Manufacturing mesenchymal stromal cells for clinical applications: A survey of Good Manufacturing Practices at U.S. academic centers

Cytotherapy, 2019; 21: 782 792

Manufacturing mesenchymal stromal cells for clinical applications: A survey of Good Manufacturing Practices at U.S. academic centers

DONALD G. PHINNEY1 & JACQUES GALIPEAU2, MSC COMMITTEE OF THE INTERNATIONAL SOCIETY OF CELL AND GENE THERAPY 1

Department of Molecular Medicine, The Scripps Research Institute Scripps Florida, Jupiter, Florida, USA, and Department of Medicine and Carbone Cancer Center, University of Wisconsin in Madison, Madison, Wisconsin, USA

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Abstract Background aims: Mesenchymal stromal cells (MSC) have gained prominence in the field of regenerative medicine due to their excellent safety profile in human patients and recently demonstrated efficacy in late-stage clinical studies. A prerequisite to achieving successful MSC-based therapies is the development of large-scale manufacturing processes that preserve the biological potency of the founder cell population. Because no standardized manufacturing process exists for MSCs, understanding differences in these processes among U.S. academic facilities would allow for better comparison of results obtained in the clinical setting. Methods: We collected information through a questionnaire sent to U.S. academic centers that produce MSCs under Good Manufacturing Practice conditions. Results: The survey provided information on the number and geographic location of academic facilities in the United States and major trends in their manufacturing practices. For example, most facilities employed MSCs enriched from bone marrow by plastic adherence and expanded in media supplemented with pooled human platelet lysate. Sterility testing and product identification via cell surface phenotype analysis were commonly reported practices, whereas initial and working cell plating densities, culture duration, product formulation and the intended use of the MSC product were highly variable among facilities. The survey also revealed that although most facilities assessed product potency, the methods used were limited in scope compared with the broad array of intended clinical applications of the product. Conclusions: Survey responses reported herein offer insight into the current best practices used to manufacture MSC-based products in the United States and how these practices may affect product quality and potency. The responses also provide a foundation to establish standardized manufacturing platforms.

Key Words: cellular therapy, Good Manufacturing Practice, mesenchymal stem cells, mesenchymal stromal cells, potency, release criteria, survey

Introduction Mesenchymal stromal cells (MSCs) have been tested as an experimental therapy for a wide array of unrelated illnesses and to date have exhibited an excellent safety profile in human patients [1,2]. Recent outcomes from late-phase clinical trials indicate that MSCs exhibit durable efficacy in treating complex perianal fistulas in patients with Crohn disease and in reducing morbidity and mortality in pediatric patients with graft-versus-host disease [3]. Despite these successes, outcomes from many other early- and late-phase clinical trials have failed to meet expectations based on preclinical studies conducted in animal models of disease. Although many factors contribute to these suboptimal outcomes, the impact of large-scale manufacturing on the

composition and biological potency of MSC populations is only now emerging [4]. For example, although it is widely accepted that the therapeutic activity of MSC-based products is paracrine in nature [5] numerous studies have shown that these activities exhibit significant inter-donor heterogeneity [6 9]. Furthermore, it is well established that conditions used to expand cells, including oxygen saturation, plating density, growth factor supplementation, and replicative age, influence the cellular composition of populations, thereby altering potency beyond intrinsic differences related to donor selection [10 12]. Consequently, methods of manufacturing have direct impact on the potency of MSC-based products. Assessing the extent of this impact is complicated by the fact that no

Correspondence: Donald G. Phinney, PhD, Department of Molecular Medicine, The Scripps Research Institute, Scripps Florida, A215, 130 Scripps Way, Jupiter, FL 33458. E-mail: [email protected] (Received 15 February 2019; accepted 3 April 2019) ISSN 1465-3249 Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jcyt.2019.04.003

MSCs for clinical applications: a survey of good manufacturing practices standardized processes exist for MSC manufacturing. Moreover, methods for producing clinicalgrade products continue to evolve at a rapid pace with respect to design, automation, and integration of cell culture technologies [13 15]. Therefore, understanding current MSC manufacturing practices is essential to evaluating their potential impact on product potency and therapeutic efficacy. Because a paucity of information exits regarding best practices for manufacturing MSCs for clinical use, we undertook a study to collect information on these practices from U.S. academic centers involved in MSC clinical research.

Methods Generation and distribution of the survey As a template, we used a survey prepared by the European Society for Blood and Marrow Transplantation (EBMT) to assess cell manufacturing practices of its members [16]. The survey was then modified for the purposes of assessing best practices for the specific manufacturing of MSCs. Survey questions were vetted by members of the MSC committee of the International Society of Cell and Gene Therapy (ISCT) and then beta tested on several committee members before distribution. Academic Good Manufacturing Practice (GMP) facilities operating in the United States were identified from a list of those accredited by the Foundation for the Accreditation of Cellular Therapy (FACT) and web-based searches. All entries were then crossreferenced against information obtained from the facilities website and literature searches (PubMed) using the director’s name as a keyword. When ambiguity existed, facility directors were contacted by e-mail or phone to clarify whether they were engaged in MSC manufacturing. When such ambiguities could not be resolved, a survey was e-mailed to the facility director to maximize inclusiveness of the process. Data were collated by the lead author with assistance from staff of the ISCT MSC Committee.

Survey content Information gathered in the survey included the intended use for the product (musculoskeletal, cardiovascular, pulmonary, hematological, immunomodulation, infection, wound healing, tissue engineering, tissue regeneration, other), tissue source (bone marrow, adipose tissue, umbilical cord blood, placental tissue, umbilical cord tissue, other), tissue disaggregation method (dissection, enzyme digestion, sieving, automated system, other), cell enrichment method (plastic adherence, fluorescence-activated cell sorting [FACS], immuno-depletion, immuno-selection, other), plating

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density (low, medium, high), culture expansion method (two-dimensional [2-D], bioreactor, bags, microcarriers, hollow fiber), and medium supplements (fetal bovine serum [FBS], human platelet lysate [hPL], synthetic, growth factors, antibiotics, other). Information about MSC product identity for clinical release included analysis of positive (CD29, CD36, CD44, CD49b, CD49c, CD49f, CD59, CD73, CD79a, CD90, CD105, CD106, CD117, CD146, CD147, CD166, CD271, HLA-abc, other) and negative (CD3, CD4, CD8, CD11b, CD14, CD19, CD31, CD34, CD45, HLA-II, other) surface markers and their cutoff threshold. Safety was addressed by evaluating practices related to sterility testing (bacteria, mycoplasma, endotoxin, viruses), quantification of cell yields, cell viability and karyotype analysis. Facilities were also queried about practices related to quantifying product potency including skeletogenic potential (adipogenesis, osteogenesis, chondrogenesis), angiogenic/angiostatic activity (endothelial cell viability, endothelial tube formation, other), immunomodulatory activity (mixed lymphocyte reaction [MLR], natural killer cell [NK] lysis, T-regulatory cell [Treg] formation, other), and omics-based analyses (quantitative polymerase chain reaction microarray, microRNA array, RNASeq, Multi-plex ELISA, Proteomics, Metabolomics, other). Finally, information on cryopreservation and final formulation of the product was also requested. Data analysis Surveys were classified as complete or incomplete based on the following criteria. All surveys that reported only their name, contact information, accreditation status, and society affiliations but no other information on manufacturing were deemed incomplete and categorized as a “partial response.” These responses were valuable because they provided information on the number and location of academic GMP facilities in the United States In a subset of these cases, the facility director specifically stated that the facility did not currently manufacture MSCs. Surveys that also contained information about specific MSC manufacturing processes were categorized as “complete” with the caveat that not all questions or sub-questions were answered in every survey. The reason for the latter was unknown, but in some cases, it was to protect proprietary methodologies. Overall, information on geographic location, accreditation status and societal affiliations was recorded from a total of 27 facilities (complete and partial responders), and specific information related to MSC manufacturing was recorded from a total of 15 facilities (complete responders). In all cases, data are reported as number of facilities and percentage of the total number of respondents for each respective category.

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Results Facilities surveyed Surveys were sent to a total of 59 academic GMP facilities believed to be engaged in the manufacturing of MSC-based products based on their FACT accreditation status, website information, and publication record of the facility director. Facilities meeting these criteria were identified in 25 states (Figure 1A). The overall survey response rate was »63% (37 of 59). Twenty-seven facilities (»46%) completed the survey (full or partial response), nine facilities (»15%) indicated they did not manufacture MSCs, and one facility (»2%) declined to complete the survey. Additionally, three facilities (»5%) were misidentified, and 19 facilities (»32%) did not respond to the survey mailing (Figure 1B). Facilities that participated in the survey (complete or partial response) represented a total of 15 states covering all regional divisions recognized by the U.S. Census Bureau including New England, Mid-Atlantic, East and West North Central, South Atlantic, East and West South Central, Mountain, and Pacific (Figure 1C). Therefore, the survey results provide a broad representation of current manufacturing practices throughout the United States. Consistent with our methodological approach, a total of 19 of the 27 facilities (70.4%) surveyed were FACT accredited, and 1 indicated that its accreditation was pending (Figure 1D). Moreover, 6 facilities (22.2%) were accredited by FACT MTMM, 8

facilities (29.6%) by AABB (formerly the American Association of Blood Banks), and 2 facilities (7.4%) by the American Association of Tissue Banks (AATB). Additionally, 3 facilities (11.1%) were certified automation professionals or registered with the U.S. Food and Drug Administration (FDA), and one facility (3.7%) also possessed a drug master file with the FDA, had ISO9001 certification or possessed a drug manufacturing license with the state of California. The facilities surveyed also held memberships in various scientific societies that support basic and clinical science; 21 facilities (77.8%) were members of the ISCT, 15 (57.6%) of the American Society of Hematology, 9 (33.3%) of the American Society of Gene and Cell Therapy, 5 (18.5%) of the International Society of Stem Cell Research and 3 (11.1%) of the American Society of Bone Marrow Transplantation. At least one facility also held a membership with the American Association for the Advancement of Science, American Association for Cancer Research, EBMT, or the European Society of Gene and Cell Therapy (ESGCT) (Figure 1E).

Clinical indications and methods of cell isolation and expansion An important finding of the survey was the broad array of clinical indications it revealed for the manufactured MSC product. For example, 10 facilities (66.7%) indicated that the intended clinical

Figure 1. Survey statistics. (A) Geographic location of academic facilities surveyed. The total number of each facility per state is also indicated. (B) Overall survey response rates broken down by categories. (C) Geographic location of facilities surveyed by regions recognized by the U.S. Census Bureau. The number of each facility per region and percentage of total facilities surveyed per region are indicated. Accreditation status (D) and academic societal affiliations (E) of the 27 total facilities that provided full or partial responses.

MSCs for clinical applications: a survey of good manufacturing practices indication of their cell product was immuno-modulation, and 7 facilities (46.7%) cited cardiovascular disease, 6 facilities (40%) reported tissue regeneration and 5 facilities (33.3%) reported pulmonary disease (Figure 2a). Other clinical indications included hematological and musculoskeletal diseases, wound healing, liver cirrhosis and tissue engineering. Furthermore, eight facilities (53.3%) indicated that the manufactured MSCs were intended for three or more indications, and three facilities (20%) reported two indications; four facilities (26.7%) manufactured MSCs for a single indication (Figure 2b). Our survey also revealed that 14 of 15 facilities (93.3%) isolated MSCs from bone marrow (Figure 2c) whereas only 4 facilities (26.7%) obtained MSCs from adipose or umbilical cord tissue, 2 facilities (13.3%) used umbilical cord blood and one facility (6.7%) used placental tissue. Overall, 10 facilities (66.7%) sourced MSCs from a single tissue, 2 facilities

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(13.3%) sourced cells from two tissue and 3 facilities (20%) used three or more tissues (Figure 2d). Methods used to disaggregate tissues were more diverse, with 7 facilities (46.7%) employing enzymatic digestion, 4 facilities (26.7%) relying on an automated system, 2 facilities each (13.3%) reporting the use of sieving (filtration) or Ficoll gradient centrifugation, and 3 facilities (20%) not responding to the question (Figure 2E). Overall, 64.3% of respondents (9 facilities) employed a single method of tissue disaggregation, 14.3% (2 facilities) employed two methods and 7.1% (1 facility) employed three or more methods (Figure 2F). Another major finding of the survey was that 14 facilities (93.3%) employed plastic adherence as the main method of MSC enrichment (Figure 3A). In contrast, only 1 facility (6.7%) reported the use of FACS, immuno-enrichment or immuno-depletion. Furthermore, 12 facilities (80%) employed 2-D

Figure 2. Distribution charts on product indication, tissue source and methods of isolation. Percentage of facilities that manufacture MSCs for a specific clinical indication (A) and total number of product indications per facility (B). Tissue material used to isolate MSCs (C) and total number of tissue sources employed by each facility (D). Methods used to disaggregate tissues before MSC isolation (E) and total number of methods employed by each facility (F). In all cases, the total number of facilities for each response are also indicated.

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Figure 3. Distribution charts on product enrichment and expansion methodologies. (A) Percentage of facilities that use the indicated methods to enrich MSCs from disaggregated tissue. (B) Percentage of facilities that employ the indicated reaction vessel to expand MSCs. (C) Percentage of facilities that employ high, medium or low initial and working plating densities to expand MSCs in 2-D flasks. Percentages are based on responses from 12 total facilities. (D) Time from initial plating density to final harvest of the clinical dose. Responses include facilities employing both 2-D and 3-D culture expansion methods. (F) Percentage of facilities that calculate population doublings (PD) and average population doubling time (DT) at final harvest for products expanded in 2-D (E) and 3-D (F) culture vessels. (G) Source of serum growth factors used in media formulations. (H) Classification of hPL with respect to donor pooling and supply sourcing. In all cases, the total number of facilities for each response are also indicated.

culture methods for MSC expansion, whereas 5 facilities (33.3%) employed hollow fiber bioreactors, 3 facilities (26.7%) employed a conventional bioreactor and 1 facility (6.7%) employed bags (Figure 3B). Overall, only 3 facilities indicated that they exclusively use three-dimensional (3-D) methods (hollow fibers, bioreactor or bags) to expand MSCs. In cases where MSCs were expanded in 2-D culture vessels, initial and working plating densities were found to vary significantly across facilities. For example, 50% of these facilities initially employed a high plating density of >2500 cells/cm2, whereas 16.7% used an intermediate density (500 2500 cells/cm2), and 33.3% used a low-density (50 500 cells/cm2) (Figure 3C). Alternatively, 66.7% of facilities employed a medium plating density (500 2500 cells/cm2) to expand cells beyond P1, and 25% used a high- and 8.3% used a low-plating density (Figure 3C). Harvest times were also highly variable

(Figure 3D). Furthermore, 66.7% of these facilities indicated they calculated total population doublings and 75% calculated average doubling times at the time of cell harvest (Figure 3E). As noted earlier, a total of 9 facilities (60%) also reported the use of 3D culture methods (Figure 3B); in these facilities, seeding densities were sparsely reported. However, of the 4 facilities using hollow fiber bioreactors, 3 reported a seeding density of 20 million cells and 1 a density of 50 100 million cells. Additionally, 4 of 5 facilities (80%) using bioreactors calculated total population doublings and all calculated average doubling times at the time of cell harvest (Figure 3F). In terms of media supplements, 11 facilities (73.3%) reported the use of hPL, 2 facilities (13.3%) used FBS, 3 facilities (20%) used lot-selected FBS and 1 facility provided no response (Figure 3B). Moreover, of those facilities that used hPL, 63.6% (7 facilities) produced it in-house, 36.4% obtained it

MSCs for clinical applications: a survey of good manufacturing practices from a commercial source and 100% generated it from pooled donors (Figure 3H). Five facilities (33.3%) reported using other media supplements, and of these, 3 facilities supplemented media with growth factors, and one used gentamicin. Phenotypic and functional characterization Most surveyed facilities (80%) reported that they validated their cellular product by cell surface marker expression analysis, and these facilities employed a variety of positive and negative epitopes. The most common positive markers included CD105 (80%), CD90 (73.3%) and CD73 (66.7%) (Figure 4A), and the most common negative markers were CD45 (86.7%), CD14 (53.3%), CD34 (46.7%) and HLA-II (46.7%) (Figure 4C). The total number of positive and negative markers used by each facility (Figure 4B,

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D) and the limits used to define the overall purity of the product based on phenotype varied markedly between facilities. In the latter case, cutoffs for negative maker expression ranged from <10% to <2%, and that for positive markers ranged from >10% to >95% (Figure 4E). A total of 13 facilities (86.7%) included sterility testing for bacteria, endotoxin and mycoplasma of the cellular product as part of the release criteria, and 1 facility (6.7%) also tested for viruses. Two facilities did not respond to the question. Quality testing of the MSC product was conducted by 12 of 13 facilities (80%) (Figure 5A), with 2 facilities (13.3%) not responding. Of the 13 respondents, 10 facilities (76.9%) recorded the total cell yield of their product, and 13 facilities (100%) recorded cell viability (Figure 5B). Furthermore, 4 facilities (30%) reported a minimal cell yield threshold for product release, and 13 facilities (100%) had

Figure 4. Distribution charts on product identity and sterility. Percentage of facilities that use the indicated positive (A) and negative (C) cell surface markers to validate product identity, and the total number of positive (B) and negative (D) markers used by each facility. (E) Limits allowed to indicate relative product purity with respect to positive and negative surface markers. (F) Percentage of facilities that measure bacteria, mycoplasma, endotoxin and viruses to assess product sterility before release. In all cases, the total number of facilities for each response is also indicated.

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Figure 5. Distribution charts on product quality testing. Percentage of facilities that perform quality testing of MSC product (A), quantify cell yield and cell viability (B) and employ threshold cutoffs values for these measurements (C). Methods used to evaluate cell viability (D) and threshold cutoff values employed for viability measurements (E). Percentages in (B E) are based on a total of 13 facilities that responded to questions on quality testing. (F) Percentage of facilities that perform karyotype analysis of the cellular product. In all cases, the total number of facilities for each response is also indicated.

a minimal viability threshold for product release (Figure 5C). A total of 11 facilities (84.6%) employed trypan blue staining to measure cell viability, and 1 facility (7.7%) employed propidium iodide staining, and another used FACS analysis (Figure 5D). However, the viability threshold value varied between 70% and 90% in these facilities (Figure 5E). Lastly, 6 facilities (40%) indicated they performed karyotype analysis of their cell product, 7 facilities (46.7%) did not, and 2 facilities (13.3%) did not respond to the question (Figure 5F). Regarding product potency, 9 facilities (60%) indicated they performed some type of potency testing, 4 facilities (26.7%) did not and 2 (13.3%) did not respond to the question (Figure 6A). Facilities that did assess product potency employed a variety of different assays; 4 facilities (26.7%) evaluated skeletogenic activity, 1 facility (6.7%) evaluated angiogenic potential via use of an endothelial tube formation assay, and 7 facilities (46.7%) evaluated immunomodulatory activity (Figure 6B). In the latter case, 6 facilities (46.2%) employed MLR-based assays to assess immuno-suppressive activity, 3 facilities (23.1%) evaluated Treg formation, 1 facility (7.7%) evaluated NK cell lysis and 1 facility (7.7%) indicated their method was proprietary (Figure 6C). Furthermore, of those facilities conducting MLR-based

assays, 5 (71%) used peripheral blood mononuclear cells (PBMNCs) and 1 (9.1%) used purified T cells as effector cells, and 6 facilities (54.5%) used antibodies against CD3 and CD28 to stimulate lymphocyte proliferation; 1 facility (9.1%) used phytohemagglutinin (PHA) (Figure 6D). Lastly, only 1 facility (6.7%) reported that they employed RNA-seq and enzymelinked immunosorbent assay to characterize their product, 12 facilities (80%) indicated they did not employ any medium to large-scale throughput analysis to characterize their product and 2 facilities (13.3%) did not respond to the question. Regarding product storage, 12 facilities (80%) cryo-banked their cellular product (Figure 6E), but the media formulation used to store cells (Figure 6f) and the methods of recovery post thaw (Figure 6G) varied considerably between facilities. Similarly, the final product formulation was also highly variable, and was reported as plasmalyte (1 facility), plasmalyte + HSA (1 facility), normosol (1 facility), DPBS (1 facility) and basal medium (1 facility). Discussion Although efforts to develop efficacious MSC-based therapies have progressed slowly, recent reports of their positive beneficial effects in clinical trials for

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Figure 6. Distribution charts on product potency testing and release formulations. (A) Percentage of facilities that perform biological potency testing of the product. (B) Percentage of facilities that evaluate the skeletogenic, angiogenic and immunomodulatory activity of the cellular product. (C) Type of test performed to assess immunomodulatory activity. (D) Source of immune effector cells and stimulant used to induce lymphocyte proliferation in MLR assays. (E) Percentage of facilities that cryo-bank their cellular product. Media formulations employed for product cryo-preservation (F) and final state of the product upon release (G). In all cases, the total number of facilities for each response is also indicated.

pediatric graft-versus-host disease [17] and Crohn disease related perianal fistulas [18] as well as the market approval of MSC-based products in Japan [3] represent a high point for the field. Results presented herein indicate that best practices used at U.S. academic current GMP facilities for manufacturing clinical grade MSC products are also evolving at a slow pace. For example, although MSCs or MSC-like cells have been identified in most tissue and organs, our survey revealed that 66.7% of facilities source MSCs from a single tissue, and for most facilities (93.3%), the preferred source was bone marrow. Although our survey did not question the motives behind this preference, the bulk of basic and translational research on MSCs has been conducted on bone marrow derived cells, and aspirates of human bone marrow are straightforward to obtain and widely available from various commercial vendors. Our survey also revealed that most academic cGMP facilities relied on plastic adherence as the main method of MSC enrichment and expanded MSCs in 2-D culture systems. Therefore, although significant advancements have been made in the development of automated and highly integrated closed, 3-D culture systems capable of producing large cell numbers, these devices were not universally

employed at the facilities we surveyed. In contrast, the use of hPL as a media supplement was widespread, which likely reflects its potent mitogenic activity on MSCs [19 21] and the FDA’s and industry’s desire to encourage development of xenogenic-free formulations for expansion of human cellbased products. Nevertheless, effects of hPL on MSC biological activity are controversial. For example, several studies have reported differences in the size, differentiation potential and immunomodulatory activity [19,21 23] of MSCs grown in hPL versus FBS, whereas others have not observed such differences [24,25]. Resolving these discrepancies will be necessary to ensure products manufactured in hPL are optimized with respect to potency for their intended disease indication. Our survey also highlighted several practices that were highly variable across facilities. These included choice of initial and working cell-plating densities, harvest times of the cellular product and methods used to assess product identity and potency. With respect to product identity, our survey indicated that the most common positive markers used to characterize the MSC product were CD73, CD90 and CD105 and those used to exclude impurities were CD45, CD14, CD34 and HLA-II. These criteria

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coincide closely with the minimal set of standard criteria established by the ISCT in 2006 [26]. However, whereas the ISCT stipulated that 95% of MSCs in a population must express each positive marker, our survey indicated that threshold criteria for marker expression varied considerably across facilities (Figure 4E). Importantly, recent studies have shown that these surface markers are not exclusive to human MSCs and are not capable of discriminating inter-donor differences in their biological activity [6,27]. Furthermore, MSCs sourced from different tissues are largely indistinguishable based on surface expression levels of CD73, CD90 and/or CD105 [28 30] despite exhibiting significant differences in growth potential, colony-forming unit fibroblast (CFU-F) activity, multipotency and angiogenic activity. Various groups have identified surface antigens that discriminate differences in the CFU-F activity, multilineage differentiation potential and hematopoiesis supporting activity of human MSCs [31 34], but none have been incorporated by the facilities we surveyed. The lack of suitable phenotypic markers has necessitated development of functional assays to measure product potency, and 60% of facilities surveyed reported that they performed potency testing of their manufactured MSCs. Furthermore, our survey revealed that immuno-modulatory activity was the most widely evaluated activity (60% of facilities), but methods used to assess it were not standardized, which makes comparing product potency across facilities difficult. Importantly, many facilities surveyed indicated that their MSC product was intended for three or more clinical indications, but whether manufacturing and product testing protocols were tailored to specific diseases was unclear. For example, of the 10 facilities that indicated their product was for immunomodulation, only 50% reported testing for this activity. Similarly, only one (25%) of four facilities producing MSCs for woundhealing applications reported testing their product for angiogenic activity, and only two (40%) of five facilities manufacturing cells for tissue engineering and musculoskeletal indications assessed the skeletogenic potential of their product. Furthermore, of the eight facilities indicating that their product was intended for three or more clinical indications, five (62.5%) sourced MSCs from a single tissue (five bone marrow, one adipose tissue). These findings suggest that a disconnect exists among manufacturing practices, product potency testing and product utilization and that manufacturing practices are not tailored toward specific disease indications. Importantly, while the survey inquired about skeletogenic potential under the guise of potency testing, in vitro differentiation assays are commonly used for characterization

purposes. Therefore, in future surveys, it will be important to clarify whether skeletogenic assays are intended for product characterization or potency testing and stipulate the nature of the assays used to assess potency. Our survey also revealed that use of omics-based approaches to characterize MSC products were rarely employed, and less than half of facilities surveyed performed karyotype analysis of their cellular product. In summary, our survey highlights current practices used in U.S. academic current GMP facilities engaged in the manufacture of MSC products for clinical use. The survey revealed several interesting and unexpected trends in the manufacturing process and identified practices and procedures that were common to and varied significantly between facilities. Comparing our data to a survey of manufacturing centers affiliated with the EBMT [16] revealed several common practices in MSC manufacturing between Europe and the United States. For example, both surveys found that most facilities sourced MSCs from bone marrow and expanded cells in hPL-supplemented media. Product characterization via flow cytometry also relied on similar subsets of surface makers, which were based on recommendations made by the ISCT, and in both cases, release thresholds for these markers varied considerably between facilities. MSC products were also routinely tested for bacteria, mycoplasma and endotoxin as part of their release criteria. Notable differences highlighted by the surveys included differences in the frequency of karyotype analysis (71% vs. 40% for Europe vs. United States, respectively) and product potency testing (29% vs. 40% for Europe vs. United States, respectively). Consequently, an important goal moving forward would be to survey manufacturing facilities in other regions of the world and collate results to assess variability. This would facilitate development of universal guidelines to better harmonize manufacturing processes to yield more homogeneous products. Another major goal moving forward would be to establish a set of standardized assays that can be used to validate the quality of MSC products produced from different tissue sources, which often exhibit similar phenotypes but disparate biological activity, as well as specialized potency assays to match each product to its proposed mechanism of action and/or specific disease indication. Developing more stringent product characterization and potency assays in manufacturing is critically needed to maximize potency and therapeutic efficacy for nonhomologous applications. Finally, future surveys should also be cognizant of the rapid pace of genetic engineering and as such should inquire whether facilities are manufacturing MSCs modified through genetic or other means.

MSCs for clinical applications: a survey of good manufacturing practices Acknowledgments The authors thank the members of the MSC Committee of the ISCT (Mauro Krampera, Katarina Le Blanc, Ivan Martin, Jan Nolta, Luc Sensebe, Yufang Shi and Sowmya Viswanathan) for supporting this endeavor, as well as Brian Poole and Sophie Bockhold from the ISCT Head Office who helped distribute the survey and collate results. Disclosure of interest: The authors have no commercial, proprietary or financial interest in the products or companies described in this article.

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