Hepatocytes are highly differentiated epithelial cells that lose their phenotype and function when removed from the in vivo environment. Given the importance of hepatic cultures for drug toxicity, bioartificial liver assist devices and basic biology studies, considerable efforts have been focused on the maintenance of hepatic function in vitro. The methods used to date include co-cultivation of hepatocytes with stromal cells, organizing these cells into spheroids and imbedding them into bioactive gels. Our team has recently demonstrated that primary rat hepatocytes confined to microfluidic channels in the absence of convection maintained the epithelial phenotype through upregulation of endogenous signals including hepatocyte growth factor (HGF). The objective of the present study was to transition from microfluidic devices, which are somewhat specialized and challenging to use, towards low volume multiwell plates ubiquitous in biology laboratories. Using a combination of 3D printing and micromolding we have constructed inserts that can be placed into standard 12-well plates and can be used to create low volume culture conditions under which primary hepatocytes maintained a differentiated phenotype. This phenotype enhancement was confirmed by hepatic function assays including albumin synthesis and expression. Importantly we confirmed upregulation of HGF inside the low volume culture plates and demonstrated that inhibition of HGF signaling degraded the hepatic phenotype in our cell culture platform. Overall, this study outlines a new cell culture system that leverages the low volume effects of microfluidic channels in a multiwell plate format. Beyond hepatocytes, such a system may be of use in the maintenance of other difficult-to-culture cells including stem cells and primary cancer cells.

中文翻译：

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使用小体积多孔板利用来自肝细胞的内源性信号。

肝细胞是高度分化的上皮细胞，当从体内环境中移出时会失去其表型和功能。鉴于肝培养物对于药物毒性，生物人工肝辅助设备和基础生物学研究的重要性，已将相当大的努力集中在体外肝功能的维持上。迄今为止使用的方法包括将肝细胞与基质细胞共培养，将这些细胞组织成球状体并将它们嵌入生物活性凝胶中。我们的团队最近证明，在不存在对流的情况下，局限于微流体通道的原代大鼠肝细胞可通过上调包括肝细胞生长因子（HGF）在内源信号来维持上皮表型。本研究的目的是从微流控设备过渡到 对于生物学实验室中普遍存在的小体积多孔板而言，它们有些专业并且难以使用。使用3D打印和微成型的组合，我们构建了可以放置在标准12孔板中的插入物，并可以用于创建低体积培养条件，在该条件下原代肝细胞保持分化的表型。通过肝功能测定，包括白蛋白合成和表达，证实了这种表型增强。重要的是，我们证实了低体积培养皿中HGF的上调，并证明了HGF信号的抑制作用会降低我们细胞培养平台中的肝表型。总的来说，这项研究概述了一种新的细胞培养系统，该系统利用了多孔板形式的微流控通道的低体积效应。除了肝细胞，