Essential protocols for animal cell culture

Maintaining cell cultures

Establishment and maintenance of animal cell cultures require standardized approaches for media preparation, feeding, and passaging (or subculturing) of the cells. Cultures should be examined regularly to check for signs of contamination and to determine if the culture needs feeding or passaging.

The cell culture protocols in this chapter have been adapted from the following sources: Culture of Animal Cells; a Manual of Basic Technique (1), Current Protocols in Molecular Biology (4), and Cells: A Laboratory Manual (2). These protocols are examples of methods for general cell culture, and have not been rigorously validated and optimized by QIAGEN. There are many alternative protocols in current use.

IMPORTANT: Potentially biohazardous materials (e.g., cells, culture medium, etc.) should be sterilized before disposal, and disposed of according to your institution’s guidelines.

Cell thawing

Heat a water bath to 37°C, and warm the growth medium into which the cells will be plated.
Add prewarmed growth medium to an appropriately sized cell culture vessel.
Remove a vial of frozen cells from liquid nitrogen, and place in the water bath until thawed.

IMPORTANT: Wear protective goggles and gloves when thawing vials that have been stored in liquid nitrogen. Vials may explode when removed from liquid nitrogen.

IMPORTANT: Proceed to step 4 as soon as the cells have thawed. Do not allow the cells to warm up before transferring them into growth medium.
Wash the outside of the vial with 70% ethanol or another suitable disinfectant.
Slowly pipet the thawed cell suspension into the cell culture vessel containing prewarmed growth medium. Swirl the vessel gently to mix the cells with the medium.

Note: Immediate removal of DMSO may sometimes be necessary, especially for suspension cells, primary cells, and sensitive cell types. For such cell types, pipet the thawed cell suspension into a sterile centrifuge tube containing prewarmed medium, centrifuge at 200 x g for 2 min, aspirate the supernatant, resuspend the cells in fresh growth medium, and then transfer to an appropriate cell culture vessel.

IMPORTANT: Thoroughly mix the cells in the cell culture vessel to ensure even distribution of the cells throughout the vessel.
Incubate cells overnight under their usual growth conditions.
The next day, replace the growth medium.

Trypsinizing cells

Trypsinization is a technique that uses the proteolytic enzyme trypsin to detach adherent cells from the surface of a cell culture vessel. This procedure is performed whenever the cells need to be harvested (e.g., for passaging, counting, or for nucleic acid isolation).

Aspirate the medium and discard.
Wash cells with PBS (phosphate-buffered saline) or HBSS (Hanks balanced salt solution) (see tables 1x PBS and 1x HBSS), aspirate, and discard. Repeat.
The volume of PBS or HBSS should be approximately the same as the volume of medium used for culturing the cells.
Add enough warmed 1x trypsin–EDTA solution (see table 1x trypsin–EDTA solution) to cover the monolayer, and rock the flask/dish 4–5 times to coat the monolayer.
Place the flask/dish in a CO₂ incubator at 37°C for 1–2 min.
Remove flask/dish from incubator and firmly tap the side of the flask/dish with palm of hand to assist detachment.

If cells have not dislodged, return the flask/dish to the incubator for a few more minutes.

IMPORTANT: Do not leave cells in 1x trypsin–EDTA solution for extended periods of time. Do not force the cells to detach before they are ready to do so, or clumping may occur.

Overly confluent cultures, senescent cells, and some cell lines may be difficult to trypsinize. While increasing the time of trypsin exposure may help to dislodge resistant cells, some cell types are very sensitive to trypsin and extended exposure may result in cell death. In addition, some cell lines will resist this treatment and will produce cell clumps.
Once dislodged, resuspend the cells in growth medium containing serum.
Use medium containing the same percentage of serum as used for growing the cells. The serum inactivates trypsin activity.
Gently pipet the cells up and down in a syringe with a needle attached to disrupt cell clumps.
If pipetted too vigorously, the cells will become damaged. Ensure that pipetting does not create foam.
Proceed as required (e.g., with passaging, freezing, nucleic acid isolation, etc.).

1x PBS

Composition
137 mM NaCl
2.7 mM KCl
4.3 mM Na2HPO4
1.47 mM KH₂PO₄

1x HBSS

Composition
5 mM KCl
0.3 mM KH₂PO₄
138 mM NaCl
4 mM NaHCO₃
0.3 mM Na₂HPO₄
5.6 mM D-glucose

1x trypsin–EDTA solution

Composition
0.05% (w/v) trypsin
0.53 mM EDTA
Dissolve trypsin and EDTA in a calcium- and magnesium-free salt solution such as 1x PBS or 1x HBSS*

Passaging cells

Many adherent cell cultures will cease proliferating once they become confluent (i.e., when they completely cover the surface of cell culture vessel), and some will die if they are left in a confluent state for too long. Adherent cell cultures therefore need to be routinely passaged, that is, once the cells are confluent, a fraction of the cells need to be transferred to a new cell culture vessel. Suspension cells will exhaust their culture medium very quickly once the cell density becomes too high, so these cultures similarly require regular passaging.

IMPORTANT: Although regular passaging is necessary to maintain animal cell cultures, the procedure is relatively stressful for adherent cells as they must be trypsinized. We do not recommend passaging adherent cell cultures more than once every 48 h.

Harvest the cells, either by trypsinization (adherent cell cultures) or by centrifugation at 200 x g for 5 min (suspension cell cultures). Resuspend the cells in an appropriate volume of prewarmed growth medium containing serum.

The volume of medium used to resuspend the cells depends on the split ratio required (see step 2) and the size of the cell culture vessel. If too small a volume is used, it may be difficult to accurately pipet the desired volume to the new culture vessel.
Conversely, if too large a volume is used, the culture vessel may be too full following transfer of the cells.

Removal of trypsin may sometimes be necessary following harvesting of adherent cells, especially for primary and sensitive cell types. Centrifuge the cells at 200 x g for 5 min, carefully aspirate the supernatant, and resuspend the cells in an appropriate volume of prewarmed medium containing serum.
Transfer an appropriate volume of the resuspended cells to a fresh cell culture vessel containing prewarmed growth medium. Swirl the vessel gently to mix the cells with the medium.

IMPORTANT: Thoroughly mix the cells in the cell culture vessel to ensure even distribution of cells.

IMPORTANT: Some cell types will not survive if too few cells are transferred. We do not recommend high split ratios for primary cells, sensitive cell types, or senescent cultures.

For adherent cells, we recommend adding enough cells so that the culture takes approximately one week to reach confluence again. This minimizes the number of times the cells are trypsinized as well as the handling time required to maintain the culture.

When determining how many cells to transfer to the new cell culture vessel, it can be helpful to think in terms of how many cell divisions will be required for the culture to reach confluence again. For example, if half the cells are transferred, then it will take the culture one cell division to reach confluency again; if a quarter of the cells are transferred then it will take 2 cell divisions, and so on. If a culture divides once every 30 h or so, then in one week it will undergo approximately 5 cell divisions. A split ratio of 1:32 (1:25) should therefore be appropriate for the cells to reach confluency in about one week. In step 1, resuspend the cells in 8 ml medium, and transfer 0.25 ml to the new cell culture vessel.
Incubate cells under their usual growth conditions.