Revisiting MSC expansion from critical quality attributes to critical culture process parameters

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Process Biochemistry xxx (2016) xxx–xxx

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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Review

Revisiting MSC expansion from critical quality attributes to critical culture process parameters Céline Martin a , Éric Olmos a , Marie-Laure Collignon b , Natalia De Isla c , Fabrice Blanchard a , Isabelle Chevalot a , Annie Marc a , Emmanuel Guedon a,∗ a Université de Lorraine, Laboratoire Réactions et Génie des Procédés, UMR CNRS 7274, 2, avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre lès Nancy, France b Université de Liège, Laboratoire de Génie Chimique, Institut de Chimie, B6c, Sart Tilman, B-4000 Liège, Belgium c Université de Lorraine, Ingénierie Moléculaire et Physiopathologie Articulaire, UMR CNRS 7365, 9, avenue de la Forêt de Haye, 54505 Vandoeuvre lès Nancy, France

a r t i c l e

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Article history: Received 8 January 2016 Received in revised form 4 April 2016 Accepted 19 April 2016 Available online xxx Keywords: Mesenchymal stem cell Multipotent stromal cell Expansion process Bioreactor Microcarrier

a b s t r a c t With over 300 clinical trials registered in 2013 and more than 20,000 hits on the NCBI database, human multipotent stromal cells (hMSC) are widely used and studied as a model for tissue engineering and regenerative medicine. This review aims to provide a general update on the latest advances regarding hMSC characterization and bioprocessing. This analysis of the recent literature once again shows the intrinsic relationship between critical quality attributes (CQA) of hMSC expanded for cell therapy and critical process parameters (CQP) used during their expansion. It hence demonstrates that process design and bulk validation will need to integrate not only proteomic but also cellular biology tools to meet regulatory authority’s requirements and limit process variability. The necessity for another streamlining effort is underlined to facilitate study comparison and settle the problematics hindering hMSC therapeutic use: the influences of cell sources, population heterogeneity and culture conditions on the final in vivo outcome. © 2016 Published by Elsevier Ltd.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Product quality: a new paradigm for upstream processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. hMSC identity and heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. hMSC as therapeutic factory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2.1. Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2.2. Secretome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. hMSC aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.1. Stemness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.2. Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.3. Malignancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Expansion process parameters: what must be investigated? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Biochemical inputs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1.1. Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1.2. Growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1.3. Feeding strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Physicochemical environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author. E-mail addresses: [email protected] (C. Martin), [email protected] (É. Olmos), [email protected] (M.-L. Collignon), [email protected] (N. De Isla), [email protected] (F. Blanchard), [email protected] (I. Chevalot), [email protected] (A. Marc), [email protected] (E. Guedon). http://dx.doi.org/10.1016/j.procbio.2016.04.017 1359-5113/© 2016 Published by Elsevier Ltd.

Please cite this article in press as: C. Martin, et al., Revisiting MSC expansion from critical quality attributes to critical culture process parameters, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.017

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3.2.1. Adhesion surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2.2. Hydromechanical stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2.3. pH, temperature and oxygen saturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusion: new concepts for classic designs or new designs for classic concepts ? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

2.1. hMSC identity and heterogeneity

Multipotent stromal cells (MSC), also called mesenchymal stem cells, have first been isolated by Friedenstein [1,2], and then conceptualized as “MSC” by Owen [3] and Caplan [4]. These cells rapidly attracted attention due to their ability to differentiate in vitro in osteocytes, chondrocytes and adipocytes [5]. They are considered since as a biological potential source for musculoskeletal tissue regeneration when differentiated in vitro in association with a biomaterial scaffold [6]. They are also progressively considered as a good candidate to remain undifferentiated as a pharmaceutical product [7], due to their capability to finely regulate secretion of chemoattractants, trophic and immunomodulatory factors [8]. Consequently, human MSC (hMSC) are currently tested in clinical trials for allogeneic as well as autologous therapies [9]. However, most of these applications require an amount of cells that cannot be provided by simple tissue aspiration. Consequently, an in vitro expansion step is required [10,11]. In fact, the cell properties and the amount of active cells are likely to vary depending on the therapeutic targeted use and on the hMSC chosen source (bone marrow, adipose tissue, Wharton jelly, etc.). However, the lack of a proper identity for hMSC makes it difficult to assess manufacturing process performance beyond cell numbers. Even though the consensus defined by the International Society for Cellular Therapy (ISCT) (adhesion, cluster of differentiation (CD) phenotype, differentiation), allowing the characterization of hMSC, was a key step to streamline the vast amount of research reported on hMSC [12], it is also admitted that these minimal criteria will be insufficient for the translation of hMSC research into clinical applications [13]. An overview of characteristics that have been identified as pertinent for the evaluation of hMSC integrity for therapeutic use is proposed in the present review in regards to their adequacy with the development of a manufacturing process. Hence, this review first considers the technical compatibility of assays currently defining hMSC attributes with process validation and lot release scheduling. Secondly, it describes how process parameters could influence these characteristics, especially in cultures with cells expanded on microcarriers.

Monitoring hMSC physiological identity and heterogeneity can be performed by quantitative methods in order to discriminate between targeted and non-targeted cell populations. As per Food and Drug Administration (FDA) guidance, the aim is to avoid a drift in the hMSC properties and set acceptable limits for culture process. Discrimination of positive and negative CD antigen surface markers is the standard method regarding hMSC characterization as defined by the ISCT. hMSC must express CD73, CD90 and CD105 (≥95%), but not CD34, CD45, CD44 or 11b, CD79␣ or 19, and HLA-DR markers (≤2%) [12]. However, expression of surface antigens is a dynamic process and can vary depending on tissue sources. For instance, monitoring of surface antigens for adipose-derived MSC includes the expression of CD44 and possibly CD34 [16]. It has also been proven that these standard sets of markers were not able to discriminate hMSC from non-stem mesenchymal cells or fibroblasts, contrary to the expression of CD24, CD108 and CD40 [17]. Looking for surface markers expression in vivo on the whole bone marrow, CD10, CD140b, CD146, CD271, and ganglioside GD2 appear as promising candidates to be also more discriminating, but this result highlights again the mixed population composing a biopsy sample [18]. The specific monitoring of CD146 expression has raised new concepts in the hMSC field. It is thought to be a marker for real hMSC in the bone marrow, here defined as skeletal stem cell, because only this population has the ability to recreate ossicles in heterotopic transplantation and keep their phenotype on subsequent passages in culture (considered as the gold standard for stemness functional assay) [19]. Pericytes also express CD146 in various fetal and adult tissues while being positive for the usual hMSC CD markers and show multipotency ability [20], which consequently might explain the ubiquitous nature of hMSC by a perivascular origin. Overall, because no unique marker has been discovered up to now to characterize hMSC, multiple surface antigens markers have to be chosen beyond those recommended by the ISCT in order to monitor possible deviations in the cell population and determine how they could actually influence the targeted biological functions.

2. Product quality: a new paradigm for upstream processing

Potency is of course closely linked to clinical efficacy, but also to manufacturing consistency and batch release, and must be taken early into account in process development [21]. Characterizing hMSC performance as therapeutic entity, i.e. characterizing their potency, is defined as “the specific ability or capacity of the product to effect a given result and must be determined by a quantitative biological assay” [22]. Two aspects have to be investigated for hMSC depending on the targeted use: tissue engineering needs the cells to be able to properly differentiate, while the secretion of adequate immunomodulatory and growth factors is looked for during MSC infusion.

hMSC attributes from a fundamental biology viewpoint, i.e. molecular identity, mode of actions (MOA) or in vivo physiological role, are highly debated and not yet well understood [14]. Because of the lack of a precise MSC characterization, two opposite concepts have emerged. One is referring MSC as a clearly defined in vivo entity limited to the bone, whereas the other considers MSC as heterogeneous and ubiquitous populations of various cells, populations that are solely defined by their common characteristics, as defined by the ISCT, through a combination of in vitro assays [15]. In fact, MSC as a product finds its roots in the latter, and the whole challenge of expansion is to maintain the main quality attributes that are critical to their in vivo potency into a defined operating window. This covers their collection, isolation, amplification and possibly banking.

2.2. hMSC as therapeutic factory

2.2.1. Differentiation The differentiation ability of hMSC has not truly been demonstrated in vivo as hMSC engraftment does not occur when they are injected undifferentiated [23,24], giving rise to discussions on their actual physiological role as progenitors.

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Their capacity to differentiate in vitro was initially the main element that drew research to study this cell population and it is still one of the fundamental criteria defining cells as MSC. This key element is generally tested in vitro using well-known induction assays. Cell differentiation is induced through cell proximity and specific culture media supplementation, that should allow MSC to acquire the adipocyte, chondrocyte and osteocyte phenotypes in 14–21 days. The obtaining of these three specific cell phenotypes is then confirmed via histochemical staining [12]. This standard procedure is however both qualitative and time-consuming. However, it is possible to use short term induction assay (3–7 days) that detects quantitative variations in either mRNA or protein expression correlated with differentiation levels [25,26]. The same methods can be used to follow specific undesirable induction events caused by the culture conditions. Indeed, the undifferentiated state of hMSC has to be verified at the end of expansion process if cells are aimed to be used as a native population. Indeed, process parameters such as mechanical stimulations or fetal calf serum (FCS) use can induce the cells towards a differentiated lineage [27]. In regards to the process analytical technology (PAT) initiative, it can be noted that preliminary evidence suggests that Raman spectroscopy could actually be of interest to follow osteogenic and adipogenic differentiations during culture process [28,29]. Taking into account the specificities of musculoskeletal tissue engineering, an induction assay is obviously performed while the cells are going through differentiation into the scaffolds. In this case, it is useful to have prospective data linking hMSC features to their ability to differentiate. Predictive models have already been developed, one based on type I collagen and osteoprotegerin expressions [30], the others on biophysical markers [31–33]. 2.2.2. Secretome When hMSC are maintained undifferentiated, their secretome is considered as the principal effector in their therapeutic efficacy. Under appropriate licensing, these cells can produce immunomodulatory, anti-inflammatory, mitotic, anti-apoptotic, anti-oxydant, anti-microbial or even angiogenic factors. This includes the secretion of cytokines (e.g. CCL-2, CCL-5, IL-6, IL-8, IL-10), growth factors (e.g. VEGF, TGF␤1, PDGF, ßFGF, HGF), enzyme (indoleamine 2,3-dioxygenase), prostaglandine (PGE2), as well as exosomes (membrane microvesicles containing protein and RNA) [34,35]. Thus, hMSC can be considered as a natural body “drugstore” [7]. These secretions can be detected with well-known proteomic physicochemical techniques (ELISA, mass spectrometry, etc.) [36], but they are not automatically correlated to in vivo hMSC efficacy depending on the targeted pathology [37,38]. Bioassays in animal models or in vitro cell cultures also exist. hMSC have been widely studied in Transwell or co-culture experiments after their capacity to suppress T-lymphocytes proliferation was demonstrated in vitro [39]. On the other hand, in vivo testing on animal models has the advantage to better link cell potency to in vivo effects, even though it cannot always be translated into clinical results in human. For instance, concerning graft versus host disease (GvHD), a lifespan increase of GvHD mice model has been linked to a decrease of T cells proliferation and IFN␥ secretion by MSC from both mouse and human sources, and then used to discriminate potencies between different MSC clones [40]. Unfortunately for process performance evaluation, these bioassays are too much time and cost consuming. Hence, in a murine wound healing model, it has been hypothesized that a matrix assay could allow the prediction of hMSC potency based on the determination of the cells growth rate, viability and proliferation [41]. Lastly, an industrial example of hMSC potency testing methodology has been disclosed: the manufacturing runs of MultiStem (manufactured by Athersys). Each cell lot goes through a pass/fail criteria testing the cells conditioned medium using ELISA assays against VEGF, CXCL5 and IL-8, knowing that their minimal

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acceptable concentrations have been previously determined using a human umbilical vein endothelial cell tube formation assay [21]. Thus, it appears that establishing manufactured hMSC as therapeutic factory is difficult and that potency quantifications have to be based on hypothesized MOA obtained from in vitro results. According to present-day knowledge, hMSC can be considered as a multifaceted cell population with several roles in vivo, consequently leading to a multiplication of needed assays. Renewed standardization efforts have been proposed, focusing on biomarkers such as chemokines or growth factors receptors [42]. Conversely, an interesting simplified procedure has been developed by the National Institute of Health using microarray global gene expression analyses [43]. Authors measured the replicative age of hMSC using a 24 genes set and compared these results to numerous release criteria based on the use of a mouse transplant model, CD surface markers, in vitro lymphocytes reactions and cytokines production [44]. This procedure could thus become, after extensive validation, a single-shot predictive assay for hMSC potency. It also underlines how the duration of a manufacturing process might impact cell quality attributes. 2.3. hMSC aging During the manufacturing of a recombinant protein, the stability of the cell line is studied from thawing the master cell bank vial to the last scale-up step. There is a necessity to determine possible cell deviation due to increasing population numbers. For hMSC therapies, the fragile cellular balance between stemness, senescence and malignancy can be altered during the manufacturing process due to artificially stimulating biological aging. Genetic stability can be followed at a molecular level in order to perform the in vitro expansion step within a safe time-frame [45]. 2.3.1. Stemness The definition of stemness has become more or less stringent depending on the studies, particularly due to common use of the term adult stem cell, MSC included, and creation of iPS cell lines (induced pluripotent stem cells). The gold standard assay actually consists of cells in vivo transplantation (see above), but it is not adequate for bioprocessing. Current characteristics of stemness are self-renewal, multipotency, clonogenicity, and expression of pluripotent markers. For hMSC, clonogenicity does not have to be proven anymore, as it is the oldest method to isolate hMSC from bone marrow [1]. The colony forming units-fibroblastic efficiency assay (CFU-F or CFE) is a simple method using a clonal seeding density and its comparison to the number of colonies obtained 14–21 days later. Results from a tissue fraction are highly dependent on culture conditions and especially on donor (age and health) [46,47]. Nevertheless, considering that these parameters are maintained, comparing CFE kinetics can be informative regarding the proportion of hMSC cells that are actively dividing [48]. It may also help to determine adequate culture conditions [49]. The expression by hMSC of embryonic pluripotent markers, i.e. stemness-related transcription factors like Oct-4, Nanog, Sox2, Rex1, or glycolipid antigenes like SSEA-3 and SSEA-4 [50] is more controversial. SSEA antigenes being surface markers, they can be detected via flow cytometry and are mainly used for the prospective isolation of hMSC subsets, displaying better self-renewal and pluripotency abilities [51,52]. For the transcription factors, low or no levels of Oct-4, Nanog, Sox2 and Rex-1 are detected in hMSC compared to embryonic stem cells [53,54]. When these factors are detected, measurements are in agreement on a general decrease of these markers over passaging [55–57]. Expression of these transcription factors are at the center of a complex balance,

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and conflicting data might be explained by epigenetic tuning such as histone acetylation [57,58] or microRNA silencing [59]. 2.3.2. Senescence Beside the loss of pluripotent markers expression over time, hMSC acquire in vitro a senescent phenotype, just as somatic primary cells do. Consequently, cell cycle is irreversibly blocked, cells do not become apoptotic and stay in quiescent state. However, they can be metabolically active for months. It has been shown that senescence impaired hMSC immunomodulatory properties and multipotency [60,61]. A late senescent state is commonly detected by morphological changes such as size increase, cell flattening and lysosomal ␤-galactosidase staining [62]. However, earlier senescent states can be detected and quantitatively measured by molecular changes [43]. The continuous telomere shortening by replicative senescence (RS) can be monitored by Southern blot, qPCR or flow cytometry [48,63]. Furthermore, stress-induced premature senescence (SIPS) is timecorrelated with increased genomic DNA damages [61]. These two senescence processes involve the cyclin dependent kinase inhibitors p16INK4A , p53, and p21WAF1 that drive the cell cycle arrest. In extensive hMSC cultures, it was reported that only p16INK4A mRNA expression was increased [64] or that p53 was downregulated [65], while others found that all three transcripts were up-regulated [66,67]. It is thereby indicated that, depending on culture conditions, donors and time of sampling, different senescence systems might be involved or detected. The telomerase reverse transcriptase (TERT) enzyme has been described as crucial to delay replicative senescence. But, as for the pluripotency markers, its activity in hMSC is as low as in primary cells and barely detected compared to pluripotent or cancerous cells [68,69]. Consequently, monitoring hTERT expression or activity might not be of interest to assess hMSC replicative senescence. However, hTERT seems to also have a role in oxidative stress protection by mitochondrial regulation, thereby protecting cells from senescence induced by oxidative stress [70], which makes its overexpression a possible objective for culture process optimization. For instance, several results showed that hypoxic environments (see below) or antioxidant chemical compounds might stabilize telomere lengths by up-regulating hTERT [71,72]. Beside these classical senescence markers, others have also been explored. For example, morphology and decreased cytoskeleton dynamics [32,73] or senescence-associated DNA methylation (SA-DNAm) [74] could be used to detect early aging of an hMSC population for quality control purposes. Senescence state may indeed become one of the most important quality attribute to monitor during hMSC expansion. Compared to stemness markers, senescence markers are more consensual and robust, and thus can more systematically reveal cell population deviation. Senescence is also the main protection against malignant cell transformation in vivo [75]. Even if a major onset of senescence has to be avoided during manufacturing, tracking the cells gradual aging may also insure the safe release of the cellular product [76]. 2.3.3. Malignancy One of the principal risks that cellular therapies have to address is tumorigenicity. Cancerous transformation of hMSC is considered as a very rare event, since no such adverse reaction has been detected in clinical trials for over 20 years [77]. However, some cases of in vitro cell immortalization were reported, but were attributed to cross-contamination of cell lines [78], except for one case [79]. While some authors advised to detect growth shifts only [80], others have striven to find specific genes expression correlated with the transformation events [79]. Recommendations from the scientific communities for preclinical settings are up to now still limited to the systematic detection of

cytogenetic abnormalities using G-banding karyotyping and fluorescence in situ hybridization (FISH) [81]. Indeed, the accumulation of chromosomal instabilities in extended hMSC cultures is common [82,83]. However, this detection is not sufficient as it has not been clearly linked to an increase in tumorigenesis [84] and can be also quite frequent in normal non-cancerous cell population [75]. While the overall agreement is that hMSC expansion should be limited to a minimum number of population doubling for safety reason, a limited time-frame is also one of the main limiting step to reach sufficient cell quantities. Some authors have suggested to induce the cells toward a malignant phenotype in order to better understand the phenomenon [85]. For instance, this has been achieved by modulating the cells biochemical environment with heavy metal salts and hypoxia [86]. Regulatory agencies, in agreement with the scientific literature, consider that hMSC are “more than minimally manipulated cells”. This is why process and analysis validations will have to be implemented to widen the hMSC use beyond clinical trials [87–89]. All hMSC quality attributes listed above will probably not be necessary, but some of them might be critical, depending on therapeutic use of hMSC (see Fig. 1). However, the consensus is that hMSC manufacturing based on better quality attributes assessment will be highly beneficial to the hMSC field [21,81]. Even more essential, a broadly used reference material would improve comparability [13]. Process standardization has to be improved at the same time [90,91], as some clinical trials failures might be due to hMSC manufacturing scale-up from limited expansion from lab- to large scale industrial expansion [92]. 3. Expansion process parameters: what must be investigated? The classical culture conditions for hMSC involve the use of culture flasks, fetal calf serum (FCS) and standard humidified incubators regulated at 37 ◦ C and 5% CO2 (pH range between 7.2 and 7.4). Optimization of these culture conditions are the main solutions for manufacturing hMSC with a reduced cost of goods per dose. Two view points have been considered to increase expansion process performance. The first one focuses on cell quality by recreating the cell micro-environment and mimicking in vitro the in vivo niche. Cultures with explants [93], aggregates [94,95], or hydrogels [96], in in vivo chambers [97] and tissue bioreactors [98] were developed for this purpose. The other investigated way is based on the assumption that hMSC in cultures are in vitro artifact per se. It focuses on cell quantity by adapting well-known bioreactor designs and culture strategies initially developed for biomolecule production with immortalized cell lines [10]. However, taking into account the complexity of quality testing, many of these studies have been devoted to increase cell quantities while complying only with the ISCT minimal quality criteria. Confronting both viewpoints supported by advances on knowledge of hMSC biology could help redefining the critical process parameters to achieve the specificities of adult stem cells therapy. 3.1. Biochemical inputs 3.1.1. Nutrients A basal culture medium provides cells with essential nutrients including carbohydrates, amino acids, vitamins and minerals. In association with human platelet lysate (hPL) or FCS, hMSC are commonly grown in DMEM, DMEM/F-12, or ␣MEM basic media [99]. The only source of carbohydrate in these media is glucose, either at physiological level around 1 g/L (␣MEM, DMEM-LG) or “hyperglycemia-like” at 4.5 g/L (DMEM). This high level of glucose has been reported to be either detrimental to hMSC [100], or having no effect on their proliferation and differentiation [101].

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Fig. 1. Prospective therapeutic use and hypothesized mode of actions (MOA) of hMSC defining current potency assays issued from clinical trials targeting various organs diseases such as acute GvHD during bone marrow transplantation [179], heart diseases [180], leg [181] and brain ischemia [182], intestine fistula [183] and maxillary bone reconstruction [184]. (CXCL5, C-X-C motif chemokine 5; GvHD, graft versus host disease; hTERT, human telomerase reverse transcriptase; IL8, interleukine 8; betaTCP, beta-tricalcium phosphate composite; IL2R␣, interleukine 2 receptor ␣; MOA, mode of action; SDF1␣, stromal cell derived factor 1␣; TNFR-1, tumor necrosis factor receptor 1; VEGF, vascular endothelial growth factor.)

Discrepancies between various studies can be explained by the hMSC heterogeneity [102] and/or the frequency of medium refreshment. Even though not essential, some amino acids might be preferentially metabolized by hMSC [103], notably asparagine or tyrosine. Media such as GMEM for instance are supplemented with nonessential amino acids [104]. Glutamine is usually considered as an essential alternative source of energy in cell culture and most culture media used for hMSC are supplemented with 2 mM glutamine. As for glucose, reports on glutamine consumption are contradictory [99,103,105]; the di-peptide L-alanyl-L-glutamine (Glutamax) allowed a better proliferation compared to glutamine, probably due to a lower ammonium accumulation in culture supernatant [106]. Ascorbic acid is present in DMEM/F12 and ␣MEM, contrary to DMEM, and this vitamin might actually be very important for hMSC growth [107,108]. Ascorbic acid is indeed known for its antioxidant properties and might delay MSC senescence in culture [109], as shown for other vitamins such as Trolox (vitamin E analog) [110]. Finally, nutrients might not impact significantly the cells phenotype outside of a transient metabolic adaptation, whereas studies detected some changes in cell surface markers expression, depending on the medium used [111,112]. Moreover, there is no report of a tailored basal media for hMSC, excepted the redefinition of a DMEM medium, composed of elements accepted in the pharmacopeia, on the hypothesis that it would be more easily accepted in human therapy [113]. 3.1.2. Growth factors Every hMSC expansion medium is composed of a basal formulation supplemented with a significant amount of growth factors such as FCS or hPL (2–5–10–20% of the final volume) or purified growth factors [114]. Despite the fact that FCS is ill-defined, it is the most common support of hMSC cell proliferation, as for many

primary cells. With the development of regenerative medicine, the demand for large and reliable volumes of FCS will likely not be met in the future [115]. Furthermore, FCS validation to GMP-grade requires expensive procedures that can neither guarantee performances of different lots, nor the absolute absence of transmissible viruses and spongiform encephalopathy agents. During clinical tests, serum may also induce immunological reactions [116] and most importantly, influence hMSC growth and phenotype, even if no effector agent present in FCS has been clearly identified and characterized yet [117,118]. Alternatives to FCS supplementation have been a major subject of interest. They include the use of human blood derivatives (serum, platelet lysate, platelet rich plasma) or the formulation of entirely defined serum-free media (SFM) by combining growth and adhesion factors [119]. The formers have consistently demonstrated better performances in culture than FCS [120–122]. However, they are also subject to variations depending on their sources and preparation methods [123,124] that might impact cells characteristics [117,120,125]. Numerous studies have demonstrated the feasibility of hMSC expansion in SFM medium as well [126,127], but a majority used undisclosed medium formulation. A full understanding of the impact of growth factors is necessary to improve hMSC expansion and the achievement of an optimal defined medium formulation will be a great step forward [107,118]. Addition of purified growth factors are already necessary to maintain the cells viability and proliferation, even in the presence of FCS or blood derivatives [128], but their effect can greatly vary depending on their concentrations and interactions (see Table 1).

3.1.3. Feeding strategies The levels of nutrients and growth factors are not usually maintained constant during cell growth in static culture. These

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Table 1 Main growth factors used in culture media for hMSC expansion and their effects on hMSC critical quality attributes (CQA). Growth factor

FGF2

MSC proliferation

+++

CQA

Ref.

Synergistic effect with Wnt-3a to preserve hMSC chondrogenic potential Synergistic effect with ascorbic acid to increase hMSC differentiation potential via PI3K/AKT and MEK/ERK activation Induction of HLA-DR Decrease of the amount of senescent cells during long-term cultures via AKT and ERK activation Change in surface markers expression and promotion of chondrogenic differentiation

[193] [108] [194] [195] [196]

TGF␤1

++

Generation of mitochondrial reactive oxygen species and senescence induction Inhibition of adipogenesis and senescence due to increased proliferation Promotion of hBMSC chondrogenesis TGF␤1 secretion induced by high glucose environment via PI3K/AKT and PKC pathways

[197] [198] [199,200] [201]

PDGF family

++

Induction of growth factor expression and generation of mitochondrial reactive oxygen species Stimulation of exosome secretion and enhancement of hMSC angiogenic potential Inhibition of hMSC osteogenesis

[202] [203] [204]

EGF

+

No effect on multipotency Overexpression of hTERT mRNA

[205,206] [207]

IGF-1

+/−

Effect on surface and stemness markers depending on MSC sources Induction of proliferation and osteogeneic gene expression only in the absence of FCS

[208] [209]

HGF

+/−

Maintenance on multipotency during long term culture

[108,195]

AKT, protein kinase B; EGF, epithelial growth factor; ERK, extracellular-signal-regulated kinase; FCS, fetal calf serum; FGF2, fibroblast growth factor 2; hBMSC, human bone marrow mesenchymal stromal cell; HGF, hepatic growth factor; HLA-DR, human leukocyte antigens-DR; hMSC, human mesenchymal stromal cell; hTERT, human telomerase reverse transcriptase; IGF-1, insulin-like growth factor 1; MEK, mitogen-activated protein kinase; mRNA, messenger ribonucleic acid; PDGF, platelet derived growth factor; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; TGF-␤1, transforming growth factor beta 1; Wnt-3a, proto-oncogene protein Wnt-3a.

components are rather replenished every 2–3 days by a full medium exchange. This method can be easily adapted to microcarrier cultivation systems by settling down carriers before carefully withdrawing spent medium. Logically, better performance of cell expansion is observed in the case of medium refreshment in comparison with batch mode [129], except when nutrient limitations or lactate/ammonia inhibition levels were not reached in batch mode [130,131]. To facilitate medium renewal, continuous perfusion systems were also investigated for hMSC expansion, in stirred-tank [127] and hollow-fiber bioreactors [132]. The first system maintained a constant flow rate of fresh medium throughout the culture when the flow rate in the other was adapted to lactate measurements. These reactors have the advantage to be closed systems and to provide slightly higher cell yields compared to the classical medium refreshment strategy [133]. Whatever the mode of medium renewal, the previous examples only investigated a complete medium feed without trying to tailor its composition throughout the culture. Some alternatives considered the use of either a concentrated nutritional medium feed [127] (undisclosed formulation), a more concentrated glucose feed within a medium refreshment [134], or a supplementation of fresh medium with serum or growth factors [135]. To circumvent the large amount of medium requested by these methods, MSC expansion was also improved with glucose released over-time by alginate microspheres [136] or FGF2-loaded microcarriers [137]. Compared to continuous cell line processes, feeding strategies for hMSC processes are still at the beginning of their development. Conflicting papers focused on nutrient limitation, growth factor stimulation or secondary metabolites inhibition needed for increased expansion efficiency. Most of the confusion can unfortunately be due to undefined media composition and donor variations. Nevertheless, none of these studies suggested an impact of feeding mode on hMSC characteristics.

example, it has already been demonstrated that these cells could not differentiate without cell-cell interaction [138], or that stiffness of surface adhesion could greatly influence hMSC future lineage [139]. Despite substantial progress in the development of serum-free expansion medium, tissue-culture treated plastics cannot sustain hMSC growth in absence of serum. As a result, coating of adhesion surfaces with extracellular matrix (ECM) proteins (usually purified fibronectin) is usually performed [140]. When true xeno-free conditions are required, the synthesis of equivalent RGD containing proteins [141] or the use of functionalized polymer plastic [142] are examined in order to mimic ECM. Tissue engineering classically requires 3D (scaffold or hydrogel) rather than 2D surface for hMSC growth, since these structures enhanced hMSC growth as well as differentiation capabilities [143] and clonogenicity [144]. Cell aggregate cultures, where cells support themselves, might be more limited in term of proliferation due to mass transfer limitation in aggregate core but can ameliorate the cells potency [145], even in serum-free conditions [146]. Microcarriers have also been extensively studied (see detailed references in Fig. 2 caption) as they seem the most promising system for culture intensification and reduction of cost of goods per dose [147]. Changing the type of adhesion surface is currently more proned to impact hMSC gene expression pattern rather than shifting from a static to a dynamic culture system [148]. Any surfaces used for hMSC expansion have to be compatible with soft cell harvesting techniques. The common detachment procedures are performed by enzymatic treatment that do not preserve the cells surface receptors [149], nor their differentiation potential [150]. Currently, efforts are made to develop alternatives with thermodegradable or thermosensitive materials [151,152]. Finally and after cell detachment, culture volumes can be filtrated in order to ensure an efficient removal of microcarriers and to concentrate cells without compromising their characteristics [153].

3.2. Physicochemical environment

3.2.2. Hydromechanical stress Since 3D cultures promote a more physiological cell organization, and keeping in mind the necessity of process scale-up, the problem of medium homogenization in the bioreactor and consequently of the local cell environment has already been addressed.

3.2.1. Adhesion surface As hMSC are adherent-dependent cells, the adhesion surface structure is thus of great importance for their culture. For

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Fig. 2. Impact of 5 main characteristics of microcarriers on hMSC expansion. Studies focused either on hMSC expansion yield depending on microcarrier choice and process parameters [130,131,185,186], including microcarrier feeding [130,187] and harvesting methods [164,188,189]; or on hMSC quality like spontaneous differentiation [190,191] or surface markers and immunomodulation [192]. (CQA, critical quality attribute; ECM, extracellular matrix; RGD, Arg-Gly-Asp tripeptide.)

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However, a compromise has to be found between homogeneity and hydromechanical forces that might induce hMSC mechanotransduction pathways (reviewed elsewhere [154]). Furthermore, stress tests in flow chambers have demonstrated that shear forces could inhibit hMSC viability and proliferation [155]. Two culture phases can be distinguished in regards to medium agitation: cell seeding (around 24 h) and expansion step (7–14 days). The seeding conditions for a cell suspension to adhere onto a surface depend on its nature. When seeding cells onto a scaffold, perfusion allows hMSC to spread more homogeneously than in static conditions, and led to a better outcome in vivo [156]. When microcarriers are used, yields of cell adhesion are favored by intermittent stirring. A decrease in cell aggregate formation is observed especially when an higher mixing intensity was applied, whereas almost no change in cell quality attributes was observed [131]. On the opposite, hydromechanical stresses applied during the expansion phase seem to direct hMSC toward an osteogenic phenotype, whatever compression [157], orbital [158], or mechanically-stirred flows were applied on cells, in a scaffold [159] or on microcarriers [150]. One specificity of microcarrier expansion process is that studies focused on applying a minimum stirring rate to reach a ‘just-suspended’ state of the particles. This parameter was indeed demonstrated as an acceptable scale-up factor [114]. In this context, the importance of choosing the appropriate impeller design to increase the Kolmogorov microscale of turbulence has been underlined in order to promote cell growth [160,161]. These studies however pointed out that an increase in microcarrier density might be deleterious, probably due to particle collisions. When these two parameters (microcarrier density and impeller design) are properly set, increasing stirrer speed does not seem to have a deleterious effect on cell expansion factor [162] other than reducing cell aggregation [163]. The Kolmogorov scale parameter was also used to design an enzymatic harvesting method combined with sufficiently high power dissipation [164].

Therapeutic use

PRE/CLINICAL DATA

PROCESS

Regulatory approval

hMSC isolation and expansion

Critical parameters

In vitro/ in vivo studies

MOA QUALITY ATTRIBUTES In vitro cell characterization Multipotency Immunomodulatory and angiogeneic properties

Identity

Senescence

Fig. 3. Development overview of a regenerative medicine applied to hMSC therapy. (MOA, mode of action.)

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3.2.3. pH, temperature and oxygen saturation pH and temperature effects have been mainly studied for cultures of rodent MSC. Two studies are in contradiction concerning the effect of temperature on rat MSC. One observed a lower cell proliferation rate at a temperature of 32 ◦ C compared to 37 ◦ C [165], while the other showed an increase in growth rate at a temperature of 33 ◦ C in comparison with 37 ◦ C [166]. Regardless, the former found that a temperature decrease led to lower stress levels (nitric oxide, reactive oxygen species and apoptosis) [165], and hence could be a way to limit SIPS. Regulating pH in mouse and human MSC cultures would apparently consist in limiting a pH rise of the medium, as it cannot sustain the cell growth under atmospheric condition if the buffer is bicarbonate based (equilibrium around pH 9) [167]. The optimal range for hMSC expansion has been identified between pH 7.47 and 8.27 [168]. However, depending on the species, alkalinity showed opposite effects on differentiation. It induced chondro- and osteogenesis in rat MSC [169], while a pH over 7.9 inhibited this differentiation potential in hMSC [168]. Oxygen saturation level in culture medium has been studied by comparing standard (21% of O2 saturation) to hypoxia (5 to 1% of O2 saturation) conditions. Results have been controversial due to the duality of cell response facing either short- (also called preconditioning) or long term exposure to hypoxia [170]. Nevertheless, oxygen reduction has an important impact on hMSC metabolism. In normoxia, the cell response appeared to be only marginally relying on oxidation for ATP production [171]. When placed under hypoxia, hMSC switched their oxydative metabolism to a glycolytic metabolism even more via the expression of hypoxia-inducible factor-1a (HIF1a) [172,173]. It has hence been demonstrated that hMSC could withstand severe hypoxia as long as the cells do not face glucose depletion [174] and that it could even significantly improve cell survival [175]. Cells would then remain in an undifferentiated multipotent state, where “stemness” genes are upregulated and differentiation inhibited [58,102,172,176]. For cultures in bioreactor, these observations allow to simplify processes by maintaining a low oxygen level with surface aeration and low stirring [177,127]. As a result, such processes would avoid damages induced by gas bubble bursting or hydromechanical stresses. 4. Conclusion: new concepts for classic designs or new designs for classic concepts ? Mammalian cell bioprocessing has more than 50 years of experience by mainly culturing continuous hybridoma and CHO cell lines [178]. However, the development of the regenerative medicine field has raised several new challenges. Indeed, hMSC appear to share only few characteristics with immortalized cell lines such as clonal heterogeneity, senescence, anaerobic metabolism, dependence to growth factors. As shown in this review, many studies dedicated to hMSC have been performed these recent years, and they picture a complex relationship between cell characteristics, their process of expansion and their final therapeutic use (see Fig. 3). It appears however that the criteria defined by the ISCT are almost never impacted by the expansion process parameters, except for the occasional induction toward a differentiated lineage. The general viewpoint is hence now to systematically combine process development to multiple cellular assays that would ensure the robustness of the hMSC expansion and ultimately lead to their therapeutic use. References [1] A.J. Friedenstein, R.K. Chailakhjan, K.S. Lalykina, The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells, Cell Tissue Kinet. 3 (4) (1970) 393–403.

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