Low Temperature Preparation Technology Study Of Human Adipose-derived Mesenchymal Stem Cells

The self-renew ability and differentiate ability of mesenchymal stem cells(MSCs)make MSCs ideal seed cells for cell therapy.Adipose-derived stem cells(ADSCs)are ideal MSCs for their easily access and abundance in adipose tissue.Currently,cryopreserved stem cell products added with cryoprotectant dimethyl sulfoxide(DMSO)were widely used in clinical treatment.DMSO is a toxic reagent,thus it would cause serious adverse reactions if these stem cell products are injected into human body.Few low temperature storage solution were reported,however,there are problems such as the use of non-clinical approval presearvation media,short duration,rapid decrease in cell viability and so on.We aimed at optimizing preservation media,durations and cell concentrations by a comprehensive evaluation system for low temperature storage in 2-8?to get a low temperature preparation solution to ensure the viability and function of ADSCs before transplantation as well as decrease the adverse reactions.Methods:1.Isolate ADSCs from human adipose tissue,and identify biological characteristics of ADSCs by cell morphology,surface markers,proliferation,osteogenic and adipogenic differentiation abilities.2.Evaluate different preservation media.Measure the viability and cluster rate,apoptosis,adhesion ability and colony-forming unit(CFU)capacity of ADSCs stored in 5%human serum albumin in multiple electrolytes(ME+HSA),5%human serum albumin in 0.9%normal saline(NS+HSA),5%glucose,and growth medium(GM).3.After the evaluation of preservation media,measure the apoptosis,adhesion ability and CFU capacity of ADSCs stored for 24h and 48h.4.After the evaluation of durations,measure the apoptosis,adhesion ability,CFU capacity,proliferation and the osteogenic and adipogenic differentiation abilities of ADSCs stored at the concentrations of1×10~6cells/ml,5×10~6cells/ml and 10×10~6cells/ml.5.After getting the best preservation medium,duration and cell concentration,store ADSCs using this solution,and determine cell surface markers,cell cycle and immunosuppressive capacity to evaluate this solution.Results:1.Cells isolated from adipose tissue were ADSCs,and these cells grew with fibroblastic spindle shape.HLA-DR,CD34 and CD45 were negatively expressed and CD73,CD90 and CD105 were positively expressed.Proliferation of cells was rapid and the osteogenic and adipogenic differentiation abilities were strong.2.ME+HSA was the best preservation medium.Cells in it had high cell viability,low cluster rate,good adhesion ability and high CFU capacity.3.Duration should be limited to 24h to ensure cell quality for transplantation.Apoptosis and non-adhesive cells increased significantly and CFU capacity decreased obviously when duration was extended to 48h.4.5×10~6 cells/ml was proper cell concentration with low late stage apoptosis,rapid proliferation and good osteogenic and adipogenic differentiation abilities.5.After the storage of ADSCs in this optimized solution,cell surface markers,cell cycle,indoleamine 2,3-dioxygenase(IDO1)gene expression and kynurenine(Kyn)concentration did not change significantly.Conclusion:5×10~6 cells/ml ADSCs suspended in ME+HSA for 24h is a good and potential stem cell preparation solution to be used in clinical therapy.