Stem cell-derived, organotypic in vitro models, known as organoids, have emerged as superior alternatives to traditional cell culture models due to their unparalleled ability to recreate complex physiological and pathophysiological processes. For this reason, they are attractive targets of tissue-engineering efforts, as constructs that include organoid technology would be expected to better simulate the many functions of the desired tissue or organ. While the 3D spheroidal architecture that is the default architecture of most organoid models may be preferred for some applications, 2D monolayer arrangements remain the preferred organization for many applications in tissue engineering. Therefore, in this work, we present a method to create monolayer organoid cultures on poly(ethylene glycol) (PEG) hydrogel scaffolds, using intestinal epithelial organoids (IEOs) as a proof-of-concept. Our process involves two steps: the hydrogel is first functionalized with a layer of poly(D-lysine) (PDL), which then allows the adsorption of pristine, unmodified basement membrane proteins. This approach successfully mediates the formation of IEO monolayer unlike conventional approaches that rely on covalent modification of the hydrogel surface with cell-adhesive peptides and basement membrane proteins. We show that these IEO monolayers recreate important physiological functions of the native intestinal epithelium, including multilineage differentiation, apical-basal polarization, and the ability to model infections with human norovirus. We also show coating of a scaffold mimicking intestinal villous topography, resulting in a 3D IEO monolayer. We expect that this protocol will be useful to researchers attempting to leverage the increased physiological relevance of organoid models to elevate the potential of their tissue-engineered constructs.

中文翻译：

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蛋白质功能化聚乙二醇水凝胶作为单层类器官培养的支架

干细胞衍生的器官型体外模型（称为类器官）因其无与伦比的重建复杂生理和病理生理过程的能力而成为传统细胞培养模型的优越替代品。因此，它们是组织工程工作的有吸引力的目标，因为包含类器官技术的构建体有望更好地模拟所需组织或器官的许多功能。虽然 3D 球状结构是大多数类器官模型的默认结构，对于某些应用来说可能是首选，但 2D 单层排列仍然是组织工程中许多应用的首选组织。因此，在这项工作中，我们提出了一种在聚乙二醇（PEG）水凝胶支架上创建单层类器官培养物的方法，使用肠上皮类器官（IEO）作为概念验证。我们的过程包括两个步骤：首先用一层聚（D-赖氨酸）（PDL）对水凝胶进行功能化，然后它可以吸附原始的、未修饰的基底膜蛋白。与依赖于细胞粘附肽和基底膜蛋白对水凝胶表面进行共价修饰的传统方法不同，该方法成功介导了 IEO 单层的形成。我们证明这些 IEO 单层重建了天然肠上皮的重要生理功能，包括多谱系分化、顶端-基底极化以及模拟人类诺如病毒感染的能力。我们还展示了模仿肠绒毛地形的支架涂层，从而形成 3D IEO 单层。我们预计该协议将对试图利用类器官模型增加的生理相关性来提高其组织工程构建体的潜力的研究人员有用。