Development of artificial liver using macroporous support

1.为了建立哺乳动物细胞的三维培养，利用一些大孔细胞支持物，如连接有肝素或细胞粘附因子的支持物和带正或负电荷的基团，进行贴壁依赖性或非贴壁依赖性细胞培养。在使用纤维素大孔载体的培养中，获得了相对高的细胞密度。研究发现，在带正电基团的纤维素载体上，细胞密度和生化产物产量最高.为构建临床应用的人工肝生物反应器，需要建立具有肝细胞特异性功能的大鼠肝细胞的长期培养体系。为此，研究了添加剂对白蛋白分泌速率（作为肝细胞特异性功能）的影响。在大鼠肝细胞培养皿中检测白蛋白分泌率作为肝细胞的特异性功能。虽然在培养过程中，包括白蛋白分泌率在内的肝细胞功能逐渐下降，但发现通过添加抗氧化剂，如氨基酸中的脯氨酸和组氨酸，酶中的过氧化氢酶和超氧化物歧化酶，抗坏血酸磷酸酯和转铁蛋白，可以获得2至3倍的高分泌率.研究了葡萄糖浓度对肝细胞特异性功能的影响。在培养皿培养物中以0.1至3.0 mM的可变水平添加古洛糖。结果表明，早期分泌速率随葡萄糖浓度的降低而增加。当通过改变培养基在不同培养天数将葡萄糖浓度从低水平（0.1mM）转移到高水平（10mM）时，也获得了高分泌率。结果发现，当4天后更换培养基时，即使在14天后仍保持2倍的高速率。这一结果表明，葡萄糖水平控制培养成为肝细胞长期培养的有效策略之一。

1. To establish a three dimensional culture of mammalian cell, anchorage dependent or independent cell culture was carried out by using some macroporous cell supports, such as a support linked with heparin or cell adhesion factor and with positive or negative charged groups. In the culture using a cellulose macroporous support, relatively high cell density was obtained. It was found that the highest value of the maximum cell density and the production of biochemicals was obtained in the cellulose support with positive charged group.2. To construct a bioreactor packed with a artificial liver for clinical use, long term cultivation of rat hepatocyte with hepatocyte specific function should be established. For this purpose, the effect of additives on albumin secretion rate as a hepatocyte specific function was studied. The albumin secretion rate as a specific hepatocyte function was checked in dish culture of rat hepatocyte. Although the hepatocyte function including an albumin secretion rate decrease gradually during cultivation, it was found that 2 to 3 times higher rate was obtained by addition of anti-oxidative agent such as proline and histidine among amino acids, catalase and superoxide dismutase among enzymes, ascorbic phosphate ester and transferrine.3. The effect of glucose concentration on hepatocyte specific function was studied. Gulucose was added at the variable levels from 0.1 to 3.0 mM in the dish culture. It was found that the secretion rate in the early stage increased with decrease of glucose concentration. When glucose concentration was shifted up from low level (0.1mM) to high level (10 mM) at various culture days by changing the medium, high secretion rate was also obtained. It was found that when the medium was changed after 4 days, 2 times higher rate was kept even through after 14 days. This result means that the glucose level controlled culture become one of the effective strategy for long term hepatocyte culture.