Missouri State University, Jordan Valley Innovation Center/Department of Biology, Springfield, MO 65806, USA.
Missouri State University, Jordan Valley Innovation Center/Department of Biology, Springfield, MO 65806, USA.
J Neurosci Methods. 2024 Feb;402:110034. doi: 10.1016/j.jneumeth.2023.110034. Epub 2023 Dec 10.
Primary neuronal cultures are used to elucidate cellular and molecular mechanisms involved in disease pathology and modulation by pharmaceuticals and nutraceuticals, and to identify novel therapeutic targets. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it can be difficult to produce high-quality consistent cultures. To overcome these issues, cryopreservation can be used to obtain standardized, high-quality stocks of neuronal cultures.
In this study, we present a simplified cryopreservation method for rodent primary trigeminal ganglion neurons and glia from Sprague-Dawley neonates, using a 90:10 (v/v) fetal bovine serum/dimethyl sulfoxide cell freezing medium.
Cryopreserved trigeminal ganglion cells stored for up to one year in liquid nitrogen exhibited similar neuronal and glial cell morphology to fresh cultures and retained high cell viability. Proteins implicated in inflammation and pain signaling were expressed in agreement with the reported subcellular localization. Additionally, both neurons and glial cells exhibited an increase in intracellular calcium levels in response to a depolarizing stimulus. Cryopreserved cells were also transiently transfected with reporter genes.
Our method is simple, does not require special reagents or equipment, will save time and money, increase flexibility in study design, and produce consistent cultures.
This method for the preparation and cryopreservation of trigeminal ganglia results in primary cultures of neurons and glia similar in viability and morphology to fresh preparations that could be utilized for biochemical, cellular, and molecular studies, increase reproducibility, and save laboratory resources.
原代神经元培养物被用于阐明与疾病病理学相关的细胞和分子机制，以及研究药物和营养保健品的调节作用，并确定新的治疗靶点。然而，从啮齿动物胚胎中制备原代神经元培养物是劳动密集型的，并且很难生产出高质量的一致培养物。为了克服这些问题，可以使用冷冻保存来获得标准化的、高质量的神经元培养物储备。
在这项研究中，我们提出了一种简化的冷冻保存方法，用于保存来自 Sprague-Dawley 新生大鼠的三叉神经节神经元和神经胶质细胞，使用 90:10（v/v）胎牛血清/二甲基亚砜细胞冷冻培养基。
储存在液氮中长达一年的冷冻保存的三叉神经节细胞表现出与新鲜培养物相似的神经元和神经胶质细胞形态，并且保持高细胞活力。涉及炎症和疼痛信号转导的蛋白质的表达与报道的亚细胞定位一致。此外，神经元和神经胶质细胞对去极化刺激均表现出细胞内钙水平的增加。冷冻保存的细胞也可以瞬时转染报告基因。
我们的方法简单，不需要特殊试剂或设备，将节省时间和金钱，增加研究设计的灵活性，并产生一致的培养物。
这种制备和冷冻保存三叉神经节的方法可获得与新鲜制备物相似的存活和形态的神经元和神经胶质细胞原代培养物，可用于生化、细胞和分子研究，提高重现性并节省实验室资源。
