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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    • "Among various CPAs, DMSO is the most widely used for cell cryopreservation (Berz et al., 2007), while FBS is routinely added to cryopreservation media as a source of nutrients and for prevention of loss of cell viability during thawing (Gonda et al., 2008;Jochems et al., 2002). In this study, 10% DMSO + 90% FBS acted as a standard CPA; this has been used to preserve many types of cells in large volume and maintain high cell viability (Li and Ma, 2012;Zeisberger et al., 2011). However, the clinical use of cells preserved with 10% DMSO have caused many adverse effects, such as neurotoxicity and respiratory depression (Benekli et al., 2000;Windrum and Morris, 2003). "
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    ABSTRACT: Cryopreservation represents an efficient way to preserve human mesenchymal stem cells (hMSCs) at early culture/passage, and allows pooling of cells to achieve sufficient cells required for off-the-shelf use in clinical applications, e.g. cell-based therapies and regenerative medicine. To fully apply cryopreserved hMSCs in a clinical setting, it is necessary to evaluate their biosafety, e.g. chromosomal abnormality and tumourigenic potential. To date, many studies have demonstrated that cryopreserved hMSCs display no chromosomal abnormalities. However, the tumourigenic potential of cryopreserved hMSCs has not yet been evaluated. In the present study, we cryopreserved human adipose-derived mesenchymal stem cells (hASCs) for 3 months, using a slow freezing method with various cryoprotective agents (CPAs), followed by assessment of the tumourigenic potential of the cryopreserved hASCs after thawing and subculture. We found that long-term cryopreserved hASCs maintained normal levels of the tumour suppressor markers p53, p21, p16 and pRb, hTERT, telomerase activity and telomere length. Further, we did not observe significant DNA damage or signs of p53 mutation in cryopreserved hASCs. Our findings suggest that long-term cryopreserved hASCs are at low risk of tumourigenesis. These findings aid in establishing the biosafety profile of cryopreserved hASCs, and thus establishing low hazardous risk perception with the use of long-term cryopreserved hASCs for future clinical applications. Copyright © 2016 John Wiley & Sons, Ltd.
    No preview · Article · Jan 2016 · Journal of Tissue Engineering and Regenerative Medicine
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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.
    Full-text · Article · Aug 2014 · Cryobiology
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    ABSTRACT: Hematopoietic stem cells (HSC) can be stored for prolonged periods at cryogenic temperatures. The techniques currently used were derived from the initial report in 1949 of cryopreservation of bovine sperm in glycerol. The addition of this penetrating cryoprotectant protected the cells from the injury associated with ice formation. Current cryopreservation techniques (with minor variations) suspend cells in an aqueous solution of salts, protein, and one or more cryoprotectants. Cells are frozen at slow rates and stored generally below -120 degrees C in mechanical freezers or nitrogen refrigerators. That these techniques are successful in maintaining HSC viability is evident from the engraftment of these cells in patients treated with marrow-lethal conditioning regimens. However, issues such as the composition of the cryoprotectant solution, cell concentration during freezing, cryoprotectant toxicity, and storage temperatures have not been adequately studied, primarily because of a lack of appropriate assays for HSC cryosurvival. HSC cryobiology will become an increasingly important subject as new HSC collection and processing techniques are developed. Improved cryosurvival of HSC using modified cryoprotectant solutions may improve engraftment kinetics and decrease the cost and morbidity of autologous transplantation.
    Preview · Article · Feb 1992 · Journal of Hematotherapy
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