The clay nano-particles gel, a novel differentiation matrix for human liver cells

Khedr, M.E.M.S., Polding, Samuel J., Abuawwad, Mahmoud, Alzoubi, Mohammad N., Mousa, Mohamed, Dawson, Jonathan, Abu-Hilal, Mohammed and Khakoo, Salim (2018) The clay nano-particles gel, a novel differentiation matrix for human liver cells. The British Association for the Study of the Liver: Annual Meeting, University of York, York, United Kingdom. 18 - 21 Sep 2018.

Abstract

Background:
Growing liver cells in 3D cultures potentially mimics the natural liver cell-cell interactions which favour long term cell viability, functionality and genetic stability. Clay nanoparticle hydrogels offer excellent biocompatibility and permeability for water-soluble metabolites, with the potential for self-organization in situ to allow delivery of growth factors. We have investigated the clay hydrogel as a novel culture matrix for culture of hepatocytes and hepatic stem/progenitor cells (HPCs).
Methods:
Liver specimens were obtained from patients undergoing liver surgery. Hepatocytes were extracted by a two-step collagenase protocol and EpCAM+ HPCs were isolated from the non-parenchymal cell population using magnetic beads cell sorting. Cells were encapsulated in clay (Laponite) nanoparticles hydrogel in Williams’ E conditioned medium. HPCs medium was supplemented with EGF, HGH and IL6 (10ng/ml each). Characterisation of HPCs was performed by immunofluorescence and analysis of mRNA expression by rtPCR. The differentiation of HPCs in clay hydrogel was compared with cells cultured in HydroMatrix peptide hydrogel, or on collagen over 5 days. Cell culture supernatant were tested for albumin, urea and LDH concentrations and MTT assays was performed.
Results:
Primary hepatocytes cultured in a clay hydrogel demonstrated low levels of LDH and survived up to 15 days. In contrast, hepatocytes cultured on collagen released high levels of LDH in the early days of culture and their survival was about 31.5% by day 3. Interestingly, hepatocytes grown in clay gels demonstrated a marked increase in albumin secretion with time (mean 100.2 ± 44.12 ng ml-1 at day 15), but cells cultured on collagen rapidly lost their ability to produce albumin (mean 26.44 ± 21.17 ng ml-1 at day 15). No marked difference was observed in urea production between both cultures.
EpCAM+HPCs cultured in clay hydrogel (without differentiation factors) showed high albumin levels, with a mean of 44.4 ±10.82 ng ml-1, at day 5 compared to 27.79 ± 16.61 ng ml-1 at day 3. Lower levels of albumin were released from cells grown on collagen or in HydroMatrix (mean 8.23 ± 0.73 ng ml-1, P = 0.282 and 14.37 ± 0.12 ng ml-1, P = 0.0003 respectively). Addition of 1% DMSO induced a minor increase in albumin levels (a mean of 54.18 ± SEM 14 ng ml-1) in the clay gel as did the wnt-3a (mean 51.05 ± SEM 11.55 ng ml-1).
Conclusion:
We show that laponite clay gels provide support for culture of primary human hepatocytes, with improved hepatocyte survival and synthesis properties.

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Contributors

Author: M.E.M.S. Khedr

Author: Jonathan Dawson

Author: Salim Khakoo

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