Three dimensional cultures of rat liver cells using a natural self‐assembling nanoscaffold in a clinically relevant bioreactor for bioartificial liver construction

Abstract

Till date, no bioartificial liver (BAL) procedure has obtained FDA approval or widespread clinical acceptance, mainly because of multifactorial limitations such as the use of microscale or undefined biomaterials, indirect and lower oxygenation levels in liver cells, short-term undesirable functions, and a lack of 3D interaction of growth factor/cytokine signaling in liver cells. To overcome preclinical limitations, primary rat liver cells were cultured on a naturally self-assembling peptide nanoscaffold (SAPN) in a clinically relevant bioreactor for up to 35 days, under 3D interaction with suitable growth factors and cytokine signaling agents, alone or combination (e.g., Group I: EPO, Group II: Activin A, Group III: IL-6, Group IV: BMP-4, Group V: BMP4 + EPO, Group VI: EPO + IL-6, Group VII: BMP4 + IL-6, Group VIII: Activin A + EPO, Group IX: IL-6 + Activin A, Group X: Activin A + BMP4, Group XI: EPO + Activin A + BMP-4 + IL-6 + HGF, and Group XII: Control). Major liver specific functions such as albumin secretion, urea metabolism, ammonia detoxification, phase contrast microscopy, immunofluorescence of liver specific markers (Albumin and CYP3A1), mitochondrial status, glutamic oxaloacetic transaminase (GOT) activity, glutamic pyruvic transaminase (GPT) activity, and cell membrane stability by the lactate dehydrogenase (LDH) test were also examined and compared with the control over time. In addition, we examined the drug biotransformation potential of a diazepam drug in a two-compartment model (cell matrix phase and supernatant), which is clinically important. This present study demonstrates an optimized 3D signaling/scaffolding in a preclinical BAL model, as well as preclinical drug screening for better drug development.

Nanostructured self-assembling peptides as a defined extracellular matrix for long-term functional maintenance of primary hepatocytes in a bioartificial liver modular device

“…The protocol of MTT assay ethoxyresorufin-O-deethylase is given in our previous report. 31 α-glutathione S-transferase (GST) was measured in the culture supernatant as a marker for hepatic damage by a commercially available enzyme immunoassay method (Biotrin Rat Alpha GST EIA; Biotrin, Dublin, Ireland).…”

“…13 Our bioreactor also allows direct contact of every individual liver cell with the oxygen supply, for enhanced oxygenation that is closer to the in vivo liver. 5,11,12 While the available image data was obtained from CLSM, basically providing the image series for our image analysis, we have restricted ourselves to 2D image-based assessments on single images taken centrally from the stacks. The reason was two-fold: first the cells considerably adhere to the ground, leading to heavy distortions so that the cells appear to be strongly flattened.…”

“…However, most experimental approaches aimed at the long-term functional maintenance of primary hepatocytes in a culture from 1 week to several weeks (42 days) have been reported by various investigators, [2][3][4] including us. [11][12][13] However, to the best of our knowledge, there is no report for the long-term functional maintenance of primary hepatocytes up to 90 days within a defined hepatic microenvironment to provide a better strategy as an alternative to in vivo animal experimentation. US Food and Drug Administration (FDA) rules require the survival potential of primary hepatocytes of a minimum of 14 days and a maximum of 90 days for in vivo toxicological experiments.…”

Idiosyncratic Drug-Induced Liver Injury (IDILI): Potential Mechanisms and Predictive Assays

“…Due to the fact that ADRs blamed on antioxidant toxicity are associated with proteins found in the mitochondrion, it is the primary indicator for liver functionality tests. Certain assays, such as the cupric ion reducing antioxidant capacity (CUPRAC) assay [146] or the MitoTracker dye [147], can rely on calculating general antioxidant capacity within a cell, or confocal microscopy to examine individual mitochondrial behavior. However, general assays are more geared towards calculating antioxidant potential associated with smaller molecules, including ascorbic acid and tocopherol.…”

Characterization of a Liver Organoid Tissue Composed of Hepatocytes and Fibroblasts in Dense Collagen Fibrils

“…[10][11][12][13] There are also some recent studies using collagen sandwich hepatocyte cultures that have achieved long culture periods and the maintenance of hepatic functions. [14][15][16] However, no studies have described the reconstruction of highly concentrated connective tissue in vitro with properties similar to endogenous liver tissue. In the liver, type I collagen fibrils serve as a primary scaffold upon which are deposited microfibrils and filaments of collagen types III, V, and VI.…”