Preparation of Primary Rat Hepatocyte Spheroids Utilizing the Liquid-Overlay Technique.
Preparation of Primary Rat Hepatocyte Spheroids Utilizing the Liquid-Overlay Technique.
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DOI：
10.1002/cptx.87
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发表时间：
2019-09
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在此，我们描述了一个协议，用于制备和分析的主要分离大鼠肝细胞的三维细胞培养格式描述为球体。肝细胞自发地自聚集成球状体，而不需要合成的细胞外基质或水凝胶。原代大鼠肝细胞（PRH）是原代分化肝细胞的现成来源，因此与普通肝细胞系相比，保留了许多所需的肝脏特异性功能标志物，并引发了天然的体内表型。我们描述了液体覆盖技术，该技术提供了一个超低附着表面，PRH可以在其上培养为球状体。版权所有2019作者。 基本方案1：琼脂糖包被板的制备基础方案2：原代大鼠肝细胞分离程序基础方案3：原代大鼠肝细胞球状体培养基础方案4：PRH球状体的免疫荧光分析
Herein, we describe a protocol for the preparation and analysis of primary isolated rat hepatocytes in a 3D cell culture format described as spheroids. The hepatocyte cells spontaneously self‐aggregate into spheroids without the need for synthetic extracellular matrices or hydrogels. Primary rat hepatocytes (PRHs) are a readily available source of primary differentiated liver cells and therefore conserve many of the required liver‐specific functional markers, and elicit the natural in vivo phenotype when compared with common hepatic cells lines. We describe the liquid‐overlay technique which provides an ultra‐low attachment surface on which PRHs can be cultured as spheroids. © 2019 The Authors. Basic Protocol 1: Preparation of agarose‐coated plates Basic Protocol 2: Primary rat hepatocyte isolation procedure Basic Protocol 3: Primary rat hepatocyte spheroid culture Basic Protocol 4: Immunofluorescent analysis of PRH spheroids