用于琼脂糖凝胶中胰岛、免疫细胞和神经元活体成像的三维共培养模型

Bio-protocol Pub Date : 2023-10-20 DOI:10.21769/p2290
Elke M. Muntjewerff, V. Josyula, G. Christoffersson
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引用次数: 0

摘要

在自身免疫性糖尿病的发病过程中,神经-免疫细胞之间的相互作用似乎起着重要作用;然而,目前还没有模型可以在体内或体外长期跟踪和干扰这些相互作用。二维体外模型无法提供足够的信息,而微流体技术或芯片上的器官通常具有挑战性。我们在这里介绍的是我们认为是第一个简单的模型,它提供了将胰岛与交感神经和免疫细胞共同培养的机会。该模型基于我们的冲压设备,可以三维打印(提供 STL 文件)。由于琼脂糖凝胶中的印记,交感神经元、胰岛和巨噬细胞可以在特定位置播种,其水平可以进行共聚焦活细胞成像。在本方案中,我们提供了在共培养模型中构建和执行活细胞成像实验的说明,包括1) 设计在凝胶中制造印记的冲压装置;2) 分离交感神经元、胰岛和巨噬细胞;3) 共培养条件;4) 如何用于活细胞成像;5) 更广泛使用该模型的可能性。总之,我们开发了一种易于使用的共培养模型,可以对交感神经、胰岛和巨噬细胞之间的相互作用进行操作和成像。这种新型共培养模型有助于研究神经-免疫细胞-胰岛之间的相互作用,并有助于确定胰腺中神经-免疫相互作用的功能相关性。主要特点 - 一种可实现交感神经元、胰岛和免疫细胞三维共培养的新型装置 - 该装置可在共培养六天后,在受控环境中捕捉小鼠交感神经元、胰岛和免疫细胞之间的活体相互作用。- 该方案在三维共培养中使用从颈上神经节中分离出来的交感神经元培养物,使用的是以前建立的方法(Jackson 和 Tourtellotte,2014 年)。- 这种方法需要三维打印我们自己设计的凝胶盖章装置(STL 打印文件提供于 SciLifeLab FigShare DOI:10.17044/scilifelab.24073062)。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Three-dimensional Co-culture Model for Live Imaging of Pancreatic Islets, Immune Cells, and Neurons in Agarose Gel
During the onset of autoimmune diabetes, nerve–immune cell interactions seem to play an important role; however, there are currently no models to follow and interfere with these interactions over time in vivo or in vitro. Two-dimensional in vitro models provide insufficient information and microfluidics or organs on a chip are usually challenging to work with. We present here what we believe to be the first simple model that provides the opportunity to co-culture pancreatic islets with sympathetic nerves and immune cells. This model is based on our stamping device that can be 3D printed (STL file provided). Due to the imprint in the agarose gel, sympathetic neurons, pancreatic islets, and macrophages can be seeded in specific locations at a level that allows for confocal live-cell imaging. In this protocol, we provide the instructions to construct and perform live cell imaging experiments in our co-culture model, including: 1) design for the stamping device to make the imprint in the gel, 2) isolation of sympathetic neurons, pancreatic islets, and macrophages, 3) co-culture conditions, 4) how this can be used for live cell imaging, and 5) possibilities for wider use of the model. In summary, we developed an easy-to-use co-culture model that allows manipulation and imaging of interactions between sympathetic nerves, pancreatic islets, and macrophages. This new co-culture model is useful to study nerve–immune cell–islet interactions and will help to identify the functional relevance of neuro-immune interactions in the pancreas. Key features • A novel device that allows for 3D co-culture of sympathetic neurons, pancreatic islets, and immune cells • The device allows the capture of live interactions between mouse sympathetic neurons, pancreatic islets, and immune cells in a controlled environment after six days of co-culturing. • This protocol uses cultured sympathetic neurons isolated from the superior cervical ganglia using a previously established method (Jackson and Tourtellotte, 2014) in a 3D co-culture. • This method requires 3D printing of our own designed gel-stamping device (STL print file provided on SciLifeLab FigShare DOI: 10.17044/scilifelab.24073062).
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