Adsorption, simple binding and complex binding of rat hepatocytes to various in vitro substrata

Abstract

Isolated hepatocytes appear to be able to attach to the culture substratum by several mechanisms. 1. 1. By direct adsorption to glass or activated (tissue culture grade) polystyrene. The adsorption is rapid, it can take place in the absence of divalent cations (but is stimulated by Ca 2+), and it is relatively temperature-insensitive. No spreading (flattening) of the cells takes place on polystyrene. Adsorption of cells to polystyrene is most easily demonstrated in protein-free media, and can be prevented by pre-saturating the polystyrene adsorption sites with albumin. At high concentrations of hepatocytes, proteins of cellular origin may similarly prevent adsorption of the cells. 2. 2. By simple binding, e.g. to a protein substratum, as exemplified by the receptor-mediated attachment of hepatocytes to adsorbed concanavalin A (ConA). This binding is stimulated by Ca 2+, but can take place in the absence of divalent cations, and the influence of temperature is small. The low temperature sensitivity indicates that the interaction between the cells and the ConA substratum is a very simple process, hence the term ‘simple binding’. Considerable cell spreading is observed on ConA. Binding of hepatocytes to adsorbed polylysine shows a low cation and temperature sensitivity similar to the attachment to concanavalin or polystyrene. The cells do not spread on polylysine. 3. 3. By complex binding of cellular receptors to certain protein substrata, such as adsorbed serum (fibronectin), collagen or asialofetuin. The binding requires divalent cations, and is maximal in the presence of both Mg 2+ and Ca 2+. However, there is considerable stimulation of the binding to asialofetuin by Ca 2+ alone (but not by Mg 2+); to collagen by Mg 2+ alone (but not by Ca 2+), or to adsorbed serum by either cation. Binding to all three substrata is strongly temperature-sensitive, which defines this type of binding as a complex process. The cells spread readily (in 2–4 h) on adsorbed serum and collagen, but not on asialofetuin.