The Isolation, Purification, Cultivation And Identification Of Neonatal Rat Muscle-derived Stem Cells

Objective: Muscle-derived stem cell (MDSCs) transplantation in thetreatment of stress urinary incontinence is attracted widely, some foreignresearchers have obtained a better effect in the clinical application. Thelong-term efficacy is not ideal with the traditional method of treatment of SUIand more complications. With the deepening of stem cell research, the stemcell transplantation has become an important therapy to repair muscle tissuedamage and also brings hopes for the treatment of SUI in which MDSCsreceived extensive attentionis. It is a highly undifferentiated pluripotent stemcells in the muscular tissue which in vitro rapidly proliferate and self-renewal,own multi-differentiation potential and a high survival rate aftertransplantation. It can be a ideal seed cells due to rich sources, easy to operateand high safety. Because of the few amount of MDSCs in muscle tissue, it isfundamental for the transplantion to isolate and cultivate MDSCs in vitro. Inthis study, MDSCs were isolated and purificated by the improved enzymedigestion and preplate technique, identified by the cell immunocytochemicalstaining and watched closely the growth conditions in vitro, in order to obtaina stable isolation and cultivation method and provide the importantexperimental evidences for the transplantation in the treatment of SUI.Methods:1The digestion and isolation of skeletal muscle cells:Clean neonatal Sprayue-Dawley rats was tested which were male orfemale.After soaked in75%alcohol10minutes and stranglrd，appendicularskeletal muscle was isolated and minced into small pieces(about1mm3) bysurgical instruments under the aseptic condition and washed with PBS buffersolution three times.Wiped off the supernate,the pieces were transferred into acentrifugation tube,then about2times volume of the mixed enzyme(2.4u/ml Dispase II、0.5%Collagenase I，2.5mmol/L CaCl2) was added.The tube wasput into water bath about1hour in37℃and theskeletal muscle cells wereisolated with the mixed enzyme. Then they were terminated to digest withgrowth medium. After filtered and centrifuged and wiped off the supernate,the mixture was resuspended and transferred into the flask and cultured at37℃in a humid atmosphere with0.05CO2in air.2The purification and cultivation of MDSCs:The adhering cells were called PP1after cultured2hours. All the cellsthat did not adhere to the flask were transferred into another flask with newgrowth medium and cultured. After24hours, the adhering cells were calledPP2. The non-adhering cells were purified by the preplate technique every24hours until acquired the PP6. PP6was cultured3days and then renewed thenew medium every2to3days.When they grew with70%~80%confluence,the cells were digested with0.25%trypsin and passaged at the ratio of1:2.3The determination of MDSCs growth curveThe MDSCs of the first, fourth and seventh generation were digestedwith0.25%trypsin and then inoculated into24-well culture plates with thedensity of3×10~4/ml.Everyday three wells were selected randomly andrecorded altogether8days, then calculated the average to draw the curve.4The effect of MDSCs growth of the different inoculating density withMTT colorimetric assayMDSCs were adjusted to different density consisted of2,4,6,8,10,12,15×10~5/ml and inoculated into96-well culture plates.After cultured48hours,the wells were added20ul MTT(5mg/ml) and cultured lucifugally4hours.Then100ul DMSO was added in every well and the crystal substance wasdissolved completely by oscillating10minutes.The96-well culture plates wasput into ELISA analyzer and the ODs were obtained in490nm.5The identification of MDSCsThe expression of desmin and stem cell antigen-1(Sca-1) were identifiedwith immunocytochemistry staining in MDSCs(PP6) and the first, fourth andseventh generation of them.The expression of desmin were identified between PP1to PP5,and the fibroblast was regarded to negative control.It wasdetermined positive expression if the brown particles were appeared clearlywithin the membrane andor cytoplasm under the microscope.We would selectrandomly different horizons to calculate the percentage of positive cells in thetotal number of cells.6Statistics processing: The experimental data was processed by theSPSS13.0softwares and analysed with statistics. It was statisticallysignificant when P<0.05was the inspection level.Results:1The digestion, isolation, purification and cell morphology of MDSCsAfter skeketal muscle was digested in the mixed enzymes, it became a mixtureincluding muscle fiber fragments, skeketal muscle cells, blood cells,fibroblasts, endothelial cells, etc. The rapidly adhering cells and slowlyadhering cells could be gradatim isolated by the preplate technique andMDSCs were acquired. PP1and PP2were included mainly fibroblast, themaximum number of cells, rapidly adhering, fast proliferating and grew totalconfluence about5days. PP3and PP4were included mainly myocyte, thenumber of cells reducing, relatively fast proliferating and grew totalconfluence about1week. PP6was adhered observably reduced and most ofcellular morphology were small round or fusiform, smaller size, betterrefraction, slowly adhering and proliferating including of a little suspendingcells. After72hours, the increased adhering cells formed small clone groupsand the fusiform cells grew dispersedly on the bottom of flask. About1weekthe number and density of adhering cells were increasing and formed manycell groups which were liked cell clusters and existed the demarcation linesbetween each cluster. The cell shape had changed the fusiform or polygoncontaining the less cytoplasm and lager nucleus. About10days the increasingcells continued proliferating and grew confluence reciprocally and the gapsbecame narrow, meanwhile the size of the long-fusiform cell cluster enlargedand linked into pieces. 2The growth curve of the MDSCs generationThe growth curve was an S-shape and included the detention period, thelogarithmic growth period and the plateau. Within2days the MDSCs fissionand proliferation were not obvious and the growth rate was slow. After3daysobviously fissioned and proliferated the growth rate was accelerated andentered the logarithmic growth period. But6days later, the cell proliferationwas inhibited, the growth rate became slow and entered the plateau. Thegrowth rate of the first generation cell was obviously faster compared with thefourth and seventh generation.3The effect of MDSCs growth of the different inoculating densityIn a certain range, the OD value was proportional to the number of cells.When the cells were inoculated in the density of1×10~6/ml, the OD value wasmaximum and explained that the number of the living cells were the largest,the cytoactive was relatively well, benefited the cells growth and subculture.4The identification of MDSCsThe expression of desmin and Sca-1was positive in MDSCs withimmunocytochemistry staining and the the membrane and cytoplasm werepainted brown under the microscope, but the fibroblasts were negative. Thepositive rate of desmin was gradually rising from PP1to PP6and both ratesreached90%in PP6. The positive rates reached80%in the first, fourth andseventh passage cells of MDSCs.Conclusion:MDSCs could be obtained by the modified the mixed enzymes digestionand preplate technique from the rebirth SD rat.Within the fourth passage thecellular fission and proliferation were obvious, the cytoactive was relativelywell and the growth rate was fast. In the appropriate inoculating density andculture condition the fission and proliferation would be best and the more cellswere obtained. MDSCs were identified using desmin and Sca-1and theseventh passage maintained a high activity.