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Cell Culturing Conditions

If the media formulation contains:

  • NaHCO3 (g/L) <1.5, it needs CO2 at 4%; 
  • NaHCO3 (g/L) 1.5–2.2, it needs CO2 at 5%; 
  • NaHCO3 (g/L) 2.2–3.4, it needs CO2 at 7%;
  • NaHCO3 (g/L) >3.5, it needs CO2 at 10% 

*There are some exceptions. Gibco™ DMEM has always been made according to Dulbecco’s original published formulation, with 3.7g/L sodium bicarbonate. Customers have been using this medium in CO2 incubators ranging from 5–10% CO2 for decades, usually with no trouble maintaining physiological pH; this also depends on the cell type. Once cells are growing, the pH will drop (due to metabolic accumulation of lactic acid).

There are a number of events that can contribute to this:

  1. Incorrect CO2 levels—monitor the level of CO2 manually with a Fyrite kit, available from Bacharach. Check if the manual readings concur with the readings displayed on the incubator. If the incubator has a trace readout, check the printout for fluctuations in CO2 level. Check the settings to insure that CO2 levels are set at appropriate levels for your cell line (usually between 5 and 10%). Check line connections frequently for leaks. Avoid frequent opening and closing of incubator doors.
  2. Temperature fluctuations in the incubator—Monitor the temperature of incubator with a good thermometer inside the incubator.
  3. Fungizone or other preventive antibiotics/antimycotics are present at toxic concentrations—use at recommended levels.
  4. Humidity is incorrect—check the water level in the water pan. Humidity is vital to appropriate gas exchange for many types of cells and media (i.e., appropriate CO2 levels are largely irrelevant for most cultures if the humidity level is not high enough).
  5. Incorrect osmotic pressure in medium—check osmolality of complete medium. Most mammalian cells can tolerate an osmolality of 260 to 350 mOsm/kg. Additions of reagents such as HEPES and drugs may affect osmolality.
  6. Contamination by microorganisms—bacterial and fungal contaminations are usually easily visible; symptoms of mycoplasma contamination are more subtle, and careful monitoring of culture morphology and regular testing are necessary to detect this type of contamination.
  7. Inappropriate medium is being used—double-check that the medium used is appropriate for your cell type and culture application. For example, ensure that the medium being used for serum-free culture is actually designed for serum-free culture; make sure that appropriate selective drugs are used at appropriate levels; check the expiration dates for the reagents being used; and store medium at appropriate temperatures in the dark.

No. Suspension cultures will quickly become oxygen limited and will only reach cell densities of 1 to 1.5 x 10E6 cells/mL. Recombinant product expression will also be greatly reduced. We recommend using plastic Erlenmeyer shake flasks with screw cap tops (not foam plugs). This allows you to cover and keep the cultures sterile and allow free gas exchange.

Humidification is only necessary for monolayer cultures and suspension cultures with volumes less than 35 mL. With shaker or spinner cultures greater than 35 mL, humidification is not necessary.

Cell Culture

Please see the possible causes and solutions we recommend below:

Reason

Solution

Cells were stored incorrectly

Obtain a new stock and store in liquid nitrogen. Keep cells in liquid nitrogen until thawing.

Homemade freezer stock is not viable

Freeze cells at a density recommended by the supplier.

Use low-passage cells to make your own freezer stocks. 

Follow procedures for freezing cells exactly as recommended by the supplier. 

Obtain a new stock.

Cells were thawed incorrectly

Follow procedures for thawing cells exactly as recommended by the supplier. 

Make sure that you thaw the frozen cells quickly but dilute them slowly using pre-warmed growth medium before plating.

Thawing medium is not correct

Use the medium recommended by the supplier. Make sure the medium is pre-warmed.

Cells are too dilute

Plate thawed cells at the highest density recommended by the supplier to optimize recovery.

Cells not handled gently

Freezing and thawing procedures are stressful to most cells. Do not vortex, bang the flasks to dislodge the cells (except when culturing insect cells), or centrifuge cells at high speeds.

Glycerol used in the freezing medium was stored in light (if applicable)

If stored in light, glycerol gets converted into acrolein, which is toxic to cells. Obtain a new stock.

Please see some common reasons below, and solutions to the issue.

Reason

Solution

Growth medium is not correct

Use pre-warmed growth medium as recommended by the supplier.

Serum in the growth medium is of poor quality

Use serum from a different lot.

Cells have been passaged too many times

Use healthy, low–passage number cells.

Cells were allowed to grow beyond confluency

Passage mammalian cells when they are in the log-phase before they reach confluency.

Culture is contaminated with mycoplasma

Discard cells, media, and reagents. Obtain new stocks of cells, and use them with fresh media and reagents.

Please see the possible causes for this rapid pH shift, and suggested solutions:

Possible cause

Suggested solution 

Incorrect carbon dioxide tension

Increase or decrease percentage of carbon dioxide in the incubator based on concentration of sodium bicarbonate in the medium. For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, use carbon dioxide amounts of 5–10%, respectively. 

Switch to carbon dioxide–independent medium. 

Overly tight caps on tissue culture flasks

Loosen caps one-quarter turn.

Insufficient bicarbonate buffering

Add HEPES buffer to a final concentration of 10–25 mM.

Incorrect salts in medium

Use an Earle’s salts-based medium in a CO2 environment and a Hanks’ salts-based medium in atmospheric conditions.

Bacterial, yeast, or fungal contamination

Discard culture and medium. Try to decontaminate culture.

The precipitate could be due to residual phosphate left over from detergent washing, which may precipitate powered medium components. Rinse glassware in deionized, distilled water several times, then sterilize. If the medium is frozen, try warming media to 37°C and swirl to dissolve. If precipitate remains, discard medium. 

This is most likely a sign of bacterial or fungal contamination. We would recommend discarding the media, and trying to decontaminate the culture.

This can occur if cells are overly trypsinized. Trypsinize for a shorter time or use less trypsin. Mycoplasma contamination could also cause this problem. Segregate your culture and test for mycoplasma infection. Lastly, check for attachment factors in the medium.

Try changing the medium or serum. Compare media formulations for differences in glucose, amino acids, and other components. Compare an old lot of serum with a new lot. Increase initial cell inoculums. Lastly, adapt cells sequentially to new medium.

Decontamination

Very often mycoplasma contamination cannot be removed from the culture so it should be discarded. You may have a unique culture that you prefer not to discard and would like to try to clean it. Ciprofloxacin and Plasmocin have reportedly been used for this application. If interested in a protocol or directions for use, check with the antibiotic supplier or published literature. Note that mycoplasma are very difficult to remove from culture and spread easily so the treated cultures should be quarantined until clear of mycoplasma, and your laboratory should be thoroughly cleaned.

Decontamination of cultures with antibiotics

When an irreplaceable culture becomes contaminated, researchers may attempt to eliminate or control the contamination.

  1. Determine if the contamination is bacteria, fungus, mycoplasma, or yeast. Read more here to view characteristics of each contaminant. 
  2. Isolate the contaminated culture from other cell lines. 
  3. Clean incubators and laminar flow hoods with a laboratory disinfectant, and check HEPA filters.

Antibiotics and antimycotics at high concentrations can be toxic to some cell lines. Therefore, perform a dose-response test to determine the level at which an antibiotic or antimycotic becomes toxic. This is particularly important when using an antimycotic such as Gibco™ Fungizone™ reagent or an antibiotic such as tylosin.

The following is a suggested procedure for determining toxicity levels and decontaminating cultures:

  1. Dissociate, count, and dilute the cells in antibiotic-free media. Dilute the cells to the concentration used for regular cell passage. 
  2. Dispense the cell suspension into a multiwell culture plate or several small flasks. Add the antibiotic of choice to each well in a range of concentrations. For example, we suggest the following concentrations for Gibco™ Fungizone™ reagent: 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 µg/mL. 
  3. Observe the cells daily for signs of toxicity such as sloughing, appearance of vacuoles, decrease in confluency, and rounding. 
  4. When the toxic antibiotic level has been determined, culture the cells for two to three passages using the antibiotic at a concentration one- to two-fold lower than the toxic concentration. 
  5. Culture the cells for one passage in antibiotic-free media.
  6. Repeat step 4. 
  7. Culture the cells in antibiotic-free medium for four to six passages to determine if the contamination has been eliminated.
Media Supplements

When L-glutamine is in a concentrated stock solution it easily precipitates when cooled. Warming the solution briefly in a 37°C water bath with gentle swirling will dissolve the precipitate. Do not use the product unless the precipitate is fully dissolved.

In all media containing GlutaMAX™ supplement dipeptides as a substitute for L-glutamine, concentration is equimolar with the L-glutamine in the original formulation.

Yes. If you suspect that this is the case, remove the medium and add fresh medium. Alternatively, you can supplement medium with growth-promoting components. It is also possible to substitute GlutaMax™ I or II for glutamine in the medium to prevent glutamine exhaustion. 

Shipping, Storage, and Shelf Life of Medium

We routinely ship media that require long-term storage in the refrigerator at room temperature. We have done studies on representative media formulations to show that media can be at room temperature for up to a week without a problem.

Generally speaking, media can be used for up to three weeks after supplementation with serum. There are no formal studies to support this, but it is the rule of thumb used by our scientists.

Liquid media made from dry powder media should be stable for as long as the same formulation sold in liquid format. This is usually 1 month provided it is stored refrigerated and protected from light.

Fetal Bovine Serum (FBS)

Gibco™ FBS is not pre-aged. When stored at 2 to 8°C, the possibility exists for various proteins and lipoproteins in serum (e.g., cold agglutinins, fibrinogen, vitronectin, etc.) to aggregate, and form either perceptible material or observed turbidity. This should not affect serum performance. We recommend that you store FBS at –20°C and avoid repeated freeze-thaw cycles.

Flocculence may appear in FBS for a variety of reasons. The most common reason is the denaturation of serum lipoproteins. You may observe fibrin, one of the clot-forming proteins present in serum, after the serum has been thawed. This should not affect product performance.

To remove the flocculence, transfer the serum to sterile tubes and centrifuge the material briefly at 400 x g. Then filter the resulting supernant along with your media. Do not attempt to filter serum containing flocculence, it may clog filters.

Our studies have shown that short-term storage of thawed FBS at 4°C for up to 28 days causes no decrease in growth or viability performance. FBS stored at 4°C for longer periods of time should be evaluated on a case-by-case basis should this storage condition be necessary.

Gibco™ Dynamis™ Medium

CD FortiCHO™ medium has proven to be an excellent single-stream medium for CHO production, clone development, and banking. In our experience, CHO clones created and selected in CD FortiCHO™ medium can be put directly into Dynamis™ medium without any adaptation. In fact, our work suggests that those cells will produce as well or better in Dynamis™ medium and are just as stable in Dynamis™ medium if used for process development and scale-up.

24.8 grams of Dynamis™ Medium powder is required for 1 liter of media.

Please refer to the sequential adaptation procedure shown below. If you have trouble getting past a certain ratio, passage the cells 2–3 more times at the previous ratio.

  1. During sequential adaptation of CHO cells grown in conventional 5–10% serum-supplemented medium or other serum-free medium, use a seeding density of 3 × 10E5–4 × 10E5 viable cells/mL. 
  2. Monitor cell growth using Countess™ Automated Cell Counter until viable cell density reaches ≥1 × 10E6 cells/mL. 
  3. Dilute cells using a 25:75 ratio of complete Dynamis™ AGT™ Medium to the original medium. We recommend maintaining backup cultures in the original ratio medium until success with the new ratio medium is achieved. At each subsequent passage, dilute cells with stepwise increasing ratios of complete Dynamis™ AGT™ Medium to original medium (25:75, 50:50, 75:25, 90:10, followed by 100% Dynamis™ AGT™ Medium). Multiple passages at each step may be needed to achieve consistent growth. 
  4. After several passages in 100% Dynamis™ AGT™ Medium, the viable cell count should reach at least 2 × 10E6 cells/mL with ≥85% viability within 3–4 days of seeding culture. At this stage, the culture is considered to be adapted to Dynamis™ AGT™ Medium. 
Cell Dissociation

Generally speaking, trypsin can be stored refrigerated for up to a week. However, this may vary depending on how often it is warmed up and used during that week. It may also depend on how tightly the cells are attached, because there will be some loss of activity each day the trypsin is not frozen.

Trypsinize this type of cells for a shorter duration or use less trypsin or Gibco™ TrypLE™ Select or Gibco™ TrypLE™ Express Trypsin Replacement. After trypsinization dilute in 2 to 5 mL of cell culture growth medium transfer the cell suspension to 15 mL centrifuge tube. Centrifuge for 5–10 minutes at 100 x g. Discard the supernatant and suspend cell pellet in 2–5 mL fresh medium. Determine the count and seed the flask according to normal protocol and cell should adhere to the surface. 

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