Much effort has been directed towards the optimization of the capture of in vivo hepatocytes from their microenvironment. Some methods of capture include an ex vivo cellular model in a
bioreactor based liver module, a micropatterned module, a microfluidic 3D chip, coated plates, and other innovative approaches for the functional maintenance of primary hepatocytes. However, none of the above methods meet US Food and Drug Administration (FDA) guidelines, which recommend and encourage that the duration of a toxicity assay of a
drug should be a minimum of 14 days, to a maximum of 90 days for a general toxicity assay. Existing innovative reports have used undefined extracellular matrices like
matrigel, rigid
collagen, or serum supplementations, which are often problematic, unacceptable in preclinical and clinical applications, and can even interfere with experimental outcomes. We have overcome these challenges by using integrated nanostructured self-assembling
peptides and a special combination of
growth factors and
cytokines to establish a proof of concept to mimic the in vivo hepatocyte microenvironment pattern in vitro for predicting the in vivo
drug hepatotoxicity in a scalable
bioartificial liver module. Hepatocyte functionality (
albumin,
urea) was measured at days 10, 30, 60, and 90 and we observed stable
albumin secretion and
urea function throughout the culture period. In parallel,
drug metabolizing
enzyme biomarkers such as
ethoxyresorufin-O-deethylase, the methylthiazol tetrazolium test, and the
lactate dehydrogenase test were carried out at days 10, 30, 60, and 90. We noticed excellent mitochondrial status and membrane stability at 90 days of culture. Since alpha
glutathione S-transferase (GST) is highly sensitive and a specific marker of hepatocyte injury, we observed significantly low alpha GST levels on all measured days (10, 30, 60, and 90). Finally, we performed the image analysis of mitochondria-cultured hepatocytes at day 90 in different biophysical parameters using confocal microscopy. We applied an automatic algorithm-based method for 3D visualization to show the classic representation of the mitochondrial distribution in double hepatocytes. An automated morphological measurement was conducted on the mitochondrial distribution in the cultured hepatocytes. Our proof of concept of a scalable
bioartificial liver modular device meets FDA guidelines and may function as an alternative model of animal experimentation for pharmacological and toxicological studies involving
drug metabolism, enzyme induction,
transplantation, viral
hepatitis, hepatocyte regeneration, and can also be used in other existing
bioreactor modules for long-term culture for up to 90 days or more.