Poster BB15 - Extracellular matrix proteins and mimics influence human hepatic organoid function and phenotype.

Introduction: Liver disease accounts for 4% of deaths worldwide.1 When patients reach end-stage liver failure, the only treatment option is receiving a transplantation. The field of liver tissue engineering seeks to alleviate this problem by creating functional engineered liver tissues that can serve as transplantable organs. A critical challenge in engineering liver tissues is that hepatocytes are highly sensitive to their microenvironment and often lose their hepatic phenotype and function when cultured in-vitro. Similarly, our lab has observed a loss of hepatic phenotype when adult human hepatocyte organoids (AHHOs) suspended in gelatin methacryloyl (GelMA), a collagen derivative, were implanted in-vivo. I hypothesize that this loss of hepatic phenotype is because these biomaterials currently do not accurately mimic the hepatocyte microenvironment in the liver. To test this hypothesis, I have incorporated peptides commonly present in the extracellular matrix (ECM) proteins of the liver with hepatocytes into hydrogels and assessed their ability to support AHHOs function and phenotype. Specifically, I investigated how arginylglycylaspartic acid (RGD), a cell-matrix adhesion peptide found in fibronectin, and aspartic acid-glycine-glutamic acid-alanine (DGEA), a peptide found in collagen, influence hepatic function and phenotype during AHHOs culture. Given that both fibronectin and collagen are prevalent ECM proteins in the liver, these peptides may play a role in the regulation of hepatic phenotype and function through integrin receptors.2 This work will ultimately enable us to preserve encapsulated hepatocyte phenotype in-vitro and in downstream in-vivo applications while shedding light on the mechanisms underlying hepatic cell-matrix interactions.

Materials and

Methods: Human hepatocytes purchased from Thermo Fisher Scientific were thawed and resuspended in basal media before being spun down at 70 xg for 5 minutes.3 Hepatocytes were diluted to a concentration of 2 million cells per mL and suspended in either 55wt% Matrigel or peptide prepolymer solution. Hanging droplets were created by pipetting 20 µL of the hepatocyte-Matrigel suspension into the center of a 48-well plate and then quickly inverted. To culture hepatocytes in peptide-conjugated hydrogels, hepatocytes were carefully resuspended in 3mM 8-arm PEG-NB, 12 mM dicysteine crosslinker, 2 mM LAP, and 1mM peptide of interest (DGEA and/or RGD or no peptides) with organoid growth media. 20 µL of the solution was transferred to each well of 48 well plates as explained earlier and polymerized at 24.5 mW/cm2 for 90 seconds using a 405 nm light projector. Afterward, 200 µL of organoid growth media was used to feed encapsulated hepatocytes in all conditions. Albumin secretion and urea activity were quantified in the collected media using ELISA and urease activity assay kit from Stanbio Labs, respectively. All condition samples were fixed, 3D cleared,6, and immuno-stained with either Hoechst + CK19 + ASGPR-1 or Hoechst + HNF4-α + E-cad antibodies in triplicate. The fluorescent area for each antibody was quantified by taking maximum intensity projections and measuring the number of pixels with signal above a set threshold. GraphPad Prism was used to generate plots and perform statistical analysis.

Results, Conclusions, and Discussions: There is an urgent need for a platform that can support hepatocyte viability, function, and phenotype in-vitro. Single-cell hepatocytes seeded directly into peptide-conjugated hydrogels formed organoids in all conditions. Although results thus far have not demonstrated differences in hepatic phenotype between DGEA and RGD peptide-conjugated hydrogels, further optimization of peptide-conjugated hydrogel composition or culture conditions may help. Impressively, these hydrogels supported hepatocyte organoid formation from single cells without stromal cell support and in a synthetic material which has not effectively been done previously. The organoid size distribution that was observed in these hydrogels was comparable to organoids grown in Matrigel, further attesting to their ability to support hepatocyte organoid growth. There was also no formation of cystic organoids with biliary phenotypes in the hydrogels, even at later culture days, while these cysts repeatedly appeared in Matrigel condition after Day 12 – suggesting that hepatocytes are transdifferentiating into Cholangiocytes. This indicates that peptide-conjugated hydrogels may maintain specific aspects of hepatocyte cell identity in long-term cell culture better than Matrigel. Nonetheless, these hydrogels are yet to show the ability to recapitulate the functional capacity observed in the Matrigel as evidenced by albumin and urea synthesis. This challenge may be because Matrigel is rich in basement membrane proteins that hydrogels lack and prompts the question of whether ECM protein concentration influences both the phenotype and function of hepatocytes. We are currently investigating this question by examining differences between biological hydrogels, incorporating other peptides that mimic essential proteins found in the liver ECM into PEG-NB, and investigating the role of non-parenchymal cells on AHHOs

Acknowledgements (Optional): Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle