Hypothermic Storage of Confluent Human Adipose Stem Cells

Cell Sheet Engineering is based on the retrieval of cells from culture dishes as extracellular matrix (ECM) rich sheets. These can then be used for the regeneration/engineering of several tissues and organs, as demonstrated in ongoing clinical trials. CS fabrication, while simple, requires cell culture facilities. Thus, ensuring adequate cell function from the fabrication site to the bedside is essential to its efficiency. We tested the ability of two commercially available compounds to preserve the viability of CS of human adipose stem cells (hASC) at 4ºC. hASC were cultured to hyperconfluence in basal medium to produce CS. 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 for all the conditions at 37ºC were established. Non-confluent cultures mirroring all the conditions were also set up. At each time point, cell viability was assessed using 7-AAD and Alamar Blue. Caspase activity and differentiation potential into adipogenic and osteogenic lineages, was also assessed. After 3 days, a sharp decrease in cell viability in 4ºC BMC was noticed, especially in non-confluent cultures. HTS and RP managed to keep viability values close to the 37ºC BMC. At day 7, few viable cells were detected in the 4ºC BMC, while none were found in their non-confluent counterparts. HTS and RP supplemented CS presented 40% of the viability of the 37ºC BMC, while non-confluent conditions presented much lower values. Although viability and cell number were severely affected in some conditions, no activation of caspase 3 was detected, suggesting cell loss by necrosis. HTS and RP where able to preserve hASC ability to differentiate into adipogenic lineage, whereas, RP demonstrated higher efficiency in preserving hASC ability to differentiate into osteogenic lineage. Summarizing, both HTS and RP demonstrated excellent hypothermic preservation of cells especially up to 3 days. Furthermore, confluence apparently confers protection against hypothermic insult. 

Acknowledgements: IF/00347/2015, SFRH/BPD/96611/2013, NORTE-01-0145-FEDER-000021, Gene2Skin Project  (H2020-TWINN-2015-692221)