Culture of Rat Primary Cortical Cells for Microelectrode Array (MEA) Recordings to Screen for Acute and Developmental Neurotoxicity

Abstract

Neurotoxicity testing of chemicals, drug candidates, and environmental pollutants still relies on extensive in vivo studies that are very costly, time‐consuming, and ethically debated due to the large number of animals typically used. Currently, rat primary cortical cultures are widely used for in vitro neurotoxicity studies, as they closely resemble the in vitro brain with respect to the diversity of cell types, their physiological functions, and the pathological processes that they undergo. Common in vitro assays for neurotoxicity screening often focus on very target‐specific endpoints such as morphological, biochemical, or electrophysiological changes, and such narrow focus can hamper translation and interpretation. Microelectrode array (MEA) recordings provide a non‐invasive platform for extracellular recording of electrical activity of cultured neuronal cells, thereby enabling the evaluation of changes in neuronal (network) function as a sensitive and integrated endpoint for neurotoxicity screening. Here, we describe an in vitro approach for assessing changes in neuronal network function as a measure for neurotoxicity, using rat primary cortical cultures grown on MEAs. We provide a detailed protocol for the culture of rat primary cortical cells, and describe several experimental procedures to address acute, subchronic, and chronic exposure scenarios. We additionally describe the steps for processing and analyzing MEA and cell viability data. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and culture of rat primary cortical cells on 48‐well MEA plates Support Protocol 1: Pretreatment and washing of 48‐well MEA plates before first use or for re‐use Support Protocol 2: Coating of 48‐well MEA plates with 0.1% PEI solution Basic Protocol 2: MEA measurements during acute exposure Alternate Protocol 1: MEA measurements during subchronic exposure Alternate Protocol 2: MEA measurements during chronic exposure Support Protocol 3: Determination of cell viability after MEA experiments Basic Protocol 3: MEA data processing Basic Protocol 4: Analyzing MEA experiments after acute and subchronic exposure Alternate Protocol 3: Analyzing MEA experiments after chronic exposure

Traumatic Brain Injury in a Well: A Modular Three-Dimensional Printed Tool for Inducing Traumatic Brain Injury In vitro

“…Dissociated neuronal cultures were obtained from cerebral cortices of newborn Sprague-Dawley rats at days P3–P4 as previously reported. 15 All experiments were approved by the local veterinary authority and the animal ethics committee of Tel Aviv University (approval ethic no. : 01-19-079) and performed in accordance with Israeli law.…”

Enterovirus D-68 Infection of Primary Rat Cortical Neurons: Entry, Replication, and Functional Consequences

“…Next, coverslips were washed four times with sterile laboratory-grade water (B. Braun, Melsungen AG, Melsungen, Germany) prior to adding 200,000 cortical cells per well of a 24-well plate. For MEA recordings, 48-well MEA plates (Axion BioSystems Inc., Atlanta, USA) were coated with 0.1% PEI, and cortical cultures were seeded at a density of 100,000 cells per well in a drop as described previously ( 40 ). For the cell viability assay of the complete network, cells were cultured at a concentration of 20,000 cells per well of a 96-well plate, coated with PEI.…”

“…The neuronal activity of EV-D68 infected primary rat cortical cultures was analyzed using MEA recordings as described previously ( 40 , 44 ). First, spontaneous baseline neuronal activity was measured for 30 min (min).…”

“…For each time point, the parameters describing neuronal (network) activity during EV-D68 or mock infection were analyzed during the last 10 min of each MEA recording and expressed as the ratio of exposure (numerator) over baseline (denominator) as a percentage of mock-infected control wells. The outlier analysis was performed as described by Gerber et al ( 40 ). The ratios of exposure were averaged per parameter (number of spikes and number of bursts), per condition (EV-D68 strains), and time point post exposure (2, 4, 8, 24, or 48 hpi).…”