Single Organoid Research Articles

Advanced roll porous scaffold 3D bioprinting technology.

Improvements in the roll porous scaffold (RPS) 3D bioproduction technology will increase print density of 10-15µm cells by ~ 20% up to ~ 1.5 × 108 cells/mL and purity of organoid formation by > 17%. The use of 360 and 1200 dpi inkjet printheads immediately enables biomanufacturing with 10-30µm cells in a single organoid with performance > 1.8 L/h for 15µm layer thickness. The spongy bioresorbable ribbon for RPS technology is designed to solve the problems of precise placement, leakage and increasing in the number of instantly useable cell types and superior to all currently dominant 3D bioprinting methods in speed, volume, and print density without the use of expensive equipment and components. The potential of RPS for parallel testing of new substances studied was not on animals, but using generated 3D biomodels "organ on a chip". Solid organoids are more suitable for personalized medicine with simultaneous checking of several treatment methods and drugs, targeted therapy for a specific patient in vitro using the 3D composition of his personal cells, and selection of the most effective ones with the least toxicity. Overcoming the shortage of organs for implantation and personal hormone replacement therapy for everyone was achieved using printed endocrine glands based on their DNA.

Compressible Hollow Microlasers in Organoids for High-Throughput and Real-Time Mechanical Screening.

Mechanical stress within organoids is a pivotal indicator in disease modeling and pharmacokinetics, yet current tools lack the ability to rapidly and dynamically screen these mechanics. Here, we introduce biocompatible and compressible hollow microlasers that realize all-optical assessment of cellular stress within organoids. The laser spectroscopy yields identification of cellular deformation at the nanometer scale, corresponding to tens of pascals stress sensitivity. The compressibility enables the investigation of the isotropic component, which is the fundamental mechanics of multicellular models. By integrating with a microwell array, we demonstrate the high-throughput screening of mechanical cues in tumoroids, establishing a platform for mechano-responsive drug screening. Furthermore, we showcase the monitoring and mapping of dynamic contractile stress within human embryonic stem cell-derived cardiac organoids, revealing the internal mechanical inhomogeneity within a single organoid. This method eliminates time-consuming scanning and sample damage, providing insights into organoid mechanobiology.

Brain Chimeroids reveal individual susceptibility to neurotoxic triggers.

Interindividual genetic variation affects the susceptibility to and progression of many diseases1,2. However, efforts to study how individual human brains differ in normal development and disease phenotypes are limited by the paucity of faithful cellular human models, and the difficulty of scaling current systems to represent multiple people. Here we present human brain Chimeroids, a highly reproducible, multidonor human brain cortical organoid model generated by the co-development of cells from a panel of individual donors in a single organoid. By reaggregating cells from multiple single-donor organoids at the neural stem cell or neural progenitor cell stage, we generate Chimeroids in which each donor produces all cell lineages of the cerebral cortex, even when using pluripotent stem cell lines with notable growth biases. We used Chimeroids to investigate interindividual variation in the susceptibility to neurotoxic triggers that exhibit high clinical phenotypic variability: ethanol and the antiepileptic drug valproic acid. Individual donors varied in both the penetrance of the effect on target cell types, and the molecular phenotype within each affected cell type. Our results suggest that human genetic background may be an important mediator of neurotoxin susceptibility and introduce Chimeroids as a scalable system for high-throughput investigation of interindividual variation in processes of brain development and disease.

Design optimization of geometrically confined cardiac organoids enabled by machine learning techniques

Stem cell organoids are powerful models for studying organ development, disease modeling, drug screening, and regenerative medicine applications. The convergence of organoid technology, tissue engineering, and artificial intelligence (AI) could potentially enhance our understanding of the design principles for organoid engineering. In this study, we utilized micropatterning techniques to create a designer library of 230 cardiac organoids with 7 geometric designs. We employed manifold learning techniques to analyze single organoid heterogeneity based on 10 physiological parameters. We clustered and refined the cardiac organoids based on their functional similarity using unsupervised machine learning approaches, thus elucidating unique functionalities associated with geometric designs. We also highlighted the critical role of calcium transient rising time in distinguishing organoids based on geometric patterns and clustering results. This integration of organoid engineering and machine learning enhances our understanding of structure-function relationships in cardiac organoids, paving the way for more controlled and optimized organoid design.

Abstract 4919: AI-enabled hit selection of drug screening on human pancreatic cancer organoids

Abstract Cancer remains one of the leading causes of death in the 21st century. Despite the latest advances in oncology, most cancer patients lack tailored therapeutic approaches with lasting benefit. Measuring the impact of anticancer compounds and their combinations is only possible on ex vivo assays. To this end, patient-derived organoids (PDOs) have been proposed as viable and efficient models for ex vivo testing. PDOs show long-term expansion potential while retaining tumor histopathology as well as cancer gene mutations. However, the translation of organoids in screening applications has so far been hampered by the lack of homogeneity and difficulties in handling and automation. Moreover, organoids are typically randomly distributed across the culture which complicates imaging and images analyses. To overcome these challenges, we set up a compound screening workflow with PDOs using Gri3D platform, comprising plates with micro-cavities suitable for high-throughput and reproducible organoid culture. Based on a standard 96 microtiter plate, each well contains a microwell array patterned in a cell repellent hydrogel. On Gri3D®, organoids are robustly generated in the microwells and are located in the same imaging plane. This greatly facilitates quantitative analyses in high content image-based screens. Furthermore, the pipetting port enables automation of cell seeding, media exchange and compound incubation with liquid-handlers, thus increasing assay reproducibility. In the presented work, we exposed human pancreatic cancer PDOs to a panel of anti-cancer compounds at different doses and followed their response to the drugs using viability dyes Calcein AM (live stain) and Ethidium Homodimer-1 (dead stain) with high-content confocal imaging. Using an AI-based approach, we efficiently detected each single organoid and extracted phenotypic features from all three channels (TL, live stain and dead stain) which correlate with cytotoxicity. The extracted features (more than 100) were first dimensionally reduced to 3 components using either PCA or UMAP, the treatments were then visualized in 3D scatter plots, ranked and clustered using machine learning. The data suggests that the compound treatment Palbociclib 50uM has significant cytotoxicity effects similar to the positive control, which is consistent with the traditional live/dead analysis. The AI approach demonstrates feasibility to perform drug screening in a robust and un-biased data analysis approach. Citation Format: Zhisong Tong, Angeline Lim, Marine Meyer, Maria Clapes, Oksana Sirenko, Nathalie Brandenberg. AI-enabled hit selection of drug screening on human pancreatic cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4919.

Oxygen metabolism analysis of a single organoid for non-invasive discrimination of cancer subpopulations with different growth capabilities.

Heterogeneous nature is a pivotal aspect of cancer, rendering treatment problematic and frequently resulting in recurrence. Therefore, advanced techniques for identifying subpopulations of a tumour in an intact state are essential to develop novel screening platforms that can reveal differences in treatment response among subpopulations. Herein, we conducted a non-invasive analysis of oxygen metabolism on multiple subpopulations of patient-derived organoids, examining its potential utility for non-destructive identification of subpopulations. We utilised scanning electrochemical microscopy (SECM) for non-invasive analysis of oxygen metabolism. As models of tumours with heterogeneous subpopulations, we used patient-derived cancer organoids with a distinct growth potential established using the cancer tissue-originated spheroid methodology. Scanning electrochemical microscopy measurements enabled the analysis of the oxygen consumption rate (OCR) for individual organoids as small as 100µm in diameter and could detect the heterogeneity amongst studied subpopulations, which was not observed in conventional colorectal cancer cell lines. Furthermore, our oxygen metabolism analysis of pre-isolated subpopulations with a slow growth potential revealed that oxygen consumption rate may reflect differences in the growth rate of organoids. Although the proposed technique currently lacks single-cell level sensitivity, the variability of oxygen metabolism across tumour subpopulations is expected to serve as an important indicator for the discrimination of tumour subpopulations and construction of novel drug screening platforms in the future.

A Multifunctional Acoustic Tweezer for Heterogenous Assembloids Patterning

Acoustic Tweezer for Heterogenous Assembloids Patterning Heterogeneous assembloids with predesigned patterns can be constructed sequentially to explore polarized tissue development or disease metastasis in multi-organs. In article 2200288, Hairong Zheng, Xiao Zhang, Teng Ma, and co-workers present a multifunctional acoustic tweezer system that manipulates single organoid to construct assembloids non-invasively and free-label—this pioneering study demonstrates the possibility of bulk-wave acoustic tweezer application in tissue engineering.

Single Cerebral Organoid Mass Spectrometry of Cell-Specific Protein and Glycosphingolipid Traits.

Cerebral organoids are a prolific research topic and an emerging model system for neurological diseases in human neurobiology. However, the batch-to-batch reproducibility of current cultivation protocols is challenging and thus requires a high-throughput methodology to comprehensively characterize cerebral organoid cytoarchitecture and neural development. We report a mass spectrometry-based protocol to quantify neural tissue cell markers, cell surface lipids, and housekeeping proteins in a single organoid. Profiled traits probe the development of neural stem cells, radial glial cells, neurons, and astrocytes. We assessed the cell population heterogeneity in individually profiled organoids in the early and late neurogenesis stages. Here, we present a unifying view of cell-type specificity of profiled protein and lipid traits in neural tissue. Our workflow characterizes the cytoarchitecture, differentiation stage, and batch cultivation variation on an individual cerebral organoid level.

Abstract A073: Multiplexed therapeutic sensitivity of patient derived pancreatic cancer organoids after neoadjuvant chemotherapy by high content imaging

Abstract Background: Therapeutic development in pancreatic ductal adenocarcinoma (PDAC) requires accurate models for assessing response. Patient-derived cancer organoids (PCOs) have been shown to faithfully recapitulate genomic features of advanced PDAC. Mechanisms of subclonal resistance or intratumoral heterogeneity cannot be characterized using well-level viability assays. Methods: A high-content imaging platform was adapted in low volume, 96-well format for using Cytation 5 (Biotek). PDAC PCOs were plated from organoid suspension in 1:1 reduced growth factor Cultrex plating 10uL per well. After 24h, physiologic chemotherapy was applied to match pharmacologic exposure of FOLFIRINOX (FFX) and gemcitabine/nab-paclitaxel (g/n-pac). Initial response was assessed by normalized change in PCO area at 72h. Subsequent viability staining was performed for single organoid level response for apoptosis (caspase-3/7-FITC) and necrosis (TO-PRO-3). Analyses were automated in Gen5.12 (Biotek) with selection for elements using circularity (>0.4) and alignment by location within <88 mm. Response was assessed using descriptive statistics and effect size (Glass’s Delta) normalized to media control. Results: PCOs were generated from patient-derived pancreatic tumor without prior treatment. Low volume plating facilitated imaging with reduced Z stack (300 mm) with an average of 14 PCOs evaluable per well. Increasing markers of apoptosis by caspase-3/7 staining stratified serial decrease in mean organoid growth by quartile [+285, +175, +42%, +28]. Increasing markers of necrosis using TO-PRO-3 staining stratified serial decrease in mean organoid growth by quartile [+259, +191, +54%, +26]. Normalized change in area was reduced with g/npac +11.9% (GD=1.43) and FFX +20.4% (GD=1.38) versus control +281%. Subclonal populations existed with normalized change in area >50% which represented 17.8% of g/npac and 18.5% of FFX treated PCOs. Individual organoids with mean area staining positive for apoptosis was increased in g/npac +67.3% (GD=2.96) and FFX +65.1 (GD=2.84) versus background control +12.8%. Individual organoids with mean with area staining positive for necrosis was increased in g/npac +47.3% (GD=1.88) as compared to both FFX +34.9 (GD=1.20, p<0.002) and background control staining of +11.1% (p<0.001). Conclusions: High content imaging in PDAC PCOs provide scalable model for response assessment. This method can track subclonal populations with residual growth after treatment with chemotherapy. Here, we provide proof of concept in patient-derived tissue for clinically relevant response assessment with increased induced necrosis with g/npac in comparison to FFX. Ongoing investigations are generating sensitivity thresholds to define subclonal populations with residual growth and viability for application in clinical prediction. Citation Format: Md Shahadat Hossan, Ethan S. Lin, Ellie E. Riedl, Austin Stram, Jamie M. Warner, Jeremy D. Kratz. Multiplexed therapeutic sensitivity of patient derived pancreatic cancer organoids after neoadjuvant chemotherapy by high content imaging [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A073.

The CellRaft AIRⓇ system: A novel system enabling organoid imaging, identification, and isolation.

Three-dimensional (3D) culture systems have been developed that can re-capitulate organ level responses, simulate compound diffusion through complex structures, and assess cellular heterogeneity of tissues, making them attractive models for advanced in vitro research and discovery. Organoids are a unique subtype of 3D cell culture that are grown from stem cells, are self-organizing, and closely replicate in vivo pathophysiology. Organoids have been used to understand tissue development, model diseases, test drug sensitivity and toxicity, and advance regenerative medicine. However, traditional organoid culture methods are inadequate because they are low throughput and ill-suited for single organoid imaging, phenotypic assessment, and isolation from heterogenous organoid populations. To address these bottlenecks, we have adapted our tissue culture consumable and instrumentation to enable automated imaging, identification, and isolation of individual organoids. Organoids grown on the 3D CytoSortⓇ Array can be reliably tracked, imaged, and phenotypically analyzed using brightfield and fluorescent microscopy as they grow over time, then released and transferred fully intact for use in downstream applications. Using mouse hepatic and pancreatic organoids, we have demonstrated the use of this technology for single-organoid imaging, clonal organoid generation, parent organoid subcloning, and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis. The results validate the ability of the CellRaft AIRⓇ System to facilitate efficient, user-friendly, and automated workflows broadly applicable to organoid research by overcoming several pain points: 1) single organoid time-course imaging and phenotypic assessment, 2) establishment of single cell-derived organoids, and 3) isolation and retrieval of single organoids for downstream applications.

Gene-Edited Fluorescent and Mixed Cerebral Organoids.

Cerebral organoids are a promising model to study human brain function and disease, although the high inter-organoid variability is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different human induced pluripotent stem cell (hiPSC) lines, which enables the identification of cells from different origin within a single organoid. The method combining gene editing and organoid differentiation offers a unique tool to study gene function in a complex human three-dimensional model. Using a CRISPR-Cas9 gene-editing approach, different fluorescent proteins were fused to β-actin or lamin B1 in hiPSCs, and mixtures of differently edited cells were seeded to induce cerebral organoid differentiation. Consequently, the development of the organoids was detectable by live confocal fluorescence microscopy of whole organoids and immunofluorescence staining in fixed samples. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, thus overcoming the inter-organoid inhomogeneity limitations. Furthermore, the approach enables mosaic analysis of mutant clones in a wild-type three-dimensional cellular environment. It paves the way for the reliable analysis of human genetic disorders using organoids and the gain of fundamental understanding of the molecular mechanisms underlying pathological conditions.

Single Organoids Droplet-Based Staining Method for High-End 3D Imaging of Mammary Organoids.

In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.

Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids.

During embryogenesis, paracrine signaling between tissues in close proximity contributes to the determination of their respective cell fate(s) and development into functional organs. Organoids are invitro models that mimic organ formation and cellular heterogeneity, but lack the paracrine input of surrounding tissues. Here, we describe a human multilineage iPSC-derived organoid that recapitulates cooperative cardiac and gut development and maturation, with extensive cellular and structural complexity in both tissues. We demonstrate that the presence of endoderm tissue (gut/intestine) in the organoids contributed to the development of cardiac tissue features characteristic of stages after heart tube formation, including cardiomyocyte expansion, compartmentalization, enrichment of atrial/nodal cells, myocardial compaction, and fetal-like functional maturation. Overall, this study demonstrates the ability to generate and mature cooperative tissues originating from different germ lineages within a single organoid model, an advance that will further the examination of multi-tissue interactions during development, physiological maturation, and disease.

Human brain organoids assemble functionally integrated bilateral optic vesicles

During embryogenesis, optic vesicles develop from the diencephalon via a multistep process of organogenesis. Using induced pluripotent stem cell (iPSC)-derived human brain organoids, we attempted to simplify the complexities and demonstrate formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day 30, brain organoids attempt to assemble optic vesicles, which develop progressively as visible structures within 60days. These optic vesicle-containing brain organoids (OVB-organoids) constitute a developing optic vesicle's cellular components, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. OVB-organoids also display synapsin-1, CTIP-positive myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photosensitive activity of OVB-organoids, and light sensitivities could be reset after transient photobleaching. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow interorgan interaction studies within a single organoid.

High-Throughput Imaging Assay for Drug Screening of 3D Prostate Cancer Organoids.

New treatments are required for advanced prostate cancer; however, there are fewer preclinical models of prostate cancer than other common tumor types to test candidate therapeutics. One opportunity to increase the scope of preclinical studies is to grow tissue from patient-derived xenografts (PDXs) as organoid cultures. Here we report a scalable pipeline for automated seeding, treatment and an analysis of the drug responses of prostate cancer organoids. We established organoid cultures from 5 PDXs with diverse phenotypes of prostate cancer, including castrate-sensitive and castrate-resistant disease, as well as adenocarcinoma and neuroendocrine pathology. We robotically embedded organoids in Matrigel in 384-well plates and monitored growth via brightfield microscopy before treatment with poly ADP-ribose polymerase inhibitors or a compound library. Independent readouts including metabolic activity and live-cell imaging–based features provided robust measures of organoid growth and complementary ways of assessing drug efficacy. Single organoid analyses enabled in-depth assessment of morphological differences between patients and within organoid populations and revealed that larger organoids had more striking changes in morphology and composition after drug treatment. By increasing the scale and scope of organoid experiments, this automated assay complements other patient-derived models and will expedite preclinical testing of new treatments for prostate cancer.

Human corneal limbal organoids maintaining limbal stem cell niche function.

Corneal epithelial stem cells reside in the limbal area between the cornea and conjunctiva. We examined the potential use of limbal organoids as a source of transplantable limbal stem cells. After treating tissue with collagenase, limbal cells were seeded onto Matrigel and cultivated using limbal phenotype maintenance medium. After 1-month, approximately 500 organoids were formed from one donor cornea. Organoids derived from vertical sites (superior and inferior limbus) showed large colony forming efficiency, a higher ratio of slow cycling cells and N-cadherin-expressing epithelial cells compared to horizontal sites. The progenitor markers Keratin (K) 15 and p63 were expressed in epithelial sheets engineered form a single organoid. Organoids transplanted in the limbus of a rabbit limbal deficiency model confirmed the presence of organoid-derived cells extending on to host corneas by immunohistochemistry. Our data show that limbal organoids with a limbal phenotype can be maintained for up to 1month in vitro which can each give rise to a fully stratified corneal epithelium complete with basal progenitor cells. Limbal organoids were successfully engrafted in vivo to provide epithelial cells in a rabbit limbal deficiency model, suggesting that organoids may be an efficient cell source for clinical use.

Injectable Therapeutic Organoids Using Sacrificial Hydrogels

SummaryOrganoids are becoming widespread in drug-screening technologies but have been used sparingly for cell therapy as current approaches for producing self-organized cell clusters lack scalability or reproducibility in size and cellular organization. We introduce a method of using hydrogels as sacrificial scaffolds, which allow cells to form self-organized clusters followed by gentle release, resulting in highly reproducible multicellular structures on a large scale. We demonstrated this strategy for endothelial cells and mesenchymal stem cells to self-organize into blood-vessel units, which were injected into mice, and rapidly formed perfusing vasculature. Moreover, in a mouse model of peripheral artery disease, intramuscular injections of blood-vessel units resulted in rapid restoration of vascular perfusion within seven days. As cell therapy transforms into a new class of therapeutic modality, this simple method—by making use of the dynamic nature of hydrogels—could offer high yields of self-organized multicellular aggregates with reproducible sizes and cellular architectures.

Abstract C55: Mapping the pharmacotranscriptomic landscape of pancreatic circulating tumor cell organoids

Abstract Introduction: Pancreatic cancer is characterized by early metastasis and resistance to chemotherapy. To elucidate the transcriptional reprogramming and drug resistance that emerges during the metastatic cascade, we set out to characterize the transcriptomic profiles and drug response of matched sets of cancer cell organoids derived from multiple sites (primary tumor, metastatic lesion, and circulating tumor cells) in tumor-bearing PKCY (p53L/wt, KrasLSL-G12D/wt, ptf1acre/wt, Rosa26LSL-YFP/LSL-YFP) mice. Methods: To establish primary and metastatic organoids, tumor lesions are excised, enzymatically digested, and plated in Matrigel. Previously described organoid cell culture media (containing R-spondin1, Wnt3A, Noggin, FGF10, and EGF) was used to maintain organoids in culture. To allow for the culture and analysis of circulating tumor cell (CTC) organoids, we developed a CTC isolation and culture protocol. Briefly, whole blood is collected from tumor-bearing mice using a terminal cardiac puncture into the right ventricle, followed by RBC lysis and plating in Matrigel. To allow for high-throughput drug screening, we developed a single organoid per 384-well approach where organoid growth and response to drug treatment can be quantified using automated fluorescent imaging. Results: We established organoid cultures from primary tumors and metastatic lesions (liver or lung) from 30 tumor-bearing PKCY mice, with a 100% success rate. In addition, we successfully established CTC organoid lines from 10 metastatic PKCY mice, with an overall success rate of 40% (10/25). The success rate was higher (60%) in mice harboring both macroscopic liver and lung metastasis, whereas mice harboring only liver or lung metastasis displayed a lower success rate (33% and 40%, respectively). We have established 5 matched sets of primary, metastatic (liver and/or lung), and CTC-derived organoids as wells as normal pancreatic ductal organoid lines from CY (ptf1acre/wt, Rosa26LSL-YFP/LSL-YFP) mice, serving as noncancer controls. Pancreatic origin of all organoids was confirmed with the YFP lineage-label. Preliminary drug testing of an 8-drug panel (gemcitabine, paclitaxel, 5FU, irinotecan/SN38, oxaliplatin, 5-azacytidin, SAHA, and EPZ6438) revealed several cases of increased drug resistance in organoids derived from metastatic lesions and CTCs relative to matched primary tumor organoids. To expand on this analysis, we are implementing a high-throughput drug screen evaluating a large library of small compound drugs and inhibitors and correlating the results to transcriptomic analysis. Conclusion: We developed an approach for mapping the pharmacotranscriptomic landscape of pancreatic cancer organoids derived from primary tumor, metastatic lesions, and CTCs. We are exploring our initial findings further using high-throughput drug screening, transcriptomic analysis, and orthotopic transplantations into syngeneic mice. Citation Format: Fredrik I. Thege, Bhargavi B. Barathi, Dhwani N. Rupani, Sonja M. Woermann, Andrew D. Rhim. Mapping the pharmacotranscriptomic landscape of pancreatic circulating tumor cell organoids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C55.

3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids.

Immunotherapy using chimeric antigen receptor (CAR)-engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models are needed that allow to test CAR-mediated cytotoxicity in a tissue-like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3-dimensional (3D) patient-derived colon organoids. Luciferase-based measurement served as a quantitative read-out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR-engineered NK-92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen-specific cytotoxicity was studied with CAR-NK-92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor-specific activity. Taken together, we report a sensitive invitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.

The Mouse Retinal Organoid Trisection Recipe: Efficient Generation of 3D Retinal Tissue from Mouse Embryonic Stem Cells.

The introduction of stem cell-based technologies for the derivation of three-dimensional retinal tissues, the so-called retinal organoids, offers many new possibilities for vision research: Organoids facilitate studies on retinal development and in vitro retinal disease modeling, as well as being valuable for drug testing. Further, retinal organoids also provide an unlimited cell source for cell replacement therapies. Here, we describe our protocol for efficiently differentiating large, stratified retinal organoids from mouse embryonic stem cells: unbiased manual dissection of the developing retinal organoid at an early stage into three evenly sized neuroepithelial portions (trisection step) doubles the yield of high-quality organoids. We also describe some useful applications of the protocol, e.g., generation of rod- or cone-enriched retinal organoids, AAV transfection, and cell birth dating. In addition, we provide details of how to process retinal organoids for single organoid gene expression analysis, immunohistochemistry, and electron microscopy.