FAQ

Q.

How does Irvine Scientific define serum-free, animal-component free, protein-free, and chemically-defined media?

A.

Here is how Irvine Scientific defines these types of media:
 

  • Serum-free media do not contain or require the addition of serum.
  • Protein-free media do not contain any proteins but can contain undefined peptides from plant hydrolysates.
  • Irvine Scientific animal-component free products are manufactured with raw materials that do not contain any animal- or human-derived products to the tertiary level and are manufactured using equipment that does not come in contact with any animal or human products. The raw material handling, storage and final product manufacture and packaging are performed in dedicated human- and animal-component free facilities and equipment.
  • Irvine Scientific chemically-defined media do not contain any undefined animal-derived raw materials and are free of any animal-derived components, including human plasma or human blood-derived components. All raw materials are defined and their exact concentrations are known, which can include recombinant proteins.
Q.

Why should I use serum-free/chemically-defined media?

A.

Serum-free and chemically-defined media allow for a more consistent culture system (reducing lot-to-lot variation) because they do not contain undefined components that may be introduced by the addition of serum. Serum-free media are lower in protein content than medium supplemented with serum, which can simplify the purification process and increase the yield of the end-product. In addition, the end-user also does not need to take time to qualify and secure lots of FBS for activity.

Q.

What does Irvine Scientific have to offer in terms of serum-free, animal-component free, and chemically-defined media?

A.

Irvine Scientific offers many serum-free media, including products for three main types of cells currently used for bioproduction and bioprocessing: Chinese Hamster Ovary (CHO) cells, HEK293 cells, and Hybridoma cells. Within each cell line, we offer a suite of growth media and feeds.


CHO cells:

BalanCD® CHO Growth Medium and Feeds are chemically-defined, animal-component free and formulated without L-glutamine, Hypoxanthine, and Thymidine to be used in a variety of applications. BalanCD® CHO Feeds are specifically designed to increase process yields of antibodies and recombinant proteins in CHO cells.

BalanCD® Transfectory™ CHO System is chemically-defined and animal-component free. The system is designed for transient transfection in CHO cells to achieve higher titers and ensure process consistency.

HEK293 cells:

The BalanCD® HEK293 system platform includes a growth medium, feed and anti-clumping supplement. The scalable platform, which is chemically-defined and animal-component free, is optimized for the production of viral vectors and transient protein expression.

Hybridoma cells:

IS MAB-CD™ is a chemically-defined, low protein medium for the growth of hybridoma and myeloma cell lines for recombinant monoclonal antibody production. This chemically-defined medium is regulatory friendly and allows for improved lot-to-lot consistency compared to other formulations that are not chemically-defined.

Q.

When adapting cells from serum-containing to serum-free media, is the direct or sequential adaptation method better?

A.

The sequential adaptation method is recommended. Sequential adaptation allows the cells to gradually adapt to their new serum-free environment. Below is an example protocol. Cell types vary, and contact us for a tailored solution.


Suggested Sequential Adaptation Protocol:

  1. Start with cultures (suspension or adherent) that are at maximum cell density and high viability (>80%). If cells are attachment dependent, trypsinization procedures should be used.
  2. Split cells 1:2 using the desired final serum-free medium as the diluent.
  3. Incubate cells until the maximum cell density is achieved.
  4. Split cells again. The ratio may vary depending on whether the cells are growing attached or in suspension culture. For attached cells, try 1:2 or 1:3 split based on confluency. For suspensions, try 1:3 to 1:5 or approximately 3-4x105 cells per mL.
  5. Continue incubation until optimal cell density is achieved. (Maximum may imply over confluency which should be avoided generally.)
  6. Monitor the growth and viability of the cells relative to cells cultured in the original or control medium. Cells that have been successfully adapted to serum-free media should have the viability greater than 85%, with a doubling time equivalent to the control medium (serum-containing or other serum-free medium) from which the culture was adapted.
  7. Low viability or poor doubling times indicate that the culture may not yet be adapted. 3 to 5 successive splits using 1:2 to 1:3 may be necessary. It is not uncommon for some cells to continue to require 0.1 to 0.5% serum.
  8. Time for adaptation to serum-free conditions will vary with the growth characteristics of the particular cell type. If at any time during the weaning process the culture viability drops below 80%, or the time to double increases significantly, weaning may have occurred too quickly. The cells may require several passages at the previous dilution before renewal of the sequential weaning process resumes.
Q.

Why was L-Glutamine eliminated from many of your formulas?

A.

L-Glutamine is easily hydrolyzed in liquid and byproducts of hydrolysis can be toxic to the cell. Additionally, the necessary concentration of L-Glutamine may be compromised after a period of time. Since L-Glutamine is an essential amino acid, it is best for the end users to supplement media with the recommended concentration of fresh L-Glutamine.

Q.

How do we prevent mycoplasma contamination in cell culture?

A.

Good aseptic techniques should be used in all laboratory applications. The best method for prevention is to test all materials that come in contact with the cells. This should be done on a regular basis to ensure that mycoplasma is not introduced into the cell culture and to identify any potential sources of contamination.

Q.

What are the minimum requirements for a custom media formula?

A.

We generally require a 100L minimum order to manufacture a custom formula. Our Manufacturing Support and Service (MSS) can make smaller non-GMP quantities to meet special investigational or development needs for our customers.

Q.

What is the timeline on a custom formula?

A.

Customs are usually delivered to the customer between 8 to 10 weeks after we receive the final signed documents with a purchase order.

Q.

Why was L-Glutamine eliminated from many of your formulas?

A.

L-Glutamine is easily hydrolyzed in liquid and by-products of hydrolysis can be toxic to the cell. Additionally, the necessary concentration of L-Glutamine may be compromised after a period of time. Since L-Glutamine is an essential amino acid, it is best for the end-users to supplement media with the recommended concentration of fresh L-Glutamine.

Q.

What effects could freezing and thawing have on my sera?

A.

We generally discourage multiple freezing and thawing of sera. Sera contain proteins, lipids, and other components that may be differentially separated or precipitated out of solution, depending upon the rate of thawing or freezing. This may adversely affect the performance of the sera. The best storage is in a freezer at -20°C or lower.

Q.

How do we prevent mycoplasma contamination in cell culture?

A.

Good aseptic techniques should be used in all laboratory applications. The best method for prevention is to test all materials that come in contact with the cells. This should be done on a regular basis to ensure that mycoplasma is not introduced into the cell culture and to identify any potential sources of contamination.

Q.

When adapting cells from serum-containing to serum-free media, is the direct or sequential adaptation method better?

A.

The sequential adaptation method is recommended. When cells are transferred to serum-free medium from a serum-rich environment, the cells are deprived of components in the serum-rich environment. Sequential adaptation allows the cells to gradually adapt to their new serum-free environment. Below is an example protocol. Cell types vary. Contact us for a tailored solution.

Suggested Sequential Adaptation Protocol:

  1. Start with cultures (suspension or adherent) that are at maximum cell density and high viability (>80%). If cells are attachment-dependent, trypsinization procedures should be used.
  2. Split cells 1:2 using the desired final serum-free medium as the diluent.
  3. Incubate cells until the maximum cell density is achieved.
  4. Split cells again. The ratio may vary depending on whether the cells are growing attached or in suspension culture. For attached cells, try 1:2 or 1:3 split based on confluency. For suspensions, try 1:3 to 1:5 or approximately 3-4x105 cells per mL.
  5. Continue incubation until maximum cell density is achieved.
  6. Monitor the growth and viability of the cells relative to cells cultured in the original or control medium. Cells that have been successfully adapted to serum-free media should have a viability greater than 85%, with a doubling time equivalent to the control medium (serum-containing or other serum-free medium) from which the culture was adapted.
  7. Low viability or poor doubling times indicate that the culture may not yet be adapted. 3 to 5 successive splits using 1:2 to 1:3 may be necessary. It is not uncommon for some cells to continue to require 0.1 to 0.5% serum.
  8. Time for adaptation to serum-free conditions will vary with the growth characteristics of the particular cell type. If at any time during the weaning process the culture viability drops below 80%, or the time to double increases significantly, weaning may have occurred too quickly. The cells may require several passages at the previous dilution before renewal of the sequential weaning process resumes.
Q.

Can you customize my serum-free media?

A.

All the formulas we offer may be tailored to meet our customers' specific requirements. Whether efforts are focused on optimizing yields or improving productivity, we can work with any lab to address specific needs.

Q.

What does Irvine Scientific have to offer in their serum-free, animal-component-free, or chemically-defined lines of media?

A.

Irvine Scientific offers many serum-free media, including products for three (3) main types of cells currently used by Biotech or Pharmaceutical companies: 293 cells, Chinese Hamster Ovary (CHO) cells, and Hybridoma cells. Within each cell line, we offer a variety of media:

HEK 293 cells:  We offer one medium formulated for optimal cell growth and viral production.

CHO cells:  We offer six (6) different formulations with three (3) feed media formulations.   All of these products are animal-component-free and are free of L-glutamine, Hypoxanthine, and Thymidine to allow for use in a variety of applications.

Hybridoma cells: We offer four (4) different formulations.

  • IS GRO™ (Catalog ID: 91105)IS PRO™ (Catalog ID: 91103) is our dual media system which comprised of low protein, serum-free media and optimal for hybridoma growth and production. IS GRO™ has been optimized for maximal hybridoma cell growth and is recommended for use during the growth phase; IS PRO™ has a very low protein concentration for use during the production phase to simplify protein purification.
     
  • HB Basal Liquid (Catalog ID: T000) is a serum-free and chemically defined medium which is designed to support the growth of murine and human hybridomas, myelomas, lymphoblastoid, and endothelial cells, as well as murine T-cell hybrids.
     
  • IS MAB-CD™(Catalog ID: 91104) is a chemically-defined, low protein medium for the growth of hybridoma and myeloma cell lines for recombinant monoclonal antibody production. This chemically-defined medium is regulatory friendly and allows for improved lot-to-lot consistency compared to other formulations that are not chemically defined.
Q.

Why should I use serum-free media?

A.

Serum-free media offer the customer better lot-to-lot consistency, because they contain fewer undefined components than media containing undefined components, such as serum. The end-user also does not need to take time to qualify lots of FBS for activity. Serum-free media are lower in protein content than medium supplemented with serum, which can simplify the purification process and increase the yield of the end-product.

Q.

What are the differences between serum-free, protein-free, and chemically-defined media?

A.

Serum-free media do not contain or require the addition of serum. Animal-component-free media are media in which none of the components are animal-derived. Protein-free media do not contain any proteins. Chemically-defined media contain components which are all known (or defined chemically).

Q.

Why would I heat-inactivate my serum?

A.

Heat inactivation is a process used to inactivate protein complement components that are present in the serum. The proteins from the complement cascade have the potential to impact or interfere with certain cellular processes.

Q.

What is the glucose concentration in the HEK 293 medium and feed?

A.

BalanCD HEK 293 medium contains 6 g/L glucose and BalanCD HEK293 Feed contains 40 g/L glucose.

Q.

How should adherent HEK 293 cells (banked in a FCS containing medium) be adapted into suspension in BalanCD HEK 293 medium?

A.

We recommend starting with adapting adherent HEK cells to suspension culture in BalanCD HEK 293 medium with serum (the same concentration the cells have been adapted to). Follow the standard protocol we suggest in our product insert (125 mL shake flask). Using the suggested protocol, customers can add additional 0.1% of poloxamer to protect cells in suspension against possible damage during stirring in shake flask. After the cells are adapted to suspension culture, take the cells in HEK 293 medium with X% FBS and gradually taper off to 0% FBS. The adaptation to 0% FBS should be quick but growth in suspension may be difficult and take a while. We highly recommend banking the cells after each adaption.

Q.

Can BalanCD HEK 293 medium be used for HEK 293 transfected cells to make stable suspension cells?

A.

Yes. BalanCD HEK 293 can be used for HEK 293 transfected cells to make stable suspension cells. The customer may need to add Anti-Clumping Supplement to prevent aggregation.

Q.

What is the preferred transfection method to be used with BalanCD HEK 293?

A.

Our recommended method is PEI-Mediated Transfection, please refer to the product insert for the general guideline. BalanCD HEK 293 medium is also compatible with other transfection methods using cationic liposomes or electroporation. Optimization of transfection parameters is highly encouraged, since optimal transfection parameters may vary depending on the application.

Q.

What is recommended feeding guide for BalanCD HEK 293?

A.

BalanCD HEK293 Feed can be evaluated with the suggested standard feed method shown in the product insert. For transient transfection, we advise to test 12-20% of the initial culture volume in total, at a maximum of 5% daily for 4 feeding events. For stable transfection, we advise to test 10-25% of the initial culture volume in total, at a maximum for 5 feeding events. Nutrient depletion analysis in the media can be used to optimize the feeding schedule if necessary. If feeds need to be used at more than 20-25% of the initial culture volume, monitor osmolality to ensure that is it not above 400 mOsm/kg.

Q.

Does BalanCD HEK293 Feed inhibit the cell doubling time but still maintain the VCD? Is the addition of valporic acid or sodium butyrate needed to inhibit cell growth?

A.

BalanCD HEK 293 Feed is used to improve cell growth and protein production and will not inhibit cell growth. Valporic acid and sodium butyrate can be added to enhance production but will arrest cell growth.

Q.

How can cell aggregation be removed in BalanCD HEK 293?

A.

If severe cell aggregation is observed, supplement with Anti-Clumping Supplement. Anti-Clumping Supplement is designed for use at a 1:200-1:1000 dilution (1-5 mL/L) range. Typically used at 2 mL/L. Supplement additional amount if necessary.

Q.

What is the Anti-Clumping Supplement? My cells are starting to clump at high cell concentrations and I consider using this supplement, but I am not sure if it will interfere with virus infection and production.

A.

Anti-Clumping Supplement may be added to culture if cells start to aggregate. If Anti-Clumping Supplement is added, it must be eliminated from the culture media prior to transfection. If cells aggregate post transfection, Anti-Clumping Supplement can be added at least one day post transfection. Up to 10 mL/L of Anti-Clumping Supplement can be added if there are larger aggregates.

Q.

What is the best way to measure pH once the powder has been hydrated in WFI? Can phenol red be added to measure the pH?

A.

We use a pH meter to measure the pH of our media. Phenol red can be added to the medium except when an assay involving fluorescence is performed since phenol red will contribute to the background.

Q.

What is the stability data of the BalanCD HEK 293 medium at room temperature (20°C) and 37°C?

A.

We currently do not have stability of this product at 20°C or 37°C in powder format. We recommend 2-8°C storage.

Q.

Does adding GlutaMAX-I improve the media quality as opposed to L-Glutamine at the same 4 mM concentration? Can GlutaMAX-I and L-Glutamine be used interchangeably?

A.

We typically recommend the addition of L-Glutamine. GlutaMAX is the dipeptide form of Glutamine which allows for longer stability in culture. The L-Glutamine form is more readily available to the cells early in culture when it is most critical. However, customers use GlutaMAX interchangeably without any negative effects.

Q.

What is the concentration of Pluronic or equivalent in BalanCD HEK 293 medium?

A.

BalanCD HEK 293 contains 0.1% poloxamer; however, an additional 0.05% to 0.1% can be supplemented if necessary.

Q.

Does BalanCD HEK 293 medium contain phenol red?

A.

No, BalanCD HEK 293 does not contain phenol red.

Q.

What is FUJIFILM Irvine Scientific’s policy for disclosing formulation information?

A.

Different levels of details can be shared depending on the request, and FUJIFILM Irvine Scientific will work with our customers to address their formulation inquiries on a case by case basis. Please contact us with your formulation requests.

Q.

How do we prevent mycoplasma contamination in cell culture?

A.

Good aseptic techniques should be used in all laboratory applications. The best method for prevention is to test all materials that come in contact with the cells. This should be done on a regular basis to ensure that mycoplasma is not introduced into the cell culture and to identify any potential sources of contamination.

Q.

Should I be concerned with mycoplasma in my cell culture?

A.

Mycoplasma contamination should always be a concern. Mycoplasma contamination can change the morphology of the cells and interfere with bioassays, cell growth, viability, and production.

Q.

What is the timeline on a custom formula?

A.

Customs are usually delivered to the customer between 6 to 8 weeks after we receive the final signed documents with a purchase order.

Q.

What are the minimum requirements for a custom media formula?

A.

We generally require a 100L minimum order to manufacture a custom formula. Our Express Media Service™ can make smaller non-GMP quantities to meet special investigational or development needs for our customers.

Q.

Can you customize my serum-free media?

A.

All the formulas we offer may be tailored to meet our customers' specific requirements. Whether efforts are focused on optimizing yields or improving productivity, we can work with any lab to address specific needs. Visit our Custom Media Services page for more details.