Differentiation of a Human Neuroblastoma Cell Line into Mature Neurons

1. General Considerations

1. See the Table of Materials/Equipment for a list of necessary reagents. Perform all steps under strict aseptic conditions.
2. Use heat-inactivated fetal bovine serum (hiFBS) for all media preparations that include FBS. To heat-inactivate, warm a 50 ml aliquot of FBS at 56 °C for 30 min, inverting every 10 min (see also Table 1).
   NOTE: When FBS is used without heat-inactivation, the epithelial-like phenotype progresses more quickly throughout cultures of SH-SY5Y cells.
3. Prior to use, allow media to warm and equilibrate in an incubator to establish a proper pH balance before every step. For example, 50 ml of media takes approximately one hour to fully equilibrate (pH 7, 37 °C, 5% CO₂).
   NOTE: This protocol uses a two-step splitting procedure that requires partially differentiated SH-SY5Y cells to be trypsinized and re-plated. This is a stressful process for these exceptionally fragile cells. Therefore, it is important to incubate the cells in trypsin for a minimal amount of time. This will allow for the preferential lift-off of neurons, leaving epithelial-like cells still attached to the dish.
4. Perform trituration of differentiated cells slowly with a 10 ml plastic pipette with the tip against the bottom of the conical tube containing the cells. Perform trituration at a slow speed, up and down no more than five times.
   

2. Thaw and Culture Undifferentiated SH-SY5Y Neuroblastoma Cells

1. Prepare Basic Growth Media.
2. Rapidly thaw frozen cells in a 37 °C water bath (approximately 2 min).
3. Resuspend cells in 9 ml Basic Growth Media in a 15 ml conical tube, and then centrifuge for 2 min at 1,000 x g.
4. Aspirate the supernatant while being careful not to disturb the pelleted cells, and gently resuspend cells in 10 ml Basic Growth Media.
5. Plate cells onto a T-25 flask or 60 mm² dish.
6. The next day, replace media to remove dead cells.

3. Day 0: Plating Cells for Differentiation

1. See Figure 1 for the differentiation schedule.
2. Rinse undifferentiated cells with 1x PBS, aspirate, and then trypsinize using 1-2 ml warmed 1x 0.05% Trypsin-EDTA.
3. When cells are in trypsin, incubate for approximately 3 min in an incubator.
4. Quench the trypsin by adding 10 ml Basic Growth Media, rinse the sides of the flask or dish, and gently triturate 1-3 times. Transfer contents to a 15 ml conical tube.
5. Centrifuge for 2 min at 1,000 x g and aspirate the media while being careful not to disturb the pellet.
6. Resuspend pellet in 5 ml Basic Growth Media and triturate 1-3 times.
7. Count cells using a hemocytometer, then dilute using Basic Growth Media to 50,000 cells/ml.
8. Plate 2 ml of cells per 35 mm² dish for a total of 100,000 cells per dish and place back into incubator.

4. Day 1: Change Media (Differentiation Media #1)

1. Aliquot 50 ml of Differentiation Media #1 (see Table 2) and incubate in a 37 °C water bath.
2. When media is warmed, allow it to equilibrate in an incubator (37 °C, 5% CO₂) for at least one hr to establish a proper pH balance prior to use.
3. Add Retinoic Acid (RA) (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.
   NOTE: Retinoic acid is light sensitive and should be stored in dark bottles at 4 °C
4. Gently aspirate off old media and discard.
5. Add 2 ml Differentiation Media #1 with RA per 35 mm² dish and return to incubator.

5. Day 3: Change Media (Differentiation Media #1)

1. Repeat Section 4 (steps 1-5)

6. Day 5: Change Media (Differentiation Media #1)

1. Repeat Section 4 (steps 1-5)

7. Day 7: Split Cells 1:1

1. Add RA to warmed and equilibrated Differentiation Media #1 immediately before adding media to dishes.
2. Gently aspirate off old media and discard.
3. Add 200 μl warmed 0.05% 1x Trypsin EDTA per 35 mm² dish and warm in an incubator for approximately 2-3 min or until cells are visibly lifted from the plate as observed under a microscope.
4. Quench the trypsin by adding 2 ml Differentiation Media #1 with RA per 35 mm² dish and use the media to rinse the remaining neuronal cells off the plate. Then transfer the contents to a 50 ml conical tube.
   NOTE: Do not trypsinize too many dishes at once during the trypsinization steps. This helps to ensure neuronal cultures are not incubated in trypsin for too long, which can be cytotoxic.
5. Combine contents from up to 10 dishes in the 50 ml conical tube and gently triturate slowly up and down no more than five times with a 10 ml plastic pipette.
6. Aliquot 2 ml cell suspension into fresh 35 mm² dishes and return to incubator.

8. Day 8: Change Media (Differentiation Media #2)

1. Add RA (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.
2. Gently aspirate off old media and discard.
3. Slowly add 2 mL Differentiation Media #2 (see Table 2) with RA per 35 mm² dish and return to the incubator. Do not allow neurons to be exposed to air for an extended period as they can dry out quickly.

9. Day 9: Prepare Extracellular Matrix (ECM) Coated Dishes

1. Thaw one vial of ECM solution on ice and dilute 1:100 into cold DMEM.
2. Dispense 2 ml of mixture into each 35 mm² dish and ensure the entire base of the dish is covered.
3. Place in an incubator (37 °C, 5% CO₂) for 1 hr or overnight.
4. Aspirate mixture and allow to air dry for approximately 1 hr in a hood. Store at room temperature for up to 2 months.

10. Day 10: Transfer Cells onto ECM Coated Plates 1:1

1. Add RA (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.
2. Gently aspirate off media and discard.
3. Add 200 μl warmed trypsin to each 35 mm² dish and allow to incubate at room temperature for approximately 1-2 min or until neurons are visibly lifted from the dish as observed under a microscope.
   NOTE: Execute this trypsinization step at room temperature to avoid over-incubating neurons with trypsin and causing damage. Neurons release from plates much faster than epithelial-like cells at this stage.
4. Quench the trypsin by adding 2 ml Differentiation Media #2 per 35 mm² dish and use the media to rinse remaining neuronal cells off the plate. Then transfer contents to a 50 ml conical tube.
5. Combine contents from up to 10 dishes in the 50 ml conical tube and gently triturate slowly up and down no more than five times with a 10 ml plastic pipette.
6. Dispense 2 ml cell suspension into ECM-coated 35 mm² dishes and return to the incubator.

11. Day 11: Change Media (Differentiation Media #3)

1. Add RA (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.
2. Gently aspirate off old media and discard.
3. Slowly add 2 ml Differentiation Media #3 (see Table 2) with RA per 35 mm² dish and return to incubator. Do not allow neurons to be exposed to air for an extended period.

12. Day 14: Change Media (Differentiation Media #3)

1. Repeat Section 11 (steps 1-3)

13. Day 17: Last Media Change (Differentiation Media #3)

1. Repeat Section 11 (steps 1-3)

14. Day 18: Neuronal Cultures Ready to Use

1. Change media to fresh Differentiation Media #3 with RA every 3 days to maintain neuron health.
   NOTE: Cells should be differentiated into neurons and exhibit a neuronal phenotype. Cultures are typically stable for up to 14 days following terminal differentiation. However, the duration of neuron viability depends on the passage number of the undifferentiated cells at the start of differentiation. Higher passage numbers yield differentiated neurons with a shorter useful lifetime.

Table 1: Stock solutions and components.

Table 2: Media recipes.