Isolation, Cultivation And Identification Of Human Bone Marrow Mesenchymal Stem Cells

Objective:Establishment of the method for the isolation, cultivation and multiplication of human bone marrow mesenchymal stem cells (MSCs) and observation of the biological characteristics of cultivation and proliferation in vitro. Flow cytometry was used to detect the cells phenotype and identify the purity of acquired bone marrow MSCs.Methods:(1) Bone marrow samples, which were aspirated5mL in the sterile condition, were mixed sufficiently with isovolumetric PBS and made into single cell suspension. Discard the fat deposit after centrifugating at a rate of1500rpm for10min. Then the single cell suspension of bone marrow was slowly added to the Ficoll separation solution with an appropriate amount of1.077g/mL and centrifuged at a rate of2500rpm for30min. Followed by collecting the mononuclear cells in the white haze of boundary layer. After that, the collected cells were diluted and washed with PBS by centrifugating at a rate of1500rpm for10min. The mononuclear cells were subsequently resuspended with LG-DMEM medium containing10%fetal bovine serum (FBS) and seeded in a25cm2flask with a density of2×105/mL. Finally the well-prepared cells were cultured in an incubator containing5%CO2at37℃. The culture medium was first replaced after3d and discarded the non-adherent cells, then the culture medium was replaced every two to three days and the cell proliferation and morphological characteristics was observed every day under the inverted microscope. When the percentage of confluence was about80～90%, the adherent cells were passaged three times in the ratio of1:2after digesting by0.25%Trypsin-0.02%EDTA (1:1, V/V).(2) The measurement of cells growth curve:The third generation of bone marrow MSCs were seeded and cultured seven days in a24holes nunclone with a density of5×104/mL. Take three holes every day to count the cells amount and get the mean value, which was used to make the cells growth curve. (3) The measurement of cells adherence rate:The third generation of bone marrow MSCs were seeded and cultured in a25cm2flask with a density of5×104/mL, then digested and count the cells amount2flasks per2h, which was used to make the cells adherence rate curve.(4)The measurement of cells phenotype:The third generation of bone marrow MSCs was digested and blow-beat to prepare single cell suspension, subsequently, the cells were centrifugated at the rate of1500rpm for5min and the supernatant was discarded. The concentration of collected cells was adjusted to5×106/mL after washing repeatedly with PBS, which was subsequently divided into several groups with each volumn of100μL and respectively added fluorescent maker antibody (i.e., anti-CD34-PE, CD44-FITC, CD29-PEcy5.5, CD105-FITC, HLA-DR-PEantibodies) and incubated in the dark at room temperature for30min. Then detected with flow cytometry after washing with PBS to check the expression of surface marker CD34, CD44, CD29, CD105and HLA-DR.Results:(1) MSCs belong to the mononuclear cells in the bone marrow, isolation and purification can be achieved by combining density gradient centrifugation with adherence cultivation. Observation of the cell morphology:round mononuclear, disuniform in size and confounding with other blood cells can be observed when initial seeded and cultivated in flask. The adherence can be observed after cultivated24h and the aggravated after48～72h. Few dispersive fusiformis and irregular polygon adherented cells emerged when renewed the culture medium firstly after culturing for72h. The growrate was fast when cultured for4-8d and appeared colony-like growth with fusiformis and protuberance morphology. Cell clone was further expanded when cultivated for10～12d and appeared big long fusiformis. The confluence percentage of adherented cells reached to80%～90%when cultivated for14～16d. A lot of cells appeared long fusiformis fiber cells-like morphology and circuiate arrangement. Passaged cells appeared big round morphology when initially seeded and fully adherented just in24h with a fusiformis morphology and fast grow rate, which can reached a confluence percentage of90%with a uniform fusiformis morphology and regular arrangement.(2) Analysis of MSCs growth curve:MSCs growth curve appeared S-shaped. The primary cells grew faster than passaged cells. The0～3rd day was growth incubation period for the primary cells seeded; the4th～12th day was logarithmic phase and then slowed down to a plateau period. The1st～2nd day was growth incubation period for passaged cells; the growth was faster from the3rd day; the6th-7th day was the vertex and plateau period appeared later.(3) Measurement of adherence rate:The adherence was rapidly for passaged cells and reached to50%after4h, and then the adherented cells over90%after12h.(4) Detection of P3cells by flow cytometry:The MSCs positive expression rates of CD29, CD44, CD105was99.82%,99.82%,97.38%respectively and the positive expression rates of CD34, HLA-DR was individually4.94%,1.87%according to flow cytometry data.Conclution:(1) MSCs with high purity and activity can be acquired from human bone marrow by combining density gradient centrifugation with aderence culture. So it is an effective and simple method for isolation and cultivation of MSCs.(2) MSCs can be preliminaryly identified according to the cells morphology、growth characteristics and cells phenotype expression.