Introduction: Cell Sheet Engineering is based on the retrieval of cells from culture dishes as extracellular matrix(ECM)-rich sheets that can then be used for the regeneration/engineering of several tissues and organs, as demonstrated in ongoing clinical trials. Cell sheet fabrication, while simple, requires cell culture facilities. Thus, ensuring adequate cell function from the fabrication site to the bedside is essential to its efficiency. Therefore, we tested the ability of two commercially available compounds to preserve the viability of sheets of human adipose stem cells (hASC) at 4ºC.

Methods: hASC were cultured in basal medium until hyperconfluence to produce cell sheets. In some plates, the medium was either exchanged by a solution of Hypothermosol® (HTS) or supplemented with Rokepie® (RP), while the rest of the plates remained with basal medium. Plates were then incubated at 4ºC for 3 and 7 days. Controls at 37ºC were established. Non-confluent cultures mirroring all the conditions were also set up. At each time point, cells were imaged for morphology and viability was assessed using flow cytometry (7-AAD) and alamar blue.

Results: After 3 days, a sharp decrease in the viability of cells cultured at 4ºC without preservation solutions was noticed when comparing with controls at 37Cº. This decrease was more noticeable in non-confluent cultures. The viability of cells cultured with HTS or RP was comparable but slightly less than controls at 37ºC. At 7 days, few viable cells were detected in the conditions of 4ºC without supplementation, while none were found in the non-confluent counterparts. HTS- and RP-supplemented cultures were comparable at around 40% of the viability of 37ºC controls. The relative viability of cells in non-confluent conditions was much lower but RP conditions managed to fare better that HTS.

Conclusions: Both HTS and RP demonstrated an excellent capability of hypothermic preservation of cell sheets for the 3-day time point, while after 7 days relative viability of cell sheets decreased to 40%. Furthermore, confluence apparently confers protection against hypothermic insult. Surface marker characterization, differentiation potential and caspase activity of cells after preservation are currently being assessed.