Joly A, Desjardins J F, Fremond B, Desille M, Campion J P, Malledant Y, Lebreton Y, Semana G, Edwards-Levy F, Levy M C, Clement B
Detoxication and Tissue Repair Unit 456, Institut National de la Santéde la Recherche Médicale, Rennes I University School of Medicine, France.
Transplantation. 1997 Mar 27;63(6):795-803. doi: 10.1097/00007890-199703270-00002.
Orthotopic liver transplantation is the most effective treatment for fulminant hepatic failure. As an alternative treatment, an efficient extracorporeal bioartificial liver should contain a large yield of functional hepatocytes with an immunoprotective barrier, for providing temporary adequate metabolic support to allow spontaneous liver regeneration or for acting as a bridge toward transplantation. Survival, proliferation, and functions of porcine hepatocytes were evaluated in primary cultures and after embedding in alginate beads, which were subsequently coated with a membrane made by a transacylation reaction between propylene glycol alginate and human serum albumin. Disruption of total pig livers by collagenase perfusion/recirculation allowed the obtention of up to 10(11) hepatocytes with a viability greater than 95%. Hepatocytes in conventional cultures or embedded in coated alginate beads survived for about 10 days, secreted proteins, particularly albumin, and maintained several phase I and II enzymatic activities, namely ethoxyresorufin-O-deethylase, oxidation of nifedipine to pyridine, phenacetin deethylation to paracetamol, glucuroconjugation of paracetamol, and N-acetylation of procainamide. Typical features of mitosis and [3H]thymidine incorporation indicated that porcine hepatocytes proliferated in both conventional cultures and alginate beads. The efficacy of the membrane surrounding alginate beads for protecting cells from immunoglobulins was tested by embedding HLA-typed human lymphocytes, which were subsequently incubated with specific anti-HLA immunoglobulin G and complement. These data show that large yields of porcine hepatocytes that are embedded in coated alginate beads remain functional and are isolated from large molecular weight molecules, such as immunoglobulins. This system represents a promising tool for the design of an extracorporeal bioartificial liver, containing xenogeneic hepatocytes, to treat acute liver disease in humans.
原位肝移植是暴发性肝衰竭最有效的治疗方法。作为一种替代治疗方法，一种有效的体外生物人工肝应含有大量具有免疫保护屏障的功能性肝细胞，以提供临时的充分代谢支持，使肝脏能够自发再生，或作为肝移植的桥梁。在原代培养以及将猪肝细胞包埋于藻酸盐珠中后，评估其存活、增殖及功能，随后用由海藻酸丙二醇酯与人血清白蛋白经转酰基反应制成的膜包被藻酸盐珠。通过胶原酶灌注/再循环破坏整个猪肝，可获得多达10¹¹个活力大于95%的肝细胞。常规培养或包埋于包被藻酸盐珠中的肝细胞存活约10天，分泌蛋白质，尤其是白蛋白，并维持多种Ⅰ相和Ⅱ相酶活性，即乙氧异吩恶唑酮 - O - 脱乙基酶、硝苯地平氧化为吡啶、非那西丁脱乙基化为对乙酰氨基酚、对乙酰氨基酚葡萄糖醛酸结合以及普鲁卡因酰胺N - 乙酰化。有丝分裂和[³H]胸苷掺入的典型特征表明猪肝细胞在常规培养和藻酸盐珠中均能增殖。通过包埋HLA分型的人淋巴细胞来测试藻酸盐珠周围的膜对保护细胞免受免疫球蛋白影响的功效，随后将其与特异性抗HLA免疫球蛋白G和补体一起孵育。这些数据表明，大量包埋于包被藻酸盐珠中的猪肝细胞仍保持功能，并与免疫球蛋白等大分子分离。该系统是设计含有异种肝细胞的体外生物人工肝以治疗人类急性肝病的一种有前景的工具。
