Common Cell Culture Problems: Poor Cell Growth

Cell growth: an overview

Phases of Cell GrowthEnsuring adequate cell growth is a critical part of collecting accurate data with cell cultures. Cells can be cultured in suspension, or as a monolayer that attaches to cultureware, such as a flask, dish or multiwell plate. The culture method is determined by the cells’ endogenous phenotype and tissue of origin — so, cells derived from blood generally grow in suspension, while those derived from solid tissues typically grow in monolayers. Co-culturing a mixed population that contains both attached cells as well as phenotypes in suspension is also possible.

For cells that grow as a monolayer, confluence is defined as the percentage of the culture vessel surface area that appears covered by a layer of cells when observed by microscopy. For example, 50% confluency means half of the surface of the culture dish, flask, etc. is covered in cells. For suspension cells, growth progress is generally measured by recommended cell density for the line or type – but ‘confluency’ may be assessed by increasing turbidity and stable medium color.

Cell growth in culture generally occurs in four phases that, when graphed as the log of the cell count over time, generates a sigmoidal (S-shaped) curve, although the amount of time spent in each phase differs between individual cell lines and cultures.

Lag phase – Cells are acclimating to culture conditions and do not divide during this phase. Cell attachment generally occurs within 24 hours after initiation of the culture. The total length of this phase is determined by the growth phase and seeding density of the cells that were used to start the culture.

Log (Logarithmic) Growth Phase – Cells are actively dividing during this phase, and this is the best time for assessing population growth as well as for general data collection. Late in the log phase is the best time to passage (subculture) cells, before overcrowding can lead to cell stress.

Plateau (Stationary) Phase – Cell growth in this phase slows as cells approach 100% confluence, and fewer than one tenth of cells are in the active cell cycle. Cells are most susceptible to injury in this phase; careful observation is therefore needed to ensure cells are passaged prior to or at the outset of this phase.

Decline Phase – As a natural part of the cell cycle, the population of live cells declines as cell death predominates in this phase.

Assessing Cell Growth

Scepter Cell CounterAccurate cell counting is of paramount importance when assessing cell growth in cultures, as faulty counts can lead to mistaken conclusions about cell health. Cell counts can be determined using a hemocytometer. However, automated cell counters that utilize the Coulter principle where cells flow, one by one, through an aperture within an electrical sensor, yield the most precise cell counts.

Common Causes of and Solutions to Poor Cell Growth

Problem/Cause Solution
No or few viable cells after thawing from stock
Stock culture was of poor quality Ensure starting culture used to generate stock has been properly identified, and is healthy, free of microbial contamination, and late in the log phase of growth (80-90% confluent).
Harvest cells gently to prevent damage.
Stock was frozen incorrectly When freezing cells in liquid nitrogen, ensure that the concentration of cells, media and other reagents  as well as the freezing protocol follows supplier’s recommendations. In general, freezing should occur slowly, at approximately 1 to 3°C per minute to minimize ice crystal formation.
Use an electronic programmable or a mechanical freezing unit to ensure a consistent and appropriate rate of freezing.
Select the most appropriate cryoprotectant for the cells. If using glycerol, do not store it in light, as light exposure converts glycerol to cytotoxic acrolein.
Stock was stored incorrectly Stock should be maintained at temperatures below -130°C at all times, ideally in liquid nitrogen, to ensure maximum viability. Storage in the vapor phase above the liquid nitrogen is preferable to storage in the liquid itself due to the risk of vial explosion during thawing if liquid nitrogen has leaked into the cryovial.
Stock was thawed incorrectly Follow the supplier’s recommended protocol when thawing cells. In general, cells should be thawed rapidly. Use pre-warmed media.
Remove media containing cryoprotectant as soon as possible to prevent a reduction in viability.
Ensure cells are handled with care. Do not vortex or centrifuge at high speeds as cells are particularly vulnerable to damage following cryopreservation.
Poor cell attachment after thawing from stock or passaging
Static electricity buildup on plastic culture vessels (especially problematic when humidity is low) Increase room humidity.
Wipe outside of culture vessels with (disinfected) damp towel.
Use antistatic device.
Inadequate mixing of cells, media, and/or other reagents Ensure solution of cells and all reagents are adequately mixed.
Rotation of roller bottles is too fast Rotate bottles at a slower speed.
Slow cell growth
Cells have been passaged too many times Obtain a new stock of cells that has been subcultured fewer times.
Cells were too confluent when harvested Start with a new stock of cells and harvest in the log phase of growth, prior to reaching 100% confluence.
Media, serum, buffers, etc. are of poor quality or incorrectly formulated Discard current reagents and use new lots.
CO2 levels do not match what is needed by the bicarbonate-based buffering system of the media being used, leading to inadequate control of pH Ensure that incubator CO2 levels match what is required by the buffering system. In general, the higher the concentration of bicarbonate in the buffer, the higher the concentration of CO2 required in the incubator gas mix.
Microbial (especially mycoplasma) contamination See Problems with Contamination for detailed best practices for preventing and eliminating microbial contamination.
Exposure of cultures to fluorescent light causes light sensitive media components (riboflavin, tryptophan, and HEPES) to be converted to cytotoxic free radicals and H2O2 Store cells and media in the dark, away from fluorescent light.
Inaccurate cell counting method leads to presumed poor cell growth   Make sure that counting sample is adequately mixed to avoid local variations in cell density that can affect the accuracy of cell counts.
Recheck all calculations used to determine counts using manual method with hemacytometer, or consider automated cell counting.
Uneven cell growth
Uneven cell growth within a vessel indicates inadequate mixing of cells and/or media Ensure cell mixture and all reagents are properly mixed via gentle vortex or pipetting to avoid local concentration variations.
Uneven cell growth between presumably identical vessels grown at the same time may be due to temperature variations within the incubator Avoid stacking culture vessels on top of one another when possible, as vessels on the bottom are closest to the metal shelves and may warm the fastest.
When vessels stored at the front of an incubator exhibit less growth than those at the back, take care to keep incubator doors closed as much as possible and relocate vessels toward the back.
Specific growth patterns:
Uneven evaporation in incubator causes reductions in media volume and poor growth in outer wells of multi-well plate Keep water reservoirs full and humidify CO2 and other incoming gases. If necessary, create a map of evaporation “hot spots” in the incubator to avoid in subsequent experiments.
Incubator vibration causes concentric rings (in dishes), parallel strips or irregular pattern (in flasks) Place incubators on sturdy surfaces not shared with other equipment in low-traffic areas. Keep other motorized equipment as far away as possible.
Incubator temperatures that are slightly too high or slightly too low can lead to patterns of dots or cross-hatching, respectively, due the metal of the shelves providing more heat than surrounding areas. Dots of higher cell density suggest better growth of cells not in contact with the metal; cross-hatching suggests cells prefer warmer areas over metal. Adjust incubator temperatures as needed and take care to minimize time that incubators are opened, which leads to heat loss. Dummy vessels filled with medium can also be used to insulate against contact with shelves.
A non-level incubator shelf can cause uneven cell density Level shelves prior to use, and after incubator cleaning and servicing.
Presence of air bubbles in media leads to clear bands (in roller bottles), spots without cell growth (in flasks and dishes) Pour or pipette media carefully to avoid air bubbles.
Condensation leads to vertical streaking (in roller bottles and flasks) Do not allow vessels to be exposed to the cooler temperatures outside the incubator longer than necessary. Remove vessels from the incubator immediately before use.
Too little media volume produces ring of increased cell density along side walls of dishes and flasks. This is due to the curve of the meniscus creating a media-poor area in the center of the flask or well. Add adequate amounts of media to culture vessels. A general guideline is 0.2-0.3 mL of media per cm2 of growth area

General tips and techniques for preventing and eliminating cell growth problems

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In addition to following good sterility practices to prevent microbial and chemical contamination, the keys to early diagnosis and treatment of cell growth problems are frequent observation of cultures and detailed recordkeeping. It’s also important to consider whether the most efficient way forward is a detailed and time-consuming search for the culprit(s) of poor cell growth, or whether it is better to simply start fresh with all new reagents, stock, and supplies.

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Observe with care

In order to fix a cell growth problem, one must first realize that a problem exists. Many growth problems are subtle, so regular, detailed observation of not just some, but all, culture vessels in an experiment is necessary. Quick assessment under the microscope may not be sufficient; many specific patterns of growth will not be apparent until cells are fixed and stained. Regularly examine media and other reagents to look for contamination.

Record with accuracy

When trying to diagnose and troubleshoot poor cell growth, having good notes about all aspects of the culture can save time, money, and uniquely modified cells such as knockout lines. For example, normalizing the cell density and recording the passage number of stock cells is vital to knowing whether or not the stock is likely to be the source of the problem. Similarly, before concluding that a particular lot of media is to blame, one must first be sure which culture dishes received that lot.

Important information to note includes the source, proof of identity and lack of contamination, passage number, cell density, and freezing and storage protocols of all stock vials. The ingredients (and concentrations), lot numbers, and dates of first opening/use for all reagents used should also be recorded. Lot numbers of all culture vessels should also be noted. Detailed protocols for all aspects of cell culture should be standardized and followed. Accurate and detailed labeling of all stock vials and culture vessels is essential. Pay attention to small details such as when incubators are cleaned and serviced, when CO2 tanks are replaced, when the culture room was last screened for mycoplasma, and note anything out of the ordinary.

Know when to cut your losses

Sometimes finding the exact source of the problem is not as important as fixing it, and it can therefore be more cost- and time-efficient to correct multiple potential causes of poor growth at once rather than attempting to isolate the exact issue. For example, it may make sense to start fresh with a new stock vial of cells and all new media, sera, buffers, etc. than to try new lots of each independently until the culprit is discovered.