Liver Tissue Engineering: A Role for Co-culture Systems in Modifying Hepatocyte Function and Viability

Abstract

A major limitation in the construction of a functional engineered liver is the short-term survival and rapid de-differentiation of hepatocytes in culture. Heterotypic cell-cell interactions may have a role to play in modulating long-term hepatocyte behavior in engineered tissues. We describe the potential of 3T3 fibroblast cells in a co-culture system to modulate function and viability of primary isolated rat hepatocytes. Over an 18-day period after isolation, hepatocytes in pure culture rapidly declined in viability, displayed sparse bile canaliculi, and lost two function markers, the secretion of albumin and ethoxyresorufin O-dealkylase (EROD) activity. In comparison, the hepatocytes within the co-cultures maintained viability, possessed well-formed canalicular systems, and displayed both functional markers. Fixed 3T3 cells or 3T3 cell conditioned medium did not substitute for the viable 3T3 cell co-culture system in preserving hepatocyte viability and functionality.

A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development

“…It is also important to keep the culture plates undisturbed for at least 4 h after plating to allow proper cell adherence. Figure 2 shows hepatocytes in culture as early as 5 h and out to 120 h. As seen by phase contrast microscopy, the morphology of the hepatocytes changes over time in culture consistent with documented morphological changes (Bhandari et al 2001;Godoy et al 2010;Li et al 2010;Talamini et al 1997: Wallace et al 2010. These cellular changes include cell polarization, de-differentiation as well as down regulation of gene expression (Bhandari et al 2001;Godoy et al 2010;Li et al 2010;Talamini et al 1997: Wallace et al 2010.…”

“…Hepatocytes isolated using the standard two step perfusion method have been used in culture as well as in cell suspension assays for many years (Klaunig et al 1981;Li et al 2010;Warner et al 2003). In these ex vivo assays, cells are used immediately after isolation to avoid changes in gene regulation, polarization and dedifferentiation (Bhandari et al 2001;Godoy et al 2010;Talamini et al 1997;Wallace et al 2010). The main downside of the current protocols is the requirement for cell purification through gradient centrifugation which extends the time that the cells are manipulated and can result in reduced cell number and viability.…”

Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes

“…8. Hepatocytes cultured in a collagen double-gel have been shown to exhibit native cell-cell contacts such as E-cadherin and bile canaliculi, 34 but do not express sinusoidal receptors such as EGF-R 34 and LDL-R. Hepatocytes cocultured with 3T3 fibroblasts exhibit hepatic cell-cell contacts such as connexin-32 44 and bile canaliculi, 24 but also do not express the EGF-R and LDL-R. On the other hand, hepatocytes cocultured with LSEC show both traditional polarity markers, 45,46 and express a high level of sinusoidal receptors (EGF-R, LDL-R) at the interface between the hepatocytes and the LSEC. At least part of the interaction between hepatocytes and LSECs has been shown to be mediated by growth factors.…”

“…This result is surprising, because the coculture of hepatocytes with 3T3-J2 has been previously shown to express a wide range of liver-specific functions in vitro. 23,24 One possibility is that LSECs, not hepatocytes, take up DiI-LDL in coculture. To detect which cell type took up the LDL, we incubated the hepatocyte-LSEC cocultures in the presence of both DiI-LDL and FITC-acLDL.…”

A two-fluid model for tissue growth within a dynamic flow environment

“…Significant research has been dedicated to the study of tissue engineering processes relevant to liver, skin, collagen (for cartilage/tendon replacement) and bone (see [15,60] for interesting reviews) and progress has been made in a number of areas. These include: maintenance of tissue-specific functions via mimicry of in vivo conditions through appropriate cell co-culture and/or three-dimensional spheroidal culture [3,26,28,41,55]; and understanding the influence of artificial scaffolds which provide mechanical support for construct growth and whose surface chemistry and pore size may be altered to encourage cell anchorage or increased population of the scaffold [30,58,65].…”