| BackgroundCurrently,cryopreservation is the only method for long-term storage of adipose-derived stem cells(ASCs).Adipose-derived stem cells with good performance after thawing and recovery are essential for the establishment of ASCs banks and clinical applications.Both clinical and basic research applications require that the quality of the stored cells provided is optimized,stable and homogeneous.However,the cell sources of ASCs banks lack uniform standards,and the cell quality is unstable and the biological activity varies greatly.To obtain frozen ASCs with optimal quality,stable and uniform biological properties,it is necessary to control the relevant factors in terms of cell source.The key factors contain donor age,site and liposuction method,which affecting the performance of adipose-derived stem cells.Due to the lack of systematic optimization studies and data for the cell sources of cryopreserved ASCs,it is not yet possible to control and optimize the above factors.Therefore,there is a need to clarify the effects of donor factors and collection methods on the biological properties of adipose-derived stem cells after cryopreservation.To understand the differences in biological characteristics of cryopreserved ASCs obtained from different ages,harvest sites and collection methods,it aims to provide basic data for optimizing adipose-derived stem cell collection indexes,and establishing standards for human ASCs banking.ObjectivesThe aim of this study is to clarify the effect of age on biological properties of cryopreserved adipose-derived stem cells;to clarify the differences in biological properties of cryopreserved adipose-derived stem cells in different donor sites;to compare the effect of Water-jet assisted liposuction and conventional liposuction on biological properties of cryopreserved adipose-derived stem cells;to explore the effect of cryopreserved ASCs from different age,donor sites,and liposuction techniques on cell-assisted lipotransfer.MethodsPart ?:Comparison of biological properties of cryopreserved ASCs of different ages.1.Biological properties of cryopreserved ASCs were examined:adipose tissue obtained from abdomen in healthy women.Samples were divided into three groups according to donor age:group A,18-29 years old;group B,30-49 years old;group C,50-65 years old(n=20).Stromal vascular fraction was isolated from adipose tissue by collagenase.The yield and viability of SVF in each group were detected by Muse cell count and viability assay;ASCs were obtained by in vitro culture at the second passage,and cryopreserved for 4 weeks.The expression level of surface markers of cryopreserved ASCs was detected by flow cytometry,proliferation ability was detected by CCK-8 assay,and cell migration ability was detected by scratch test.The adipogenic differentiation potential of ASCs was analyzed by induction of lipid formation in vitro.The expression levels of PPAR-y and CEBP-? genes were detected by RT-PCR assay.2.Cryopreserved ASCs-assisted fat graft animal:BALB/c nude mice were randomly divided into 4 groups;1 × 106 cryopreserved ASCs from 3 different age groups were mixed with 0.3 ml of fat tissue and injected subcutaneously on both sides of the back spine of nude mice;control group:10?l PBS was mixed with 0.3 ml of fat tissue.The weight and volume of fat grafts were analyzed among the groups,HE staining was performed to assess the integrity of adipocytes and the proportion of necrotic tissue,Immunofluorescence staining of Perilipin was performed to analyze the survival of adipocytes within the grafts,and CD31 immunohistochemical staining was performed to evaluate the density of neovascularization after fat grafting.Part ?:Comparison of biological properties of cryopreserved ASCs from different donor sites.1.Detection of biological properties of cryopreserved ASCs:20 ml of aspirated adipose tissue harvested from four sites of healthy volunteers:upper limbs,abdomen,waist and thighs,respectively.SVF was separated by collagenase.The yield and viability of SVF in adipose tissue of each site were detected by Muse cell count and viability assay.The percentage of adipose-derived stem cells in SVF of each site was detected by flow cytometry.ASCs were cultured in vitro and cryopreserved for 4 weeks.The biological properties of ASCs from the four donor sites were compared after cryopreservation:MSC surface marker expression;proliferation ability by CCK-8 assay and cell migration ability by scratch test;in vitro adipogenesis,osteogenesis and chondrogenesis induction to assess the differentiation potential of cryopreserved ASCs from each site.The expression levels of lipogenesis-related genes(PPAR-y and CEBP-?),osteogenesis-related genes(ALP and OPN),and chondrogenesis-related genes(COL2A1 and COMP),were measured by RT-PCR.2.Cryopreserved ASCs-assisted lipotransfer models:fat grafts were co-transplanted with frozen ASCs from 4 different donor sites and injected subcutaneously into nude mice,4 experimental groups and control group(n=10),harvested 3 months after transplantation.Compare the weight and residual volume of fat grafts in each group,HE staining and histological evaluation,analyzing the integrity of adipocytes in each group,cysts/vacuoles,inflammatory tissue,and fibrosis;CD31 immunohistochemical staining was performed to observe the neovascularization of fat grafts in each group.Part ?:Comparison of biological properties of Cryopreserved ASCs obtained by the water-jet assisted liposuction and conventional liposuction.1.Analysis of biological properties of cryopreserved ASCs:50ml of lipoaspirates was harvested randomly by water-jet assisted liposuction technique and conventional liposuction,respectively.The yield and viability of SVF in adipose tissue of each site were detected by Muse cell count and viability assay.Adipose-derived stem cells were expanded and cultured in vitro,and then cryopreserved for 4 weeks.The MSC surface markers of cryopreserved ASCs were detected by flow cytometry,the morphology of each group was observed by live/dead cell staining.The proliferation ability was detected by CCK-8 assay,and the migration ability was detected by scratch test.The adipogenic differentiation potential of ASCs was analyzed by induction of lipid formation in vitro.The expression levels of PPAR-y and CEBP-a genes were detected by RT-PCR assay.2.Cryopreserved ASCs-assisted lipotransfer models:fat grafts were co-transplanted with cryopreserved ASCs from two liposuction techniques on the back of nude mice,respectively,two experimental groups and the control group(n=10).The grafts were collected 3 months after transplantation,and the weights and volumes of the grafts in the3 groups were measured;paraffin sections were stained by HE,Perilipin and Masson to evaluate the distribution of adipose tissue and fibrosis of grafts in each group,and CD31 immunohistochemical staining was used to observe the neovascularization of the grafts in each group.Western blot was applied to detect the expression level of Annexin V protein to compare the apoptosis in each group.ResultsPart ?.1.There was no significant difference in the SVF yield among the age groups,and the cell viability of SVF showed a decreasing trend with age.There was no difference in the levels of positive MSC surface markers of cryopreserved ASCs of each group.The proliferative capacity and migratory capacity of ASCs cells gradually decreased with age.The adipogenesis differentiation in vitro showed no difference in the absorbance values of oil red O staining among the age groups,and no significant difference in the expression levels of adipogenesis-related genes PPAR-? and CEBP-?.2.After three months,the survival rate of fat grafts in nude mice gradually decreased with age when comparing the graft weight and remaining volume of each group.Immunofluorescence staining showed that more Perilipin-positive adipocytes were observed in the ASCs-assisted fat grafting in each age group,with uniform distribution.The density of neovascularization was similar in all age groups,and the differences were not statistically significant.Part ?.1.Adipose tissue isolated from the thigh had the highest cell yield and viability of SVF.The percentage of adipose-derived stem cells was highest in the thigh,followed by the abdomen;Cryopreserved ASCs from the thigh had the highest recovery rate.Donor site did not affect the cell phenotype and morphology of cryopreserved ASCs.Cryopreserved ASCs from the thigh site had a higher cell proliferation and migration capacity,followed by the abdomen.The adipogenesis differentiation potential in vitro of cryopreserved ASCs was similar in the upper limbs,abdomen,waist and thighs.Cryopreserved ASCs in the abdomen and thighs had better in vitro osteogenic differentiation potential;in vitro chondrogenic differentiation potential of cryopreserved ASCs in the thighs was better than other sites.2.The survival rate of fat grafts was higher in the abdomen and thigh groups than in the upper limb and waist groups;the fat integrity was significantly higher in the abdomen and thigh groups than in the upper limb and waist groups;the degree of cysts/vacuoles,inflammatory infiltration,and fibrosis were significantly lower in the abdomen and thigh groups;and there was no statistical difference in neovascularization among the co-transplantation groups by CD31 immunohistochemical staining of fat grafts.Part ?.1.The yield and cell viability of SVF isolated from adipose tissue harvested by the water-jet assisted liposuction were greater than that of the conventional liposuction.There were no significant differences in cell morphology and MSC marker expression levels between the two groups of cryopreserved ASCs.The cell proliferation and migration abilities of frozen ASCs in the water-jet assisted group were higher than those in the conventional group;the cryopreserved ASCs in the water-jet assisted group had better in vitro adipogenesis differentiation potential.2.The survival rate of fat grafts in the water-jet assisted group was higher than that in the conventional group.The integrity of adipocytes in the water-jet assisted group was higher than that in the conventional group,and the proportion of fibrosis in the water-jet assisted group was lower than that in the conventional group,and the differences were statistically significant;the density of blood vessels in the grafts in the water-jet assisted group was significantly higher than that in the conventional group;the level of apoptosis of fat grafts in the water-jet assisted group was lower than that in the conventional group.Conclusions1.The cell proliferation and migration ability of human cryopreserved ASCs showed a decreasing trend with age,but the age factor did not affect the adipogenesis differentiation potential.Cryopreserved ASCs of different ages effectively improved the survival rate of fat grafts in nude mice,and the effect of cryopreserved ASCs-assisted fat transplantation in the young population was better than that in the elderly.2.Cryopreserved ASCs from thighs and abdomen had more significant cell proliferation,migration ability and multipotential differentiation ability compared to upper limbs and waist,and adipose-derived stem cells from thighs and abdomen maintained better biological properties after cryopreservation.cryopreserved ASCs from the thigh and abdomen rich in fat grafts resulted in better survival.3.Cryopreserved ASCs obtained by water-jet assisted liposuction technique have better biological properties.Their cell proliferation and migration ability,in vitro adipogenesis differentiation potential,and the survival rate of fat transplantation were better than those of the conventional group. |
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