How fast is the average growth rate of cells?

Well-grown cultured cells, fresh culture medium, DMSO (Sigma D-2650), sterile plastic cryopreservation tube (Nalgene 5000-0020), 0.4% (w/v) trypan blue (gibco brl15250-061), blood cell counting disk and cover.

2, frozen storage method:

(1) traditional method: place the cold storage tube at 4℃10min →-20℃ 30min →-80℃16 ~18h (or overnight) → store it in a liquid nitrogen tank for a long time. The temperature of -20℃ should not exceed 1 hour, so as to avoid a large number of cell deaths caused by excessive ice crystals. You can also skip this step and put it directly in the refrigerator at -80℃, but the survival rate decreases slightly.

(2) programmed cooling: using a programmed isokinetic cooling machine, the temperature is lowered from room temperature to (below -80℃)-120℃ at a speed of-1 ~-3℃/min, and then it is stored in a liquid nitrogen tank for a long time. Suitable for the preservation of suspended cells and hybrids.

Step 3:

(1) Change half or all of the culture medium the day before freezing, and observe the cell growth.

(2) Preparation of frozen preservation solution (preparation before use): DMSO is added to the fresh culture medium with the final concentration of 5 ~ 10%, mixed evenly, and placed at room temperature for later use.

(3) According to the operation of cell subculture, collect cultured cells, take a small amount of cell suspension (about 0. 1ml), and count the cell concentration and the survival rate before cryopreservation.

(4) Centrifuge, remove the supernatant, add an appropriate amount of cryopreservation solution to make the cell concentration 1 ~ 5× 106 cells /ml, mix well, and sub-package in completely labeled cryopreservation tubes 1ml/ bottle, and take a small amount of cell suspension for pollution detection.

4. Precautions:

(1) The cells to be frozen should be in a state of good logarithmic phase and high survival rate, and the density is about 80-90%.

(2) Before freezing, test whether the cells still retain their unique properties. For example, hybridomas should be tested for antibody production one to two days before freezing.

(3) Pay attention to the quality of cryoprotectant. DMSO should be reagent-grade, sterile and colorless (sterile product filtered with 0.22micron FGLP Telflon or directly purchased, such as Sigma D-2650), packed in a small volume of 5 ~ 10 ml, stored at 4℃ in the dark, and not thawed for many times. Glycerol should also be reagent grade, sterilized by high pressure steam and stored in the dark. If used within one year after opening, long-term storage will cause toxicity to cells.

(4) Cell concentration of cryopreservation:

① Normal human fibroblasts: 1 ~ 3× 106 cells/ml

② hybridoma: 1 ~ 3× 106 cells /ml, and the cell concentration should not be too high. Due to the high freezing concentration, some hybrids will die after thawing for 24 hours.

③ Adhesion tumor strain: 5 ~ 7× 106, depending on the cell type. Adenocarcinoma needs a higher concentration after thawing, while HeLa only needs 1 ~ 3× 106 cells /ml.

④ Other suspensions: 5 ~ 10× 106 cells /ml, and the human rhythm should be at least 5× 106 cells /ml.

(5) The concentration of cryoprotectant is 5 or 10% DMSO. If the freezing conditions of cells are uncertain, backup culture should be carried out at the same time of freezing to prevent freezing failure.

Second, the activation of frozen cells

1. The activation principle of frozen cells is to thaw quickly to avoid the damage and death of cells caused by recrystallization of ice crystals.

2. After cells are activated, it will take several days, or one or two generations, for their cell growth or characteristics to return to normal (such as producing monoclonal antibodies or other protein).

3. Materials: 37℃ constant temperature water tank, fresh culture medium, sterile straw/centrifuge tube/culture bottle, liquid nitrogen or dry ice container.

4. Step:

(1) Operators should wear protective masks and gloves to prevent freezing pipes from bursting.

(2) Take out the freezing tube from the liquid nitrogen or dry ice container and check whether the cover is tightened. Because of the process of thermal expansion and cold contraction, the cover is easy to loosen at this time.

(3) Put the fresh culture medium into a water tank at 37℃ for heating, spray 70% alcohol for wiping after heating, and move it into a sterile operating table.

(4) Take out the frozen tube, immediately put it into a water tank at 37℃ for quick thawing, gently shake the frozen tube to make it completely melt within 65438 0 minutes, wipe the outside of the tube with 70% alcohol, and move it into a sterile operating table.

(5) Take out the thawed cell suspension, slowly add it into a culture container with culture medium (dilution ratio:1:/kloc-0 ~1:15), mix it evenly, and put it into a CO2 incubator for culture. Take 0. 1 ml thawed cell suspension for survival test.

(6) Whether cryoprotectants (such as DMSO or glycerol) are removed immediately after thawing depends on the cell type. Generally speaking, it is not necessary to remove antifreeze immediately. However, if it needs to be removed immediately, add the thawed cell suspension into a centrifuge tube containing 5- 10ml culture medium, centrifuge at 1000rpm for 5 minutes, remove the supernatant, add fresh culture medium, mix well, and put it in a CO2 incubator for culture.

(7) If the cryoprotectant does not need to be removed immediately, change the culture medium every other day after thawing culture.

Third, cell counting and survival test.

1, principle:

(1) The number of cells can be automatically counted by blood cell counting disk or Coultercounter particle counter.

(2) Generally, the blood cell counting disk has two chambers, each chamber is carved with nine squares 1mm2, and the four corners are carved with squares 16 cells, with a depth of 0.1mm.. When the cover glass is covered above the cavity, the volume of each large square is1mm2× 0.1mm =1.0x10-4 ml. When in use, count the number of cells in each square, multiply it by the dilution multiple, and then multiply it by 104, which is the number of cells per milliliter.

(3) The step of the survival test is dye exclusion method. Dyes will infiltrate dead cells and change color, while living cells will not change color, because the cell membrane is intact and dyes cannot penetrate. Generally, blue trypan blue dye is used. If cells are not easy to absorb trypan blue, red trypan blue dye is used.

2. Materials:

0.4%w/v trypan blue (gibco br15250-061); Red pigment blue stain; 0. 1 g erythrosine blue (Sigmae-9259) and 0.05g preservative methyl paraben (Sigmah-3647) are dissolved in 100 ml of ca++/mg++ free saline; Blood cell counting disk and cover glass; A counter; Low power inverted microscope; Particle counter (Coulter Electronics).

Step 3:

(1) Take 50 μ l cell suspension and 50μl trypan blue, and mix them evenly in a small centrifuge tube of1.5ml.

(2) Add a little mixed liquid (about 15μl) from the groove above the cavity of the blood cell counting disk, cover it with a cover glass, and observe it under an inverted microscope with a magnification of 100. Living cells are not stained, and dead cells are blue (or red-erythrosine blue).

(3) Count the total number of cells in four large squares, divide by 4, multiply by the dilution multiple (at least 2, because it is mixed with trypan blue in equal volume), and finally multiply by 104, which is the number of cells in each milliliter of cell suspension. If the cell is in the row, only the cells in the row and the right row (or the cells in the row and the left row) are counted.

Note: total number of 4 cells × dilution multiple × 104/4= number of cells/ml; The volume of each grid = 0.1cm× 0.1cm× 0.01cm =10-4 ml.

4. Example:

T75 monolayer culture was made into 10ml cell suspension. The 0. 1ml solution and 0. 1ml trypan blue were mixed evenly in the test tube, and a little mixed solution was added to the blood cell counting plate to count the number of cells in four squares.

Number of living cells/grid: 55,62,49,59; Number of dead cells/grid: 5, 3, 4, 6; Total number of cells =243

Average cell number/square = 60.75; Dilution multiple =2

Cell number /ml: 60.75× 104× 2 (dilution multiple) = 1.22× 106.

Cell count/vial (10ml):1.22×106×100ml =12.2×106.