Autonomously regulated microenvironments generated by oil overlay enhance primary mouse trigeminal ganglia cultures

• Austin Safadi, Medical Diagnostics, University of Delaware

• Austin Keeler, Biological Scienes, University of Delaware

Title: Autonomously regulated microenvironments generated by oil overlay enhance primary mouse trigeminal ganglia cultures

Authors: Austin Safadi*, Miriam Gerges*, Shrutee Gupta*, Yulia Skrypniuk*, Nicole Guarino#, Austin Keeler#

Abstract: Neurons evolved to function in a tightly regulated, relatively hypoxic environment that differs substantially from atmospheric conditions. While atmospheric air contains approximately 20.9% oxygen, neurons in the brain are typically exposed to only about 0.5–8% oxygen, depending on the brain region and local metabolic activity. This difference presents a challenge for in vitro experiments because neurons are commonly cultured under atmospheric oxygen levels, which can alter cellular physiology through increased oxidative stress and changes in metabolism. To address this issue, we investigated whether different oil overlays could create a controlled diffusion barrier to produce an autonomously regulated oxygen microenvironment (AROM) that better mimics the physiological oxygen environment experienced by neurons in vivo. We cultured adult mouse trigeminal ganglia, the neural tissue housing primary somatosensory neurons that innervate the face. Unlike most neural tissue, the trigeminal ganglia is readily accessible and the neural cells survive culturing even in adults. We then tested AROMs produced by a set of different oils, assessing cell growth and survival after a week in culture. Silicone oil AROMs resulted in increased cell number by approximately 70% compared to no-AROM controls and other oil AROMs. Since AROMs shift the available oxygen levels, we assessed mitochondrial membrane potential. We found a profound sex difference in controls that was reduced in the AROM cultures. Together, we found that silicone oil overlays produce a cheap and reproducible AROM that changes the cellular states of adult mouse trigeminal ganglia cultures.