Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium

Abstract

Background: We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells.

Methods: Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry.

Results: Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages.

Conclusion: Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.

Figure 1. Characterization of intestinal subepithelial myofibroblast phenotype.

A–C: Immunofluorescent staining against α-smooth muscle actin (A), vimentin (B), and desmin (C). Scale bar 100 µm (100x magnification). D: Gene expression by reverse transcriptase polymerase chain reaction. ACTA2: α-smooth muscle actin. DES: desmin. VIM: vimentin.

Figure 2. In vitro growth of human intestinal enteroids co-cultured with intestinal subepithelial myofibroblasts in collagen gel.

Scale bar 100 µm (40x magnification).

Figure 3. Hematoxylin and eosin stained sections of human small intestinal epithelium in collagen gel.

Both enteroids and monolayer epithelial sheets are noted (A), with confluence of these two domains at areas where the enteroids have grown or migrated to the surface of the collagen gel (B). Scale bar 100 µm (200x magnification).

Figure 4. Immunohistochemical evidence of intestinal epithelial cell lineage differentiation in collagen.

A: Cdx2 (caudal type homeobox 2). B: E-Cadherin. C: CD10. D: Periodic Acid-Schiff. E: Chromogranin A. F: α-smooth muscle actin. Scale bar 100 µm (200x magnification).

Figure 5. Gene expression profile of enteroids co-cultured with intestinal subepithelial myofibroblasts, relative to native small intestine, on a logarithmic scale.

Reverse transcriptase polymerase chain reaction was performed on mRNA isolated from co-cultures after 1 week in vitro, using GAPDH as an internal calibrator gene. N = 7 for Matrigel, and N = 6 for collagen. Error bars denote standard deviation, and p-values are reported for Student's t-test comparing individual genes. CDH1: E-Cadherin. CDX2: caudal type homeobox 2. VIL1: villin 1. ALPI: intestinal alkaline phosphatase. CHGA: chromogranin A. LYZ: lysozyme. LGR5: leucine-rich repeat-containing G-protein coupled receptor 5.

Figure 6. In vitro growth of human intestinal epithelium in monoculture with exposure to myofibroblast conditioned medium.

A: Monocultured enterospheres appear as simpler, thinner-walled cysts. B: Hematoxylin and eosin-stained cross-section of an enterosphere. C: Cdx2 staining. D: E-Cadherin staining. Scale bar 100 µm (A: 40x magnification; B–D: 200x magnification).

Figure 7. A: Sub-culturing of monocultured enterospheres with exposure to myofibroblast conditioned medium.

‘P’ denotes passage number; passages 8–10 are omitted for brevity. B: Large enteroid formed by fusion of passaged enterospheres. Scale bar 100 µm (40x magnification).

Figure 8. Gene expression profile of enterospheres monocultured in collagen gel, with exposure to myofibroblast conditioned medium.

Reverse transcriptase polymerase chain reaction was performed on mRNA using GAPDH as an internal calibrator gene relative to native small intestine. ‘P’ denotes passage number; N = 3 for each passage. Error bars denote standard deviation, and p-values are reported for single factor analysis of variance (ANOVA) comparing individual genes across all passages. CDH1: E-Cadherin. CDX2: caudal type homeobox 2. VIL1: villin 1. ALPI: intestinal alkaline phosphatase. CHGA: chromogranin A. LYZ: lysozyme. LGR5: leucine-rich repeat-containing G-protein coupled receptor 5.