Cryoprotective effect of dimethyl sulfoxide in cell cryopreservation for animal experiments

Why cells are afraid of freezing

In animal experiments, cells taken from blood or tissue often cannot be used immediately and need to be preserved first. If cells are placed directly into a low-temperature freezer, the water inside the cells will form ice crystals. These ice crystals are like tiny needles and may puncture the cell membrane and internal structures, causing massive cell death after thawing. To avoid this situation, a class of substances called cryoprotectants needs to be added. Dimethyl sulfoxide is one of the more widely used ones. It can enter the interior of cells, change the freezing process of aqueous solutions, and make ice crystals smaller and more uniform, thereby reducing mechanical damage to cells.

Common concentration of dimethyl sulfoxide in freezing solution

In cell cryopreservation for animal experiments, dimethyl sulfoxide is usually not used alone, but is combined with culture medium, serum, etc. to form a freezing solution. The common final concentration is generally between 5% and 10%, and the specific ratio will be adjusted according to cell type and laboratory experience. For example, some more fragile primary cells may be more sensitive to high concentrations of dimethyl sulfoxide, and operators may tend to use lower concentrations; while for cell lines with stronger tolerance to cryopreservation, a conventional concentration of about 10% may be used. The prepared freezing solution generally needs to be used immediately after preparation or stored in aliquots to avoid repeated freezing and thawing affecting the protective effect.

Why processing should be done as soon as possible during recovery

Dimethyl sulfoxide can protect cells at low temperatures, but it has certain toxicity to cells at room temperature. Therefore, after cells are removed from liquid nitrogen or an ultra-low temperature freezer, the recovery operation generally needs to be completed within a relatively short time. The usual practice is to quickly thaw the cell suspension, then dilute it with culture medium as soon as possible, then remove the supernatant containing a relatively high concentration of dimethyl sulfoxide by centrifugation, and finally resuspend the cells in fresh culture medium. This is done to reduce the contact time between dimethyl sulfoxide and cells at room temperature and help the cells recover their viability. Ordinary experimenters can understand it as: at low temperature it is a "protective umbrella," and after returning to room temperature it must be "withdrawn" in time.

Small details easily overlooked in operation

After the freezing solution is prepared, it should be mixed gently to avoid producing too many bubbles.

After opening, dimethyl sulfoxide easily absorbs moisture from the air, so it is recommended to aliquot it into small bottles and store them sealed.

Before the recovery process, prepare the warm water bath, centrifuge tubes, and culture medium in advance to reduce waiting time.

During operation, gloves and goggles can be worn to avoid contact of dimethyl sulfoxide with skin or eyes.

These seemingly simple details often affect the survival state of cells after recovery. For animal experiments, only when the cell state is stable are the subsequent inoculation, drug administration, or detection results more valuable as references.