Study Of The Adipose Tissue-derived Stem Cells In Peripheral Neural Recovery

Part 1:Culture and Characterisation of Adipose tissue-derived stem cells of SD ratsObjectives To harvest Adipose tissue-derived stem cells (ADSCs) of SD rats in vitro, and identify the cell.Study the method of culture ADSCs.Methods Adipose tissue was harvested from 4-6-weeks-old SD rat. After euthanasia, the inguinal fat pad was carefully dissected and minced using a sterile razor blade. Tissue was then enzymatically dissociated for 30 min at 37℃using 0.1%(w/v) collagenase typeⅠ.The solution was passed through a 70-μm filter. The undissociated tissue was re-dissociated by 0.1% collagenase typeⅠ. Then the solution was neutralized by the addition of DMEM containing 10%(v/v) foetal bovine serum (FBS) and centrifuged at 1500rpm for 7 min. The cellular pellet was resuspended in DMEM/10% FBS, and transferred to culture flasks. Cultures were maintained at subconfluent levels in a 37℃incubator with 5% CO2. After 24 h, the nonadherent cells were removed by replacing the medium. The cells were performed CCK-8 array and flow cytometry to study the characterisation.Results The cells had typical morphological characters of stem cells, and proliferated quickly which passaged at the third day after culture. They kept a highly growth rate. The cells were positive in the ADSCs'marks CD29, CD44, CD54 and CD105, negative in CD34, CD45 and CD 106.Conclusions The adipose tissue-derived stem cells could easily isolated and cultured by enzymatic method,and had a highly growth rate. Part 2:Study of Adipose tissue-derived stem cells (ADSCs) differentiated into Schwann cellObjectives To explore the possibilities of rat adipose-derived stem cells (ADSCs) to differentiate into Schwann cells followed by a series of chemical inducers in vitro.Methods The passage 3 of ADSCs obtained from SD rats were treated with beta-mercaptoethanol followed by retinoic acid,and a mixture of glial growth factors (forskolin, bFGF,PDGF-AA and heregulin) were added to induce ADSCs' differentiation. Schwann Cell markers, S-100 and GFAP were used to discriminate the induced ADCs'properties by immunofluorescent staining.Results The ADSCs after induced adopted a spindle-like morphology similar to Schwann cells.Immunocytochemical staining indicated that the treated cells expressed the glial markers, S-100 and GFAP, indicative of differentiation.Conclusions These results suggested that ADSCs had the capabilities to differentiate into Schwann cell with a high differentiation rate. Part 3:Biocompatibility Study of Schwann Cells Differentiated From Adipose Tissue-Derived Stem Cells and Nerve ScaffoldObjectives To explore the biocpmpatility of the differentiated ADSCs and poly(lactic acid-co-glycolic acid) (PLGA) in vitro.Methods The biocompatibility study included two part:direct biocompatibility study and indirect biocompatibility study.In the direct biocompatibility study,Schwann cells differentiated Adipose Tissue-Derived Stem Cells and Schwann cells obtained from the nerve of neonatal postnatal day 3 SD rat were co-cultured with different concentration of the extraction of PLGA scafold, in comparison with those of the cells co-cultured with the polyvinyl chloride(PVC) used as the positive poisonal control. At 24 h after cell seeding, the viability of the attached cells on the different substrates were tested by the MTS tetrazolium cytotoxicity assay.Then in the indirect compatibility study the cells were transseeded into PLGA scaffold to culture and the growth curve was tested in comparison with the growth curve of the dADSCs cultured olone.After 7 days,the morphology of the cells seeded on the PLGA scafford were investigated by SEM.Results The MTS tetrazolium cytotoxicity assay shown that the ADSCs and SCs grew well in the various concentration of the extraction of PLGA scaffold without statistical difference,while they both had statistical difference with the poisonal groups respectively. The SEM images shew the cells attached on the film surfaces rather well and had good morphology with a high density.Conclusions These results suggested that ADSCs after differentiated into Schwann cells had good biocompatility with PLGA scafold.