【摘要】 为建立番鸭胚成纤维细胞(MDEF)的体外培养、传代和冻存技术,选用11胚龄番鸭胚,2.5g/L胰蛋白酶消化,原代MDEF于含100 mL/L血清的DMED培养液中培养;研究热、冷两种消化方法对MDEF消化密度及活率的影响,探讨不同血清浓度对MDEF生长、冻存活性的影响,以及离心对MDEF传代和复苏时细胞活率的影响。结果显示,胰酶消化法获得了MDEF,细胞贴壁紧密,生长良好;冷消化得到的原代MDEF密度和活率均高于热消化;血清浓度在20mL/L~100mL/L范围内,浓度越高MDEF生长速度越快;血清浓度显著影响MDEF复苏的活率;MDEF冻存液中DMSO∶血清∶培养液比例为1∶2∶7冻存效果良好;在MDEF传代和复苏过程中去除离心操作,不仅可简化操作,细胞活率也有一定提高。本研究建立了MDEF的原代分离和培养方法,为适用于番鸭相关研究的体外细胞培养体系奠定基础。更多还原

【Abstract】 In order to establish the primary culture,subculture and cryopreservation techniques of muscovy duck embryo fibroblasts(MDEF),11day-age muscovy duck embryoes were selected,2.5g/L trypsin digestion of the embryoes and primary MDEF were cultured in DMEM containing 100 mL/L serum.The effects of hot and cold digestion methods on MDEF density and viability were studied.The effects of different serum concentrations on MDEF growth and cryopreserved cell viability,and the effects of centrifugation on MDEF viability in subculture and resuscitation were investigated.The results showed that this methods could be used for isolation and pure culture of MDEFin vitro.The density and viability of MDEF by cold digestion were higher than the hot digestion.The cells grew faster with the higher serum concentration range of 20mL/L-100mL/L.Serum concentrations significantly affected MDEF recovery viability;DMSO∶serum∶medium ratio of 1∶2∶7was good for MDEF frozen liquid.It may simplify operations and increase a certain cell viability if the centrifugation in MDEF subculture and recovery process was removed.This study established primary isolation and culture methods of MDEF,and laid the foundation for in vitro cell culture system suitable for study of MDEF.更多还原