2. Resources
  3. Articles
  4. How to Cryopreserve and Thaw Cells More Effectively?
How to Cryopreserve and Thaw Cells More Effectively?
The process of cryopreserving cells and thawing cells is critical in cell culture, as it directly determines cell survival and viability. This, in turn, affects the success of subsequent cellular experiments as intended. To ensure optimal efficacy of cell cryopreserving and thawing procedures, follow this essential principle: slow freezing and rapid thawing!
“Slow freezing and rapid thawing”?

As cells freeze, ice crystals form both inside and outside the cells. When cells are immediately frozen in liquid nitrogen at -196℃, the extracellular solution freezes first, gradually concentrating the solution outside the cell. This in turn increases the concentration of cell electrolytes, induces changes in osmotic pressure, triggers dehydration, and induces protein denaturation. Such changes can cause mechanical damage to the cells, compromising their integrity.

• During cryopreservation, the water in the external environment of the cells freezes. Different cooling rates can cause different degrees of cell shrinkage due to osmotic dehydration. The osmotic pressure of the external environment of the cells continuously increases as the water forms ice crystals. The slower the cooling rate, the longer it takes for the water in the cells to move out of the cells by osmosis. Therefore, very slow cooling can lead to cell death due to excessive dehydration (A), rapid cooling (C) can lead to the formation of ice crystals inside the cells, causing the cells to die upon rewarming. An intermediate cooling rate (B) can balance the risks of osmotic dehydration and intracellular ice formation, usually resulting in maximum cell survival.

Fig 1. Effect of different cooling rates on cell viability
Fig 1. Effect of different cooling rates on cell viability[1].

When a cryoprotectant such as DMSO is added to the freezing medium, it helps to lower the freezing point and stabilize the osmotic pressure of the cells. This addition mitigates the dehydration shrinkage that occurs during slow cooling, reduces ice crystal formation during slow freezing, and thereby prevents cell damage[1].

Recommendations for cell cryopreservation

Upon revival, a rapid temperature rise followed by dilution within 1-2 minutes is essential. At this point, no significant ice crystals have formed inside or outside the cells, nor have the cells been exposed to high concentration electrolyte solutions for an extended period of time. These precautions help to avoid cell damage, thereby increasing cell survival rates.

What is the optimal time to freeze cells? What is the appropriate cell concentration? How much cryopreservation medium should ideally be used? And how can we ensure a "slow freezing" of the cells? Please refer to the table below.

Table 1. Recommendations for cell cryopreservation[2].
Optimal time Cells that are in the logarithmic growth phase, are in good health, and have produced successful experimental results are optimal for freezing. It is advisable to begin freezing cells within two weeks of revival, while reserving a portion for ongoing cultivation as needed for subsequent experiments.
Cryopreservation medium • Cryopreservation medium should be freshly prepared on the day of the experiment. Standard formulations for this medium typically follow either a 7:2:1 ratio of culture medium to serum to DMSO or a 9:1 ratio of serum to DMSO.
• DMSO is commonly used as a cryoprotectant, and concentrations of 5-10% are sufficient to preserve most cell types. Because DMSO is hygroscopic, it's important to store it in a dry environment to avoid using a supply that has been opened for more than a year.
• Incorporation of serum into the cryopreservation medium facilitates cell recovery after thawing. However, with the increasing use of serum-free culture media, it's necessary to freeze cells without serum. Commercially available serum-free cryopreservation media are a viable alternative in such cases.
Cell Concentration The recommended cell concentration is approximately 1x106 cells/mL. Depending on the specific cell type, this may vary from 5x10^5 to 1x10^7 cells/mL. A higher cell concentration will not result in a higher number of viable cells after thawing. Instead, it may inadvertently compromise cell viability due to an accumulative effect stimulated by the high concentration.
Volume of cell freezing medium Ideally, a standard 2 mL cryovial should contain 1 mL of cryopreservation solution. Avoid storing too large a volume per sample, as inconsistent cooling rates across the sample could potentially compromise cell viability.
Cooling Rate For the majority of cells, a cooling rate of -1℃/min can greatly preserve cell viability. Programmable freezers or gradient cooling can be used. The recommended gradient cooling steps are 4℃ for 20 min → -20℃ for 30 min → -80℃ overnight → storage in liquid nitrogen. Alternatively, commercially available non-programmable cell cryopreservation solutions that allow direct storage at -80°C can be used.
Storage Conditions For short-term storage, cells can be safely stored at -80°C. However, for long-term storage, the cells must be stored in liquid nitrogen. Prolonged storage at -80°C may reduce cell viability and functionality.
Labeling and Record Keeping Ensure that the cryovial is clearly labeled with pertinent information such as cell name, generation, person responsible for freezing the cells, and date of freezing. In addition, record the cell information along with the exact storage location in a record book. Such meticulous record keeping ensures that the correct cells can be conveniently retrieved when needed for thawing.
Cell Revival – Restoring Cells to Full Vitality

Cell Retrieval

Minimize the time your sample passively warms between removal from liquid nitrogen or -80℃ storage and placement in the thawing device to avoid significant impact on cell vitality. If the storage and thawing devices are physically separated, a thermal container filled with dry ice (-80°C) or liquid nitrogen (-196°C) can be used for temporary storage and transport of cells.

(Note: Always wear goggles and gloves when removing cells from liquid nitrogen to prevent frostbite and possible cryovial explosion!)

Thawing Method

a) During thawing, cells require rapid heating (50~100℃/min) to maximize their vitality; an optimal way to achieve this is to use a 37℃ water bath[3]. After removing the frozen cells from liquid nitrogen or -80℃, unscrew the vial cap slightly to prevent the cryovial from bursting during thawing. Place in a 37℃ water bath and shake vigorously for 1-2 minutes until thawed.

It's generally recommended to stop thawing when visible ice has melted but the sample remains ice cold (~0℃). A common practice is to remove the sample from the water bath when only a small piece of ice remains. Warming to room temperature or higher increases the cytotoxicity of the cryoprotectant and severely compromises cell viability[1].

b) It is not recommended to thaw cells at room temperature, as this may precipitate cell death.

c) To reduce the risk of contamination, ensure that the mouth of the cryovial remains above the water level during thawing in a water bath to prevent water from entering the cryovial. The sterile water in the water bath should also be changed regularly.

Dilution

Dilute cells as soon as possible after thawing to reduce DMSO cytotoxicity. Add culture medium at a ratio of culture medium: cryopreservation medium ≥ 10:1 for dilution, then centrifuge and replace the culture medium to resuspend the cells. This may reduce cell stress caused by osmotic pressure changes.

Medium Change

Change the medium and continue culturing after cell attachment or after 24 h, depending on cell status.

Question & Answer

Q: Why aren't my revived cells adhering at all?

A: It could be due to poor growth status of the cells before freezing or slow operations during revival. Therefore, it's crucial to freeze cells that are in good growth status and exponential growth phase, and the process should follow the principle of slow freezing and rapid thawing!

Q: Why did my revived cells die completely when they were clearly adhered the day before?

A: This could be due to over-digestion before cell freezing, causing cell apoptosis. Therefore, it's essential to strictly control the digestion time when processing cells!

Q: If only a very small number of cells adhere after revival, do I need to revive them again?

A: Don't rush to discard them! You can supplement with serum and cell factors, or change fresh growth medium every 2-3 days. After 2-3 media changes, most cells should show noticeable proliferation. At this point, normal passaging operations can be followed. Of course, if you have a large supply of cells or are in a hurry for an experiment, you can revive a new batch of cells~

Q: I prepared too much cryopreservation medium and don't want to waste it, can I save it for next time?

A: The prepared cryopreservation medium can be temporarily stored at 4℃ for 1 week and at -20℃ for up to one month. However, it's best to prepare and use it as needed, avoiding repeated freeze-thaw cycles.

Related Products

Serum/Protein-Free Cell Freezing Medium

A complete ready-to-use cryopreservation medium for the cryopreservation of conventional mammalian cells and serum-free cultured cells.

DMEM (High Glucose, L-Glutamine, Pyruvate, Phenol Red, no HEPES)

DMEM (Dulbecco's Modified Eagle Medium) is a widely used basal medium for supporting the growth of many different mammalian cells. Cell lines successfully cultured in DMEM include Hela, 293, Cos-7, and PC-12, as well as primary fibroblasts, neurons, glial cells, HUVECs, and smooth muscle cells.

RPMI 1640 (L-Glutamine, Phenol Red, no HEPES)

RPMI 1640 contains glutathione and high concentration of vitamins, also contains biotin, vitamin B12, 4-Aminobenzoic acid (PABA) not found in MEM and DMEM, as well as high concentration of inositol and choline chloride.

Cell Counting Kit-8

Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays.

Hygromycin B, Sterile

MCE Hygromycin B, Sterile is an aminoglycosidic antibiotic purified from Streptomyces hygroscopicus. It acts by binding to the 70S subunit of the bacterial ribosome and inhibiting protein synthesis, leading to the death of bacteria, fungi and mammalian cells.