for a fever during therapy. 
If fistula closure is not achieved after 12 weeks from the second procedure, the patient will 
receive standard therapy. 
 
 
 
 

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Identification of patient 
with fistula by GI or CT 
Surgery 
RN consult for history 
review and 
inclusion/exclusion criteria 
Patient meets inclusion 
criteria and informed 
consent is obtained 
Yes 
Endoscopic assessment 
and picture. 
Fistula measuring > 15 
mm 
Patient excluded. 
Standard therapy 
Fistula measuring 4-15 
mm 
Lipoaspirationappointm
ent scheduled 
SVF processing 
Endoscopic 
administration of final 
d
No 
Patient excluded. 
Standard therapy 

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3.2 
Primary Study Endpoints 
SVF harvested without complication and a treatment regimen is established for applying the SVF 
suspended in fibrin glue in the fistulous tract.  
 
 
3.3 
Secondary Study Endpoints 
 
• Fistulous tract reduction and/or closure within 16 weeks of therapy documented by 
measurement and photography of the fistula in endoscopic/bronchoscopic evaluation. 
• Cardinal symptoms to be reduced, documented by history and physical examination. 
• Fistula recurrence to be less, documented by long term follow up. 
 
3.4 
Primary Safety Endpoints 
 
Study procedures will be stopped if adverse reactions occur with either the SVF, the fistula 
itself, or the lipoaspiration wound and the patient will be referred for standard therapy. This 
would include but not limited to: 
a. Signs of intravascular application of TISEEL which can lead to intravascular coagulation, 
thromboembolic events and acute hypersensitivity reactions. 
b. Signs of adverse reactions to the lipoaspiration procedure include an allergy to the 
Hunstad solution, infection at the puncture wound site or risk of bleeding at the puncture 
wound site. Risks include bleeding or infection at the site and bruising for approximately 
one week. No adverse consequences of fluid overload have been associated with low 
volume lipoaspiration (< 5 liters).   
c. Signs of increased fistula size and/or aggravation of symptoms associated with fistulous 
tract such as paroxysmal cough, dysphagia, wheezing, abdominal distention, chest pain, 
dyspnea, pneumonia. 
 
For documented contamination or positive culture determined once the product has already been 
administered, patient will undergo a standard clinical assessment to include vital signs, symptom 
history, serum electrolytes, liver function tests, CBC and blood cultures x 2.  If clinically appropriate, 
the patient would be admitted to Mayo Clinic Hospital for inpatient overnight observation until 
documented as medically stable by qualified medical personnel 
4 Subject Selection Enrollment and Withdrawal 
4.1 
Inclusion Criteria 
• Adults ≥ 18 years old 
• Adults ≤ 90 years old 
• Aerodigestive fistula opening size between 2 mm and 15 mm in diameter (as measured 
by comparing its size with the open mouth of a biopsy forceps) 
• Inpatient or outpatient setting 
• Recurrent or de novo fistulas 
• A prior diagnosis of ADF in which the standard therapy has failed or is not applicable 
• Fistula location may include: 
o Tracheopharyngeal 

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o Tracheoesophageal 
o Bronchoesophageal 
• Fistula etiology may include: 
o Secondary to previous malignancy with complete remission 
o Secondary to radiotherapy as long as complete remission for 5 years has been 
achieved and documented 
o Congenital  with or without previous treatment 
o Secondary to surgical interventions or endoscopic therapies such as dilation and 
esophageal manipulation 
o Post prolonged tracheal intubation 
o Secondary to foreign body ingestion 
o Secondary to thoracic trauma/crush injuries 
o Secondary to caustic ingestion 
o Secondary to pneumonectomy or mechanical ventilation 
o Esophagomalacia 
• The ability of subjects to give appropriate consent or have an appropriate representative 
available to do so 
• The ability of subjects to return for follow up endoscopic assessment as established.  
 
4.2 
Exclusion Criteria 
 
• Exposure to any investigational drug or procedure within 3 months prior to study entry. 
• Patients with allergy to fibrin glue (TISSEEL) or anesthetics 
• Patients with active/ongoing malignancy such as esophageal, lung, tracheal, thyroid, 
oropharyngeal or gastric cancer 
• Patients on active regimen of chemotherapy 
• Patients receiving radiation 
• Diabetics with poor glucose metabolic control exhibited by an HbA1c > 9 
•  If there is evidence, in endoscopy, of dysplastic-appearing mucosa such as Barrett’s 
dysplasia near the fistula, this will be excluded.Patients that require surgical intervention 
at the fistula area for any reason 
• BMI of <16 (may difficult lipoaspiration procedure) 
• Women who are pregnant or nursing or women of childbearing potential who are 
unwilling to maintain contraceptive therapy for the duration of the study 
• Clinical signs of respiratory tract or pleuro-pulmonary infections 
• Prolonged (> 6 months) use of steroids 
• Drug or alcohol dependence 
• Active infectious disease positive for HIV, HTLV, HBV, HCV, CMV (IgM > IgG) 
and/or syphilis 
• End of life 
 
4.3 
Subject Recruitment, Enrollment and Screening 
 
Recruitment efforts will include the following: 
• From the Co-Investigators’ clinical practices. 

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• Referring physicians. 
• Word of mouth 
 
Enrollment will include the evaluation and documentation of inclusion/exclusion criteria done 
with the consent. Family members identified by the patient will be included in the education and 
informed consent process if the patient prefers so. Subjects of child bearing potential are required 
to use appropriate contraceptive method(s). 
After the patient and family agree, the study RN will examine the patient for the feasibility and 
appropriateness of the patient to include: 
• Review of inclusion and exclusion criteria 
• Review of current symptoms. 
• Prior fistula treatment and management will be documented. 
 
If patient is eligible, an appointment for endoscopic/bronchoscopic evaluation with the study 
investigators will be scheduled. During this evaluation: 
 
• A picture of the fistula will be obtained. 
• Fistula characteristics such as size, location and appearance will be documented.  
• If the fistula measures up to 1.5 cm the patient will continue to be eligible. 
 
Qualified study personnel will be consulted to review the patient for the following: 
• Assessment of the ability to harvest adipose tissue. The abdomen will be used as the 
primary site however if unable to obtain an adequate amount, the hips will be used.  
 
4.4 
Early Withdrawal of Subjects 
4.4.1 When and How to Withdraw Subjects 
 
A subject may be withdrawn from the study prior to that subject completing all of the study 
related procedures. Some reasons include: 
• Signs of intravascular coagulation, thromboembolic events and acute hypersensitivity 
reactions secondary to TISSEEL. 
• Signs of adverse reactions to the lipoaspiration procedure including allergy to the 
Hunstad solution, infection at the puncture wound site, or bleeding at the puncture wound 
site. 
• Signs of increased fistula size and/or aggravation of symptoms associated with fistulous 
tract such as paroxysmal cough, dysphagia, wheezing, abdominal distention, chest pain, 
dyspnea, or pneumonia. 
• Failure of subject to adhere to protocol requirements. 
• Clinical signs that the primary research intervention is exposing the subject to an 
unacceptable level of risk. 
• Subject decision to withdraw from the study (withdrawal of consent). 
 
Sudden study treatment termination would occur for any noted signs of increasing infection, 
pulmonary complications or cardiac arrest. Conventional treatment, as appropriate, would 
continue with any subjects withdrawn from the study group.  

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Those participants that withdraw or are withdrawn from the study will be referred to their 
primary care physician for continued follow-up. A summary of the clinical procedures and 
patient response will be written and sent to the participant’s provider of choice. 
 
 
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 
Safety and feasibility information will be collected for the withdrawn subjects up to the date of 
withdrawal. Documentation of withdrawal will include whether the withdrawal resulted from a 
decision by the subject or by the investigator, the reasons for the withdrawal, if known and 
whether the withdrawal is from all components of the research study or just the primary 
interventional component. If follow-up information is required, permission will be obtained from 
the subject. 
 
5 Study Drug  
5.1 
Preparation and Administration of Study Drug 
 
The investigational agent is autologous, uncultured stromal vascular fraction. The fistulous area 
will be estimated upon consent.  Patients with a fistula measuring up to 1.5 cm in diameter will 
have 80-100mL of fat aspirated.  Lipoaspiration will be performed in the Regenerative Medicine 
Therapy Suites and processed in the Kiso located on the 3rd floor of the same building.  
 
The product dispensed by the KISO machine (SVF pellet) will be diluted in 2 ml lactated ringers 
solution. A total of 10 million MNCs (containing MSCs cells) are expected to be contained in 
this pellet-LR Solution combination.  A 0.5ml sample will be reserved for gram stain, culture, 
endotoxin testing, cell count and viability. The remaining product will be combined with 1 ml of 
Thrombin Solution of  TISSEEL (500 IU Thrombin/ml) to be injected with Sealer Protein 
Solution (3000 KIU/ml; as  per  manufacturer guidelines).  The solution as injectate would be 
delivered through a [Wilson Cook; varies] 19 or 22 gauge endosonographic needle 
circumferentially 3-5 mm from the fistulous tract in 0.1 to 0.3 ml aliquots [either sequentially or 
suspension followed by Sealer]. During surgical closure of fistula endoscopically, the 
product/TISSEEL combo will be injected around the fistula, then topically over the fistula.   
 
5.2 
Treatment Regimen  
Patients will receive approximately 5.0x106 mononuclear cells (MNCs) of SVF per cubic 
centimeter of fistulous area.  The fistula upper limit diameter will be 1.5 cm. Therefore, the total 
area of the fistula would measure 1.8 cm2 at the most. Most aerodigestive fistulas are contiguous 
in nature. We estimate that the fistulous tract will measure 1 cm at the most. Fistula volume will 
have an upper limit volume of 1.8 cm3. As recommended by the manufacturer, the volume of 
TISSEEL to be utilized will be determined by dividing the fistula volume by 4 to yield the final 
amount of TISSEEL needed to re-suspend the SVF.  
Fistulous Area 
Required TISSEEL amount 
1.8cm2 
0.5ml 
 

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5.3 
Isolation of Stromal Vascular Fraction (SVF) using KISO: 
The lipoaspirated tissue KISO system is used in this study for automated SVF cell isolation 
from human lipoaspirate. The KISO system employs disposable tissue processing bag kit, 
Collagenase Solution (LRS). The sterile disposable tissue processing bag kit contains all-in-one 
3 chamber tissue processing bag; plastic connection tubings; stopcock manifold controlling 
delivery of fluid reagents; a standard spike designed for connection with standard clinical LRS 
bag; and standard contamination-free safe environment, consistency between multiple isolations, 
and compact size of the device. It contains chambers for (1) adipose tissue digestion, and 
separation of crude SVF from adipocytes; (2) waste collection; (3) further SVF purification by 
filtration, concentration, standard catheter tip syringe, minimizing potential contamination of the 
sample. Movement of mixing cell suspension in the bag with external agitator and separating 
SVF cells by filtration within the waste chamber. After digestion SVF cell suspension is 
delivered to another chamber with embedded mesh (mesh-in-the-bag). Smaller size mesh retains 
cell clamps and adipocytes which passed the mesh in tissue digestion chamber. Eventually, 
filtered SVF cell suspension will be incubated, mixed and deconstructed, using a standard 
protocol under sterile conditions.  
 
The KISO system employs disposable tissue processing bag kit, SERVA cGMP Collagenase 
(SERVA, Cat. No. 17454) enzyme, and a standard bag of Lactate Ringers Solution (LRS). The 
sterile disposable tissue processing bag kit contains all-in-one 3 chamber tissue processing bag; 
plastic connection tubings; stopcock manifold controlling delivery of fluid reagents; a standard 
spike designed for connection with standard clinical LRS bag; and standard luer lock 60 mL 
output syringe. All-in-one 3 chamber tissue processing bag provides contamination-free safe 
environment, consistency between multiple isolations, and compact size of the device. It contains 
chambers for (1) adipose tissue digestion, and separation of crude SVF from adipocytes; (2) 
waste collection; (3) further SVF purification by filtration, concentration, and collection. 
Adipose tissue is delivered to the processing chamber via input nozzle with a standard catheter 
tip syringe, minimizing potential contamination of the sample. Movement of fluids between the 
chambers in such 3 chamber tissue processing bag is mediated by gravity eliminating the need 
for a pump, and is controlled by single stopcock valve integrated in processing bag. Adipose 
tissue processing chamber with embedded mesh (mesh-in-the-bag) allows performing multiple 
steps for SVF separation in one compartment: two consecutive washings of the adipose tissue, 
enzymatic digestion, phase separation (an aqueous fraction of tissue and a fatty fraction of the 
digested tissue sample) thus maintaining the same compact dimensions, minimizing 
contamination risk, and further facilitating fat tissue disruption by mixing cell suspension in the 
bag with external agitator and separating SVF cells by filtration. 
 
External agitator and heating plate provide the best optimized environment for adipose tissue 
digestion and SVF cell separation. All the wastes are separated and collected automatically 
within the waste chamber. After digestion SVF cell suspension is delivered to another chamber 
with embedded mesh (mesh-in-the-bag). Smaller size mesh retains cell clamps and adipocytes 
which passed the mesh in tissue digestion chamber. Eventually, filtered SVF cell suspension is 
drawn into a standard luer lock 60 mL output syringe and centrifuged (600g, 10 min) to remove 
the fluid and recover SVF cell pellet. The resulting fluid with stromal vascular fraction (SVF) 
will be centrifuged for 10 minutes. Total preparation time for the SVF is expected to 
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Ringer’s solution.  This product is then combined with the recommended quantity of TISSEEL 
fibrin glue (TISSEEL). The sample of SVF in Lactated Ringers is the final product that will be 
administered, along with TISSEELvia a minimally invasive endoscopic procedure into and 
around the patient’s fistula. 
 
5.4 
Subject Compliance and prior therapy 
To continue in the study, the subject must comply with all components of the study including 
every follow up appointment for assessment of fistula closure.  
Patient history related to fistula management will be documented at the initial visit.  Fistula 
therapies outside of the study protocol will not be permitted. We would exclude patients who 
require further endoscopic or surgical intervention for management of the fistula.  
 
5.5 
Packaging 
No special packaging will be involved in this study. After cell preparation as outlined above, the 
TISSEEL applicator containing the cells will be labeled with " CAUTION: NEW DRUG – 
LIMITED BY FEDERAL LAW TO INVESTIGATIONAL USE. FOR AUTOLOGOUS USE 
ONLY, NOT EVALUATED FOR INFECTIOUS SUBSTANCES and will be placed in a cooler 
for transportation of the therapy room located in the same building. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5.6 
Blinding of Study  
This is an open-label pilot study in which every participant will receive the treatment of interest 
and will be aware of it. 
 
 
5.7 
Receiving, Storage, Dispensing and Return 
5.7.1 Receipt of Drug Supplies 
Lipoaspirate will be received at the Regenerative Medicine Therapy Suites following protocol 
for “Receipt of Biospecimens for Clinical Trials”. 
Mayo Clinic Cell Therapy Laboratory  
4500 San Pablo Road S, Jacksonville, FL 32224  
(904) 956-1867  
 
Stromal Vascular Fraction          IRB #_______________________ 
Lot Number: XXXXXXX 
Expiration Date: XXXXXX            Subject #______________________ 
Cell Dose: XXXXXX 
 
Subject Name_________________ 
Released for Clinical Use: DATE 
CAUTION: NEW DRUG – LIMITED BY FEDERAL LAW TO 
INVESTIGATIONAL USE. 
FOR AUTOLOGOUS USE ONLY, NOT EVALUATED FOR INFECTIOUS 
SUBSTANCES 
 

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Study supplies (KISO and TISSEEL kits) will be received at the Regenerative Medicine Therapy 
Suites per protocol “Receiving Reagents & Supplies into the Cell Therapy Laboratory”. Supplies 
will be reviewed and checked for expiration prior to use in this study. 
 
5.7.2 Storage 
Excess SVF product will be disposed of according to appropriate handling of bio-hazardous 
waste. 
 
5.7.3 Dispensing of Study Drug 
 
Regular study SVF product reconciliation will be performed to document cells assigned, cells 
dispensed, cells returned, and cells remaining.  This reconciliation will be logged on the SVF 
product reconciliation form, which will be signed and dated by the study team. 
 
 
5.7.4 Return or Destruction of Study Drug 
 
At the completion of the study, there will be a final reconciliation of SVF product dispensed and 
remaining product. This reconciliation will be logged on the SVF product reconciliation form, 
which will be signed and dated.  Any discrepancies noted will be documented and investigated, 
prior to destruction of unused study cells. Cells destroyed will be documented in the study files. 
 
6 Study Procedures 
6.1 
Visit 1 
1. Screening to identify interested and qualified subjects. 
2. When an interested, qualified subject is identified, a study RN consult will be initiated.  
3. The study RN will assess patient eligibility. 
4. If inclusion criteria are met, participation in this pilot clinical trial will be offered.   
5. A subsequent visit will be scheduled to obtain radiographic studies for baseline assessment of 
the fistula if not available already.  
6. If the patient already has radiographical evidence and assessment of the fistula meeting the 
study criteria and the patient agrees to participation, a delegated study team member will 
obtain written consent.  
7. The endoscopist/cardiothoracic surgeon will assess the radiographical fistula characteristics, 
and thoroughly document them on the medical records. Undermining and/or tunneling will 
be assessed in centimeters, when present. 
8. Initial assessment information will be obtained and documented on the case report form 
(CRF).   
9. Visit 2 will be scheduled for the lipoaspiration, endoscopy procedure, and SVF application. 
 
6.2 
Visit 2 
1. Qualified study personnel will prepare the patient for the lipoaspiration procedure to obtain 
the adipose tissue.   
2. 80-100 cc’s of adipose tissue will be removed by lipoaspiration and inserted into a sterile 
specimen container. The specimen will be labeled with the patient’s name and Subject ID 
#.A sticker indicating “Research Patient” will also be applied.  

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3. The adipose tissue will be processed and stromal cells isolated.  
4. The study RN will be paged to deliver the product and return to the endoscopist/CT surgeon 
for application per the study procedure. Patient will be transported to the endoscopy suites. 
5. Post-procedural measures will include an immediate contrast study if no contraindications. 
Other radiographic studies for bronchopleural fistula can be performed within the next 24 
hours if clinically indicated.  
6. If a stent is placed over the repaired fistula, the patient will be scheduled for stent removal in 
1 month. 
  
6.3 
Visit 3 
1. The patient will be evaluated by the physician who performed the procedure. 
2. If a stent was placed over the repaired fistula after the SVF administration, the stent will be 
removed during this visit. 
3. If clinically indicated, a radiographic study such as but not limited to barium swallow will be 
obtained in order to assess fistula evolution. Fistula measurements will include length, width, 
and depth in centimeters. Undermining and/or tunneling will be assessed in centimeters, 
when present. 
4. Assessment of the lipoaspiration site will be completed. 
5. All assessment findings and the treatment provided will be documented in the EMR and in 
the CRF by the study RN and/or physician who performed the procedure. 
6. Every image will be available and/or uploaded to QREADS in accordance with the Mayo 
Clinic Photography policy.  
7. Assessment for presence of adverse events will be completed and documented in the CRF. 
Any question of potential adverse events will be referred to the consultant.  
8. Patient will receive a trimestral return visit until study endpoints are reached. 
 
 
6.4 
Follow-up Visits 
Follow-up visits will be scheduled at month 4, 7, 10 and yearly for 5 years, to obtain information 
about fistula healing. Assessment for presence of adverse events will be completed and 
documented in the CRF. Any question of potential adverse events will be referred to the 
consultant. Each visit will include clinical assessments, exams, education, and imaging as 
clinically indicated. 
 
 
 

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6.5 
Study Visit Table 
 
Study Activity 
Visit 1 
Screening  
(two weeks 
prior to Day 
1) 
Visit 2  
Day 0   
Visit 3 
1 
month 
 
Visit 4 
4 
month 
  
Visit 5 
7 
month 
Visit 6 
10 
month 
Visit 7  
Year 1 
Year 2-
5 
follow-
up 
Inclusion/Exclusion 
criteria review  
X 
 
 
 
 
 
 
 
Review med list 
X 
 
 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
Medical History 
X 
X 
 
 
 
 
 
 
Physical exam  
(BMI, Ht, Wt, BP) 
X 
X 
 
 
 
 
 
X 
Temperature 
X 
X 
X 
X 
X 
X 
X 
X 
Clinical Assessment 
X 
X 
X 
X 
X 
X 
X 
X 
Fistula assessment with 
imaging 
X 
X 
X 
X 
X 
X 
X 
X 
Lipoaspiration 
 
X 
 
 
 
 
 
 
Application of SVF  
 
X 
 
 
 
 
 
 
Adverse event evaluation  
 
X 
X 
X 
X 
X 
X 
X 
Patient education 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
7 Statistical Plan 
7.1 
Sample Size Determination 
The size of the pilot study was based on factors of patient recruitment, time, and available 
resources. Patient recruitment determination was based on a review of the number of patients 
seen annually by Gastroenterology, Cardiothoracic Surgery and Otolaryngology with a diagnosis 
of aerodigestive or bronchopleural fistulae. We estimate that each of the latter departments treat 
around 5 patients with potential inclusion criteria for our study every year (15 patients per year in 
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7.2 
Statistical Methods 
Analysis will proceed according to the specific objectives following intention-to-treat principles.  
Sample sizes are too small for any formal comparisons; therefore all data will be summarized 
numerically and displayed graphically as appropriate.   
 
The primary objective relates to the safety and feasibility of implementing the harvesting and 
application of SVF on aerodigestive fistulae and no statistical analysis will be conducted related 
to this objective except to assist in summarizing the proportions of patients for whom 
complications occur that may impact the feasibility of conducting a future trial. 
The secondary objective relates to the efficacy of SVF regarding time to fistula closure.  For this 
aim, we will graphically display the size of each patient’s fistula until closure is achieved.  
Separately we will also summarize the number and percent of patients with the following 
secondary endpoints: (1) greater than 20% fistula healing within 4 weeks after start of treatment; 
(2) greater than 60% fistula healing within 4 months after start of treatment and (3) in-vitro cell 
characterization, differentiation and quantification. 
 
All study results will be taken into consideration when determining whether the use of SVF for 
treating aerodigestive fistulae warrants further study in a larger randomized control trial.    
 
8 Safety and Adverse Events 
8.1 
Definitions 
 
Adverse Event 
An untoward or undesirable experience associated with the use of a medical product (i.e. drug, 
device, biologic) in a patient or research subject. 
 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets the following three criteria:  
• Serious: Serious problems or events that results in significant harm, (which may be 
physical, psychological, financial, social, economic, or legal) or increased risk for the 
subject or others (including individuals who are not research subjects). These include: (1) 
death; (2) life threatening adverse experience; (3) hospitalization - inpatient, new, or 
prolonged; (4) disability/incapacity - persistent or significant; (5) birth defect/anomaly; (6) 
breach of confidentiality and (7) other problems, events, or new information (i.e. 
publications, DSMB reports, interim findings, product labeling change) that in the opinion 
of the local investigator may adversely affect the rights, safety, or welfare of the subjects 
or others, or substantially compromise the research data, AND 
• Unanticipated: (i.e. unexpected) problems or events are those that are not already 
described as potential risks in the protocol, consent document, not listed in the 
Investigator’s Brochure, or not part of an underlying disease. A problem or event is 
"unanticipated" when it was unforeseeable at the time of its occurrence. A problem or 
event is "unanticipated" when it occurs at an increased frequency or at an increased 
severity than expected, AND 
• Related: A problem or event is "related" if it is possibly related to the research procedures. 
 

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All adverse events that do not meet any of the criteria for serious, should be regarded as non-
serious adverse events.  
 
Adverse Event Reporting Period 
For this study, the study treatment follow-up period is defined as the first application of the SVFs 
to the fistula until the end of the first year follow-up visit to assess the fistula status. Adverse 
events may fall into three areas: 
1.  A positive culture of the study product.  
2. Fistula complication e.g. infection, further progression, stent migration with fistula 
recanalization, TISSEEL intravascular injection with DIC, perforation, TISSEEL allergy. 
2.  Lipoaspiration complications e.g. infection at site of incision. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting 
condition.   
The NIH Common Terminology Criteria for Adverse Events (CTCAE) version 4 will be used to 
grade the adverse event.  
At the end of the study, any new clinically significant findings/abnormalities that meet the 
definition of an adverse event will be recorded and documented as an adverse event.  
 
Post-study Adverse Event 
All unresolved adverse events will be followed by the sponsor-investigator until the events are 
resolved, the subject is lost to follow-up, or the adverse event is otherwise explained.  At the last 
scheduled visit, the sponsor-investigator will instruct each subject to report, to the sponsor-
investigator, any subsequent event(s) that the subject, or the subject’s personal physician, 
believes might reasonably be related to participation in this study.  
 
8.2 
Recording of Adverse Events 
At each contact with the subject, the study team will seek information on adverse events by 
specific questioning and, as appropriate, by examination.  Information on all adverse events will 
be recorded immediately in the source document, and also in the appropriate adverse event 
section of the case report form (CRF).  All clearly related signs, symptoms, and abnormal 
diagnostic, laboratory or procedure results will be recorded in the source document. 
 
All adverse events occurring during the study period will be recorded.  The clinical course of 
each event should be followed until resolution, stabilization, or until it has been ultimately 
determined that the study treatment or participation is not the probable cause.  Serious adverse 
events that are still ongoing at the end of the study period will be followed up, to determine the 
final outcome.  Any serious adverse event that occurs after the study period and is considered to 
be at least possibly related to the study treatment or study participation will be recorded and 
reported immediately. 
 
 
8.3 
Reporting of Serious Adverse Events and Unanticipated Problems 
When an adverse event has been identified, the study team will take appropriated action 
necessary to protect the study participant and then complete the Study Adverse Event Worksheet 

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and log.  The sponsor-investigator will evaluate the event and determine the necessary follow-up 
and reporting required. 
Should any of the tested samples yield a positive culture, the Regenerative Medicine Therapy 
Suites Medical Director, Dr. Shapiro, and the PI, Dr. Woodward will be notified. An immediate 
assessment of results will be performed to determine if the source is from the product (True 
positive) or due to a lab contaminant. Colony count, identity of the organism, if immediately 
available, and specimen handling and process will all be evaluated when determining the source 
of the microbial contamination.  
 
If during follow-up visits, the fistulous tract increases in size or if there are clinical or 
radiological signs of a worsening condition, perforation or infection, the PI will be notified, the 
patient will be informed and offered standard of care. 
No study subject will be further treated until root-cause analysis is performed and appropriate 
actions to address the findings are completed.  
 
8.3.1 Sponsor-Investigator reporting: notifying the Mayo IRB 
The sponsor-investigator will report to the Mayo IRB any UPIRTSOs and Non-UPIRTSOs 
according to the Mayo IRB Policy and Procedures within 24 hours.  
Information will be collected on the adverse event worksheet (and entered in the REDCap 
database):  
• Subject’s name:  
• Medical record number:  
• Disease/histology (if applicable):  
• The date the adverse event occurred:  
• Description of the adverse event:  
• Relationship of the adverse event to the research (drug, procedure, or intervention):  
• If the adverse event was expected:  
• The severity of the adverse event: (use a table to define severity scale 1-5) 
• If any intervention was necessary:  
• Resolution: (was the incident resolved spontaneously, or after discontinuing treatment) 
• Date of Resolution:  
 
The sponsor-investigator will review all adverse event reports to determine if specific reports 
need to be made to the IRB and FDA.  The sponsor-investigator will sign and date the adverse 
event report when it is reviewed.  For this protocol, only directly related SAEs/UPIRTSOs will 
be reported to the IRB. Non-UPIRTSOs will be reported to the IRB at the continuing review.  
 
8.3.2 Sponsor-Investigator reporting: Notifying the FDA 
The sponsor-investigator will report to the FDA all unexpected, serious suspected adverse 
reactions according to the required IND Safety Reporting timelines, formats and requirements. 
 
Unexpected fatal or life threatening suspected adverse reactions where there is evidence to 
suggest a causal relationship between the study drug and the adverse event, will be reported as a 
serious suspected adverse reaction.  This will be reported to the FDA on FDA Form 3500A, no 
later than 7 calendar days after the sponsor-investigator’s initial receipt of the information about 
the event. 

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Other unexpected serious adverse reactions where there is evidence to suggest a causal 
relationship between the study drug and the adverse event and any clinically important increase 
in the rate of serious adverse reactions will be reported as a serious adverse reaction.  This will 
be reported to the FDA on FDA Form 3500A, no later than 15 calendar days after the sponsor-
investigator’s initial receipt of the information about the event. 
 
8.4 
Stopping Rules  
Study enrollment and treatment procedures will be suspended in the event that two successive 
patients experience a Serious Adverse Event during the procedure or within the first 2 weeks 
after treatment. The study may also be suspended or terminated at the direction of the DSMB 
based on the review criteria outlined in the Data Safety Monitoring Plan (DSMP). The study 
would only be resumed after a thorough review of the incidents and any corrective and 
preventive actions have been put in place along with consultation between the study team and the 
IRB. 
 
8.5 
Medical (Clinical) Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study at his site.  
This safety monitoring will include careful assessment and appropriate reporting of adverse 
events as noted above, as well as the construction and implementation of a site data and safety-
monitoring plan (see section 10  “Study Monitoring, Auditing, and Inspecting”).  Medical 
monitoring will include a regular assessment of the number and type of serious adverse events. 
 
8.5.1 Internal Data and Safety Monitoring Board 
The internal DSMB will review the study data as outlined in section 3.4 related to the primary 
safety outcomes. This internal board will be comprised of (Albert G. Hakaim, M.D., Brian Lacy, 
M.D., Ph.D, and Frank Lukens, M.D.). They will evaluate the study according to the DSMP. 
 
9 Data Handling and Record Keeping 
9.1 
Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  
Those regulations require a signed subject authorization informing the subject of the following:  
• What protected health information (PHI) will be collected from subjects in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization.  For subjects that have revoked authorization to collect or use PHI, attempts 
should be made to obtain permission to collect at least vital status (long term survival status that 
the subject is alive) at the end of their scheduled study period. 
Hard copy data such as consent forms will be stored in locked file cabinets; electronic data will 
be stored in secure web-based database (REDCap) that will be designed with the help of the 
statistician. 

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9.2 
Source Documents 
Source data is all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  Source 
data are contained in source documents.  Examples of these original documents and data records 
include: hospital records, clinic and office charts, laboratory notes, memoranda, evaluation 
checklist, recorded data from automated instruments, photographic images, subject files, and 
records kept at the laboratories involved in the clinical trial. 
 
9.3 
Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the study.  All 
data requested on the CRF will be recorded in the REDCap database.  A printed REDCap source 
document will be used to capture the subject’s clinical data. All missing data will be explained.  
If a space on the CRF is left blank because the procedure was not done or the question was not 
answered, it will be documented as “N/D”.  If the item is not applicable to the individual case, it 
will be documented as “N/A”.  Errors will be identified by a strike-through and corrected data.   
 
Data Management 
Study data will be managed in a study specific REDCap system. 
 
Data Security and Confidentiality 
The REDCap system has built in system for control of access, data integrity and audit trails. 
Access and confidentiality are controlled in a manner similar to other institutional systems.  
 
Data Quality Assurance 
The principal investigator will be accountable to verify the accuracy, completeness, and validity 
of the data recorded.  
 
Data Clarification Process 
A report will be run on the REDCap program to identify missing elements in the data capture.  A 
designated study team member will make corrections in the data and the principal investigator 
will verify. 
 
 
9.4 
Records Retention 
The sponsor-investigator will maintain records and essential documents related to the conduct of 
the study.  These will include subject’s source documents and case report form, REDCap 
database and regulatory documents. 
The sponsor-investigator will retain the specified records and reports for; 
1. Up to 2 years after the marketing application is approved for the drug; or, if a marketing 
application is not submitted or approved for the drug, until 2 years after shipment and 
delivery of the drug for investigational use is discontinued and the FDA has been so 
notified OR 
2. As outlined in the Mayo Clinic Research Policy Manual –“Access to and Retention of 
Research Data Policy” http://mayocontent.mayo.edu/research-policy/MSS_669717  
whichever is longer. 

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10 Study Monitoring, Auditing, and Inspecting 
10.1 Study Monitoring Plan 
The investigator will allocate adequate time for such monitoring activities.  The Investigator will 
also ensure that the monitor or other compliance or quality assurance reviewer is given access to 
all the study-related documents and study related facilities (e.g. Regenerative Medicine Therapy 
Suites, etc.), and has adequate space to conduct the monitoring visit. 
 
This study will be monitored on a routine basis during the conduct of the pilot.  The Mayo Clinic 
Office of Research Regulatory Support can provide assistance with clinical monitoring for the 
pilot support the sponsor-investigator.  Clinical trial monitoring requires review of the study data 
generated throughout the duration of the study to ensure the validity and integrity of the data 
along with the protection of human research subjects.  This will assist sponsor-investigators in 
complying with Food and Drug Administration regulations. 
 
Study participants will have follow-up visits at 4 months, 7 months, 10 months, and 1 year as 
well as yearly for 5 years after treatment to assure they continue to heal and there are no 
questions or complications.  
 
10.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, the 
sponsor, and government regulatory agencies, of all study related documents (e.g. source 
documents, regulatory documents, data collection instruments, study data etc.).  The investigator 
will ensure the capability for inspections of applicable study-related facilities (e.g. Cell Therapy 
Laboratory, etc.). 
Participation as an investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable compliance offices. 
 
11 Ethical Considerations 
This study is to be conducted according to all regulations and Mayo Clinic research policies and 
procedures. 
 
This protocol and any amendments will be submitted to a properly constituted local Institutional 
Review Board (IRB), in agreement with local legal prescriptions, for formal approval of the 
study.  The decision of the IRB concerning the conduct of the study will be made in writing to 
the sponsor-investigator before commencement of this study. 
 
All subjects for this study will be provided a consent form describing this study and providing 
sufficient information for subjects to make an informed decision about their participation in this 
study.  This consent form will be submitted with the protocol for review and approval by the IRB 
for the study.  The formal consent of a subject, using the Approved IRB consent form, must be 
obtained before that subject undergoes any study procedure.  The consent form must be signed 
by the subject or the subject’s legally authorized representative, and the individual obtaining the 
informed consent. 
 

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12 Study Finances 
 
12.1 Funding Source 
This pilot study will be financed either by external grants or internal grants from Mayo Clinic.  
 
12.2 Conflict of Interest 
None 
 
13 Publication Plan 
 
The Principal Investigator will be responsible for manuscript preparation and submission to a 
suitable journal for publication of results.  
Registration on ClinicalTrials.gov will be completed as appropriate.  
 
 
14 References 
 
 
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for wound healing applications. Ann Plast Surg. Feb 2011;66(2):210-215. 
2. 
G. Vantrappen JH. Diseases of the esophagus2012. 
3. 
Hyman S. Abrams MT. Esophagorespiratory Fistulae. JAMA Archives of Otolaryngology. 
1954;60(3):371-374. 
4. 
Murthy S, Gonzalez-Stawinski GV, Rozas MS, Gildea TR, Dumot JA. Palliation of malignant 
aerodigestive fistulae with self-expanding metallic stents. Dis Esophagus. 2007;20(5):386-389. 
5. 
Balazs A, Kupcsulik PK, Galambos Z. Esophagorespiratory fistulas of tumorous origin. Non-
operative management of 264 cases in a 20-year period. Eur J Cardiothorac Surg. Nov 
2008;34(5):1103-1107. 
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Richter GT, Ryckman F, Brown RL, Rutter MJ. Endoscopic management of recurrent 
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Park KJ, Ryoo SB, Kim JS, et al. Allogeneic adipose-derived stem cells for the treatment of 
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Lee WY, Park KJ, Cho YB, et al. Autologous adipose tissue-derived stem cells treatment 
demonstrated favorable and sustainable therapeutic effect for Crohn's fistula. Stem Cells. Nov 
2013;31(11):2575-2581. 
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Sanz-Baro R, Garcia-Arranz M, Guadalajara H, de la Quintana P, Herreros MD, Garcia-Olmo D. 
First-in-Human Case Study: Pregnancy in Women With Crohn's Perianal Fistula Treated With 
Adipose-Derived Stem Cells: A Safety Study. Stem Cells Transl Med. Jun 2015;4(6):598-602. 
10. 
Mizushima T, Takahashi H, Takeyama H, et al. A clinical trial of autologous adipose-derived 
regenerative cell transplantation for a postoperative enterocutaneous fistula. Surgery Today. 
2016;46(7):835-842. 
11. 
Aho JM, Dietz AB, Radel DJ, et al. Closure of a Recurrent Bronchopleural Fistula Using a Matrix 
Seeded With Patient-Derived Mesenchymal Stem Cells. Stem Cells Transl Med. Oct 
2016;5(10):1375-1379. 
12. 
Diaz-Agero Alvarez PJ, Bellido-Reyes YA, Sanchez-Giron JG, Garcia-Olmo D, Garcia-Arranz 
M. Novel bronchoscopic treatment for bronchopleural fistula using adipose-derived stromal cells. 
Cytotherapy. Jan 2016;18(1):36-40. 

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13. 
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Casteilla L. Adipose-derived stromal cells: Their identity and uses in clinical trials, an update. 
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