A Human Colonic Crypt Culture System to Study Regulation of Stem Cell-Driven Tissue Renewal and Physiological Function

Abstract

A human colonic crypt culture system to study regulation of stem cell-driven tissue renewal and physiological function AbstractThe intestinal epithelium is one of the most rapidly renewing tissues in the human body and fulfils vital physiological roles such as barrier function and transport of nutrients and fluid.Investigation of gut epithelial physiology in health and disease has been hampered by the lack of ex vivo models of the native human intestinal epithelium. Recently, remarkable progress has been made in defining intestinal stem cells and in generating intestinal organoid cultures.In parallel, we have developed a 3D culture system of the native human colonic epithelium that recapitulates the topological hierarchy of stem cell-driven tissue renewal and permits the physiological study of native polarised epithelial cells. Here we describe methods to establish 3D cultures of intact human colonic crypts and conduct real-time imaging of intestinal tissue renewal, cellular signalling and physiological function, in conjunction with manipulation of gene expression by lentiviral or adenoviral transduction. Visualisation of mRNA-and protein-expression patterns in cultured human colonic crypts, and crossvalidation with crypts derived from fixed mucosal biopsies, is also described. Alongside studies using intestinal organoids, the near-native human colonic crypt culture model will help to bridge the gap that exists between investigation of colon cancer cell lines and/or animal (tissue) studies, and progression to clinical trials. To this end, the near native human colonic crypt model provides a platform to aid the development of novel strategies for the prevention of inflammatory bowel disease and cancer.Human Colonic Crypt Culture 3

Nutrient-Gene Interaction in Colon Cancer, from the Membrane to Cellular Physiology

“…Later, they adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. The three-dimensional culture system of the native human colonic epithelium recapitulates the topological hierarchy of stem cell–driven tissue renewal, opening the methodological door for ex vivo studies designed to examine the effects of bioactive dietary compounds on colonic crypt metabolism (153). …”

Organoids, organs-on-chips and other systems, and microbiota

“…Transwell culture is limited however, as cultured epithelial cells do not produce functional specialised cell types [12,43]. Consequently, a substantial amount of research has focused on the development of a 3D culturing technique, initially using individual murine intestinal stem cells [44,45] and also patient-derived tissues and human stem cells [46][47][48] that grow into selfrenewing organoids that encompass the structure, and all cell types of the in vivo intestinal crypt, providing a more human-relevant model.…”

Long-Term Culture of Intestinal Cell Progenitors: An Overview of Their Development, Application, and Associated Technologies

“…Thus, the organoids maintain their integrity, unlike classical 2D cell culture with its inherent loss of heterogeneity and the genomic rearrangements associated with the culture ‘crisis’/cellular senescence events that occur during cellular adaption. This maintenance of cell identity and genetic integrity within ICP containing organoid cultures makes them the current gold standard tool for interrogating basic and diseased intestinal biology ex vivo and the protocols for isolation of human intestinal progenitor cells from resected surgical samples and biopsies are now well established [18, 19]. Indeed the derivation of ICP organoid cultures from normal tissue and tumour material is carried out in such a way that cells are never grown directly upon culture plastic, as opposed to spheroid or tumoursphere culture models that are generated from established 2D cell lines.…”

“…The use of ICP culture and technology to alter gene behaviour can elicit a myriad of cellular biology; thus, there is a requirement for techniques that can be used to analyse and quantify relevant parameters within complex 3D cellular environments [19]. This section sets out the cell culture analysis tools that can be used with 3D organoid cultures to quantify the complexity of organoid systems that display a wider range of biology compared with ‘classical’ 2D culture models.…”