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High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays

ABSTRACT: Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at industrial scale. Here, we report the fabrication of microengineered cell-culture devices and scalable and automated methods for the suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix significantly reduces organoid heterogeneity, as we show for mouse and human gastrointestinal organoids. We used the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and high-content image-based phenotypic analyses to reveal insights into drug-action mechanisms. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.

ORGANISM(S): Homo sapiens

PROVIDER: GSE148347 | GEO | 2020/04/10

REPOSITORIES: GEO

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Project description:Background & Aims Patient-derived gastrointestinal organoids are powerful tools for studying human physiology and disease. However, generating organoids requires prompt isolation and culture of fresh tissue, which is labor and time intensive, placing restraints on basic and clinical research. For example organoid biobanking efforts, or the ability to obtain specimens from distant locations that lack the expertise to culture organoids, is limited by current approaches. We sought to develop a method by which tissue biopsies from patients could be cryo-preserved, stored, or shipped, and later thawed in order to establish live organoid cultures. Methods A method for freezing and recovery, along with straightforward isolation methods using readily available materials were developed. Epithelial isolation and culture conditions were optimized to promote cell survival and the establishment of long-term culture post-thawing. Results Patient biopsies from stomach, duodenum, ileum, colon and from adenomateous colonic polyps were frozen, subsequently thawed and used to successfully establish patient-specific epithelium-only organoid cultures with 100% efficiency (n=31 independent patient biopsies from different regions of the GI tract). Frozen tissue could be shipped internationally and across the United States and used to establish successful organoid cultures. Organoid cultures could be expanded, passaged and frozen down for long-term storage. RNA-sequencing demonstrates that organoids derived from fresh tissue or from frozen biopsies are >99% transcriptionally identical. Conclusions Cryo-preservation of human tissue biopsies followed by the establishment of long-term, expandable cultures has negligible effect on transcription when compared to freshly prepared organoids, and will be of immense benefit to research and for clinical applications. This technique will allow for the establishment of biobanks of human tissues that can be revived and cultured in the future as well as transported across the globe for research, therapeutic or diagnostic use in remote labs and/or clinics.

Project description:In Rspondin-based three-dimensional cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. Here we report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely resemble the original tumor. The spectrum of genetic changes within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. Self-renewal of the intestinal epithelium is driven by Lgr5 stem cells located in crypts. We have recently developed a long-term culture system that maintains basic crypt physiology. Wnt signals are required for the maintenance of active crypt stem cells. Indeed, the Wnt agonist R-spondin1 induces dramatic crypt hyperplasia in vivo. R-spondin-1 is the ligand for Lgr5. Epidermal growth factor (EGF) signaling is associated with intestinal proliferation, while transgenic expression of Noggin induces a dramatic increase in crypt numbers. The combination of R-spondin-1, EGF, and Noggin in Matrigel sustains ever-expanding small intestinal organoids, which display all hallmarks of the original tissue in terms of architecture, cell type composition, and self-renewal dynamics. We adapted the culture condition for long-term expansion of human colonic epithelium and primary colonic adenocarcinoma, by adding nicotinamide, A83-01 (Alk inhibitor), Prostaglandin E2 and the p38 inhibitor SB202190. Of note, a two-dimensional culture method for cells from normal and malignant primary tissue has been described by Schlegel and colleagues. Here, we explore organoid technology to routinely establish and phenotypically annotate ‘paired organoids’ derived from adjacent tumor and healthy epithelium from CRC patients.