DMSO-based CryoMed-M medium is developed for suspension freezing and storage of cultured cells in an extended temperature range from -40oC to -197OC. The high survival rate of preserved cells is ensured by synergistic action of 2 types of cryoprotectors. Provides effective preservation of working batches of cell cultures in conditions of low-temperature cooling devices (-70oC) for 1-4 months. The medium does not contain serum and organic components of indeterminate composition. It has a balanced ionic composition.
This product is intended for research purposes.
Instructions for use
1. Take a culture with a monolayer density of 70-80% (for cells of attached cultures), or 50-70% of suspension density (for cells of suspension cultures).
2. Remove the cells of the attached cultures from the substrate using dissociating Trypsin-EDTA solution of 0.05% (0.25%) for 5 minutes at 37oC (pre-rinse cell monolayer with an isotonic Ca2+ and Mg2+ ions-free solution). The cells of suspension cultures are pipetted and collected in a centrifuge tube.
3. Inactivate trypsin using: a medium containing 2-10% serum (for serum cultures) or working solution of a trypsin inhibitor from soy fruits (0.2 mg/ml) – (for serum-free or serum cultures).
4. Precipitate the cells by centrifuging at 1000 rpm for 5-7 minutes. Remove the supernatant without affecting the cellular sediment.
5. Resuspend cells in the "Cryomed-M" medium at the rate of 0.5-2.0 million/ml.
6. Pour the prepared cryosuppression into cryoprobes, seal the frozen samples hermetically.
7. Place the frozen samples in a special cryocontainer with a lid, you can put it in a styrofoam box and place it in a freezer (- 20oC) for slow cooling for 0.5 - 1.0 hours.
8. Transfer the sample box to the deep freeze freezer (-70oC) for 8-12 hours or for operational storage for 1 to 4 months.
9. Quickly transfer the sample box to liquid nitrogen (-197OC) for long-term storage.
Defrosting of cells:
* Completely defrost the contents of cryoprobes at 37oC.
* Gently resuspend the cells and wash them once in a culture medium or isotonic solution (1 ml of cryogenic sample / 4 ml of medium, solution). It is optimal to incubate the cells in a washing solution at room temperature for 15 minutes. Centrifugate 1000 rpm for 5-7 minutes.
* Resuspend the cells in the culture medium and plant them in a culture vial of 25 cm2.
• For maximum removal of DMSO, it is recommended to completely replace the medium 1 day after the start of cell culture.
• If necessary, before the passage, the number of viable cells in the seed material is evaluated by staining with trypan blue.
Shelf life: 1 year
Storage and transportation conditions:
Store at temperature of 2-8 ° C in a place protected from light.
Transportation by all types of covered transport at temperature of 2-8 oC.
