A consensus introduction to serum replacements and serum-free media for cellular therapies

Karnieli, Ohad; Friedner, Oryan Makler; Allickson, Julie; Zhang, Nan; Jung, Sunghoon; Fiorentini, D.; Abraham, Eytan; Eaker, Shannon; Yong, Tan; Chan, Allan; Griffiths, Sarah; Wehn, Amy K.; Oh, Steve Kah‐Weng

doi:10.1016/j.jcyt.2016.11.011

Cited by 153 publications

(170 citation statements)

References 48 publications

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“…When cell products are intended for utilization as an ATMP, GMP guidelines must be fulfilled [2, 21]. Beside environmental safety requirements, characteristics of consumables and additives utilized for cell expansion are regulated by GMP guidelines [1].…”

Section: Discussionmentioning

confidence: 99%

“…As stated in the European Regulation 1394/2007, ATMPs must be obtained in compliance with good manufacturing practice (GMP) guidelines [1]. Employment of validated and standardized animal and xeno-free compounds for cell manufacturing process is crucially important to preserve clinical safety of the final product [2]. Fetal bovine serum is the most common source of growth factors, and it is routinely used in research protocols as culture medium additive to promote cell expansion.…”

Section: Introductionmentioning

confidence: 99%

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Standardization of platelet releasate products for clinical applications in cell therapy: a mathematical approach

et al. 2017

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BackgroundStandardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined.MethodsSupernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20% CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools.ResultsGrowth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)–CV); VEGF: 950–81.4; FGF-b: 27–74.6; PDGF-AA: 7883–28.8; PDGF-AB: 107834–32.5; PDGF-BB: 11142–48.4; Endostatin: 305034–16.2; Angiostatin: 197284–32.9; TGF-β1: 68382–53.7; IGF-I: 70876–38.3; EGF: 2411–30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20%. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential.DiscussionResults deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-017-1210-z) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Standardization of platelet releasate products for clinical applications in cell therapy: a mathematical approach

et al. 2017

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“…The most common source of growth factors to support rapid in vitro expansion of MSCs is animal serum, such as fetal calf serum (FCS). However, FCS is a potential source of pathogens, can elicit immunological reactions in humans, is prone to variable composition, and has a limited production capacity that cannot support commercialized cell therapies . Human blood derivatives, including serum, platelet‐rich plasma, and platelet lysate or fractions thereof, are animal‐free alternatives to FCS but are also subject to source variations that exacerbate the cost of goods .…”

Section: Introductionmentioning

confidence: 99%

Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence

Wiese

Ruttan

Wood

et al. 2019

Stem Cells Translational Medicine

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In preclinical studies, mesenchymal stromal cells (MSCs) exhibit robust potential for numerous applications. To capitalize on these benefits, cell manufacturing and delivery protocols have been scaled up to facilitate clinical trials without adequately addressing the impact of these processes on cell utility nor inevitable regulatory requirements for consistency. Growing evidence indicates that culture‐aged MSCs, expanded to the limits of replicative exhaustion to generate human doses, are not equivalent to early passage cells, and their use may underpin reportedly underwhelming or inconsistent clinical outcomes. Here, we sought to define the maximum expansion boundaries for human umbilical cord‐derived MSCs, cultured in chemically defined xeno‐ and serum‐free media, that yield consistent cell batches comparable to early passage cells. Two male and two female donor populations, recovered from cryostorage at mean population doubling level (mPDL) 10, were serially cultivated until replicative exhaustion (senescence). At each passage, growth kinetics, cell morphology, and transcriptome profiles were analyzed. All MSC populations displayed comparable growth trajectories through passage 9 (P9; mPDL 45) and variably approached senescence after P10 (mPDL 49). Transcription profiles of 14,500 human genes, generated by microarray, revealed a nonlinear evolution of culture‐adapted MSCs. Significant expression changes occurred only after P5 (mPDL 27) and accumulated rapidly after P9 (mPDL 45), preceding other cell aging metrics. We report that cryobanked umbilical cord‐derived MSCs can be reliably expanded to clinical human doses by P4 (mPDL 23), before significant transcriptome drift, and thus represent a mesenchymal cell source suited for clinical translation of cellular therapies. Stem Cells Translational Medicine 2019;8:945&958

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“…In the stem cell field, a great deal of research is targeted at the use of adult and mesenchymal stem cells to fix damaged tissues. The wound healing response has provided the basis, for example, for the use of platelet lysates in MSC cell culture . We too have observed that platelet lysate is an excellent replacement for serum in the growth of cells differentiated from iPSCs .…”

Section: Discussionmentioning

confidence: 99%

Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions

Gandhi

Knudsen

Hill

et al. 2019

Stem Cells Translational Medicine

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Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)‐derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.g., laminin) for expansion in adherent cell culture for use in cell therapy. To address this, we investigated the potential of culturing iPSCs on fibrin hydrogels for three‐dimensional applications and further examined the use of fibrinogen, the soluble precursor protein, as a coating substrate for traditional adherent cell culture. iPSCs successfully adhered to and proliferated on fibrin hydrogels. The two‐dimensional culture with fibrinogen allows for immediate adaption of culture models to a nonxenogeneic model. Similarly, multiple commercially available iPSC lines adhered to and proliferated on fibrinogen coated surfaces. iPSCs cultured on fibrinogen expressed similar levels of the pluripotent stem cell markers SSea4 (98.7% ± 1.8%), Oct3/4 (97.3% ± 3.8%), TRA1‐60 (92.2% ± 5.3%), and NANOG (96.0% ± 3.9%) compared with iPSCs on Geltrex. Using a trilineage differentiation assay, we found no difference in the ability of iPSCs grown on fibrinogen or Geltrex to differentiate to endoderm, mesoderm, or ectoderm. Finally, we demonstrated the ability to differentiate iPSCs to endothelial cells using only fibrinogen coated plates. On the basis of these data, we conclude that human fibrinogen provides a readily available and inexpensive alternative to laminin‐based products for the growth, expansion, and differentiation of iPSCs for use in research and clinical cell therapy applications. stem cells translational medicine 2019;8:512–521

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A consensus introduction to serum replacements and serum-free media for cellular therapies

Cited by 153 publications

References 48 publications

Standardization of platelet releasate products for clinical applications in cell therapy: a mathematical approach

Standardization of platelet releasate products for clinical applications in cell therapy: a mathematical approach

Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence

Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions

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