Dorris David M, Hauser Caitlin A, Minnehan Caitlin E, Meitzen John
Department of Biological Sciences, North Carolina State University, Raleigh, NC, USA.
Department of Biological Sciences, North Carolina State University, Raleigh, NC, USA; W.M. Keck Center for Behavioral Biology, Center for Human Health and the Environment, and Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC, USA.
J Neurosci Methods. 2014 Dec 30;238:1-10. doi: 10.1016/j.jneumeth.2014.09.017. Epub 2014 Sep 22.
Ex vivo acute living brain slices are a broadly employed and powerful experimental preparation. Most new technology regarding this tissue has involved the chamber used when performing electrophysiological experiments. Alternatively we instead focus on the creation of a simple, versatile aerator designed to allow maintenance and manipulation of acute brain slices and potentially other tissue in a multi-well cell culture plate.
Here we present an easily manufactured aerator designed to fit into a 24-well cell culture plate. It features a nylon mesh and a single microhole to enable gas delivery without compromising tissue stability. The aerator is designed to be individually controlled, allowing both high throughput and single well experiments.
The aerator was validated by testing material leach, dissolved oxygen delivery, brain slice viability and neuronal electrophysiology. Example experiments are also presented, including a test of whether β1-adrenergic receptor activation regulates gene expression in ex vivo dorsal striatum using qPCR.
Key differences include enhanced control over gas delivery to individual wells containing brain slices, decreased necessary volume, a sample restraint to reduce movement artifacts, the potential to be sterilized, the avoidance of materials that absorb water and small biological molecules, minimal production costs, and increased experimental throughput.
This new aerator is of high utility and will be useful for experiments involving brain slices and other potentially tissue samples in 24-well cell culture plates.
离体急性活脑切片是一种广泛应用且强大的实验准备。关于这种组织的大多数新技术都涉及进行电生理实验时所使用的腔室。相反，我们专注于创建一种简单、通用的曝气器，旨在允许在多孔细胞培养板中维持和操作急性脑切片以及潜在的其他组织。
在此，我们展示一种易于制造的曝气器，其设计可适配24孔细胞培养板。它具有尼龙网和单个微孔，以实现气体输送而不影响组织稳定性。该曝气器设计为可单独控制，允许进行高通量和单孔实验。
通过测试材料浸出、溶解氧输送、脑切片活力和神经元电生理对曝气器进行了验证。还展示了示例实验，包括使用qPCR测试β1 - 肾上腺素能受体激活是否调节离体背侧纹状体中的基因表达。
主要差异包括对向含有脑切片的各个孔中输送气体的控制增强、所需体积减小、减少运动伪影的样品约束、可灭菌的潜力、避免吸收水和小生物分子的材料、生产成本最低以及实验通量增加。
这种新型曝气器具有很高的实用性，将对涉及24孔细胞培养板中的脑切片和其他潜在组织样本的实验有用。
