Hello Zeynep Çapraz

I haven’t work with SIM-A9 microglial cells, but if you follow the instructions provided below carefully, I think you may be able to solve your problem. While thawing any cell line, it is essential to optimize the thawing speed, media composition, and post-thaw handling in order to minimize the damaging effects of the cryoprotectant. SIM-A9 cells are naturally loosely adherent, which makes gentle handling crucial throughout the process.

1. Before retrieving your cells, warm the complete growth medium for SIM-A9 cells to 37°C.

2. Rapid thawing is essential to prevent the formation of damaging intracellular ice crystals as cells rehydrate. After removal of the cryovial from liquid nitrogen storage, immediately immerse it in a 37°C water bath. Swirl the vial gently until a small ice crystal is all that remains.

3. Transfer the thawed cells dropwise into a conical tube containing 9 mL of the pre-warmed complete growth medium. Adding the medium slowly helps prevent osmotic shock, which can damage the cell membrane.

4. Centrifuge the cell suspension at a low speed (e.g., 125 x g for 5–7 minutes) to pellet the cells. A gentle centrifugation prevents stress on the delicate cells.

5. Carefully aspirate the supernatant containing the cytotoxic cryoprotectant (DMSO), making sure not to disturb the cell pellet. Resuspend the pellet gently in fresh, pre-warmed complete growth medium.

6. Microglia survive best in higher density cultures that facilitate cell-to-cell contact. Resuspend the cells in the appropriate volume of media and seed them at a higher density.

7. During passaging and media changes, handle SIM-A9 cells gently to prevent accidental detachment. Avoid vigorous pipetting during media changes. Also, avoid agitating or shaking the flask when detaching cells for subculture.

Successful thawing will depend on the quality of the cells before freezing. If you have followed the below steps correctly, reviving the cells after thawing will be much easier.

a. Only freeze cells that are healthy and in their logarithmic growth phase, typically when they are about 70–80% confluent.

b. Use optimal freezing media containing 10% DMSO and 90% serum. This formulation helps protect the cells from ice crystal damage during freezing.

c. Freeze the cells slowly at a controlled rate of about 1°C per minute until they reach -80°C. Using an insulated container like a Mr. Frosty can help achieve this.

d. For long-term storage, transfer the vials from the -80°C freezer to liquid nitrogen after 24 hours. Store them in the vapor phase of liquid nitrogen to ensure a colder and more stable temperature. Storing at -70°C will result in loss of viability.

Good Luck!

Hi Zeynep,

• Quick-thaw, dilute DMSO fast, and plate at higher density; do a gentle medium change 4–6 h post-thaw to remove DMSO and debris.
• Drop amphotericin (and minimize antibiotics) for the first 48 h — it often suppresses attachment/proliferation; use fresh, heat-inactivated serum.
• Pre-coat surfaces (PDL/PLL 10–50 µg/mL, or collagen/fibronectin) and avoid harsh spins/trituration; SIM-A9 attach and spread much better on treated plates.
• For consistently stronger adhesion and recovery, use DendroTEK dPGA | Protease-Resistant Culture Matrix as the base layer (Instead of PDL/PLL above) you can still overlay with ECM like laminin if needed.

Best of luck, Jason Hamlin

Source: I work at DendroTEK Biosciences. www.dendrotek.ca