Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

Abstract

Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

Figure 1

Scanning electron microscopy. Porous foamed hydroxyapatite scaffolds (a) were used for cell culture. After 28 days of culture, numerous cells attached to the scaffold could be observed in cultures with collagen-1 (b), whereas considerably less cells were attached to scaffolds in culture with hyaluronan (c).

Figure 2

Numbers of liver cells in culture. Cells were cultured for 5, 15, and 28 days with (w) or without (wo) hydroxyapatite scaffolds embedded in hyaluronic acid gel (HyA), collagen-1 gel (C1), or no gel (NG), and cell numbers were determined by correlation with DNA concentration. Data are given as means from 6 biological repeats ± standard deviation.

Figure 3

Albumin secretion of liver cell cultures. Cells were cultured for 5, 15, and 28 days with (w) or without (wo) hydroxyapatite scaffolds embedded in hyaluronic acid gel (HyA), collagen-1 gel (C1), or no gel (NG), and albumin secretion was measured by ELISA. Data are given as means from 6 biological repeats ± standard deviation.

Figure 4

Immunocytochemistry of liver cell cultures. Liver cells were cultured for 28 days and stained, and images were taken by confocal microscopy. (a) Cells cultured in collagen-1 gel; (b) cells cultured in collagen-1 gel on hydroxyapatite scaffold; (c) cells cultured on hydroxyapatite scaffold only. Blue = DAPI, red = phalloidin, and green = CK19.