This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.
This protocol facilitates the use of human pluripotent stem cell-derived neurons for high content screening applications. It is particularly well suited for assaying synapses, which in human neurons require lengthy culture periods. We demonstrate the replating of neurons from large format dishes into HCS-compatible multi wells in a way that preserves their viability.
Counter-intuitively, we show that extending protease incubation prior to resuspending and replating yields improved neuronal survival. Human pluripotent stem cell-derived neurons are increasingly relevant in the areas of basic research, drug development, and degenerative medicine. This gentle titration method can be used to resuspend any large mono-layer of cells having a thick network of processes.
In addition to human iPSCs, it can be used to replate culture of primary neurons or to resuspend them for fact sorting or single cell sequencing. It is important to follow the protocols steps carefully and frequently monitor the protease mediated detachment of the neurons from the plate. The optimal incubation time may vary, depending on the culture.
Visual demonstration enables even inexperienced investigators to reproduce this procedure. Start by gently rinsing the plate of differentiated neurons with PBS. Disperse the PBS down the wall of the plate and not directly onto the cells to avoid disrupting them.
Aspirate the PBS from the edge of the dish while tipping it, taking care not to touch the cells. Apply at least one milliliter of proteolytic enzyme per 10-centimeter plate. And return the cells to the incubator for 40 to 45 minutes.
During the incubation, check neurons on a phased contrast microscope and continue the protease treatment until the neural network completely detaches from the plate and starts to break apart. When the neurons have detached, stop digestion by adding five milliliters of fresh DEMEM media for every one milliliter of protease. Use a serological pipette to gently titrate the cells against the plate five to eight times, making sure to not apply too much pressure.
Strain the cells through a 100 micrometer mesh into a 50 milliliter conical tube, drop by drop. And rinse the strainer with an additional five milliliters of fresh DMEM media. Use a bench-top centrifuge to spin the cells down at 1000 times G for five minutes.
After centrifugation, return the conical tube to the biosafety cabinet and aspirate most of the media, leaving 250 microliters to keep the cells moist. Gently resuspend the cells in two milliliters of fresh DMEM media then pass them through the end of a five milliliter serological pipette. Finally, invert the tube two to three times.
Use a hemocytometer and tripan blue to count the viable cells. And then DMEM to the desired concentration. Add appropriate supplements to the tube, depending on the requirements of the specific cell line and gently mix the cells by tilting the conical tube two to three times.
Aspirate laminin coating from a 24-well plate and rinse it once with PBS. Aspirate the PBS. And apply cell solution to each well in a figure eight motion to avoid clumping.
When finished plating the cells, return the plate to the incubator set at 37 degrees Celsius and 5%carbon dioxide. Briefly vortex the stock solution of the early cell death reporter and add it to the wells according to the manuscript directions. Wait 20 minutes.
And then image the live and dead cells on glass bottom multi wells. Prolonging the protease incubation allows loosening of the neuronal network within the lifted sheet of cells. This results in reduced cell death at the time of dissociation as well as results in more efficient recovery over the following days.
Using the extended enzyme incubation procedure, the cultures exhibit an approximate doubling of cell viability during the days after replating and a lower density of dead or dying cells. The transition phase of neuronal differentiation takes place over several days, during which the cells gradually express late-stage neuronal markers. These markers are immediately detected in cultures that were replated after four weeks of pre-differentiation.
The extended protease protocol also moderately enhances neuride outgrowth. Both total neuride length and dendrite growth are improved. Replating is also useful for studying synapses.
Just one week after replating, markers for pre-and post-synaptic proteins were observed. Moreover, electrical activity from spontaneous depolarization and synaptically-driven currents is detectable using calcium imaging or multielectrode arrays. This replating procedure can be adapted to many types of applications.
In addition to high content screening to identify potential therapeutic compounds, it could be used for imaging growth cones or multielectrode array recordings. Mechanical dissociation of neurons that have already formed long processes is stressful. Elongated enzymatic incubation and gentle titration of cells are essential steps for the success of this procedure.
