ExpiFectamine™ CHO Transfection Kit, For 1 L of culture - FAQs

ExpiFectamine reagents generally does not transfect well after being frozen, as the freezing disrupts the lipid micelles. Therefore, we do not recommend using this kit.

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No, we do not offer this ExpiFectamine CHO Transfection Kit component as a standalone product.

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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.

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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.

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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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

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 ExpiFectamineCHO 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 polythyleneimine (PEI) for the transfection reagent, while using 50% less DNA in both methods.

For the majority of antibodies tested, a 1:1 ratio has been found to be ideal for the ExpiCHO system. The optimal heavy- to light-chain plasmid ratio is dependent upon the rate at which the two chains are expressed when cotransfected into the same cell, and may be antibody specific in some instances. We recommend starting with this ratio and then modifying as necessary for specific molecules. We recommend cloning the heavy- and light-chain subunits separately into the Invitrogen pcDNA3.4 TOPO vector initially, and then optimizing the ratios of the two plasmids. We have also tested single plasmids incorporating both heavy- and light-chain genes with comparable results, so in the end, the answer will come down to user experience and preference.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

ExpiFectamine CHO is highly efficient for reagent transfection, enabling use of significantly lower levels of plasmid DNA for expression runs. Higher levels of DNA can be more stressful to the cells. The volume of ExpiFectamineCHO reagent specified in the kit protocol will account for using plasmid DNA in the range from 0.5 to 0.8 µg/mL; if using less DNA than this, the amount of ExpiFectamineCHO reagent should be reduced proportionately for best results. It is recommended to use 0.6-0.8 µg/mL for most proteins. For some proteins that are aggregate-prone or otherwise difficult to express, lower DNA levels may benefit expression.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

If it is not possible to immediately add diluted ExpiFectamine CHO 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 media 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. Mix 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

For best results, it is recommended to perform DNA complexation in the following manner:

1. Dilute plasmid DNA into cold Gibco OptiPro medium.
2. At the time of use, dilute the ExpiFectamine CHO reagent with cold OptiPro medium and then immediately add to the diluted plasmid DNA.
3. Mix by gentle pipetting 1-2 times and/or inversion; do not vortex or pipet vigorously.
4. Complexation time should take 30 seconds - 5 minutes.

Avoid elongated hold times for the diluted ExpiFectamine CHO reagent or the ExpiFectamine CHO reagent-plasmid DNA complexation reaction.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

ExpiCHO-S Cells should be subcultured at a density of 3-4 x 10E6 cells/mL one day prior to transfection to obtain a cell density of approximately 7-10 x 10E6 cells/mL on the day of transfection. These cells should be diluted to a final density of 6 x 10E6 cells/mL with fresh media and gently swirled to mix prior to transfection. Discard any remaining cells; do not reuse high-density cells for seeding of maintenance flasks.

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).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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.

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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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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).

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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

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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)

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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.

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.

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.