Cell Growth and Propagation

Animal Cell Culture Guide Cell Growth and Propagation

Table of Contents

Passage Number and Population Doubling Level

Primary cultures are generally subcultured at a 1:2 ratio (they are split in half with each passage). Most continuous cell lines replicate at higher rates and are subcultured at a much higher split ratio. Passage number is generally the number of times the cells have been subcultured into a new vessel. For diploid cultures, passage number is roughly equal to the number of population doublings (or population doubling level, PDL) since the culture was started. This is not the case for continuous cell lines as they are passaged at higher split ratios. Consequently the PDL is not determined for continuous cell lines. In most cases, the PDL is an estimate as it does not account for any cells that were lost due to death from necrosis or apoptosis or cells which are nearing senescence and no longer divide. Calculate the population doubling level with the following formula:

PDL = 3.32 (log Xe - log Xb) + S

Xb is the cell number at the beginning of the incubation time.
Xe is the cell number at the end of the incubation time.
S is the starting PDL.

Growth curve for cells grown in culture

Figure 1. Growth curve for cells grown in culture. Cells should be subcultured while still in the exponential phase.

Calculate the population doubling time, or the time required for a culture to double in number, with the following formula:

DT=T ln2/ln(Xe/Xb)

T is the incubation time in any units.
Xb is the cell number at the beginning of the incubation time.
Xe is the cell number at the end of the incubation time. (See: NOTE)

ATCC tracks the PDL and passage number for many adherent cell lines when the depositor supplies this information at the time of deposit. See the Product Information Sheet for the specific cell line for the passage number and/or PDL as part of the batch-specific information supplied.

NOTE:

Cells grow at different rates in each of the different phases of the growth cycle and the calculated doubling time may be a composite of growth during more than one of these phases. Growth during exponential growth or log phase is fairly constant and reproducible for a given set of growth conditions.

Adapting to a New Medium or Serum

To ensure that the characteristics of your cell line remain constant, maintain your cells in the same medium, serum, and supplements with the same subculturing regimen used to establish the culture. Any change to the culturing conditions has the potential to change the characteristics of the cell line.

Be particularly cautious when working with a new cell line as media formulations vary among suppliers, even for media with similar or identical names. Read descriptions, formulations, and labels carefully to ensure that the appropriate medium is used or the cell line may be inadvertently adapted to a new medium. All ATCC cell lines come with information on their growth medium. In most cases, the recommended medium and serum can be purchased from ATCC along with the cell line.

Use the following procedure to adapt a cell line to a new medium:

  1. Subculture the line at a 1:2 split ratio (split the culture in half) into two vessels. Maintain one with the original medium and continue to subculture these cells for the entire adaptation process. Use a 1:1 mix of the original and new medium in the second vessel. The culture grown in the original medium serves as a reference point as well as a safeguard in case the adapting cells do not survive the process. The low split ratio helps mitigate the stress associated with subculturing as well as with the new medium.
  2. Monitor cell growth in the two media and watch for any change in morphology or growth rate. If they are identical, subculture the adapting cells at the next passage with a 1:2 split ratio in a 1:3 medium mix (25% original, 75% new).
  3. Monitor the growth rate and morphology of the original and adapting cultures. At the next passage, split the adapting cultures 1:2 in a 1:7 medium mix (12.5% original, 87.5% new).
  4. Monitor the growth rate and morphology of the original and adapting cultures. If the cells are identical, then at the next passage split the adapting cells 1:2 in 100% new medium. At this point, the culture should be adapted to the new medium.

To confirm complete adaptation to the new medium, perform functional tests on cells derived from the original and new medium. If at any point in the process the adapting culture fails to perform as well as the reference culture, then allow the adapting culture more time and a few more passages in their current medium mix (e.g., 1:3, 1:7) until they match the reference cells.

The same approach can be used to adapt cells to serum-free medium; simply decrease the serum level in the medium by half with each passage until a 0.06% (or lower) serum level is reached. At this point, the cells can be maintained in serum-free medium. If at any point the growth rate declines, then the serum level should be increased to the level where the cells grew normally. In this procedure, start with the “serum-free” medium supplemented with serum so that only the level of serum changes with each passage.

Temperature

Most animal cell lines require 37°C for optimum growth. Insect and amphibian cells require lower temperatures (such as 28°C) as do some animal cell lines which are temperature sensitive for their phenotypic characteristics. While cultured cells can withstand considerable drops in temperature and most can survive for several days at 4°C, few can tolerate even a few hours at more than 2°C above their optimal temperature. (See: NOTE)

NOTE:

Regularly calibrate the temperature control system of incubators and use an alarm system when possible to warn against temperature increases above the optimum setting.

Examination of Cultures

Observe the morphology and viability of cultures regularly and carefully. Examine the medium in the vessel for macroscopic evidence of microbial contamination. This includes unusual pH shifts (yellow or purple color from the phenol red), turbidity, or particles. Also, look for small fungal colonies that float at the medium-air interface. Specifically check around the edges of the vessel as these may not be readily visible through the microscope.

With an inverted microscope at low power (40×), check the medium for evidence of microbial contamination and the morphology of the cells. Bacterial contamination will appear as small, shimmering black dots within the spaces between the cells. Yeast contamination will appear as rounded or budding particles, while fungi will have thin filamentous mycelia. For nonadherent cells grown in flasks, such as hybridomas, this is a simple matter of viewing the flask directly on the microscope. For cells grown in spinner flasks or bioreactors, a sample of the cell suspension will need to be withdrawn and loaded into a microscope slide or hemocytometer for observation.

Most adherent cells should be attached firmly to the surface. In some cases, healthy cells will round up and detach somewhat during mitosis and appear very refractile. Following mitosis, they will reattach. Some of these will float free if the culture vessel is physically disturbed. In contrast, dead cells often round up and detach from the monolayer and appear smaller and darker (not refractile) than healthy cells. Cells in suspension culture grow either as single cells or as clusters of cells. Viable cells appear round and refractile whereas dead cells appear smaller and darker. Occasionally, a portion of the cells will attach and grow on the side of the culture vessel and appear round or flattened. The percentage of attached cells varies with the culture conditions and the cell density. Cellular debris may also be observed in healthy cell populations. Some cell lines grow as mixed adherent and suspension cultures.

As a reference, photomicrographs for some ATCC cell lines are available on the respective product details page on the website. Cells are shown at two different densities: just after subculturing (low) and just before they need to be subcultured (high).

In addition to daily examinations, periodically test a sample of the culture for the presence of fungi, bacteria, and mycoplasma. There are several methods that can be used to check for these contaminants. For additional information, refer to the section on microbial contamination.

CCL-61 at high density

ATCC® CCL-61™ at high density.

CCL-61 at low density

ATCC® CCL-61™ at low density.

Cell Counting

Cell counts are necessary in order to establish or monitor growth rates as well as to set up new cultures with known cell numbers. Hemocytometers (also spelled hemacytometers) are commonly used to estimate cell number and determine cell viability. A hemocytometer is a fairly thick glass slide with two counting chambers, one on each side. Each counting chamber has a mirrored surface with a 3 × 3 mm grid of 9 counting squares. (See Figure 2) The chambers have raised sides that will hold a coverslip exactly 0.1 mm above the chamber floor. Each of the 9 counting squares holds a volume of 0.0001 mL.

Hemocytometer grid with Neubauer ruling

Figure 2. Hemocytometer grid with Neubauer ruling.

Count cells as follows: 

  1. Clean, thoroughly dry, and assemble the hemocytometer with the cover slip. 
  2. Transfer a small amount of cell suspension to the edge of each of the two counting chambers. Allow the cell suspension to be drawn into the counting chamber by capillary action. 
  3. Place the hemocytometer under an inverted microscope and view the cells at 100× magnification. 
  4. Focus on the quadrants, labeled 1, 2, 3, and 4 in Figure 2. 
  5. Record the number of cells in each section. Average the number of cells, and multiply by the dilution factor. If the cells have not been diluted, this factor will be 104 cells/mL. Any dilution of the sample after it was removed from the cell suspension, such as using vital stain, needs to be included in the calculation. 

For example, if the four counts are 60, 66, 69, and 75, the concentration would be 68 × 104 cells/mL for the sample that was loaded into the hemocytometer. For best results, adjust the concentration of the suspension so that 50 to 100 cells are in each of the four sections. Most cultures will grow at an initial inoculum cell concentration ranging from 103 to 104 cells/cm2. Faster growing cultures are usually set up at lower concentrations. Some cultures do not grow well unless a minimum concentration of cells is initially added; see the Product Sheet for details.

Cell Viability

Viability assays measure the number of viable cells in a population. When combined with the total number of cells, the number of viable cells provides an accurate indication of the health of the cell culture. The most common and rapid methods rely upon the integrity of the cell membrane as an indicator of cell viability. Both trypan blue and erythrosin B stains are actively excluded by viable cells but are taken up and retained by dead cells, which lack an intact membrane.

While both stains are used in the same way, ATCC recommends erythrosin B in place of trypan blue for hematopoetic cells. When using trypan blue, incubate cells for two to five minutes prior to use. If not counted within this time, the cells will begin to deteriorate and take up the dye. Erythrosin B does not require an incubation period.

Erythrosin B stain generates more accurate results with fewer false negatives and false positives. Erythrosin B stain solution provides a clear background and does not bind serum proteins as avidly as trypan blue, making stained cells more distinct and easier to identify. Also, microbial contamination or precipitates in the cell culture are more readily apparent. Finally, trypan blue is toxic and a potential carcinogen.

For either stain use the following directions:

  1. Mix the cell suspension 1:1 with a 0.1% erythrosin B solution in PBS or 0.4% trypan blue solution in PBS.  
  2. Load the cells in the erythrosin B solution directly into a clean, dry hemocytometer, but incubate the trypan blue solution for two to five minutes before loading. 
  3. Nonviable cells will be stained red (erythrosin B) or dark blue (trypan blue). Cell viability is calculated as the number of unstained or viable cells divided by the total number of cells and expressed as a percentage.

Subculturing Monolayer Cells

Anchorage-dependent cell lines growing in monolayers need to be subcultured at regular intervals to maintain them in exponential growth. When the cells are near the end of exponential growth (roughly 70% to 90% confluent), they are ready to be subcultured. The subculturing procedure, including recommended split-ratios and medium replenishment (feeding) schedules, for each ATCC cell line is provided on the Product Information Sheet.

Subcultivation of monolayers involves the breakage of both intercellular and intracellular cell-to-surface bonds. For some cells that are loosely attached, a sharp blow with the palm of your hand against the side of the flask can dislodge them. Many require the digestion of their protein attachment bonds with proteolytic enzymes such as trypsin/EDTA. For some cell lines mechanical forces such as scraping to dislodge the cells is preferred. After the cells have been dissociated and dispersed into a single-cell suspension, they are diluted to the appropriate concentration and transferred into fresh culture vessels with the appropriate growth medium where they will reattach, grow and divide.

The procedure below is appropriate for most adherent cell lines. However, since every cell line is unique, incubation times and temperature, number of washes or the solution formulations may vary. In all cases, continually observe the cells with a microscope during the dissociation process to prevent damage by the dissociation solution. The amounts used in this procedure are for a 75-cm2 flask. Adjust volumes as appropriate for different sized vessels.

Monolayer Subculturing

  1. Bring the trypsin-EDTA solution (ATCC® 30-2101™), balanced salt solution [Dulbecco’s Phosphate Buffered Saline without calcium or magnesium, ATCC® 30-2200™], and complete growth medium to the appropriate temperature for the cell line. In most cases, this is the temperature used to grow the cells (usually 37°C). For some sensitive cells, the trypsin-EDTA solution may need to be used at room temperature or 4°C. 
  2. Remove and discard the cell culture medium from the flask. 
  3. Rinse the cell monolayer with Dulbecco’s PBS without calcium or magnesium and remove. 
  4. Add 2 mL to 3 mL of the trypsin-EDTA solution and incubate at the appropriate temperature. Check the progress of cell dissociation by microscopy. To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for them to detach. 
  5. Once the cells appear to be detached (5 to 15 minutes for most cell lines; they will appear rounded and refractile under the microscope), add 6 to 8 mL of complete growth medium with a pipette to the cell suspension to inactivate the trypsin. Gently wash any remaining cells from the growth surface of the flask. Check the cells with the microscope to be sure that most (>95%) are single cells. If cell clusters are apparent, continue to disperse the cells with gentle pipetting. (See: NOTE)
  6. Add 12 mL to 15 mL of fresh culture medium to a new flask and equilibrate this medium to the appropriate pH and temperature. 
  7. Count the cells in suspension and determine their viability or simply divide them according to a routine split ratio and dispense them into the medium of the newly prepared flask. Do not add a concentrated cell suspension to an empty culture vessel as this can result in uneven cell attachment and growth. 
  8. Place the flask back into the incubator. Examine the culture the following day to ensure the cells have reattached and are actively growing. Change the medium as needed; for most actively growing cultures two to three times per week is typical.

NOTE:

For serum-free or low-serum medium, remove the trypsin-EDTA solution by gentle centrifugation (10 minutes at 125 × g) and then resuspend the cells in 6 mL to 8 mL of fresh medium. In some cases, the trypsin will need to be inactivated with a trypsin inhibitor.

Troubleshooting Monolayer Cell Subculturing

Cells are difficult to remove.
  • Inhibitors in the medium (such as serum) have inactivated the dissociating agents. Rinse the cell monolayer twice with Dulbecco’s PBS without calcium or magnesium before adding the dissociating solution. Or use the trypsin-EDTA solution in place of the Dulbecco’s PBS for the first rinse of the monolayer. 
  • The dissociating solution was too weak. Use higher enzyme concentrations, higher EDTA concentrations, or different and/or additional enzymes (e.g., dispase, collagenase). Or incubate the cells at 37°C to increase the activity of the dissociating solution. 
  • The cells have been confluent for too long and the cell-to-cell junctions are so tight they prevented the dissociation agents from reaching the substrate-cell interface. In the future, subculture the cells before they become confluent. 

Cells form clumps after dissociation. 

  • The dissociation procedure was too harsh and genomic DNA was released from lysed cells. Either the pipetting was too vigorous or the dissociating solution was too strong or too toxic (i.e., the pH or osmolality of the buffer was incorrect). Add a drop of sterile DNAse (1 mg/mL in water) to the cell suspension to break down the DNA strands. In the future, treat the cells more gently during pipetting, shorten the incubation period, use a weaker dissociation solution (lower the enzyme concentration or remove the EDTA), or incubate at a lower temperature. 
  • The cells aggregated before dilution and dispersion into the medium. Hold the cell suspension on ice if there will be a delay between removing the cells from the flask growth surface and seeding a new flask. 
  • The cells were centrifuged too hard or too long when removing excess dissociation solution. Be sure to use gentle centrifugation (10 minutes at 125 × g). 

Cells have difficulty reattaching to the flask. 

  • The dissociation procedure was too long and stripped away necessary attachment proteins from the cell membrane.
  • Insufficient serum or attachment factors were present in the medium (common with serum-free medium). Add attachment factors to the medium and/or use a protein-coated flask (collagen, poly-Llysine, fibronectin, gelatin, etc.). 
  • The dissociating solution was not inactivated or removed by centrifugation. Add additional serum or specific enzyme inhibitors (e.g., soybean trypsin inhibitor) to the neutralizing medium or centrifuge (5 minutes at 125 × g) the cells down from the dissociation solution and resuspend in fresh medium. 

Viability is lower than expected. 

  • The dissociating procedure was too harsh. 
  • The pH or osmolality of the balanced salt solution containing the dissociation agents is incorrect. Check these directly and/or use a fresh bottle. 
  • The dispersed cell suspension was left too long at too high a cell concentration prior to reseeding. Keep the cells on ice. 
  • The medium was faulty. Use the recommended formulation and make sure it contains all of the required additives.

Suspension Cells

Most primary cultures, finite cell lines, and continuous cell lines are anchorage dependent and thus grow in monolayers attached to a surface. Other cells, particularly those derived from hematopoietic or certain tumor tissues, are anchorage independent and grow in suspension.

Cell propagation in suspension has several advantages over propagation in monolayer. Subculturing is a simple matter of dilution. There is little or no growth lag after splitting a suspension culture as there is with a monolayer culture, because there is none of the trauma associated with proteolytic enzyme dispersal. Suspension cultures require less lab space per cell yield, and scale-up is straightforward. Cells can be propagated in bioreactors similar to the fermentors used for yeast or bacteria cultures. 

Depending upon the cell type, suspension cultures are seeded at densities from 2 × 104 to 5 × 105 viable cells/mL and can attain densities of 2 × 106 cell/mL. If cells are seeded at too low a density they will go through a lag phase of growth, grow very slowly, or die out completely. If cell densities are allowed to become too high, the cells may exhaust the nutrients in the medium and die abruptly. Recommended seeding and subculturing densities, media replenishment (feeding) schedules, and medium formulations for each ATCC cell line are provided on the Product Sheet as well as in the catalog description on the website.

Suspension Cell Subculturing

  1. Bring the complete growth medium to the appropriate temperature (usually 37°C) in a water bath. 
  2. Thoroughly mix the cell/medium suspension; use a pipette to suspend cells grown in stationary flasks. Remove a small amount of the cell suspension to determine the cell density and viability using a hemocytometer and vital stain (Figure 2). 
  3. Calculate the volume of cells required to re-seed the flask at the minimum density for that cell line, taking into consideration the amount of fresh medium that will be used. 
  4. Add the appropriate volume of medium to the culture vessel and then add the cell suspension. Do not add the concentrated cell suspension to an empty flask. The same culture vessel can be reused, but the chances of contamination increase with each reseeding due to the buildup of small spills of medium on the flask opening. 
  5. If necessary, “gas” the atmosphere of the flask with sterile-filtered CO2, seal the flask, and then incubate at the appropriate temperature. 

It is generally not necessary to completely change the medium unless the cells attain a very high density or the medium has an acidic pH (yellow in color from the phenol red). To completely replace the medium, centrifuge the cells gently (10 minutes at 125 × g), decant the medium, and then resuspend the cells in fresh medium at the lower seeding density.

Troubleshooting Suspension Cell Subculturing

Viability is lower than expected. 
  • The cell suspension was left too long at too high a cell concentration prior to subculture. In this case, the medium will have a low pH and be yellow in color. Completely change the medium by gently centrifuging the cells and resuspend in fresh medium at the lower seeding density. 
  • The cell suspension was diluted below the recommended cell density range. Recover the cells by centrifugation and resuspend in fresh medium at the appropriate cell density. 
  • The harvesting procedure was too harsh (pipetting too vigorous, cells were centrifuged too hard or too long, cells damaged during scraping or banging). 
  • The medium was faulty. Use the recommended formulation and make sure it contains all of the required additives.

Adapting a Monolayer Cell Line to Grow in Suspension

Some cell lines such as L-929 (ATCC® CCL-1™), HeLa (ATCC® CCL-2™) and BHK-21 (ATCC® CCL-10™) can be adapted to grow in suspension. With time, a population of cells can be selected that does not selfaggregate or adhere to a growth surface as readily as the parental line. However, the newly selected line may have lost or acquired characteristics that are different from the original cell population. In most cases it will be necessary to maintain the culture in suspension with mechanical stirring. Keep in mind that most anchorage-dependent cells will grow in suspension only with the use of microcarrier beads. 

The procedure below was developed for BHK-21 cells,4 but can be used as a starting point for most cell lines.

  1.  Dissociate the cell monolayer using standard procedures. Centrifuge and resuspend the cell suspension in an appropriate spinner medium such as Joklik’s modified Eagle’s Minimum Essential Medium (EMEM). Spinner media have reduced levels of calcium and magnesium. 
  2. Count the cell suspension, and then seed two or more spinner flasks with 5 × 105 cells/mL. This density may need to be adjusted for your particular cell line. The sides of the culture flask may need to be siliconized to prevent the cells from sticking to the glass. 
  3. Observe the cultures daily. Remove samples and record the number of viable cells for each flask. 
  4. Every three days, collect the cells growing in suspension by centrifugation (10 minutes at 125 × g). Count, and re-seed a fresh flask with fresh medium at 2.5 × 105 cells/mL. Depending on how well (or not) the cells adapt to growth in suspension, they may need to be combined with cells from different flasks to achieve the necessary cell density. 
  5. If there is a significant amount of cells attached to the walls of the culture vessel, particularly at the surface of the medium, remove them with trypsin-EDTA and discard them. If the cells in suspension are badly clumped, they can be dispersed with the trypsin-EDTA solution, collected by centrifugation, and then re-seeded into the flask as the appropriate density. This treatment may be necessary for the first few subcultures. 
  6. Continue to monitor the cells and subculture them every three days. Over time, they should adapt to growth in suspension and attain a constant growth rate. 

A complete growth medium consists of a basal cell culture medium supplemented with ingredients such as sera, growth factors, trace elements, and hormones. There are numerous formulations ranging from simple, basic mixtures containing the minimum requirements for growing many cell lines to complex serum-free mixtures specific for growing a single fastidious cell line. The choice of a medium for a particular cell line is somewhat empirical. Many medium formulations are available commercially in powder or liquid form. (See: NOTE) 

ATCC lists complete medium formulations, plus all handling and passage information, for all ATCC cell lines both in the online catalog description and on the Product Sheet that accompanies the cell line when shipped. Additionally, ATCC offers a full line of media, sera, and reagents for culturing cells. These are the same reagents used at ATCC for cell growth and propagation. See the Complete Growth Media section for descriptions of ATCC cell culture products.

NOTE:

Formulations can vary widely among suppliers, even for media with similar or identical names. Be sure to read catalog descriptions, formulations, and medium labels carefully to ensure that the appropriate medium is used. For best results start cell cultures in the same medium used and distributed by ATCC (listed on the Product Sheet).