Cryopreservation Impacts Cell Functionality of Long Term Expanded Adipose Derived Stem Cells
Objective: Adipose Derived Stem Cells (ADSCs) are less immunogenic cells and have a heterogenic cytokine secretion profile making them a better candidate for cell based immunotherapy. Even innately or stimulated, Interleukin 6 (IL 6) and Toll like Receptor 2 (TLR2) secreted by ADSC were modulated and led to different inflammatory mechanism pathways through different inflammatory factors secretion. These properties can be very useful in the treatment of inflammation associated chronic diseases. To be used, ex vivo ADSC expansion is a critical issue prior to transplantation or cryopreservation. Functional cell changes have been reported during culture expansion being susceptible to interact with freezing/thawing effects leading to doubt on cell therapeutic outcomes. The aim of this study is to identify the effect of freezing/thawing at the different time point of expansion culture on IL 6 and TLR2 secretion.Methods: ADSC were collected from young female donors, expanded in culture and cryopreserved in Foetal Bovine Serum (FBS) and Dimethylsulfoxide (DMSO) after each passage for 6 months to a year. ADSC was then tested for proliferation, clonogenicity, cytokine gene expression and assessment before cryopreservation (fresh) and after thawing and cultured until confluence (frozen/thawed) ADSC preserved at Passage 0 (P0) were thawed and tested after confluence at P1.Results: Cryopreserved ADSC as P1 resulted in increased clonogenicity, total RNA and protein secretion compared to the fresh ones. Relative Quantification (RQ) and cytokine assessment of IL 6, IL 10, Tumor Necrosis Factor (TNF) a and TLR2 revealed a moderate up regulation of TLR2 while significantly higher IL 6 secretion levels were observed in long term expanded and cryopreserved ADSC.Conclusion: Our results suggested that cryopreserved ADSC long term expanded in culture were functionally different and might have impaired immunosuppressive properties through modulation of the inflammatory responses by IL 6 and TLR2 activation.