Hypothermic preservation of confluent Human Adipose Stem Cells

Cell Sheet (CS) Engineering is based on the retrieval of hyperconfluent cultured cells as ECM- rich sheets that can be used therapeutically for the regeneration of several tissues. CS fabrication, while simple, requires cell culture facilities. Thus, ensuring adequate cell function from the fabrication site to bedside is essential to its efficiency. We tested the ability of two commercially available compounds to preserve the viability of confluent cultures of human adipose stem cells (hASC) at 4oC. hASC were cultured to confluence in basal medium for 3 days to mimic CS fabrication conditions. Culture medium was then either replaced by a solution of Hypothermosol®(HTS) or supplemented with Rokepie®(RP) and cells were maintained at 4oC for 3 and 7 days. Basal medium controls (BMC) at 4oC and 37oC were established together with non- confluent cultures mirroring all culture and storage conditions. At each time point, cell condition was assessed using 7-AAD, Annexin V and Alamar Blue. After 3 days of storage , a sharp decrease in cell viability in BMC at 4oC was noticed, especially in non-confluent cultures. HTS and RP managed to keep cell viability values close to the BMC at 37oC. At day 7 of storage, few viable cells were detected in BMC at 4oC, while none were found in their non-confluent counterparts. HTS- and RP-supplemented CS presented 40% of the viability of the BMC at 37oC, while non-confluent conditions presented much lower values. Summarizing, both HTS and RP demonstrated excellent hypothermic preservation of cells especially up to 3 days of 4oC storage. Furthermore, confluence apparently confers protection against hypothermic insult. Surface marker characterization, differentiation potential and caspase activity after preservation are currently being assessed.

Acknowledgements: Funding project: POCI-01-0145-FEDER-007038. Funding to RPP: SFRH/BPD/101886/2014, MTC: SFRH/BPD/96611/2013, AFC: SFRH/BPD/109595/2015.

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