Bader A, Knop E, Kern A, Böker K, Frühauf N, Crome O, Esselmann H, Pape C, Kempka G, Sewing K F
Institut für Allgemeine Pharmakologie und Toxikologie, Medizinische Hochschule Hannover, Wuppertal, Germany.
Exp Cell Res. 1996 Jul 10;226(1):223-33. doi: 10.1006/excr.1996.0222.
Models for cocultures of parenchymal (PC) and nonparenchymal cells (NPC) of the liver relied on mixing the cells in a two-dimensional configuration or on establishing spheroidal aggregates. In vivo hepatic nonparenchymal cells, such as endothelial cells and Kupffer cells, are separated from parenchymal cells by extracellular matrix (ECM). Due to their location outside of the space of Disse they can form a barrier toward the sinusoid. Hepatocytes are attached to ECM of the space of Disse via two opposing sinusoidal surfaces. No three-dimensional coculture model reflecting this specific microenvironment of the liver cell plates in vivo has been available to date. We designed a three-dimensional model by positioning NPC on top of PC enclosed as a monolayer within a collagen sandwich. A gas-permeable membrane support can be used to allow the supply of oxygen to the resulting cell plate also from underneath the cell layers. Morphological analysis was performed by inverse and cross-sectional studies by light microscopy, scanning, and transmission electron microscopy of the coculture model. Cuboidal hepatocytes formed confluent layers below the NPC layer. They regularly expressed bile canaliculi at intercellular contact zones. Both sinusoidal surfaces expressed microprojections. Characteristic NPC including endothelial cells, Kupffer cells, and Ito cells completely covered the second matrix layer within a week. Kupffer cells were located on top of endothelial cells. Ito cells were intermingled and could be identified by their intracytoplasmic lipid droplets. LPS stimulation of cocultures resulted in a depression of albumin secretion. Phase I and phase II metabolites of the cytochrome P-450 1A1 substrate ethoxyresorufin were generated independently from the presence of cocultured NPC. This study describes the development of a novel three-dimensional coculture model, which intends to mimic more closely the microenvironment of the hepatic sinusoid by respecting the specific plate structure of the liver parenchyma. The model could serve as a complex tool to study potential collaborations between PC and NPC of the liver.
肝脏实质细胞（PC）与非实质细胞（NPC）共培养的模型，依赖于在二维结构中混合细胞或形成球形聚集体。在体内，肝脏非实质细胞，如内皮细胞和库普弗细胞，通过细胞外基质（ECM）与实质细胞分离。由于它们位于狄氏间隙之外，所以能够对肝血窦形成一道屏障。肝细胞通过两个相对的肝血窦表面附着于狄氏间隙的ECM。迄今为止，尚无反映体内肝细胞板这种特定微环境的三维共培养模型。我们设计了一种三维模型，将NPC置于单层封闭在胶原三明治内的PC之上。可使用透气膜支架，以便从细胞层下方也能向生成的细胞板供应氧气。通过对共培养模型进行光学显微镜、扫描电子显微镜和透射电子显微镜的反向及横断面研究来进行形态学分析。立方形肝细胞在NPC层下方形成汇合层。它们在细胞间接触区域规则地表达胆小管。两个肝血窦表面均表达微突起。包括内皮细胞、库普弗细胞和伊托细胞在内的特征性NPC在一周内完全覆盖了第二层基质。库普弗细胞位于内皮细胞之上。伊托细胞相互交织，可通过其胞质内脂滴加以识别。对共培养物进行脂多糖刺激会导致白蛋白分泌减少。细胞色素P - 450 1A1底物乙氧基试卤灵的I相和II相代谢产物的生成与共培养NPC的存在无关。本研究描述了一种新型三维共培养模型的构建，该模型旨在通过尊重肝实质的特定板层结构，更紧密地模拟肝血窦的微环境。该模型可作为研究肝脏PC与NPC之间潜在协作关系的复杂工具。
