Adam Figarski
have You tried just full culture medium +10% DMSO? Be gentle with detachment! But neural crest my adhere much more than NES!
0,5mM EDTA in DPBS to up to 10 min could be fine but might not be enough
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Jaba Tkemaladze
Culture Preparation:Ensure NCCs have been passaged at least once (P1) before cryopreservation. Allow cells to reach 80–90% confluency, typically 2–4 days after seeding. Cell Detachment:Warm an appropriate amount of StemPro® Accutase® Cell Dissociation Reagent to 37°C. Aspirate the spent medium and add pre-warmed Accutase® to the culture vessel (e.g., 1 mL per well of a 6-well plate). Incubate for 3–5 minutes at 37°C until cells detach. Gently dislodge cells by pipetting or using a cell scraper. Cell Collection and Counting:Transfer the cell suspension to a 15 mL conical tube. Add 1 mL of Dulbecco’s Phosphate-Buffered Saline (DPBS) to the culture vessel to collect any remaining cells and combine with the initial suspension. Gently pipette the suspension to break up cell clumps. Centrifuge at 300 × g for 4 minutes. Aspirate the supernatant and resuspend the cell pellet in DPBS. Repeat centrifugation and resuspend the pellet in Neural Expansion Medium. Determine cell concentration using a hemocytometer or automated cell counter. Preparation for Freezing:Dilute the cell suspension in Neural Expansion Medium to a concentration of 2 × 10⁶ to 4 × 10⁶ cells/mL. Mix an equal volume of Neural Expansion Medium containing 20% dimethyl sulfoxide (DMSO) to achieve a final concentration of 10% DMSO. Aliquoting and Freezing:Dispense 1 mL of the cell suspension into each cryovial. Place cryovials in a controlled-rate freezing container (e.g., Nalgene® Mr. Frosty®) and store at –80°C overnight to ensure a gradual cooling rate of approximately –1°C per minute. Transfer the vials to liquid nitrogen storage for long-term preservation. Thawing:Quickly thaw cryovials in a 37°C water bath until only a small ice crystal remains. Disinfect the outside of the vials with 70% ethanol before opening in a biosafety cabinet. Cell Recovery:Transfer the thawed cell suspension to a 15 mL conical tube. Slowly add pre-warmed Neural Expansion Medium to the tube to minimize osmotic shock. Centrifuge at 300 × g for 5 minutes and aspirate the supernatant. Resuspend the cell pellet in Neural Expansion Medium supplemented with 5 μM ROCK inhibitor Y27632 to enhance post-thaw survival. Plating:Plate cells onto Geltrex® matrix-coated culture vessels at a density of 0.5 × 10⁵ to 1 × 10⁵ cells/cm². After overnight incubation, replace the medium with fresh Neural Expansion Medium without ROCK inhibitor. Continue medium changes every other day until cells reach the desired confluency.
Thawing and Recovery:
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