Method for cryopreservation of brainstem pons and medulla oblongata tissue from Sprague Dawley rats for establishing primary mixed neuron-glia cell cultures

Primary cultures of brainstem tissue can be used to investigate cellular and molecular mechanisms involved in disease pathology and to identify novel therapeutic targets that modulate neuron and glial cell activities. However, preparation of primary cultures from rodent embryos or neonatal animals 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 brainstem neuronal and glial cells. We present a simplified cryopreservation method for establishing primary cell cultures of pons and medulla oblongata tissue from Sprague-Dawley neonates, using a 90:10 (v/v) fetal bovine serum/dimethyl sulfoxide cell freezing medium. Cryopreserved brainstem cells stored for up to one year in liquid nitrogen exhibited similar neuronal and glial cell morphology, cell ratios, and viability when compared to fresh cultures. The expression of proteins in neurons and glial cells implicated in pain signaling and central sensitization agreed with their reported subcellular localization. Elevated intracellular calcium levels were observed in neurons and glia in response to ATP. This method for the preparation and cryopreservation of brainstem cells for establishing primary neuron-glia cultures similar to fresh preparations, is straightforward, can be utilized for biochemical, cellular, and molecular studies, increases reproducibility, requires no special equipment or reagents, saves laboratory resources including time and money, reduces the number of animals used in research, and increases flexibility in study design.