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Gibco™ Recovery™ Cell Culture Freezing Medium

Product Code. 11560446
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Product Code. 11560446 Supplier Gibco™ Supplier No. 12648010

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187.15 EUR valid until 2026-04-30
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Description

Complete, ready-to-use freezing medium for cryopreservation of a wide variety of mammalian cells, including CHO-S, CHO-K1, HEK 293, Jurkat, and NIH 3T3

For many years, researchers have used a common recipe for making freezing medium: High-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% serum and 10% DMSO. Recovery Cell Culture Freezing Medium is an optimized version of this classic formulation, using Gibco Bovine Serum and Fetal Bovine Serum to provide improved cell recovery and viability after thawing.

  • Suitable for a wide variety of mammalian cells
  • Can be used to freeze most mammalian cells cultured in DMEM, DMEM/F-12, MEM, RPMI 1640, and Ham's F-12
  • Quality tested for pH, osmolality, sterility, and endotoxin
  • Each lot is performance tested using CHO-K1 cells

Cell Counting, Viability & Cryopreservation, Cell Culture, Mammalian Cell Culture, Gene Expression Analysis & Genotyping, In Vitro Transcription, PCR & Real-Time PCR

Order Info

Shipped on dry ice.

Compliance

Manufactured at a cGMP-compliant facility registered with the FDA as a medical device manufacturer and certified to ISO 13485 standards.

TRUSTED_SUSTAINABILITY

Specifications

Cell Type Mammalian Cells
Form Liquid
Product Type Freezing Medium
Serum Level Standard Serum Supplementation
Sterility Sterile-filtered
With Additives High Glucose, Phenol Red, DMSO (10%)
Without Additives No Phenol Red
Product Line Gibco, Recovery
Quantity 50 mL
Shipping Condition Dry Ice
Purity or Quality Grade Research Grade
Content And Storage Storage conditions: -5 to -20°C. Protect from light.
Shipping conditions: Frozen
Shelf life: 12 months from date of manufacture
Culture Type Mammalian Cell Culture
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Frequently Asked Questions (FAQs)

What is the procedure for cryopreserving mammalian cells?

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.

Is there any difference between your Cell Culture Freezing Medium-Dimethylsulfoxide(DMSO) and the standard method using DMSO? Does it depend on the cell types? What protocol should I use for freezing cells?

Our Cell Culture Freezing Media is composed of DMEM, FBS, calf serum, and 10% DMSO. This is useful for many mammalian cells for freezing and is the same percentage of DMSO used in most methods. This product will work fine for most adherent cell lines grown with serum. A good general protocol for freezing cells can be found on our website by searching "Freezing cells protocol" from the home page.

Is it important to centrifuge and aspirate Recovery Cell Culture Freezing Medium before seeding into the flask?

Recovery Cell Culture Freezing Medium is meant for general cell culture applications where a wide range of growth media will be used. We recommend that you aspirate Recovery Cell Culture Freezing Medium before seeding, although other protocols can be substituted (this will need to be determined by the end user).
Can Recovery Cell Culture Freezing Medium be stored at 2 to 8 degrees C?

Recovery Cell Culture Freezing Medium should be stable at 2 to 8 degrees C for 1 week, although we have no data to support this.

Can Recovery Cell Culture Freezing Medium undergo multiple freeze thaw cycles?

There have not been any bench studies at this time. It is best to aliquot this product.
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.

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.

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.

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).
How do I freeze my cells?

Please see our protocol here for freezing cells (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/freezing-cells.html).

Safety and Handling

compliance-icons compliance-icons
Product Identifier
  • 3-Methyl-1-butanol
 Signal Word
  • Warning
 Hazard Category
  • Acute toxicity Category 4
  • Serious eye damage/eye irritation Category 2
  • Flammable liquid Category 3
  • Skin corrosion/irritation Category 2
  • Specific target organ toxicity Category 3
 Hazard Statement
  • H226-Flammable liquid and vapour.
  • H315-Causes skin irritation.
  • H319-Causes serious eye irritation.
  • H332-Harmful if inhaled.
  • H335-May cause respiratory irritation.
  • EUH066-Repeated exposure may cause skin dryness or cracking.
 Precautionary Statement
  • P210-Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking.
  • P280-Wear protective gloves/protective clothing/eye protection/face protection.
  • P302+P352-IF ON SKIN: Wash with plenty of water/soap.
  • P304+P340-IF INHALED: Remove person to fresh air and keep comfortable for breathing.
  • P332+P313-If skin irritation occurs: Get medical advice/attention.
  • P337+P313-If eye irritation persists: Get medical advice/attention.
 Supplemental information
  • MIXTURE LIST-Contain: 3-Methyl-1-butanol

For Research Use Only. Not for use in diagnostic procedures.

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