Cell Culture Techniques | Lonza (2024)

• What species?Non-human and non-primate cell lines usually have less biosafety restrictions, your experiments will dictate what cell line species to use.
• Which functional characteristics?For example, liver- and kidney-derived cell lines may be more suitable for toxicity studies (ADMET).
• Finite or continuous?Choosing a primary (finite) cell may relate better to the in vivo situation, continuous cell lines are often easier to clone and maintain.
• Normal or transformed?Genetically transformed cell lines usually have an increased growth rate and higher plating efficiency, the counter part is they have undergone a permanent change in their phenotype through a transformation, no longer an exact copy of the original.
• Which growth conditions?For example, to express a recombinant protein in high yields, you might want to choose a cell line with a fast growth rate and an ability to grow in suspension.
• Other criteria?If you are using a finite cell line, be sure the cell line is well characterized or you have to perform the validation yourself.
  Read about the risks of using misidentified, unauthenticated cell lines in a publication by Hughes et al (2007).

We advise against using cultures from other laboratories because:

• There is a high risk of contamination (e.g. mycoplasma)
• They may not be the cell type which is on the label (we recommend to characterize them)

Regardless of their source (purchased or borrowed), make sure that allnew cell lines are tested for mycoplasma contamination before you begin to use them.

Watch this webinar on mycoplasma testing and learn about potential impacts of a contamination on cell cultures and how you can easily uncover and remove it.

View webinar

Cell Passage

Passaging a cell line is a cell culture technique where the cell culture medium is removed and cells are transferred from a previous culture into fresh growth medium, a cell culture technique that enables the further growth of the cell line.

The growth of cells in culture proceeds from the lag phase following seeding to the log phase, where the cells proliferate exponentially. When the cells in adherent cultures occupy all the available substrate and have no room left for expansion, or when the cells in suspension cultures exceed the capacity of the medium to support further growth, cell proliferation is reduced or ceases entirely. To keep the culture at an optimal density for continued cell growth and to stimulate further proliferation, the culture has to be divided and fresh medium supplied.

For some cell culture techniques like transfection, it is important to have the cells in the log phase, this will improve the transfection efficiency.

Clarification on Nomenclature in Cell Culturing

A passage involves trypsinizing the cells off of a vessel to reseed on another vessel (aka. subculture).
A population doubling is when the cells double in count during culture (~2-3 pop doublings per passage).

Primary cells will have a passage number, the earlier the passage number the better, which is also valid for cell lines:

• P0 – Cells never plated, cryopreserved immediately
• P1 – Cells that have been plated into a flask after initial isolation
• P2 – Cells that have been plated into a flask after initial isolation, grown to confluence, re-plated into a second flask

Perform the following steps before you begin media or cell preparation:

1. Prepare a sterile surface
   • A sterile field consist of a Class II biological safety cabinet with a front access opening and filtered laminar airflow, or an equivalent device.


2. Determine the amount of medium required
   • Review the growth area of Common Plastic ware table (see table on the right) to determine the amount of medium to be used.


3. Sterile instruments and vessels required
   • Sterile disposable serological pipettes
   • Micropipettes and sterile pipette tips
   • Adjustable multichannel pipette or repeating pipette.
   • Sterile reservoir for use with multichannel pipette
   • Sterile 15mL centrifuge tubes
   • Cell culture flasks, or multiwall, flat-bottom tissue culture plates
   • Hemacytometer or cell counter



4. Other required supplies

   • 70% alcohol (ethanol or isopropanol)
   • Growth medium (cell type specific)
   • Protective gloves and garments
   • Trypan Blue
   • Check the calibration on the humidified incubator. Incubator should be humidified and set to 5% CO₂, 95% air and 37⁰C


5. Check the calibration on the humidified incubator. Incubator should be humidified and set to 5% CO₂, 95% air and 37⁰C.

Counting cells by use of a hemacytometer is a convenient and practical method of determining cell numbers in suspension culture or from dispersed monolayer cultures.

The hemacytometer consists of two chambers, each of which is divided into nine 1.0 mm squares. A cover glass is supported 0.1 mm over these squares so that the total volume over each square is 1.0 mm × 0.1 mm or 0.1 mm³, or 10⁴ cm³. Since 1 cm³ is approximately equivalent to 1 mL, the cell concentration per mL will be the average count per square × 10⁴.

Hemacytometer Counts Are Subject to the Following Sources of Error:

• Unequal cell distribution in the sample
• Improper filling of chambers
• Failure to adopt a convention for counting cells in contact with boundary lines or with each other
• Statistical error.

With careful attention to detail, the overall error can be reduced to about 15%. It is assumed that the total volume in the chamber represents a random sample. This will not be a valid assumption unless the suspension consists of individual separated cells. Cell distribution in the hemacytometer chamber depends on the particle number, not particle mass. Thus, cell clumps will distribute the same as single cells and can distort the final result. Unless 90% or more of the cells are free from contact with other cells, the count should be repeated with a new sample. Cells that are difficult to obtain in uniform suspensions, or in which extensive clumping cannot be avoided, may be counted by separating nuclei. This method is more time-consuming than direct counting and is subject to additional error if the population contains multinucleate cells. A sample will not be representative if the cells are permitted to settle before a sample is taken. Always mix the cell suspension thoroughly before sampling.

With a 10X objective and a 10X ocular, one square (1 mm²) will approximately fill the microscope field (the circle on the representation of a hemacytometer grid). The cell suspension should be diluted so that each such square has between 20 and 50 cells (2–5 × 10⁵ cells/mL). A total of 300 to 400 cells should be counted since the counting error is approximated by the square root of the total count. A common convention is to count cells that touch the middle line (of the triple lines) to the left and top of the square, but not to count cells similarly located to the right and bottom (see diagram).

In order to fill the hemacytometer chamber properly by capillary action, the cover slip, chamber, and the pipette used to fill the chamber must be scrupulously clean. The chamber and cover slip are cleaned first with distilled water, then with absolute ethanol, and wiped dry. Hemacytometer counts do not distinguish between living and dead cells. A number of stains are useful to make this distinction. Trypan Blue, among others (erythrosin B, nigrosin), is excluded by the membrane of the viable cells, whereas the nuclei of damaged or dead cells take up the stain. Although this distinction has been questioned, it has the virtue of being simple and giving a good approximation. If more than 20% of the cells are stained, the result is probably significant.

Materials

1. Clean hemacytometer and glass coverslip
2. Pasteur pipettes
3. Hanks’ Balanced Salt Solution (HBSS)
4. Trypan Blue, 0.4% in BSS
5. Microscope
6. Tubes (12 × 75 mm)
7. Hand counter
8. Cell suspension

Procedure

1. Dilute 0.2 mL of Trypan Blue with 0.8 mL of HBSS
2. Place glass coverslip over hemacytometer chamber
3. Transfer 0.5 mL of agitated cell suspension to a 12 × 75mm tube and add 0.5 mL of diluted Trypan Blue
4. With Pasteur pipette, fill both chambers of the hemacytometer (without overflow) by capillary action. Cells will settle in the tube and in the pipette by gravity within a few seconds. Work quickly.
5. Using a microscope with a 10X ocular and a 10X objective, count the cells in each of 10 squares (1 mm 2 each). If over 10% of the cells represent clumps, repeat entire sequence. If fewer than 200 or more than 500 cells are present in the 10 squares, repeat with a more suitable dilution factor.
6. Calculate the number of cells per mL, and total number of cells in the original culture as follows: Cells/mL = average count per square × 10⁴ × dilution factor (i.e., 2, if 0.5 mL of cells plus 0.5 mL of Trypan Blue is used)
   Total cells = cells/mL × total volume of cell preparation from which sample was taken.
7. Repeat count to check reproducibility

NOTE: Not applicable to Clonetics^® and Poietics^® Primary Human or Animal Cells.

In cell culture there is frequently the need to subculture cells. In doing so, cells can be propagated for the purposes of increasing cell numbers or providing cells in a culture vessel suitable to one’s needs. There are a number of ways to remove cells from one culture vessel and pass them to another vessel.

Cells may be removed from surfaces on which they are attached by:

• Mechanical means (scraping)
• Chelating agents, ethylenediaminetetraacetic acid (EDTA)
• Enzymes (trypsin, pronase, collagenase)

Enzymes and chelating agents are often used in combination. Trypsin is an aqueous crude extract prepared from porcine pancreas. It is the most common means used for removal of cells from surfaces and from intact tissue. Trypsin is, to some extent, a misnomer because in addition to trypsin, the preparation contains other proteases, lipases, and carbohydrases. The multitude of digestive enzymes produced by the pancreas would be expected to be found in trypsin preparations. Pure crystalline trypsin can be used, but it is more expensive than crude trypsin and often does not work as well, especially when preparing cells from intact tissue.

The optimum conditions for trypsin activity are a pH range of 7.6–7.8 and a temperature of 37°C. The effect of trypsin is to break down the intracellular matrix that binds cells to each other or to a substrate surface. There are no chemical standards for trypsin activity. We conduct quality assurance tests on trypsin to determine its capacity to detach cells from a substrate surface in a standard time period without damage. This is in addition to the usual tests for sterility.

Trypsin is typically used at concentrations between 0.05% and 0.25%, although some applications may require concentrations outside this range. Versene^®Solution (EDTA) enhances trypsin action, and therefore lowers the required trypsin concentration for effective performance. Concentrated trypsin (2.5%) should be diluted in calcium- and magnesium-free balanced salt solution (BSS) or Dulbecco’s Phosphate Buffered Saline. Dilution in water is not recommended since the solution will be hypotonic and produce cell damage. Dilution in saline alone is also damaging to cells.

Trypsinization Procedure

Cell cultures are normally subcultured (“split”) when the cultures are at or near confluency. As a general rule, the longer the time frame between when confluency is first achieved and subculturing, the longer it will take for the trypsin to act.

1. Decant medium from the culture vessel. Serum inhibits trypsin activity, so complete removal of serumcontaining medium is necessary.

2. Rinse the cell sheet with BSS without calcium and magnesium before addition of Trypsin/Versene^®. The monolayer should be thoroughly covered with BSS. This rinse is instantaneous but the BSS can remain on the cell sheet for up to 4 hours, if desired.

3. Pour off rinse medium. Trypsin/Versene^® is to be added to each vessel as follows:
75 cm²→2.5 mL to 5.0 mL
150 cm²→ 5.0 mL to 10.0 mL
850 cm² roller bottle→ 10.0 mL to 20.0 mL

4. Cover the monolayer thoroughly with Trypsin/Versene^®. Since different lots of Trypsin/Versene^® may vary in strength, it is acceptable to monitor the trypsinization process at room temperature for the first 30 seconds.This will ensure that the trypsinization process is not occurring too rapidly.

5. The culture vessel should then be moderately hit against the palm of the hand to see if the cells are being dislodged. Hold the vessel up to a light in a vertical position and look for signs of the cell sheet sloughing off of the surface. If the entire monolayer is dislodged, proceed to step #6. If not, incubate at 37°C and observe the vessel every minute for dissociation. The culture vessel should again be hit against the palm of the hand to ensure all cells have been dislodged. Remove culture vessel from the incubator.

6. Immediately transfer dissociated cells to a vessel containing medium supplemented with 10% serum. All of the cells should be removed. Aspirate the medium plus cells with a pipette onto the surface to remove all remaining cells. It is essential that this aspiration be done as completely as possible with a small bore pipette so as to obtain individual, dispersed cells. If the cells are not separated, the new culture will contain numerous microcolonies. Cells added to the vessel should be stirred with a magnetic stir bar at a speed that avoids vortexing (approximately 100–200 rpm), or agitated frequently. It is important at this point to add medium containing serum at least 10 times the volume of Trypsin/Versene^® used. This will ensure that the digestive agent is inhibited.

7. Add suffcient fresh medium to the aspirated suspension so that the total volume will cover the surface of two culture vessels, each having the same surface area as the original culture vessel (or use a single culture vessel having twice the floor area of the original vessel). This is a 1:2 split. Other split ratios can be used for vigorously growing cell populations.

8. Incubate the culture vessel (or vessels) at 37°C.

9. When making 1:2 splits, subculturing of human diploid cell cultures should be done on a rigid 3 or 4 day schedule, at which time confluent sheets should occur. Surplus cells can be frozen and stored in liquid nitrogen.

10. Populations that can be cultivated indefnitely can be subcultured serially each time confluency is reached. If the culture is a diploid population with a finite doubling capacity, increase the population doubling level (PDL) number by one at each 1:2 subculturing (split).

11. By making repeated 1:2 splits (twice a week) it can be seen that the number of culture vessels can be built up geo metrically (1, 2, 4, 8, 16, 32, 64, etc.) in a short period of time for the production of large quantities of cells for various purposes.

12. Although the line will be eventually lost as a continuously passaged line, it will not be lost for use since frozen ampoules can be obtained at almost every passage and thus the line can be restored to continuous passage again, up to a cumulative total of about 50 population doublings. By repeating this procedure, the number of cells that can be obtained is almost unlimited for all practical purposes.

13. A human embryonic diploid line has an in vitro life span of about fifty 1:2 subcultivations, or population doublings, at which time the cells will cease to divide and eventually die.

14. Using split ratios higher than 1:2 results in the advantage of minimizing the number of manipulations necessary to obtain a specific cell density or number of culture vessels. Since human embryonic diploid cell lines pass through a finite number of population doublings in vitro, it is necessary to keep a record of the number of population doublings that have elapsed. With a 1:2 split ratio this is achieved by simply adding “1” to each split since this ratio yields one population doubling. Larger split ratios can be used. For example, a split ratio of 1:4 would yield 2 doublings per 1:4 split; a 1:8 split ratio would yield 3 doublings per 1:8 split. In order to have knowledge of the approach of cessation, it is essential to keep records of the number of elapsed population doublings.

15. Since human diploid cells multiply by fission, the increase in population may be expressed per cell as follows:

References

1. Hayflick, L. and Moorhead, P.S. (1961) The serial cultivation of human diploid cell strains. Exp. Cell Research 25:585.
2. Hayflick, L. (1970) Aging under glass. Exp. Geront. 5:291.
3. Hayflick, L. (1965) The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37:614.
4. Hayflick, L. (1968) Human cells and aging. Scientific American 218:32.
5. Hayflick, L. (1973) Subculturing human diploid _broblast cultures. Methods and Applications of Tissue Culture Eds. Patterson, M.K. and Kruse, P.F., Academic Press, N.Y.
6. Freshney, R.I. (1983) Culture of Animal Cells: A Manual of Basic Technique. Alan R. Liss, Inc., New York

Several factors, or a combination of factors, can contribute to low cell count and low cell viability. If cell yield or viability is unsatisfactory, use the following information to increase the success rate of future cultures.

Improving Cell Yield

If your cell yield is low (less than 50%), determine the cause(s) and possible solution(s) using the table below. Then subculture one or more flasks applying the appropriate solution(s).

Improving Cell Viability

If your cell viability is low (less than 50%), determine the possible cause(s) and solution(s) using the table below. Then subculture one more flask applying the appropriate solution(s).