I am looking for different methods of mammalian stem cells freezing and thawing. Both in industrial and laboratory scale. Would greatly appreciate your input.
Different media are commonly used to freeze cells and could be serum-containing or serum-free media. Re-suspend cells in freezing medium to a concentration of 5 x 10^6 to 1 x 10^7 cells/mL. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min. Cells should be frozen slowly at 1°C/min.
To thaw, remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial. Also visit the link for details: https://www.thermofisher.com/ng/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cryopreservation-of-mammalian-cells.html
JOVE has several videos covering the basic cell culture techniques and freeze-media types.
However, a lot depends on the actual cells. I work with cells that are extra-sensitive to the procedure, and other cells that I could thaw overnight at 4°C without any problems.
Randolph B Caldwell and Maurice Ekpenyong
Thanks for your answers. I am quite familiar with conventional cell thawing and and freezing (including using water bath and heat block) . I mostly looking for other ways. specially in CHO cells.
May be one of other ways is using some types of nanoparticles:
Stefanic M., Ward K., Tawfik H., Seemann R., Baulin V., Guo Y., Fleury J.B., Drouet C. Apatite nanoparticles strongly improve red blood cell cryopreservation by mediating trehalose delivery via enhanced membrane permeation. Biomaterials. 2017 Sep;140:138-149.
Wang T., Zhao G., Liang X.M., Xu Y.P. Numerical simulation of the effect of superparamagnetic nanoparticles on microwave rewarming of cryopreserved tissues // Cryobiology. – 2014. – Vol. 68,№2. –P. 234-243.
Khosla K., Wang Y., Hagedorn M., Qin Z., Bischof J. Gold Nanorod Induced Warming of Embryos from the Cryogenic State Enhances Viability // ACS Nano. – 2017. – Vol.11, №8. – Р. 7869-7878.
Zhou X., Li W., Zhang D., Dai J. Hydroxyapatite nanoparticles improved survival rate of vitrified porcine oocytes and its mechanism // Cryo Letters. – 2015. – Vol.36, №1. – Р.45-50.
Thanks Nikolay Bondarovich and Gintautas Saulis
The links were quite useful.
Jonathan Ko Yes we use DMSO at 10% and 90% media. I think the 90% FBS is quite old school! are people still using this?
Hahaha, I'm old. And, switched fields. I just remember a colleague exclaiming, "DMSO is magic!"
Due to the fact that it is not advisable to use DMSO in the clinic for freezing cord blood cells in one of the works, PEO was used:
BABIYCHUK L.A., ZUBOV P.M., ZUBOVA O.L., RYAZANTSEV V.V. Whole Cord Blood Cryopreservation with PEO-1500: Assessment of Apoptotic Stages in Nucleated Cells. Problems of cryobiology Vol. 22, 2012, №3. P.312
But it seems to me that DMSO as the cryoprotector is the best.
Hello
The following protocol describes a general procedure for thawing cryopreserved cells.
1. Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath.
2. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial.
3. Transfer the vial it into a laminar flow hood. Before opening, wipe the outside of the vial with 70% ethanol.
4. Transfer the thawed cells dropwise into the centrifuge tube containing the desired amount of pre-warmed complete growth medium appropriate for your cell line.
5. Centrifuge the cell suspension at approximately 200 × g for 5–10 minutes. The actual centrifugation speed and duration varies depending on the cell type.
6. After the centrifugation, check the clarity of supernatant and visibility of a complete pellet. Aseptically decant the supernatant without disturbing the cell pellet.
7. Gently resuspend the cells in complete growth medium, and transfer them into theappropriate culture vessel and into the recommended culture environment.
https://www.stembook.org/node/720
http://www.els.net/WileyCDA/ElsArticle/refId-a0002561.html
Hello Hadi,
We've been using RCCFM for years, and it's been working great for us.
You could look it up with the following link.
https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2F12648010_cell_culture_freeze_media_PI.pdf&title=UmVjb3ZlcnkgQ2VsbCBGcmVlemluZyBNZWRpYQ==
Best,
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