Formation of functional, extended bile canaliculi, and increased bile acid production in sandwich-cultured human cryopreserved hepatocytes using commercially available culture medium.

Drug-induced cholestasis results in drug discontinuation and market withdrawal, and the prediction of cholestasis risk is critical in the early stages of drug development. Animal tests and membrane vesicle assay are currently being conducted to assess the risk of cholestasis in the preclinical stage. However, these methods have drawbacks, such as species differences with humans and difficulties in evaluating the effects of drug metabolism and other transporters, implying the need for a cholestasis risk assessment system using human hepatocytes. However, human hepatocytes hardly form functional, extended bile canaliculi, a requirement for cholestasis risk assessment. We previously established a culture protocol for functional, extended bile canaliculi formation in human iPSC-derived hepatocytes. In this study, we modified this culture protocol to support the formation of functional, extended bile canaliculi in human cryopreserved hepatocytes (cryoheps). The production of bile acids, which induces bile canaliculi extension, increased time-dependently during bile canaliculi formation using this protocol, suggesting that increased bile acid production may be involved in the extended bile canaliculi formation. We have also shown that our culture protocol can be applied to cryoheps from multiple donors and that bile canaliculi can be formed stably among different culture batches. Furthermore, this protocol enables long-term maintenance of bile canaliculi and scaling down to culture in 96-well plates. We expect our culture protocol to be a breakthrough for in vitro cholestasis risk assessment.

Human intestinal organoid-derived PDGFRα + mesenchymal stroma enables proliferation and maintenance of LGR4 + epithelial stem cells.

BACKGROUND: Intestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture. METHODS: We established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies. RESULTS: The proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture. CONCLUSIONS: The high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

Construction of extended and functional bile canaliculi using long-term sandwich-cultured cryopreserved human hepatocytes and the application of hepatocytes for predicting the biliary excretion of pharmaceutical and food-related compounds.

The biliary excretion of pharmaceutical and food-related compounds is an important factor for assessing pharmacokinetics and toxicities in humans, and a highly predictive in vitro method for human biliary excretion is required. We have developed a simple in vitro culture method for generating extended and functional bile canaliculi using cryopreserved human hepatocytes. We evaluated the uptake of compounds by hepatocytes and bile canaliculi, and the biliary excretion index (BEI) was calculated. After 21 days of culture, the presence of extended and functional bile canaliculi was confirmed by the uptake of two fluorescent substrates. Positive BEIs were observed for taurocholic acid-d4, rosuvastatin, pitavastatin, pravastatin, valsartan, olmesartan, and topotecan (reported biliary-excreted compounds in humans), but no difference in BEI was observed for salicylic acid (a nonbiliary-excreted compound). Furthermore, 8 of 21 food-related compounds with specific structures and reported biliary transporter involvement exhibited positive BEIs. The developed in vitro system was characterized by functional bile canaliculus-like structures, and it could be applied to the prediction of the biliary excretion of pharmaceutical and food-related compounds.

Construction of a culture protocol for functional bile canaliculi formation to apply human iPS cell-derived hepatocytes for cholestasis evaluation.

Cholestatic toxicity causes the failure of pharmaceutical agents during drug development and, thus, should be identified at an early stage of drug discovery and development. The formation of functional bile canaliculi in human hepatocytes is required for in vitro cholestasis toxicity tests conducted during the early stage of drug development. In this study, we investigated the culture conditions required for the formation of bile canaliculi using human-induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps). When hiPSC-Heps were sandwich-cultured under the condition we established, extended bile canaliculi were formed on the whole well surfaces. Biliary efflux transporters were localized in the formed bile canaliculi structures which had junctional complexes. After the model substrates of the biliary efflux transporters were taken up into cells, their subsequent excretion into the bile canaliculi was observed and was found to be impeded by each inhibitor of the biliary efflux transporter. These findings suggest that bile canaliculi have transporter-specific bile excretion abilities. We will continue to study the application of this culture protocol to cell-based cholestasis assay system. As a result, the culture protocol could lead to a highly predictable, robust cell-based cholestasis assay system because it forms functional bile canaliculi reproducibly and efficiently.

Consideration of Commercially Available Hepatocytes as Cell Sources for Liver-Microphysiological Systems by Comparing Liver Characteristics.

In recent years, microphysiological systems (MPS) have been developed to shorten the test period and reduce animal experiments for drug development. We examined cell sources for the liver-MPS, i.e., MPS mimicking liver function. For liver-MPS, liver-like cells with high liver functions are required. Cryo-preserved hepatocytes (cryoheps), the gold standard hepatocytes for in vitro drug development, present several disadvantages, including differences between lots due to individual donor variations or a limited cell supply from the same donor. As such, alternatives for cryoheps are sought. Hepatocyte-like cells derived from human induced pluripotent stem cells (hiPSC-Heps), hepatocytes derived from liver-humanized mice (PXB-cells), and human liver cancer cells (HepG2 cells) were examined as source candidates for liver-MPS. Gene expression levels of the major cytochrome P450 of hiPSC-Heps, PXB cells, and HepG2 cells were compared with 22 lots of cryoheps, and the activities of hiPSC-Heps were compared with 8 lots of cryopreserved hepatocytes. A focused DNA microarray was used for the global gene analysis of the liver-like characteristics of hiPSC-Heps, PXB-cells, cryoheps, and HepG2 cells. Gene expression data from the focused microarray were analyzed by principal component analysis, hierarchical clustering, and enrichment analysis. The results indicated the characteristics of individual hepatocyte cell source and raised their consideration points as an alternative cell source candidate for liver-MPS. The study contributes to the repetitive utilization of a robust in vitro hepatic assay system over long periods with stable functionality.

Immortalization of human hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing.

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

Upregulations of metallothionein gene expressions and tolerance to heavy metal toxicity by three dimensional cultivation of HepG2 cells on VECELL 3-D inserts.

The VECELL 3-D insert is a new culture scaffold consisting of collagen-coated ePTFE (expanded polytetrafluoroethylene) mesh. We analyzed the effects of VECELL 3-D inserts on the functionality of HepG2, a human hepatocellular carcinoma cell line. HepG2 cells cultured on VECELL 3-D inserts maintained a round shape, while those cultured on a standard culture plate or collagen-coated cell culture plate showed a flattened and cubic epithelial-like shape. HepG2 cells cultured on VECELL 3-D inserts had showed upregulated expression of metallothionein genes and in turn a higher tolerance to toxicity induced by heavy metals. These results suggest that HepG2 cell functions were changed by the cell morphology that is induced by culturing on a VECELL 3-D insert.

Global gene expression changes including drug metabolism and disposition induced by three-dimensional culture of HepG2 cells-Involvement of microtubules.

Constitutive upregulation and a higher degree of induction of drug metabolism and disposition-related genes were found in a three-dimensional HepG2 culture. The upregulated genes are believed to be regulated by different regulatory factors. Global gene expression analysis using the Affymetrix GeneChip indicated that altered expression of microtubule-related genes may change the expressed levels of drug metabolizing and disposition genes. Stabilization of microtubule molecules with docetaxel, a tubulin-stabilizing agent, in the two-dimensional culture showed gene expression patterns similar to those found in the three-dimensional culture, indicating that the culture environment affects drug metabolism functions in HepG2 cells.

Change in broth culture is associated with significant suppression of Escherichia coli death under high magnetic field.

When Escherichia coli B was cultivated under an inhomogeneous magnetic field of 5.2-6.1 T, a significant 100,000-fold suppression of cell death was observed [Bioelectrochemistry 53 (2001) 149]. The limited magnetic field exposure for 12 h after logarithmic growth phase was sufficient to observe similar suppressive effects on cell death [Bioelectrochemistry 54 (2001) 101]. These results suggest some possible changes in either the medium or the cells during the magnetic field exposure. When the cell-free filtrate of the broth cultured under the magnetic field for 10 h and the cells of E. coli cultivated under the geomagnetic field for 30 h were mixed, and the mixture was subsequently cultivated under the geomagnetic field, the number of cells observed in the filtrate exposed to the high magnetic field was 20,000 times higher than that in the filtrate exposed to the geomagnetic field. When the cells cultivated under the magnetic field for 10 h and the cell-free filtrate of the broth culture exposed to the geomagnetic field were mixed, only a 50-fold difference in the number of cell between under the magnetic field and under the geomagnetic field was observed. This suggests that the filtrate of the broth culture exposed to the magnetic field is primarily responsible for the cell death suppression. It was also revealed that the small difference in pH of the filtrates of the broth culture between under the magnetic field and under the geomagnetic field was critical for the cell death suppression.