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INTRODUCTION: Liver diseases are becoming a significant public medical burden[1]. To overcome the lack of donor livers and high cost of therapy, this project aims to exploit a long-term culture system that maintains hepatocyte specific functions [2] and cell viability for liver regeneration using self-assembled peptide hydrogels to mimic the normal hepatic extracellular matrix (ECM). Previous work [3] proved that a three-dimensional cell culture environment could keep differentiated hepatocytes compared with in vitro cell culture on a flat surface.
METHODS: All tested self-assembled peptide hydrogels, PeptiGel® Alpha1, Alpha2, Alpha4, Alpha5, Alpha6 and Alpha7 were purchased from Manchester BIOGEL. HepG2 cells (1x106) were suspended in 166μl fresh media then encapsulated within 1ml peptide hydrogel to get a homogeneous cell-gel mixture. 100μl of cell-gel mixture were placed into 24-well inserts, with fresh media added to surround each insert and on top of the sample. The viscoelastic properties of peptide hydrogels and the porcine liver tissue were measured using rheometry. The Cell viability at days 1, 3, 7 and 14 were investigated using LIVE/DEAD® cell viability assay with confocal microscopy. QuantIT Picogreen assay was performed to compare the cell proliferation. To investigate hepatic functions, the cell culture medium was collected every 48 hours to test albumin production and urea secretion by using Human Albumin ELISA Kit (ab179887) and Urea Assay Kit (ab83362). Cytochrome P450 Metabolism and drug response were analysed using qPCR at days 1, 3, 7 and 14. Histology staining was used to observe the morphology of HepG2 cells encapsulated within peptide gel.
RESULTS: The theoretical net charge and mechanical stiffness of peptide hydrogels were investigated. A cell viability assay showed that over 14 days in vitro culture, HepG2 cells proliferated within Alpha5 and Alpha7, would survive within Alpha1, Alpha2, Alpha6. HepG2 cells displayed different viability in pH9 Alpha1 and ph4 Alpha1. During 14 days in vitro culture, ELISA assay revealed a significant increase of Albumin production; HepG2 cells maintained urea secretion and the level increased after day7. qPCR determined increased level of RNA expression for CYP1A2, CYP2C9, CYP3A4 and Albumin but no significant difference of CYP2E1 gene level, from day1 to day14. It was observed that cells produced fibronectin, laminin, collagen I and intercellular albumin according to histology staining.
DISCUSSION AND CONCLUSIONS:
Hepatocytes could maintain cell viability and proliferation when encapsulated within negative-charged peptides rather than within positive-charged peptides. Hepatocytes can maintain the main hepatic functions within Alpha7 gel over 14 days. The results show the potential of 3D peptide hydrogels for drug toxicity prediction and liver regeneration. The investigations of hepatocytes drug metabolism are underway.
REFERENCES:
[1] L. Rolfe et al., Lancet, vol. 391, no. 10125, pp. 1097–1107, 2017.
[2] O. Ogoke, J. Oluwole, and N. Parashurama, Journal of Biological Engineering, vol. 11, no. 1. 2017.
[3] C. T. Nicolas et al., Stem Cells, vol. 35, no. 1, pp. 42–50, 2017.
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