This article describes a method for preparing neuronal cultures using polyornithine and laminin to enhance cell attachment. Neural stem cells are then added to the coated coverslips, promoting their differentiation into neurons.
In a culture plate, coat a coverslip with polyornithine, a positively charged polymer, and incubate.
Polyornithine forms a mesh-like layer that helps negatively charged cells stick to the coverslip.
Remove the solution and wash the coverslips with a buffered salt solution to remove unbound polyornithine.
Add a solution containing an extracellular matrix protein called laminin and incubate.
Laminin forms a matrix over the polyornithine-coated coverslip, enhancing cell attachment.
Discard the solution and take a small number of neural stem cells suspended in a growth factor-free medium.
Add this low-density culture over the prepared coverslip in a slow rotating movement to evenly distribute the cells.
Initially, growth factor deprivation promotes cell division.
Over time, stem cells differentiate into neurons with long projections
Eventually, neurons produce spine-like structures over their projections, like the cortical pyramidal neurons.
To prepare neuronal cultures, place glass coverslips into six-well culture dishes, and add polyornithine. Incubate overnight under a flow hood. The following day, use DPBS to wash the coverslips three times and add laminin. Incubate under a flow hood for at least 10 hours.
Prepare culture medium containing the following components. Then, use the medium to plate and dispatch neural stem cells or NSCs at a density of 50,000 cells per square centimeter, pipetting with slow rotating movements in order to reduce cell clustering.
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