Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells

Abstract

Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the study of intestinal physiology as well as many disease conditions. These organoids present numerous advantages compared to immortalized cell lines, but working with them requires dedicated techniques. The protocols described in this article provide a basic guide to establishment and maintenance of human intestinal organoids derived from small intestine and colon biopsies. Additionally, this article provides an overview of several downstream applications of human intestinal organoids. © 2020 The Authors. Basic Protocol 1: Establishment of human small intestine and colon organoid cultures from fresh biopsies Basic Protocol 2: Mechanical splitting, passage, and expansion of human intestinal organoids Alternate Protocol: Differentiation of human intestinal organoids Basic Protocol 3: Cryopreservation and thawing of human intestinal organoids Basic Protocol 4: Immunofluorescence staining of human intestinal organoids Basic Protocol 5: Generation of single-cell clonal intestinal organoid cultures Support Protocol 1: Production of Wnt3A conditioned medium Support Protocol 2: Production of Rspo1 conditioned medium Support Protocol 3: Extraction of RNA from intestinal organoid cultures.

Keywords: adult stem cells; human intestinal organoids; organoid cryopreservation; organoid culture establishment; organoid differentiation; organoid immunofluorescence; organoid passage; single-cell clonal organoid culture; specialized organoid reagents.

Figure 1

Establishment of human colon organoid lines from fresh biopsy of normal tissue. Representative images from two organoid lines. Cells are shown after establishment of cultures (left), before the first split (middle), and after the first split (right). P indicates passage number; d indicates days after seeding or passage; scale bars, 0.5 mm.

Figure 2

Mechanical splitting and expansion of human intestinal organoids. (A) Schematic of culture procedure. (B) Glass Pasteur pipette connected to a 10‐μl tip used for mechanical disruption of organoids. (C) Representative bright‐field microscopy images of organoid cultures at days 0, 1, 2, and 4 after mechanical split. Scale bars, 2 mm. (D) Examples of a healthy organoid (top), composed mostly of stem/transit amplifying cells, and a suboptimal organoid (bottom), with signs of differentiation (thickening of wall). Scale bars, 0.4 mm.

Figure 3

Differentiation of human intestinal organoids. (A) Representative images of organoids differentiated towards the enteroendocrine cell lineage as in Beumer et al. (2020). Scale bars, 0.2 mm. (B) Representative images of organoids differentiated towards the enterocyte lineage. Scale bars, 0.4 mm. Inset: Detail of differentiated organoid with enterocytes (elongated cells, arrowhead). Scale bar, 0.1 mm.

Figure 4

Confocal immunofluorescence images of human intestinal organoids cultured under expansion conditions. DAPI, nuclei; KI67, proliferation marker; phalloidin, F‐actin staining. Scale bar, 0.1 mm.

Figure 5

Clonal expansion of human intestinal organoid cultures. (A) Schematic of process. (B) Gating strategy for sorting of living cells used for clonal organoid outgrowth. Cells in P3 gate were sorted and seeded. (C) Single‐cell organoid outgrowth after 10 days. Arrowhead indicates organoid of proper size for picking. Scale bar, 2 mm. (D) Organoid fragments after trypsinization of a single picked organoid. Scale bars, 1 mm (left) and 0.4 mm (inset at right).