Isolation and characterization of hepatic stem cells, or "oval cells," from rat livers

Abstract

The pace of research on the potential therapeutic uses of liver stem cells or "oval cells" has accelerated significantly in recent years. Concurrent advancements in techniques for the isolation and characterization of these cells have helped fuel this research. Several models now exist for the induction of oval cell proliferation in rodents. Protocols for the isolation and culture of these cells have evolved to the point that they may be set up in any laboratory equipped for cell culture. The advent of magnetic cell sorting has eliminated reliance on expensive flow cytometric sorting equipment to generate highly enriched populations of oval cells. Our laboratory has had much success in using the oval cell surface marker Thy-1 in combination with magnetic sorting to produce material suitable for testing the influence of a myriad of chemical signaling molecules on the oval cell phenotype. This chapter will describe our basic strategy for oval cell induction and isolation. Additionally, two in vitro procedures are described which the reader may find useful in the early stages of developing an oval cell research project.

Fig. 24.1

Partial hepatectomy: (a) Normal anatomy of the rat abdominal viscera. (b) Following incision, three lobes should be clearly visible. (c) The median and left lateral lobes of the liver should be externalized, through the loop of silk suture, by pressure from the thumbs. The index fingers brace the lower portion of the rib cage.

Fig. 24.2

Two step collagenase perfusion: (a) Mid-line incision from just below the diaphragm to the groin. (b) Make cuts perpendicular to the main incision to expose viscera. (c) Identify the IVC and portal vein. (d) Diagram showing the correct placement of the silk sutures and catheter.

Fig. 24.3

Immunomagnetic sorting.

Fig. 24.4

Oval cell cytospin: immunomagnetic sorting of Thy-1⁺ oval cells yields a cell population, which is almost entirely Thy-1⁺/OV-6⁺ (both are oval cell markers) while almost completely devoid of CD45+ leucocytes.

Fig. 24.5

Schematic representation of transwell in chamber for migration assay.

Fig. 24.6

Example of migration assay, where three different doses of a specific factor are tested.