Isolation of Primary Mouse Hepatocytes and Non-Parenchymal Cells from a Liver with Precancerous Lesions.

Abstract

In the early stages of liver carcinogenesis, rare hepatocytes and cholangiocytes are transformed into preneoplastic cells, which can progressively acquire a neoplastic phenotype, favored by the failure of natural antitumor immunosurveillance. The detailed study of both hepatic parenchymal (e.g., hepatocytes) and non-parenchymal cells (NPCs), such as immune cells, could help understand the cellular microenvironment surrounding these pre-cancerous and neoplastic lesions.Cultures of primary hepatocytes are of interest in various biomedical research disciplines, serving as an ex vivo model for liver physiology. Obtaining high viability and yield of primary mouse hepatocytes and other liver cell populations is technically challenging, thus limiting their use. In the first section of the current chapter, we introduce a protocol based on the two-step collagenase perfusion technique (by inferior vena cava) to isolate hepatocytes and, to a lower extent, NPCs and detailed the different considerations to take into account for a successful perfusion. The liver is washed by perfusion, hepatocytes are dissociated with collagenase, and different cell populations are separated by centrifugation. Various techniques have been described for the isolation of healthy and malignant hepatocytes; however, the viability and purity of the isolated cells is frequently not satisfactory. Here, we significantly optimized this protocol to reach improved yield and viability of the hepatocytes and concomitantly obtain preserved NPC populations of the liver.Within NPCs, tissue-resident or recruited immune cells are essential actors regulating hepatocarcinogenesis. However, simultaneous isolation of hepatic leukocytes together with other cell types generally yields low immune cell numbers hindering downstream application with these cells. In the second section of this chapter, as opposed to the first section primarily aiming to isolate hepatocytes, we present a tissue dissociation protocol adapted to efficiently recover leukocytes from non-perfused bulk (pre-)cancerous livers. This protocol has been optimized to be operator-friendly and fast compared to other liver processing methods, allowing easy simultaneous sample processing to retrieve hepatic (tumor-infiltrating) immune cells.