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View additional product information for ExpiCHO-S™ Cells - FAQs (A29127, A29132)
41 product FAQs found
ExpiCHO-S cells are biosafety level 1.
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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.
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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.
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We have provided optimal shake speeds for various formats. For most flasks, there is a drop-off in expression when you go too fast or slow because of cell shear stress or insufficient aeration. The effect is sharper in plates where there is a sharper transition between static and moving fluid in wells.
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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
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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.
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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.
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Yes. Please visit our website (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression/expicho-expression-system.html?SID=fr-expichosystem-main) where you will find short videos (including https://www.youtube.com/watch?v=l2emhdSx0e4) showing exactly how ExpiCHO experiments are performed in our laboratories. This is an excellent resource for getting started or for troubleshooting.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
This drop in viability tends to indicate that some aspect of the cell culture conditions are not optimal during the expression run. If this is observed, the shaking speed of the flasks should be verified to be within protocol specifications; the volume and size of the flask should be appropriate; and care should be taken when handling the ExpiCHO-S Cells ahead of the expression run to ensure that cells are not stressed by vigorous mixing, etc.
In some instances, flask-to-flask variability has been observed using the high- and max-titer protocols at the 125 mL flask scale. Here, it is recommended to increase the shaking speed to 130 rpm for 25 mm shakers and
140 rpm for 19 mm shakers.
Alternatively, we have found that baffled flasks work well for the 125 mL scale to correct any flask-to-flask variability—in such an instance, to account for the baffles, the volume of cells to be transfected should be increased from 25 mL to 35-40 mL to allow for optimal flow over the baffles, and the volumes of other reagents should be scaled accordingly.
Lower transfection volumes (i.e., 30-35 mL) can also be used with baffled flasks; however, shaking speeds must be reduced slightly to account for the baffles (i.e., 115 rpm for 19 mm shakers and 110 rpm for 25 mm shakers). We have not found baffled flasks to be necessary or beneficial at other flask sizes.
Yes. For expression in 24- and 96-deep well blocks and 50 mL mini bioreactor tubes, please refer to the online protocol (https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Protein%20Bio%20-%20Thermo/pdf/ExpiCHO-Protocols-Deep-Well-Blocks-Mini-Bioreactor-Tubes.pdf)
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Because the ExpiCHO system uses components that differ from that of HEK 293-based expression systems such as the Gibco Expi293 system, the supernatant may be more difficult to process through standard bottle top filters in some instances. To remedy this, we recommend centrifuging the supernatant first at ~5,000 x g for 30 minutes followed by filtration using a 0.22 µm filter. Alternative filters (such as depth filters) provide superior filtration for supernatants, especially at larger scales, as these filters are specifically designed to handle supernatant from CHO-derived expressions.
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The time of harvest is highly dependent upon the nature of your protein. For stable proteins such as antibodies, we generally recommend harvesting on day 7-8 posttransfection (using the standard protocol), on day 9-10 (using the high-titer protocol, and on day 11-12 (using the max-titer protocol. Cell viability should still be high at this time, ideally 70-80% or greater. Intracellular proteins may require earlier harvesting, between days 4-7. For proteins that may be susceptible to degradation, a time course of harvesting should be considered to identify the optimal takedown time. In all instances, maintaining high cell viability at the time of harvest is ideal for both protein quality and purification.
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When shifting the temperature for the high-titer and max-titer protocols, it is recommended that a dedicated 32 degrees C incubator be used, as simply changing the temperature of a 37 degrees C incubator to 32 degrees C may take a prolonged period of time to cool down and may limit the effectiveness of the temperature shift. Cooling the incubator by opening the door may result in contamination.
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ExpiCHO Feed and ExpiFectamine CHO Enhancer should be added 18-22 hours posttransfection for best results. These solutions may be added to the flasks without prewarming. ExpiCHO Feed and ExpiFectamine CHO Enhancer may also be premixed ahead of addition to flasks to reduce the number of steps required. If using the standard protocol at 37 degrees C, it is recommended to add the feed and enhancer closer to the 18-hour timepoint.
Find additional tips, troubleshooting help, and resources within our Protein Expression 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.
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.
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As a quick check, ExpiCHO-S Cells can be seeded at 0.3 x 10E6 cells/mL in 30 mL of ExpiCHO medium in a 125 mL non-baffled flask, and the viability and viable cell density checked on days 5, 6, 7 postseeding. Typically, ExpiCHO-S Cells will reach maximal density around day 6 postseeding and should attain a density in the range of 20 x 10E6 cells/mL, and then will die off on day 7 and beyond. Determining the final viable cell density will be dependent upon the method used to count cells. Significant variability can be observed from different counting methods. If cells are exhibiting significantly different growth profiles, optimization of culture conditions should be performed. In such instances, it is typically useful to test multiple different shaking speeds simultaneously to determine which speed provides optimal cell growth and then start with this speed for your protein expression runs.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
If cells significantly overgrow the target of 4-6 x 10E6 cells/mL (i.e., growing to 8-10 x 10E6 cells/mL), simply subculture the cells to a higher cell density than normal (i.e., seed new flasks at ~0.5 x 10E6 cells/mL) to allow the cells to recover. Since ExpiCHO-S Cells belong to a high-density cell line, it is not recommended to subculture the cells if they have not yet reached log phase of growth at 4-6 x 10E6 cells/mL, as this can impair growth over time.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
ExpiCHO-S Cells should be passaged at least twice post-thaw and be growing within the ranges specified in the ExpiCHO system manual, prior to transfection. Cells should maintain consistent performance for at least 20 passages if maintained in accordance with the cell culture maintenance guidelines in the manual.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
ExpiCHO-S Cells do not reach log phase of growth until approximately 3 x 10E6 cells/mL cells, thus ExpiCHO-S Cells should be allowed to attain a density of 4-6 x 10E6 cells/mL at the time of subculturing to ensure the cells have reached log phase of growth. As cells approach 6 x 10E6 cells/mL, some very small cell clumps may be visible; do not try to break up the clumps, simply let them settle and remove the suspended cell solution for subculturing. For all cell manipulations, simply swirl flasks to resuspend the cells. Do not shake or pipet the cells vigorously to mix, as this can lead to decreased performance, especially just prior to transfection, when cells have attained very high densities.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
In instances where ExpiCHO-S Cells are thawed and do not start to grow as noted above—attaining only relatively low densities in culture—one common solution is to verify the temperature of the cultures to ensure that the equipment settings are not generating too much heat. CHO cells are very tolerant to lower temperatures, but are more sensitive to elevated temperatures compared to HEK293 cells. Incubators, even when set to 37 degrees C, in conjunction with the heat generated from the shakers, can elevate the temperature in the culture flasks above optimal for CHO cells.
The optimal temperature of the media in the flask for ExpiCHO-S Cells should be around ~36.5 degrees C. If overheating is suspected, reduce the temperature of the incubator to reach optimal operating temperatures in the flasks. In such instances, it is best practice to thaw a new vial of cells rather than attempt to recover cells from elevated temperature conditions. Also, non-baffled flasks are recommended for use at all scales for both routine subculturing and protein expression runs.
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Within 1-2 passages post-thaw, ExpiCHO-S Cells should be growing with a doubling time of approximately 18 hours. There should be minimal cell clumping in the flasks, with only small clumps visible when the cells approach higher cell densities (i.e., ~6 x 10E6 cells/mL). When cells are cultured at 0.2-0.3 x 10E6 cells/mL, or 0.1-0.2 x 10E6 cells/mL, viable cell density should be approximately 4-6 x 10E6 cells/mL within 3 or 4 days, respectively. If cells are not growing within these approximate ranges, cell culture conditions will require further optimization.
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Upon receipt of the cells on dry ice, it is best to either thaw the cells immediately or place the vials into liquid nitrogen storage for ~72 hours to allow cells to acclimate until the time of thaw; do not store cells at -80 degrees C. Once thawed and transferred into prewarmed media in a vented, non-baffled shake flask, cells should be incubated at 37 degrees C with 8% CO2 on a shaker platform set to 120 ±5 rpm for a shaker with a 25 mm orbital diameter or 125 ±5 rpm for a 19 mm orbital diameter. Cells should have high viability at the time of thaw and should recover quickly post-thaw, reaching their normal 18-hour doubling time within 1-2 passages.
Cells that are very clumpy or stringy in appearance or do not recover into a normal growth pattern within 1-2 passages may have been compromised during shipping or receiving, and shouldn't be used.
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Yes. Please visit the ExpiCHO page at thermofisher.com/expicho where you will find short videos showing exactly how expression with the ExpiCHO system is performed in our laboratories. This is an excellent resource for getting started or for troubleshooting.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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).
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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.
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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.
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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.