Multiple sequential periods of DNA synthesis and quiescence in primary hepatocyte cultures maintained on the DMSO‐EGF on/off protocol

Abstract

Repeated periods of DNA synthesis activity (each period consisting of two to three cycles) separated by intervals of quiescence in primary rat hepatocytes can be stimulated by sequential addition and removal of 2% dimethyl sulfoxide (DMSO) in the presence of epidermal growth factor (EGF). Hepatocytes can be kept in nonproliferating cultures for 7 days in media supplemented with 2% DMSO and EGF. If DMSO is removed while EGF is maintained, rat and human hepatocytes enter a 3 to 4 day period of DNA synthesis that declines rapidly by days 4 and 5. If DMSO is reintroduced into cultures at that point, kept on for 3 more days and removed again, hepatocytes reenter into proliferation with another self-limited response of 3 to 4 days. Similar phenomena can seen with hepatocytes maintained in the presence of 3 mM phenobarbital. These protocols demonstrate that loss of responsiveness to mitogens in primary hepatocyte cultures is not an irreversible process. They also raise the possibility that signals for termination of DNA synthesis in hepatocytes emanate from hepatocytes themselves. These studies also suggest for the first time the possibility of designing in vitro systems that will allow clonal expansion of differential hepatocytes.

Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes

“…Enterchange between these two culture systems should contribute to studies on hepatocyte activities in relation with cell shape and the cell cycle. Chan et al (1989) also reported that multiple cycles of sequential DNA synthesis and quiescence could be induced in monolayers maintained on the DMSO-EGF onloff protocol. But our results indicated that spheroid culture is better than monolayer culture a t high cell density for maintaining liver-specific functions and that cell-cell contact and a cuboidal morphology are important in regulation of liver functions.…”

Thyroid Hormone Receptor β Agonist Induces β-Catenin-Dependent Hepatocyte Proliferation in Mice: Implications in Hepatic Regeneration

“…This was strengthened by the observation that DMSO delivery by itself affected hepatocyte proliferation. Indeed, the role of DMSO in inhibiting hepatocyte proliferation, especially in cultures, has been known for a long time 25 . When GC-1 was administered to mice via diet, hepatocyte proliferation was increased to a notably greater extent, although it was less so than that induced with T3 diet.…”

TNFα‐mediated extracellular matrix remodeling is required for multiple division cycles in rat hepatocytes†

“…Other groups have shown that small and mature hepatocytes could undergo several rounds of DNA synthesis in presence of 1% to 2% DMSO 32,33 or nicotinamide, 34-37 2 conditions known to favor survival and differentiation stability. However, these studies evidenced neither net increase in number of mature hepatocytes, requirement of complementary effect of TNF␣ and mitogen as in vivo, nor described the alternative proliferation and quiescence periods.…”