Cryopreservation of rat cortical cells for brain-on-chip research.

BACKGROUND: Primary cortical cultures from neonatal rat brain are widely used in in vitro models of neuronal network development and disease, but depend on freshly isolated tissue, which limits experimental flexibility and increases animal use. Cryopreservation may offer a solution, but primary cortical cells from P1 rat pups have proven difficult to freeze successfully with standard protocols. NEW METHOD: We evaluated CryoStor CS10 for cryopreservation of P1 rat cortical cells. Following storage in liquid nitrogen for up to two years, thawed cells were compared with freshly isolated cells for viability, cellular composition, and electrophysiological network function in multi-electrode array cultures, including inhibitory responses to picrotoxin. RESULTS: Although post-thaw recovery was lower than for freshly prepared material, cryopreserved cells generated cultures with similar astrocyte-to-neuron ratios, similar fractions of inhibitory neurons, and largely comparable electrophysiological network properties. Within our workflow and regulatory context, our method reduced the number of animals required per experiment by approximately 90%. COMPARISON WITH EXISTING METHOD(S): CryoStor CS10 has previously been validated for cryopreservation of embryonic cortical neurons of mice and sensitive stem-cell populations. We show that CryoStor CS10 also preserves the capacity of mixed postnatal rat cortical cultures to form functional neuron-astrocyte networks with maintained spontaneous and inhibitory network activity. CONCLUSION: We present a practical cryopreservation method for primary rat cortical cells that increases experimental flexibility and can substantially reduce the number of animals required for in vitro brain-on-chip research.