Journal Article

Type I Collagen as an Extracellular Matrix for the In Vitro Growth of Human Small Intestinal Epithelium

Jabaji, Ziyad, Khalil, Hassan A., Dunn, James C. Y., Sears, Connie M., Lewis, Michael, Brinkley, Garrett J., Lei, Nan Ye, Martín, Martín G., Stelzner, Matthias

2014

Overview

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. 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. 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. 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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Actin / Alkaline phosphatase / Basement Membrane - cytology / Basement Membrane - metabolism / Bioengineering / Biology and Life Sciences / CDX2 protein / Cell culture / Chelation / Coculture Techniques / Collagen / Collagen (type I) / Collagen - chemistry / Collagen Type I - chemistry / Collagen Type I - metabolism / Conditioning / Crypts / Drug Combinations / E-cadherin / Engineering schools / Epithelial cells / Epithelium / Ethylenediaminetetraacetic acids / Extracellular matrix / Extracellular Matrix - chemistry / Gastroenterology / Growth / Histology / Humans / Immunohistochemistry / In Vitro Techniques / Intermediate filament proteins / Intestinal Mucosa - cytology / Intestinal Mucosa - growth & development / Intestinal Mucosa - metabolism / Intestine / Intestine, Small - cytology / Intestine, Small - growth & development / Intestine, Small - metabolism / Laminin - chemistry / Light microscopy / Lysozyme / Maintenance / Markers / Medicine / Medicine and Health Sciences / Monoculture / Muscle proteins / Muscles / Myofibroblasts - cytology / Nuclei / Nuclei (cytology) / Optical microscopy / Ostomy / Pathology / Pediatrics / Phosphatase / Polymerase chain reaction / Proteoglycans - chemistry / Rodents / Small intestine / Smooth muscle / Stem cells / Studies / Surgery / Tissue culture / Tissue engineering / Vimentin