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What is the best way to thaw cells? Hand-warming vs. water bath immersion vs. automated cell thawing

Lab Academy

Ask the expert - Answer by Ines Hartmann, Application Specialist Cell Handling at Eppendorf

This article appeared first in “ Inside Cell Culture ”, the monthly newsletter for cell culture professionals.

When removing a cryovial from the precious liquid nitrogen stock to start a fresh culture, one strives for a good and reproducible cell recovery. A generally accepted method in cryopreservation is a slow freeze and rapid thaw approach [1]. Rapid thawing can be achieved by simple warming the cryovial in the hand, immersing it in a water bath or by using an appropriate thawing device. All methods result in thawing times within an acceptable range of a few minutes [2]. However, the methods have different pros and cons.

Hand-warming efficiency is strongly person-dependent. Letting three different persons thawing a cryovial by hand-warming may result in large variations in thawing time and temperature profile in the cryovial [2]. This can be explained by the difference in hand size and temperature. The method can hardly be standardized, hence, is not optimal for reproducible cell thawing.

Thawing by immersing the cryovial in a water bath is the most common and by far the fastest method among the three compared [2]. However, the water bath is a well-known source of contamination. One should make sure that the cap of the vial is tightly closed, avoid submerging the cap area and wipe the vial thoroughly with alcohol 70% prior opening it. Also, stick with one immersion practice. A change such as one time moving, one time not moving the vial in the water or other time using a floater may affect thawing times and temperature profiles, hence reproducibility [2].

A dedicated thawing device works water-free, which minimizes the risk of a contamination. Thawing occurs within an acceptable time frame and is highly reproducible [2]. Another advantage is the small footprint facilitating an easy integration into the workspace next to the biosafety cabinet. It has been shown to be suitable for cell lines as well as for sensitive stem cells [2,3].

To sum up, we recommend either using a water bath with the precautions described above or an automated cell thawing device.


[1] Thompson ML, Kunkel EJ, Ehrhardt RO (2014). Cryopreservation and Thawing of Mammalian Cells. In eLS, John Wiley & Sons, Ltd (Ed.)

[2] Tacheny A, Tejerina Vargas S, Chandelier N, Hoet JF, Karow K, Hartmann I. Standardized and Water-free Cell Thawing using the Eppendorf ThermoMixer® C with the Eppendorf SmartBlock cryo thaw, APPLICATION NOTE I No. 437

[3] Tacheny A, Tejerina Vargas S, Chandelier N, Hoet JF, Vanbellinghen B, Hartmann I. Standardized and Water-free Cell Thawing of Stem Cells using the Eppendorf ThermoMixer® C with the Eppendorf SmartBlock cryo thaw, APPLICATION NOTE I No. 452

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Related files:

  • White Paper: Cell Thawing Protocol Standardization – Guide for More Reproducible Cryo-preservation Results

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