Product FAQ

Can the Cryopreservation Medium from PSC Cryopreservation Kit be aliquoted and frozen again?

Answer

We have not tested this. However, the medium is stable when stored at 4 degrees C for up to 6 months. There are no components that R&D would be concerned about during a freeze thaw; however, this was not formally tested.

Answer Id: E12462

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Product FAQ

If the Synth-a-Freeze Cryopreservation Medium is not suitable for melanocytes, how should I freeze melanocytes?

Answer

We recommend using DMEM plus 10% FBS and 10% DMSO, or the Recovery Cell Culture Freezing Medium. You can try the same general recommendations for freezing cells, just changing the freezing solution.

Answer Id: E14944

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Citations & References

Biological impact of xeno-free chemically defined cryopreservation medium on amniotic epithelial cells.

  • Authors: Miki T, Wong W, Zhou E, Gonzalez A, Garcia I, Grubbs BH
  • Journal: Stem Cell Res Ther 2016; (7): 8-8
  • PubMed ID: 26758986
Catalog #

Product FAQ

Do I need to spin the cells out of the cryopreservation medium to plate them?

Answer

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.

Answer Id: E11966

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Manual / Product Insert

Synth-a-Freeze® Cryopreservation Medium

Version: 02/08
Catalog #
  • R00550(Discontinued)

Product FAQ

Is there a Drug Master File for the Synth-a-Freeze Cryopreservation Medium?

Answer

There isn’t a Drug Master File, but there is a Device Master File on file with the FDA. Please contact our Licensing team at outlicensing@thermofisher.com in order to obtain instructions for referencing this Device Master File.

Answer Id: E14943

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Manual / Product Insert

Synth-a-Freeze, Cryopreservation Medium

Version: MAN0007380 1.00 (24 May 13)
Catalog #

Product FAQ

What can I use Synth-a-Freeze Cryopreservation Medium for?

Answer

Synth-a-Freeze Cryopreservation Medium can be used with any standard freezing protocol. It offers performance comparable to that of our standard, serum-containing cryopreservation medium for cyropreserving a variety of cell types including human keratinocytes, embryonic stem cells, neural stem cells, and mesenchymal stem cells.

Answer Id: E14942

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Product FAQ

What is the PSC Cryopreservation Kit?

Answer

The PSC cryopreservation kit contains xeno-free PSC Cryopreservation Medium, which is a ready-to-use solution for the cryopreservation of early passage pluripotent stem cells (PSCs), and Gibco Revitacell Supplement (100X), a chemically defined recovery supplement for use in the post-thaw culture medium. When used in combination, these reagents help minimize loss of cell viability, maximize post-thaw recovery, and minimize unwanted differentiation of PSCs. This kit can also be used to cryopreserve and recover peripheral blood mononuclear cells (PBMCs) to improve post-thaw cell viability and recovery.

Answer Id: E12461

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Product FAQ

Can I expand your cells and re-freeze them? If so, how?

Answer

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.

Answer Id: E11967

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Product FAQ

How should melanocytes be frozen?

Answer

Do not use Synth-a-Freeze Cryopreservation Medium to cryopreserve melanocytes. We recommend using DMEM with 10% FBS and 10% DMSO, or the Recovery Cell Culture Freezing Medium.

Answer Id: E11999

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Product FAQ

What is the procedure for cryopreserving mammalian cells?

Answer

Mammalian cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous cell lines, and to avoid aging and transformation in finite cell lines. Before cryopreservation, cells should be characterized and checked for contamination.

There are several common media used to freeze cells. For serum-containing medium, the constituents may be as follows:
1) Complete medium containing 10% DMSO (dimethylsulfoxide)
2) 50% cell-conditioned medium with 50% fresh medium with 10% DMSO

Cryopreservation media generally consists of a base medium, cryopreservative, and a protein source. The cryopreservative and protein protect the cells from the stress of the freeze-thaw process. A serum-free medium has generally low or no protein, but one can still use it as a base for a cryopreservative medium in the following formulations:

1) 50% cell-conditioned serum free medium and 50% fresh serum-free medium containing 7.5% DMSO
2) Fresh serum-free medium containing 7.5% DMSO and 10% cell culture grade BSA

Protocol for Suspension Cultures:
1. Count the number of viable cells to be cryopreserved. Cells should be in log phase.
2. Centrifuge the cells at ~200 to 400 x g for 5 min to pellet cells.
3. Using a pipette, remove the supernatant down to the smallest volume without disturbing the cells.
4. Resuspend cells in freezing medium to a concentration of 1 x 10E7 to 5 x 10E7cells/ml for serum-containing medium, or 0.5 x 10E7to 1 x 10E7 cells/ml for serum-free medium.
5. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 minutes.
6. Cells are frozen slowly at 1°C /min. This can be done by programmable coolers or by placing vials in an insulated box placed in a -70°C to -90°C freezer, then transferring to liquid nitrogen storage.

Protocol for Adherent Cultures:
1. Detach cells from the substrate with dissociation agents. Detach as gently as possible to minimize damage to the cells.
2. Resuspend the detached cells in a complete growth medium and establish the viable cell count.
3. Centrifuge at ~200 x g for 5 min to pellet cells.
4. Using a pipette, withdraw the supernatant down to the smallest volume without disturbing the cells.
5. Resuspend cells in freezing medium to a concentration of 5 x 10E6 to 1 x 10E7 cells/ml. Aliquot into cryogenic storage vials.
6. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min.
7. Cells are frozen slowly at 1°C /min. This can be done by programmable coolers or by placing vials in an insulated box placed in a -70°C to -90°C freezer, then transferring to liquid nitrogen storage.
Reference: Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 220, Alan R. Liss,Inc., New York.

Thawing of Cryopreserved Cells:
Cryopreserved cells are fragile and require gentle handling. Thaw cells quickly and plate directly into complete growth medium. If cells are particularly sensitive to cryopreservation (DMSO or glycerol), they are centrifuged to remove cryopreservative and then plated into growth medium. The following are suggested procedures for thawing cryopreserved cells:

- Centrifugation Method: Remove cells from storage and thaw quickly in a 37°C water bath. Place 1 or 2 ml of frozen cells in ~25 ml of complete growth medium. Mix very gently. Centrifuge cells at ~80 x g for 2 to 3 min. Discard supernatant. Gently Resuspend cells in complete growth medium and perform a viable cell count. Plate the cells. Cell innoculum should be at least 3 x 10E5 cells/ml.
- Direct Plating Method: Remove cells from storage and thaw quickly in a 37°C water bath. Plate cells directly with complete growth media.

Answer Id: E4295

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Product Literature

Thermo Scientific HyCryo-STEM: A chemically defined and serum-free freeze media for stem cell cryopreservation

Product Literature

Skin and eye primary cell sourcebook