Rat liver ECM incorporated into electrospun polycaprolactone scaffolds as a platform for hepatocyte culture

Abstract

Liver disease is expanding across the globe; however, health-care systems still lack approved pharmaceutical treatment strategies to mitigate potential liver failures.Organ transplantation is the only treatment for liver failure and with increasing cases of liver disease, transplant programs increasingly cannot provide timely transplant availability for all patients. The development of pharmaceutical mitigation strategies is clearly necessary and methods to improve drug development processes are considered vital for this purpose. Herein, we present a methodology for incorporating whole organ decellularised rat liver ECM (rLECM) into polycaprolactone (PCL) electrospun scaffolds with the aim of producing biologically relevant liver tissue models. rLECM PCL scaffolds have been produced with 5 w/w% and 10 w/w% rLECM:PCL and were analyzed by SEM imaging, tensile mechanical analyses and FTIR spectroscopy. The hepatocellular carcinoma cell line, HepG2, was cultured upon the scaffolds for 14 days and were analyzed through cell viability assay, DNA quantification, albumin quantification, immunohistochemistry, and RT-qPCR gene expression analysis.

A Unification of Nanotopography and Extracellular Matrix in Electrospun Scaffolds for Bioengineered Hepatic Models

“…While we did see increases within the culture periods, we did not see differences between groups. A contradictory finding has been shown previously, where we found a statistical increase in viability in the dECM scaffolds compared to the control group. ,, This in part may be explained by the comparatively decreased protein addition in this current study, incorporating an amount 10 times lower.…”

“…In particular, albumin, which is involved in the transport of various hormones, enzymes, and vitamins and maintains an appropriate osmotic pressure of blood, had an increasing trend on both 0.07% DFECMS and 0.14% DFECMS. Our findings agree with previous research where the addition of dECM into the PCL electrospun fiber scaffold improved the production of albumin over time. ,, The increasing trend might also occur due to the increased cell–cell interaction on both dECM scaffolds (Figure ), as suggested previously . In addition, apart from albumin gene expression, we also looked at protein production.…”

“…dECM is water-insoluble, and only a few solvents can be used to dissolve it for electrospinning purposes . Hexafluoroisopropanol (HFIP) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) is one of the mainly used solvents for ECM dissolution in electrospun scaffolds. ,,− However, in our study, in order to create the surface depression structure, different solvent systems (CFM/DMSO) needed to be used. dECM is poorly soluble in CFM because components like collagen and elastin are hard to break down due to their cross-linking of amino acid chains. , Our method of solubilizing dECM enzymatically in pepsin/HCL provides a favorable way to break down the insoluble components into smaller peptides. , Enzymatic pepsin digestion in an acidic buffer is the gold standard in solubilizing dECM, though the process can cause the loss of integrity to some ECM components. , Furthermore, the homogenization provided a good mechanical force to break down the big junctions left in the digested dECM, which was blended with PCL in our solvent system.…”

“…The cell-seeded samples ( N = 5) were placed in 500 μL of Tri-Reagent (Sigma) and stored at −80 °C. RNA was extracted from the thawed samples using CFM and purified using Qiagen’s RNeasy spin column system as per a previously described method and the manufacturer’s instructions . Nanodrop (Thermo Scientific NanoDrop 2000c spectrophotometer) was used to measure the nucleic acid concentration of each sample, and then RNA was reversed-transcribed to complementary DNA (cDNA) using reverse transcriptase (Promega) according to the manufacturer’s instructions.…”

Research progress in decellularized extracellular matrix hydrogels for intervertebral disc degeneration

“…Currently, many research theories about animal tissues or organs used as dECM have been established, but most focus on porcine SIS, bladder, heart, fat, nucleus pulposus and peritoneum, [85][86][87][88] bovine heart, 89,90 goat liver, 91,92 rat liver and heart. 93,94 However, heterologous dECM may be associated with a risk of viral infection, 95 and the composition, mechanical properties, and degradability of heterologous dECM may differ from those of human organs and tissues. 96 Humanderived dECM remains the preferred biomedical material to avoid viral contamination and immunogenic reactions, 97 but this type of dECM is limited in its application due to the scarcity of organs and tissues of human origin.…”