The Study Of Zwitterionic Betaine As A Cryoprotectant For MSC Cryopreservation

Mesenchymal stem cells(MSCs)have promising potential in medical treatments,such as tissue damages,cancer,diabetes and immune deficiency due to their abilities of self-renewing,multi-lineage differentiation and immunoregulation.Cryopreservation is one of the relable technologies for long-term storage of MSCs,because cellular metabolism can be suspended at ultralow temperature.After warming in water bath,the cryopreserved cells can recover their original biological activity.Commonly,MSC cryopreservation utilizes dimethyl sulfoxide(DMSO)as a cryoprotectant(CPA)to protect MSCs from cryoinjuries.However,DMSO can affect the gene expression and cause abnormal differentiation of MSCs.Its intrinsic toxicity may cause life-threatening adverse reactions of patients,such as allergies,arrhythmias,organ failure,etc.Therefore,it is of great significance to explore novel CPA with great biocompatibility and cryoprotection efficiency for MSCs.In this thesis,a DMSO-free cryopreservation method of MSCs is developed by utilizing betaine as a sole CPA combining with reversible electroporation technology.Firstly,we investigate the potential of betaine to reduce cryoinjuries.The ice inhibition tests show that betaine can increase the content of nonfreezing water and depress the freezing point of pure water,which is expected to reduce the damage to cells caused by ice formation.The osmotic regulation tests present that betaine can enter cells via membrane transporters to balance osmotic pressure,thereby protecting the MSCs from osmotic injury.Besides,betaine can inhibit excessive production of reactive oxygen species(ROS)and scavenge ABTS free radicals,indicating its antioxidative activity.Secondly,we investigate the cryoprotection efficacy of betaine to MSCs.Betaine as the sole CPA cannot cryopreserve MSCs.With an incubation with betaine for 4 h prior to cryopreservation,the cryopreservation efficacy can be improved to approximately 50%.More importantly,by combining with reversible electroporation technology,the cell viability can be further improved to approximately 85%.This is mainly because temporary hydrophilic pores produced by reversible electroporation technology improve the nonspecificly permeablility of cells,thus promoting the uptake of betaine and enhancing the intracellular and extracellular cryoprotection to cells.The in vivo homing distribution of MSC is closely related to the efficiency of cell therapies.Green fluorescent protein(GFP)and firefly luciferase(Fluc)gene are integrated to MSCs(GFP-Fluc-UCMSCs)by lentiviral transfection and flow cytometry sorting.Betaine combined with reversible electroporation effectively cryopreserve GFP-Fluc-UCMSCs.The post-thawed GFP-Fluc-UCMSCs maintain high cell survival rate and normal expression of reporter genes.The bioluminescence imaging tests demonstrate that post-thawed GFP-Fluc-UCMSCs exhibit the same homing distribution pattern in mouse with that of fresh MSCs in mouse.