| Culture Medium |
Claycomb medium, 2 mM glutamine, 10% FBS, 0.1 mM norepinephrine in L-ascorbic acid (We do not sell)
Prepare 0.1 mM norepinephrine in L-ascorbic acid:
Prepare a 100-fold stock solution = 10 mM norepinephrine.
1. Prepare a 30 mM L-ascorbic acid solution (50 mL)
2. Add 160 mg of norepinephrine to the ascorbic acid solution (50 mL) = 10 mM norepinephrine
3. Sterilely filter the solution through a 0.2-µm filter cartridge.
4. Fill 1-mL aliquots of the 100-fold norepinephrine stock solution into sterile vials and store at −20 °C. Wrap in aluminum foil; must be protected from light. The solution is stable for approximately 1 month at −20 °C.
5. Add 1 mL of this solution to 100 mL of Claycomb medium. The medium is stable for only 1 month. |
| Supplements |
Supplement the medium with 10% FBS, 2 mM glutamine, 0.1 mM norepinephrine in L-ascorbic acid |
| Dissociation Reagent |
Accutase |
| Doubling time |
approx. 24 to 36 hours |
| Subculturing |
Remove the old medium from the adherent cells and wash them with PBS that lacks calcium and magnesium. For T25 flasks, use 3-5 ml of PBS, and for T75 flasks, use 5-10 ml. Then, cover the cells completely with Accutase, using 1-2 ml for T25 flasks and 2.5 ml for T75 flasks. Let the cells incubate at room temperature for 8-10 minutes to detach them. After incubation, gently mix the cells with 10 ml of medium to resuspend them, then centrifuge at 300xg for 3 minutes. Discard the supernatant, resuspend the cells in fresh medium, and transfer them into new flasks that already contain fresh medium. |
| Split ratio |
1 to 3 |
| Seeding density |
2 to 4 x 104 cells/cm2 |
| Fluid renewal |
Daily (Claycomb Medium changed every 24 hours to maintain contractile phenotype) |
| Post-Thaw Recovery |
After thawing, plate cells onto fibronectin/gelatin-coated flasks (pre-coat with 12.5 µg/ml fibronectin and 0.02% gelatin; incubate 37°C for ≥1 hour, aspirate before seeding). Allow 24–48 hours for adherence and recovery before the first medium change. Feed daily with supplemented Claycomb Medium thereafter. |
| Freeze medium |
As a cryopreservation medium, we use complete growth medium (including FBS) + 10% DMSO for adequate post-thaw viability, or CM-1 (Cytion catalog number 800100), which includes optimized osmoprotectants and metabolic stabilizers to enhance recovery and reduce cryo-induced stress. |
| Thawing and Culturing Cells |
Confirm that the vial remains deeply frozen upon delivery, as cells are shipped on dry ice to maintain optimal temperatures during transit.
Upon receipt, either store the cryovial immediately at temperatures below -150°C to ensure the preservation of cellular integrity, or proceed to step 3 if immediate culturing is required.
For immediate culturing, swiftly thaw the vial by immersing it in a 37°C water bath with clean water and an antimicrobial agent, agitating gently for 40-60 seconds until a small ice clump remains.
Perform all subsequent steps under sterile conditions in a flow hood, disinfecting the cryovial with 70% ethanol before opening.
Carefully open the disinfected vial and transfer the cell suspension into a 15 ml centrifuge tube containing 8 ml of room-temperature culture medium, mixing gently.
Centrifuge the mixture at 300 x g for 3 minutes to separate the cells and carefully discard the supernatant containing residual freezing medium.
Gently resuspend the cell pellet in 10 ml of fresh culture medium. For adherent cells, divide the suspension between two T25 culture flasks; for suspension cultures, transfer all the medium into one T25 flask to promote effective cell interaction and growth.
Adhere to established subculture protocols for continued growth and maintenance of the cell line, ensuring reliable experimental outcomes.
|
| Incubation Atmosphere |
37°C, 5% CO2, humidified atmosphere. |
| Flask Coating |
The cell culture bottles must be coated with fibronectin and gelatin. 25 µg fibronectin, dissolved in 2 ml of 0.02% gelatin in water, are placed in a T25 and incubated overnight at 37°C |
| Shipping Conditions |
Cryopreserved cell lines are shipped on dry ice in validated, insulated packaging with sufficient refrigerant to maintain approximately −78 °C throughout transit. On receipt, inspect the container immediately and transfer vials without delay to appropriate storage. |
| Storage Conditions |
For long-term preservation, place vials in vapor-phase liquid nitrogen at about −150 to −196 °C. Storage at −80 °C is acceptable only as a short interim step before transfer to liquid nitrogen. |
