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TRANSLATIONAL AND CLINICAL RESEARCH Concise Review: Adipose-Derived Stromal Vascular Fraction Cells and Stem Cells: Let’s Not Get Lost in Translation JEFFREY M. GIMBLE,a BRUCE A. BUNNELL,b ERNEST S. CHIU,c FARSHID GUILAKd aStem Cell Biology Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA; bCenter for Stem Cell Research and Regenerative Medicine and cDivision of Plastic and Reconstructive Surgery, Department of Surgery, Tulane University Medical Center, New Orleans, Louisiana, USA; dOrthopaedic Research Laboratory, Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA Key Words. Adipose • Adipogenesis • Adult stem cells • Cellular therapy • Stem cell transplantation • Stromal cells • Clinical translation • Current Good Manufacturing Practices ABSTRACT Subcutaneous fat has emerged as an alternative tissue source for stromal/stem cells in regenerative medicine. Over the past decade, international research efforts have established a wealth of basic science and preclinical evi- dence regarding the differentiation potential and regener- ative properties of both freshly processed, heterogeneous stromal vascular fraction cells and culture expanded, relatively homogeneous adipose-derived stromal/stem cells. The stage has been set for clinicians to translate adipose-derived cells from the bench to the bedside; how- ever, this process will involve ‘‘development’’ steps that fall outside of traditional ‘‘hypothesis-driven, mechanism- based’’ paradigm. This concise review examines the next stages of the development process for therapeutic appli- cations of adipose-derived cells and highlights the current state of the art regarding clinical trials. It is recom- mended that the experiments addressing these issues be reported comprehensively in the peer-review literature. This transparency will accelerate the standardization and reproducibility of adipose-derived cell therapies with respect to their efficacy and safety. STEM CELLS 2011;29:749–754 Disclosure of potential conflicts of interest is found at the end of this article. INTRODUCTION—WHAT IS THIS ABOUT? Subcutaneous fat is an abundant and accessible source of both uncultured/heterogeneous stromal vascular fraction (SVF) cells and cultured/relatively homogeneous adipose-derived stromal/ stem cells (ASCs). The peer-reviewed literature focusing on SVF cell and ASC research has expanded exponentially over the past decade. This body of work has excited the international stem cell community as demonstrated by the registration of 36 clinical trials in 11 different countries on the NIH (http://www.clinicaltrials. gov) identified with the key words ‘‘adipose stem cells’’; this com- pares to 143 studies under the term ‘‘mesenchymal stem cell.’’ Regulatory authorities require more than mechanism-based evi- dence before authorizing ‘‘investigational new drug’’ (IND) stud- ies with cell-based therapies. Additional ‘‘development’’ studies must be provided to complete the ‘‘research and development’’ required to support IND proposals. Despite the collection of this information in both public (academic) and private (biotech) sector, little of this data has appeared in the scientific literature. Increased distribution of such data through peer-reviewed papers would accelerate the pace of translation for ASCs and SVF cells to the clinic. Such studies would document the reproducibility of out- comes-based evidence regarding adverse events, safety, and effi- cacy from independent sources. Disseminating information on iso- lation and culture methods, surgical approaches, challenges, and their solutions would foster international cooperation and stand- ardization. Despite financial incentives and intellectual property concerns to the contrary, all parties in the stem cell community could benefit from a greater public awareness of the development side of the picture. This concise review evaluates current and future experiments designed to minimize the likelihood that the clinical value of SVF cells and ASCs will get ‘‘lost in translation.’’ PRECLINICAL SAFETY AND EFFICACY DATA— WHAT (AND HOW) HAVE WE BEEN DOING? Regulations The regulations controlling the delivery of adipose-derived cell therapeutics to the clinic parallel many of those devel- oped for the pharmaceutical industry [1]. Guidelines govern- ing the development of cell-based products can be found on websites for the U.S. Food and Drug Administration (FDA: http://www.fda.gov/), the European Medicines Agency Author contributions: J.M.G.: conception and design, financial support, administrative support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; F.G., B.A.B., E.S.C.: conception and design, final approval of manuscript. Correspondence: Jeffrey M. Gimble, M.D., Ph.D., Stem Cell Biology Laboratory, Pennington Biomedical Research Center, Louisiana State University System, 6400, Perkins Rd, Baton Rouge, Louisiana 70808, USA. Telephone: 225-763-3171; Fax: 225-763-0273; e-mail: gimblejm@pbrc.edu Received January 5, 2011; accepted for publication February 10, 2011; first published online in STEM CELLS EXPRESS March 23, 2011. VC AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.629 STEM CELLS 2011;29:749–754 www.StemCells.com (EMEA: http://www.ema.europa.eu/ema), and related govern- mental regulatory authorities. Similarly, the United States Pharmacopeia (USP) is an internationally recognized resource defining the currently accepted industry standards for product purity, potency, and quality assurance (http://www.usp.org/). The use of USP-based assays for each step in the ASC and SVF cell manufacturing process ensures the reproducibility and reliability of the final product. To date, most laboratories use several common steps to process cells from adipose tissue [2]. These are: (a) washing; (b) enzymatic digestion/mechani- cal disruption; (c) centrifugal separation for isolation of SVF cells which can used directly, cryopreserved, or (d) culture expanded for the generation of ASCs (see figure in [3]). GLP, cGMP, and Standard Operating Procedures Most academic research laboratories do not produce adipose stem cells in accordance with the criteria for either Good Lab- oratory Practices (GLP) or the more stringent current Good Manufacturing Practices (cGMP). Both GLP and cGMP require strict operational and certification records relating to all laboratory equipments used in the cell manufacture process [1, 4]. Additionally, all operational procedures, from the mop- ping of floors to the maintenance of incubators and biological safety cabinets, must be performed and recorded routinely in accordance with defined and validated standard operating pro- cedures. Lot specific manufacturing records should be devel- oped to ensure standard practices and provide a written docu- ment validating quality assurance and quality control by the operators. In a recent manuscript, Sensebe´ et al. [5] provide a comprehensive and thorough review of this topic. Since any adipose cell–based therapeutics destined for clinical use must meet cGMP standards, there are multiple reagents and proce- dures that merit special attention. Closed System Manufacturing Devices Contamination by infectious agents presents a fundamental challenge to any cell or tissue product. Several companies have developed self-contained lipoaspirate processing devices that collect, wash, digest, and separate cells without exposing them to the environment (http://www.cytori.com and http:// www.tissuegenesis.com/) [6]. Closed culture/expansion sys- tems have been developed to exchange medium in large capacity tissue culture flasks using stopcocks and gravity-based flow to minimize the risk of operator error during the culture expansion process [4]. Bioreactors with controlled flow rates and built in monitors for cell viability, lactate production, pH/ pO2, and glucose levels need to be made efficient, practical, and scalable to current and future needs [7]. The use of auto- mated, computer-controlled devices has the potential to reduce the risk of operator error during the culture period. Donor Considerations The age, depot site, and sex of the adipose tissue donor have the potential to impact the functionality and quality of the derived cells. For example, a recent murine study found that a subpopulation of adipocyte progenitor cells are most frequent in visceral as opposed to subcutaneous adipose depots, increase with advancing age, and are more frequently observed in female donors [8]. A limited number of human studies provide similar findings. While an analysis of breast tissues specimens from >180 women donors aged 16–73 did not observe an age dependent difference in stromal cell num- bers or adipogenesis, increased body mass index correlated significantly with reduce cell numbers and differentiation [9]. Clinical studies examining subcutaneous adipose tissue from 12 to 52 donors have reported reduced ASC adipogenesis, angiogenesis, osteogenesis, and/or proliferative capacity as a function of advancing donor age [10–12]. Similarly, a detailed comparison of five different subcutaneous depots determined that ASC isolated from the arm and thigh best maintained adi- pogenic potential as a function of advancing age [12]. Further studies in larger cohorts will be necessary before patient dem- ographics can be used to predict the functionality and recov- ery of SVF cells and ASCs from donors as well as the rela- tive utility of specific depot sites. Besides, future studies will need to compare the efficacy of SVF cells versus ASCs from the same donor based on function in vivo. Sourcing of Reagents The quality of all cell-processing reagents must be validated by in-house assays. Each lot of growth factor, medium, or serum (e.g., fetal bovine serum; FBS) should be tested for potency using quantifiable metrics such as cell proliferation rates, viability, and/or differentiation potential. Even if a sole source provider is used for a particular reagent, there is no guarantee that they will not make changes in their manufac- turing process to reduce costs or in response to another customer. Indeed, there is a risk to using a single supplier for any particular reagent. Changes in ownership, ordering back- logs, or other uncontrollable external factors can prevent access to critical materials. Enzyme Quality Collagenase, dispase, and hyaluronidase are some of the enzymes used to disrupt lipoaspirate tissue. In their crude form, these reagents often contain contaminating amounts of endotoxin, other peptidases, and xenoproteins [13]. The steps involved in the manufacture of ‘‘sterile’’ or cGMP grade enzymes increases their cost by over 10-fold. The develop- ment of an efficient and reproducible mechanical-based tissue disruption process would remove the need for enzyme reagents and merits further investigation. There is evidence that functional ASCs can be expanded directly from lipoaspi- rate fluids without the need for collagenase digestion [14]. Similarly, multiple groups routinely use porcine-derived tryp- sin to passage plastic adherent ASCs, and recent studies have documented the equivalent performance of bacterial-derived or corn-derived trypsin products [15–17]. Thus, the removal of enzyme reagents is achievable. Serum Alternatives Historically, ASCs have been expanded in culture medium supplemented with FBS. The European regulatory agencies have particular concerns regarding any use of FBS due to the widespread presence of bovine spongiform encephalopathy (BSE). While rare cases of BSE have been identified in North American cattle herds, the use of irradiated FBS is allowed for cell expansion. Nevertheless, it is likely a matter of time before FBS will be phased out for use in clinical products. There is evidence that the presentation of FBS proteins, such as albumin, to the recipient immune system results in subse- quent antibody-based responses with the risk of serum sick- ness [18, 19]. A number of laboratories have found that human serum or platelet-derived supplements can serve as alternatives (reviewed in [20]). Some groups have relied on autologous serum, donated by the subject at the time of tissue collection, for ASC expansion [16]. The future may witness the development of commercial grade, infectious agent-free allogeneic serum sources for the generation of cGMP cell products. An optimal human serum reagent would be depleted of antibodies and complement proteins to reduce the risk of cell damage or adverse events. There is the possibility of removing serum entirely from the culture medium [15]. The Regea Institute has demonstrated the use of a commercially 750 Adipose-Derived SVF Cells and ASCs available xenoprotein-free product for ASC expansion [15]. While the proprietary nature of the medium leaves the public with questions about its active ingredients, the deposition of a confidential master file with a regulatory agency (FDA and EMEA) would address this concern. Product Definition There remains some dispute over the criteria defining an SVF cell or an ASC. While there is a general consensus that the SVF cells are a heterogeneous population, no specific ranges for each subpopulation have been agreed upon formally. The International Society for Cell Therapy (ISCT) has provided guidelines for the definition of mesenchymal stromal cells (MSCs) based on their plastic adherent properties, immuno- phenotype (CD73þ CD90þ CD105þ CD11b/14� CD19/ CD73b� CD34� CD45� HLA-DR�), and multipotent differ- entiation potential (adipogenic, chondrogenic, and osteogenic) [21]. While some have attempted to apply these criteria to ASC, there is a reason to doubt their applicability because early passage ASCs are routinely CD34þ [22, 23]. Investiga- tors continue to search for ASC specific surface markers. Some have used the protein Pref1, first identified on murine 3T3-L1 preadipocytes, as a putative ASC marker [24]. Others have reported the use of pericytic markers such as platelet- derived growth factor receptor b and 3G5 [23, 25–28]. Finally, combinatorial phage display approaches have associ- ated the presence of a5 b1 integrin with ASCs [29]. It is rec- ommended that the ISCT, the International Federation of Adi- pose Therapeutics and Science, or an equivalent society establish a task force of academic, biotechnology, and regula- tory agency representatives to issue a consensus statement on minimal acceptance criteria for both SVF cells and ASCs. These criteria should be based on cell viability and/or prolif- eration rates, immunophenotype, and differentiation potential. Wherever possible, criteria that can be collected in process and without the destruction of the final cell product should be considered, for example, measurement of secreted proteins in the conditioned medium. Additional parameters based on tran- scriptomic or proteomic approaches can be considered. Finally, the criteria must be practical, reproducible, and robust to meet future industry and manufacturing demands. Contamination Testing Assays must document that all cell products for human clini- cal applications are free of bacterial, endotoxin, mycoplasma, and viral (B19, cytomegalovirus, Epstein-Barr virus, hepatitis B and C, human immunodeficiency viruses 1 and 2, as well as human T-cell leukemia viruses 1 and 2) contamination. The adipose tissue donors may themselves be carriers of in- fectious agents or these can be introduced during the manu- facturing process despite precautions implemented under the cGMP process. For example, the inclusion of antibiotics and antimycotics in the culture medium can mask the presence of contaminants. There is reduced, but not absent, concern for infectious agents, when autologous adipose-derived cells are used. All allogeneic cell products must be defined rigorously as infectious agent-free and this introduces considerable cost and time to the manufacturing process. Cryopreservation Long-term storage will be critical to ensure a reliable supply and delivery of ASCs and SVF cells to point of care pro- viders. The majority of published ASC and SVF cell cryopre- servation procedures rely on the use of dimethyl sulfoxide (DMSO) as a cryoprotectant agent (CPA), often in combina- tion with serum protein components. While DMSO is used routinely with blood cell products, it has potential adverse effects on the recipient and may not be optimal for all cells. Alternative CPAs for ASCs and SVF cells include hydroxyethyl starch, trihelose, and polyvinyl and some can be used under serum free conditions [30–32]. These alternative options should be explored, validated as reproducible, and considered as future industry standards. While most academic laborato- ries store cryopreserved cells submerged in liquid nitrogen, cGMP grade products must be maintained in liquid nitrogen vapor phase storage containers. This removes any risk of cross-contamination between individual containers. It is unlikely that hospitals and clinics will routinely have access to liquid nitrogen storage containers at the point of care. Instead, it is likely that cell products will be kept at �70�C to �80�C and further data on the shelf life of adipose cell prod- ucts at these temperatures is needed. Shipping It is not only unlikely but also financially undesirable to maintain GMP facilities at all hospitals and clinics for the preparation of either SVF cells or ASCs. Consequently, adi- pose tissue and cell products will be shipped between the do- nor/recipient site and the processing laboratory. Data suggests that viable and functional ASC can be recovered from adipose tissue stored for up to 24 hours after liposuction [33, 34]. Studies need to be published relating to the viability of SVF cells and ASCs after shipment by either vehicle or air freight for extended periods of time. All studies must monitor the ambient temperature of the product. Outcome measures should include the minimal acceptance criteria for the cell products outlined above. Animal Studies There is a wealth of published evidence in animal models evaluating the safety and efficacy of adipose-derived cells (reviewed in [35]). The majority involves the use of rodents but a substantial number have used canine, ovine, porcine, and other large animal models. Nevertheless, is this body of evidence sufficient to satisfy regulatory authorities? The drug industry must perform trials in large numbers of male and female animals of varying ages monitored over periods rang- ing from a few days to �1 year. Monitoring studies need to evaluate the migration of cell implants to major organs (brain, heart, liver, lung, and kidney). Animal recipients must be monitored closely for evidence of tumor formation. There are few if any long-term, large animal studies with adipose- derived cells reported in the literature and this literature needs to be expanded in the near future. Needless to say, all animal studies require veterinary oversight and must be reviewed and approved by an institutional animal care and safety committee before implementation. Tumorigenesis There is precedent documenting the ability of human ASCs to transform during in vitro passage based on karyotypic changes in genotype and the development of nonadherent growth char- acteristics in agar cultures [36]. Furthermore, when these transformed ASC were implanted in immunodeficient mice, they formed sarcomas in vivo. Similar evidence from work using bone marrow MSCs has led to a policy statement by the ISCT regarding tumorigenesis [37]. While there is evidence suggesting that not all reported transformations in culture were actual events, it is incumbent on the stem cell community to take the most conservative approach with respect to patient safety. Studies should be published that spe- cifically monitor for the absence or presence of tumorigenesis using SVF cells and ASCs.