Neuron Isolation on the CytoR1™ Platform: A Customized Workflow for Subpopulation Enrichment

KE Degen, JD Joyce, T Harrell, AS Bertke, AR Hyler. Neuron isolation on the CytoR1 Platform: a customized workflow for subpopulation enrichment. American Society for Cell Biology Annual Meeting, Washington D.C. December 2022.

The isolation of primary neurons from peripheral ganglia of adult animals results in a dense mixture of axon debris, satellite glial cells (SGCs), and neurons. Conventional clean up techniques utilize density gradients, which achieve roughly 50-75% removal of SGCs and debris. Here, a novel workflow was developed using the Cyto R1 Platform to deplete debris and satellite glial cells to achieve more purified downstream neuron cultures. The Cyto R1 employs label-free, biophysical enrichment (dielectrophoresis) to separate live cells from debris by trapping cells with intact membranes. Using a disposable microfluidic cartridge, Cyto Chip, neurons can be enriched in a sterile system in which trapping occurs at stationary pillars in the Chip. In order to generate the appropriate electrical fields for enrichment, we transferred samples into an ultra-low conductivity buffer, Cyto Buffer. For this workflow development, a customized buffer formulation was needed to support neuron viability. Murine trigeminal ganglia (TG) neurons were exposed to 6 novel buffers in suspension for 30 min at room temperature, plated on Matrigel and maintained in Neurobasal A medium with neurotrophic factors. Neuron viability was assessed immediately prior to plating, then 24 hours, 5 days and 14 days after plating. One buffer out-performed the others, maintaining neuronal viability on par with the control neurons in supplemented Neurobasal A media under the same conditions. Neurons exposed to this buffer were able to reestablish normal biophysical architecture once plated and remained viable for at least 14 days in post-exposure culture. With the adjusted formulation of buffer, additional TG suspensions were then enriched on the Cyto R1 Platform at 400 kHz and 640 Vpp in 5-minute batches. After enrichment, 85% recovery of purified neurons was achieved. Additionally, we observed variable trapping efficiencies as different flow rates, voltages, and frequencies suggesting that even finer sorts of cell subpopulations will be possible. The Cyto R1 provides a versatile platform for isolation of purified primary neurons from adult animal peripheral nervous system tissues for downstream culture or assays.

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