Preliminary Studies On Culture And Proliferation Of Macaca Fascicularis Ear Fibroblast Cells In Vitro

Macaca fascicularis was selected as experiment material in this study.By the means of tissue block culture,we aimed to establish an isolation method and culture system for Macaca fascicularis ear fibroblast cells in vitro.The biological characteristics of the ear fibroblast cells were also studied.In addition,the effects of supplementation of different concentration of EGF, insulin,IGF-I for cell proliferation of Macaca fascicularis ear fibroblast cells in the 5%and 10%FBS concentration were also evaluated by colorimetric MTT assay.The results showed that:1,Primary culture of Macaca fascicularis ear fibroblast cells could be obtained through tissue block culture successfully.2,Using 0.25%trypsin+0.02%EDTA as digested medium and DMEM containing 10%FBS as culture medium for subculture of medium,and the cells were passaged to the 25^thpassage.The pured fibroblast cells could be obtained by passed 3～5 subcultures.The cultured cells were typical fibroblast morpha, which grooved as irregular triangle or shuttle,appearing as radioactive blaze and helix in shape. 3,The fibroblast cells were frozen in DMEM containing 10%FBS and 10%DMSO as freezing medium with the manual methods,and the cell freezing denisity was approximately 5×10⁶ cell/ml.The cells survival rate of the cells was observed by trypan blue staining method.The results showed that the cells might be affected by many experiment factors during cryopreservation,but there was more than 90%of cells survived after frozen and thawed.The cells before and after freezing had not showed obvious changes in cell morphology and growth speed.4,The growth curve appeared in "S" shape.The cells understood latent phase,logarithmic growth phase and stagnate phase.The result accorded with the cells growth regularity in vitro.5,The number of chromosome is 2n=42 in Macaca fascicularis on the 10^th,15^th,20^thand 25^thpassages cells.Results of chromosome ploidy analysis indicated that cells are diploid,and there had no significant difference on the ratio of diploid cells among these 4 passages(P＞0.05).6,when the serum concentration was 5%in DMEM,the addition of EGF (20～80 ng/ml),insulin(1～5μg/ml)and IGF-I(50～100 ng/ml)had significant promoting effects on the growth of the cells respectively(P＜0.05).7,when the serum concentration was 10%in DMEM,the addition of EGF (10～40 ng/ml),insulin(10～15μg/ml)and IGF-I(25～100 ng/ml)had significant promoting effects on the growth of the cells respectively(P＜0.05).