Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes

Abstract

Primary hepatocytes are commonly used as liver surrogates in toxicology and tissue engineering fields, therefore, maintenance of functional hepatocytes in vitro is an important topic of investigation. This paper sought to characterize heparin-based hydrogel as a three-dimensional scaffold for hepatocyte culture. The primary rat hepatocytes were mixed with a prepolymer solution comprised of thiolated heparin and acrylated poly(ethylene glycol) (PEG). Raising the temperature from 25 degrees to 37 degrees C initiated Michael addition reaction between the thiol and acrylated moieties and resulted in formation of hydrogel with entrapped cells. Analysis of liver-specific products, albumin and urea, revealed that the heparin hydrogel was non-cytotoxic to cells and, in fact, promoted hepatic function. Hepatocytes entrapped in the heparin-based hydrogel maintained high levels of albumin and urea synthesis after three weeks in culture. Because heparin is known to bind growth factors, we incorporated hepatocyte growth factor (HGF)-an important liver signaling molecule - into the hydrogel. HGF release from heparin hydrogel matrix was analyzed using enzyme linked immunoassay (ELISA) and was shown to occur in a controlled manner with only 40% of GF molecules released after 30 days in culture. Importantly, hepatocytes cultured within HGF-containing hydrogels exhibited significantly higher levels of albumin and urea synthesis compared to cells cultured in the hydrogel alone. Overall, heparin-based hydrogel showed to be a promising matrix for encapsulation and maintenance of difficult-to-culture primary hepatocytes. In the future, we envision employing heparin-based hyrogels as matrices for in vitro differentiation of hepatocytes or stem cells and as vehicles for transplantation of these cells.

Figure 1

Diagrams describing gel chemistry and cell cultivation experiments. (A) Heparin gel was formed by Michael addition reaction of thiolated heparin and acrylated poly(ethylene glycol) (PEG). (B) Primary rat hepatocytes were mixed with liquid gel precursors at room temperature and became encapsulated in the gel upon increasing the temperature to 37°C. Hepatocyte growth factor (HGF) was added into the gel precursor solution along with cells.

Figure 2

Characterizing viability of primary rat hepatocytes entrapped in heparin and PEG hydrogel. (A–B) Representative brightfield images of hepatocytes in the form of single cells (A) and spheroids (B) entrapped in a gel. Scale bars are 200 μm. (C–D) Live/dead staining of hepatocytes entrapped in heparin hydrogel as single cells (C) and spheroids (D) for one day. Viability exceeded 70%. (E–F) Live/dead staining of hepatocytes entrapped in PEG hydrogel as single cells (E) and spheroids (F) for one day. In both cases viability was ~20%. (G) Confirming hepatocyte viability with WST-1 assay. In both cases of the single cells and spheroids, higher viability was observed for hepatocytes entrapped in heparin hydrogels compared to PEG hydrogels. (**) p<0.001 (n=4).

Figure 3

The effects of heparin gel concentration on function of hepatocyte spheroids. Albumin (A) and urea (B) production of hepatocyte spheroids suggested that 10 wt % heparin hydrogel was optimal. (*) p<0.05 and (#) p<0.0001 (n=4).

Figure 4

In vitro release profile of HGF release from heparin (●) and PEG (○) hydrogels. HGF was mixed at 1 μg/ml concentration with gel precursor solution. More than 60% of HGF was retained in heparin hydrogels after 30 days while PEG hydrogels completely released HGF in 15 days (n=3).

Figure 5

The function of primary hepatocytes encapsulated in heparin and PEG hydrogels as single cell with or without HGF. The levels of hepatic production of albumin (A) and urea (B) were significantly higher in heparin hydrogel compared to PEG hydrogel. The addition of HGF to heparin hydrogel further enhanced albumin and urea synthesis of hepatocyte. In contrast, incorporation of HGF into PEG hydrogel had no significant effect on hepatic function. (*) p<0.05, (**) p<0.001, and (#) p<0.0001 (n=4).

Figure 6

The function of hepatocyte spheroids encapsulated in heparin and PEG hydrogels with or without HGF. The albumin (A,B) and urea (C) synthesis of hepatocyte spheroids was maintained at a high level after 20 days inside heparin hydrogel and was enhanced even further by incorporating HGF into the gel. In contrast, hepatocyte spheroids in PEG gel had much lower albumin and urea production and were unaffected by addition of HGF. (**) p<0.001 and (#) p<0.0001 (n=4). (B) Immunofluorescence staining for albumin in hepatocyte spheroids after 14 days of cultivation in heparin hydrogel. Hepatocytes in heparin hydrogels showed strong staining for albumin (red). Staining of cell nucleus with DAPI (blue) was used to demonstrate localization of albumin in the cytoplasm. All scale bars are 50 μm.