By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry, and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system.

中文翻译：

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小鼠脾脏器官型培养的优化，用于染色和功能测定。

通过保持细胞活力和三维定位，有机型培养在细胞培养的最新领域中脱颖而出。它已成功地用于研究瘤形成的疾病，其中肿瘤细胞利用周围的基质促进自身的增殖和存活。在免疫系统的背景下，器官型培养变得尤为重要，免疫系统的细胞以复杂而动态的方式相互交谈以引发生产反应。然而，器官型培养对于原发性和继发性淋巴器官尚不完善。在这里，我们详细描述了适用于有效切除小鼠脾脏的协议的开发过程，该协议克服了与特殊器官纹理有关的技术难题，并优化脾切片的器官型培养。此外，我们使用显微镜，免疫荧光，流式细胞仪和qRT-PCR来证明脾切片中的大多数细胞在切开后的几天内仍然活着并在器官结构中保持原始位置。该协议的发展代表了在涉及免疫系统的生理和病理条件下研究淋巴微环境的重大技术进步。