以小型肝細胞培養技術應用於肝臟晶片的開發

Abstract

肝臟具備合成代謝解毒等功能，然而肝臟的in vitro study 常受限於無法完全長期評估體內情況，因此如何建立一個能精準模擬體內肝臟環境且能進一步作為藥物/毒物檢測的體外平台是所有肝臟研究者的共同願景。 本研究擬以微流道系統 (Microfluidic system) 建立一個新型的Liver-on-chip 的培養平台，並使用大鼠的小型肝細胞 (Small hepatocyte) 作為細胞來源，小型肝細胞是肝臟前驅細胞 (Liver progenitor cell) 的一種，具有良好的分化與生存能力。由實驗結果得知小型肝細胞在 2D 培養環境下會形成大小約為300-400 μm 的肝細胞聚落 (Colony) ，且肝細胞生存活性會由原本的 5-7 天提升至 3-4 周；另一方面，以 Quantitative real-time PCR (qPCR) 方式進行基因表現檢測也發現小型肝細胞的白蛋白 (Albumin) 表現量隨著培養時間上升約 3 倍，且 Follistatin (小型肝細胞的 Marker) 隨著培養時間降低約 0.4 倍；而以次世代定序 (Next generation sequencing，NGS) 來進行基因表現檢測亦發現 Cytokeratin 18 (CK 18 ，成熟肝細胞的 Marker) 及 Cytokeratin 19 (CK 19 ，具膽管分化的肝細胞的 Marker) 表現量隨著培養時間皆升約 2 倍，且 Cluster of designation 44 (CD 44 ，小型肝細胞的 Marker) 降低約 7 倍；我們以免疫染色方式亦發現 CK18 之表現隨著培養時間上升。以上結果都證實了小型肝細胞除能分化為成熟肝細胞之外亦能於體外培養的環境下長時間維持其肝機能。 除了小型肝細胞的培養技術開發之外，本研究亦利用聚甲基丙烯酸甲酯 [(Poly(methyl methacrylate) ，PMMA] 開發一套僅需利用市售微波爐及雷射加工即可製作供細胞培養的微流道系統，其製作方式不但簡單容易，且比起傳統以聚雙甲基矽氧烷 [Poly(dimethylsiloxane) ，PDMS] 需花費 1~2 天才能製備而成的微流道而言約，PMMA 的微流道製程可以大幅度縮減至僅需 3~4 小時即可從微流道設計到微流道晶片實體完成；此外，我們亦成功於 PMMA 之微流道上來培養小型肝細胞，而細胞生存率更相較傳統 2D 培養提升約 27 % 。 利用小型肝細胞以及 PMMA 作為肝臟晶片 (Liver-on chip) 平台的開發不但製作簡單、亦具有潛力做為肝臟晶片之開發平台，相信也更能模擬真正的器官功能，因此能幫助我們能在體外建立藥物於肝臟代謝後的毒理數據。我們之後更將進一步對藥物，化學藥品和農藥於此肝臟平台進行安全性評估。相信這樣的技術對於未來肝臟晶片的研究開發有著拋磚引玉之效。

The liver is an organ with vital functions, including energy storage, secretion protein synthesis, and especially metabolism of pharmaceutical drugs. However, in vitro studies of drug test are usually limited to precisely evaluate the real influences on hepatic tissue because it is an obstacle to develop a platform which can sophisticatedly mimic in vivo hepatic environment. Thus, in this study we established a microenvironment-mimicking liver-on-chip (LOC) platform for in vitro hepatotoxicity test. Small hepatocytes, which have been identified in primary hepatocyte cultures with high potential for proliferation and differentiation into mature hepatocytes, was used as cell source for LOC platform. The result shows that small hepatocytes can survive in 2D primary cultures, and form 300-400 μm colonies for maintaining hepatocyte functions. Compared to primary hepatocytes, which normally maintain their function for about 7 days, small hepatocytes can survive at least 4 weeks. We analyzed the gene expression of small hepatocytes by q-PCR. Expression of albumin and Tryptophan 2,3-dioxygenase (marker of primary hepatocytes) are 3 times and 120 times increase, whereas Follistatin (marker of small hepatocytes) expression is 0.4 times decrease, after 2 weeks of culture. We also analyzed the RNA expression by NGS. The expression of CK18 and CK19 increase 2 times whereas CD44 decreases 7 times, after 2 weeks of culture. On the other hand, poly(methyl methacrylate) was utilized to fabricate microfluidic devices. The substrates are patterned using the laser cutter, and bonded in a commercial microwave.Compared to the traditional PDMS fabrication process (usually needs 1~2 days), this bonding process is very simple and can therefore save more time (only 3~4 hours)). Besides, the viability of small hepatocytes in poly(methyl methacrylate)-microfluidic devices is 27% higher than that in 2D primary cultures. In summary, the small hepatocytes-derived liver-on-chip platform was successfully developed and therefore can simulate the real environment in model animals, and also build toxicology database and make safety assessment of drugs, chemicals and pesticides in the future.