Trypsinizing and Subculturing Mammalian Cells

Tissue cultured technology Has found wide application in the field of cell biology. However, cultured cells require regular maintenance to stay healthy. As cells reach co fluency, they must be sub cultured or passaged.

Failure to do so results in reduced growth and eventually cell death. In this video, we demonstrate how to maintain tissue culture cells for both adherent and suspension cell lines. Hi, I'm Ricky here at Dr.LAN's Laboratory at Molecular Pathology Laboratories Network in East Tennessee.

Today I want to show you two techniques for packaging suspension and adhering cells. To passage a plate of Cells. We'll want to start with a primary culture grown to co fluency in a 60 millimeter Petri dish or a 25 centimeter squared tissue culture flask containing five mils tissue culture medium.

We start by removing all of the medium from the primary culture with the sterile pipette. Then we wash the adhering cell monolayer once or twice with a small volume of 37 degrees Celsius HBSS without calcium and magnesium. To remove any residual fetal bovine serum that may inhibit the action of the trypsin enzyme that will add in the next step.

Next, add enough warm trypsin EDTA solution to the culture so that you cover the adherent cell layer. Then you can place the plate on a 37 degree Celsius warming tray for one to two minutes. After the incubation, tap the bottom of the plate on a flat surface to dislodge the cells.

Check the culture with an inverted microscope to be sure that the cells are rounded up, which indicates that they are detached from the surface. If the cells are not sufficiently detached, return the plate to the warming tray for an additional minute or two. After looking through the scope, rinse the dish in complete medium and pipette the suspension into a 15 mil conical tube containing two mils of complete medium.

Spin the cells down to pellet them and resuspend the pellet in complete medium. Now, add an equal volume of cell suspension to each of the fresh plates that have been appropriately labeled. Alternatively, the cells can be counted using a hemo cytometer and diluted to the desired density so a specific number of cells can be added to each plate.

Be sure to label every plate with the date of the subculture and the passage number. After adding the cell suspension, place one mil of fresh medium to each new culture. Now incubate the plates in a humidified 37 degrees Celsius 5%CO2 incubator.

After you've incubated the cells overnight, add fresh medium to the plates and put them back at 37 degrees. Once the plate has grown to con fluency, you can passage the plate again by repeating this procedure and continue to passage as necessary. Now we'll show you how to passage cells in suspension culture.

Packaging cells in suspension culture is simpler than for adherence cells. Before packaging suspension cultures, the cells must be maintained by feeding every two to three days until they reach co fluency, which is when the cells clump together in the suspension and the medium appears turid. When the flask is swirled.

To do this, remove the flask of suspension cells from the incubator, taking care not to disturb those that have settled to the flask. Bottom aseptically, remove and discard about one third of the medium from the flask and replace it with an equal volume of prewarm medium at 37 degrees Celsius. After you've changed the medium, swirl the flask and return it to the incubator, which is at 37 degrees Celsius, 5%CO2.

If there's less than 15 mils, a medium in the flask incubate the flask in a horizontal position to enhance cell to medium contact. Otherwise, the flask can be incubated vertically on the days the cultures are not being fed. Check them by swirling the flats to resuspend the cells.

Be sure to observe any color changes in the medium that indicate good metabolic growth. Once the cultures are confluent, which should be about 2.5 million cells per mil, you can passage them. Once again, We've just shown you how to passage cultured cells for both adherent and suspension cultures.

So that's it. Thanks for watching and good luck with all your experiments.