While hepatic cell lines are mainly used for in vitro drug induced toxicity studies, they exhibit limited functionalities. To overcome this, the authors have employed genetically engineered mouse hepatoma cells, Hepa/8F5, wherein expression of liver enriched transcription factors is induced by doxycycline leading to increased functionality. Further enhancement in functionality is achieved by spheroid culture in a previously developed 3D cell culture platform. Cells are seeded in presence of temperature-responsive poly(N-isopropylacrylamide) on poly(N-isopropylacrylamide--co-gelatin) cryogel scaffold based high throughput platform. Cells seeded in presence of poly(N-isopropylacrylamide) and induced with doxycycline exhibited highest functionalities. There is an increase of ≈26, 36, and 39% in albumin secretion, ammonia removal, and CYP3A4 activity, respectively. Morphological analysis showed arrest in cell proliferation and enlarged nucleus in presence of doxycyline and spheroid formation in presence of poly(N-isopropylacrylamide). Drug induced liver toxicity studies revealed that cells induced with doxycycline are resistive to tamoxifen but sensitive to acetaminophen whereas, cultures initiated in presence of poly(N-isopropylacrylamide) are resistive to both the drugs which is indicative of diffusional barrier of the spheroids. The authors conclude that Hepa/8F5 cells show enhanced functionality in cryogel based spheroid culture platform which can be successfully used for high throughput screening of hepatic toxicity in vitro.

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Hepatoma cells are a candidate cell source for bio-artificial livers. However, they exhibit reduced liver functions compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells were created by transduction with vectors mediating inducible overexpression of eight liver-enriched transcription factors. Upon the induction of the liver-enriched transcription factors transduced, the cells expressed both phenotypic and genotypic liver functions at high levels. In the present study, we performed three-dimensional culture of these cells using macroporous gelatin beads. When immobilized on the macroporous gelatin beads, these cells exhibited further enhancement in liver functionality, including increased albumin secretion, ammonia removal and cytochrome P450 activity. The levels of these functions were significantly enhanced compared to monolayer culture. The method is simple and scalable, and provides highly functional cells that can be used in basic and applied fields of hepatic research.

Tightly regulated and robust mitochondrial activities are critical for normal hearing. Previously, we demonstrated that Fus1 KO mice with mitochondrial dysfunction exhibit premature hearing loss. Molecular analysis of the cochlea revealed hyperactivation of the mTOR pathway, oxidative stress, altered mitochondrial morphology and quantity, suggesting compromised energy sensing and production. Here, we investigated whether pharmacological modulation of metabolic pathways using rapamycin (RAPA) or 2-deoxy-D-glucose (2-DG) supplementation can protect against hearing loss in female Fus1 KO mice. Additionally, we aimed to identify mitochondria- and Fus1-dependent molecular pathways and processes critical for hearing. We found that inhibiting mTOR or activating alternative mitochondrial energetic pathways to glycolysis protected hearing in the mice. Comparative gene expression analysis revealed dysregulation of critical biological processes in the KO cochlea, including mitochondrial metabolism, neural and immune responses, and cochlear hypothalamic–pituitary–adrenal axis signaling system. RAPA and 2-DG mostly normalized these processes, although some genes showed a drug-specific response or no response at all. Interestingly, both drugs resulted in a pronounced upregulation of critical hearing-related genes not altered in the non-treated KO cochlea, including cytoskeletal and motor proteins, and Ca2+-linked transporters and voltage-gated channels. These findings suggest that pharmacological modulation of mitochondrial metabolism and bioenergetics may restore and activate processes critical for hearing, thereby protecting against hearing loss