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

Abstract

BackgroundWe 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.MethodsHuman 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.ResultsCollagen 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.ConclusionType 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.

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.…”

Take a deep breath and digest the material: organoids and biomaterials of the respiratory and digestive systems

“…[5,[14][15][16][17][18][19][20][21][22] Intestinal organoids have been used to model epithelial barrier function, interactions with microbes, infectious diseases and cancer. [23][24][25][26][27][28][29][30][31][32] Intestinal organoids derived from human samples were first grown from isolated small intestinal or colonic crypts, which are localized at the base of the intestinal and colonic epithelium, respectively.…”

“…In the crypt, an adult stem cell population can be found with the capacity to replenish all intestinal epithelial cells, including absorptive and secretory cells (goblet cells, enterocytes/colonocyte, enteroendocrine cells and Paneth cells in the small intestine). [14][15][16][17]33] Once placed in threedimensional culture, whole crypts or individual intestinal stem cells form cystic or budded organoids that contain the differentiated intestinal epithelial cell types while maintaining the stem cell population. In addition to healthy crypts, patient tissue with specific mutations and cancer tissue has been used to derive organoid models in order to conduct drug screens.…”

“…[14,[27][28][29][30][34][35][36] Both the healthy and diseased organoids were maintained in Matrigel, [14][15][16]33] yet was also shown that intestinal organoids derived from primary crypts can be maintained in ECM protein, Collagen I. [17] Matrigel is the most common ECM scaffold used for the culture and transplantation of organoids including intestinal organoids (Table I). Matrigel is derived from mouse tumor cells that secrete basement membrane and consists of ECM proteins (enriched for laminin) and numerous undefined growth factors.…”

“…[38] For these reasons, there has been an effort to define the matrix and materials suitable for intestinal organoids. For example, organoids can be grown in purified Collagen I [17] ; Natural materials, such as purified collagen or alginate, or synthetic materials (e.g., PEG) have more well-defined components, along with the potential to modified the physical and chemical properties (Table I).…”