Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium.
Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium.
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I型胶原蛋白作为人类小肠上皮体外生长的细胞外基质。
DOI：
10.1371/journal.pone.0107814
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Dunn JC
中科院分区：
文献类型：
--
作者：
Jabaji Z;Brinkley GJ;Khalil HA;Sears CM;Lei NY;Lewis M;Stelzner M;Martín MG;Dunn JC
我们以前曾报道过使用Matrigel（一种专有的基底膜产品）体外维持和增殖人小肠上皮。存在对基于基质胶的方法用于未来人类疗法的适用性的担忧。我们研究了I型胶原作为培养人肠上皮细胞的替代品。人小肠取自新鲜的外科病理标本。使用EDTA螯合分离小肠隐窝。肠上皮下肌成纤维细胞从儿科样本中分离，并在体外扩增。在基质胶或I型胶原凝胶中悬浮后，在肌成纤维细胞的汇合层上方共培养隐窝。隐窝也在暴露于肌成纤维细胞条件培养基的单一培养物中生长;这些细胞随后在体外传代培养并以1∶2的分流比扩增。用光学显微镜、RT-PCR、组织学和免疫组织化学对培养物进行评估。胶原蛋白在体外原代培养中支持活的人上皮至少一个月。传代培养的上皮细胞在60天内扩增12代。组织切片显示极化柱状细胞，顶端刷状边界和基底外侧位于核。基于胶原的培养物在凝胶-液体界面处产生单层上皮片，这在Matrigel中未观察到。免疫组化染色鉴定了分化的肠上皮和肌成纤维细胞的标记物。RT-PCR显示肌成纤维细胞表达α-平滑肌肌动蛋白和波形蛋白，上皮细胞表达E-Cadherin、CDX 2、villin 1、肠碱性磷酸酶、嗜铬粒蛋白A、溶菌酶和Lgr 5。这些标记物在几代中得以保持。I型胶原蛋白凝胶支持体外长期维持和扩增完全精心制作的人肠上皮细胞。基于胶原蛋白的方法产生熟悉的肠样结构以及新的片状生长模式，并且它们消除了体外人肠上皮生长对基质胶的需要。未来的研究需要进一步开发这种细胞培养系统的组织工程应用。
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.
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影响因子：
--
作者：
Nagaoka M;Si-Tayeb K;Akaike T;Duncan SA
通讯作者：
Duncan SA
影响因子：
2.9
作者：
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通讯作者：
MARTIN, GR
DOI：
10.1007/s10856-008-3415-4
发表时间：
2009-12-01
影响因子：
3.7
作者：
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通讯作者：
Werkmeister, Jerome A.
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作者：
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影响因子：
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通讯作者：
Clevers, Hans