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, 3 (10), 2502-2513

In Vitro Generation of Mouse Colon Crypts

Affiliations

In Vitro Generation of Mouse Colon Crypts

Yuli Wang et al. ACS Biomater Sci Eng.

Abstract

Organoid culture has had a significant impact on in vitro studies of the intestinal epithelium; however, the exquisite architecture, luminal accessibility, and lineage compartmentalization found in vivo has not been recapitulated in the organoid systems. We have used a microengineered platform with suitable extracellular matrix contacts and stiffness to generate a self-renewing mouse colonic epithelium that replicates key architectural and physiological functions found in vivo, including a surface lined with polarized crypts. Chemical gradients applied to the basal-luminal axis compartmentalized the stem/progenitor cells and promoted appropriate lineage differentiation along the in vitro crypt axis so that the tissue possessed a crypt stem cell niche as well as a layer of differentiated cells covering the luminal surface. This new approach combining microengineered scaffolds, native chemical gradients, and biophysical cues to control primary epithelium ex vivo can serve as a highly functional and physiologically relevant in vitro tissue model.

Keywords: differentiation; gradient; intestinal epithelial stem cells; intestine-on-a-chip; microfabrication; tissue mimics.

Conflict of interest statement

Notes The authors declare the following competing financial interest(s): N.L.A., Y.W., C.E.S., S.T.M., and S.J.B. have a financial interest in Altis Biosystems LLC.

Figures

Figure 1
Figure 1
Culture of freshly isolated mouse colon crypts results in rapid loss of crypt shape, crypt polarity, and luminal accessibility. (a) Properties of a freshly isolated crypt. Shown in the left two panels are a schematic and an SEM image. The middle panel is a fluorescence image of a crypt from a Sox9-EGFP/CAG-DsRed mouse. The right two panels are a fluorescence image of mucin 2 (Muc2) immunostaining (red) and a bright-field image following incubation with a colorimetric substrate for alkaline phosphatase (ALP) (red). DNA within nuclei was stained with Hoechst 33342 (blue) in the Muc2 image. (b) A crypt embedded in Matrigel rapidly converts into an organoid when cultured in a growth-factor-rich medium (stem medium (SM)). The crypt lumen closes off, and Sox9-EGFP+ progenitor cells expand across the organoids. Colon crypts from CAG-DsRed/Sox9-EGFP mouse were used in this panel. (c, d) Forced lineage differentiation of mouse colon organoids by incubation in medium altering Wnt and Notch signaling intensity. Abbreviations: SM, stem medium; DM, differentiation medium; I, IWP-2; B, sodium butyrate; G, γ-secretase inhibitor LY-411575. (c) Schematic of the differentiation steps. Green, red, and blue indicate stem/proliferative cells, goblet cells, and absorptive colonocytes, respectively. (d) Fluorescence (left two columns) and bright-field (right column) images of organoids cultured under SM (for 3 days), DM-IB (3 days), or DM-IG (3 days). In the left two columns, EdU incorporation, Hoechst 33342 labeling, and Muc2 immunostaining are marked as green, blue, and red, respectively. In the right column, a colorimetric assay marks ALP in red. Scale bars = 100 μm.
Figure 2
Figure 2
Development of a monolayer from primary mouse colonic epithelial cells. (a) Schematic showing the strategy for in vitro crypt construction. (i) In vivo crypts were isolated and used to recreate in vitro monolayers of both stem/proliferative and differentiated cells. SM, stem medium; DM, differentiation medium. (ii) Stem/proliferative cells were grown into a folded monolayer by culturing the cells on scaffolds with an array of microwells. (iii) The in vitro-formed crypts were polarized to create differentiated-cell and stem/proliferative-cell regions by application of a gradient of Wnt-3A (W) and R-spondin (R). (b) Fluorescence images of 1.5 mm-sized regions of the monolayers cultured under different media. Cells were cultured in stem medium (SM) for 2 days followed by either SM or differentiation medium (DM, DM-IB, or DM-IG) for 2 days. The monolayers were assayed for incorporation of EdU (green, 3 h pulse), alkaline phosphatase (red, ALP), mucin 2 (yellow, Muc2), and DNA (blue, Hoechst 33342). Patterning of the various cells as described previously is apparent. (c) Percentages of the Hoechst-positive surface area displaying fluorescence from the EdU, ALP, or Muc2 stains. **, p < 0.005; *, p < 0.05.
Figure 3
Figure 3
Properties of the chemically differentiated monolayer. (a) Fluorescence images of a 10 μm-thick cross section through the monolayer. Actin was stained using phalloidin (green), mucin was stained with anti-Muc2 antibody (red), and DNA within nuclei was labeled with Hoechst 33342 (blue). The monolayers were cultured in SM for 2 days, followed by either SM, DM-IB, or DM-IG for an additional 2 days. (b) Cross sections of the monolayers visualized by TEM. The monolayers were cultured under the same conditions as in (a). (c) Apical surface topography of mouse colonic monolayer inspected by SEM. The upper two panels show low- and high-magnification images of a monolayer cultured under SM for 4 days. Sparse microvilli are present. The middle two panels show low- and high-magnification images of a monolayer cultured under SM for 3 days followed by DM-IB for 3 days. The cells differentiated into enterocytes with a high density of microvilli on their apical surface. The bottom two panels show low- and high-magnification images of a monolayer cultured under SM for 3 days followed by DM-IG for 3 days. The cells possessed large secretory goblets characteristic of goblet cells.
Figure 4
Figure 4
Micromolded collagen scaffold to support in vitro crypt formation. (a) Schematic of the components of the modified insert. The drawing is not to scale and is for illustrative purposes only. A nonpermeable COC film was attached to the back side of the porous membrane to reduce the transport area from 12 mm to 3 mm diameter. (b) Schematic of the micromolding process used to generate a shaped cross-linked collagen scaffold on a modified insert. (c) Image of the insert with shaped scaffolding. To demonstrate that trans-scaffold transport occurred only at the central area of the 3 mm diameter, the insert was placed on a 12-well plate and briefly exposed to 0.1% toluidine blue placed in the bottom compartment while PBS was loaded into the top compartment. The toluidine blue stained only the collagen within the central 3 mm-diameter area in contact with the basal compartment. (d) Top view of an array of microwells created in the collagen scaffold. (e) Geometry of the PDMS stamp that was used to micromold collagen. (f) SEM image of the stamp (left) and fluorescence microscopy image showing a side view of a fluorescein-labeled collagen scaffold (right). (g) Simulated model of the concentration of Wnt-3A (40 kDa molecule) across the scaffold when 0 and 30 ng/mL of the molecule are placed into the luminal and basal reservoirs, respectively. (h) Concentration of fluorescein–dextran (MW = 40 kDa) measured experimentally or predicted by the COMSOL model in the luminal (▲, experimental; △, simulation) and basal (■, experimental; □, simulation) reservoirs at 24, 48, and 72 h. At 0 h, 0.5 mL of PBS was added to the luminal reservoir and 1.5 mL of fluorescein–dextran (100 μg/mL) was added to the basal reservoir. (i) Concentration profile of Wnt-3A along the z axis of the scaffold predicted by the COMSOL model using the conditions in (h). On the x axis, the zero point marks the bottom of the microwell or crypt while the 250 μm point marks the luminal surface of the crypt.
Figure 5
Figure 5
Strategy to fold and polarize the monolayer of colonic epithelial cells. (a) Schematic showing how the microwell scaffold guides the folding of the cell monolayer into a cryptlike geometry, followed by application of a chemical gradient to polarize the crypt. Top row: side views of a slice through the array. Bottom row: top views looking down onto the array and into the crypts. Fragments of a precultured monolayer (indicated with *) were allowed to settle onto the scaffold, attach, proliferate, and spread to cover the entire scaffold surface, including lining the walls of the microwells (indicated by →). (b) Time-lapse images showing the propagation of cells across the surface of the microwell array. Microwells appeared darker in these bright-field images as their walls were lined with cells. (c) Side view of in vitro crypts. (d) Bright-field image of a 10 μm-thick cryosection through an in vitro crypt demonstrating the open lumen and a monolayer of cells covering the microwell wall. (e) A z slice through an in vitro crypt obtained by confocal fluoresecence imaging. The cells were stained with Hoechst 33342 (blue) and propidium iodide (red). Scale bars = 100 μm.
Figure 6
Figure 6
Formation of polarized in vitro colon crypts. (a) Crypts were not polarized in the absence of a gradient of Wnt-3A and R-spondin (top row), while crypts were polarized in the presence of a gradient of Wnt-3A (0 ng/mL on the luminal side and 30 ng/mL on the basal side) and R-spondin (0 ng/mL on the luminal side and 75 ng/mL on the basal side) for 2 days (bottom row). Left column: projected views of confocal slices through in vitro crypts so that the entirety of each crypt is superimposed onto a plane in the microscopy image (see the schematic of the top view in Figure 5a). Right column: side views of crypts that were detached from the scaffold and then placed on their sides for imaging. L, luminal; B, basal. Green = EdU, blue = DNA. (b) Box plots depicting the relative positions of EdU+ cells along the basal–luminal axis of the crypts. Zero represents the basal end of the crypt, while 1 marks the luminal end of the crypt. (c–e) Effect of gradient steepness on the location of EdU+ cells within the crypt. On the luminal side, [Wnt] = 0 ng/mL and [R-spondin] = 0 ng/mL. On the basal side, varying [Wnt-3a] and [R-spondin] were used: [Wnt-3a]:[R-spondin] = 60:150, 30:75, 15:37, and 7:18 (ng/mL:ng/mL). (c) Side views of crypts removed from the scaffold and imaged lying on their sides. Shown are representative crypts under different gradient steepness conditions. (d) Box plots showing the relative positions of EdU+ cells along the basal–luminal axis of the crypts. On the y axis, the zero point marks the bottom of the microwell or crypt while the 1.0 point marks the luminal surface of the crypt. (e) Box plots representing the number of EdU+ cells per crypt. n ≥ 20 crypts were quantified for (b), (d), and (e). **, p < 0.005; *, p < 0.05. Scale bars = 100 μm.
Figure 7
Figure 7
Arrays of polarized colon crypts. (a) Wide-field (left) and close-up (right) top views of a 3 mm-diameter array stained for ALP (red), Sox9 (green), and DNA (blue). The images were obtained with a low-magnification, large-depth-of-field objective so that the crypt regions are superimposed into a single plane (see the schematic of the top view in Figure 5a). (b) Side views of a crypt removed from the scaffold and imaged lying on its side. Shown is a representative in vitro crypt that was stained for Sox9, ALP, and DNA. The image labeled “BF” is a bright-field image. Polarity is demonstrated in the merged image. (c) Wide-field (left) and close-up (right) top views of a 3 mm-diameter array stained for ALP (red), EdU (green), and DNA (blue). The images were obtained as in panel (a). (d) Confocal fluorescence images of crypts stained for ALP (red), EdU (green), and DNA (blue). Left: side view of a 3D reconstructed image. Right: images of xy slices through the crypts at three locations in the crypt (L, luminal; M, middle; B, basal). (e) Distribution of EdU incorporation along the crypt axis. The y-axis value of 0 marks the crypt base, while 1 denotes 250 μm from the base. (f) Quantification of polarity of in vitro crypts. Shown are the ratios of the fluorescence intensities of the stains for EdU, Sox9, and ALP in the luminal half of the crypt to those in the basal half. n ≥ 20 crypts were quantified for (e) and (f). **, p < 0.005; *, p < 0.05. Scale bars = 100 μm for all images except the wide-field views in (a) and (c) (scale bars = 1 mm).

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