模拟微重力培养肝细胞的形态特点

Morphological features of simulated microgravity cultured hepatocytes

目的 模拟微重力方法培养大鼠原代肝细胞 ,初步分析其形态学特点及其意义。方法 改良Seglen原位胶原酶灌注法获得大鼠肝脏单细胞悬液 ,2 .2× 10 5个 /ml加微载体Cytodex 3(4 g/L)接种 ,采用旋转细胞培养系统 (RCCS)进行模拟微重力培养。第 0、6、2 4、72、12 0、168小时取样 ,相差、体视显微镜观察活细胞形态 ,第 2 4小时标本苏木素 伊红 (HE)染色观察组织学形态 ,电镜观察超微结构。结果 模拟微重力培养中肝细胞 2 4h内贴附微载体并出现三维结构 ,2 4～ 72h发展为独特的肝细胞 微载体聚球体。电镜下可见细胞膜的 3种不同形态 ,其分布与功能相一致。结论 模拟微重力培养方法能使肝细胞形成分化的三维类组织结构 。

Objective To culture primary rat hepatocytes by means of simulated microgravity,describe their basic morphological characteristics and illustrate the significance primarily.Methods SD rat hepetocytes were separated by the modified Seglen's two step in situ collagenase perfusion method and inoculated at 2.2×10 5 cells/ml with microcarrier Cytodex 3 (4 g/L) as the solidial support materials.Simulated microgravity environment was created by Rotary Cell Culture System (RCCS Synthecon.com USA).The samples were collected on 0,6,24,72,120 and 168 h and morphological studies were made by means of phase contrast microscope,hematoxylin and eosin (HE) dyed paraffin sections and electron microscope (TEM).Results In the RCCS,hepatocytes started to grow on the surface of microcarrier on the 1st day and then form three dimensiongal polylayer structure during 24 to 72 h.TEM revealed three different kinds of membranes among simulated microgravity cultured cells,whose distribution were in accordance with their functions.Conclusion We successfully cultured primary rat hepatocytes in simulated microgravity condition.Initial morphological study shows that this new culture technology can establish a three dimensional structure in vivo,suggesting this method may be potentially valuable in areas of tissue engineering.