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Cell cryopreservation program introduction and steps
Time:2017-11-29
Cell cryopreservation program introduction and steps!
In cell culture testing experiments in laboratory equipment, culture, etc. work to spend a lot of manpower and physical strength. Fortunately, a long time ago, the Supreme has invented the cell preservation of this experimental step, the extra cells temporarily frozen to preserve the vitality of cells.
 
First, the cells were cryopreserved
1. Material:
Well-grown cells, fresh medium, DMSO (Sigma D-2650), sterile plastic cryopreservation tubes (Nalgene 5000-0020), 0.4% (w / v) trypan blue (GibcoBRL 15250-061) Disk and cover glass, cell cryopreservation program (also known as the program cooler, the brand extension of Shanghai)
2. Cryopreservation method:
(1) The traditional method: cold storage tube placed at 4 ℃ 10 minutes ---> -20 ℃ 30 minutes ---> 80 ℃ 16 ~ 18 hours (or overnight) ---> liquid nitrogen tank vaporphase long-term storage.
-20 ℃ can not exceed 1 hour, in order to prevent excessive intracellular ice crystals, resulting in a large number of cell death, you can skip this step directly into the -80 ℃ refrigerator, but the survival rate slightly lower.
(2) Program cooling: Using the isobaric cell cryopreservation program which has been programmed, the temperature is decreased from room temperature to below -80 ℃ -120 ℃ at -1 ~ -3 ℃ / min, vaporphase long-term storage. Suitable for the preservation of suspension cells and hybridoma.
3. Steps:
(1) 24-48 hours before freezing, replace half or full medium, so that the cells in the exponential growth phase.
(2) Preparation of cryopreservation solution (prepared before use): Another centrifuge tube was added to the medium, serum, dimethylsulfoxide (DMSO) was added dropwise to a concentration of 20% , Placed at room temperature stand-by.
(3) Collect the cultured cells by centrifugation, resuspend the cells with serum-added medium, and take a small amount of cell suspension (about 0.1 ml) to count the cell concentration and the pre-thaw survival rate.
(4) Take the same amount of cryopreservation solution as the cell suspension, slowly add the cell suspension dropwise, and shake the test tube to prepare a cell suspension (final concentration of DMSO is 5 to 10%) so that the cell concentration is 1 ~ 5 × 106cells / ml, mixed evenly, aliquoted in the cryopreservation tube labeled completely, 1 ~ 2ml / vial, and take a small amount of cell suspension for contamination detection. Strict seal, specify the cell name, algebra, date. Then freeze.
    Program-controlled freezing device commonly known as: program cooling device, also known as the program freezer, embryo freezer, cell freezing device, programmed cooling device; widely used in frozen cells, embryos and so on. Microcomputer control technology, special software, can more accurately control the amount of liquid nitrogen in order to ensure that the frozen biological products at a suitable freezing rate cooling and freezing. The instrument is easy to operate, human-machine interface is clear.