Dataset Information

Cryopreservation of Mesenchymal Stem Cells Using Medical Grade Ice Nucleation Inducer.

ABSTRACT: Mesenchymal stem cells (MSCs) can differentiate into multiple different tissue lineages and have favourable immunogenic potential making them an attractive prospect for regenerative medicine. As an essential part of the manufacturing process, preservation of these cells whilst maintaining potential is of critical importance. An uncontrolled area of storage remains the rate of change of temperature during freezing and thawing. Controlled-rate freezers attempted to rectify this; however, the change of phase from liquid to solid introduces two extreme phenomena; a rapid rise and a rapid fall in temperature in addition to the intended cooling rate (normally -1 °C/min) as a part of the supercooling event in cryopreservation. Nucleation events are well known to initiate the freezing transition although their active use in the form of ice nucleation devices (IND) are in their infancy in cryopreservation. This study sought to better understand the effects of ice nucleation and its active instigation with the use of an IND in both a standard cryotube with MSCs in suspension and a high-throughput adhered MSC 96-well plate set-up. A potential threshold nucleation temperature for best recovery of dental pulp MSCs may occur around -10 °C and for larger volume cell storage, IND and fast thaw creates the most stable process. For adhered cells, an IND with a slow thaw enables greatest metabolic activity post-thaw. This demonstrates a necessity for a medical grade IND to be used in future regenerative medicine manufacturing with the parameters discussed in this study to create stable products for clinical cellular therapies.

SUBMITTER: Wragg NM

PROVIDER: S-EPMC7696797 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Cryopreservation of Mesenchymal Stem Cells Using Medical Grade Ice Nucleation Inducer.

Wragg Nicholas M NM Tampakis Dimitris D Stolzing Alexandra A

International journal of molecular sciences 20201113 22

Mesenchymal stem cells (MSCs) can differentiate into multiple different tissue lineages and have favourable immunogenic potential making them an attractive prospect for regenerative medicine. As an essential part of the manufacturing process, preservation of these cells whilst maintaining potential is of critical importance. An uncontrolled area of storage remains the rate of change of temperature during freezing and thawing. Controlled-rate freezers attempted to rectify this; however, the chang ...[more]

PMID: 33203028

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Project description:Cell culture plays a critical role in biomedical discovery and drug development. Primary hepatocytes and hepatocyte-derived cell lines are especially important cellular models for drug discovery and development. To enable high-throughput screening and ensure consistent cell phenotypes, there is a need for practical and efficient cryopreservation methods for hepatocyte-derived cell lines and primary hepatocytes in an assay-ready format. Cryopreservation of cells as adherent monolayers in 96-well plates presents unique challenges due to low volumes being susceptible to supercooling, leading to low recovery and well-to-well variation. Primary cell cryopreservation is also particularly challenging due to the loss of cell viability and function. In this study, we demonstrate the use of soluble ice nucleator materials (IN) to cryopreserve a hepatic-derived cell line (HepG2) and primary mouse hepatocytes, as adherent monolayers. HepG2 cell recovery was near 100% and ∼75% of primary hepatocytes were recovered 24 hours post-thaw compared to just 10% and 50% with standard 10% DMSO, respectively. Post-thaw assessment showed that cryopreserved HepG2 cells retain membrane integrity, metabolic activity, proliferative capacity and differentiated hepatic functions including urea secretion, cytochrome P450 levels and lipid droplet accumulation. Cryopreserved primary hepatocytes exhibited reduced hepatic functions compared to fresh hepatocytes, but functional levels were similar to commercial suspension-cryopreserved hepatocytes, with the added benefit of being stored in an assay-ready format. In addition, normal cuboidal morphology and minimal membrane damage were observed 24 hours post-thaw. Cryopreserved HepG2 and mouse hepatocytes treated with a panel of pharmaceutically active compounds produced near-identical dose-response curves and EC50 values compared to fresh hepatocytes, confirming the utility of cryopreserved bankable cells in drug metabolism and hepatotoxicity studies. Cryopreserved adherent HepG2 cells and primary hepatocytes in 96 well plates can significantly reduce the time and resource burden associated with routine cell culture and increases the efficiency and productivity of high-throughput drug screening assays.

Dataset Information

Cryopreservation of Mesenchymal Stem Cells Using Medical Grade Ice Nucleation Inducer.

Publications

Cryopreservation of Mesenchymal Stem Cells Using Medical Grade Ice Nucleation Inducer.

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