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Use of Collagen Gel As An Extracellular Matrix for the In Vitro Growth of Human Small Intestinal Epithelium

Sunday, October 21, 2012: 9:20 AM
Versailles Ballroom (Hilton Riverside)
Ziyad B. Jabaji, M.D.1, Connie Martin, B.S.1, Stephanie Tung, B.A.2, Michael Lewis, M.D.3, Matthias Stelzner, M.D.1, Martin Martin, M.D.2 and James C. Y. Dunn, M.D., Ph.D.1, (1)Department of Surgery, University of California, Los Angeles, Los Angeles, CA, (2)Department of Pediatrics, University of California, Los Angeles, Los Angeles, CA, (3)Department of Pathology, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA

Purpose

We recently reported the capacity for long-term in vitro maintenance and proliferation of human small intestinal epithelium. Previous methods have relied on the use of Matrigel®, a proprietary basement membrane protein enriched product. However, there are numerous concerns over the potentially applicability of Matrigel-based cell culture methods for future human therapies. We investigated the ability to use collagen gel as an extracellular matrix substitute.

Methods

Human small intestine samples were procured from pancreatico-duodenectomy and small bowel resection surgical pathology specimens. Subepithelial myofibroblasts were isolated from neonatal C57BL/6 mice as well as a subset of the pediatric surgical samples and expanded in in vitro culture. Small intestinal crypts were isolated from the remainder of the specimens using EDTA chelation.  After suspension in either Matrigel or collagen gel, cells were co-cultured on top of a confluent layer of myofibroblasts.  Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry.

Results

Collagen was able to support viable epithelial structures for up to one month in vitro.  Histologic sections revealed polarized columnar cells, with an apical brush border and basolaterally located nuclei.  Further, collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel.  Immunohistochemistry studies identified the presence of intestinal epithelial markers, as well as markers of myofibroblasts.  Assessment with RT-PCR demonstrated expression of CDX2, E-Cadherin, Villin, Chromogranin A, Lysozyme, LGR5, α-Smooth Muscle Actin, and Vimentin.

Figure 1: Histologic sections of human small intestinal enteroids and surface epithelial sheets in vitro.  Scale bar: 100 µm.  A: hematoxylin and eosin, B: hematoxylin and eosin showing junction of enteroid with epithelial sheet, C: CDX2, D: E-cadherin, E: CD10, F: periodic acid-Schiff, G: chromogranin A, H: smooth muscle actin

Conclusion

Collagen gel is able to support the long-term in vitro maintenance and expansion of a fully elaborated human intestinal epithelium. The use of collagen yields familiar enteroid structures as well as a new pattern of sheet-like growth.  Collagen based methods eliminate the need for Matrigel for in vitro human intestinal epithelial growth.  Further research needs to be directed at developing this human cell culture system for tissue engineering applications.