乳猪肝细胞的分离、培养及功能检测

Isolation, incubation and function of suckling pig liver cells

目的 为构建生物人工肝进行肝细胞的准备。方法 采用胶原酶半原位灌流法分离单个乳猪肝细胞,并对其活力及单层和聚集培养后的白蛋白、尿素合成功能进行检测。结果 采用本方法从每头乳猪中分离到的单个肝细胞数为(3.1±1.5)×10^(10),活性超过95％。在加入激素和生长因子的培养基中单层培养时,肝细胞功能良好,可维持2周左右。而在未加入激素和生长因子的培养中肝细胞虽能存活1周,但功能于24h后即丧失。球形聚集培养可实现肝细胞的大量培养,且生物学活性较单层培养显著提高。结论 采用胶原酶半原位灌注法所得单个乳猪肝细胞基本能满足构建生物人工肝对肝细胞数量的要求。聚集培养接种密度大,细胞生物学活性高,可用于构建生物人工肝。

Objective To prepare suckling pig liver cells for the bioartificial liver. Methods The semi-in situ perfused technique with collagenase was used to obtain discrete liver cells from the suckling pig liver, and the vitality and synthetic function of albumin and urea of cells growing in monolayer culture and those cells growing in spheroid aggregation were determined. Results We obtained (3. 1 ±.5)×1010 liver cells from each suckling pig, and the cell vitality was more than 95% . Hepatocytes growing in culture medium with hormones and growth factors maintained good function for two weeks. Without hormones or growth factors, hepatocytes survived only one week, and their function vanished in 24 hours. Spheroid aggregation culture fulfilled the massive culture of liver cells. Cells growing in spheroid aggregation possessed higher biological activity than those growing in monolayer culture. Conclusion The quantity of discrete liver cells in our preparation obtained by the semi-in situ perfused technique with collagenase basically meets the requirement for the bioartificial liver. Cells growing in aggregation can be applied in establishing the bioartificial liver because of their higher density inoculation and biological activity.