Cell subculture

From Molecular Biology Wiki

Cell subculture is necessary to prevent overgrowth of cell culture.

Main steps in Cell Subculture

Step1) After subculture, cells are sparsely attached to the cell culture flask/plate.

Step 2) After a few hours-days (depending on the cell line), cells grow into the gaps of the plate, and reach confluency. At this stage, they need to be split.

Step 3) Addition of trypsin to the plate after washing away the serum digests away the protein-cell contacts allowing the cells to break away from their contacts.

Step 4) After trypsinization, most of the cells are removed from contact with the plate, and are floating.

Step 5) After subculture, most of the cells are removed and either discarded, or added to other flasks. Fresh Fetal bovine serum (FBS) and media is added.

Detailed Cell Subculture Protocol - Adherent Cells

Subculture at 1:3 or higher ratio for up to several passages (sometimes up to 40) before discarding cell line. At this stage you must thaw fresh aliquots from the liquid nitrogen stocks and start over.

Step 1. Aspirate media from flask containing monolayer of adherent cell culture, and rinse the cells with PBS

(PBS wash not necessary but helps increase the activity of the trypsin added at later stage - saving you money as you can use less!).

Step 2 . Aspirate PBS, and pipette trypsin-EDTA onto the monolayer in the flask. Incubate at 37°C for 2-5 min. Bump the side of the plate against the palm of the hand to help detach the cells. Wait until most cells are not only detached, but are not in clumps any more.

Step 3A. You can then either transfer the floating cells to a centrifuge tube or simply remove most of the cells. Rinse the culture vessel with HBSS to collect remaining cells, and add to centrifuge tube. Neutralize trypsin action by adding FBS, approximately one-half the volume of trypsin used. Centrifuge cells at 150g, 7 min Resuspend cell pellet in fresh growth media and seed the cells onto a new flask.

Step 3B. Take an aliquot of the cells and add them into fresh media in a new flask.