漂浮或贴壁肝细胞球形培养使用微孔芯片与聚乙二醇或聚酰亚胺表面。

Sae Yokomine, Tomomi Makino, Emiko Nagao, Kohji Nakazawa
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引用次数: 0

摘要

微孔芯片培养是一种很有前途的控制球体尺寸和生产大量均匀球体的技术。在本研究中,我们重点研究了芯片材料与肝细胞球体性质的关系。基本芯片设计为397个圆形微孔,每个直径为400µm。制备了两种类型的微孔芯片,在底表面涂覆聚乙二醇(PEG芯片)或聚酰亚胺(PI芯片)。肝细胞逐渐聚集,在PEG芯片的每个微孔内形成漂浮球体,而在PI芯片的每个微孔内形成粘附球体。这种漂浮和粘附的球体形态至少维持了一个月的培养。对球体形成机制的一种解释是,纤溶酶原激活剂/纤溶酶和基质降解/重塑系统在粘附球体的形成中被激活。此外,在贴壁球形培养中,除了细胞间连接的发展外,细胞-基质连接的形成也得到了促进。肝细胞球状体的白蛋白分泌和药物代谢活性高于传统单层肝细胞,PI芯片中的贴壁球状体比PEG芯片中的漂浮球状体保持更高的功能表达。在此基础上,通过基因表达分析评估肝细胞功能特性、关键代谢酶葡萄糖6-磷酸酶(糖代谢)、色氨酸2,3 -双加氧酶(氨基酸代谢)、精氨酸酶1(尿素循环)、细胞色素P450 7a1(脂代谢)和细胞色素P450家族(药物代谢)的表达。这些关键酶在肝细胞中的表达在球形培养中高于一般单层培养,并且贴壁球形细胞的功能优于漂浮球形细胞。这些结果表明,微孔芯片的材料特性是调节肝细胞球体形态和功能特性的重要因素。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Floating or adherent hepatocyte spheroid cultures using microwell chips with polyethylene glycol or polyimide surfaces.

Microwell chip culture is a promising technique for controlling spheroid size and producing a large number of homogeneous spheroids. In this study, we focused on the relationship between chip material and the properties of hepatocyte spheroids. The basic chip design was 397 circular microwells, each 400 µm in diameter. Two types of microwell chips were fabricated, coating the bottom surface either with polyethylene glycol (PEG chip) or polyimide (PI chip). Hepatocytes gradually aggregated and formed floating spheroids within each microwell in the PEG chip but formed adherent spheroids within each microwell of the PI chip. Such floating and adherent spheroid morphologies were maintained for at least one month of culture. An explanation for the spheroid formation mechanism is that the plasminogen activator (PA) /plasmin and matrix degradation/remodeling systems were activated in the formation of adherent spheroids. Furthermore, in adherent spheroid cultures, the formation of cell-matrix junctions was promoted, in addition to the development of intercellular junctions. The albumin secretion and drug metabolism activities of the hepatocyte spheroids were higher than those of traditional monolayer hepatocytes, and the adherent spheroids in the PI chip maintained a higher functional expression than the floating spheroids in the PEG chip. Further to this, functional properties of hepatocytes, the expressions of key metabolic enzymes, glucose 6-phosphatase (sugar metabolism), tryptophan 2, 3-dioxygenase (amino acid metabolism), arginase 1 (urea cycle), cytochrome P450 7a1 (lipid metabolism), and cytochrome P450 families (drug metabolism) were evaluated by gene expression analysis. The expression of these key enzymes in hepatocytes was higher in spheroid culture than in general monolayer culture, and the functions of adherent spheroids were superior to those of floating spheroids. These results indicate that the material properties of the microwell chips are important factors that regulate the morphological and functional characteristics of hepatocyte spheroids.

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