Dear Scientists of Research Gate,
Not too long ago I started working with an L929 cell line we need to keep in-house, and got the passaging conditions figured out. I even started cytotoxicity, but I seem to really struggle with storage.
Does anyone have any suggestions to a good cryopreservation protocol for these cells as well as a quality, reliable dewar intended for storage? I bought a cheap one, but I guess you get what you pay for - it leaks and kills my cells :)
Any feedback would be greatly appreciated.
Best regards,
-Tim M Seyidov
Dear Tim!
Please You look at the protocol:
https://www.nature.com/articles/s43246-021-00118-1#Sec13
Molecular mechanisms of cell cryopreservation with polyampholytes studied by solid-state NMR
Cell culture
L929 (mice fibroblast cell line, American Type Culture Collection, Manassas, VA, USA) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS). Cell culturing was carried out at 37 °C under 5% CO2 in a humidified atmosphere. When the cells reached 80% confluence, they were removed using 0.25% (w/v) trypsin containing 0.02% (w/v) EDTA in PBS (−) and seeded on a new tissue culture plate for subculturing.
Cryopreservation protocol
The cryopreservation solutions were prepared as follows: COOH-PLL was dissolved in DMEM without FBS at a concentration of 7.5% (w/w), and the pH was adjusted to 7.4 using 5 M HCl or NaOH. The osmotic pressure was measured using an osmometer (Osmometer 5520; Wescor, Inc. UT, USA) and was adjusted to ~600 mOsm for cryopreservation solutions of PLL derivatives using a solution of 10% NaCl. The cells were counted and re-suspended in 1 mL volumes of saline solutions of COOH-PLL, PEG, BSA, and DMSO with various concentrations at a density of 1 × 106 cells/mL in 1.9 mL cryovials (Nalgene, Rochester, NY) and stored in a −80 °C freezer after cooling at a rate of −1 °C/min in a controlled freezing container (Mr. Frosty, Nalgene) over 1 w.
Cell viability assay
Individual vials were thawed in a water bath at 37 °C with gentle shaking, after which the thawed cells were diluted in DMEM. Following centrifugation, the supernatant was removed, and the cells were re-suspended in DMEM (5 mL). To determine the cell survival, the medium was collected, and all cells were stained with trypan blue and counted using a hemocytometer immediately after thawing. The reported values are the ratios of living cells to the total number of cells.
Article Polyampholytes as Cryoprotective Agents for Mammalian Cell C...
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