Culture of Rat Retinal Ganglion Cells  

作　　者：徐志蓉 姜发纲 曾艳彩 Hamed TM Alkhodari 陈飞 

机构地区：[1]Department of Ophthalmology,Union Hospital,Tonggi Medical College,Huazhong University of Science and Technology [2]Department of Information,Wuhan General Hospital,Guangzhou Military Area Command

出　　处：《Journal of Huazhong University of Science and Technology(Medical Sciences)》2011年第3期400-403,共4页华中科技大学学报（医学英德文版）

基　　金：supported by a grant from National Natural Sciences Foundation of China(No.30700922)

摘　　要：This study aimed to modify the mixed and purified culture of rat retinal ganglion cells（RGCs） in vitro.The retinae of 1-3 day old Sprague-Dawley（SD） rats were separated bluntly into two layers：inner layer and outer layer,under a surgical microscope.Retinal cells isolated from different layers（inner layer,outer layer and whole retinal tissue） by using enzyme dissociation method were cultured in F12/DMEM medium containing 15% FBS.After 3-day culture,the RGCs in the retinal cells obtained from mixed culture of inner,outer,and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1,and the rate of RGCs to retinal cells（RGCs%） was calculated.Two monoclonal antibodies,anti-macrophages/granulocytes（OX-41） against rat macrophage and antibody against rat Thy-1.1（OX-7）,were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure（purification in situ）.Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15% FBS.Immunocytochemical staining for Thy-1.1,MTT,and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs.The results showed：（1） Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs% was（19.9±1.2）%,（0.5±0.2）%,and（6.2±1.7）% respectively in the mixed culture of inner,outer,and whole retinal tissue,with differences being significant（P0.05）;（2） fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs%,RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method;（3） the viability of purified RGCs seeded at high density was in-creased and the cells developed complex intercellular networks.The viability of RGCs was declined with the decreasing seeding density,and most cells presented round or oval in shape with thin neurites.It was concluded that：（1） RThis study aimed to modify the mixed and purified culture of rat retinal ganglion cells（RGCs） in vitro.The retinae of 1-3 day old Sprague-Dawley（SD） rats were separated bluntly into two layers：inner layer and outer layer,under a surgical microscope.Retinal cells isolated from different layers（inner layer,outer layer and whole retinal tissue） by using enzyme dissociation method were cultured in F12/DMEM medium containing 15% FBS.After 3-day culture,the RGCs in the retinal cells obtained from mixed culture of inner,outer,and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1,and the rate of RGCs to retinal cells（RGCs%） was calculated.Two monoclonal antibodies,anti-macrophages/granulocytes（OX-41） against rat macrophage and antibody against rat Thy-1.1（OX-7）,were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure（purification in situ）.Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15% FBS.Immunocytochemical staining for Thy-1.1,MTT,and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs.The results showed：（1） Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs% was（19.9±1.2）%,（0.5±0.2）%,and（6.2±1.7）% respectively in the mixed culture of inner,outer,and whole retinal tissue,with differences being significant（P0.05）;（2） fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs%,RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method;（3） the viability of purified RGCs seeded at high density was in-creased and the cells developed complex intercellular networks.The viability of RGCs was declined with the decreasing seeding density,and most cells presented round or oval in shape with thin neurites.It was concluded that：（1） R

关 键 词：retinal ganglion cells microscopic separation in situ purification seeding density culture models 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]