Figure 2. Application of bioprinted organoids for compound testing in 96-well format.
A. Bioprinted day 7 + 18 organoids within a 96-well Transwell format (1× 105 cells per organoid). B. 96-well plate within plate holder on print stage. C. Quality control assessment of cell number per organoid and viability across a 96-well plate. Error bars show SD for cell number measurements (see Methods). D. Immunofluorescence after 10μM Doxorubicin treatment depicts podocytes (MAFB), apoptotic cells (cleaved caspase 3 [CC3]), distal tubules (cytokeratin 8/18; CCK8/18), proximal tubules (LTL) and nuclei (DAPI). Images representative of n = 3 experiments, Scale bars = 50μm. E. Expression of kidney injury molecule-1 (HAVCR) and apoptosis genes (CASP3, BAX) after Doxorubicin treatment (n = 3 per treatment group, n = 2 for controls). 10μM Doxorubicin increases HAVCR (p < 0.0001) and BAX (p = 0.04) expression. F. Expression of podocyte (NPHS1, PODXL) and proximal tubule (CUBN) genes after Doxorubicin treatment (n = 3 per treatment group, n = 2 for controls). NPHS1 and PODXL are decreased at both doses (p < 0.0001 for all). CUBN expression is decreased with 10μM treatment (p = 0.0019). For E. and F significance was determined by two-way ANOVA, with Dunnett’s multiple comparison test. **** = p < 0.0001, ** = p < 0.01. Error bars = SD, shaded bars = mean. G. Cell viability 72 hours after Doxorubicin treatment in 6-well (green) or 96-well (blue) format. 6-well data are n = 6 (control) or n = 3 (treatment) per dose from 3 independent experiments. 96-well data are minimum n = 1, maximum n = 3 per dose from a single experiment. Error bars = SD, shaded bars = mean. Curves represent a non-linear fit for each plate type, total n = 27 (6-well) or n = 22 (96-well). H. Application of 96-well bioprinted organoids for testing viability in response to aminoglycoside antibiotics. Curves represent a non-linear fit for each compound; n = 19 (Amikacin), n = 24 (Tobramycin), n = 30 (Gentamycin), n = 30 (Neomycin), n = 22 (Streptomycin).