Maintenance of human hepatocyte function in vitro by liver-derived extracellular matrix gels

Abstract

Tissue engineering and regenerative medicine (TE&RM) approaches to treating liver disease have the potential to provide temporary support with biohybrid-liver-assist devices or long-term therapy by replacing the diseased liver with functional constructs. A rate-limiting step for TE&RM strategies has been the loss of hepatocyte-specific functions after hepatocytes are isolated from their highly specialized in vivo microenvironment and placed in in vitro culture systems. The identification of a biologic substrate that can maintain a functional hepatocyte differentiation profile during in vitro culture would advance potential TE&RM therapeutic strategies. The present study compared two different biologic substrates for their ability to support human hepatocyte function in vitro: porcine-liver-derived extracellular matrix (PLECM) or Matrigel. Because Matrigel has been shown to be the most useful matrix for static, traditional hepatocyte culture, we directly compared PLECM with Matrigel in each experiment. Albumin secretion, hepatic transport activity, and ammonia metabolism were used to determine hepatocyte function. Hepatocytes cultured between two layers of PLECM or Matrigel showed equally high levels of albumin expression and secretion, ammonia metabolism, and hepatic transporter expression and function. We conclude that like Matrigel, PLECM represents a favorable substrate for in vitro culture of human hepatocytes.

FIG. 1.

Schematic of functional assays used to profile PHHs. Timeline represents days in culture. PHHs, primary human hepatocytes.

FIG. 2.

Macroscopic view of decellularized porcine-liver-derived extracellular matrix (PLECM) tissue is shown in (A). After solubilization and gelation the material has a smooth topography (B, 10 × ) and uniform consistency. The surface of Matrigel is shown for comparison (C, 10 × ). Color images available online at www.liebertonline.com/ten.

FIG. 3.

PHHs maintained for 10 days under different ECM conditions (100 × ). PHHs were cultured (A) on type-I collagen; (B) between two layers of Matrigel; (C) or between two layers of PLECM. PHHs cultured in a Matrigel (MG) or PLECM sandwich formed chord-like arrays. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Mean DNA content of cell cultured from six livers at the time of final harvest (Day 10). Values represent mean ± standard deviation (SD).

FIG. 5.

Average albumin mRNA expression of PHHs cultured in an MG or PLECM sandwich at day 5. Values represent mean ± SD of three livers.

FIG. 6.

Albumin daily secretion of PHHs cultured in an MG or PLECM sandwich at days 4, 6, 8, and 10. Values represent mean ± SD of three livers.

FIG. 7.

NTCP (A) and BSEP (B) mRNA expression levels of PHHs cultured in an MG or PLECM sandwich at day 5. Values represent mean ± SD of four livers. NTCP, sodium taurocholate cotransporting polypeptide; BSEP, bile salt export pump.

FIG. 8.

Hepatic transport activity of PHHs. (A) [3H]Taurocholate uptake and (B) [3H]taurocholate efflux of PHHs. Values represent mean ± SD of four livers.

FIG. 9.

Ammonia metabolism of PHHs cultured for 5 days in an MG or PLECM sandwich. All data were normalized to PHHs cultured on type-I collagen. Values represent mean ± SD of four livers.