Yadav Anita, Sharma Anu, Moulick Mohini, Gavande Parmeshwar V, Nandy Aparajita, Xuan Yi, Sen Chandan K, Ghatak Subhadip
McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
Nat Protoc. 2025 Sep 12. doi: 10.1038/s41596-025-01238-5.
Extracellular vesicles are a heterogeneous group of membrane-bound vesicles involved in cell-cell communication, formed at the plasma membrane (ectosomes) or by endocytosis (exosomes). Most exosome studies so far have focused on in vitro systems or exosomes derived from bodily fluids, while tissue-derived exosomes remain underexplored. Here we present a protocol using cell-type-specific promoter-driven reporter constructs for the targeted labeling and subsequent isolation of exosomes from specific cell types in vivo from mouse tissues. The differentiation between exosomes and ectosomes remains challenging due to limitations of current isolation techniques that are primarily based on size, density or surface markers. To address this issue, our approach leverages genetic engineering to mark exosomes specifically, enabling their precise identification and isolation from a complex biological pool of heterogenous extracellular vesicles. The isolated cell-type-specific exosomes are characterized by electron microscopy, nanoparticle tracking analysis, antibody exosome array assay and other established techniques. The labeling and isolation of exosomes spans 2-3 days and is designed to be accessible to researchers with fundamental laboratory competencies. This protocol facilitates the study of exosome-mediated cellular communication by enabling the isolation of cell-type-specific exosomes from either individual cell types or multiple cell types in combination. Most experiments within the protocol have used murine wound-edge skin tissue, but the protocol can, in principle, also be applied to other tissues to isolate exosomes, with a few modifications as required. This methodology opens new avenues for exploring the functional roles of cell-type-specific exosomes in intercellular communication.
细胞外囊泡是一类异质性的膜结合囊泡，参与细胞间通讯，在质膜处形成（外泌体）或通过内吞作用形成（外泌体）。迄今为止，大多数外泌体研究都集中在体外系统或源自体液的外泌体上，而源自组织的外泌体仍未得到充分探索。在这里，我们展示了一种方案，使用细胞类型特异性启动子驱动的报告构建体进行靶向标记，随后从小鼠组织的体内特定细胞类型中分离外泌体。由于目前主要基于大小、密度或表面标志物的分离技术存在局限性，外泌体和外泌体之间的区分仍然具有挑战性。为了解决这个问题，我们的方法利用基因工程特异性标记外泌体，使其能够从异质性细胞外囊泡的复杂生物池中精确识别和分离。分离出的细胞类型特异性外泌体通过电子显微镜、纳米颗粒跟踪分析、抗体外泌体阵列分析和其他成熟技术进行表征。外泌体的标记和分离跨越2 - 3天，设计为具有基本实验室能力的研究人员可以使用。该方案通过能够从单个细胞类型或多种细胞类型组合中分离细胞类型特异性外泌体，促进了对外泌体介导的细胞通讯的研究。该方案中的大多数实验都使用了小鼠伤口边缘皮肤组织，但原则上该方案也可以应用于其他组织以分离外泌体，只需根据需要进行一些修改。这种方法为探索细胞类型特异性外泌体在细胞间通讯中的功能作用开辟了新途径。
