title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Ziyad
authors.initials: Z
authors.lastname: Jabaji
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Garrett J
authors.initials: GJ
authors.lastname: Brinkley
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Hassan A
authors.initials: HA
authors.lastname: Khalil
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Connie M
authors.initials: CM
authors.lastname: Sears
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America; Department of Bioengineering, Henry Samueli School of Engineering, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Nan Ye
authors.initials: NY
authors.lastname: Lei
authors.affiliation: Department of Pathology, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Lewis
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America; Department of Surgery, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America.
authors.forename: Matthias
authors.initials: M
authors.lastname: Stelzner
authors.affiliation: Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America; Eli and Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Martín G
authors.initials: MG
authors.lastname: Martín
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America; Department of Bioengineering, Henry Samueli School of Engineering, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: James C Y
authors.initials: JC
authors.lastname: Dunn
mesh_terms: D001485:Basement Membrane / Q000166:cytology / Q000378:metabolism; D018920:Coculture Techniques; D003094:Collagen / Q000737:chemistry; D024042:Collagen Type I / Q000737:chemistry / Q000378:metabolism*; D004338:Drug Combinations; D005109:Extracellular Matrix / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D007421:Intestine, Small / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D007797:Laminin / Q000737:chemistry; D058628:Myofibroblasts / Q000166:cytology; D011509:Proteoglycans / Q000737:chemistry
chemical_list: D024042:Collagen Type I; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen