Development of Artificial Cultured Liver

哺乳动物的肝脏作为动物摄取的所有物质的代谢处理中心，发挥着许多重要的功能。这一事实不允许我们为这个器官严重受损的人制作肝脏替代品。肝脏替代物的这种情况与心脏或肾脏替代物的情况形成对比。制造人工肝的唯一方法是利用肝细胞作为功能关键单元。为此，我们开发了一种在健康条件下长时间培养肝细胞的方法。我们从以下几个方面寻找肝细胞培养的最佳条件。1.引入细胞外基质（ECM）作为培养基质。2.最佳气相。3.肝细胞与非实质细胞的相互作用。4.灌注培养的发展5.发展立体文化。1. ECM。1.1.胶原在胶原纤维上培养的肝细胞DNA合成速率明显受到抑制。然而，在相同的底物上，白蛋白的分泌增强。1.2.肝基质分离。通过用尿素灌注肝脏来提取肝脏的ECM。发现该提取物是肝细胞的有效底物。2.肝细胞培养物的气相。肝细胞需要高氧（40-50%分压）以获得最佳粘附、铺展和培养活力。3.成纤维细胞需要长期培养。当肝细胞与成纤维细胞（3 T3或肺成纤维细胞）共培养时，它们在形态和功能上保持健康状态40天。4.灌注培养。我们开发了一种培养设备，其中肝细胞可以保持在灌注培养基中，将pH和氧浓度控制在所需水平。5.立体文化。在胶原凝胶基质中三维培养肝细胞。该技术为我们提供了一种将肝细胞以致密块的形式包裹的方法。

The liver of mammals plays many important functions as a center of metabolic processing of all materials taken up by the animal. This fact does not allow us make a liver substitute for the person who had severe demages to this organ. This situation of the liver substitute contrasts to that of a heart or renal substitute. The only way to make an artificial liver is to utilize hepatocytes as a functional key unit. For this aim, we developed a way for culturing hepatocytes in a healthy condition for a long period of time. We sought for the optimal condition for hepatocytes cultures from the following points of view. 1. Introduction of extracellular matrices (ECM) as a culture substrate. 2. Optimal gas phase. 3. Interactions of hepatocytes with non-parenchymal cells. 4. Development of perfusion culture 5. Development of three-dimensional culture. 1. ECM. 1.1. Collagen. The rate of DNA synthesis of hepatocytes was surpressed markedly when cultured on collagen fibrils. However, the secretion of albumin was enhanced on the same substrates. 1.2. Separation of liver matrix. ECM of the liver was extracted by perfusing the liver with urea. This extract was found to be an effective substrate for hepatocytes. 2. Gas phase of the hepatocyte cultures. Hepatocytes required heperoxia (40-50% in partial presure) for the optimal adhesion, spreading and viability in culture. 3. Requirement of fibroblasts for a long term culture. When hepatocytes were co-cultured with fibroblasts (3T3 or lung fibroblasts), they were maintained in morphologically and functionally healthy conditions over a term of 40 days. 4. Perfusion culture. We developed a culture equipment, where hepatocytes can be maintained in a perfused media, controlling pH and oxygen concentrations at a desired level. 5. Three-dimensional culture. Hepatocytes were cultured three-dimensionally in a matrix of collagen gels. This technique provides us a method to immobilize hepatocytes in a compact mass.