Bioprocessing of Cryopreservation for Large-Scale Banking of Human Pluripotent Stem Cells

Department of Chemical and Biomedical Engineering, Florida State University , Tallahassee, Florida.
BioResearch open access 10/2012; 1(5):205-14. DOI: 10.1089/biores.2012.0224
Source: PubMed


Human pluripotent stem cell (hPSC)-derived cell therapy requires production of therapeutic cells in large quantity, which starts from thawing the cryopreserved cells from a working cell bank or a master cell bank. An optimal cryopreservation and thaw process determines the efficiency of hPSC expansion and plays a significant role in the subsequent lineage-specific differentiation. However, cryopreservation in hPSC bioprocessing has been a challenge due to the unique growth requirements of hPSC, the sensitivity to cryoinjury, and the unscalable cryopreservation procedures commonly used in the laboratory. Tremendous progress has been made to identify the regulatory pathways regulating hPSC responses during cryopreservation and the development of small molecule interventions that effectively improves the efficiency of cryopreservation. The adaption of these methods in current good manufacturing practices (cGMP)-compliant cryopreservation processes not only improves cell survival, but also their therapeutic potency. This review summarizes the advances in these areas and discusses the technical requirements in the development of cGMP-compliant hPSC cryopreservation process.

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Available from: Yan Li, Sep 29, 2015
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    • "It is presently unknown whether post-thaw MSCs retain the same potential for regenerative therapeutic applications as their non-cryopreserved counterparts. The response of stem cells to cryopreservation can include a reduction in cell viability due to cold-shock and/or the toxic effects of DMSO, and changes in the expression of stem cell-related markers, cytoskeletal disassembly, delayed apoptosis, and osmotic and oxidative stresses [27]. These factors may have an influence on the functionality of MSCs and reduce their applicability for regenerative therapies. "
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    ABSTRACT: The effects of cryopreservation on mesenchymal stem cell (MSC) phenotype are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation (10% dimethyl-sulfoxide) on the morphology, viability, gene-expression and relative proportion of MSC surface-markers on cells derived from rat adipose, bone marrow and dental pulp. Cryopreservation significantly reduced the number of viable cells in bone marrow and dental pulp cell populations but had no observable effect on adipose cells. Flow cytometry analysis demonstrated significant increases in the relative expression of MSC surface-markers, CD90 and CD29/CD90 following cryopreservation. sqRT-PCR analysis of MSC gene-expression demonstrated increases in pluripotent markers for adipose and dental pulp, together with significant tissue-specific increases in CD44, CD73 and CD105 following cryopreservation. Cells isolated from different tissue sources did not respond equally to cryopreservation with adipose tissue representing a more robust source of MSCs.
    Cryobiology 08/2014; 69(2). DOI:10.1016/j.cryobiol.2014.08.003 · 1.59 Impact Factor
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    ABSTRACT: Because of their multi/pluripotency and immunosuppressive properties mesenchymal stem/stromal cells (MSCs) are important tools for treating immune disorders and for tissue repair. The increasing use of MSCs has led to production processes that need to be in accordance with Good Manufacturing Practice (GMP). In cellular therapy, safety remains one of the main concerns and refers to donor validation, choice of starting material, processes, and the controls used, not only at the batch release level but also during the development of processes. The culture processes should be reproducible, robust, and efficient. Moreover, they should be adapted to closed systems that are easy to use. Implementing controls during the manufacturing of clinical-grade MSCs is essential. The controls should ensure microbiological safety but also avoid potential side effects linked to genomic instability driving transformation and senescence or decrease of cell functions (immunoregulation, differentiation potential). In this rapidly evolving field, a new approach to controls is needed.
    Human gene therapy 10/2010; 22(1):19-26. DOI:10.1089/hum.2010.197 · 3.76 Impact Factor
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    ABSTRACT: Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as anefficient culture configuration, enabling rapid and controlled large scale expansion. Aggregate-based PSC cryopreservation facilitates theintegrated processof cell expansion and cryopreservation, but its feasibility has not been demonstrated. The goals of current study are to assess the suitability of cryopreserving intact mouse embryonic stem cell (mESC)aggregates and investigate the effects ofaggregate size and the formulation of cryopreservation solution on mESC survival and recovery. The results demonstratedthe size-dependent cell survival and recovery of intact aggregates. In particular, the generation of reactive oxygen species(ROS) andcaspaseactivationwere reducedfor small aggregates (109±55 µm)compared to medium (245 ± 77 µm) and large (365 ± 141 µm) ones, leading to the improved cell recovery.In addition, adefinedprotein-free formulation was tested and found to promotetheaggregate survival, eliminating the cell exposure to animal serum. The cryopreserved aggregates also maintained the pluripotentmarkers and the differentiation capacity into three-germ layers after thawing. In summary,the cryopreservation of small PSC aggregates in a defined protein-free formulationwas shown to be asuitable approachtowards a fully integrated expansion and cryopreservation process at large scale. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012.
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