Systematic parameter optimization of a Me2SO- and serum-free cryopreservation protocol for human mesenchymal stem cells

Freimark, Denise; Sehl, Constanze; Weber, Christian; Hudel, Klaus; Czermak, Peter; Hofmann, Nicola; Spindler, Ralf; Glasmacher, Birgit

doi:10.1016/j.cryobiol.2011.05.002

Cited by 54 publications

(35 citation statements)

References 30 publications

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“…Multicomponent solutions have also been explored and factorial experiments to identify favorable combinations and freezing protocols have shown increased success. 5,6 Previously published work by this group 7 describes algorithm optimization of multicomponent cryopreservative solutions, including molecules that target different aspects of cell protection during freezing.…”

Section: Introductionmentioning

confidence: 99%

Combinations of Osmolytes, Including Monosaccharides, Disaccharides, and Sugar Alcohols Act in Concert During Cryopreservation to Improve Mesenchymal Stromal Cell Survival

Pollock

Moller-Trane

et al. 2016

Tissue Engineering Part C: Methods

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There is demand for non-dimethyl sulfoxide (DMSO) cryoprotective agents that maintain cell viability without causing poor postthaw function or systemic toxicity. The focus of this investigation involves expanding our understanding of multicomponent osmolyte solutions and their ability to preserve cell viability during freezing. Controlled cooling rate freezing, Raman microscopy, and differential scanning calorimetry (DSC) were utilized to evaluate the differences in recovery and ice crystal formation behavior for solutions containing multiple cryoprotectants, including sugars, sugar alcohols, and small molecule additives. Postthaw recovery of mesenchymal stem cells (MSCs) in solutions containing multiple osmolytes have been shown to be comparable or better than that of MSCs frozen in 10% DMSO at 1°C/min when the solution composition is optimized. Maximum postthaw recovery was observed in these multiple osmolyte solutions with incubation times of up to 2 h before freezing. Raman images demonstrate large ice crystal formation in cryopreserved cells incubated for shorter periods of time (*30 min), suggesting that longer permeation times are needed for these solutions. Recovery was dependent upon the concentration of each component in solution, and was not strongly correlated with osmolarity. It is noteworthy that the postthaw recovery varied significantly with the composition of solutions containing the same three components and this variation exhibited an inverted U-shape behavior, indicating that there may be a ''sweet spot'' for different combinations of osmolytes. Raman images of freezing behavior in different solution compositions were consistent with the observed postthaw recovery. Phase change behavior (solidification patterns and glass-forming tendency) did not differ for solutions with similar osmolarity, but differences in postthaw recovery suggest that biological, not physical, methods of protection are at play. Lastly, molecular substitution of glucose (a monosaccharide) for sucrose (a disaccharide) resulted in a significant drop in recovery. Taken together, the information from these studies increases our understanding of non-DMSO multicomponent cryoprotective solutions and the manner by which they enhance postthaw recovery.

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Section: Introductionmentioning

confidence: 99%

Combinations of Osmolytes, Including Monosaccharides, Disaccharides, and Sugar Alcohols Act in Concert During Cryopreservation to Improve Mesenchymal Stromal Cell Survival

Pollock

Moller-Trane

et al. 2016

Tissue Engineering Part C: Methods

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“…Optimal concentration of DMSO in freezing medium for O. dancena embryonic cell lines was 10 %, in which sufficient number of cells for further use were able to be retrieved from a small number of post-thaw cells after 2 days of culture suggesting that slow freezing program employing DMSO can be well applied to O. dancena embryonic cell lines for effective cryopreservation. FBS is a major component of the freezing medium since it plays a key role in reducing oxidative stress (Freimark et al 2011;Gutteridge and Quinlan 1993). In this study, addition of FBS to freezing medium under 10 % DMSO did not influence post-thaw cell viability.…”

Section: Discussionmentioning

confidence: 72%

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena)

et al. 2014

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This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7 days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10 % DMSO, 20 % FBS, and 0.1 M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57 days) and (2) freezing of short-term cultured cells from other batches for 7 days. The viability of postthaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7 days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.

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“…The standard is a slow freezing rate of 1°C/min down to -80°C and a quick thawing rate (2 min at 37°C for 2 mL vials) using 5-20% dimethylsulfoxide (DMSO) in serum as a CPA [72]. Neither DMSO nor serum are suitable for ATMPs [69,108]. For other stem cells, successful cryopreservation has been achieved using 5% DMSO in 5% human albumin [109].…”

Section: Formulation and Storage Of Stem Cellsmentioning

confidence: 99%

“…Based on promising preliminary studies [69], ectoin and proline have been investigated as alternative CPAs for hMSC-TERT cells [108]. They were compared to commercially available Biofreeze SFM lacking DMSO (Biochrom, Germany [113]) combined with methylcellulose supplemented PBS.…”

Section: Formulation and Storage Of Stem Cellsmentioning

confidence: 99%

The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

Elseberg¹,

Leber²,

Weidner³

et al. 2017

New Insights Into Cell Culture Technology

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In the field of cell therapy, allogenic human mesenchymal stromal cells (hMSCs) are often used in clinical trials, creating a demand for cell mass production using efficient dynamic bioreactor systems. As an advanced therapy medicinal product (ATMP), such cells should meet certain special requirements, including product specifications requiring a production process compatible with good manufacturing practice (GMP). The development of processes in which the cells are the product therefore remains a significant challenge. This chapter describes the requirements at different steps in the upstream and downstream phases of such dynamic processes. Potential solutions are presented and future prospects are discussed, including the selection of media and carriers for the strictly adherent growing cells, allowing efficient cell adhesion and detachment. Strategies for dynamic cultivation in bioreactors are described in detail for fixed-bed and stirred-tank reactors based on GMP requirements and the integration of process analytical technology (PAT). Following cell harvest, separation and purification, the formulation and storage of the product are also described. Finally, the chapter covers important cell quality characteristics necessary for the approval of ATMPs.

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Systematic parameter optimization of a Me2SO- and serum-free cryopreservation protocol for human mesenchymal stem cells

Cited by 54 publications

References 30 publications

Combinations of Osmolytes, Including Monosaccharides, Disaccharides, and Sugar Alcohols Act in Concert During Cryopreservation to Improve Mesenchymal Stromal Cell Survival

Combinations of Osmolytes, Including Monosaccharides, Disaccharides, and Sugar Alcohols Act in Concert During Cryopreservation to Improve Mesenchymal Stromal Cell Survival

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena)

The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

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