+10,000 cells per 25 l drop of culture media on a lidWe have been studying the function of group 2 innate lymphoid cells 323 days10,000 B16F10 melanoma cells/drop7 days5 ml of PBS in a culture plate7 days incubation with change of medium every 3rdday Transfer to an appropriate plate for experiments of interest(ILC2), which are capable of producing large amounts of type 2 cy-tokines, such as IL-4, IL-5 and IL-13. Although ILC2s are rare in secondary lymphoid organs relative to other immune cells, they harbor a unique location within non-lymphoid tissues, especially skin and mucosal barriers (i.e., respira-tory and intestinal mucosa), and in fat-associated lymphoid clusters (FALCs) in the visceral adipose tissue. Despite intensive studies on the role of ILC2 in allergic disorders, their role in anti-tumor immunity has been obscure and controversial. It is generally believed that type 2 immune responses are benefi-cial for tumor growth. However, the action of ILC2 on tumor cells seems to be context dependent, and several studies have shown that IL-5-mediated eosino-philia can effectively control the growth of certain tumors such as melanoma. To examine the interaction between a solid tumor and immune cells, tumor cell culture in two-dimensional (2D) monolayers has limitations. In light of the increasingly recognized role of interactions between tumor and immune cells and to better characterize tumor-immune cell interactions, we have established a three-dimensional (3D) tumor organoid system using skin melanoma cells. This is a simple, fast and easy technique for generating 3D spheroids through the use of a hanging drop culture method. Within the hanging drop, cells have no direct contact with substratum and due to gravity accumulate at the free liquid-air interface to form a single spheroid (see Figure). Pre-established spheroids can be co-cultured directly or indirectly with different populations of immune cells. Our method provides a tumor microenvironment that resembles micrometastases and replicates many features of solid tumors. As in the non-proliferating regions of avascular solid tumors, tumor cells within the inner regions of the spheroids usually demonstrate perturbed gene and protein ex-pression, altered metabolism, cell cycle arrest, and necrotic death. Using this method, we have discovered an immunosuppressive activity imposed on ILC2s by tumor cells through the accumulation of lactic acid in the tumor microenvi-ronment.Figure: A simple, fast and easy technique for generating 3D spheroids through the use of a hanging drop culture method(A) Ten thousand tumor cells per drop of cell culture medium are placed on the lid of a PBS containing dish and incubated for 7 days. Pre-established spheroids are collected and co-cultured indirectly or directly with an immune cell population of interest. (B) Representative images of skin melanoma spheroids. B16F10 melanoma cells (10,000 cells per 25 µl drop of cell culture medium) placed on the lid of a PBS containing dish and incubated for 3 and 7 days.Recent Major PublicationsWagner M, Ealey KN, Tetsu H, Kiniwa T, Motomura Y, Moro K, Koyasu S. Tumour-derived lactic acid contributes to the paucity of intratumoural ILC2s. Cell Rep 30, 2743-2757 (2020)Wagner M, Koyasu S. A 3D Skin melanoma spheroid-based model to assess tumor-immune cell interactions. Bio Protoc 10, e3839 (2020)Nakamura R, Yoshizawa A, Moriyasu T, Deloer S, Senba M, Kikuchi M, Koyasu S, Moro K, Hamano S. Group 2 innate lymphoid cells exacerbate amebic liver abscess in mice. iScience 23, 101544 (2020)ABLaboratory for Immune Cell SystemsTeam Leader: Shigeo Koyasu
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