小型肝細胞培養技術應用於可拆式肝臟晶片的開發

Abstract

開發一套能精準模擬體內器官的體外平台一直是科學界研究的目標，這是因為在傳統的新藥試驗方法過程中，往往需要大量的動物實驗以及臨床實驗來確認藥物毒性對人體是否有害，然而這樣的方法不但需要大量研究經費與花費長期的時間，而且最終能成功通過試驗的新藥比例卻相當有限。由於微流道模型不但能夠有效模擬人體內器官結構與環境、且亦能利用高通量的檢測來縮短試驗時間、細胞更能進一步在相似體內結構的環境下生長並進行測試，這樣的做法不但提高新藥檢測效率也提升了試驗結果的可靠度，因此微流道器官晶片的研究在近年來受到很大的注目。 在體內的器官當中，最大的藥物代謝相關器官是肝臟，他同時具有合成、排泄、解毒等功能。肝臟主要由四種細胞組成：成熟肝細胞 (Primary hepatocytes) 、內皮細胞 (Liver sinusoidal endothelial cells) 、星狀細胞 (Stellate cells) 巨噬細胞 (Kupffer cells) 等，這些細胞藉由細胞與細胞之間的相互作用而組成一個具有功能的肝臟器官。本篇所研究的可拆卸式肝臟微流道晶片，在細胞來源的選擇方面使用大鼠具有分化能力的前驅幹細胞 (Liver progenitor cell) ；而在流道的選擇方面，本研究使用具有生物相容性的聚二甲基矽氧烷 (Polydimethylsiloxane，PDMS) 作為流道的材料，最後再將小型肝細胞培養於可拆卸式肝臟微流道晶片 (Detachable microfluidic liver platform) 上。將小型細胞培養於模擬體內肝臟的微流道晶片上，以此微流道晶片來當作研發新藥物的體外細胞測試平台。我們除了定期記錄培養期間的細胞型態變化之外、 亦利用酵素免疫分析 (Enzyme-linked immunosorbent assay，ELISA) 方法和定量連鎖聚合酶反應 (Quantitative real-time PCR，qPCR) 來檢測細胞白蛋白 (Albumin) 之實際分泌量及基因表現量；另外，我們也使用細胞免疫染色法來觀察細胞分化的過程 (Cytokeratin 18，成熟肝細胞標記) ，並同時檢驗肝細胞於微流道裝置中的尿素代謝之功能。 本研究結果得知：(1) 相較初代成熟肝細胞在培養 1 周後會失去細胞特性，小型肝細胞，且可以在體外環境下長時間培養至少 3 周，並且維持細胞聚落 (Colony) 型態；(2) 使用可拆卸式的PDMS 微流道來培養小型肝細胞，其小型肝細胞在第 9 天後仍能維持白蛋白分泌量，且隨著培養時間的增加其肝機能的分泌仍然保持穩定；(3) 小型肝細胞在使用注射幫浦的微流道系統以製造動態養分供給的培養環境下，會比起不使用注射幫浦的靜態微流道系統提升至少 2 倍以上的白蛋白分泌量及至少 1 倍以上的尿素代謝量。 另外一方面，於此可拆卸式肝臟微流道晶片上再加入離胺酸 (Lysine) 培養後，本研究結果亦顯示：(1) 使用含量 0.1 mg/mL 的離胺酸培養下，小型肝細胞不論在動態培養或是靜態培養下，皆可以維持 Colony 型態到第 14 天；(2) 而在培養第 3、6 天培養下的離胺酸動態白蛋白分泌量，高出離胺酸靜態培養下約 39%、34%。(3) 在培養的第 3、6、10 天，離胺靜態培養下的白蛋白分泌量比未添加離胺酸的靜態培養提升約 6倍 、19倍 、18倍。(4) 在培養的第 3、6、10 天，添加離胺酸動態培養下的白蛋白分泌量比未添加離胺酸的動態培養提升 4 倍、16 倍 、17 倍。 而於此可拆卸式肝臟微流道晶片上再另外加入單寧酸 (Tannic acid) 培養後，本研究結果證明：(1) 使用含量 1 μg/mL 的單寧酸培養下，小型肝細胞不論在動態培養或是靜態培養下，皆可以維持 Colony 型態到第 14 天；(2) 而在培養第 7、 10、 14 天培養下的單寧酸動態白蛋白分泌量，高出單寧酸靜態培養下約 3~4 倍。 此外，若在針對上述條件在培養到第 7 天後的現象看來，(1) 添加單寧酸之靜態培養下的白蛋白分泌量，會比未添加單寧酸的靜態培養提升約 4 倍以上、也比成熟肝細胞的靜態培養提升約 100 倍；(2) 添加單寧酸之動態培養下的白蛋白分泌量比未添加單寧酸的動態培養提升約 15 倍以上、 也比成熟肝細胞的靜態培養提升約 500 倍。 綜觀以上的結果，我們相信這樣的研究方向與目的對於未來肝臟晶片的建立以及長時間藥物測試的體外平台發展，能夠具有一定的參考價值和意義。

Developing a microfluidics platform which is efficient and time-saving for drug toxicity assessment is always a goal that scientists have been working on it. However, in vitro study of drug tests usually takes lots of time and money but put less new therapy in the market. Thus, we believe that organ-on-a-chip can be a convenient and easy way to test drug toxicity and safety in the future. The appearance of organ-on-a-chip provides a great opportunity for in vitro drug toxicity assessment without performing animal experiments. Additionally, the largest organ which has metabolism and detoxification activity in the human body is the liver, which has the function of metabolic activity, secretion of protein synthesis and energy production. Physiologically, the liver is composed of four kinds of cells – Kupffer cells, stellate cells, the liver sinusoidal endothelial cells (LSECs), and hepatocytes. Primary rat small hepatocytes are progenitor cells that have a tendency to differentiate into specific cells. In this paper, we cultured primary rat small hepatocytes on a detachable microfluidic device and used a syringe pump to mimic the blood flow condition in vivo, the prototype of liver-on-a-chip is formed. Our microfluidic platform is made of polydimethylsiloxane (PDMS) as material and can assemble easily and disassemble conveniently in order to collect cells and medium to test liver function. The PDMS microfluidic chip is also reusable, so it can lessen experiment materials and shorten the experiment time which is used to produce abandon single-used microfluidic chips. The results exhibit that (1) small hepatocytes can survive in 2D culture and form 300-400μm colonies in order to maintaining hepatocyte functions. Different from primary hepatocytes which normally maintain their morphology and function for only 7 days, small hepatocytes have great performance on both morphology and liver-specific function for at least 21 days. (2) Moreover, we analyzed the gene expression of the equipment by quantitative real-time PCR (qPCR) and the expression of albumin and Tryptophan 2,3-dioxygenase (a marker of primary hepatocytes) are increased. On the other hand, albumin secretion by enzyme-linked immunosorbent assay (ELISA) was increased after culturing 9 days. (3) Small hepatocytes which were cultured in the detachable microfluidic system which was connected to syringe pump secreted at least 2 times higher albumin and about 1 times higher urea than without syringe pump. All of these results indicated that this liver-on-a-chip had well performance in vitro culture condition and primary rat small hepatocytes performance well liver-specific function, so we thought it can be a convenience system for hepatocyte-related experiments and it can be used to drug experiments to test toxicity. Then, we choose lysine and tannic acid as drug components to test this detachable microfluidic system. First results showed that small hepatocytes cultured by 1 mg/mL lysine-contained DMEM/F12 medium represents higher albumin secretion, urea secretion and higher gene expression of albumin than cultured by DMEM/F12 medium only. (1) Small hepatocytes cultured in flow system or static system both maintained morphology of colony after culturing 14 days. (2) Albumin secretion in flow system were about 39% and 34% higher than static condition while small hepatocytes were cultured 3 and 6 days. (3) Albumin secretion of small hepatocytes cultured by lysine-contained medium in static system were about 6 times ,19 times, and 18 times higher than DMEM/F12 medium in static system after culturing 3 days, 6 days and 10 days. (4) Albumin secretion of small hepatocytes cultured by lysine-contained medium in flow system were about 4 times, 16 times and 17 times higher than by DMEM/F12 medium in flow system after culturing 3 days, 6 days and 10 days. Second results showed that small hepatocytes cultured by 0.1 μg/mL tannic acid-contained DMEM/F12 medium represents higher albumin secretion, urea secretion and higher gene expression of albumin than cultured by DMEM/F12 medium only. (1) Small hepatocytes cultured in flow system or static system both maintained morphology of colony after culturing 14 days. (2) Albumin secretion in flow system were about 3-4times higher than static condition while small hepatocytes were cultured 7, 10 and 14 days. On the other hand, when we focused on the results after small hepatocytes cultured 7 days. (1) Albumin secretion of small hepatocytes cultured in static system with tannic acid-contained DMEM/F12 medium increased about 4 times than small hepatocytes cultured in static system with DMEM/F12 medium only, and also increased about 100 times than mature hepatocytes cultured with WE medium in the static system. (2) Albumin secretion of small hepatocytes cultured in flow system with tannic acid-contained DMEM/F12 medium increased about 15 times than small hepatocytes cultured in flow system with DMEM/F12 medium only, and also increased about 500 times than mature hepatocytes cultured with WE medium in the static system. In conclusion, we hope that such research purpose provides a well value of build-up liver-on-a-chip system and in vitro long-time drug test platform in the future.