Hello everyone! Today I bring you a very practical topic: how to use serum-free cell cryopreservation solution to keep our cell babies safe during the long "hibernation period". I believe that many friends will encounter the situation of needing to preserve cells for a long time when doing experiments. At this time, it is particularly important to master the correct cell cryopreservation skills! Let's take a look at the specific steps below!
1. Preparation
First, we must ensure that the cell babies are in the best condition. It is best to choose to cryopreserve them during their rapid growth period. Next, we need to accurately count the number of these little guys and ensure that the cell density in each cryopreservation tube is moderate, about 1-5 million cells/ml, so as to ensure the best cryopreservation effect.
Next, it is to prepare the cryopreservation solution. Serum-free cell cryopreservation solution is safer because it does not contain serum components and will not introduce unnecessary variables. Prepare enough cryopreservation solution according to the proportion in the instructions, and remember to put it in the refrigerator to cool it down in advance.
2. Cryopreservation process
1. Prepare cell suspension: Gently shake the culture bottle to evenly disperse the cells, and then take a portion of the cell suspension into the centrifuge tube.
2. Add cryopreservation solution: slowly add the cooled serum-free cell cryopreservation solution to the cell suspension, gently shaking while adding to mix the two thoroughly.
3. Dispense into cryopreservation tubes: evenly distribute the mixed liquid into the cryopreservation tubes prepared in advance, remember to move gently to avoid bubbles.
4. Precooling treatment: put the cryopreservation tubes containing cell suspension into a pre-set refrigerator or freezer box, and slowly cool down according to the recommended cooling rate, which can reduce the damage of ice crystals to cells.
3. Thawing method
When you need to use these cells, follow the steps below to thaw:
1. Rapid thawing: After taking out the cryopreservation tube from the refrigerator or liquid nitrogen tank, immediately put it in 37°C warm water for rapid thawing until the liquid inside is completely melted.
2. Dilute cells: quickly pour the thawed liquid into a centrifuge tube containing preheated complete culture medium, so that the protective agent in the cryopreservation solution can be diluted.
3. Centrifugal washing: Centrifuge at an appropriate speed (such as 1000 rpm for 5 minutes), then pour off the upper liquid and resuspend the cells with fresh culture medium.
4. Inoculation culture: Finally, transplant these treated cells into a new culture container, add an appropriate amount of fresh culture medium, and return to the incubator for continued culture.
Tips & Notes
· Avoid repeated freezing and thawing: Try not to let the cells experience multiple freezing and thawing, which will cause great harm to them.
· Control the freezing speed: The appropriate cooling speed is very important. Too fast or too slow is not conducive to cell survival.
· Clear labeling: Each cryopreservation tube must indicate important information such as date and cell type for easy future search.
· Regular inspection: Occasionally check the status of the frozen cells and update or replace problematic samples in time.
*Content generated by GPT-4o