Human Neurons

PLO/Laminin Coating

1. Dilute Poly-L-Ornithine (PLO) Coating Solution (0.1 mg/ml; TM062) in sterile PBS to a final concentration of 5 µg/ml. Coat the culture surface and incubate for 2 hours.

2. Remove PLO and wash the surface 3 times with sterile PBS.

3. Dilute laminin in sterile PBS or the recommended coating buffer to 20 µg/ml. Coat the surface evenly and incubate for 2 hours.

4. Remove the coating solution immediately before cell seeding. For optimal      performance, use freshly coated culture vessels.

Thawing and Cell Recovery

1. Pre-warm only the volume of complete Human Neuron Maintenance Medium (TM230) required for immediate use in 37°C water bath. Do not warm the entire bottle.

2. Rapidly thaw the vial in a 37°C water bath with gentle agitation until only a      small ice crystal remains (~2 minutes). Disinfect the vial with 70% ethanol before placing it in the biosafety cabinet.

3. Transfer the cell suspension dropwise into a sterile 15 ml tube containing 9 ml      pre-warmed Human Neuron Maintenance Medium (TM230). Mix gently.

4. Centrifuge at 200 × g for 5 minutes and carefully remove the supernatant.

5. Gently resuspend the cell pellet in 1 ml fresh Human Neuron Maintenance Medium (TM230).

Cell Seeding

1. Seed cells onto PLO/laminin-coated plates at 60,000–80,000 cells/cm².

2. Gently rock the plate in a cross pattern to distribute cells evenly.

3. Incubate at 37°C with 5% CO₂. Avoid disturbing the culture during initial      attachment.

Post-Thaw Recovery

1. Do not disturb the cells for 12–24 hours after seeding.

2. At 24 hours post-thaw, carefully replace 50% of the medium.

3. Monitor cell morphology, neurite extension, attachment, and overall culture      health. 

Maintenance

1. Replace the culture medium every 2 days with pre-warmed Human Neuron Maintenance Medium (TM230). Pre-warm only the volume of complete maintenance medium required for immediate use. Do not warm the entire bottle.

 2. During medium changes, aspirate slowly from the side of the vessel without directly disturbing the neuronal layer. Add fresh medium gently along the wall of the culture vessel.

3. Maintain cultures at 37°C and 5% CO₂ and monitor regularly for attachment, neurite extension, cell density, aggregation, debris, and contamination. Use the cells for downstream applications or continue to maintain them in culture.

Important: Do not allow the culture surface to dry completely during media changes or handling, as drying may damage neuronal cells and neurites. Handle cultures gently and avoid direct pipetting onto the cells.

Expected Morphology: Healthy human neurons should exhibit phase-bright cell bodies with extending neurites and progressively interconnected neuronal networks. As cultures mature, neurite length and network complexity may increase.

Note: Human Neurons are differentiated cells intended for maintenance and downstream applications rather than routine expansion by subculturing. Routine trypsinization and passaging are not recommended.