Mammalian Cell Culture Basics Support—Getting Started

Yes, we are ISIA Tracability Certified effective February 12, 2014. Please read more here.

Please use our media formulation search tool.

Yes. Store animal sera at –5 to –20°C. Store media at 2 to 8°C; use within recommended shelf life. Store complete media (supplemented) at 2 to 8°C, and for complete medium the recommended shelf life is 2 to 4weeks. Additionally, minimize exposure of sera and media to light.

Please see the comparison chart here.

The regulations and recommendations for biosafety in the United States are contained in the document Biosafety in Microbiological and Biomedical Laboratories, prepared by the Centers for Disease Control (CDC) and the National Institutes of Health (NIH), and published by the U.S. Department of Health and Human Services. The document defines four ascending levels of containment, referred to as biosafety levels 1 through 4, and describes the microbiological practices, safety equipment, and facility safeguards for the corresponding level of risk associated with handling a particular agent. 

Biosafety Level 1 (BSL-1): BSL-1 is the basic level of protection common to most research and clinical laboratories, and is appropriate for agents that are not known to cause disease in normal, healthy humans. 

Biosafety Level 2 (BSL-2): BSL-2 is appropriate for moderate-risk agents known to cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. Most cell culture labs should be at least BSL-2, but the exact requirements depend upon the cell line used and the type of work conducted. 

Biosafety Level 3 (BSL-3): BSL-3 is appropriate for indigenous or exotic agents with a known potential for aerosol transmission, and for agents that may cause serious and potentially lethal infections. 

Biosafety Level 4 (BSL-4): BSL-4 is appropriate for exotic agents that pose a high individual risk of life-threatening disease by infectious aerosols and for which no treatment is available. These agents are restricted to high containment laboratories.

For more information about the biosafety level guidelines, refer to Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, which is available for download at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.

Most normal mammalian cell lines grow well at pH 7.4, and there is very little variability among different cell strains. While most researchers usually use 5–7% CO₂ in air, 4–10% CO₂ is common for most cell culture experiments. Read more about buffering conditions here.

Aseptic technique, designed to provide a barrier between the microrganisms in the environment and the sterile cell culture, depends upon a set of procedures to reduce the probability of contamination from these sources. The elements of aseptic technique are a sterile work area, good personal hygiene, sterile reagents and media, and sterile handling. Click here to read more about aseptic technique or view our checklist.

There are two basic systems for growing cells in culture, as monolayers on an artificial substrate (i.e., adherent culture) or free-floating in the culture medium (suspension culture). The majority of the cells derived from vertebrates, with the exception of hematopoietic cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading (i.e., tissue-culture treated). However, many cell lines can also be adapted for suspension culture. Similarly, most of the commercially available insect cell lines grow well in monolayer or suspension culture. 

Cells that are cultured in suspension can be maintained in culture flasks that are not tissue-culture treated, but as the culture volume to surface area is increased beyond which adequate gas exchange is hindered (usually 0.2–0.5 mL/cm²), the medium requires agitation. This agitation is usually achieved with a magnetic stirrer or rotating spinner flasks. 

Find a comparison chart between the two here.

Subculturing, also referred to as passaging, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain. Read more about cell growth and subculturing here

Please see our protocol here for freezing cells.

Please see our protocol here for thawing frozen cells.

Please see the general directions here regarding how to use the hemocytometer.

The following procedure will enable you to accurately determine cell viability.

You can find a protocol on our web site. In the main search box type "Media Preparation from Powder and Concentrates" and hit Enter or click Search. This should take you to a detailed protocol page.

Gibco™ cell culture media is formulated using water meeting all USP monograph requirements for Water For Injection.

No, you do not need to adjust the pH of the liquid media when you receive it. It should be in the correct pH range for use. After you add your supplements (if needed), you can check the pH again and adjust if necessary. However, usually it does not change significantly. It would be necessary to pH media that you make up yourself from a powder, with the exception of AGT™ media where pH and osmolality are auto-adjusted. 

This water is purified by distillation or by reverse osmosis. It contains no added substances. It cannot be used for vivo injection because it is not packaged in a single-dose glass or plastic container to protect the water from contamination. It is intended for use as a solvent for the preparation of parenteral solutions, such as media and reagents.

These two products are the same water. However, Water for Injection (WFI) for Cell Culture has undergone more testing, including conductivity measurement as well as testing for nitrate and total organic carbon.

Gibco™ Cell Culture Bags are convenient, pre-filled bags in a variety of formulations and sizes, specifically designed to increase productivity and reduce costs. The bags can be formatted up to 1,000 L. Read more about these bags here.

Based on feedback from customers, we designed a media bag portfolio that uses a consistent film for all bag sizes. This film, called PE film, is superior in function to other commercially available films. Click here to read more about the PE Film specifications.

The commercially available film is a very clear, multi-layered, single web film with a polyethylene contact surface that can be made into a wide range of bag sizes (250 mL to 1,000L). In addition, the film is a market leader with exceptional gas barrier properties and very low extractable and leachable results. Click here to read more about the PE Film specifications.

Each bag size will arrive in a specific heavyweight corrugated box designed for shipment: 

 5 L: 12” x 14.25” x 5.5” 

 10 L: 13” x 23.5” x 6.125” 

 20 L: 15” x 24” x 6.125” 

Yes. Although we feel that the PE film exceeds many of the technical properties of the other available films, we will continue to offer and support our other currently available films through custom orders.

Almost all Gibco™ cell culture basal media products can be customized using the Gibco™ Media Configurator. To check on a particular product, use the Media Configurator page to search by Gibco™ catalog number, or browse our offering by description. For the customization of non-media Gibco™ products, such as buffers and growth factors, contact us at custommedia@lifetech.com. 

No, the Gibco™ Media Configurator cannot be used to request pricing on non-Gibco cell culture products. However, we are very experienced at manufacturing customer-owned media formulations. Please visit www.thermofisher.com/custommedia or contact us at custommedia@lifetech.com for more information. 

Using the Gibco™ Media Configurator, you can view specific customization options for your cell culture product, online, at any time. Also, this tool provides faster pricing for custom media, generally within 48 business hours. 

Yes, there are price breaks for most configurations based on the volume purchased. 

Yes, a Gibco™ catalog number is required to use the Gibco™ Media Configurator, as the customization options are tailored to a specific formulation. 

If you are not currently using a similar Gibco™ formulation, browse our selection of Gibco™ products using the link found on www.thermofisher.com/mediaconfigurator. 

No, it is not a requirement to be logged in to use the Gibco™ Media Configurator; however, it may expedite your request. In addition, if you are logged in, your quote requests will be stored in your history, facilitating future requests. 

You will be notified if the product you need is already available as a stocked catalog product. All custom media requests undergo a comprehensive review by our feasibility engineers prior to production—it benefits both of us if your product can be shipped right away! 

We have a dedicated team of Gibco™ Custom Media Specialists who can partner with you during customization, manufacturing, and delivery. Please email custommedia@lifetech.com or call the local phone number listed on www.thermofisher.com/mediaconfigurator with inquiries about your submission. 

For questions related to website effectiveness, please contact System Support through the toll-free or local phone number. 

Yes. If you were logged into the website while requesting your Gibco™ Media Configurator quotation, simply log back into thermofisher.com, retrieve the form from your Favorites, adjust it, and resubmit. If you were not logged in, please contact the Gibco™ Custom Media Specialist team for assistance. 

Yes. Our Gibco™ Custom Media Specialists and R&D staff can provide advice related to the addition or elimination of media components. For more comprehensive services, our PD-Direct™ Bioprocess Services team specializes in media development and optimization. 

 The minimum order volume is generally 1 L for non-cGMP manufacturing (Gibco™ Media Express™) and 10 L for cGMP production. There may be exceptions, which will be displayed by the Media Configurator. 

When you are ready to place your first order, contact a Gibco™ Custom Media Specialist or your local Account Manager. Regional contact information for our Gibco™ Custom Media Specialists can be found at www.thermofisher.com/mediaconfigurator. 

Yes. If you are reordering the same formulation, in the same quantity and packaging, simply use the Quick Order link found at the top of every page of thermofisher.com, or visit your Order History page and click on the Reorder box. 

Contact your local Account Manager or your Custom Media Specialist for questions related to pricing. Custom price quotes are valid for 90 days, unless stated otherwise. 

Manufacturing lead times can vary based on formulation, availability of raw materials, batch size, QC test duration, and overall customer demand. We monitor lead times closely, and we will provide you with an estimated time frame for delivery when you place an order. You can also discuss delivery requirements with your Gibco™ Custom Media Specialist or Account Manager. 

Please view the following page to browse the cell culture antibiotics we offer.

Amphotericin B is the generic version of Fungizone. Fungizone is a trademark of E.R. Squibb & Sons, LLC.

The serum concentration will vary with the cell line and basal medium used. Please click here to see our recommended sera supplementations for tested cell lines.

None. The term "reduced serum" means that one can reduce the amount of serum they use with classical cell culture media. When working with Opti-MEM™ I medium, most cells that are routinely cultured in serum-supplemented medium may be transferred directly into Opti-MEM™ I medium with a minimum of 50% reduction in serum.

The Advanced media allow a reduction in serum usage from 50 to 90%. In the majority of cell lines, a 50% reduction can be achieved with no weaning procedure. Further reductions can be realized by weaning cells gradually. The amount of reduction in serum percentage beyond 50% will be cell line dependent. The advantages of using less serum include cost savings, particularly during those times when serum prices increase, extending the life of your serum lot, and reduced variability.

For more information on the Advanced DMEM and Advanced MEM, please search "Advanced Media" from our website home page.

Fetal Bovine Serum (FBS) was once known as Fetal Calf Serum (FCS). They are one and the same thing.

Remove serum from the freezer and allow it to thaw in the refrigerator at 2–8°C overnight. The thawing process may then be completed at room temperature. The serum must be regularly mixed during this process. Warning: Do not incubate FBS at 37°C for extended periods of time. The product may become cloudy, and performance could be affected due to the sensitivity of many serum components. Once it is thawed, you can use the product immediately.

No, there is no need to filter the serum before use. Gibco™ serum products are manufactured using the most stringent processes, including membrane filtration.

Yes. We offer a wide range of custom options. Please contact our Sera Specialists to discuss your application and research needs. We will work with you to supply sera specially processed and tested to meet your individual requirements. Some examples of our custom capabilities are heat inactivation, gamma irradiation, and testing for tetracycline residues.

Gamma irradiation is recognized as an effective method for inactivating viruses in animal-origin material. Based on USDA regulations for the general requirements for antibody products (9CFR, Section 113.450), the minimum dosage for blood derivatives of animal origin is 25 kGy. Certain European countries require products to be treated prior to importation with a minimum dose of 25 kGy.

We will gamma-irradiate serum on request. We have validated a process for utilizing gamma irradiation in the range of 30–45 kGy to inactivate the most common bovine viruses and mycoplasmas that may be present in FBS. The level of inactivation is 6–8 logs for viruses and 6–7 logs for mycoplasmas. We have also demonstrated that physiochemical properties and cell culture performance of serum is not altered by gamma irradiation at levels of 30–45 kGy. 

Gamma irradiation is generally recognized by regulatory agencies as the best method of virus inactivation and for reducing the risk of viruses that are naturally present in animal sera. We offer a small range of gamma-irradiated products ex-stock. Gamma irradiation is also available on request as a custom service for most standard catalog products. We offer a range of fully validated custom processes covering many dose ranges.

The heating process inactivates portions of the complement cascade. Complement occurs in the following events: cytolytic activities, contraction of smooth muscle, release of histamine from mast cells and platelets, enhanced phagocytosis, chemotaxis, and activation of lymphocytic and macrophage cell types.

You can incubate the thawed product at a thermostatically controlled temperature of 56°C for 30 minutes. Serum can be heat-inactivated in both our plastic and glass bottles. Warning: Do not attempt to heat-inactivate at a higher temperature for prolonged periods as this may compromise the product’s performance through protein denaturation.

Gibco™ FBS is available in 100 mL, 500 mL, and 1,000 mL bottles and by special order in 3.5 L and 4 L volumes.

BVD stands for bovine viral diarreah. It is one of the most common viral infections in cattle. It is estimated that 70 to 90 percent of the world's cattle population is seropositive for BVD. This virus can cause abnormalities and fetal abortions in cattle. Several strains of BVD exist, some of which are non-pathogenic. 

In cell culture applications, the Code of Federal Regulations (9CFR) has requirements for cell lines as well as ingredients used for the production of biologicals. It requires that the serum used for the production of biological products have a negative result for BVD virus.

BVD virus testing on Gibco™ serum is performed according to 9CFR protocols. An in-house specification of 0 to 4 is in place. A result of 0 indicates that a lot of serum is negative for the virus when tested according to 9CFR protocols. A result of 1–4 indicates that the lot of serum is positive for the virus. A +1 indicates low fluorescence and a +4 indicates high fluorescence for the virus. The word 'tested' is reported on the certificate of analysis for this test and not the actual BVDV fluorescent antibody value.

A lot of serum may have a 0 result when tested according to 9CFR protocols but be positive when tested using newer and more sensitive technologies such as PCR. To guarantee that a lot of serum be 'truly' negative for BVD virus, serum treatment such as gamma irradiation should be employed.

FBS is tested for tetracyline. The specification is “None Detected”, and this is listed on the COA. Minimum level of tetracycline that can be detected by the assay is 19.7 ng/mL. 

It is dialyzed by tangential flow filtration against 0.15 M NaCl using a 10,000 molecular weight cut-off membrane until the glucose level is less than 5 mg/dL. Since this is not exhaustive dialysis, low–molecular weight dialyzable components, such as amino acids may not be totally removed. Exhaustive dialysis is not performed because it can result in precipitation and inactivation of serum peptides.

It is a quality indicator that reflects rapid collection and processing.

Usually the best way to find out is to go to the source you obtained your cells from. For example, ATCC will have serum requirements for the cells they sell. Most insect cell lines and embryonic stem cell lines require heat-inactivated serum. 

Phenol red in tissue culture is a weak estrogen. Implications concerning study of estrogen-responsive cells in culture are discussed in Proc Natl Acad Sci U S A 83:2496 (1986). 

Phenol red is used as a true ion-front tracking dye for the very small pore–size gels, especially Tricine gels. Phenol red is smaller than Coomassie™ Blue G-250. In high percentage gels, molecules in this size range are resolved on the basis of size, such that phenol red runs with the ion front while G-250 can run as much as 2 cm behind the ion front. In gels suitable for resolving larger proteins, including the NuPAGE™ gels, G-250 runs with the ion front and is generally the more useful tracking dye.

Yes, please visit this page for a list of our phenol red–free media.

Sodium pyruvate serves as an additional energy source for cells in culture. It is often added to low-glucose formulations (1.0 g/L glucose) and is sometimes added to higher-glucose formulations as well. Cells can become "hooked" on sodium pyruvate however, and if it is withdrawn suddenly from the media, they may experience a short lag in growth.

Sodium pyruvate (Cat. No. 11360070) is supplied as a liquid supplement for cell culture media at 100 mM (100X) and a concentration of 11,004 mg/L.

GlutaMAX™-I supplement is an improved cell culture supplement that can be used as a direct substitute for L-glutamine in your cell culture medium. Please click here to read more about the properties of GlutaMAX™ supplement.

The MEM Vitamin Solution (Cat. No. 11120-052 or 11120-037 depending on your country) is a growth supplement for cell culture medium. This solution is formulated to contain 100X the vitamins found in standard MEM. Find the complete formulation here.

The MEM Amino Acids Solution is used as a growth supplement for cell culture medium, to help increase cell growth and viability. It is formulated to contain 50X the essential amino acids (except L-glutamine) found in the standard MEM. Find the complete formulation here. 

Granulated cell culture media is an easy-to-use, granular form of dry media that is produced by applying fluid bed granulation technology to cell culture nutrient media manufacturing. The patented process allows production of complete formulations of a variety of serum-free, protein-free, and chemically defined media in a dry format. 

AGT™ offers the benefits of liquid media without the cost, storage, and transportation issues. It is a complete medium that is pH and osmolality pre-adjusted. The granules dissolve instantly for faster media preparation time than conventional dry powder media. AGT™ media can therefore help reduce total cycle costs, decreasing time involved in raw material planning, procurement, and testing as well as media preparation. These benefits are all realized while maintaining comparable cell growth to liquid media.

Complete formulations 

• Homogeneous distribution of minute components 
• Complete complex formulations in a dry format, including serum-free, protein-free, and chemically defined nutrient media 
• Pre-adjusted to the appropriate pH and osmolality 
• Require only standard supplementation with L-glutamine for L-glutamine dependent systems 
• Can be customized for modified catalog and customers’ proprietary formulations 

Granular format 

• Dissolves instantly for faster media preparation times 
• Dust-free granules reduce mess and environmental contamination 
• Performance equivalent to liquid media 
• Workflow improvement 
• Helps reduce total cycle costs due to lower number of components, decreasing time involved in raw material planning, procurement, and testing 
• Faster and easier media preparation, with less potential for error 
• Easy to scale up from research through production, in batch sizes from 2 kg to 6,000 kg 
• Available in various packaging options

MEM Non-Essential Amino Acids are used as a supplement for cell culture medium, to increase cell growth and viability. MEM Non-Essential Amino Acids contains the same non-essential amino acids found in the standard Minimum Essential Medium (MEM) at a strength of 100X. The complete formulation is available here.

Once reconstituted at 1X, 2X, and 3X concentrations, GlycanTune Total Feeds can be stored at room temperature (21-22 degrees C), in the dark, where they are stable for approximately 30 days.

The AGT dry format has a 12 month shelf-life.

We can provide DMF (drug master file) support or other support as needed.

It is a feed that allows you to shift the glycan profile from 90% G0F to as low as 45% G0F without significant loss of performance.

You just need to add water according to the instructions provided in the manual (https://tools.thermofisher.com/content/sfs/manuals/MAN0014673_glycantune_c_totalfeed_PI.pdf). No pH adjustment is needed

We recommend using this product for exosome-specific assays, but you may also use it to culture the cells prior to the assay to minimize background signaling in your cells. We recommend determining the concentration to use, but 5–10% FBS is the most common.

Exosome-depleted FBS undergoes full sterility testing like our other sera products. It is subjected to additional tests for exosome depletion and a specific cell culture performance test.

The Exosome-depleted FBS that we ship to you will contain ≤10% of the exosomes present in the starting material FBS.  We use an in-house fluorescence assay specifically developed for exosome quantification to confirm this level of depletion in every lot. 

Yes, this has been tested. The product can be thawed into aliquots and refrozen.

We have developed a proprietary process to remove exosomes and maintain as much of the growth performance as possible.

We have specifically tested Jurkat, MCF-7, HeLa, PC3, HEK293, and A549 cells, all of which exhibited acceptable performance. HeLa cells showed the most sensitivity to the presence of Exosome-depleted FBS. Adjustments may be needed for the concentration of Exosome-depleted FBS to match the requirements of the specific cell line and system. We recommend starting at 10% Exosome-depleted FBS, and adjusting up or down from there as necessary. Also, the Exosome-depleted FBS has been tested and validated for a single passage of cells for 48 hours (and we have also tested HeLa, MCF-7, and HEK293 cells successfully up to 96 hours of culture).

Dynamis™ medium was designed to be more scalable in AGT™ dry format than CD FortiCHO™ liquid medium. Although there are many formulation similarities, the key differences are equimolar substitutions of alternate forms of some components as well as addition of a small amount of a component found in many other basal media.

When using a CHO cell line other than Lonza’s GS Gene Expression System™, addition of 2–8 mM L-glutamine or similar amount of GlutaMAX™ Supplement is recommended. Additionally, we recommend that you do not allow glucose levels to fall below 2 g/L in culture by supplementing with glucose as needed (see product insert for more details).

When culturing cells without complete feeds for fed-batch production, we recommend that you do not to allow glucose levels to fall below 2 g/L in culture by supplementing with glucose as needed (see product insert for more details). We have found that EfficientFeed™ A+, B+, and/or C+ supplements work well with Dynamis™ medium to maximize titers. We recommend trying EfficientFeed™ C+ 1X and 2X first, and if specific productivity drops off late in culture try the addition of FunctionMAX™ Titer Enhancer. No supplements should be added before reaching the day when half-peak cell density is reached for cultures not fed with complete feeding supplements like the EfficientFeed™ supplements

We have done some studies to show comparability of results between the two media, and that information may be filed with agencies in support of any work your Regulatory Team feels is necessary to establish transitioning from the one medium to the other. It is possible that characterization of your expressed protein after you see no other titer changes will be viewed no differently than a CMC update or minor modification to feeding profile during technical transfer to manufacturing. Of course our Regulatory Team would be happy to have a discussion with your team if it makes this transition any simpler for you.

Please use this selection chart that compares our cell dissociation reagents.

TrypLE™ reagent is free of animal- and human-derived components, producing an exceptionally pure reagent that is gentle on cells. Inactivation with trypsin inhibitors is not required. TrypLE™ reagent is ideal for dissociating a variety of attachment-dependent mammalian cell lines both in serum and in serum-free conditions, and can be directly substituted for trypsin in your current protocol.

TrypLE™ reagent is room-temperature stable and ready to use when you need it. TrypLE™ cell dissociation reagents remain stable for 24 months at room temperature, making storage and handling easier and more convenient.

Both of the products contain rProtease, non-animal, trypsin-like enzyme formulation used for the dissociation of attachment-dependent cell lines. This alternative for porcine trypsin is a recombinant enzyme derived from microbial fermentation.

The Express and Select products differ in their production and testing methods and in the documentation provided. TrypLE™ Select is for the industrial market and has release-testing both for endotoxins and enzyme activity. This material also has a Drug Master File on record. It is manufactured in AOF-dedicated equipment and has Intended Use for Research Use/Further Cell Culture Manufacturing.

TrypLE™ Express is manufactured without use of dedicated equipment and is intended for Research Use Only. Not for use in diagnostic procedures.

To read more about TrypLE™ and cell release times using this reagent, please click here.

Yes, the TrypLE™ products contain rProtease, a non-animal, trypsin-like enzyme used for the dissociation of attachment dependent cell lines. TrypLE™ enzyme has demonstrated the ability to dissociate cells cultured both in serum-free and serum-supplemented systems. The catalog numbers for TrypLE™ Select are 12563-011 (100 mL) and 12563-029 (500 mL). Some example catalog numbers for TrypLE™ Express include 12604-013 without Phenol red (100 mL) and 12605-010 with Phenol red (100 mL). Both formulations are also available in additional sizes. All these products are animal origin–free.