Was the tissue cryopreserved prior to isolation of primary cells?
No, the tissue was not cryopreserved.
Can I expand your cells and re-freeze them? If so, how?
When either Gibco or Invitrogen cryopreserved or proliferating cultures are purchased from us, they may be expanded and cryopreserved again. However, the cryopreservation process may result in altered growth performance of the cells. The following protocol provides a basic guideline for the cryopreservation of cells using Synth-a-Freeze medium, a defined, protein-free cryopreservation medium available from us.
Please note: Due to differences in cryopreservation equipment and individual techniques, we cannot guarantee that cells cryopreserved using this protocol will be viable upon recovery from cryopreservation, and we do not provide a warranty for cells cryopreserved in an investigator's laboratory.
1. Thaw Synth-a-Freeze medium in a 37 degrees C water bath or overnight at 4 degrees C.
2. If thawed in a water bath, do not exceed 37 degrees C and do not leave the product at 37 degrees C for an extended period of time.
3. Synth-a-Freeze medium should be equilibrated to 4 degrees C prior to use. For optimal results, the use of a controlled-rate freezer is recommended. In the absence of a controlled-rate freezer, a cell cryopreservation container (e.g., Thermo Scientific Mr Frosty container) may be useful.
4. If enzymatic agents are used to remove the cells from a culture surface, resuspend the cells in a solution that will neutralize the effects of the enzyme.
5. Pellet the cells by centrifugation.
6. After removing the supernatant, resuspend the cell pellet in cold Synth-a-Freeze medium at a concentration of 5 x 10E5 to 3 x 10E6 cells/mL.
7. Distribute the cell suspension in an appropriate number of cryopreservation vials.
8. Cool the vials of cells to 4 degrees C as quickly as possible.
9. If using a controlled-rate freezer: freeze the material by reducing the temperature 1degrees C per minute until the temperature reaches -40 degrees C. Then reduce the temperature at a rate of 2 degrees C per minute until the temperature reaches approximately -90 degrees C.
10. If using a cell cryopreservation container: Prepare the container according to the manufacturer's instructions.
For best results we recommend transferring the vials to the vapor phase of a liquid nitrogen storage facility as soon as possible after the cells have reached -80 degrees C.
As a substitute for Synth-a-Freeze medium, the recommended basal medium for the cell type being cryopreserved, supplemented with 10% fetal bovine serum (FBS) and 10% DMSO, may be used. Please note that Synth-a-Freeze medium is NOT recommended for the cryopreservation of human epidermal melanocytes.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Do I need to spin the cells out of the cryopreservation medium to plate them?
We do not recommend spinning cells out of cryopreservation medium prior to plating. Centrifugation can be harmful to cells, particularly if inappropriately high speeds are used. Experience in our in-house cell culture laboratory has shown that cells do not suffer deleterious effects if the DMSO concentration is sufficiently low. Therefore, our product instructions include a detailed protocol that involves diluting the cells into culture medium such that the final DMSO concentration is less than 0.4% (v/v) at the recommended seeding density and volume of medium.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
How do I establish a culture from cryopreserved cells?
The procedure given below is a sample protocol for establishing cultures from the contents of one vial.
1. Prepare a beaker of water at 37 degrees C.
2. Remove a vial of cells from liquid nitrogen storage, taking care to protect hands and eyes.
3. Loosen the cap on the vial 1/4 turn for 10 seconds to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
4. Dip the lower half of the vial into the 37 degrees C water to thaw.
5. When the contents of the vial have thawed, wipe the outside of the vial with disinfecting solution and move to a Class II, type A laminar flow culture hood.
6. Open the vial and pipette the suspension up and down with a 1 mL pipette to disperse the cells.
7. Remove 20 µL from the vial and dilute the cell suspension in 20 µL of trypan blue solution (for example: Gibco Trypan Blue, Cat. No. 15250-061).
8. Use a hemacytometer to determine the number of viable cells per mL.
9. Dilute the contents of the vial (1 mL) to the concentration recommended by the product instructions (for example 1.25 X 10E4 viable cells/mL for Gibco neonatal human epidermal keratinocytes ).
10. Add 5 mL of cell suspension to each 25 cm2 culture flask or 15 mL of cell suspension to each 75 cm2 culture flask.
11. Following inoculation, swirl the medium in the flasks to distribute the cells. Many cell types attach to culture surfaces quickly, and if the medium is not distributed immediately following inoculation, the cells may grow in uneven patterns.
12. Incubate the cultures in a 37 degrees C, 5% CO2/95% air, humidified cell culture incubator. For best results, do not disturb the culture for at least 24 hours after the culture has been initiated.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
How I do thaw frozen cells?
Please see our protocol here for thawing frozen cells (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/thawing-cells.html).
