Cell passage

1. Adherent cells

Operations (take a T25 flask for example):

A. Suck out the original culture medium in the flask.

B. Add about 2ml PBS, and gently shake to infiltrate cells. Suck out PBS and discard it.

C. Add about 1ml 0.25% trypsin (including EDTA), gently shake the flask to infiltrate all cells, and put the flask into the incubator at 37℃ for dissociation.

D. The dissociation time varies according to cellular characteristics. Dissociation can be terminated if the cells are observed to separate obviously and become rounded under the microscope, or if the cells can slide down the wall when gently shaking the flask. Do not pat the flask during the whole procedure.

E. Add 3ml medium which contains serum to terminate dissociation. Pipette up and down repeatedly to flush down and separate the cells, making them a single-cell suspension. Observe the cell state under the microscope and see whether they are single.

F. Collect cell suspension and centrifuge at 1200rpm (about 250g) for 3 min. Then pipette out the supernatant and discard it.

G. Add fresh medium, and gently pipette to fully mix the cells. Seed the cells into a new flask as needed and replenish the medium. Loosen the cap or use the breathable cap for culture.

H. Make sure the water tray, CO2 concentration, and temperature are in the right condition.  

2. Suspended cells

Operations (take a T25 flask for example):

A. Gently pipette up and down to separate the suspended cells. Some loosely attached suspended cells can be suspended by flushing directly at the bottom of the flask. Collect the cell suspension into a sterile centrifuge tube, centrifuge at 1200rpm (about 250g) for 3 minutes. After centrifugation, suck out the supernatant and discard it.

B. Add an appropriate amount of fresh medium to re-suspend the cells. Seed the cells into the flask at a proper ratio. If the ratio is uncertain, count the cell number before seeding.

C. Normally, it’s recommended to seed the cells at 3 ~ 5×105 cells/ml in a new flask. For example, in a 25T flask, the medium volume is 5mL. In order to seed the cell at 5×105 cells/ml, the total cell number needed will be 2.5×106. When they grow to 2×106 cells/ml, it’s time to passage.

D. It is suggested to count the cell number before a passage for the first several generations. Then the passage can be run at a ratio based on experience.  

E. Make sure the water tray, CO2 concentration, and temperature are in the right condition.  

3. Semi-adherent cells

Operations (take a T25 flask for example):

A. Suck out the culture medium in the flask with a pipette and transferred it to a sterile centrifuge tube.

B. Infiltrate the cells with 1ml PBS. Suck out the PBS and collect it into the centrifuge tube used in step 1.

C. Add 1 ml 0.25% trypsin with EDTA into the flask and put it into the incubator at 37℃ for dissociation.

D. The dissociation time varies according to cellular characteristics. Dissociation can be terminated if the cells are observed to separate obviously and become rounded under the microscope, or if the cells can slide down the wall when gently shaking the flask. Do not pat the flask during the whole procedure.

E. Add 3ml medium which contains serum to terminate dissociation. Pipette up and down repeatedly to flush down and separate the cells, making them a single-cell suspension. Collect the suspension into the centrifuge tube used in step 1.

F. Centrifuge the tube at 1200rpm (about 250g) for 3min. After centrifugation, discard the supernatant and add new medium for re-suspension. Seed the cells into a new flask as needed and replenish the medium. Loosen the cap or use the breathable cap for culture.

G. Put the flasks in CO2 incubator. Make sure the water tray, CO2 concentration, and temperature are in the right condition.  

H. Check the carbon dioxide, temperature and water tray of the incubator.

Note:

A. Before discarding any liquid, think about whether there might be cells in the liquid to avoid losing cells.

B. Cell type, cell density, cell state, and  even the performance of trypsin and its opening time can affect the dissociation time of cells. The dissociation time in the instructions can only be used for reference. It’s better to determine the time by the state of cells under the microscope. For example, if the reference dissociation time of a cell is at 37℃ for 2-3 minutes, it can be taken out at 1 and a half minutes and observed under the microscope whether the cells shrink and become round and flow. If not, it can be put back into the incubator. Continue to observe every half minute or one minute until the cells shrink and become round and flow. At this moment the dissociation can be terminated.

C. The concentration of trypsin with EDTA used in the dissociation process is usually 0.25%. But the passage steps of some cell lines or primary cells are different, please refer to the cell characteristics and instructions for details.

D. Cell passage ratio is usually based on containers with an equal bottom area. If passed to different containers, it needs to be converted. For example, cells from a T25 flask are transferred to a 10cm dish. The ratio will not be 1:1. As the bottom area of a T25 flask is 25cm2, and the bottom area of a 10cm dish is about 55cm² (the inner diameter of the dish is about 8.4cm, the bottom area can be calculated as 55cm² according to the inner diameter), so the actual passage ratio is 1:2.2.

E. In centrifuge experiments, the standard centrifuge conditions should be measured by RCF (relative centrifugal force). But in actual experiments, rpm is usually used to express the rotational speed per minute. RCF is expressed by the multiple of gravity acceleration g (980.66cm/s2). RCF=11.2×Radius×(RPM/1,000)2 (the distance between the center of the centrifuge shaft and the center of the centrifugal tube is the radius, expressed in cm). Therefore, the centrifuge speed setting in each laboratory is different. The setting is generally recommended at 1200rpm, which is about 250g.  

F. Regarding changing the medium after resuscitation or passage, if the color of the medium is clear, we can change the medium once every 2-3 days; if the medium starts to turn yellow within 24 hours, we can add a small amount of complete medium to ensure nutrition, and then change the medium after about 48 hours; if the medium turns purple, it means that the pH of the medium changes, and it is necessary to replace the correct complete medium in time and find out the cause of the change.

G. All reagents should be rewarmed before being added to cells (generally at about 25℃). Medium and PBS can be reheated in a 37℃ incubator or water bath. When the condensate no longer appears after drying, and the bottle feels at normal temperature, it means the reheating is completed. Trypsin needs to be rewarmed at room temperature, so it is recommended to take it out in advance. Rewarming of a small bottle of trypsin can generally be completed in about 1 hour. A larger bottle will take longer. When the bottle feels at normal temperature, it means the reheating is completed. Shake the trypsin before use.