How to keep up with cell culture - Introduction to essential methods

Introduction

Different cell types require different conditions for cell growth and maintenance. However, several characteristics and techniques used for the maintenance of mammalian cell culture are universal. All in vitro cell cultures share some common characteristics such as; slow cell growth, complex metabolism, and the need for complex media. 

There are primarily two types of cell lines found in cell culture laboratories. The techniques for maintaining them vary slightly, but the concepts behind why we employ them are the same. In this post, we will take you through the two different types of cell cultures and how to keep them alive!

1. Different types of cell cultures

Cells can be grown in culture in two ways: as monolayers on an artificial substrate (adherent culture) or free-floating in the culture media (suspension culture).

• Suspension cell culture 

Suspension culture is a type of cells, that are able to grow in the media without having to attach to a surface. They are grown in flasks where the volume to surface area ratio does not allow for sufficient gas exchange, so mixing is needed for their growth. 

The most widely used suspension cultures are mammalian Chinese Hamster Ovary cells (CHO) and Sf9 insect cells. Suspension cultures are predominantly used in the industry for protein production, as they are more easily adapted for growth in a bioreactor.

• Adherent cell culture 

Adherent cell culture consists of cells that are anchorage-dependent. These kinds of cells, will only grow and survive in culture when attached to a surface such as glass or plastic. The flasks in which such cells grow are specifically treated to allow cell adhesion. The cells grow in a monolayer until they reach a 100% confluence (completely covering the flask surface), after which they usually stop proliferating. As the surface-to-volume ratio is optimal for gas exchange, there is no need for mixing the culture. The most widely used adherent cultures are MDCK, HEK 293, and Vero cells amongst others. 

As you can see, there is a big variety in cell cultures. 

1. How to handle cell culture?

Before doing anything with your cells, you first need to examine them! Observe the morphology and viability of the cells by looking at any signs of cell death and/or contamination. The signs are usually visible by the naked eye, such as turbidity and floating particles, however, you should also make sure by looking through the inverted microscope (especially at the edges of the flasks 😉). 

Since you are already familiar with the first steps in cell culture (if not, check out our blog on the first steps in cell culture), we can now jump straight into the techniques, you will definitely use during your time in the cell culture lab!

• Why, when, and how to subculture cells?

When a cell culture is first initiated, cells will start to grow in the flasks. After a period of time, the cell density will increase, nutrients in the medium will get depleted and toxic metabolite production will lead to cell death. So before this happens, cells need to be split or subcultured, creating new cultures with lower cell density. The cells are supplied with fresh nutrients and hazardous metabolites are removed when the medium is withdrawn and the cells are transferred to a fresh growth medium, allowing the culture to be maintained for a long time. This is referred to as a passage. Each passage number indicates the number of times cells have been subcultured into a new flask.

Cell lines must be kept in the exponential phase to maintain their viability and stability of the cell line. This means the best time for cells to be subcultured on a frequent basis is between the log and stationary phase, before the monolayer reaches 100% confluency, or before a suspension reaches the maximum suggested cell density. To determine the optimal point for subculturing, the best approach is to create a growth curve for each cell line.

• Lag phase —Right after seeding of the culture flask, the cells slowly start to recover from the subculturing stress and grow. 
• Log or exponential phase — The cells enter a period of exponential growth that lasts until they reach confluency or maximum medium capacity.
• Stationary phase — Cell growth slows and reaches a plateau. 
• Decline/death phase — If the cells are not subcultured and replenished with fresh culture medium the cells start dying. 

Cell culture conditions and subculture methods vary for each cell type. Below the basic steps in the workflow of adherent and suspension cell cultures are described. Note that during the process it is essential to work in aseptic conditions.

There are some differences between subculturing suspension or adherent cell cultures. 

If the cells are adherent and growing in a monolayer follow the next steps: 

1. Aseptically remove all of the old media and wash the cells with phosphate buffer to remove dead cells.
2. Afterwards add warm trypsin-EDTA (a proteolytic enzyme) to detach cells from the flask surface and incubate at 37°C with 5% CO₂ (optimum T and CO2 concentration for most cell lines, but make sure to check for the specific cell line you are using) for 1-3 min or until the cells are detached. 
3. Firmly tap the flask to assist in detaching the cells and pipette the desired amount of detached cells into a new flask with a fresh pre-heated medium.
4. Incubate the cells till further use.

TIP: Constant passaging is required to sustain animal cell cultures, however, adherent cells must be trypsinized, causing cell stress. To avoid poor growth subculturing adherent cell cultures more than once per 48 hours is not a good idea.

If the cells are growing in suspension follow these steps: 

1. Aseptically transfer the entire contents of the flask to a centrifuge tube. 
2. Centrifuge at 125 × g for 5 to 10 minutes. 
3. Remove all of the supernatant and resuspend the cells in a fresh medium.
4. Aseptically transfer the desired amount of resuspended cells to a new flask.
5. Incubate the cells in a shaking incubator till further use.

• How to properly count the cells? 

To follow the growth rates and the status of your cell line you will need to count them. The most used counting technique is using the improved Neubauer’s counting chamber or hemacytometer. They are a type of cell counter with a glass slide and two counting chambers, one on each side. 

The chamber is first cleaned with 70% ethanol and dried. The cell sample is taken from the trypsinized cells or the suspension, mixed thoroughly, and then the counting chamber is filled with ~15µL of the sample. The slide is taken to the microscope under which, the total number of cells is counted. The numbered big squares in the picture need to be counted. So big squares indicated with 1, 2, 3, and 4 are counted, which consist of 4x4 smaller squares. Counting of the big square should start at the top-left and continue in a zig-zag manner through all the small squares, just as in the picture. Counting may be enhanced with the microscope’s illumination reduced. Four big squares need to be counted and the average number can be used for the calculations.

Certain guidelines must be followed to ensure that cells on or near the limit lines are not counted twice or missed during the count. To avoid counting cells twice, just two limit lines (usually the left limit line and the upper limit line) must be taken into account per square. The cells in the square can be counted after the cells on these two lines have been counted.

To calculate the number of cells per mL you have to take into account the size of your chamber. Since one large square (16 smaller squares) is 1mm2 and 0.1mm in depth, the volume is 0.1µL (equals 1x104 µL). 

So the formula is as follows:

To ensure the reliability of your counting it is wise to count 4 squares.

NOTES on counting:

• The difference of the number of cells between the large squares and the difference between the group squares must not be more than 10 cells. 
• For all cell counts, double-checking is required. The bottom counting net is counted in the same way.
• The difference between the total counts for the two counting nets cannot be more than ten cells.

Conclusion

You made it through the first 2 essential methods you will master during your time in the cell culture lab! Subculturing and counting are done so often that you will know the protocols by heart soon enough without even trying.

And remember! Never start your work without your lab coat! Still need one? Check out our shop.

References

• Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 6.2, Growth of Animal Cells in Culture.  
• Verma, Anju et al. “Animal tissue culture principles and applications.” Animal Biotechnology (2020): 269–293. doi:10.1016/B978-0-12-811710-1.00012-4
• Freshney, R.I. (1993) Culture of Animal Cells, A Manual of Basic Technique, 3rd ed., New York: Wiley-Liss.
• Spector, D., Goldman, R.R., and Leinwand, L.A., eds. (1998) Cells: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
• How to do a Proper Cell Culture Quick Check | Science Lab | Leica Microsystems (leica-microsystems.com)
• Animal Cell Culture Guide.pdf