Muntjewerff Elke M, Josyula Vijay S, Christoffersson Gustaf
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Bio Protoc. 2023 Oct 20;13(20):e4852. doi: 10.21769/BioProtoc.4852.
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).
在自身免疫性糖尿病发病过程中，神经 - 免疫细胞相互作用似乎起着重要作用；然而，目前尚无模型可用于在体内或体外随时间追踪和干预这些相互作用。二维体外模型提供的信息不足，而微流控技术或芯片上器官通常操作起来具有挑战性。我们在此展示了我们认为的首个简单模型，该模型提供了将胰岛与交感神经和免疫细胞共培养的机会。此模型基于我们的冲压装置，该装置可以进行3D打印（提供了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接种到特定位置，达到共聚焦活细胞成像所需的水平。在本方案中，我们提供了在我们的共培养模型中构建和进行活细胞成像实验的指导，包括：1）冲压装置的设计以在凝胶中形成印记，2）交感神经元、胰岛和巨噬细胞的分离，3）共培养条件，4）如何将其用于活细胞成像，以及5）该模型更广泛应用的可能性。总之，我们开发了一种易于使用的共培养模型，可用于操纵和成像交感神经、胰岛和巨噬细胞之间的相互作用。这种新的共培养模型对于研究神经 - 免疫细胞 - 胰岛相互作用很有用，并将有助于确定胰腺中神经 - 免疫相互作用的功能相关性。关键特性 • 一种新型装置，可实现交感神经元、胰岛和免疫细胞的3D共培养 • 该装置能够在共培养六天后，在可控环境中捕捉小鼠交感神经元、胰岛和免疫细胞之间的实时相互作用。 • 本方案在3D共培养中使用了先前建立的方法（Jackson和Tourtellotte，2014）从颈上神经节分离培养的交感神经元。 • 此方法需要3D打印我们自己设计的凝胶冲压装置（SciLifeLab FigShare DOI: 10.17044/scilifelab.24073062上提供了STL打印文件）。
