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Operation method and precautions of cell cryopreservation

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Cryopreservation. Biobanking technician in protective gear working with biological samples

When cells cold below zero, organelles dehydration, soluble substance concentration in the cell increases, and in the formation of ice crystals in the cell, the size of the ice crystals have an effect on cell is different, large ice crystals cause damage to the cell membrane, organelle and burst, thus will cause our recovery of cell survival rate, status and cryopreserved in front of the state. So how do we solve these problems?

First, we need to reduce the formation of ice crystals, especially large ice crystals during cryopreservation. By freezing cells slowly, we can avoid the formation of large ice crystals inside cells. So how do we freeze cells?

  • Slow freezing cells
  1. Routine procedures: Use a programmed cooling box

When the temperature is above -25℃, 1-2℃/min.

When the temperature is below -25℃, 5-10℃/min.

When the temperature reaches -100℃, it can be quickly transferred into liquid nitrogen.

  1. Simple Procedure: The mouth of the frozen tube is upward and put into the gauze bag. The gauze bag is connected with a string. The gauze bag is fixed to the mouth of the liquid nitrogen tank by the string, and drops to the surface of the liquid nitrogen overnight in 40 minutes at the rate of 1-2℃ per minute.
  2. Traditional Procedures: The refrigerated tube is placed at 4℃ for 10min, -20℃ for 30min, -80℃ for 16-18h (or overnight), and the liquid nitrogen is stored for a long time.
  • low temperature protective agent

The commonly used cryoprotective agent is DMSO, which is a kind of osmotic protective agent, can quickly penetrate into the cell, improve the permeability of the cell membrane to water, reduce the freezing point, delay the freezing process, can make the water inside the cell before freezing out of the cell, form ice crystals outside the cell, reduce the ice crystals inside the cell, so as to reduce the damage of ice crystals to the cell.

  • Cell cryopreservation methods
  1. Pre-prepared cryopreservation fluid: the proportion of cryopreservation fluid is varied, and the proportion of cryopreservation fluid can be adjusted according to the characteristics of cells: 5%-10% DMSO + 20%-90% serum + 0%-70% basal culture fluid;
  2. Cells at logarithmic growth stage were taken, the old culture medium was removed, and cleaned with PBS for 1-2 times; Remove the residual serum in the culture flask;
  3. Add appropriate trypsin (concentration: 0.25%) to cover the whole bottle and put it into the incubator for digestion;
  4. The cells were digested for 0.5-2min (the specific time was determined according to the morphology under the microscope). The cells were observed under the microscope.
  5. The cells were gently blown with a Pasteurized pipette to form cell suspension, which was centrifuged for 3-5min at about 1000r/min;
  6. Discard the supernatant, add an appropriate amount of pre-prepared cryopreservation liquid, and gently blow the cells with a Pasteurized pipette to make the cells uniform and count. Adjust the final density of cells in the cryopreservation liquid to 5×106/ml ~ 1×107/ml;
  7. Divide the cells into cryopreservation tubes, 1-1.5ml for each tube;
  8. Cryopreservation tube marks cell name, cryopreservation time, operator and cell algebraic information. In the case of suspension cell cryopreservation, centrifugal cells were collected directly, and the steps of trypsin digestion were removed, otherwise the same.

matters need attention

  1. The cells that need cryopreservation should grow well and have a high survival rate, with a density of about 80-90%. Cell viability should be ensured before cryopreservation without pollution.
  2. In the process of cell cryopreservation, the resulting ice crystals do great damage to cells, so the process must be slow. Freeze storage or resuscitation is best with a newly prepared culture medium.


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