If you want freeze your cells you have, after trypsination and counting your cells in your current culture medium, to determinate the appropriate volume necessary to make x aliquots at 2x10 6 in cryopreservation medium
After determinated it, you centrifugate another time your cells. You resuspend the cell pellet in the previously determinated volume (cf before) in DMSO cell freezing medium ( ref C6164 SIGMA). Make aliquots of 1ml at 2x10 6 in sterile cryotubes
Put your preparated cryotubes in Dr Frosty isopropanol device at -80 C . After respecting the temperature falling time (generally 4 hours or more) you put your cryotubes in nitrogen (cryoconservator or equivalent apparatus) for long time conservation.
Sincerly yours
If you want freeze your cells you have, after trypsination and counting your cells in your current culture medium, to determinate the appropriate volume necessary to make x aliquots at 2x10 6 in cryopreservation medium
After determinated it, you centrifugate another time your cells. You resuspend the cell pellet in the previously determinated volume (cf before) in DMSO cell freezing medium ( ref C6164 SIGMA). Make aliquots of 1ml at 2x10 6 in sterile cryotubes
Put your preparated cryotubes in Dr Frosty isopropanol device at -80 C . After respecting the temperature falling time (generally 4 hours or more) you put your cryotubes in nitrogen (cryoconservator or equivalent apparatus) for long time conservation.
Sincerly yours
@Gizem,
Jean's protocol is mostly used and reliable method for croypreservation of cultured mammalian cells (primary or cell line).
Recently one advanced method is available in literature where cryopreservation of Normal Human Dermal Fibroblasts has been described using an advanced CryoStor (Sigma Aldrich) cryopreservation procedure without using traditional cell freezing media consisting culture media/serum/DMSO.
The basic procedure is as follows:
1. Preparing Cells for Cryopreservation:
To prepare for cryopreservation, place cells into suspension by mechanical or enzymatic dissociation. Next, centrifuge cells to obtain a cell pellet. After centrifugation, remove as much of the culture media supernatant as possible, to reduce dilution of the CryoStor solution, which will be added in the next step.
Before opening the bottle of cold CryoStor solution, wipe down the outside of the container with 70% ethanol.
ISOLATION: Add cold CryoStor to obtain cell concentrations of 0.5-10 x 106 cells/mL.
PRE-FREEZE: Incubate the cell suspension at 2-8°C for approximately 10 minutes.
2. Freezing and Storing Cells:
To freeze most mammalian cell systems, use a standard slow rate controlled cooling protocol with a freezing device or isopropanol container pre-cooled to 2-8°C.
NUCLEATION: Freeze samples at -80°C.
After approximately 10 min. at -80°C, initiate ice nucleation within the sample (seeding) at approximately -5°C using either a liquid nitrogen burst program setting on a controlled rate freezer or mechanical agitation (flick or tap) of the cryovial/sample container.
Freeze the cells for 3-4 hours (for isopropanol container).
STORAGE: For long-term storage, place samples at liquid nitrogen temperatures, below -130°C. Sample storage at -80°C is only recommended for short-term storage of weeks to months.
3. Thawing Cells:
Frozen samples should be thawed quickly in a 37°C water bath. Gently swirl the sample(s) until all visible ice has melted. The approximate thaw time for a 1 mL sample in a cryovial is approximately 3 minutes.
DO NOT allow the sample(s) to warm above chilled temperatures (0-10°C). When removed from the water bath, the cryovial(s) should be cool to the touch.
Dilute the cell/CryoStor mixture immediately with culture media that is between 20°C and 37°C, using a dilution ratio of 1:10 or greater of sample to media.
Plate cells in the appropriate configuration.
Place cells into culture conditions or utilize immediately.
Twenty four hours post-thaw, assess the cells for viability.
As a ready reference I am sharing the link with video demonstration for the above-mentioned protocol published in JOVE.
http://www.jove.com/video/2206/cryostor-cryopreservation-protocol-advertisement
The author,,Aby Mathew also compared the recovery of human fibroblasts following cryopreservation in traditional culture media/serum/DMSO with the serum-free and protein-free intracellular-like CryoStor solution. The data show increased post-thaw viability (70%) of fibroblasts compared to the conventional cell freezing media (40%) .
To freeze my primary cell (chondrocyte, myoblast), I use 95%SVF 5%DMSO. It works very well. Becareful about the antibiotics. It might be toxic when used during cell freezing. DMSO is highly toxic. During cell thaw, limit the delay in presence of DMSO.
Thank you everybody for all suggestions. @.Amritral, I will try your suggestion for freezing and thawing primary cells. I hope, it will work.
Hi Van,
I was freezing those cells by using 7.5%DMSO and 20%FBS. Freezing procedure:
+4C for 10 min
-20 for 2h.
-80 for overnight
Liquid nitrojen.
It worked for primary cells, you can try.
I was freezing those cells by using 10%DMSO and 40%FBS. Freezing procedure:
-20 for 2h.
-80 for overnight
Liquid nitrojen.
It worked for primary cells, you can try.
Will primary fibroblasts be okay left in a Dr. Frosty at -80 for a couple of days before transfer to liquid nitrogen?
Abigail Vikstrom That is usually no problem for most cells. I sometimes leave the cells in for a week and I have thawed them without a problem. I also did this with primary fibroblasts both human and mouse derived.
Abigail Vikstrom I would not suggest storing primary cells at -80 for any length of time.
Keeping them in Mr. Frosty overnight to freeze them is the only exception.
The primary fibroblasts might still look okay right after thawing, but there might be problems later. For example, they are quite likely to behave differently in experiments or stop proliferating earlier.
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