Recent advances in the directed differentiation of human pluripotent stem cells to kidney organoids advances the prospect of drug screening, disease modelling, and even restoration of renal function using patient-derived stem cell lines. Here, we demonstrate the successful adaptation of our directed differentiation protocol to the NovoGen Bioprinter® MMX technology to achieve automated, rapid fabrication of self-organizing kidney organoids. Bioprinted organoids were found to be equivalent to those previously reported via manual generation at both the level of morphology and component cell types, as well as gene expression patterns and cell clusters revealed by single cell transcriptional profiling. Utilization of a bioprinter allows for the generation of large numbers of uniform and highly reproducible organoids in reduced time (approximately 20x faster) compared to manual processes. Treatment of bioprinted kidney organoids cultured in conventional 6-well format with doxorubicin exhibited concentration-dependent morphological changes consistent with cell injury and degeneration. Consistent with clinical observations, doxorubicin showed distinct glomerular toxicity with marked increases in cleaved caspase 3 mRNA and protein, accompanied by loss of podocyte-specific cell markers. Proof of concept high-throughput toxicity screening was achieved with bioprinted kidney organoids in 96-well Corning® Transwell® plates treated for 72 hours with a range of doxorubicin concentrations (0.2 to 25 µM). Analysis of 6-well and 96-well cell viability data suggested that organoids printed in both multi-well plate formats were similarly sensitive to doxorubicin. The doxorubicin IC50 for organoids bioprinted in 6-well plates was 3.9 ± 1.8 µM (value ± S.E.), while the calculated IC50 for organoids bioprinted in a 96-well plate was 3.1 ± 1.0 µM. Collectively, these results suggest that bioprinted kidney organoids are functionally equivalent to those prepared manually and thus are likely to be useful for toxicity screening, the development of iPSC-based approaches for the interrogation of complex disease phenotypes, and the scaling needed for clinical restoration of renal function with patient-derived iPSCs.
Cells isolated from donors with nonalcoholic fatty liver disease exhibit disease phenotypes in 3D bioprinted human liver tissue
The identification of targets and biomarkers and development of therapeutics for nonalcoholic fatty liver disease (NAFLD) may be accelerated by the use of well-characterized primary cell and tissue reagents, as well as improved in vitro human cell-based disease models, including three-dimensional (3D) bioprinted liver tissue. The characteristics of donors from which the cells are isolated, and especially their stage on the NAFLD continuum, are likely to influence the resulting performance in two-dimensional (2D) and 3D models. Evaluation of individual cell type characteristics, and their performance when combined in a tissue coculture model, could enable development of in vitro models more representative of specific patient populations and disease phenotypes.
RNA sequencing (RNA-seq) was performed on (5) non-diseased and (5) NAFLD liver tissues with NAFLD Activity Score (NAS) of 3 or more, revealing clear separation of non-diseased vs. NAFLD tissues and differential expression of fibrosis related genes. Histological analyses performed on tissue microarrays revealed consistent altered distribution patterns of hepatic stellate cells (HSC), with differential activation of HSC. Hepatocytes and non-parenchymal cells (NPC) (HSC, endothelial cells, and Kupffer cells), were isolated from non-diseased donors and from donors with NAS of 3 or more. The isolated cells were characterized with respect to viability, growth kinetics, cytokine production, and phenotype. 3D bioprinted liver tissue was generated using either NPCs isolated from diseased donors combined with non-diseased hepatocytes, or hepatocytes isolated from diseased donors combined with non-diseased NPCs. 3D bioprinted liver tissue generated using NPCs from a diseased donor exhibited accelerated collagen deposition (by trichrome stain) and HSC activation (by α-SMA staining) in comparison to bioprinted liver tissue generated with non-diseased tissue donors. Tissue generated using hepatocytes from a diseased donor exhibited steatosis induction.
Characteristics of the tissue of origin for cells used for in vitro models, including disease status, influence the performance of the cells and the utility of the resulting model. Thus, characterization of cell donors could enable development of in vitro models more representative of specific patient populations and disease phenotypes.
Bioprinted pluripotent stem cell-derived kidney organoids provide opportunities for high content screening
Abstract
Recent advances in the directed differentiation of human pluripotent stem cells to kidney brings with it the prospect of drug screening and disease modelling using patient-derived stem cell lines. Development of such an approach for high content screening will require substantial quality control and improvements in throughput. Here we demonstrate the use of the NovoGen MMX 3D bioprinter for the generation of highly reproducible kidney organoids from as few as 4,000 cells. Histological and immunohistochemical analyses confirmed the presence of renal epithelium, glomeruli, stroma and endothelium, while single cell RNA-Seq revealed equivalence to the cell clusters present within previously described organoids. The process is highly reproducible, rapid and transferable between cell lines, including genetically engineered reporter lines. We also demonstrate the capacity to bioprint organoids in a 96-well format and screen for response to doxorubicin toxicity as a proof of concept for high content compound screening.
Organovo is collaborating with the University of Virginia to develop 3D bioprinted tissues for volumetric muscle loss injury
Organovo Holdings, Inc. (NASDAQ:ONVO) (“Organovo”), a three-dimensional biology company focused on delivering scientific and medical breakthroughs using its 3D bioprinting technology, today announced a collaboration with the University of Virginia to develop 3D bioprinted tissues for volumetric muscle loss (“VML”) injury. The research will take place in the laboratory of George J. Christ, Ph.D., professor of biomedical engineering and orthopaedic surgery at UVA.
Organovo is collaborating with the National Center for Advancing Translational Sciences and the National Eye Institute of Health to develop better and more clinically predictive tissue models using 3D bioprinted functional eye tissue
Organovo announced today that they are joining together with two institutes from the National Institutes of Health (NIH) to help scientists develop more reliable tools for bringing safer, more effective treatments to patients on a faster timeline. Organovo is collaborating with the National Center for Advancing Translational Sciences (NCATS) and the National Eye Institute (NEI) to develop better and more clinically predictive tissue models using Organovo’s NovoGen MMX Bioprinter®. Organovo will collaborate with NCATS and NEI in using the NovoGen Bioprinting platform to create three-dimensional, architecturally correct, functional living tissues.
Organovo and Yale School of Medicine, Department of Surgery have formed a collaboration to develop bioprinted tissues for surgical transplantation research
Organovo Holdings, Inc. (NYSE MKT: ONVO) (“Organovo”), a three-dimensional biology company focused on delivering breakthrough 3D bioprinting technology, and Yale School of Medicine, Department of Surgery have formed a collaboration to develop bioprinted tissues for surgical transplantation research, made possible by a generous gift from the Methuselah Foundation.
Modeling tumor phenotypes in vitro with three-dimensional bioprinting
The tumor microenvironment plays a critical role in tumor growth, progression, and therapeutic resistance, but interrogating the role of specific tumor-stromal interactions on tumorigenic phenotypes is challenging within in vivo tissues. Here, we tested whether three-dimensional (3D) bioprinting could improve in vitro models by incorporating multiple cell types into scaffold-free tumor tissues with defined architecture. We generated tumor tissues from distinct subtypes of breast or pancreatic cancer in relevant microenvironments and demonstrate that this technique can model patient-specific tumors by using primary patient tissue. We assess intrinsic, extrinsic, and spatial tumorigenic phenotypes in bioprinted tissues and find that cellular proliferation, extracellular matrix deposition, and cellular migration are altered in response to extrinsic signals or therapies. Together, this work demonstrates that multi-cell-type bioprinted tissues can recapitulate aspects of in vivo neoplastic tissues and provide a manipulable system for the interrogation of multiple tumorigenic endpoints in the context of distinct tumor microenvironments.
Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis
Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure time frame. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 μM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents.
Optimizing drug discovery by investigative toxicology: Current and future trends
Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an “Investigative Toxicology Think-Tank”, which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology’s position by identifying key challenges and perspectives.
Modeling Liver Biology and the Tissue Response to Injury in Bioprinted Human Liver Tissues
Kelsey Retting, Dwayne Carter, Candace Crogan-Grundy, Chirag Khatiwala, Leah Norona, Elizabeth Paffenroth, Umesh Hanumegowda, Alice Chen, Lisa Hazelwood, Lois Lehman-McKeeman, and Sharon Presnell. Appl In Vitro Toxicol 4:3 (2018).
