Gibco™ ExpiCHO™ Expression Medium

Product Code.	Quantity	unitSize
15421783	1000 mL	1000mL
15492163	6 x 1 L	6 L
15471044	20 L	20 L
15461044	10 L	10 L

Product Code. 15421783 Supplier Gibco™ Supplier No. A2910001

Description

A GMP-manufactured, chemically-defined, serum-free, protein-free, animal origin–free medium specifically designed for high-density growth and transfection of suspension-adapted Chinese hamster ovary (CHO) cells.

ExpiCHO Expression Medium is specifically designed for high-density growth and transfection of suspension-adapted Chinese hamster ovary (CHO) cells. This medium is GMP-manufactured, chemically-defined, serum-free, protein-free, and animal origin–free. It is a core component of the ExpiCHO Expression System Kit (Cat. No. A29133) for scalable transient protein production. To achieve the highest levels of transient protein expression, the medium is matched to a culture feed, ExpiCHO Feed, also included in the system kit and transfection kits.

With ExpiCHO Expression Medium you can:

• Grow suspension CHO cultures to densities of over 2 x 10⁷ cells per mL, with >95% cell viability
• Achieve high transfection efficiencies of >90% when coupled with ExpiFectamine CHO transfection reagent
• Transiently transfect suspension CHO cells at a density of 6 x 10⁶ cells per mL
• Achieve yields up to 3 g per L of recombinant protein of high-density transiently transfected cultures by sustained, high-level expression
• Grow and transfect CHO cells at volumes of <1 mL up to 5 L in culture formats from multiwell plates up to 5 L shake flask

ExpiCHO Expression Medium is ready to use. It is formulated with GlutaMAX-I reagent and does not require supplementation. It does not contain phenol red. The chemically-defined, animal origin free formulation results in high reproducibility and lot-to-lot reliability. ExpiCHO Expression Medium is not recommended for adherent cell cultures.

Specifications

• Cell Line: ExpiCHO-S™, CHO-S, SFM Adapted
• Cell Type: Chinese hamster ovary (CHO) cells
• Classification: Animal Origin-free, Chemically-defined, Protein-free, Serum-free
• Concentration: 1X
• Form: Liquid
• Product Type: CHO Expression Medium
• Serum Level: Serum-free
• Sterility: Sterile
• Sterilization Method: Sterile-filtered
• With Additives: GlutaMAX
• Without Additives: No Phenol Red
• Product Line: ExpiCHO
• Quantity: 1000 mL
• Shipping Condition: Room Temperature
• Format: Bottle
• Content And Storage: Storage conditions: in the dark at 2-8°C
  Shipping conditions: ambient
  Shelf life: 12 months from date of manufacture
• Culture Type: Suspension Cell Culture
• Expression System: ExpiCHO-S™

Frequently Asked Questions (FAQs)

My ExpiCHO Expression Medium (Cat. No. A2910002) was accidentally frozen. Can I still use it?

This product should be stored at 2-8 degrees C and should not be frozen. The biggest issue with accidentally freezing this product is its solubility. If this product was accidentally frozen, we recommend placing it in a 2-8 degree environment and allowing it to slowly thaw overnight. If the product is fully thawed and shows no signs of precipitation, then it should still be usable, but we cannot guarantee effectiveness.

When subculturing ExpiCHO the day before a transfection to 3-4 x 10E6/mL, would you recommend pelleting the cells and resuspending them in completely fresh media, or simply replacing some of the media to split them down to the desired cell density?

Before transfecting ExpiCHO cells, there is no need to change out the media during the subculturing. This would become expensive and the conditioned media is not necessary.

For how many passages would you recommend using ExpiCHO cells before replacing them with new cells that were frozen at earlier passages?

For optimal performance with respect to protein production, we recommend thawing ExpiCHO cells and letting them grow for 2-3 passages prior to transfection. Overall, we do not recommend using the cells beyond ˜20 passages.

How many days should I wait before harvesting my protein with the ExpiCHO system?

Optimal time to harvest protein will depend on the specific properties of the protein being expressed and the protocol chosen. Typical harvest times to reach maximum titers for the various protocols are as follows:
- Standard Protocol: 8-10 days post-transfection
- High Titer Protocol: 10-12 days post-transfection
- Max Titer Protocol: 12-14 days post-transfection

I didn't add the enhancer with the ExpiCHO system. Will I still get good protein expression?

The Enhancer is designed to work with the ExpiCHO system for maximal expression. If the enhancer is not added, there will be a greater than 50% loss in expression.

Can I use a different transfection reagent with the Expi-CHO Expression System?

The ExpiFectamine CHO Transfection Reagent, ExpiFectamine CHO Enhancer and ExpiCHO Feed are optimized to work together to provide maximal protein expression levels and are provided as a single kit for convenience. The ExpiFectamine CHO Transfection Reagent provides high transfection efficiency of high density cultures, superior to any other transfection reagent. Expression levels greater than 30-fold higher are obtained using the ExpiCHO kit as directed versus substitution of PEI as a transfection reagent, while also using 50% less DNA.

For transfection of ExpiCHO cells, can I really use only 0.5-0.8 µg/mL plasmid DNA? Can lower DNA doses benefit hard-to-express proteins?

ExpiFectamine CHO Transfection Reagent is a highly efficient transfection reagent and as such significantly lower levels of plasmid DNA not only can, but in most instances should, be used for expression runs. Higher levels of DNA can be more stressful to the cells. The volume of ExpiFectamine CHO Reagent dictated in the kit protocol will account for using plasmid DNA in the range of 0.5-0.8µg/mL; if using less DNA than this, the amount of ExpiFectamine CHO Reagent should be reduced proportionately for best results. We recommend that you use 0.6-0.8 µg/mL for most proteins. For some proteins that are aggregate-prone or otherwise difficult to express, lower DNA doses may benefit expression.

What if I cannot add the diluted ExpiFectamine CHO Transfection Reagent to the diluted plasmid DNA immediately?

If it is not possible to immediately add diluted ExpiFectamine CHO Transfection Reagent to diluted plasmid DNA, we recommend diluting your plasmid DNA in the total complexation volume that would be used according to the kit protocol (i.e., the total volume of OptiPro Reagent that would normally be used for diluting both the ExpiFectamine CHO Reagent and the plasmid DNA) and then adding non-diluted ExpiFectamine CHO Reagent directly to the diluted plasmid DNA and then mixing by gentle pipetting 1-2 times and/or inversion. This method is useful for automation or small-scale transfections where it is impossible or undesirable to add the ExpiFectamine CHO Reagent to plasmid DNA immediately after dilution.

Can I substitute other media for ExpiCHO medium for culturing ExpiCHO-S Cells?

No. The ExpiCHO Expression System is able to achieve very high titers due to the way the components of the system have been optimized to work together for maximal protein expression. ExpiCHO medium is a transfection-compatible, high-density growth medium specifically matched to Gibco ExpiCHO Feed and ExpiFectamine CHO Enhancer. Other media are not compatible with the ExpiCHO system and may inhibit protein expression altogether.

I left my DNA:Expifectamine CHO complexes sitting longer than 5 minutes. Will they be okay?

The optimal complexation time is 5 minutes. We have observed a small drop in protein yield (approximately 20% reduction) if complexes sit up to 10 minutes; for 20 minutes or longer yield will be drastically reduced (>50% reduction in yield).

I am getting lower yield than I expected with the ExpiCHO expression system. What should I do to improve my results?

Yield can vary greatly from protein to protein. We strongly recommend expressing the rabbit antibody positive control (Cat. No. A14662) to determine if the low yield is due to a low expressing protein, a problem with the system components, or transfection and culturing conditions. If you are not achieving the expected yield with the rabbit IgG positive control, we recommend checking the following:

- Ensure that cells are >95% viable during normal passaging and at time of transfection
- Have a doubling time of approximately 17 h
- Recover cells rapidly post-thaw (within 3-4 days post thaw); if not, verify you are using the culturing guidelines provided in the manual and thaw a new vial of cells if necessary.
- We recommend gentle swirling at all handling steps for ExpiCHO-S cells (avoid vigorous swirling, shaking, and pipetting up and down). Verify that your shake speed is about 110-125 rpm for 25 mm throw shakers and about 125 for 19 mm throw shakers. Test a flask with containing water and a thermometer to determine whether the shaker is putting off excess heat; reduce the incubator temperature setting as necessary to achieve a final temperature of 37 degrees C for your cultures. All of our shake speed recommendations are provided for Corning non-baffled flasks; if you are using a different culture vessel, additional shake speed optimization may be required.

I notice my ExpiCHO-S cells are clumping. What should I do?

Some mild cell clumping is normal during ExpiCHO-S cell passaging and is okay as long as the cells have >95% viability. At each passage you may allow the clumps to settle to the bottom by letting the flask rest for 30 sec to 1 min, then passage the suspended cells. If the cells have a lot of clumping paired with viability < 95%, this indicates a problem either with the culture conditions or the cells themselves. We recommend verifying that the cells are: cultured according to the recommendations in the manual, below passage 20, and free of contamination. Thaw a new vial of cells as necessary.

What method do you recommend for quantifying the rabbit Antibody-Expressing Positive Control Vector (Cat. No. A14662)?

The formation of intact IgG molecules may be quantitated using a sandwich ELISA designed to capture and detect rabbit IgG. Besides the rabbit IgG positive control, reagents, and consumables that are included in the kit, you will also need purified rabbit IgG to be used as a standard, F(ab')2 goat anti-rabbit IgG HRP conjugate (Cat. No. A10547), Protein A-coated plates (Cat. No. 15130 for clear plates used in colorimetric detection), TMB colorimetric substrate (Cat. No. 34021), SuperBlock (TBS) Blocking Buffer (Cat. No. 37581), and PBS or TBS buffer for washes. There is an example procedure in our Protein A-coated plates manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011310_Pierce_ProteinA_G_AG_Coat_96Well_UG.pdf). Please note, our R&D scientists determine titer values from crude cell culture supernatants using a Pall Life Sciences FortéBio Octet instrument equipped with a protein A biosensor.

What viability is typical for ExpiCHO-S cells, and what method do you recommend for monitoring cell viability of ExpiCHO-S cells?

For both normal passaging and at the time of transfection for ExpiCHO-S cells, greater than 95% viability is critical for best results with the ExpiCHO expression system. We recommend for monitoring cell viability to use a trypan blue exclusion method (either manual or automated using a Vi-CELL Cell Viability Analyzer or Cedex Analyzer). Trypan blue stain is available for purchase (Cat. No. 15250061).

How does the protein expression yield of the ExpiCHO expression system compare to FreeStyle CHO or Expi293 expression systems?

With several test proteins in the ExpiCHO-S expression system, we see approximately 25-160 fold higher expression than FreeStyle CHO expression system and approximately 2-4 fold higher expression than Expi293 expression system. Please see Figure 1 on the ExpiCHO web page at the following link for the data: http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression/expicho-expression-system.htmL?icid=npiGP-MJ-NP01-expicho-expression-system-20150908

Can ExpiCHO-S Cells be used to create stable clones?

Our ExpiCHO-S Cells are derived from our cGMP banked CHO-S cells (Cat. No. A1155701), thus stable CHO-S selection methods are applicable to these cells. We recommend you follow the protocol outlined in the User Bulletin for the Creation and Scale up of a stable cell ine using ExpiCHO Products (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017764_CreatScaleup_StableCellExpiCHO_UB.pdf)

Can I substitute a different medium for ExpiCHO Expression Medium in my Expifectamine CHO transfection?

We do not recommend substituting another medium for ExpiCHO Expression Medium because it likely cannot support the same viable density as ExpiCHO Expression Medium, and may also contain components that inhibit the transfection using ExpiFectamine CHO.

I don't have ExpiCHO-S cells. Can Expifectamine CHO transfection reagent and enhancer be used with other CHO cell lines than the ExpiCHO-S cell line?

Yes, you may be able to adapt your CHO cells into ExpiCHO medium. Long-term adaptation in ExpiCHO medium may increase productivity of your CHO cells, and should sustain high-density growth. However there is no guarantee that CHO lines other than Expi CHO-S cell line will achieve the same levels of expression as the ExpiCHO-S cells. In limited testing, we have found other CHO subclones to be less easy to transfect than ExpiCHO-S cells in large-scale suspension format.

Do I need to change the media after the ExpiFectamine CHO transfection?

No. The ExpiCHO Expression System is designed to run without media exchanges. There is no need to remove transfection complexes or to change growth medium following transfection, however there are an enhancer addition and 1-2 optional feed additions for improved yield.