3D bioprinting of liver tissue models

National Center For Advancing Translational Sciences

Investigators

Abstract

The groups at UPitt and NCATS have been working on re-engineering the current 3D, chip-based, 4 liver cell vLAMPS NAFLD disease model developed at UPitt to a bioprintable 12-well transwell Liver Acinus model (BpLAMPS) for use as a medium and high throughput drug screening platform. So far, the UPitt group has optimized and verified stellate cell activation as a NAFLD disease progression marker when cultured 8-10 days in the presence of exogenously added molecular drivers of liver disease. In response to the addition of disease drivers including fatty acids, increased glucose, TGF-, and LPS, stellate cells become fibrogenic, indicated by increased smooth muscle actin (SMA) expression. High Content Screening (HCS) and immunofluorescence will monitor SMA expression. Alternate fibrosis markers (stellate cell proliferation, pro-collagen secretion) were also evaluated and shown expected responses. The group at UPitt have demonstrated the expected pharmacological effects of control drugs to reduce fibrogenic transformation consistent with a reversal or slowing of disease progression, including Sorafenib, Cenicriviroc, Simtuzumab and Selonsertib, which have been developed and tested in the clinic as antifibrotic agents in the BpLAMPS model. The PItt group is now working with NCATS to adapat the assay to 96-well plate in preparation of HTS at NCATS. The NCATS 3DTBL is using 3D liver spheroids generated with human primary hepatocytes, liver endothelial cells, stellate cells and kupffer cells, and combinations of hepatocytes with the different cells, and assess the toxic effects of ~ 200 known liver toxicants, as measure by cell viability, lipid accumulation, and generation of reactive oxygen species. The data will be used as input to generate in silico predictive models of DILI. This is a collaboration with the NIEHS Division of Translational Toxicology (DTT) and the NCATS Tox21 group to use 3D liver spheroid models to investigate drug-induced liver injury (DILI) and develop DILI in silico predictive models, as part of a funded NIH Directors Award. Liver spheroids have been generated using primary hepatocytes, stellate cells, Kupffer cells and endothelial cells and assay readout for cell viability and mitochondria membrane potential have been developed in 384-well plate. A screen of the DILI collection has been implemented using the cell viability assay and for one patient, and we are planning to implement for spheroids with cells from different patients and using additional readouts. We are also working with the NCATS PaVe-GT team to using liver spheroids and transwell models to create models of liver acidemia and test the efficacy of AVV-vectors being develop for clinical testing. So far, we have shown that liver spheroids formed using primary cells are transducible with AAV2 and AVV5 vectors. We have received iPSC from PCCA patients and are currently working with the Automated Cell Technologies group to create PCCA iPSC-derived hepatocytes to create PCCA liver models.