鼠胚成纤维细胞的培养条件及滋养层制备

Culture conditions of mouse embryonic fibroblasts and preparation of feeder layer

背景：鼠胚成纤维细胞经丝裂霉素C处理或γ射线照射阻断其有丝分裂后，铺层的细胞可保持活力但不增殖，并能够在生长过程中产生促进胚胎干细胞生长的因子和抑制胚胎干细胞分化的因子，但其生命期有限。目的：探讨分离小鼠胚胎成纤维细胞的最适胚龄与培养条件，以及用其制备细胞饲养层的效果。设计、时间及地点：细胞学体外观察，于2005-11／2006--07在广东省计划生育专科医院实验室完成。材料：清洁级昆明系孕7．5，10．5，13．5，16．5，19．5d雌鼠各5只。方法：无菌取上述各孕龄小鼠胚胎分离鼠胚成纤维细胞，调整细胞浓度为1×10^7L^-1、1×1097L^-1、1×10^11L^-1，胰酶消化传代。当胚胎成纤维细胞生长并相互接触时，加入丝裂霉素C作用2-4h，用无钙无镁PBS配制的胰蛋白酶液消化2-5min，终止后用吸管吹打皿底，使细胞充分分离，以含10％胎牛血清的DMEM液调整细胞浓度为1×10^8L^-1，将该悬液移入明胶处理的培养皿内常规培养，制备鼠胚成纤维细胞饲养层。主要观察指标：胎龄、细胞浓度、传代次数对鼠胚成纤维细胞生长增殖的影响。不同胎龄鼠胚成纤维细胞饲养层制备效果。结果：7．5-19．5d鼠胚均可分离出成纤维细胞，但7．5d，10．5d鼠胚分离所得成纤维细胞数量少，寿命短，且混有大量杂细胞；13．5d鼠胚分离所得成纤维细胞数量多，增殖快，所含杂细胞少：16．5～19．5d鼠胚分离所得成纤维细胞生长状态不良，增殖缓慢，杂细胞较多。同等条件下与1×10^7L^-1、1×10^11L^-1浓度比较，1×10^9L^-1浓度的鼠胚成纤维细胞生长状况良好，铺层时间适中，细胞寿命最长（P〈0.05）。以13．5d鼠胚成纤维细胞经初代培养后传4代，成功建立了成纤维细胞株，相同条件下14代细胞形态、体积、生长状况无明显差别。7．5～19．5d鼠胚成纤维细胞制备的饲养层，铺层时间基本相似（P〉0．05），细胞寿命均可维持一二周。结论：分离小鼠胚胎原代成纤维细胞的最适胎龄及细胞浓度分别为13．5d与1×10^9L^-1，4代以内传代次数不影响鼠胚成纤维细胞的生长增殖，且不同胎龄所制备的饲养层细胞寿命无差别。

BACKGROUND： Mitochysis of mouse embryonic fibroblasts is inhibited by mitomycin C treatment or γ irradiation. Cells keep the activity, but dose not proliferate, which can produce factors enhancing the growth of embryonic stem cells and inhibiting the differentiation of embryonic stem cells, but the life span is limited. OBJECTIVE： To investigate the isolation proper age of mouse embryonic fibroblasts and the preparation of embryonic fibroblast feeder layer. DESIGN, TIME AND SETTING： The cytology in vitro experiment was performed at the Laboratory of Guangdong Family Planning Special Hospital from November 2005 to July 2006. MATERIALS： Clean pregnant Kunming rats （7.5, 10.5, 13.5, 16.5, 19.5 days of pregnancy） （five each） were used in this study. METHODS： Mouse primary embryonic fibroblasts were sterilely prepared from mouse fetus of different embryonic ages. Cells at densities of 1×10^7L^-1, 1×10^9L^-1,1×10^11L^-1 were digested in trypsin. When embryonic fibroblasts grew and contacted each other, and then treated with mitomycin C for 2-4 hours, trypsin solution supplemented with Ca2＋-free and Mg2＋-free phosphate buffered saline for 2-5 minutes. Cells were blown and hit with a pipette to isolate cells. Subsequently, cells were inoculated in DMEM containing 10% fetal bovine serum into a density of 5 ×10^8L^-1. This suspension was inoculated in gelatin-treated culture dish to prepare fibroblast feeder layer. MAIN OUTCOME MEASURES： Effects of embryonic age, cell concentration and passage times on the proliferation of mouse embryonic fibroblasts. Effects of feeder layer preparation of mouse embryonic fibroblasts at different embryonic ages. RESULTS： Fibroblasts were harvested from pregnant mice at embryonic days 7.5-19.5. Number of fibroblasts were few and with short life span in mice at embryonic day 7.5 and day 10.5, and with many other types of cells. Number of fibroblasts was more and with rapid proliferation in mice at embryonic day 13.5, with few other types of cells. Fibroblasts grew poorly, and proliferated slowly, with many other types of cells in mice at embryonic days 16.5-19.5. Under the same condition, fibroblasts at a density of 1×10^9L^-1 grew well, with suitable monolayer confluence and long life span in mice （P 〈 0.05）. Mouse embryonic fibroblasts at embryonic day 13.5 were cultured at fourth passage. Fibroblast strain was successfully established. Under the same condition, the morphology, volume and growth condition of 1st-4th passages of cells had no significant difference. The feeder layer of mouse embryonic fibroblasts at embryonic day 7.5-19.5 had similar monolayer confluence （P 〉 0.05）, and the cell life span could maintain for 1-2 weeks. CONCLUSION： The optimal embryonic age and cell concentration are respectively 13.5 days and ×10^9L^-1 in mouse embryonic fibroblasts. Passage time within the fourth passage cannot affect the proliferation of mouse embryonic fibroblasts. No significant difference is detected in cells in feeder layer at different embryonic days.