分离肝细胞纯化培养方法的比较研究

目的 评价一种可提高肝细胞纯度和存活率的分离培养方法。方法 以体外两步胶原酶灌流法分离肝细胞,然后将其分成两组,对照组在接种培养前不经进一步处理,试验组则在应用适宜的Percoll梯度液离心纯化之后再行培养。藉台盼蓝拒染法比较两组肝细胞的存活率,采用MTT法动态比较两组肝细胞的增殖状态,在相差显微镜下观察细胞的纯度和形态。结果 未经进一步纯化处理的猪肝细胞存活率为90％±5％,鼠肝细胞存活率为80％±5％,两者纯度均约90％;经Percoll梯度液离心纯化后,其高活力肝细胞比率均提高至98％±2％,纯度可达99％以上。从开始接种到大部分肝细胞贴壁生长,试验组比对照组肝细胞的时间有所缩短。结论 用Percoll梯度液纯化新分离肝细胞,可提高肝实质细胞的活力与纯度。

Comparison of the methods for isolation and purification of hepatocytes

Objective To evaluate the method of obtaining vigorous and purified hepatocytes. Methods Hepatocytes were harvested by two - step perfusion with collagenase using an extracorporeal perfusion apparatus. Working Percoll solution was prepared. Hepatocytes were purified by adding cell suspension into working percoll solution. Cells was collected by centrifugation, then the hepatocytes were resuspended and washed for three times. The cellular morphologies were examined during culture periods. Viability of cells was determined using standard trypan blue exclusion, and proliferation of hepatocytes was tested by MIT assay. Results Before the isolated cells were purified, pig hepatocytes viability was 90%+ 5% , and rat hepatocytes viability was 80% + 5% . After purification, both viabilities were 98% + 2% , and non - parenchyma cells was hardly seen under phase contract microscope. The time from inoculation to attachment was shortened in purified hepatocytes. Conclusions The viability and purification of hepatocytes could be enhanced by Percoll grade centrifugation.