Presentation Information
[1Chemi-19]Engineered Production of Hypoosmotic Stimulation-Induced Migrasome-Like Vesicles Using Thermoresponsive Substrates
○Koki Yoshikawa1, Shogo Saito1, Mina Okochi1 (1. Institute of Science Tokyo (Japan))
Keywords:
Extracellular vesicles,Migrasome
[Purpose]
Migrasomes are extracellular vesicles that form on retraction fibers of migrating cells and mediate intercellular communication. However, their low yield and limited controllability remain challenges for systematic studies and translational exploration. In this study, we investigated the formation of migrasome-like vesicles induced by hypoosmotic stress and developed a method to reproducibly generate and isolate such vesicles.
[Method]
Human neuroglioma H4 cells and other adherent cell lines were cultured on fibronectin-coated glass substrates or thermoresponsive poly(N-isopropylacrylamide) (pNIPAM)-modified substrates. Hypoosmotic stress was applied by diluting the culture medium with sterile water, and cell morphology and retraction fiber behavior were monitored by fluorescence microscopy. Migrasome-associated features were assessed using a GFP-tagged TSPAN4, wheat germ agglutinin (WGA) membrane labeling, and cholesterol staining. To establish an efficient workflow, cells cultured on pNIPAM-modified substrates were subjected to various cooling protocols to induce retraction fiber formation, followed by hypoosmotic stimulation to generate vesicles. Vesicles were harvested by gentle mechanical detachment without enzymatic treatment and characterized by fluorescence imaging, size distribution analysis, Western blot analysis, and transmission electron microscopy.
[Results and Discussion]
Hypoosmotic stress rapidly induced the formation of migrasome-like vesicles along retraction fibers within seconds. These vesicles exhibited migrasome-like characteristics, including sizes of 0.5–3.0 µm and enrichment of cholesterol and the migrasome-associated protein TSPAN4. They also contained cytoplasmic components. Building on these findings, we established a reproducible workflow using pNIPAM-modified substrates. A stepwise cooling treatment (4 °C followed by 20 °C) induced retraction fiber formation in a contraction-dependent manner, enabling efficient vesicle generation upon subsequent hypoosmotic stimulation. The resulting vesicles, termed osmotically-induced migrasomes (OsMigs), were strongly labeled by WGA and enriched in migrasome-associated markers including integrin α5 and TSPAN4. This workflow was applicable across multiple adherent cell lines and increased vesicle yield by approximately 50-fold in normal human dermal fibroblasts compared with spontaneously formed migrasomes. These results suggest that hypoosmotic stimulation provides a rapid trigger for vesicle formation on retraction fibers and that combining this stimulus with pNIPAM-based cell detachment enables enzyme-free recovery of OsMigs. This OsMigs platform offers a controllable system to investigate vesicle cargo and potential biological activities.
Migrasomes are extracellular vesicles that form on retraction fibers of migrating cells and mediate intercellular communication. However, their low yield and limited controllability remain challenges for systematic studies and translational exploration. In this study, we investigated the formation of migrasome-like vesicles induced by hypoosmotic stress and developed a method to reproducibly generate and isolate such vesicles.
[Method]
Human neuroglioma H4 cells and other adherent cell lines were cultured on fibronectin-coated glass substrates or thermoresponsive poly(N-isopropylacrylamide) (pNIPAM)-modified substrates. Hypoosmotic stress was applied by diluting the culture medium with sterile water, and cell morphology and retraction fiber behavior were monitored by fluorescence microscopy. Migrasome-associated features were assessed using a GFP-tagged TSPAN4, wheat germ agglutinin (WGA) membrane labeling, and cholesterol staining. To establish an efficient workflow, cells cultured on pNIPAM-modified substrates were subjected to various cooling protocols to induce retraction fiber formation, followed by hypoosmotic stimulation to generate vesicles. Vesicles were harvested by gentle mechanical detachment without enzymatic treatment and characterized by fluorescence imaging, size distribution analysis, Western blot analysis, and transmission electron microscopy.
[Results and Discussion]
Hypoosmotic stress rapidly induced the formation of migrasome-like vesicles along retraction fibers within seconds. These vesicles exhibited migrasome-like characteristics, including sizes of 0.5–3.0 µm and enrichment of cholesterol and the migrasome-associated protein TSPAN4. They also contained cytoplasmic components. Building on these findings, we established a reproducible workflow using pNIPAM-modified substrates. A stepwise cooling treatment (4 °C followed by 20 °C) induced retraction fiber formation in a contraction-dependent manner, enabling efficient vesicle generation upon subsequent hypoosmotic stimulation. The resulting vesicles, termed osmotically-induced migrasomes (OsMigs), were strongly labeled by WGA and enriched in migrasome-associated markers including integrin α5 and TSPAN4. This workflow was applicable across multiple adherent cell lines and increased vesicle yield by approximately 50-fold in normal human dermal fibroblasts compared with spontaneously formed migrasomes. These results suggest that hypoosmotic stimulation provides a rapid trigger for vesicle formation on retraction fibers and that combining this stimulus with pNIPAM-based cell detachment enables enzyme-free recovery of OsMigs. This OsMigs platform offers a controllable system to investigate vesicle cargo and potential biological activities.
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