Culturing Human Mesenchymal Stem Cells On Microcarriers And Inducively Culturing Human Mesenchymal Stem Cells In Vitro In Large Scale On Microcarriers

Objective: To establish a method to culture human bone-marrow mesenchymal stem cells (bMSCs) and to establish a method to induce bMSCs into osteoblast on microcarriers in vitro in large scale in order to meet the need for the cell quantities and quality in the treatment of bone deficiency by means of tissue-engineeing technology. Methods: 1. Culturing bMSCs on microcarriers: Human bone marrow samples was aspirated from iliac crest of normal adult volunteer ranging in age from 20 to 50 yeas old . The mesenchymal stem cells were isolated by density gradient centrifugation and inoculated in ordinary tissue culture flask in supplemented with 15% Fetal bovine serum(FBS), and were digested by trypsin when conflunced approximately 80%. The third generation of bMSCs cultured and amplied in ordinary tissue culture flask were divided into two portion, one of which amount to 1×107 was cultured in stiring bioreactor filled with 100ml culture medium in combination with 0.5g Cytodex3 microcarriers, stiring 2 minutes at the speed of 40r/min every half hour in the first 6 hours and subsequently duratively stiring at the same speed. The other portion was cultured all the same by ordinary tissue culture flask. Examples was taked respectively to monitored the growth condition of the bMHCs on the microcarriers by the phase-contrast photomicrography at the 1th, 3th, 5th, 7th, 9th, 11th day respectively, and cell growth curves were drawed. The cell proliferation rate of the two methods were compared with each orther. 2. Inducively culturing bMSCs into osteoblast on microcarriers: The third generation of bMSCs cultured and amplied in ordinary tissue culture flask were divided into two portion, one of which amount to 1×107 was cultured in stiring bioreactor filled with 100ml Dulbecco,s modified Eagle,s medium (DMEM) supplemented with 15% Fetal bovine serum and osteogenesis supplements (OS) including 10-8M/L dexamethasone, 10mM/Lβ-glycerphosphate+50mM/L ascorbic acid in combination with 0.5g Cytodex3 microcarriers, stiring 2 minutes at the speed of 40r/min every half hour in the first 6 hours and subsequently duratively stiring at the same speed. The other portion was cultured all the same by ordinary tissue culture flask in DMEM supplemented with 15% Fetal bovine serum and osteogenesis supplements (OS) including 10-8M/L dexamethasone, 10mM/Lβ-glycerphosphate+50mM/L ascorbic acid. Examples were taken at day 2, 4, 6, 8, 10, 12, 14 respectively to measure the Alkaline phosphatase(ALP) activity of the cultivated cells, which was compared with that of common flask culture. The growth condition of the bMHCs was monitored, and the cell growth curves were drawed. The cell proliferation rate of the two methods were compared with each orther. In the end the expression of ALP, collagenⅠand osteocalcin of the induced cells were studied in order to investigate the phenotype expression. Results: 1. Culturing bMSCs on microcarriers: 24 hours after inoculation approximately 87.1% the hMSCs attached to and spread on the microcarrieres. 3 days after inoculation the cell growth speeded up, and 9 days the cell density reached a peak. The final cell density was 15-20 times of that of inoculation. The cell proliferation rate of microcarrier method was approximately 3 times of that of the ordinary method, and there was a very significant difference between these two methods(P<0.01). 2. Inducive culturing bMSCs into osteoblast on microcarriers: 24 hours after inoculation approximately 85.9% the hMSCs attached to and spreaded on the microcarrieres. 3 days after inoculation the cell growth speeded up, and approximately 9 days the cell density reached a peak. The final cell density was 15~20 times of that of inoculation. The cell proliferation rate of microcarrier method was approximately 3 times of that of the ordinary method, and there was a very significant difference between these two methods (P<0.01). The ALP activity of the induced cells reached a peak value at day 12, and there was no significant difference between these two methods (P...