Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture
Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture
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DOI：
10.1038/nprot.2017.095
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发表时间：
2017-11-01
期刊：
影响因子：
14.8
通讯作者：
Lutolf, Matthias P.
中科院分区：
文献类型：
--
作者：
Gjorevski, Nikolce;Lutolf, Matthias P.
在细胞外基质样 3D 凝胶中培养细胞是许多细胞类型离体生长所必需的，或者可以改善许多细胞类型的离体生长。在这里，我们描述了一种用于生成用于培养肠道干细胞（ISCSC）和肠道类器官的明确基质的协议。这些基质包含聚（乙二醇）（PEPEG）水凝胶骨架，用最小的粘附线索进行功能化，包括足以 ISCSC 扩张的 RGD (Arg-Gly-Asp) 和类器官形成所需的层粘连蛋白-111。因此，水凝胶提供了一个明确的、可重复的、可调节的环境，允许研究人员操纵物理和化学参数，并检查它们对 ISCSC 和类器官生长的影响。水凝胶是通过带有谷氨酰胺和赖氨酸肽的多臂 PEPEG 前体的酶促交联反应形成的。含有稳定或可水解降解部分的 PEPEG 前体用于生产机械软化水凝胶，分别用于 ISCSC 的扩张或类器官的形成。我们还提供对这些基质中生长的细胞结构进行免疫荧光分析的方案，以及用于下游使用的细胞解离和回收的方案。可以生产水凝胶前体并表征其机械性能，以确定 5-7 天内的刚度。 ISCSC 扩张或类器官形成的水凝胶形成需要 1-2 小时。这里描述的材料可以很容易地适应其他类型的正常或转化的类器官结构的培养。
Growing cells within an extracellular matrix-like 3D gel is required for, or can improve, the growth of many cell types ex vivo. Here, we describe a protocol for the generation of well-defined matrices for the culture of intestinal stem cells (ISCSCs) and intestinal organoids. These matrices comprise a poly(ethylene glycol) (PEPEG) hydrogel backbone functionalized with minimal adhesion cues including RGD (Arg-Gly-Asp), which is sufficient for ISCSC expansion, and laminin-111, which is required for organoid formation. As such, the hydrogels present a defined and reproducible, but also tunable, environment, allowing researches to manipulate physical and chemical parameters, and examine their influence on ISCSC and organoid growth. Hydrogels are formed by an enzymatic cross-linking reaction of multiarm PEPEG precursors bearing glutamine-and lysine-containing peptides. PEPEG precursors containing either stable or hydrolytically degradable moieties are used to produce mechanically softening hydrogels, which are used for the expansion of ISCSCs or the formation of organoids, respectively. We also provide protocols for immunofluorescence analysis of cellular structures grown within these matrices, as well as for their dissociation and retrieval of cells for downstream use. Hydrogel precursors can be produced and their mechanical properties characterized to ascertain stiffness within 5-7 d. Hydrogel formation for ISCSC expansion or organoid formation takes 1-2 h. The materials described here can be readily adapted for the culture of other types of normal or transformed organoid structures.