随着体细胞重编程为诱导多能干细胞 (iPSC)、基因编辑技术以及干细胞定向分化为异质组织的方案的最新进展，iPSC 衍生的肾脏类器官已成为研究多能干细胞过程的有用手段。肾脏发育和疾病。在基本肾脏发育信号通路知识的指导下，通过使用外源形态发生素在体外生成更坚固的肾样组织，已经取得了相当大的进展。然而，生化和生物物理微环境因素对组织发育和自组织有重大影响。在微环境生物物理方面的工程背景下，使用水凝胶细胞外支架进行类器官研究已引起人们的兴趣。两个水凝胶家族最近受到了广泛关注：自组装肽水凝胶（SAPH），其是完全合成且化学成分确定的，以及明胶甲基丙烯酰（GelMA）水凝胶，其是半合成的。两者都可以用作生长肾脏类器官的支持基质。根据我们最近发表的工作，我们重点介绍了描述人类 iPSC (hiPSC) 衍生的肾脏类器官的生成及其在 SAPH 和 GelMA 水凝胶中成熟的方法。我们还详细介绍了使用免疫荧光成像表征此类类器官所需的方案。总之，这些协议应该使用户能够在这种水凝胶中生长 hiPSC 衍生的肾脏类器官，并评估水凝胶提供的生物物理微环境对肾脏类器官成熟的影响。 © 2024 作者。当前协议由 Wiley periodicals LLC 出版。基本方案 1：人类诱导多能干细胞 (hiPSC) 定向分化为肾类器官，并在机械可调自组装肽水凝胶 (SAPH) 内成熟。 替代方案：将第 9 天的肾单位祖细胞聚集在明胶甲基丙烯酰 (GelMA) 水凝胶中。支持方案 1：人类诱导多能干细胞 (hiPSC) 培养。支持方案 2：用多聚甲醛 (PFA) 固定类器官 基本方案 2：肾脏类器官的整体免疫荧光成像。基本方案 3：类器官冷冻切片的免疫荧光。© 2024 作者。当前协议由 Wiley periodicals LLC 出版。

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.© 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.