Gap junction-mediated intercellular communication in a long-term primary mouse hepatocyte culture system

Abstract

Gap junction-mediated intercellular communication (GJIC) is critical for maintaining integral cellular processes including differentiation and growth control. The disruption of GJIC has been correlated with aberrant function in many cell types, including hepatocytes in vivo; therefore it is imperative that cellular model systems support intercellular communication to simulate normal cellular functions. Functional GJIC has been shown in long-term primary rat hepatocyte cultures, which have been implemented widely to study various aspects of hepatocellular function; however, the onset of transgenic technology in murine species has necessitated the development of a primary mouse hepatocyte system. In this report, we analyze GJIC in a dimethylsulfoxide (DMSO)-containing long-term primary mouse hepatocyte culture system. The cells retain morphologic and biochemical characteristics of differentiated hepatocytes through day 30 post plating, including liver-specific gene expression. We further show that connexin32 and connexin26 expression and gap junction plaque formation increase over time in culture concomitant with an increase in GJIC between adjoining primary mouse hepatocytes. In conclusion, the findings described in this study make it possible to maintain differentiated primary mouse hepatocytes that also show GJIC in long-term culture for 30 days. In addition, this system has the potential to be extended to study primary mouse hepatocytes isolated from genetically engineered mice. T he development of in vitro systems to study hepatocellular function largely has been impaired by the inability to maintain hepatocytes in a differentiated state in long-term culture. Previous reports have shown that primary rat hepatocytes, plated on collagencoated dishes and cultured in a chemically defined, serum-free medium supplemented with dimethylsulfoxide (DMSO), retain morphologic and biochemical characteristics of differentiated hepatocytes in long-term culture. 1 DMSO maintains primary rat hepatocytes in a differentiated state over long periods of time (Ͼ60 days), negating the need to coculture the hepatocytes with another cell type. 1 Although the rat system has been invaluable for studying various facets of liver biology in vitro, 1-7 the onset of transgenic technology in murine species has necessitated the development of a primary mouse hepatocyte culture system. Thus, the establishment of a long-term culture system for studying primary mouse hepatocytes (PMHs) would be highly important because hepatocytes could be isolated from genetically engineered mice as well as wild-type mice. Many laboratories have attempted to establish PMH culture systems. Short-term cultures using PMHs have been developed, but these cultures typically are maintained for 24 to 72 hours and therefore either are recovering from cellular isolation procedures or in the process of dedifferentiation. Importantly, gap junctionmediated intercellular communication (GJIC) rapidly declines within the first few days of hepatocyte culture. 8 Previous attempt...

Keratinocyte serum-free medium maintains long-term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors

“…The presence of connexin proteins in liver is associated with normal liver function. The induction of Cx32 and Cx26 proteins in DMSO-containing primary mouse hepatocytes cultures was also observed and confirmed the importance of connexin proteins in differentiating hepatocytes (Stoehr & Isom, 2003). In the present study, Cx32 was shown to be expressed in hepatocytes maintained in KDS culture medium for up to 3 weeks (Fig.…”

An artificial liver sinusoid with a microfluidic endothelial‐like barrier for primary hepatocyte culture

“…primary hepatocytes were allowed to form extensive cell-cell contacts in solution, multicellular spheroids were observed that preserved viability and metabolic activity over many weeks (Hamilton et al, 2001;Landry et al, 1985). The activation of signaling pathways resulting from hepatocyte contact can be mediated through membrane proteins (Corlu et al, 1991) or via gap junction communication (Stoehr and Isom, 2003). While the advantages of hepatocyte aggregates for in vitro culture are well known, this approach has been limited due to the lack of suitable technology for controlling the size and location of the spheroids.…”

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

“…Hepatocytes were isolated from untreated HB-EGF WT male mice, 6–8 weeks of age, by a modification of previously described methodology (23). Hepatocytes were resuspended in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin, plated on 60-mm BD Biocoat Collagen I Culture Dishes (BD Biosciences, Bedford, MA, USA), and maintained at 37°C in a humidified atmosphere of 5% CO 2 /95% air.…”