Kim S S, Sundback C A, Kaihara S, Benvenuto M S, Kim B S, Mooney D J, Vacanti J P
Laboratory for Transplantation and Tissue Engineering, Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Tissue Eng. 2000 Feb;6(1):39-44. doi: 10.1089/107632700320874.
Our laboratory has investigated hepatocyte transplantation using biodegradable polymer matrices as an alternative treatment to end-stage liver disease. One of the major limitations has been the insufficient survival of an adequate mass of transplanted cells. This study investigates a novel method of dynamic seeding and culture of hepatocytes in a flow perfusion system. In experiment I, hepatocytes were flow-seeded onto PGA scaffolds and cultured in a flow perfusion system for 24 h. Overall metabolic activity and distribution of cells were assessed by their ability to reduce MTT. DNA quantification was used to determine the number of cells attached. Culture medium was analyzed for albumin content. In Experiment II, hepatocyte/polymer constructs were cultured in a perfusion system for 2 and 7 days. The constructs were examined by SEM and histology. Culture medium was analyzed for albumin. In experiment I, an average of 4.4 X 10(6) cells attached to the scaffolds by DNA quantification. Cells maintained a high metabolic activity and secreted albumin at a rate of 13 pg/cell/day. In experiment II, SEM demonstrated successful attachment of hepatocytes on the scaffolds after 2 and 7 days. Cells appeared healthy on histology and maintained a high rate of albumin secretion through day 7. Hepatocytes can be dynamically seeded onto biodegradable polymers and survive with a high rate of albumin synthesis in the flow perfusion culture system.
我们实验室研究了使用可生物降解聚合物基质进行肝细胞移植，作为终末期肝病的一种替代治疗方法。主要限制之一是移植细胞足够数量的存活率不足。本研究调查了一种在流动灌注系统中动态接种和培养肝细胞的新方法。在实验I中，将肝细胞流动接种到聚乙醇酸（PGA）支架上，并在流动灌注系统中培养24小时。通过其还原MTT的能力评估细胞的总体代谢活性和分布。DNA定量用于确定附着细胞的数量。分析培养基中的白蛋白含量。在实验II中，将肝细胞/聚合物构建体在灌注系统中培养2天和7天。通过扫描电子显微镜（SEM）和组织学检查构建体。分析培养基中的白蛋白。在实验I中，通过DNA定量平均有4.4×10⁶个细胞附着在支架上。细胞保持高代谢活性，并以13 pg/细胞/天的速率分泌白蛋白。在实验II中，SEM显示在2天和7天后肝细胞成功附着在支架上。在组织学上细胞看起来健康，并在第7天维持高白蛋白分泌率。肝细胞可以动态接种到可生物降解聚合物上，并在流动灌注培养系统中以高白蛋白合成率存活。
