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In Vitro Technologies is committed to supporting reproducible life science research in Australia and New Zealand. The lack of reproducibility typically results from cumulative errors & flaws, so we recognise that “gold standard” research require robust protocols with methods, reagents and techniques that are easily transferable. To support your cell culture techniques, In Vitro Technologies in collaboration with our cell culture partners (ATCC, Bio-techne, Corning & HyClone) present a range of Top 50 cell culture tips to ensure that you have all the information you need to be assured of ‘happy cells’ from culture initiation to cryopreservation.

While In Vitro Technologies uses reasonable efforts to include accurate and up-to-date information in this technical help section, we make no warranties or representations as to its accuracy. In Vitro Technologies is not responsible for any errors or omissions or for the results obtained from the use of this information. Content of the site is subject to change without notice.

Cell Growth and Propagation

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1. What are the consequences of using high passage cell lines?
2. What is the difference between the passage number of a cell line and its population doubling level (PDL)?
3. How do I determine the correct initial seeding density for my cells?
4. What is the best way to monitor the growth of monolayer cultures?
5. How should I refresh the medium or feed a monolayer culture?
6. What is the protocol for enzymatic dissociation of monolayer cultures?
7. Why is the viability of my cells lower than expected after subculture?
8. Do some cell lines grow in suspension?
9. How do I subculture my suspension cell line?
10. What are the correct temperature and CO2 settings for tissue culture incubators?
11. Should flasks be incubated horizontally or vertically?
12. My cell culture has failed to attach. How can I get the cells to attach?

Complete Growth Media

cell fulture faq tiles

 

 

 

13. What is the general composition of cell culture medium?
14. What is meant by the phrase "complete medium?"
15. How does the sodium bicarbonate-carbon dioxide system buffer the pH of cell culture medium?
16. Are there tissue culture media that do not require using a CO2 incubator?
17. What is insulin and why is it used in cell culture media?
18. Why are antibiotics or antimycotics added to cell culture medium?
19. What are the advantages of serum-free media over regular, serum-containing media?
20. What is conditioned medium? Why is it recommended for my cells?
21. What medium formulation should be used for culturing cell lines?
22. How do I determine the amount of medium to add or the split ratio when culturing suspension cell lines?
23. Why are NZ and AU sourced FBS preferred over other FBS sources?
24. Are you wasting your time with Heat Inactivated FBS?
25. What are Endotoxins?
26. How do endotoxins affect my cell culture?
27. Why is L-Glutamine stability a concern to Cell Biologists?
28. Is it okay to use a different medium than ATCC recommends?

Advanced Surfaces

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29. What is an extracellular matrix?
30. Why are Extracellular Matrices (ECM) used in Cell culture?
31. What is a basement membrane extract (BME)?
32. Is the time right for High Throughput 3D Cell Culture Assays?

Primary Cells and hTERT Cell Lines

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33. What are the advantages of ATCC Primary Cell solutions?
34. What can I do to ensure that I have a healthy population of ATCC primary cell cultures for use in my growth assays and to make my own frozen stocks?
35. What is the best method for freezing the ATCC primary cells I have in culture?
36. What is an hTERT cell line?
37. What is the difference between using hTERT and using Epstein Barr Virus (EBV) to immortalize cells?
38. What is the difference between primary microvascular endothelial cells and the hTERT immortalized microvascular cell lines?

Cell Line Contamination and Authentication

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39. How can microbial contamination in cell cultures be prevented?
40. How can I cure a fungal contamination?
41. Can a cell line be cured of mycoplasma contamination?
42. How does contamination affect cell culture?
43. What are some of the assessments that ATCC perform to authenticate cell lines?

Cryopreservation

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44. What is the process for Cryopreservation?
45. What is “best practice” Cryopreservation?
46. Why does the ATCC advise against thawing cultures at room temperature?
47. What are some causes of poor viability immediately after cells are thawed?
48. Is it necessary to remove the cryoprotective agent before putting the cells into culture?
49. Should cryopreservation be used for short-term storage, or will it shock the culture?
50. What is the best way to achieve the optimal cooling rate for cryopreservation?

1. What are the consequences of using high passage cell lines?

The consequences on experiments of using over sub-cultured or high-passage cell lines remain. It is well documented that cell characteristics can change when cell lines are cultivated for extended periods. Cell lines that have been over-sub-cultured can experience phenotypic as well as genotypic changes (genetic drift). It is also true that stocks of commonly used cell lines maintained in many laboratories have been Passaged hundreds of times and should not be considered true models of the original source material.

A frequently asked question about cell lines is “How many passages of a cell line are too many?” Unfortunately, the answer is not that simple. 

For example, would you expect the transfection efficiency of a cell line to increase or decrease, with increasing passage number?

Actually, the answer is “both”.  Transfection efficiency can increase AND decrease.  As you can see from this quick experiment, the outcome is cell line dependent.  There are some cell lines like the human colon cancer line, Caco-2, that will show an INCREASE in transfection efficiency after more than 25 passages… whereas other cell lines like the human breast cancer line, MCF-7, that will have a DECREASE in transfection efficiency after more than 25 passages.

More Info?

ATCC Animal Cell Culture GuideATCC Animal Cell Culture Guide

Passage number effectsATCC Passage number effects

2. What is the difference between the passage number of a cell line and its population doubling level (PDL)?

The passage number simply refers to the number of times the cells in the culture have been sub-cultured, often without consideration of the inoculation densities or recoveries involved. The population doubling level (PDL) refers to the total number of times the cells in the population have doubled since their primary isolation in vitro. This is usually an estimate rounded off to the nearest whole number. A formula to use for the calculation of population doublings is as follows: n = 3.32 (log UCY - log l) + X, where n = the final PDL number at end of a given subculture, UCY = the cell yield at that point, l = the cell number used as inoculum to begin that subculture, and X = the doubling level of the inoculum used to initiate the subculture being quantitated (Hayflick 1973).

More Info

happy cellsKeeping Cells Happy - Topics in Cell Health Maintenance & Viability

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3. How do I determine the correct initial seeding density for my cells?

For best results, follow the instructions in the product information sheet and on the batch specific sheet when initiating a cell culture.  The batch specific instructions for ATCC cell lines are printed on the last page of the product information sheet, and the specific lot information found on the batch specific instructions will show the recommended dilution ratio or initial seeding density.  The recommended dilution ratio or initial seeding density is based on the recovery tests we perform on the lot during post-cryopreservation quality control characterization and should be followed to initiate the cell culture.

The two most common size flasks used to initiate our frozen cell lines are T-25 and T-75 flasks.  At ATCC, we use 10 ml when seeding a T-25 flask and 15 ml when seeding a T-75 flask.  The size of flask chosen should depend on the cell density and the cell line.  It is also important to use the correct amount of medium in the flask to allow for good gas exchange and to prevent rapid nutrient depletion.

Here are some general guidelines for initiating cultures of different cell types.

  1. Hybridomas are usually initiated at 2 x 105 viable cells/ml\
  2. Leukemias or lymphomas can be seeded at 3-4 x 105 viable cells/ml
  3. Normal diploid fibroblasts are usually seeded at 1-2 x 106 cells/25 cm2
  4. Tumor lines are usually initiated at 2-4 x 106 viable cells/25cm2
  5. Contact inhibited lines should be seed at 5 x 103 viable cells/cm2
  6. Myoblast lines can be seeded at 1-2 x 104 viable cells/cm2

If the product information sheet does not specify seeding density or your cells do not perform well using the general guidelines above, you may need to do a growth curve to determine the optimum seeding density for that specific cell line.

More Info

ATCC Animal Cell Culture Guide2
ATCC Animal Cell Culture Guide 

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4. What is the best way to monitor the growth of monolayer cultures?

Anchorage-dependent cell lines growing in monolayers need to be sub-cultured at regular intervals to maintain the culture in logarithmic growth.  Examine the flask using an inverted microscope every 1 to 3 days, depending on the growth rate of the culture and the size of the vessel.  When the cells are near the end of exponential growth (roughly 70 to 90% confluent), they are ready to be sub-cultured.  Many anchorage-dependent cell lines will stop proliferating if the culture is allowed to reach 100% confluence.  The cells may be sub-cultured even if the culture is 100% confluent, but there may be a lag before the cells reach logarithmic growth again.  The sub-culturing procedure, including recommended split-ratios and medium replenishment (feeding) schedules, for each ATCC cell line is provided on the product information sheet.

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 5. How should I refresh the medium or feed a monolayer culture?

Two different possibilities exist when refreshing/feeding monolayer cultures. The cells in the culture may be well attached or the cells may be poorly attached. The culture must be handled differently according to how well the cells are attached.

If the monolayer is well attached:

  • Pre-warm medium in water bath to the appropriate growth temperature.
  • Remove the old medium from the flask by aspirating with a pipet hooked to a vacuum suction.
  • Replace with fresh medium. Use the appropriate volume for the vessel.
  • For a T-75, if the cells are less than 50% confluent use 15ml. If cells are greater than 50% confluent, then use 30mls.

If the monolayer is poorly attached:

  • Pre-warm medium in water bath to the appropriate growth temperature.
  • Add fresh medium instead of performing a complete fluid change.
  • Or, remove all but a few milliliters of the spent medium, collect the cell suspension in a centrifuge tube and gently pellet the cells at 125 x g for 5 minutes. Re-suspend the pellet in fresh medium and add back into the flask with the adherent cells

More Info

ATCC Animal Cell Culture Guide2
ATCC Animal Cell Culture Guide 

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6. What is the protocol for enzymatic dissociation of monolayer cultures?

Refer to the product sheet included with your ATCC cell line for the sub-culturing procedure recommended for that particular cell line. Anchorage-dependent cell lines are usually sub-cultured by disaggregation of the cell sheet with proteolytic enzymes such as trypsin. Ethylene diamine tetra acetic acid (EDTA), a chelating agent, may be added to the dissociation solution to enhance the activity of the trypsin by removing calcium and magnesium from the surfaces of the cells. An appropriate solution for general use is a solution of 0.25% (w/v) trypsin to 0.03% (w/v) EDTA prepared in saline without divalent cations (such as calcium- and magnesium-free phosphate buffered saline).

  • Rinse the cell sheet with the trypsin solution (5.0 to 10.0 ml/75 cm2 flask) and remove the rinse.
  • Add a small amount of trypsin solution to cell sheet (2.0 to 5.0 ml/75 cm2 flask) and observe until the cells begin to round up and lift from the surface (usually within 5 to 15 minutes). To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Monolayers that are particularly difficult to detach can be placed at 37°C to facilitate dispersal.
  • Once the cells begin to lift from the surface of the flask, add growth medium containing serum to the flask. Serum contains proteins that will suppress the activity of the trypsin. Note: it is not usually necessary to centrifuge the cells at this step. However, if either serum-free or low serum medium is used, centrifuge the cells at 125 x g for 5 minutes to remove the residual dissociation solution.
  • Aspirate the cells by pipetting gently, and then disperse the cells to new flasks. The subculture ratio will vary from 1:2 to 1:20 or greater depending on the cell line. Consult the product sheet for the recommended dilution ratio.

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7. Why is the viability of my cells lower than expected after subculture?

Enzymatic dissociation and mechanical dissociation (scraping) are both fairly harsh procedures.  It is not unusual to lose some cells during subculture.  Here are some possible reasons for lower than expected viability following subculture.

  • The dissociating procedure was too harsh.  To prevent excessive cell damage during dissociation, (a) use the correct concentration of dissociating agent, (b) avoid vigorous pipetting and small-bore pipets, and (c) entrifuge the cells gently (125 x g for 5 minutes).
  • 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 and dilute them as soon as possible.
  • Mechanical dissociation was too harsh.  When scraping is used to subculture cells, gently move the scraper in one direction across the flask.  Cover each section of the flask only one time, rather than scrub back and forth across the surface.  Pipet the cells gently to disperse them.
  • The medium was faulty. Use the recommended formulation and make sure it contains all of the required additives.

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8. Do some cell lines grow in suspension?

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 x 104 to 5 x 105 viable cells/ml and can attain densities of 2 x 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.

The growth characteristics, recommended seeding and sub-culturing densities, media replenishment (feeding) schedules, and medium formulations for each ATCC cell line are provided on the product information sheet as well as in the catalog description on ATCC website.

More Info

ATCC Animal Cell Culture Guide2
ATCC Animal Cell Culture Guide 

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9. How do I subculture my suspension cell line?

Subculture a suspension cell line using the following procedure:

  • Bring the complete growth medium to the appropriate temperature (usually 37°C) in a water bath.
  • 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 such as trypan blue.
  • 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.
  • 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.
  • If you are using a closed system, 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 min at 125 x g), remove the supernatant, and resuspend the cells in fresh medium at the lower seeding density.

More Info

ATCC Animal Cell Culture Guide2
ATCC Animal Cell Culture Guide  

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10. What are the correct temperature and CO2 settings for tissue culture incubators?

The temperature and carbon dioxide levels in an incubator are important for optimal conditions of growth.  The correct incubator settings will depend on the type of cells and the type of medium used to culture the cells.  Most animal cell lines require a temperature of 37°C for incubation.  Some cell lines, including insect and amphibian cell lines, require lower temperatures, e.g. 28°C.  Temperatures set higher or lower than the temperature recommended for a cell line can produce detrimental results.  The carbon dioxide (CO2) atmosphere of the incubator also plays an important role in the performance of a cell line.  The use of 5% or 10% CO2 in incubators should correlate to the amount of sodium bicarbonate present in the recommended culture medium.

In summary, both the temperature and carbon dioxide levels recommended on the product information sheet provide optimal conditions for the cell line.  Please review these conditions before thawing and initiating a cell line.  If you encounter problems during cultivation, and these two factors were confirmed before cultivation, it is a good idea to check the incubator itself for any possible fluctuations and/or recalibrate the settings to confirm that the readings are accurate.

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11. Should flasks be incubated horizontally or vertically?

It is recommended that flasks be placed flat (horizontal), rather than standing on end (vertical). This is generally a requirement to achieve proper gas exchange for cell growth. The usual ratio of medium volume to surface area is 0.2 - 0.5 mL/cm2. The upper limit is set by gaseous diffusion through the liquid layer, and the optimal diffusion depends on the oxygen requirements of the cells. Cells with a high O2 requirement do better in shallow medium (e.g., 2 mm), and those with a low requirement do better in deep medium (e.g., 5 mm). If the depth of the medium is greater than 5 mm, then gaseous diffusion may become limiting (Freshney 2005).

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ATCC Primary Cell Culture Guide
ATCC Primary Cell Culture Guide

ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide

12. My cell culture has failed to attach.  How can I get the cells to attach?

Cell attachment depends on many factors:

  • Check the product sheet sent with the item to see if the cells are listed as suspension or as partially attached. If cells in suspension are normal, be sure not to throw them out (e.g. when changing the medium).
  • Check the product sheet to see if coating the flask is recommended (e.g. collagen, pig gelatin, etc.). Also note that some cells attach more efficiently when grown in a flask, rather than in a dish.
  • Make sure that the exact medium formulation recommended by ATCC is being used. Check the product sheet that was shipped with the culture to verify that you are using the correct medium formulation.
  • Any fluctuation in environmental conditions, such as temperature, humidity or pH may cause cells to detach. Make sure your incubator is properly calibrated and check for any fluctuations in temperature, gas content, or humidity. Cells shipped as a growing flask may also detach at ambient shipping temperatures. Once resuspended  in the proper volume of medium, the cells should reattach after incubation at the recommended temperature.
  • Check the product sheet for the recommended cell density. Cells may detach if the culture is overly confluent.
  • Before you discard any floating cells, check the viability (e.g. trypan blue or Erythrosin B Stain Solution ATCC® 30-2404 ™) to ensure they are dead.

More Info

Complete Growth Media

13. What is the general composition of cell culture medium?

Cell culture media is composed of a complex mixture of salts, carbohydrates, vitamins, amino acids, metabolic precursors, growth factors, hormones, and trace elements. Formulations range 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. Carbohydrates are supplied primarily in the form of glucose. In some instances, galactose is used in place of glucose as it is metabolized at a slower rate which decreases the amount of lactic acid build up. Other carbon sources include amino acids (particularly L-glutamine) and pyruvate.

More Info

ATCC media
ATCC Media Brochure

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14. What is meant by the phrase "complete medium?"

Most cell culture media are sold as basal media. Basal medium is a complex mixture of salts, carbohydrates, vitamins, amino acids, metabolic precursors and trace elements. To enhance cell growth, you must add growth factors, hormones, and other proteins to the basal medium. Often this is accomplished by supplementing the basal medium with serum. Serum should be added to the basal medium at the concentration specified on the product sheet for the cell culture you are growing.

In some cases additional supplements must be added as indicated on the cell culture's product sheet or in the catalog description. In general, if the medium formulation states that the medium has been adjusted to contain certain components, these components have already been added to the medium at the stated concentrations. However, if the medium formulation states that it is to be supplemented with certain components, then those components must be added to the medium by the user.

More Info

ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide

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15. How does the sodium bicarbonate-carbon dioxide system buffer the pH of cell culture medium?

Sodium bicarbonate is a buffer used to stabilize pH. Cells in culture produce CO2 but require only small amounts of the compound for growth and survival. CO2 affects the pH of medium. Increasing atmospheric CO2 decreases the pH of the medium. Decreasing the atmospheric CO2 increases the pH of the medium. As cells metabolize, they produce CO2 thus causing the media to become more acidic. If the medium contains phenol red, the color of the medium becomes more yellow. In culture media, dissolved CO2 is in equilibrium with bicarbonate ions and many cell culture media formulations take advantage of this CO2/bicarbonate reaction to buffer the pH of the media. CO2 dissolves freely into the culture media and reacts with water to form carbonic acid. As the cells metabolize and produce more CO2, the pH of the medium decreases (becomes more acidic).

Sodium bicarbonate, NaHCO3, is used as a buffer. Sodium bicarbonate dissociates into sodium and bicarbonate ions. By increasing the bicarbonate ions, the buffer drives the top equation to the left and thus increases the pH. The concentration of the sodium bicarbonate in the medium must be matched with the level of CO2 in the atmosphere above the medium. For media containing 1.5 to 2.2 g/L sodium bicarbonate, use 5% CO2. For media containing 3.7 g/L sodium bicarbonate, use 10% CO2. If the concentration of sodium bicarbonate is too high for the CO2 atmosphere in the incubator, the media becomes more alkaline (the pH increases).

More Info

ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide

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16. Are there tissue culture media that do not require using a CO2 incubator?

Some cell lines may be maintained satisfactorily on an alternative medium such as:

  • CRCM-30 medium
  • L-15 medium.
  • CO2-independent medium (Invitrogen Life Technologies Cat. No. 18045088).

You can usually determine if a medium is satisfactory by using it with the cell line in question for 3 to 5 passages. However, cultures established at very low concentrations (e.g., cloning) usually require CO2 in the gas phase. An alternative to using a CO2 incubator is to have a 5% CO2 gas tank at your work site and stream filtered CO2 into the gas phase above the medium prior to sealing the flask.

More Info

ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide

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17. What is insulin and why is it used in cell culture media?

Insulin is a polypeptide hormone found in both vertebrates and invertebrates. It is a growth hormone that promotes uptake of glucose and amino acids. The Dictionary of Cell Biology defines insulin as:

1. Hormone found in mammalian serum.

2. Secreted by B cells of the pancreas in response to high blood sugar levels.

3. A mitogen-activator, which is any chemical specifically stimulating a eukaryotic cell to enter S phase of the cell cycle, that commits the cell to G2 and mitosis.

4. Has sequence homologies with other growth factors.

5. Frequent addition to cell culture media for demanding cell types.

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18. Why are antibiotics or antimycotics added to cell culture medium?

Antibiotics and/or antimycotic agents are added to cell culture media as a prophylactic to prevent contamination, as a cure once contamination is found, to induce the expression of recombinant proteins, or to maintain selective pressure on transfected DNA.

ATCC does not use antibiotics or anti-mycotics for routine cell culture. Long-term use of antibiotics or anti-mycotics may mask the presence of low levels of microbial or mycoplasma contamination. In addition, some antibiotics and anti-mycotics are toxic and may affect the recovery and proliferation of some cell lines.

However, one may elect to introduce antibiotics for short periods to primary cultures or as a safeguard while propagating specific valuable stocks to produce working stocks. If you do elect to use an antibiotic in your medium, ATCC recommends using a Penicillin-Streptomycin solution at a final concentration of 50-100 I.U./ml penicillin and 50-100 µg/ml streptomycin. ATCC offers a Penicillin-Streptomycin solution (ATCC® 30-2300 ™). This sterile solution can be added at 0.5 to 1 ml of solution per 100 mL of cell culture media for a final concentration of 50 to 100 I.U./mL penicillin and 50 to 100 µg/mL streptomycin.

While ATCC avoids use of the following two agents, commonly used concentrations are as follows. Gentamicin sulfate is an antibiotic and is used at 50 to 100 µg/ml culture medium. The anti-mycotic amphotericin B is used at 2.5 µg /ml culture medium. See Chapter 7 in Methods in Enzymology: Cell Culture, (1979) Vol. 58, W. B. Jacoby and I. H. Pasten, eds. (Academic Press, New York).

More Info

ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide

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19. What are the advantages of serum-free media over regular, serum-containing media?

Many factors make serum-free media an attractive alternative to media containing serum:

  • Serum is an undefined media supplement. The components in the serum, such as growth factor and hormones, may vary from lot to lot. In addition, inhibitors such as endotoxins and hemoglobin may be higher in some lots than others.
  • Serum-free media maintains physiological consistency - all the components are known and remain unchanging. In contrast, media containing sera varies depending on the different batches of serum.
  • Availability of the media is not dependent on cattle conditions. Drought and disease often affect the availability of serum, but serum-free media is unaffected.
  • There is no danger of contamination from viruses in the serum. While these viruses are often harmless to cell culture, they represent an additional unknown factor outside the operator's control.
  • Serum-free media creates the ability to make selective media. A medium can be specifically designed for a particular cell type.
  • Serum-free media enables the regulation of proliferation and differentiation. Serum contains factors which promote growth and factors which inhibit growth and it is impossible to regulate how much of each is present. With serum-free media, it is possible to create two different media and to switch from growth-enhancing media for propagation to differentiation-inducing media by altering the concentration and types of growth factors and other inducers.

More Info

ATCC media
ATCC Media Brochure

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20. What is conditioned medium? Why is it recommended for my cells?

Conditioned medium is spent media harvested from cultured cells. It contains metabolites, growth factors, and extracellular matrix proteins secreted into the medium by the cultured cells. Examples of each might include: metabolites such as glucose, amino acids, and nucleosides; growth factors such as interleukins, EGF (epidermal growth factor), and PDGF (platelet-derived growth factor); and matrix proteins such as collagen, fibronectin, and various proteoglycans. Most cell lines requiring conditioned medium are dependent on at least one major constituent found in the conditioned medium

For example, one of the major growth factors present in LADMAC conditioned medium is CSF-1 (colony stimulating factor 1). CSF-1 assists with macrophage progenitor proliferation and differentiation. LADMAC conditioned medium is obtained from the LADMAC cell line ATCC® CRL-2420 ™ and is added to the growth media used to propagate ATCC® CRL-2467 ™, ATCC® CRL-2468 ™, ATCC® CRL-2470™, and ATCC® CRL-2471™, all of which express colony stimulating factor 1 receptors.

Mouse embryonic stem cells (ESC) also benefit from growth-promoting components found in conditioned medium. LIF (leukemia inhibitory factor) is secreted into medium by near-primary cultures of mouse embryonic fibroblasts (MEF), and is an essential component in maintaining the undifferentiated state of ESCs. For example, ATCC® SCRC-1002 ™ requires both the use of a mouse embryonic fibroblast feeder layer, as well as additional supplementation with purified LIF (Chemicon, ESG1107) to maintain the undifferentiated state of the embryonic stem cells.

It is important to bear in mind that omission of conditioned medium from a cell line requiring this type of supplementation should be carefully evaluated. While most cell lines are, in fact, dependent on at least one major constituent found in the conditioned medium, replacing the conditioned medium with a recombinant, or purified, form of this one, single growth factor may yield unfavorable results such as altered expression phenotypes, slowed growth, quiescence or even complete loss of the culture. Any of these responses would indicate that the conditioned medium contains more than just a single growth factor essential to the healthy maintenance and growth of the cells (Freshney RI, 2005).

More Info

ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide

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21. What medium formulation should be used for culturing cell lines?

ATCC generally lists in the product description catalog and product sheet either the medium recommended by the originator of the cell line or a standard medium formulation that has been found to be effective otherwise.  Media formulations vary widely among commercial suppliers even for media with similar, if not identical names. A change in media or absence of an additive from the recommended formula could affect growth, recovery and/or function of the cell line. It is very important to read catalog descriptions and media bottle labels carefully since media mix-ups are a leading cause of cell culture problems.  On the ATCC website, the product page of the cells, in the "Culture Method" tab, you will find the recommended media listed for the cell line.

The formulations to the ATCC media can be on the ATCC website, www.atcc.org on the media's product page. Or you may contact media manufacturers for complete formulations of the most common cell culture media.

Hyclone media  formulations of the most common cell culture media can be found at https://promo.gelifesciences.com/gl/hyclone/products/classical-liquid-media.html

ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide

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22. How do I determine the amount of medium to add or the split ratio when culturing suspension cell lines?

Whether you add medium or split your suspension culture will depend on the optimal density range for logarithmic growth, the number of cells in your flask and the amount of medium needed to reduce the cell density.  To determine how much medium to use, follow these steps:

  • Perform a cell count and determine the viability by dye exclusion on the day that you are going to add medium or split the culture.
  • Calculate how many viable cells/ml you have and then how many milliliters of fresh medium should be added in order to lower the cell density toward the minimum end of the density range.
  • Depending on the volume you must add, you can choose to simply add fresh complete medium to the same culture vessel or to split the suspension into multiple vessels.

For example, assume your suspension cell line should be maintained between 2 x 105 and 1 x 106 viable cells/ml.  If your medium volume is 10 ml, and your cell count is only about 3 x 105 viable cells/ml, then, you have several choices.

  • You could wait another day before manipulating the culture.
  • You could centrifuge and resuspend the cells in the same volume.
  • You could simply add 5 ml of fresh medium to bring the concentration back down to 2 x 105 viable cells/ml.

If you chose option #3, your vessel would now hold 15 ml of medium.  Depending on the vessel size, that is probably okay.  However, if you do your cell count and determine that the cell density is about 1 x 106 viable cells/ml, then you would have to add 40 mls to bring the cell density back to 2 x 105 viable cells/ml.  This would result in a total of 50 ml and the culture would probably have to be split into multiple vessels.

More info

ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide

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23. Why are NZ and AU sourced FBS preferred over other FBS sources?

FBS is the serum of choice for most cell culture research as it has the richest source of growth factors. Within the FBS selection there is undoubtedly a global preference for New Zealand and Australian sourced characterized FBS. This preference is associated with the geographical and biological benefits of an island continent, that makes animal disease and control management easier than in most areas of the world.

New Zealand’s islands are isolated from many outside influences with the benefit that New Zealand has the fewest reported bovine diseases in the world, making Characterized FBS, New Zealand Origin an excellent source of bovine serum as it offers greater security.

More Info

hyclone serum
Hyclone Serum Products

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24. Are you wasting your time with Heat Inactivated FBS?

Heat inactivating serum was originally developed when only serum from adult animals was available for cell culture. Adult serum contains various immune factors, particularly serum complement, which may inhibit or destroy cells under certain conditions. As such, heat-inactivation (heating to 56°C for 30 minutes) arose to inactivate complement, a group of proteins present in sera that are part of the immune response. This is sometimes important for cells that will be used to prepare or assay viruses, or cells that are used in cytotoxicity assays or other systems where complement may have an unwanted influence.  Heat-inactivation is also recommended for growing embryonic stem cells (Rudnicki and McBurney, 1987) as well as for many insect cell lines (Weiss, S.A., et al. 1995).

Heat has also been used to destroy mycoplasma in serum. However, because most serum suppliers filter through 0.1 µm filters to remove mycoplasma before distribution, this is not usually necessary.

However, note that ATCC does not routinely heat-inactivate serum unless specifically noted in the medium formulation for a specific cell culture. Heat inactivation will reduce or destroy serum growth factors, and should only be done when there is a compelling reason. Currently, heat- inactivated (HI) serum is often preferred without any evidence of beneficial effect, simply because an earlier protocol reported that HI serum was used.

More info?

HEAT INACTIVATIONAre you wasting your time with Heat Inactivated Serum?

Choosing the right Serum
Choosing the right Serum

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25. What are Endotoxins?

Endotoxin is a variable component of serum products which, as the name suggests, is toxic to cells and inhibits proliferation. It is produced by gram-negative bacteria and is sometimes found in serum due to bacterial contamination of the serum before the filtration step. Endotoxins are polysaccharides and may remain in the serum following filtration. High quality serum contains low levels of endotoxin as shown by the certificate of analysis. The endotoxin level of high quality serum should be less than 10 EU/ml.

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26. How do endotoxins affect my cell culture?

Endotoxin contamination of cell-based products is a major concern to the pharmaceutical industry, since endotoxin in vaccines and other cell-based parenteral drug products render them unfit for use. Depending on the cell type and culture conditions, endotoxins can have a variety of effects on cell growth and function including altered cell growth, differentiation, contractility and protein production, as well as being a source of significant variability. This is especially true when using cells known to be sensitive to the low endotoxin levels, and that are commonly found in cell culture systems. Therefore, unless you are confident that endotoxin has no effect on your cultures and will not be a potential source of variability in your experiments, every effort should be made to reduce the risk of endotoxin related problems. Some basic considerations include using cell culture media, sera and plasticware that are certified by their manufacturers to be non-pyrogenic. An additional precaution is an assessment of the laboratory water source.

More info:

Endotoxins
Corning Endotoxins

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27. Why is L-Glutamine stability a concern to Cell Biologists?

L-glutamine is one of several amino acid components that is essential to cell culture media. However, L-glutamine can degrade in liquid medium in cell culture. Maintaining required levels of L-glutamine in cell culture often requires the repeated supplementation of the medium with additional L-glutamine. This procedure can lead to toxic levels of ammonia build-up as well as potential contamination making glutamine decomposition in cell culture medium a concern to many researchers who are seeking to increase culture longevity and density while preventing the compromise of viability and growth.

As such HyClone recommend storing liquid medium containing glutamine at 2°C–8°C, or purchasing medium without glutamine and adding it at the time of use.

More Info

Glutamine Stability in Cell Culture Media
Glutamine Stability in Cell Culture Media

L Glutamine stability
L-Glutamine Stability

28. Is it okay to use a different medium than ATCC recommends?

ATCC generally lists either the medium recommended by the originator of the cell line or a standard medium formulation that has been found to be effective otherwise. Sometimes donors of cell lines do not determine if there is an absolute need for each and every component of the medium and sometimes chose a formula simply because it has proven in the past to be successful with similar cell lines. While other unspecified media may also be entirely satisfactory, a change in media or absence of a component from the recommended formula could affect growth, recovery and/or function of the cell line. ATCC conforms to the standards set by the donor in order to produce a uniform and high quality product that we are confident will be the most usable for our customers. The standards of growth must be maintained by ATCC and we can only assure customers optimum growth of cells with the conditions that are known. Therefore, our warranty requires that ATCC cultures must be grown in the medium recommended on the product information sheet.

If you would prefer to use an alternative medium, it is prudent to recover the cells and prepare a seed stock using the originally prescribed medium and growth conditions before attempting a substitution. Once a seed stock has been created, two approaches can be used to introduce an alternative medium. The simplest method is to simply change the medium to the desired alternative formula and then pass the cell line 3 to 5 times to force it to adapt to the new conditions. A more gentle approach is to subculture the cells at a 1:2 split ratio into two identical culture vessels, with one vessel containing the original medium and the other containing a mixture of 50% original medium and 50% new medium. The effects of the new medium formulation on the cells can then be easily observed by comparing the growth in the cultures with the medium mixture with the culture containing only the original medium formulation. Once the cells in the mixed culture have become confluent, the process can be repeated by again splitting the mixed culture at a 1:2 ratio into two identical vessels. This time one vessel should contain 50% original medium and 50% new medium while the second culture has 25% original medium and 75% new medium. Once this second culture has become confluent it can be sub-cultured into 100% new medium.

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Advanced Surfaces

29. What is an extracellular matrix?

The extracellular matrix (ECM) consists of an organized, complex network of locally secreted macromolecules that provide the structural framework for cell migration, adhesion, proliferation, and differentiation within the tissues of an organism. The ECM is composed primarily of different glycosaminoglycans and fibrous proteins, such as collagen, laminin, and fibronectin. The fibrous proteins of the ECM regulate cell structure, adhesion, and mediate interactions within the ECM, while the glycosaminoglycans (GAGs), such as hyaluronan or protein-linked GAGs, form a porous hydrated gel that is essential for mechanical support and signaling between cells. The properties of isolated ECM components or heterogeneous extracts can be exploited for use as cell culture reagents and tools for cell-based assays.

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30. Why are Extracellular Matrices (ECM) used in Cell culture?

A wide range of animal, human, and synthetic ECMs are available for researchers to support improved cell attachment, propagation, differentiation, and migration. Matrigel, Cultrex, Collagen and Fibronectin are products that are commonly used to create a more in vivo like environment during in vitro cell culture.

More info?

Matrigel FAQ
Matrigel FAQs

3D matrices Assay Kits
Cultrex 3D Matrices and Assay Kits

31. What is a basement membrane extract (BME)?

A basement membrane matrix is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins to include laminin (a major component), collagen IV, heparan sulfate proteoglycans, and entactin/nidogen. The BME also contains TGF-beta, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogen activator, and other growth factors which occur naturally in the EHS tumor.

The basement membrane can act as a selective barrier, affect cell polarity, metabolism, or migration, and induce cellular differentiation. Basement membranes can be reconstituted in vitro using basement membrane extracts and components of the ECM. 

More Info

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32. Is the time right for High Throughput 3D Cell Culture Assays?

The use of 3D cell culture models has dramatically increased since its inception more than two decades ago. Evidence strongly suggests that 3D cell cultures establish physiological cell-cell and cell-ECM interactions, and, as a result, they can mimic the specificity of native tissue with greater physiological relevance than conventional 2D cultures. This is particularly evident in applications such as stem cell culture and differentiation, cancer biology, drug and toxicity screening, and tissue engineering.

More Info?

3D matrices Assay Kits
Cultrex 3D Matrices and Assay Kits

Spheroid MP
Spheroid Microplates

3D tissue Modeling
ATCC 3D Cell Culture Webinar

Primary Cells and hTERT Cell Lines

33. What are the advantages of ATCC Primary Cell Solutions?

Primary cell cultures more closely mimic the physiological state of cells in vivo and generate more relevant data representing living systems. Primary cultures consist of cells that have been freshly derived from a living organism and are maintained for growth in vitro. Once adapted to in vitro culture conditions, primary cells undergo a limited, predetermined number of cell divisions before entering senescence.

ATCC has provided a solution to help investigators overcome the high cost and inconsistency found in routine primary cell culture with the development of ATCC Primary Cell Solutions, a standardized cell culture system that includes high quality cells, media, supplements, reagents and protocols. ATCC Primary Cell Solutions focuses on providing researchers with superior quality from a trusted source.

More Info?

Primary Cell Solutions collection
ATCC Primary Cell Solutions

ATCC Primary Cell Culture Guide
ATCC Primary Cell Culture Guide

34. What can I do to ensure that I have a healthy population of ATCC primary cell cultures for use in my growth assays and to make my own frozen stocks?

There are several things you can optimize to ensure that your primary cell cultures are healthy:

ATCC primary cell cultures are tested to assure growth for a minimum of either 10 or 15 population doublings. However, since longevity studies are not performed for these items, we cannot say for sure how long the cells may continue to divide before reaching senescence. ATCC generally recommends minimizing the passage of primary cells in vitro to avoid the complications that are most often associated with long-term propagation; e.g. genotypic or phenotypic variation, increased risk for microbial contamination, and added opportunity for cellular cross-contamination to take place.

It is very important to monitor the population doubling level (PDL) for all primary cell cultures. PDL refers to the exponential number of times the cells from a single starting population have increased since their initial isolation or initiation. PDL is different from passage number. 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.

 It is important to use appropriate solutions that are approved for general use with primary cells during subculture. We recommend using a dissociating solution approved for primary cell use, such as 0.05% (w/v) Trypsin and 0.02% EDTA in phosphate buffered saline without calcium, magnesium or phenol red (ATCC® PCS-999-003 ™). Do not use higher concentrations of enzymatic dissociating agents as this could eventually affect cell recovery and growth with each subculture, if it is not adequately removed or neutralized prior to seeding. We also recommend using Trypsin Neutralizing Solution (ATCC® PCS-999-004 ™) to effectively neutralize this enzymatic dissociation reagent. It is best to subculture primary cell cultures before reaching 100% confluence, since post-confluent cells may undergo differentiation and exhibit slower proliferation after passaging.

Avoid repeated warming of the complete growth media. If using a small volume of media (50 ml or less) aliquot the media or only warm the volume needed in a sterile conical tube each time. Be sure to pre-equilibrate the tissue culture vessels containing the required volume of growth media in a 37°C, 5% CO2 humidified incubator for thirty minutes prior to adding any cells.  Make sure that the % CO2 in the tissue culture incubator is within +/- 1% CO2 of the recommended level; primary cells are particularly sensitive to fluctuations in media pH. ATCC recommends leaving out antimicrobial supplements from the complete culture medium if possible. 

Primary cells are not intended for re-freezing. Once a primary cell culture has been passaged and frozen by an end user, it is no longer primary, and may lose some characteristics associated with primary cells. Unlike continuous cell line cultures, primary cells are intended for one-time applications. If re-freezing is unavoidable, most primary cell cultures can be cryopreserved in a mixture of 80% complete growth medium supplemented with 10% FBS and 10% DMSO. The freezing process should be slow and controlled at a rate of -1°C per minute, to minimize the formation of ice crystals within the cells.

ATCC Primary Cell Culture Guide
ATCC Primary Cell Culture Guide

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35. What is the best method for freezing the ATCC primary cells I have in culture?

Primary cells are not intended for re-freezing and ATCC has not validated any method for doing this. Once a primary cell culture has been passaged and frozen by a customer, it is no longer primary, and may lose some characteristics associated with primary cells. Unlike established cell cultures, primary cells are intended for one-time applications.

ATCC Primary Cell Culture Guide
ATCC Primary Cell Culture Guide

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36. What is an hTERT cell line?

An hTERT cell line is one that has been immortalized by inserting the hTERT (human telomerase reverse transcriptase) gene into the cells' DNA. This gene immortalizes cells by maintaining telomere ends. This prevents the telomere shortening of a cell's DNA which would otherwise cause the cells to senesce.

hTERT Cell Culture Guide
hTERT Culture Guide

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37. What is the difference between using hTERT and using Epstein Barr Virus (EBV) to immortalize cells?

During normal cell division, the telomeres, or ends of DNA, gradually shorten. Eventually, the telomeres shorten so much that the content of the DNA is jeopardized. The p53 gene monitors the conditions of the DNA, and when the telomeres become too short, the p53 gene initiates apoptosis in the cell.

Epstein Barr Virus is a virus which interferes with the functionality of the p53 gene. When a cell is infected with EBV, it can no longer undergo apoptosis. As a result, it continues to reproduce and divide, regardless of the shortening telomeres. Although infection with EBV results in an immortal cell line, the shortening of the telomeres eventually alters the DNA and causes chromosomal abnormalities and dramatically different cellular phenotypes.

hTERT, on the other hand, is a gene which maintains telomere ends. When inserted into a cell, the hTERT gene repairs telomere ends during cellular reproduction and division. As a result, the telomeres do not shorten and the cell is able to continue reproducing without genotypical or phenotypical change.

More Info?

hTERT Immortalized Cell Lines
hTERT Immortalized Cell Lines

hTERT Cell Culture Guide
hTERT Culture Guide

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38. What is the difference between primary microvascular endothelial cells and the hTERT immortalized microvascular cell lines?

Primary microvascular endothelial cells may grow for 15 population doublings (PD) until they become senescent and undergo growth arrest.

The hTERT-immortalized microvascular cell lines, for example TIME (ATCC® CRL-4025 ™) and TIME-GFP (ATCC® CRL-4045 ™), are able to grow for more than 25 PD without changes in endothelial cell characteristics such as expression of CD31 and the ability to uptake AcLDL.  Immortalized microvascular cell lines do not undergo grow arrest at later passages.

hTERT Cell Culture Guide
hTERT Culture Guide

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Cell Line Contamination and Authentication

39. How can microbial contamination in cell cultures be prevented?

Always practice strict aseptic techniques when culturing cells.  It is best to culture the cells in a laminar flow hood to ensure a sterile culture environment.  If possible, use disposable pipets and disposable culture vessels.

Do not bring outer clothing into the laboratory. It is always a possibility that your clothing is harboring bacteria and/or fungi. You may want to wear sleeve guards over your lab coat along with the rubber gloves. If possible, use disposable lab coats and change them frequently. Clean the water bath on a regular schedule depending on usage (preferably weekly, but at least every month). When starting a new culture, sterilize the outside of ampoules by submerging in a fresh beaker of 70% ethanol after thawing. Wrap the decontaminated ampoule in a square of sterile gauze to blot excess ethanol. Open ampoules with sterile gauze. Always use a pipet to transfer the contents of the ampoule into a prepared flask.

Occasionally, airborne fungal contamination can become chronic and severe. Once contamination of the original samples and operator error have been ruled out, using dry rather than the usual humidified incubators may help cure the problem. Growing cells in tissue culture flasks rather than in Petri dishes in dry CO2 incubators prevents the problem of evaporation of medium, although, even when using flasks, care must be taken that significant levels of medium are not lost by evaporation. If a humidified incubator is being converted to dry operation, the CO2 regulation has to be recalibrated (Freshney, 2005). Alternatively, a closed system can be used in place of CO2 incubators. Flasks are gassed with a mixture of 5% CO2 in air by means of a sterile pipette (with cotton plug) attached to tubing that is attached to a 5% CO2 in air tank. The tubing is first attached to an Erlenmeyer filtering flask that contains sterile, double distilled water. After gassing the vessel enough to replace the airspace with 5% CO2 in air, close the cap tightly. This gassing takes place at every fluid-change. In rare cases, contamination may result from a contaminated or malfunctioning laminar flow hood. If all other attempts to prevent microbial contamination fail, it would be prudent to have a service decontaminate the hood and clean the vents.

More Info?

Mycoplasma Detection Mycoplasma Detection

Mycoplasma Whitepaper Mycoplasma Whitepaper

trusted partnerTrusted Partner

Corning Contamination
10 ways to reduce contamination

ACEA Culture and Monitoring of Animal Cells Basic Techniques
ACEA Culture and Monitoring of Animal Cells Basic Techniques

40. How can I cure a fungal contamination?

Try treating cells with elevated amphotericin B concentrations. It is very important to test several concentrations of antifungal in the complete medium to find a concentration that kills the contaminant but not the cells.

  • As a starting point, add amphotericin B at a final concentration of 2.5 ug/ml that is typical for primary culture of skin fibroblasts.
  • Treat for 2 weeks in antifungal medium.
  • After treating the cell culture, continue culturing in antibiotic/antimycotic free medium and retest to make sure that the culture is clean.

Continual observation and periodic testing is necessary to make sure that the contaminant does not reappear. Since many antibiotics may be toxic to cells, a selected population that no longer exhibits qualities of the parental line may result. It may be necessary to examine the cured culture to assure that it retains properties sufficiently similar to the original line.

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41. Can a cell line be cured of mycoplasma contamination?

If your cell culture is contaminated with mycoplasma, it is best to discard the culture and start over. Curing cell lines of mycoplasma contamination is time consuming and does not always work.

When attempting to cure a cell line of mycoplasma contamination, first identify the contaminant and select a suitable antibiotic, preferably by testing the contaminating mycoplasma for its antibiotic sensitivity. Culture the cells for 1 to 2 weeks in the presence of the antibiotic, and then culture them without antibiotic for 1 to 2 months. Then retest the line to make sure that the culture is clean. A very sensitive testing method should be used. Periodic retesting is necessary to make sure that the contaminant does not reappear. Since many antibiotics may be toxic to cells, a selected population that no longer exhibits qualities of the parental line may result. It may be necessary to examine the cured culture to determine if it is sufficiently similar to the original line.

More Info 

Mycoplasma Whitepaper
Mycoplasma White Paper

Mycoplasma Detection
Mycoplasma Detection

42. How does contamination affect cell culture?

Low grade infections may not cause deterioration of the culture and may be held back by low concentrations of antibiotics and/or frequent fluid changes. If the culture is allowed to sit without fluid changes/or antibiotics added, the contamination often becomes obvious within a few days. At this point cell cultures will usually deteriorate.

cell line authentication
Cell Line Authentication
STR Essentials
ATCC Cell Authentication STR Essentials
Solving Identity Crisis
Solving Identity Crisis in Animal Cells – ATCC Webinar 

 

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43. What are some of the assessments that ATCC perform to authenticate cell lines?

All ATCC cell lines undergo authentication tests during the accessioning process. This process is described in the online ATCC brochure Maintaining High Standards in Cell Culture. Characterizations that are applied to final seed and distribution stocks of cell lines for certification include testing viability of the cell population just prior to freezing and immediately after thawing by trypan-blue dye-exclusion test.

  • Observations of recovery and growth are recorded along with morphological appearance.
  • Isoenzymology and/or the Cytochrome C subunit I (COI) PCR assay is performed for confirmation of species.
  • Human cell lines have their identity confirmed by STR analyses.
  • Characterizations of hybridoma cell lines involve testing for antibody production and confirmation of the isotype. Confirmation of antibody production is checked by the ouchterlony diffusion method and in some cases ELISA. Mouse antibodies are isotyped and confirmed by the use of an Isostrip.

The ATCC does have some cell lines that report such characteristics as karyotype, tumorigenicity, biochemical traits, drug susceptibility, and receptor or antigen expression. ATCC does not routinely perform functional assays or characterize our cell lines based on these aspects. Information regarding these descriptions can be further examined utilizing the references that are named within the product description of our cell lines. In most instances the depositor of the cell line provides ATCC with this information and description.

More Info?

cell line authentication
Cell Line Authentication
STR Essentials
ATCC Cell Authentication STR Essentials
Solving Identity Crisis
Solving Identity Crisis in Animal Cells – ATCC Webinar 

trusted partner
Trusted Partner

Cyropreservation

44. What is the process of cryopreservation?

As the cell suspension is cooled below the freezing point, ice crystals form and the concentration of the solutes in the suspension increases. Intracellular ice can be minimized if water within the cell is allowed to escape by osmosis during the cooling process. A slow cooling rate, generally -1°C per minute, facilitates this process. However, as the cells lose water, they shrink in size and will quickly lose viability if they go beyond a minimum volume. The addition of cryo-protective agents such as glycerol or dimethylsufloxide (DMSO) will mitigate these effects. The standard procedure for cryopreservation is to freeze cells slowly until they reach a temperature below -70°C in medium that includes a cryo-protectant. Then, transfer the vials to a liquid-nitrogen freezer to maintain them at temperatures below -130°C.

The recovery of cryopreserved cells is straightforward: Cells are thawed rapidly in a water bath at 37°C, removed from the freeze-medium by gentle centrifugation and/or diluted with growth medium, and seeded in a culture vessel in complete growth medium. There are numerous factors that affect the viability of recovered cells. It may be necessary to modify the procedure for each cell line to attain optimal cell viability upon recovery. Some of the critical parameters for optimization include the composition of the freeze medium, the growth phase of the culture, the stage of the cell in the cell cycle and the number and concentration of cells within the freezing solution. ATCC provides information on cryopreservation for all of our cell lines in the product information sheet. Most of our cell lines are frozen with a cryopreservation medium consisting of 5% DMSO and complete growth medium.

Cryopreservation
Cryopreservation
ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide
atcc cryo
ATCC Best Practices in Cryopreservation – ATCC Webinar

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45. What is “best practice” Cryopreservation?

Cryopreservation is the use of very low temperatures to structurally preserve intact living cells and tissues. Normally, the freezing of water in cells causes catastrophic damage to cellular structure by physical damage of ice formation and increased imbalance of solutes. Cryopreserving cells with the proper cryo-protectants and techniques will maximize viability of cells for cell culture. The best practices for cryopreservation are based on determining optimal freezing rates and cryo-protectants, selecting proper containment units, managing a biorepository, and handling cells post-thaw.

Key Points:

  • For the most accurate administration of DMSO, use Serum Free Cell Freezing Media already formulated with exactly 10% DMSO
  • A controlled cooling rate (1 – 3°C /min) is necessary to maximize cell viability when freezing down cell lines

More Info?

CryopreservationCryopreservation

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46. Why does the ATCC advise against thawing cultures at room temperature?

Thawing cultures at room temperature allows ice formation within the culture, which can rupture cell membranes and kill the cells. To prevent ice crystal formation, the ATCC recommends thawing cultures quickly in a 37 °C water bath (or the recommended temperature on the product sheet) and immediately transferring the entire thawed contents of the vial to the appropriate medium.

More Info?

ATCC Animal Cell Culture Guide2
ATCC Animal Cell Culture Guide

Cryopreservation
Cryogenic Preservation of Animal Cells

 47. What are some causes of poor viability immediately after cells are thawed?

DMSO (dimethyl sulfoxide) is a penetrating agent commonly used in cryopreservation.  It functions by crossing the cellular membrane to displace water from within the cells.  A slow freezing process of -1 degree Celsius per minute using a penetrating agent, like DMSO, not only freezes cells, but also dehydrates the cells.  While the cryopreservation process should be slow to prevent the formation of ice crystals inside or outside the cells, thawing frozen ampoules should be done rapidly in a 37°C water bath (or the recommended incubation temperature for the cells) to help prevent damage from the ice crystals.

However, even following the best cryopreservation techniques, one of the most troubling problems that a cell culturist will encounter is reduced post-freeze viability resulting from osmotic shock.  This phenomenon usually occurs when a liquid, be it reagent, dye, or complete growth media, is quickly added to thawed cells (or, vice versa).  Freshney states, "The cell suspension should be diluted slowly after thawing as rapid dilution reduces viability.  This gradual process is particularly important with DMSO, with which sudden dilution can cause severe osmotic damage and reduce cell survival by half" (Freshney, 2005).

It is also important to note that cells which have been exposed to DMSO have undergone a change in membrane permeability.  Trypan blue is an exclusion dye that is usually only taken up by non-viable cells due to damage to the cellular membrane.  However, cells exposed to DMSO might also inadvertently take up the dye, giving a false indication of low viability.  Centrifuging the cells to remove the DMSO prior to counting with an exclusion dye might help to circumvent this problem.  However, a better solution is to allow the cells some time in culture to recover from the stressful effects of cryopreservation before assaying viability and counting the cells.

More Info

Cryopreservation
Cryopreservation
ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide
atcc cryo
ATCC Best Practices in Cryopreservation – ATCC Webinar

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48. Is it necessary to remove the cryoprotective agent before putting the cells into culture?

When DMSO is used as the cryoprotectant, we recommend removing the DMSO prior to initiating the culture.  DMSO (dimethyl sulfoxide) is a powerful solvent and penetrating agent that can be toxic to some cell types.  If the concentration of DMSO in the cryopreservation medium is 5%, diluting the thawed culture to 15 ml total volume with complete growth medium may dilute the DMSO below the toxic concentration.  However, some cell types may be sensitive to even low levels of DMSO.  Therefore, we recommend removing the DMSO prior to initiating the culture.

  • Thaw the cells as instructed on the product information sheet and add the cells to a centrifuge tube containing 9 ml of complete medium.
  • Centrifuge the cells at 125 x g for 5 minutes.  It is important to centrifuge the cells gently.  Cells that have been cryopreserved in DMSO are porous and centrifugation at high speeds may damage the recovering cells. 
  • Aspirate the supernatant.
  • Gently resuspend the cells in 1-2 ml of the recommended complete medium and seed into the recommended tissue culture flask containing pre-warmed complete medium.

More Info

Cryopreservation
Cryopreservation
ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide
atcc cryo
ATCC Best Practices in Cryopreservation – ATCC Webinar

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49. Should cryopreservation be used for short-term storage, or will it shock the culture?

Cryopreservation is an excellent means of both long-term and short-term storage. It will not shock the culture, because cryopreserved cells stored at the coldest possible temperature are stable. As long as the cryopreservation technique is correct for the particular type of culture, cryopreservation is the most stable form of preserving cells. In addition, the genetic stability of the cells is better when the cells are kept in cryopreservation and thawed as needed than when the cells are kept in passage constantly.

More Info

Cryopreservation
Cryopreservation
ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide
atcc cryo
ATCC Best Practices in Cryopreservation – ATCC Webinar

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50. What is the best way to achieve the optimal cooling rate for cryopreservation?

There are several means to achieve a cooling rate of -1°C per minute. The best method uses a computer controlled, programmable electronic freezing unit (e.g., CryoMed Freeze) which rigorously maintains this rate of cooling. This is the method used exclusively at ATCC. Such equipment is expensive and is only absolutely necessary for the most sensitive cells.

A less costly approach is to place the cryopreservation vials into an insulated chamber and cool for 4 to 24 hours in a -80°C (or lower) mechanical freezer. The CoolCell LX is an alcohol-free, insulated freezing container and is available from ATCC, catalog number ACS-6000.  The CoolCell LX chamber maintains the optimal controlled cooling rate of -1°C per minute and allows you to transfer the vials from the CoolCell LX chamber into the liquid nitrogen tank after 4 hours at -80°C.

There are other commercially available freezing chambers that may achieve a cooling rate very close to the ideal -1°C per minute.  Many of these other freezing chambers use alcohol or other fluids to produce the slower cooling rate.

As a last resort, the vials can be placed into a Styrofoam box with 15-mm (3/4 inch) thick walls and 1-liter capacity packed with paper, cotton wool, or Styrofoam peanuts for insulation.

More Info

Cryopreservation
Cryopreservation
ATCC Animal Cell Culture Guide
ATCC Animal Cell Culture Guide
atcc cryo
ATCC Best Practices in Cryopreservation – ATCC Webinar

 atcc cool cells
ATCC CoolCell

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In Vitro Technologies and ATCC, use reasonable efforts to include accurate and up-to-date information in this technical help section, we make no warranties or representations as to its accuracy. In Vitro Technologies and ATCC are not responsible for any errors or omissions or for the results obtained from the use of this information. Content of the site is subject to change without notice. Any update on these FAQs related to ATCC technical advice will be available on the ATCC website