Preparation of Three-Dimensional (3D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation

摘要

The use of three-dimensional (3D) in vitro culture systems (spheroids; organoids) in biomolecular and drug discovery research has become increasingly popular. The popularity is due, in part, to a diminished reliance on animal bioassays and a desire to develop physiologically relevant cell culture systems that simulate the in vivo tissue microenvironment. Most evaluations of 3D cultures are by confocal microscopy and high-content imaging; however, these technologies do not allow for detailed cellular morphologic assessments or permit basic hematoxylin and eosin (H&E) histologic evaluations. There are few studies that have reported detailed processes for preparing 3D cultures for paraffin embedding and subsequent use for histochemical or immunohistochemical staining. In an attempt to do so, we have developed a protocol to paraffin-embed human liver spheroids that can be sectioned with a microtome and mounted onto glass slides for routine histochemical and immunohistochemical staining, and light microscopic evaluations.