Enhanced Functional Maturation of Fetal Porcine Hepatocytes in Three-Dimensional Poly-L-lactic Acid Scaffolds: A Culture Condition Suitable for Engineered Liver Tissues in Large-Scale Animal Studies
Enhanced Functional Maturation of Fetal Porcine Hepatocytes in Three-Dimensional Poly-L-lactic Acid Scaffolds: A Culture Condition Suitable for Engineered Liver Tissues in Large-Scale Animal Studies
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DOI：
10.3727/000000006783981486
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发表时间：
2006-09
影响因子：
3.3
通讯作者：
Hongyun Huang;S. Hanada;N. Kojima;Y. Sakai
中科院分区：
文献类型：
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作者：
Hongyun Huang;S. Hanada;N. Kojima;Y. Sakai
为了设计具有临床意义大小的肝组织，在进行人体临床试验之前，有必要使用大规模动物研究进行体内性能评估。由于猪是最合适的候选者，因此开发适合猪肝细胞祖细胞的培养条件对于设计猪肝组织等效物非常重要。因此，我们研究了聚l -乳酸（PLLA）三维（3D）支架在不同生物因子组合下对胎猪肝细胞功能成熟的影响。细胞是从当地屠宰场获得的猪胎儿中分离出来的，并在单层和PLLA支架中培养15天。虽然培养15 d后，单层PLLA和3D PLLA的增殖率几乎相同（约为5倍），但添加肝细胞生长因子（HGF, 10 ng/ml）和丁酸钠（Sb, 1 mM）的PLLA培养显著增强了胎猪肝细胞的各种肝脏特异性功能。将第1天固定细胞的数量与第1天单层细胞的数量进行比较，得出最终的功能；白蛋白分泌增加191倍，细胞色素P450 IA1/2容量增加70.5倍，氨去除增加20.9倍，尿素合成增加18.0倍。这些功能比同一时期的单层培养高2.0 - 3.3倍。此外，在第15天获得的最终单位细胞功能几乎与单层和三维球形培养的成年猪肝细胞的水平相当。这些结果表明，使用生物可降解聚合物为基础的3D培养物和适当的生物因子组合是一种很有前途的方法，可以最大限度地提高大型动物肝细胞祖细胞的功能成熟。此外，所建立的培养条件值得用于在大型动物临床前研究中为猪设计大型肝组织等量物。
To engineer liver tissues with a clinically significant size, in vivo evaluation of performance using large-scale animal studies are necessary before proceeding to human clinical trials. As pigs are the most suitable candidates, the development of culture conditions suitable for porcine hepatocyte progenitors is very important to engineer pig liver tissue equivalents. We therefore investigated the efficacy of poly-L-lactic acid (PLLA) three-dimensional (3D) scaffolds on the functional maturation of fetal porcine hepatocytes in the presence of various combinations of biofactors. Cells were isolated from pig fetuses obtained from a local slaughterhouse, and cultured for 15 days both in monolayer and PLLA scaffolds. Although 15 days of culture resulted in almost the same ratio of proliferation (about fivefold) in both monolayer and 3D PLLA culture, the PLLA culture with hepatocyte growth factor (HGF, 10 ng/ml) and sodium butylate (Sb, 1 mM) remarkably enhanced various liver-specific functions of fetal porcine hepatocytes. The final attained functions based on the numbers of immobilized cells on day 1 compared with those of day 1 monolayers; 191fold increase in albumin secretion, 70.5-fold increase in cytochrome P450 IA1/2 capacity, 20.9-fold increase in ammonia removal, and 18.0-fold increase in urea synthesis were obtained. These functions were 2.0–3.3-fold higher than those obtained by the same period of monolayer culture. In addition, final attained unit cell-based functions on day 15 were almost comparable to the levels reported for cultures of adult porcine hepatocytes in both monolayer and 3D spheroid cultures. These results demonstrate that the use of a biodegradable polymer-based 3D culture with an appropriate combination of biofactors is a promising approach to maximize functional maturation of hepatocyte progenitors from large animals. In addition, the established culture conditions are worth using to engineer large liver tissue equivalents for pigs in large-animal-based preclinical studies.