Replating Differentiated Neurons Derived from Human Pluripotent Stem Cells

Start with a large culture plate containing human pluripotent stem cell-derived differentiated neurons with a dense neuronal network.

Incubate these neurons with mild proteolytic enzymes to detach them from the plate, lifting them as a sheet.

With continued incubation, enzymes partially disrupt the cell connections, loosening the neuronal networks.

Add a culture medium to stop the enzyme activity.

Repeatedly pipette the detached neurons to separate them from each other.

Filter the neurons through a cell strainer to remove cell clumps and collect the individual neurons.

Centrifuge and remove the supernatant. Resuspend them in a growth medium.

For replating, seed these neurons onto a polymer and an adhesion protein-coated multi-well plate. Incubate to allow their attachment.

Neurons utilize nutrients from the medium to remain viable. Replace the culture medium regularly to maintain nutrient levels.

Incubate further to allow neuronal maturation and formation of connections between them.

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 1 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 phase-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 5 milliliters of fresh DMEM media for every 1 milliliter of protease.

Use a serological pipette to gently triturate the cells against the plate 5 to 8 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 5 milliliters of fresh DMEM media.

Use a benchtop centrifuge to spin the cells down at 1,000 times g for 5 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 2 milliliters of fresh DMEM media, and pass them through the end of a 5-milliliter serological pipette.

Finally, invert the tube 2 to three times. Use a hemocytometer and trypan blue to count the viable cells, and then dilute the cells with fresh 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 2 to 3 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.