Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix

Abstract

Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 +/- 0.37 and 9 +/- 2.7 nmol glucose/h/microg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 +/- 4 nmol urea/h/microg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 +/- 152 pmol/microg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and glutathione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-alpha and -beta, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I).

Transcytosis: Crossing Cellular Barriers

“…As the importance of the extracellular matrix in maintaining gene expression and promoting cell repolarization became apparent, investigators began culturing hepatocytes on different matrix components in various geometries and physical states. The most successful reconstitution of polarity has come from the use of collagen gels or Matrigel in a sandwich configuration (194,328). Despite these advances, the limited lifespan of primary hepatocytes and the difficulties in reproducibly obtaining such polarized cultures prompted the use of secondary hepatocyte cell lines.…”

A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro

“…Studies have shown that gene expression profiles of 3D-cultured cells are dramatically different to monolayer cultures and more closely resemble the profiles of the tissues of origin than their 2D counterparts. [11][12][13][14] 3D multicellular aggregate (MCA) structures also contain many features absent in 2D cultures that are present in primary tissues. For example, in MCAs of tumor cells hypoxic and necrotic areas can develop, regions that are commonly found in primary tumors and known to be associated with chemoresistance.…”

Perspectives on whole-organ assembly: moving toward transplantation on demand

“…Research into these cell-cell interactions is further confounded by the diversity of supportive cell types that are found in whole organs. Nearly all cells in the body require cues from a truly 3D environment to assemble relevant physiological tissue structures that functionally mimic authentic organs, including appropriate cell adhesion, migration, contraction, metabolic function, and differentiation (107)(108)(109)(110)(111)(112)(113). Because ECM scaffolds largely retain the 3D tissue architecture and composition of the tissue from which they are isolated (23,26,28), they can provide the physiological 3D anatomical structures of the native organ, including vascular conduits, which are difficult to manufacture in vitro (5,9,42,114,115).…”