Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays

Abstract

Objective: The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualised medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (ie, barrier function and host-microbial interactions).

Design: We created a large panel of human gastrointestinal epithelial cell lines (n=65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays.

Results: We obtained epithelial lines from all accessible tissue sites within 2 weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3-1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarised monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli.

Conclusions: This culture system will facilitate the study of interindividual, functional studies of human intestinal epithelial cells, including host-microbial interactions.

Keywords: CELL BIOLOGY; DIFFERENTIATION; E. COLI; EPITHELIAL CELLS; INTESTINAL EPITHELIUM.

Figure 1

Establishment of human epithelial spheroid cultures from multiple regions of the gastrointestinal tract. (A) Representative images showing the procedure for establishment of intestinal spheroid culture. Ruler lines above biopsy samples indicate 1 mm. Bars, 100 μm. (B) Diagram illustrating the numbers of spheroid and gastric lines established to date. The first number reports the total number of lines established from the indicated region, and the number in parenthesis indicates the number of those lines established from IBD patients.

Figure 2

Robust growth of cultured intestinal spheroids in 50% L-WRN CM. (A) Representative images demonstrating the qualitative growth of an individual spheroid imaged daily over a 3-day period. Bars, 100 μm. (B) Spheroids were seeded (Day 0) into 96-well format and growth was assessed at 24-hr intervals by MTT assay on Days 1, 2 and 3. The reduction of MTT (yellow color) to formazan (purple color) by metabolically active cells was measured by absorbance at 595 nm with a plate reader. The background subtracted results for 3 rectal (gray bars) and 3 ileal (red bars) cell lines are presented as fold change (mean ± s.e.m.) compared to Day 1 for each region (n = 9 per line at each time point). ***, P = 0.004 by Student’s t test. (C) qRT-PCR gene expression analysis for (A) LGR5, (B) VIL1 and (C) VIM in rectal and ileal epithelial cell cultures. Fresh biopsy samples were used as a positive control (white bars). Data are presented as fold change relative to the biopsy samples (mean ± s.e.m.; n = 3 samples per bar). n.d., not detected.

Figure 3

Human intestinal epithelial spheroids grown in low percentages of L-WRN CM exhibit decreased Wnt signaling activity. (A) Human rectal and ileal spheroids (n = 3 lines per region) were cultured for 2 days in 50%, 20%, 10%, 5% or 0% L-WRN CM. Gene expression of AXIN2, LGR5, OLFM4, MKI67 and MYC was determined by qRT-PCR (n = 3 experiments per line). A dose-dependent decrease was observed for AXIN2 (canonical Wnt target), mouse intestinal stem cell markers LGR5 and olfactomedin 4 (OLFM4), and proliferative markers MKI67 and MYC. Both 0% and 5% L-WRN CM showed a significant decrease for all of these markers compared to 50% L-WRN CM. As some assays required cells with some potential to divide, we used cells grown in 5% L-WRN CM for downstream applications. Data were presented as fold change (mean ± s.e.m.) compared to 50% L-WRN CM for each experiment. *, P <0.05; **, P<0.01; ****, P<0.0001 by 1-way ANOVA followed by a Dunnett’s post test with 50% L-WRN CM set as the control group: F = 17.58, P <0.0001 (A, AXIN2); F = 449.60, P <0.0001 (A, LGR5); F = 113.70, P <0.0001 (A, OLFM4); F = 56.13, P <0.0001 (A, MKI67); F = 87.09, P <0.0001 (A, MYC); F = 49.48, P <0.0001 (B, AXIN2); F = 1570.00, P <0.0001 (B, LGR5); F = 156.70, P <0.0001 (B, OLFM4); F = 63.81, P <0.0001 (B, MKI67); F = 125.20, P <0.0001 (B, MYC). (B) Spheroids were passaged (Day 0) and allowed to recover overnight in 50% L-WRN CM. Media was replaced daily, with some cultures changed to 5% or 5% L-WRN CM plus 5 μM DAPT for the indicated number of days. On Day 3, the spheroids were treated with EdU for 2 hr, dissociated, stained and sorted by flow cytometry based on EdU incorporation and SYTOX AAD (DNA content marker). An average of 22.2% of rectal cells and 16.8% of ileal cells were in S-phase when grown in 50% L-WRN CM. One day of culture in 5% L-WRN CM was not sufficient to reduce the proportion of cells in S-phase; however, after 2 days, a decrease in the proportion of cells in S-phase was observed for both rectal and ileal lines. Addition of 5 μM DAPT to the 5% L-WRN CM rapidly decreased the proportion of S-phase cells within 1 day and led to greatly diminished proportions of S-phase cells (1.6–2.9%) after 2 days.**, P <0.01; ***, P <0.001; ****, P <0.0001 by 1-way ANOVA followed by a Dunnett’s post-test with 50% L-WRN CM set as the control: F = 43.01, P <0.0001 (Rectal); F = 54.13, P <0.0001 (Ileal).

Figure 4

Region-specific differentiation of rectal and ileal spheroids. (A-D) qRT-PCR gene expression analysis for (A) CA1, (B) SI, (C) MUC2, (D) DEFA5, (E) TRPV6 and (F) SLC10A2 in rectal (gray bars), ileal (red bars) or duodenal (blue bars) spheroids (n = 3 lines per region). RNA was collected from spheroids that had been passaged, allowed to recover overnight in 50% L-WRN CM and then treated with 50% or 5% L-WRN CM with or without the addition of 5 μM DAPT for an additional 2 days, with media replaced daily. Data are presented as fold change relative to rectal or duodenal 50% L-WRN CM, except for DEFA5, which is presented as fold change relative to ileal 50% L-WRN CM (mean ± s.e.m.; n = 3 per line). n.d., not detected. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001 by 1-way ANOVA followed by a Tukey’s post-test: F = 7.23, P = 0.0008 (A, Rect); F = 3.73, P = 0.0210 (A, Ile); F = 3.53, P = 0.0256 (B, Rect); F = 6.24, P = 0.0019 (B, Ile); F = 4.99, P = 0.0060 (C, Rect); F = 6.16, P = 0.0020 (C, Ile); F = 9.42, P = 0.0001 (D, Ile); F = 9.05, P =0.0002 (E, Duod); F = 0.67, P = 0.5777 (E, Ile); F = 17.33, P < 0.0001 (F, Duod); F = 4.39, P = 0.0151 (F, Ile).

Figure 5

Dissociated spheroid cells form polarized monolayers on Transwell membranes. (A, B) Spheroids were dissociated and seeded onto coated Transwell membranes to form epithelial monolayers. After 3 days, monolayers were used for measurement of transepithelial resistance (TER) and/or fixed and paraffin-embedded. Paraffin sections from rectal (A) and ileal (B) epithelial monolayers were stained for H&E or immunostained for VIL1 and ZO-1, MUC2 or CHGA (with UEA-1 lectin for ileal cells) to visualize differentiated cell types. (a) and (b) denote insets. Bars, 10 μm. (C) TER measurements are shown as the resistance x area product (Ω•cm²) for 3 lines per intestinal region (mean ± s.e.m.; n = 4 transwells per line). (D) Representative confocal Z-stack images of ileal monolayer (most epithelial cells are GFP-positive, green) and fluorescent beads (red). The top image is a transverse view with white dashed lines denoting the apical surface of the epithelial layer and range of suspended beads and a yellow solid line indicating the measured mucus thickness. The lower image shows a tilted view of the transverse image. Bars, 10 μm. (E) Graph of mucus thickness measurements in rectal and ileal monolayers before (−) and after (+) disruption of the mucus layer by pipetting (mean ± s.e.m.; n = 4–7 Transwells per bar). **P <0.01; ***P <0.001 by Student’s t test comparing mucus thickness before and after disruption from the same region.

Figure 6

Development of a bacterial adherence assay for human intestinal spheroid cells. Rectal and ileal spheroids were grown on glass chamber slides for 3 days to promote two-dimensional cell growth prior to assessment, which allows for the visualization of adherent bacteria. For adherence assays, rectal and ileal spheroid-derived epithelial cells were incubated with the indicated strains for 1 hr followed by several washes to remove non-adherent E. coli. Similarly treated HeLa cells served as a positive control for adherence. (A) Visual assessment of adherence phenotype was assessed by crystal violet staining (adherent bacteria are dark purple). Clustered and scattered adherence of the O157:H7 strain was observed with spheroid-derived epithelial cells (arrows). Bar, 30 μm. (B) qPCR was used to determine the relative adherence index (RAI) for the EAggEC, EPEC and EHEC strains (mean ± s.e.m.; n = 4 per strain per cell type). The RAI for each wild-type E. coli is the ratio of malB to GAPDH divided by this ratio for ORN172 (represented by dashed line in EHEC strain graph), which is a non-adherent control. *, P <0.05; **P <0.01; ***P <0.001 compared to HeLa cells by 1-way ANOVA followed by a Tukey’s post-test: F = 4.41, P = 0.0463 (EAEC042); F = 26.73, P = 0.0002 (UD792); F = 10.66, P = 0.0042 (B171); F = 27.35, P = 0.0001 (E2348/69); F = 19.04, P = 0.0006 (O157:H7).