Abstract
Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids, but this technique presents challenges that impede scalable culture expansion, experimental manipulation, high-throughput screening and diagnostic applications. We have developed a low-viscosity matrix (LVM) suspension culture method that enables efficient establishment and propagation of organoids and tumoroids from the human large intestine. Organoids and tumoroids cultured in LVM suspension recapitulate the morphological development observed in solid matrices, with tumoroids reflecting the histological features and genetic heterogeneity of primary colorectal cancers. We demonstrate the utility of LVM suspension culture for organoid and tumoroid bioreactor applications and biobanking, as well as tumoroid high-throughput drug sensitivity testing. These methods provide opportunities for the study and use of patient-derived organoids and tumoroids from the large intestine.
Highlights
Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids, but this technique presents challenges that impede scalable culture expansion, experimental manipulation, high-throughput screening and diagnostic applications
Organoids were grown in commercially available IntestiCult Organoid Growth Medium, tailored to the niche requirements for growth of normal intestinal epithelium[5,6], while tumoroids were grown in reduced medium (DMEM/F12, HEPES, B27 supplement, N2 supplement, nicotinamide, N-acetyl-Lcysteine, bFGF, EGF, penicillin-streptomycin, and normocin) lacking Wnt agonists, BMP, TGF-β, and p38 inhibitors[2,8,10]
Our results show that solid support matrices can be replaced with a low-viscosity matrix (LVM) suspension preparation to enable efficient establishment, propagation, and expansion of organoids and tumoroids from the human large intestine
Summary
Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids, but this technique presents challenges that impede scalable culture expansion, experimental manipulation, high-throughput screening and diagnostic applications. Cells grow into mature organoids comprising stem and differentiated cells, organized into a sphere-shaped lumen and cryptlike protrusions similar to normal colorectal epithelium[2,7] These conditions support the growth of human colorectal adenoma and carcinoma tumoroids, tumor niche factor requirements are more adaptable, with Wnt ligands commonly omitted to enable selective outgrowth of Wnt pathway mutated tumor cells (~90% of cases)[8,9]. While cell embedment into a solid support matrix is considered essential for intestinal epithelial organoid and tumoroid culture, this presents technical challenges that impede culture expansion, experimentation, and high-throughput screening applications. We demonstrate the utility of this suspension culture approach for organoid and tumoroid establishment, propagation, scalable expansion, and biobanking, as well as tumoroid high-throughput drug screening and diagnostic testing
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