Cell Freezing Media

Cell cryopreservation is a common method of cell culture technology for the preservation and long-term storage of cells. Cell freezing media, a solution that must be used for cell lyophilization, is used for cell preservation and plays a key role in the purchase, dispatch, exchange, and delivery of cells. Therefore, it is especially important to choose a good cell culture freezing medium.

Researchers often need to freeze cells and preserve them for subsequent experiments or clinical use. The basic process is fairly simple: freeze the cells slowly, place the cryopreservation tubes in liquid nitrogen, and thaw them quickly when needed. Cell freezing media supports the cells and prevents ice crystals from forming. However, the results of using different freezing media can be very different.

Conventional cell freezing media add a protective agent to the culture medium, which is typically 10% dimethyl sulfoxide (DMSO) and 10%-90% bovine serum. The problems encountered when freezing cells in conventional cell lyophilization solution are summarized as follows.

a. Procedural cooling (4°C → -20°C → -80°C → liquid nitrogen). This step is tedious and time-consuming.

b. Containing serum with a higher risk of cell contamination (viruses, mycobacteria, mycoplasma, etc.).

c. Differences in serum batch and quality lead to batch differences in cell freezing media.

d. Liquid nitrogen freezing is required for high freezing requirements.

e. Microvolume and in situ (e.g., well plates) cannot be lyophilized.

The following three serum-free cell lyophilization products are all serum-free and non-programmed cooling and can be placed directly in the -80°C refrigerator for long-term freezing and storage.

a. Free of serum and animal-derived proteins to reduce contamination by viruses, mycobacteria, and mycoplasma.

b. Special formula can effectively improve the survival rate of cell freezing and recovery rate.

c. Ready-to-use, no on-site preparation required.

d. Universal type, which is suitable for freezing and storing various animal and human cell lines and primary cells.

e. Clear composition, no batch-to-batch variation.

f. No programmed cooling and no liquid nitrogen required. It can be placed directly at -80℃ for long-term freezing and storage.

g. Micro and in situ freezing (e.g. hybridoma cell freezing) is possible, saving time and efficiency.

a. High safety, no serum, and animal-derived ingredients, low possibility of contamination by viruses, mycobacteria, and mycoplasma. Higher product quality consistency from batch to batch.

b. Low DMSO with 5% DMSO (v/v), which greatly reduces the potential toxic effects on cells.

c. High cell viability with no batch differences.

d. Completely lyophilized liquid formulation, ready for direct use, convenient and simple. It can be stored directly in a -80℃ refrigerator for freezing and storage without going through the time-consuming process of programmed cooling (save time, effort, and money).

e. It can be frozen in situ, such as subclones of hybridoma cells, which can massively reduce workload and avoid contamination.

a. High safety, no serum, and animal-derived ingredients, low possibility of contamination by viruses, mycobacteria, and mycoplasma. Higher product quality consistency from batch to batch.

b. No DMSO, no potential toxic effects on cells.

c. High cell viability with no batch differences.

d. Complete lyophilization solution formulation, can be used directly, convenient and simple.

e. It can be stored directly in a -80℃ refrigerator for freezing, without going through the time-consuming procedure of cooling (save time, effort, and money). No need to use ☆ programmed cooling instrument, and no need to place in -196℃ liquid nitrogen.

f. It can be frozen in situ, such as subclones of hybridoma cells, which can massively reduce workload and avoid contamination.

The basic principle of cell freezing and recovery is slow freezing and fast thawing. Experiments have shown that this can maximize the preservation of cell viability.

Currently, cell freezing uses glycerol or dimethyl sulfoxide as a protective agent. These two substances can improve the permeability of the cell membrane to water, plus slow freezing can make the water inside the cell seep out of the cell, reducing the formation of intracellular ice crystals, and thus reducing the cell damage caused by the formation of ice crystals. Resuscitation of cells should be done by rapid thawing, which can ensure that the extracellular crystals melt in a very short time and avoid damage to cells due to slow thawing that allows water to seep into the cells to form intracellular recrystallization.

Cell cryopreservation is an important technical tool for cell culture, seed introduction, seed preservation, and smooth experimentation. It is important to freeze primary cells promptly in cell culture and lineage establishment. In the process of hybridoma monoclonal antibody preparation, the freezing and seed preservation of hybridoma cells, subclonal cells obtained from each cloning is often an essential experimental operation. Without the establishment of a stable cell line or a stable antibody-secreting cell line, the cell culture process may fail at any time due to contamination of cells, loss of antibody-secreting ability or genetic mutation, etc. Without the freezing of the original cells, the experiment will be lost due to the above-mentioned accidents.

When the cells grow well and the supply exceeds the demand, the cells can be considered to be frozen and stored for later use. When the temperature is below -70℃, the enzymatic activity inside the cell stops and the metabolic activity stops, so the purpose of long-term storage can be achieved. When freezing cells, as the temperature decreases, the water inside and outside the cells will crystallize, and the ice crystals formed will cause the destruction of cell membranes and organelles, thus causing cell death, especially at the stage of -20-0℃, the ice crystals are needle-like and easy to cause cell damage.

Use cell freezing media. The common cell culture freezing medium is glycerol or dimethyl sulfoxide (DMSO), which can quickly penetrate the cell membrane to enter the cell, lower the freezing point, delay the lyophilization process, and increase the permeability of the cell membrane, increase the concentration of intracellular ions, reduce the formation of intracellular ice crystals, and thus achieve the purpose of cell protection.

Preparation of lyophilization solution (usually 10% DMSO + 90% FBS with culture medium). Or cell freezing medium: serum: DMSO=7:2:1.

Select cells with good viability and a density of about 80%, when the cells are in the logarithmic growth phase and are most suitable for lyophilization. Cell lyophilization is still divided into wall cell lyophilization and suspension cell lyophilization.

Observe the cell status under the microscope. If the cell density was high and the medium turned yellow, it was necessary to change the liquid for 4h before the lyophilization operation. After the cells were fully grown, the medium in the culture dish was carefully aspirated with a gun, and then PBS was rinsed once or twice, and try to aspirate the rinsed PBS, and trypsin were added to digest the cells. Digestion must pay attention to the control of trypsin action time, digestion of cells due to each cell adhering to the wall is different, and digestion time will vary greatly. Trypsin digestion is a delicate operation, not only to fully digest the cells and break them into a single-cell suspension for uniform inoculation but also to avoid severe cell damage caused by excessive digestion. The trypsin activity, digestion steps, and reagents used by each customer are different, so the digestion time cannot be fully specified, and the customer's experience prevails. For the digestion step, if the customer has little experience with the cell and cannot accurately control the trypsin action time, we suggest the customer follow the following operation: firstly, add the cell after the trypsin is rewarmed to room temperature, put it into 37℃ incubator, and then blow the cell every 30 seconds, if the cell can be blown apart (not digested until the cell is completely floating), it means that the cell digestion is completed and the digestion can be terminated. After cell digestion, mix the cells as gently as possible to not produce too many bubbles. If 30 seconds of blowing does not work, wait 30 seconds and repeat the operation.

After digestion, add 6-8 ML of complete medium to terminate the digestion, mix the cells, collect the cell suspension by centrifugation at 900-1000 rpm for 3 min, discard the supernatant, add the prepared lyophilization medium, gently blow with a pipette to make the cells homogeneous, divide the cells into lyophilization tubes, 1-1.5 ml per tube, label the lyophilization tubes with the name of the cells, the lyophilization time and the operator, and put the lyophilization tubes with the cells into thick-walled foam boxes. Cells were placed in a thick-walled foam box in a 4°C refrigerator for 10-30 min and then placed in a -80°C refrigerator overnight together with the foam box.

If you are interested in our cell freezing media or have any questions, please write an e-mail to [email protected], we will reply to you as soon as possible.