Local remodeling of synthetic extracellular matrix microenvironments by co-cultured endometrial epithelial and stromal cells enables long-term dynamic physiological function.
Local remodeling of synthetic extracellular matrix microenvironments by co-cultured endometrial epithelial and stromal cells enables long-term dynamic physiological function.
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DOI：
10.1039/c6ib00245e
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发表时间：
2017-04-18
期刊：
影响因子：
--
通讯作者：
Griffith LG
中科院分区：
文献类型：
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作者：
Cook CD;Hill AS;Guo M;Stockdale L;Papps JP;Isaacson KB;Lauffenburger DA;Griffith LG
粘膜屏障组织由一层紧密结合的上皮细胞组成，与含有成纤维细胞和免疫细胞的富含基质的基质进行密切的分子通信，是抗感染、慢性炎症和其他疾病过程的药物的重要靶点。尽管这类屏障的人体体外模型是机制研究和药物开发所必需的，但上皮细胞和基质细胞对细胞外基质(ECM)需求的差异阻碍了建立此类模型的努力。在这里，以子宫内膜为例，我们描述了一种适合3D功能共培养的合成、模块化的ECM水凝胶，其特点是可以由细胞重塑的成分，并动态反应隔离每种细胞类型的局部细胞分泌的ECM特征。合成的水凝胶将多肽与现成的试剂结合在一起，因此细胞生物学实验室可以使用。具体地说，我们首先利用2D半经验筛选确定了一种适合子宫内膜上皮细胞和间质细胞初始附着的单肽。然后，使用凝胶包裹的基质细胞顶部培养的上皮细胞的共培养系统，我们发现在水凝胶中包含ECM结合肽以及整合素结合肽，导致在培养的两周内基质细胞在上皮下的基底膜增加和更多的纤维状胶原基质组装。重要的是，由细胞系或原代细胞组成的子宫内膜共培养显示出激素介导的分化，根据形态变化和分泌蛋白的产生进行评估。对顶端细胞因子和生长因子分泌的多重分析，比较了细胞系和原代细胞，发现了截然不同的模式，强调了使用原代细胞模型在分析细胞-细胞通讯网络中的重要性。总之，我们定义了一种“一刀切”的合成ECM，它能够在黏膜屏障形式下对上皮细胞和基质细胞进行长期的、生理反应的共培养。合成的3D基质，结合细胞分泌的基质，概括了子宫内膜生理性上皮-间质组织结构、激素反应和已知的细胞因子的产生。
Mucosal barrier tissues, comprising a layer of tightly-bonded epithelial cells in intimate molecular communication with an underlying matrix-rich stroma containing fibroblasts and immune cells, are prominent targets for drugs against infection, chronic inflammation, and other disease processes. Although human in vitro models of such barriers are needed for mechanistic studies and drug development, differences in extracellular matrix (ECM) needs of epithelial and stromal cells hinder efforts to create such models. Here, using the endometrium as an example mucosal barrier, we describe a synthetic, modular ECM hydrogel suitable for 3D functional co-culture, featuring components that can be remodeled by cells and that respond dynamically to sequester local cell-secreted ECM characteristic of each cell type. The synthetic hydrogel combines peptides with off-the-shelf reagents and is thus accessible to cell biology labs. Specifically, we first identified a single peptide as suitable for initial attachment of both endometrial epithelial and stromal cells using a 2D semi-empirical screen. Then, using a co-culture system of epithelial cells cultured on top of gel-encapsulated stromal cells, we show that inclusion of ECM-binding peptides in the hydrogel, along with the integrin-binding peptide, leads to enhanced accumulation of basement membrane beneath the epithelial layer and more fibrillar collagen matrix assembly by stromal cells over two weeks in culture. Importantly, endometrial co-cultures composed of either cell lines or primary cells displayed hormone-mediated differentiation as assessed by morphological changes and secretory protein production. A multiplex analysis of apical cytokine and growth factor secretion comparing cell lines and primary cells revealed strikingly different patterns, underscoring the importance of using primary cell models in analysis of cell-cell communication networks. In summary, we define a “one-size-fits-all” synthetic ECM that enables long-term, physiologically responsive co-cultures of epithelial and stromal cells in a mucosal barrier format. Synthetic 3D matrix, that binds cell-secreted matrix, recapitulates endometrial physiologic epithelial-stromal tissue architecture, hormone responsiveness, and known cytokine production.