Apr 29, 2026
Thawing and freezing cell stocks
This protocol is a draft, published without a DOI.
- 1Discovery Research Platform for Hidden Cell Biology
Protocol Citation: Georg Kustatscher 2026. Thawing and freezing cell stocks. protocols.io https://dx.doi.org/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: June 21, 2024
Last Modified: April 29, 2026
Protocol Integer ID: 102227
Keywords: stocks of rpe1 cell, freezing cell stocks protocol, rpe1 cell, freezing stock, warm reagent, cell stocks protocol, liquid nitrogen, cell
Abstract
Protocol for thawing and freezing stocks of RPE1 cells from liquid nitrogen
Notes: warm reagents to 37oC before culturing
Materials
DPBS, no calcium, no magnesium (Gibco, 14190094)
Trypsin-EDTA (0.5%), no phenol red (Gibco, 15400054)
-> dilute to 0.05% in DPBS without calcium and magnesium for use
Trypan blue (BIO-RAD, 1450021)
Cell Counting Slides for TC10‱/TC20‱ Cell Counter (BIO-RAD, 1450015)
DMSO (Sigma, D4540-100ML)
Dialysed FBS (Gibco, 26400044)
Mr Frosty (Thermo, 10110051)
Fisherbrand‱ Externally and Internally Threaded Cryogenic Storage Vials 2ml (Fisher scientific, 11787939)
Thawing
Obtain cells from liquid nitrogen, keeping them on dry ice until ready to thaw. Update liquid nitrogen storage map after thawing.
Thaw cells using bead bath or warmed autoclaved water until there is only a small ice crystal left
- DMSO is toxic to cells at high concentrations, so it is important to thaw quickly
Add a few drops of warmed media to complete thawing
Transfer cells to a T75 with 12ml pre-warmed media
- We thaw into a T75 so that DMSO concentration is <1%
Change media the next day to remove DMSO
Freezing
Trypsinise at late log phase (approaching confluence) following subculturing protocol
After quenching trypsinised cells with media, perform a cell count using trypan blue exclusion
Aliquot appropriate number of cells, spin down 300g 5 min
- e.g over 25 million cells for 10x aliquots of 2.5 million
Remove supernatant
Resuspend cells in freezing media (30% L media, 60% dFBS, 10% DMSO) to required concentration for 1 ml aliquots.
- E.g 2.5 million cells/ml, suitable for thawing in a T75
Make 1ml aliquots in cryovials
Transfer the vials to Mr Frosty (chamber must be filled with isopropanol)
- This is to decrease temperature by 1ºC per minute to preserve cells during the freezing process
Store at -80ºC overnight, then remove from Mr Frosty and transfer to liquid nitrogen storage. Update liquid nitrogen storage map.