2023 3D Cell Culture User Day

Senior Lecturer, King's College London

3D In Vitro Disease Models to Study Pathological Mechanosensing

The onset and progression of aging-associated pathologies is highlighted by continuous local extracellular matrix (ECM) transformation. The contribution of ECM remodelling to disease is largely overlooked when predictive in vitro models are proposed. Following an ischemic insult or in chronic disease, ECM pathological remodeling results in dramatic changes in the chemistry, local stiffness, and nanostructure of the organ scaffold. Deposited by the tissue fibroblasts, the newly produced matrix displays impaired compliance and aberrant signalling, thus hinders cell functionality. The response of tissue cells to the ensuing biomechanical stress leads to the alteration of their transcriptional and post-transcriptional landscape, with the aim of preserving organ function.

Together with others, our group has been active in the field of pathological mechanosensing for a few years, trying to dissect the processes driving ECM remodelling in disease and unveil the molecular pathways highlighting cell response to the new conditions. Cardiac diseases represent a paradigmatic example of how ECM pathological remodeling can hinder cell functionality, here affecting muscle contractility and organ pumping function. By using human specimens, induced pluripotent stem cell (iPSC)-derived 3D organoids and a combination of advanced microscopy and molecular techniques, we identified reproducible modifications in the 3D nanostructure, interconnected porosity, and mechanics of heart ECM that contribute to organ failure.

ECM remodelling is also key to tumorigenesis, so that altered matrix stiffness is considered predictive of malignancies. We therefore analyzed primary prostate cancer tissues obtained from numerous patients undergoing radical prostatectomy to underline reproducible structural changes in the tumor ECM and promoting the loss of the glandular architecture. Starting from patient cells, we established prostate cancer tumoroids (PCTs) that mimic key features of the native tumor microenvironment (TME) and proceeded to investigate the cooperative role of TGF-β signalling and ECM desmoplasia in prompting prostate cell epithelial-to-mesenchymal transition (EMT) and fostering tumor progression and dissemination.

Ph.D. Student, University of Zurich

Single-cell Full-Length Isoform Sequencing of Patient-derived Organoid Cells of Clear cell Renal Cell Carcinoma

Clear cell Renal Cell Carcinoma (ccRCC) is a heterogenous, lethal, and aggressive cancer that arises in kidney tubules. Late-stage ccRCC patients tend to be resistant to various chemotherapies. Therefore, identifying new drug targets and predictive biomarkers is urgently needed. Studies investigating the alternative splicing landscape in ccRCC used short-read sequencing data for assessing the expression of transcripts and exon-junctions. To get a more reliable overview of full-length transcripts in ccRCC and search for pathogenic and druggable splicing events, we have utilized single-cell long-read sequencing in 4 patient-derived ccRCC organoids. Multiplexed Arrays Sequencing (MAS-Seq) is a new single-cell full-length isoform sequencing technology that can accurately detect full-length transcripts across single cells in high-throughput. In MAS-Seq, multiple transcripts are concatenated to a single long cDNA fragment and sequenced as high-fidelity reads (HiFi) on PacBio’s SMRT Flowcells. The concatenation typically results in a 16x fold increase in data output, allowing us to gain in-depth insights into the transcriptome of single cells. In collaboration with the Functional Genomics Center Zurich, we have applied the MAS-Seq technology on patient-derived organoid cells of ccRCC samples and assessed their alternative splicing landscape. In addition, to gain a deeper view of the transcriptome of each cell, we attempted to barcode as few cells as possible with the 10x Chromium Next GEM Single Cell 3ʹ Kit v3.1. Here, we will share our single-cell full-length isoform sequencing results. Our results and analyses will likely reveal new diagnostic and predictive biomarker candidates and give ccRCC patients new hope for novel precision medicine treatments against their detrimental disease.

Research Director, The French National Centre for Scientific Research (CNRS)

Using 3D Cell Culture to Decipher Virus-Brain Interactions

Viral infection of the central nervous system (CNS) leads to a wide range of clinical outcomes, ranging from barely detectable to deadly. Viral neuroinvasion with fatal consequences represents a major health concern. Yet, neurotropic viruses also impact CNS functioning in very subtle ways, causing psychological and psychiatric disorders that might be more difficult to pinpoint. Our lab aims to determine the molecular mechanisms involved in virus-brain interactions, with a particular focus on how viruses cross the so-called “blood-brain barrier” and the impact of viral neuroinvasion on the CNS. We use animal-free strategies to address these questions, including stem cell-derived cortical organoids, ex vivo organotypic culture of human brain slices and live cell imaging. This focuses on recent techniques we implemented in the lab to determine the molecular mechanisms associated to virus neuroinvasion.

Senior Scientist, University of Basel

Establishing a Living Biobank of the Digestive Tract From Oncology Patients for the Identification of Actionable Targets and New Therapeutic Avenues

With a strategic position in Pathology Institute, at the interface with the University Hospital, our lab is dedicated to implementing a biorepository of human tissues from patients with visceral tumours (CRC, PDAC, HCC, etc.). The collection of multiple cohorts, stratified by tissue type, location, primary or metastatic origin and treatment options, allow us to accomplish multi-layered retrospective analyses based on omics profiling, multiplexing imaging and drug screening, in a personalised fashion. Alongside tissue biobank, we extract multiple components of the tumour microenvironment, including resident immune, stromal and cancer cells, cultured as organoids in a 3D matrix. To interrogate not-yet answered questions in tumour biology and how these translate clinically, we use back-to-back complementary approaches: 1) a bottom-up approach where we use tumoroids alone or in co-culture with other constituents of the TME to study the contribution of each in cancer progression; 2) a top-down approach where we deep-dive in the tissue complexity to decipher how heterotypic cells communicate locally, influence each other cellular state and how they are spatially arranged.

Senior Scientific Applications Manager, Americas, Corning Life Sciences

Complementary Technologies to Advance 3D Development & Disease Modeling

When it comes to modeling human development and disease, traditional 2D cell culture models are often unable to capture complex biological properties observed in vivo. Animal models attempt to capture these inadequacies but involve a significant investment of time and resources while not accurately capturing human development and disease states. Cells grown in 3D more closely mimic in vivo behavior in tissues and organs. These models continue to be broadly adopted for their importance in attaining in vivo-like conditions to study developmental pathways, disease state, diagnostics, and therapeutic possibilities. Organoid models are derived from stem or organ progenitor cells and offer the unique ability to study human development and mechanisms of disease progression. To generate robust organoid model systems, there is a need to identify and implement complementary technologies. This highlights 3D cell culture applications utilizing this complementary approach to advance development and disease modeling.

Director, Enterprise Customers & Cellomics Proteomics, Tecan

Automated 3D Cell Culture Using Corning Microplates on the Tecan Fluent^®

3D cell culture has allowed the industry to produce more physiologically accurate models from which we can develop drugs and fight disease. Here we would like to present the latest development between Corning and Tecan – Automating the Corning ULA surface plates on the Fluent Liquid handler.

Director of Research and Development, HUB Organoids

HUB Organoids: A Translatable Patient-derived In Vitro Platform for Disease Modelling and Drug Development

HUB Organoid Technology is based on the groundbreaking discovery of Lgr5+ adult stem cell in the intestine which subsequently led to the development of the first “mini gut in a dish”. Since their original discovery, adult stem cells have been identified in most epithelial organs, including the gastrointestinal tract, liver, pancreas, breast, and lung. The technology that allows for the 3D in vitro cultures of these mini organs or organoids has been patented and further optimized for the aforementioned organ types. HUB Organoids^® possess exceptional expansion capabilities, maintain genetic and phenotypic stability, and preserve the biological and functional properties of the original tissue. By mimicking the response of the original tissue to external stimuli, HUB Organoids serve as an invaluable in vitro models for drug development, diagnostics, and patient stratification.

Over the years HUB has built a comprehensive living biobank of well-characterized patient-derived organoids from both healthy and diseased tissues which has been extensively used to develop assays and provide services aimed at preclinical drug discovery, toxicity assessment, validation, and personalized medicine. Pharmaceutical and Biotech partners can leverage this platform to evaluate their therapeutics' efficacy, mode of action, and adverse effects. HUB expertise has been applied to advance multiple agents to the clinic including targeted therapies, bi-specific antibodies, and engineered immune cells, for immuno-oncology, inflammatory and genetic disorders.

Ph.D. Student, Friedrich Miescher Institute for Biomedical Research (FMI)

Elucidating Human Intestinal Regeneration Using Patient-derived Organoids

Biopsy-derived human intestinal organoids hold significant potential for biomedical applications, as they can replicate aspects of regeneration, disease progression, and normal biological function in an accessible model. They provide an opportunity to study human physiology and pathology in ways that are challenging or impossible to conduct in patients. To unlock the full potential of human intestinal organoids, we conducted multiple high-content imaging screens to engineer a medium-regime which results in organoids which are highly similar to their in vivo counterpart and regenerate from single cells with high efficiency. Transcriptomic analysis revealed initial dedifferentiation and regeneration trajectories from all major lineages before homeostasis is re-established. To demonstrate the medium regime's broad applicability, we generated a multiplexed imaging dataset consisting of approximately 1,000,000 organoids from 51 healthy individuals and patients, enabling exploration of interpatient variability during organoid maturation. This developed in vitro workflow, combined with a planned CRISPR-KO screen, offers a unique opportunity to investigate the mechanisms of human intestinal regeneration.