Application of Open Porous Microspheres in Hepatic Tissue Cultivation

一直以來肝臟疾病對於台灣境內及許多其他國家的人民都構成莫大的生命威脅，無論是酒精、藥物、病毒引起的肝炎或是肝癌等等都會造成肝臟受損，當肝臟功能喪失時可能會導致嚴重的肝衰竭。在臨床上相對來說較有效的肝衰竭治療方法為肝臟移植，例如急性肝衰竭(acute liver failure; ALF)的患者若無法及時進行肝臟移植手術，致死率可能高達百分之八十；然而肝臟來源一直都是供不應求的，因此近幾十年來便開始發展肝臟組織工程希望能提供更多替代方式，其中包括了肝細胞移植(hepatocyte transplantation)、組織工程移植物(tissue-engineered grafts)和人工肝輔助裝置(liver assist devices, LADs)。肝臟組織工程自發展以來遇到最大的難題之一是肝細胞在體外無法長時間維持其活性且會快速的去分化(de-differentiation)，近來的研究指出選擇適當的支架、加入特定生長因子和以非肝細胞株共同培養之方式可望改善此問題。 本實驗利用氣體發泡(gas foaming)法結合雙重乳化(double emulsion)法製備出具開放性孔洞之聚乳酸-甘醇酸微球體當作支架，微球體平均大小約為430μm，球體表面孔洞則集中在10-20μm。與一般使用實心微球體作為培養支架相比，肝細胞培養於具開放性孔洞的微球體可提高其細胞承載量，且能夠促進養份、氧氣和代謝物的交換。此外其具備可注射性的優點，解決了一般具孔洞性支架必須以開放性手術方式植入生物體內的問題。為了延長肝細胞在體外的功能性表現，實驗中利用纖維母細胞、間葉系幹細胞和人類臍靜脈內皮細胞分別與肝細胞進行共同培養，並以肝細胞產生的白蛋白及尿素濃度來評估其功能性；結果顯示非肝細胞株可能藉由細胞-細胞接觸的方式或者分泌特定的生物因子使得肝細胞在培養期間能夠維持其在體外的功能性。 研究結果證實，具開放性孔洞之聚乳酸-甘醇酸微球體能夠做為肝細胞培養的支架；此外利用非肝細胞共同培養的系統可延長肝細胞在體外的功能性表現長達兩週。雖然影響的原因是由細胞接觸或生物因子所導致仍須進一步確認，但結合具開放性孔洞之微球體、肝細胞及非肝細胞共同培養的系統在應用於肝臟組織工程上是有潛力的。Each year liver diseases have threatened people’s lives in Taiwan and many other countries. Alcohol, drug, virus-induced hepatitis, and malignant tumor can cause liver damage and result in liver failure. In the treatment of liver failure, liver transplantation has been established as an effective final option. For instance, the mortality rate of patients who suffered from acute liver failure (ALF) was reported as high as 80% without liver transplantation. However, due to the shortage of donated organ supply, hepatic tissue engineering, which includes hepatocyte transplantation, tissue-engineered grafts and liver assist devices (LADs), has been investigated for several decades in attempt to provide alternative approaches. One of the major obstacles of hepatic tissue engineering was the short-term viability and rapid de-differentiation of hepatocytes in vitro. Recent studies have indicated that combination of appropriate scaffold, factors, and co-culturing with other cell types may overcome this problem. In this study, open porous poly (lactic-co-glycolic acid) (PLGA) microspheres were fabricated using gas foaming in a W1/O/W2 double emulsion method, with a mean size of approximately 430μm and an average pore diameter mostly between 10-20μm. Comparing with nonporous microspheres as scaffold, culturing hepatocytes with open porous microspheres provided suitable microenvironments for higher cell seeding density and exchange of nutrients, oxygen, and metabolites. Unlike porous scaffolds requiring open surgery process for implantation in the body, open porous microspheres can deliver cells with a syringe in an injectable manner, which is more patient-friendly. For further improvement in viability and specific hepatic functions, nonparenchymal cells (rat fibroblasts, rat mesenchymal stem cells and human umbilical venous endothelial cells) were each co-cultured with mature hepatocytes. Quantification of albumin secretion and urea synthesis showed that nonparenchymal cells may support hepatocellular functions due to cell-cell interactions or secretion of soluble factors. This study suggested that open porous PLGA microspheres were successfully developed and used as the hepatocytes culturing scaffold. Moreover, the hepatocellular specific functions sustained up to 2 weeks in the support of co-culturing with nonparenchymal cells. Although the cell-cell interactions and soluble factors between hepatocytes and nonparenchymal cells requires further understanding, the combination of open porous microspheres, hepatocyes, and nonparenchymal cells co-culturing system has the potential in hepatic tissue engineering application