Replating Differentiated Neurons Derived from Human Pluripotent Stem Cells

1. Differentiation Period Prior to Replating

1. Differentiate neurons on 10 cm dishes, using a protocol of choice, until neurons have formed a thick network with their processes and express not only early neuronal markers such as MAP2 or TuJ1, but also late markers such as NeuN.
2. Change half the medium of choice every 4 days during the neuronal differentiation process.
   NOTE: More extensive or frequent medium changes dilute essential trophic factorsaq and could disfavor maturation.
   1. For the iPSC-derived WT126 neurons, use the following post-differentiation culturing media: 5 mL 100x N2 (neural) supplement, 10 ng/mL brain-derived neurotrophic factor (BDNF), 10 ng/mL glial cell line-derived neurotrophic factor (GDNF), 1 µg/mL laminin, 200 µM ascorbic acid, 1 µM dibutyryl-cAMP and 10 mL SM1 for 500 mL Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12). Gradually, using half-media changes, replace with neural basal medium (Table of Materials) and the same supplements.
   2. For the induced pluripotent stem cell or iPSC-derived CVB WT neurons, use the following post-differentiation culturing medium: 500 mL neural basal A medium (Table of Materials) and 500 mL DMEM/F12 medium with 2 µg/mL laminin, 10 mL glutamine supplement (Table of Materials), 0.75 mg/mL sodium bicarbonate, 5 mL minimum essential medium (MEM) nonessential amino acids, 0.2 mM ascorbic acid, 10 ng/mL BDNF, 20 mL 50x B27, and 10 mL 100x N2 supplement.

2. Coating Multiwells

1. The day before replating, coat with poly-L-ornithine (PLO). Dissolve PLO in sterile water to make a stock solution (10 mg/mL). Store this stock at -20 °C. Dilute PLO 1:100 in water to yield a concentration of 50 µg/mL when coating glass and 1:1,000 ratio (10 µg/mL) when coating plastic.
2. Apply the coating directly to the target plates. Use the volume of coating appropriate to the plate size (i.e., for a 24-well plate apply 500 µL of PLO solution per well).
3. Allow plates to sit in the dark overnight at room temperature.
4. Retrieve coated plates on the day of replating and transfer to a sterile biosafety cabinet.
5. Aspirate the PLO solution and rinse twice with sterile water.
6. Dilute laminin (1.15 mg/mL) in phosphate-buffered saline (PBS) at 1:400 dilution.
   NOTE: Thaw laminin at 4 °C and quickly add to PBS to avoid aggregation of laminin and uneven coating.
7. Aspirate sterile water and apply 500 µL of laminin coating to wells previously coated with PLO.
8. Place plates in a 37 °C incubator for a minimum of 4-6 h. Use longer incubations, up to 16 h, for glass surfaces. Use consistent incubation times.

3. Replating Differentiated Neurons

1. Rinse the plate of differentiated neurons with PBS once gently. Disperse PBS gently down the wall of the plate, and not directly onto the cells, to avoid disrupting them.
2. Gently aspirate PBS, being careful to avoid touching the cells directly but to aspirate from the edge of the dish while tipping it towards the researcher.
3. Apply a minimum of 1 mL of the proteolytic enzyme (Table of Materials) per 10 cm plate and return the cells to the incubator. Add slightly higher volumes if the tissue culture room exhibits a high evaporation rate due to low humidity.
4. Incubate cells with proteolytic enzyme for 40-45 min in order to detach them from the plate and detach them from other neurons within the neuronal network.
   NOTE: Timing at this step is critical. Quenching the protease too early can lead to increased cell death after replating. The proteolytic enzyme manufacturer recommends that temperatures much lower than 37 °C be used with longer incubation periods for passaging cell lines (e.g., overnight at 4 °C). However, handling neurons at 4 °C should be avoided, as they often show poor survival after cold exposure. The manufacturer also states that a 60-minute incubation with the enzyme at 37 °C leads to its enzymatic inactivation. However, in the authors' experience, a 40-45 min incubation of hiPSC-derived neuronal cultures at 37 °C is sufficient for efficient dissociation and excellent neuronal survival upon replating.
5. Check neurons on a phase-contrast microscope during the incubation time and allow protease treatment to continue until the neural network completely detaches from the plate and starts to break apart into smaller sheets upon briefly shaking the plate under the microscope.
6. Quench the protease activity using 5 mL of fresh DMEM media per 1 mL of protease in the 10 cm plate to stop the digestion. Gently triturate cells against the plate 5-8 times to disrupt the network, using serological pipettes. Be careful not to apply too much pressure when triturating, as differentiated neurons are fragile. Do not use a P1000 tip, as the end is too sharp and narrow, and thus can sheer or damage the neurons.
7. Apply solution with cells through a cell strainer with 100 µm diameter mesh into a fresh 50 mL conical tube drop by drop.
8. Rinse the strainer with an additional 5 mL of fresh DMEM media, after cells have filtered through.
9. Spin cells in a benchtop centrifuge at 1,000 x g for 5 min.
10. Return the conical tube to the biosafety cabinet and aspirate most of the media, leaving around 250 µL to ensure cells retain moisture.
11. Resuspend cells gently in 2 mL of fresh DMEM media. Do not pipette the pellet against the side of the tube. Instead, gently invert the conical tube 2-3 times and pass cells through the end of a 5 mL serological pipet to dislodge them.
12. Apply 10 µL of resuspended neurons onto the edge of a hemocytometer.
13. Add 8-10 µL of trypan blue to the droplet of cells to assess cell viability during this resuspension step. Apply 10 µL of this mixture to the hemocytometer or other automatic cell counters. Assess as viable those cells that are phase bright and exclude the trypan blue dye.
14. Determine the amount of viable cells/mL, and prepare to dilute the contents of the conical tube according to the desired cell density.
15. Plate ~10,000 cells per well for a 384-well plate; plate ~150,000-200,000 cells per well for a 24-well plate.
16. Add fresh DMEM to the conical tube of resuspended cells to achieve appropriate dilution and add additional appropriate supplements such as B27 and/or BDNF, depending on the requirements of the specific cell line.
17. Gently tilt the conical tube to mix 2-3 times.
18. Aspirate laminin coating from the 24-well plate or from the 384-well multiwell plate using a 16-channel pipet and rinse once with PBS.
19. Aspirate PBS using a P1000 for the 24-well plate or the 16-channel pipette for 384-well plates.
20. Apply cell solution to each well in a figure-eight motion to avoid clumping. Plating uniformity might also be optimized by using automated liquid handling devices.
    NOTE: The addition of laminin to the media starting 2-4 days post-replating also helps maintain a homogenous distribution of the cells.
21. Repeat steps 3.19 and 3.20 for each well.
22. Return the plate to the incubator, set at 37 °C and 5% carbon dioxide.
23. After 2 days, start changing half the medium every 4 days using the post-differentiation culturing media described in section 1.2.