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title The correct procedure of cell cryopreservation

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Provider: Hengyuan immunology third party testing center Release time: 2020 / 7 / 30 reading times: 33 times >>Enter the company's stand
We know that in the cell culture detection experiment, we have to spend a lot of manpower and physical strength on the experimental equipment, culture medium and so on. Fortunately, a long time ago, some experts invented the experimental step of cell preservation, freezing the extra cells temporarily to preserve the cell vitality to the greatest extent.

1. Materials:
The results showed that there were good growth cells, fresh medium, DMSO (sigma d-2650), sterile plastic cryopreservation tube (Nalgene 5000-0020), 0.4% (w / V) trypan blue (gibcobrl15250-061), blood cell counting plate and cover glass, kryo10 Series II

2. Cryopreservation method:

① Traditional method: cold storage tube is stored at 4 ℃ for 10 minutes - > - 20 ℃ for 30 minutes - > - 80 ℃ for 16-18 hours (or overnight) -- > - liquid nitrogen tank vapor phase for long-term storage. -It should not be more than 1 hour at 20 ℃ in order to prevent the excessive ice crystal in the cell and cause a large number of cell death.

② Program cooling: the temperature is reduced from room temperature to below (- 80 ℃) - 120 ℃ at the speed of - 1 ~ - 3 ℃ / min, and then stored in liquid nitrogen tank for long-term storage. It is suitable for the preservation of suspension cells and hybridoma.

Step 3

① 24-48 hours before freezing, half or full amount of medium was changed to make the cells in exponential growth phase.

② Preparation of cryopreservation solution (prepared before use): take another centrifuge tube, add culture medium and serum, add dimethyl sulfoxide (DMSO) drop by drop to 20% concentration, then make double cryopreservation solution and keep it at room temperature.

③ Centrifugation was used to collect the cultured cells. A small amount of cell suspension (about 0.1 ml) was taken to count the cell concentration and survival rate before freezing.

④ Take the same amount of cryopreservation solution as the cell suspension, slowly add the cell suspension drop by drop, and shake the test tube to prepare the cell cryopreservation suspension (the final concentration of DMSO is 5-10%), make the cell concentration of 1-5 × 106 cells / ml, mix evenly, and put them into the completely labeled cryopreservation tube, 1-2 ml / vial, and take a small amount of cell suspension for contamination detection. After sealing tightly, the cell name, algebra and date should be indicated. Then frozen.

4. Precautions:

① The cells to be cryopreserved should be in the state of good growth (logphase) and high survival rate, about 80-90% density. For example, hybridoma should be tested for antibody production one to two days before cryopreservation.

② The cells can be frozen in liquid nitrogen for a long time without affecting cell viability, and can be stored for several months at - 70 ℃.

③ Pay attention to the quality of cryoprotectants. DMSO should be reagent grade, sterile and colorless (filtered with 0.22micron fglp Telflon or directly purchased sterile products, such as sigma d-2650), packed in small volume of 5-10 ml, stored in dark at 4 ℃, and not thawed for many times. Glycorol should also be reagent grade and stored away from light after autoclaving. It should be used within one year after opening because it will be toxic to cells after long-term storage. In this method, double cryopreservation solution was prepared first, which could avoid the damage of cells by heat released when DMSO was directly added. Slowly adding cell suspension dropwise can make the cells adapt to hyperosmolarity gradually and reduce cell damage. DMSO may induce the differentiation of some leukemia cell lines, which can be cryopreserved with 10% glycerol.

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