Cryopreservation Of Human Adipose-derived Stem Cells

Background:Reliable schemes for cryopreservation of human adipose-derived stem cells(hADSCs)is crucial for the establishment of cell banks and clinical application of hADSCs.However,there is still a lack of research in the field of cryopreservation of hADSCs.Objectives:To evaluate the effect of isolation and purification methods of hADSCs on their biological characteristics.To provide the cryobiological information of hADSCs during freezing.To evaluate the effect of the key freezing factors on biological characteristics of hADSCs.To explore the role of nano graphene oxide in the cryopreservation of hADSCs.Methods:1.Studies on the isolation and purification of hADSCs before cryopreservation.1.1 The cell yields and biological characteristics of stromal/stem cells(SVF)from lipoaspirate with different digestion loading ratio.The mixture of lipoaspirates and collagenase were divided into four groups according to loading volume ratios:0.2 group,0.4 group,0.6 group,0.8 group.SVF were obtained from each group,then total cell counts,viability and viable cell counts were compared.hADSCs were harvested,whose immunophenotypes,proliferation,and tri-differentiation abilities were compared.1.2 Effects of ammonium chloride(NH4C1)-based erythrocyte lysis isolating method on biological characteristics of hADSCs.Lipoaspirates were obtained and divided into two groups.For lysis group,SVF was purified with NH4Cl-based erythrocyte lysis.For control group,non-lysis group,SVF was suspended with Low-glucose DMEM.SVF were obtained from each group,then cell yields and viability were compared.hADSCs were harvested,whose viability,degree of apoptosis,immunophenotypes,proliferation,adipogenic and osteogenic differentiation abilities were compared.1.3 Effects of NH4Cl-based erythrocyte lysis on cryopreservation of hADSCs.Lipoaspirates were obtained and divided into lysis and non-lysis groups as described earlier.hADSCs were isolated from processed SVF and cryopreserved for approximately 2 weeks.Then,the viability,immunophenotype,degree of apoptosis and proliferation of fresh and post-thawed cells of each group were compared.2.Water-transport and intracellular ice formation of hADSCs during freezing.Systematic freezing experiments were conducted under a cryo-microscope system to investigate the cryoinjury mechanism for hADSCs at different cooling rates.By simultaneously fitting morphological change data to the numerical models,the plasma membrane hydraulic conductivity(Lpg),activation energy(ELp),kinetic factor(ΩOSCN)and thermodynamic factor(kOSCN)were determined.Moreover,the optimal cooling rate was also predicted by using mathematical model method.3.Studies on freezing process of hADSCs.3.1 Effects of cooling rates on biological characteristics of hADSCs.hADSCs were freezed at the cooling rates of 0.5℃/min,1℃/min,10℃/min,25℃/min,50℃/min,80℃/min and 100℃/min.After cryopreserved for 3 months,the viability,degree of apoptosis,immunophenotypes,proliferation,reactive oxygen species(ROS)and tri-differentiation ability of post-thawed cells of each group were compared.3.2 Effects of cell concentrations for cryopreservation of hADSCs.hADSCs were cryopreserved with cell concentrations of 0.5×106/mL,1×106/mL,2×106/mL,5×106/mL and 10×106/mL.After 2 weeks of cryopreservation,the viability,immunophenotypes,proliferation and tri-differentiation ability of post-thawed cells of each group were compared.4.Effects of nano-graphene oxide in cryopreservation of human adipose-derived stem cells.In vitro experiment:The cryoprotectant of CPA-C group was 90%FBS+10%DMSO,and CPA-GO group was FBS+10%DMSO+5μg/mL GO.hADSCs were cryopreserved for 2 weeks.The viability,immunophenotypes,proliferation,tri-differentiation ability and cell apoptosis of fresh and post-thawed cells of each group were compared.In addition,residues and cytotoxicity of GO were evaluated.In vivo experiments:after cryopreserved for 3 months,the viability,immunophenotypes,proliferation,and tri-differentiation ability of post-thawed cells of each group were compared to verify the biological characteristics of hADSCs.An animal model of fat transplantation assisted by hADSCs was designed:three treatment groups were set:PBS group,CPA-C group and CPA-GO group.The mixture of human adipose and hADSCs/PBS was transplanted on the neck of nude mice.After 3 months,graft weight,the survival and origin of adipocytes,the number and origin of blood vessels,the expression level of the genes related to lipogenesis and apoptosis,the infiltration of macrophages and the expression of inflammatory factors in the graft were compared.Results:1.Studies on the isolation and purification of hADSCs before cryopreservation.1.1 The cell yields and biological characteristics of SVF/hADSCs from lipoaspirate with different digestion loading ratio.0.4 loading volume ratio provided the highest cell yield(2.65±0.98×105 per mL fat).The 0.2 loading volume ratio group had the highest viability(76.20±4.91%),and 0·4 loading ratio was the second(72.96±4.64%).There was no significant difference in other biological characteristics of hADSCs among groups.1.2 Effects of ammonium chloride(NH,Cl)-based erythrocyte lysis isolating method on biological characteristics of hADSCs.Compared with the lysis group,non-lysis group had a higher cell yield and viability in SVF.NH,C1-based erythrocyte lysis induced apoptosis and reduced proliferation of hADSCs.There was no significant difference in other biological characteristics of hADSCs between groups.1.3 Effects of NH4Cl-based erythrocyte lysis on cryopreservation of hADSCs.After cryopreservation,the apoptosis of hADSCs was more likely to occur in the lysis group.NH,Cl-based erythrocyte lysis reduced proliferation of both fresh and cryopreserved hADSCs.2.Water-transport and intracellular ice formation of hADSCs during freezing.The combined-fitting biotransport parameters were Lpg=3.79×10-14 m/s/Pa(0.23 μm/min/atm)and ELp=154.94 kJ/mol(37.01 kcal/mol).After incorporating the biotransport parameters into the Generic Optimal Cooling Rate Equation(GOCRE)model,the optimal cooling rate was calculated to be 4.30℃/min.The average kinetic parameter ΩOSCN was 7.79×108 m-2s-1,and the average thermodynamic parameter kOSCN was 2.93×109K5.3.Studies on freezing process of hADSCs.3.1 Effects of cooling rates on biological characteristics of hADSCs.The post-thawed viability increased with the decreasing of cooling rates from 50℃/min to 0.5℃/min.The viability in the 0.5℃/min group was 93.17 ± 2.29%.The curve of ROS level against the cooling rate was like a inverted U-shape,and the cooling rate corresponding to the peak ROS level was 25℃/min.The 80℃/min group maintained a better chondrogenic ability.3.2 Effects of cell concentrations for cryopreservation of hADSCs.The post-thawed viability of hADSCs improved with the increasing of cell concentrations from 0.5 to 10 ×106/mL.The viability was 91.28±2.57%in the 5×106/mL group and 93.87±1.80%in the 10×106/mL group.There was no significant difference in other biological characteristics of hADSCs among groups.4.Effects of nano-graphene oxide in cryopreservation of hADSCs.In vitro experiment:cryoprotectant with 5 μg/mL GO can improve the viability and recovery rate of post-thaw hADSCs,and reduce the degree of apoptosis.No cytotoxicity was found in 0.01-100μg/mL GO.No obvious GO residue was found in post-thawed cells.In vivo experiment:The number of mature adipocytes and blood vessels were the most in the CPA-GO group.The VEGF level in the CPA-GO group was higher than that in the PBS group.The inflammatory response is much lighter in hADSC-assisted transplantation group than that in the PBS group.Conclusion:1.Studies on the isolation and purification of hADSCs before cryopreservation.0.4 may be the optimal loading volume ratio for hADSCs isolation from lipoaspirate by enzymatic digestion.It is not recommended to implement NH4Cl-based erythrocyte lysis during the isolation of hADSCs.2.Water-transport and intracellular ice formation of hADSCs during freezing.hADSCs demonstrated a relative lower permeability of the cell membrane to water.And intracellular ice formation is easy to appear in relatively high temperature range.The cooling rate should be slow enough to avoid intracellular ice formation.3.Studies on freezing process of hADSCs.The cooling rate of 0.5℃/min achieve better viability and biological characteristics of hADSCs.5X 106/mL-10×106/mL may be the suitable cell concentrations for the cryopreservation of hADSCs.4.Effects of nano-graphene oxide in cryopreservation of hADSCs.Nano-GO plays a protective role in the cryopreservation of hADSCs.