Reconstruction of liver organoid using a bioreactor
Reconstruction of liver organoid using a bioreactor
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DOI：
10.3748/wjg.v12.i12.1881
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发表时间：
2006-03-28
影响因子：
4.3
通讯作者：
Braet, Filip
中科院分区：
文献类型：
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作者：
Saito, Masaya;Matsuura, Tomokazu;Braet, Filip
目的：目的：探索生物人工肝体外器官重建的有效技术。方法：采用径向流生物反应器（Radial-Flow Bioreactor，RFB）建立了一个三维高密度培养体系。目前，我们使用功能性人肝细胞癌细胞系（FLC-5）作为肝细胞，小鼠永生化肝窦内皮细胞（SEC）系M1和小鼠永生化肝星状细胞（HSC）系A7作为RFB中的非实质细胞，重建了肝类器官。在RFB中孵育2 × 10（7）个FLC-5细胞。5天后，以类似方式加入2 × 10（7）A7细胞，5天后再加入10（7）M1细胞。灌注三天后，收获一些具有贴壁细胞的纤维素珠。最后的孵育期包括用200 nmol/L swinholide A灌注2 h，然后从RFB收获剩余的纤维素珠沿着粘附细胞。透射电镜（TEM）和扫描电镜（SEM）观察细胞形态。为了评估肝细胞的功能，我们比较了尿素循环酶的mRNA表达以及FLC-5在单层培养物中白蛋白的合成相比，在RFB.RESULTS的单一类型的文化和共培养物：通过透射电子显微镜，FLC-5，M1，和A7安排在有关的灌注侧的肝脏样组织。FCL-5细胞间可见类似胆小管的结构。扫描电子显微镜显示SEC表面上的窗孔。当我们在RFB中引入肌动蛋白结合剂swinholide-A 2 h时，泡-空泡细胞器（WO）和窗孔的数量增加。关于肝功能，尿素被发现在培养基中，和表达的mRNAs编码的琥珀酸合成酶和脱氢酶增加时，这三种细胞类型的RFB中共培养。结论：RFB系统共培养可显著改变各种细胞的结构和功能，包括肝细胞的功能特性。我们的系统证明了使用生物人工肝重建肝脏类器官的有效性。(C)2006年，WJG出版社。All rights reserved.
AIM: To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver.METHODS: We previously reported that a radial-flow bioreactor (RFB) could provide a three-dimensional high-density culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line (FLC-5) as hepatocytes together with mouse immortalized sinusoidal endothelial cell (SEC) line M1 and mouse immortalized hepatic stellate cell (HSC) line A7 as non parenchymal cells in the RFB. Two x 10(7) FLC-5 cells were incubated in the RFB. After 5 d, 2 x 10(7) A7 cells were added in a similar manner followed by another addition of 10(7) M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB.RESULTS: By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles (WO) and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased.CONCLUSION: Co-culture in the RFB system can dramatically change the structure and function of all cell types, including the functional characteristics of hepatocytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver. (C) 2006 The WJG Press. All rights reserved.