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Vanga, M. G.

Publications and source records attributed to Vanga, M. G..

2 recordsLinked to original sources
bioRxiv2024

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays

Human liver organoids (HLOs) differentiated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can recapitulate the structure and function of human fetal liver tissues, thus being considered as a promising tissue model for liver diseases and predictive compound screening. However, the adoption of HLOs in drug discovery faces several technical challenges, which include the lengthy differentiation process with multiple culture media leading to batch-to-batch variation, short-term maintenance of hepatic functions post-maturation, low assay throughput due to Matrigel dissociation and HLO transfer to a microtiter well plate, and insufficient maturity levels compared to primary hepatocytes. To address these issues, expandable HLOs (Exp-HLOs) derived from human iPSCs were generated by optimizing differentiation protocols, which were rapidly printed on a 144-pillar plate with sidewalls and slits (144PillarPlate) and dynamically cultured for up to 20 days into differentiated HLOs (Diff-HLOs) in a 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for in situ organoid culture and analysis. The dynamically cultured Diff-HLOs exhibited greater maturity and reproducibility than those cultured statically, especially after a 10-day differentiation period. In addition, Diff-HLOs in the pillar/perfusion plate were tested with acetaminophen and troglitazone for 3 days to assess drug-induced liver injury (DILI) and then incubated in an expansion medium for 10 days to evaluate liver recovery from DILI. The assessment of liver regeneration post-injury is critical to understanding the mechanism of recovery and determining the threshold drug concentration beyond which there will be a sharp decrease in the livers regenerative capacity. We envision that bioprinted Diff-HLOs in the pillar/perfusion plate could be used for high-throughput screening (HTS) of hepatotoxic compounds due to the short-term differentiation of passage-able Exp-HLOs, stable hepatic function post-maturation, high reproducibility, and high throughput with capability of in situ organoid culture, testing, staining, imaging, and analysis. Graphical abstractThe overall process of dynamic liver organoid culture and in situ analysis in the 144PillarPlate/144PerfusionPlate for high-throughput hepatotoxicity assays. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/586638v3_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1f2902aorg.highwire.dtl.DTLVardef@1d6e074org.highwire.dtl.DTLVardef@2a0dc0org.highwire.dtl.DTLVardef@eccde0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗
bioRxiv2024

Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15 - 18%, without any detrimental effect on cell viability. Despite utilizing 10 - 50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 {+/-} 1.6 {micro}M and 25.4 {+/-} 8.3 {micro}M, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

bioengineering↗