Use of Collagen Gel as an Alternative Extracellular Matrix for the In Vitro and In Vivo Growth of Murine Small Intestinal Epithelium

Abstract

Methods for the in vitro culture of primary small intestinal epithelium have improved greatly in recent years. A critical barrier for the translation of this methodology to the patient's bedside is the ability to grow intestinal stem cells using a well-defined extracellular matrix. Current methods rely on the use of Matrigel TM , a proprietary basement membrane-enriched extracellular matrix gel produced in mice that is not approved for clinical use. We demonstrate for the first time the capacity to support the long-term in vitro growth of murine intestinal epithelium in monoculture, using type I collagen. We further demonstrate successful in vivo engraftment of enteroids co-cultured with intestinal subepithelial myofibroblasts in collagen gel. Small intestinal crypts were isolated from 6 to 10 week old transgenic enhanced green fluorescent protein (eGFP +) mice and suspended within either Matrigel or collagen gel; cultures were supported using previously reported media and growth factors. After 1 week, cultures were either lysed for DNA or RNA extraction or were implanted subcutaneously in syngeneic host mice. Quantitative real-time polymerase chain reaction (qPCR) was performed to determine expansion of the transgenic eGFP-DNA and to determine the mRNA gene expression profile. Immunohistochemistry was performed on in vitro cultures and recovered in vivo explants. Small intestinal crypts reliably expanded to form enteroids in either Matrigel or collagen in both mono-and co-cultures as confirmed by microscopy and eGFP-DNA qPCR quantification. Collagen-based cultures yielded a distinct morphology with smooth enteroids and epithelial monolayer growth at the gel surface; both enteroid and monolayer cells demonstrated reactivity to Cdx2, E-cadherin, CD10, Periodic Acid-Schiff, and lysozyme. Collagen-based enteroids were successfully subcultured in vitro, whereas pure monolayer epithelial sheets did not survive passaging. Reverse transcriptase-polymerase chain reaction demonstrated evidence of Cdx2, villin 1, mucin 2, chromogranin A, lysozyme 1, and Lgr5 expression, suggesting a fully elaborated intestinal epithelium. Additionally, collagen-based enteroids co-cultured with myofibroblasts were successfully recovered after 5 weeks of in vivo implantation, with a preserved immunophenotype. These results indicate that collagen-based techniques have the capacity to eliminate the need for Matrigel in intestinal stem cell culture. This is a critical step towards producing neo-mucosa using good manufacturing practices for clinical applications in the future.

Using primary murine intestinal enteroids to study dietary TAG absorption, lipoprotein synthesis, and the role of apoC-III in the intestine

“…Previous studies in enteroids have focused on stem cell biology, lineage tracing, and Na + /H + exchange (23,(26)(27)(28). We asked whether enteroids were able to accomplish TAG absorption, lipoprotein synthesis, and lipoprotein secretion, which are major functions of the intestine.…”

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…More recently, several independent groups published exciting novel culture protocols allowing the long-term culture of primary intestinal epithelial cells in vitro as intestinal organoid structures (Jabaji et al, 2013;Sato et al, 2009). In this respect, mouse and human intestinal crypts are embedded in a three dimensional matrix such as Matrigel or Collagen I covered with medium basically mimicking the intestinal stem cell niche by providing factors of the Wnt-, EGF-, BMP-, and Notch-signaling pathways (Cao et al, 2013;Farin et al, 2012).…”

The effect of polymeric formula on enterocyte differentiation

“…An alternative method that involved the culture of a primary enterocyte monolayer on collagen was investigated as a means to explore the effect of PF treatment [46,54].…”

“…These This is important because in vivo these nutrients would be readily available through the local blood supply and/or directly from the intestinal lumen. For example, stem growth factors are derived from myofibroblasts beneath the basement membrane [46]. One of the most important of these is Wnt, which is present in low concentrations at the tips of villi and high concentrations in the cypts in order to prevent the stem cells from differentiating.…”