1. Materials

1) Cell growth medium

2) 37℃ water bath

3) 37℃ incubator

4) Flasks, plates, or dishes

5) Balanced salt solution(PBS containing no calcium, magnesium)

6) Dissociation reagent(trypsin)

7) Equipment to cell counts

8) Shaking platform

2. Cell Resuscitation

1) Preheat the medium in advance, prepare the cell culture dish and centrifuge tube, and add 2mL of preheated medium to the tube.

2) Thawing cells: Take out cells from -80°C or liquid nitrogen, put them directly into a 37℃ water bath, thaw while shaking, about 5min, pay attention to the mouth of the cryogenic vial facing up, so as to prevent water from entering the cryogenic vial.

3) After thawing, wipe the cryogenic vial with alcohol and move it into a biological safety cabinet, then add the liquid in the cryogenic vial to the centrifuge tube containing the 2mL culture solution, and blow evenly.

4) Seal the centrifuge tube with a sealing film, centrifuge at 800r for 5min, discard the medium after centrifugation, add 1ml of complete medium to resuspend the cells, and inoculate them into a 6cm2 culture dish.

5) Observe the cells the next day, and change the medium if necessary.

Notes:

1) After taking out the frozen cells, put them in a 37℃ water bath to thaw as soon as possible, and the whole recovery process should be fast;

2) Then putting the cryogenic vial into or taking it out of the liquid nitrogen container, it is necessary to do a good job of protection to avoid frostbite;

3) Mix the cells by flicking gently, do not repeatedly pipette.

Fig. 1 Cell Resuscitation Flowchart

3. Cell Culture

Adherent Cells Culture

1) Wash the cells

Take the cell culture dish out of the carbon dioxide incubator, and suck out the medium in the bottle in the ultra-clean workbench. Add 2mL of 1*PBS, swirl the dish gently to wash the cells, aspirate and discard 1*PBS.

2) Cell Dissociation

(1) Add 25mL of 0.25% trypsin solution to a 50mL centrifuge tube and shake gently for 15 min in a warm environment or in a 37℃ water bath shaker at a speed of about 180r/min.

(2) Observing the state of cells through a microscope, transfer the liquid containing the suspended cells to a sterile 50mL centrifuge tube, add raw serum to the tube according to the ratio of 1mL calf serum per 10mL supernatant to inactivate trypsin.

3) Preparation of Single Cell Suspension

(1) Centrifuge the mixed cell suspension at 1,200rpm for 5min, discard the supernatant.

(2) Resuspend the pellet with fresh sterile PBS, then centrifuge at 1,000-1,200rpm for 5min.

(3) Repeatedly wash the cells with PBS until the supernatant is clear, then centrifuge according to the first step, discard the supernatant after centrifugation, and repeatedly blow the pellet with a small amount of cell culture medium to make a cell suspension.

4) Cell Seeding

(1) Count cells using the conventional method.

(2) Suspended cells 1:2-1:5 using culture medium, after which 10 ml are seeded to each 100 mm dish.

5) Cell culture

(1) Make a mark on the outside of the culture dish (indicating the name and time of the cultured cells).

(2) Cultured in a 37 ℃ CO2 incubator.

(3) It can be observed according to doubing time.

Suspension Cells Culture

1) Cell Dissociation

Gently pipette the cell culture suspension to disperse cells in the dish, then transfer the solution to a centrifuge tube.

2) Repeat steps 3)-5) above

4. Freezing Cells

1) Prepare freezing medium. To prevent ice crystals from forming in a solution when it is frozen, the solution should contain a cryoprotective agent such as DMSO or glycerol. Put it store at 2-8℃ until use.

2) Resuspend cells in the medium required for their growth and maintenance.

3) Centrifuge at 4℃, 1,000 rpm, 5min. Remove the supernatant with an aspirator.

4) Resuspend the cell pellet in freezing medium. The cell density is 5*10^5-1*10^7 cells/mL.

5) Transfer the cell suspension into cryopreservation tubes.

6) Store the tubes at -80℃.

Fig. 2 Freezing Cells Flowchart