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View additional product information for ExpiCHO™ Expression System Kit - FAQs (A29133)
64 product FAQs found
这种细胞存活率的突然降低往往提示蛋白表达过程中某些细胞培养条件不在最佳状态。发现这种问题时，应确认摇床速度在操作规程规定范围内；培养体积与培养瓶尺寸应相配；开始蛋白表达前操作 ExpiCHO-S 细胞时应小心，确保细胞不会因剧烈混匀动作而发生应激，等等。
有时，按照高滴度或最高滴度操作规程使用 125 mL 培养瓶培养时，不同瓶之间会有差异。此时，建议将 25 mm 摇床和19 mm 摇床的摇床速度分别提高到 130 rpm 和 140 rpm。
另外，我们发现如果使用 125 mL 培养瓶，那么有挡板培养瓶(baffled flask) 的效果更好，可消除不同瓶之间的差异——此时，考虑到挡板的影响，应将转染的细胞悬液量从 25 mL 增加到 35–40 mL，使其在挡板上达到最佳流动效果，同时其他试剂量也应相应调整。
使用有挡板培养瓶时也可使用较少的转染体积 (即 30–35mL)；但此时考虑到挡板的影响，应稍微降低摇床速度 (即：将19 mm 摇床和 25 mm 摇床的速度分别设为 115 rpm 和 110rpm)。我们发现对于其他尺寸的培养瓶，使用有挡板培养瓶既无必要，也无益处。
可以。如果使用 24 和 96 孔深孔板以及 50 mL 迷你生物反应管进行蛋白表达，请参照以下在线操作规程(https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Protein%20Bio%20-%20Thermo/pdf/ExpiCHO-Protocols-Deep-Well-Blocks-Mini-Bioreactor-Tubes.pdf)。
由于 ExpiCHO 系统所用组分不同于以 HEK 293为基础的表达系统 (如：Gibco Expi293 系统)，因此有时上清液可能较难通过普通的瓶顶过滤器进行处理。 解决这一问题时，我们建议首先将上清液以大约 5,000 x g 的离心力离心 30 分钟，然后再用 0.22 μm 过滤器过滤。其他过滤器 (如深层过滤器) 能提供更出色的上清液过滤性能，尤其是对较大的培养规模，因为此类过滤器专为处理 CHO 类表达系统的上清液而设计。
收集时间高度取决于蛋白的性质。对于抗体等稳定蛋白，如果采用标准操作规程我们一般建议在转染后第 7–8 天收集，如果采用高滴度操作规程建议在第 9–10 天收集，如果采用最高滴度操作规程建议在第 11–12 天收集。此时细胞存活率应该仍然很高，理想状况下可达 70–80% 或更高。细胞内蛋白可能需要提早在第 4–7 天时收集。对于易于降解的蛋白，应该考虑设置不同的时间点收集，以确定最佳的收集时间。任何情况下，保持收集时细胞高存活率对蛋白质量和纯化都有好处。
按照高滴度和最高滴度操作规程调整温度时，建议使用一台专用的 32°C 培养箱，因为将 37°C 培养箱的温度改为 32°C后，培养箱的温度需要很长时间才能降下来，从而影响温度调整的效果。打开培养箱门降温可能会导致污染。
ExpiCHO 养料和 ExpiFectamine CHO 增强剂应在转染后 18–22 小时加入，以获得最佳结果。上述试剂可直接加入培养瓶中，无需预热。或者，也可以先将 ExpiCHO 养料和ExpiFectamine CHO 增强剂混合，再加到培养瓶中，以减少操作步骤。如果 使用37°C 条件的标准操作规程操作，建议添加养料和增强剂的时间更靠近 18 小时的时间点。
我们建议使用 pcDNA3.4 TOPO TA 载体 (货号：A14697)，以获得最佳表达效果。该载体包含全长 CMV 启动子，在克隆位点下游有一个土拨鼠转录后调控元件 (WPRE)。但是，研究发现其他以 CMV 为基础的载体表达效果与 pcDNA3.4 TOPOTA 载体类似，因此任何以 CMV 为基础的载体都是ExpiCHO系统的理想选择。
ExpiFectamine CHO 转染试剂、ExpiFectamine CHO 增强剂和 ExpiCHO 养料经过优化，可互相配合，达到最高的蛋白表达水平，而且为了方便使用，三者以一套试剂盒的形式提供。ExpiFectamine CHO 转染试剂对高密度培养的细胞有很高的转染效率，优于任何其他转染试剂。与使用聚乙烯亚胺 (PEI) 作为转染试剂相比，使用 ExpiCHO试剂盒按照说明书操作可以获得 30 倍高的表达水平，同时这两种方法中使用减少 50% 的 DNA 用量。
对于大多数测试过的抗体，1:1 的比例最适合 ExpiCHO 系统。重链/轻链质粒最佳比例取决于二者共转染到同一细胞后的表达速度，有时则要取决于具体的抗体。我们建议按此比例开始，然后针对具体的分子进行必要的调整。我们建议首先将重链和轻链亚基分别克隆到 InvitrogenpcDNA3.4 TOPO 载体中，然后再优化这两种质粒的比例。我们也测试了一个质粒同时包含重链和轻链基因的方法，结果与前一种方法类似，因此最终的答案要取决于用户的经验和偏好。
ExpiFectamine CHO 是一种高效的转染试剂，可以使用显著较低的质粒 DNA 用量进行蛋白表达。较高浓度的 DNA 会对细胞产生更大的应激。试剂盒操作规程中规定的 ExpiFectamine CHO 试剂体积对应的质粒 DNA使用浓度为 0.5–0.8 μg/m；如果 使用比这少的DNA ，则应按比例相应减少 ExpiFectamine CHO 试剂的用量，以获得最佳效果。对大多数蛋白，建议使用 0.6–0.8 μg/mL 的浓度。对于一些易于聚集或者难以表达的蛋白，更低的 DNA 用量可能有利于蛋白的表达。
如果不能立即将稀释的 ExpiFectamine CHO 试剂加入到稀释的质粒 DNA 中，我们建议您按照试剂盒操作规程将质粒DNA 稀释到将要使用的总复合物体积 (即：正常情况下稀释ExpiFectamine CHO 试剂和质粒 DNA 所需的 OptiPro 培养基总量)，然后将未稀释的 ExpiFectamine CHO 试剂直接加到稀释的质粒 DNA 中。轻轻吹打 1–2 次并且/或者颠倒容器进行混匀。该方法适用于自动化操作或者小规模转染，因为上述情况下不可能或者不方便稀释后立即将 ExpiFectamine CHO 试剂加到质粒 DNA 中。
为了获得最佳效果，建议按以下方法进行 DNA 复合物形成：
1.用低温 Gibco OptiPro 培养基稀释质粒 DNA。
2.使用时，用低温 OptiPro 培养基稀释 ExpiFectamine CHO试剂，然后立即将其加入到稀释的质粒 DNA 中。
3.轻轻吹打 1–2 次并且/或者颠倒容器进行混匀；不要涡旋振荡或剧烈吹打。
4.复合物形成时间应在 30 秒至 5 分钟范围内。
避免延长存放稀释后的 ExpiFectamine CHO 试剂或者ExpiFectamine CHO 试剂-质粒 DNA 复合物反应体系。
ExpiCHO-S 细胞应于转染前一天以 3–4 x 106 个细胞/mL 的密度传代，以便在转染当天达到约 7–10 x 106 个细胞/mL 的密度。转染前用新鲜培养基将该细胞稀释至 6 x 106 个细胞/mL 的最终密度，并轻轻摇晃混匀。剩余细胞应丢弃；不要用高密度细胞继续接种培养。
不可以。ExpiCHO 表达系统能够达到极高的产物滴度，这是因为该系统的组分经过优化，互相配合，达到最佳的蛋白表达效果。ExpiCHO 培养基是一种兼容转染的高密度生长培养基，专门用来与 Gibco ExpiCHO 养料和 ExpiFectamine CHO 增强剂搭配使用。其他培养基无法与 ExpiCHO 系统匹配，可能最终会抑制蛋白表达。
有一个快速的检查方法，即：将 ExpiCHO-S 细胞以 0.3x106 个细胞/mL 的密度接种到装有 30 mL ExpiCHO 培养基的 125 mL 无挡板培养瓶 (non-baffled flask) 中，然后在接种后第 5、6、7 天检查细胞存活率和活细胞密度。通常，ExpiCHO-S 细胞大约会在接种后第 6 天达到最大细胞密度，接近 20 x106 个细胞/mL 的密度范围，然后从第 7 天开始逐渐死亡。最终活细胞密度的测定结果将取决于细胞计数方法。不同计数方法获得的结果可能有较大误差。如果细胞呈现显著不同的生长模式，则应优化培养条件。此时，通常可以同时测试多种摇床速度，以确定最适宜细胞生长的速度，然后以此速度开始蛋白表达工作。
如果细胞密度大大超过 4–6 x 106 个细胞/mL (即：达到 8–10x 106 个细胞/mL)，将细胞传代至较正常传代更高的密度(即：以大约 0.5 x 106 个细胞/mL 的密度接种新的培养瓶)，待细胞恢复。由于 ExpiCHO-S 细胞属于高密度细胞系，不建议在细胞未达到 4–6 x 106 个细胞/mL 的对数生长期前进行传代，因为这样会逐渐损害细胞的生长性能。
ExpiCHO-S 细胞解冻后至少应传代两次，并且应达到ExpiCHO 手册中所述的正常生长状态，方可进行转染。如果按照手册中的细胞培养维持指南进行培养，该细胞的性能至少可稳定保持 20 代。
ExpiCHO-S 细胞生长到约 3 x 106 个细胞/mL 的密度时才会到达对数生长期，因此，应在 ExpiCHO-S 细胞达到 4–6 x106 个细胞/mL 的密度时进行传代，以确保细胞已达到对数生长期。细胞密度接近 6 x 106 个细胞/mL 时，肉眼可见一些微小的细胞团块；不要试图吹散这些团块，只需待这些团块沉淀后取出细胞悬液进行传代即可。进行所有细胞操作时，只要简单地晃动培养瓶使细胞重悬即可。不要剧烈摇晃或吹打细胞进行混匀，因为这可导致细胞性能受损，特别是转染前已经达到很高密度的细胞。
如果 ExpiCHO-S 细胞解冻后未按上述模式生长——仅仅达到相对较低的细胞密度——通常的解决办法是检查培养物温度，确认设备设置未导致培养体系温度过高。CHO 细胞对较低的温度有极强的耐受性，但是对高温却比 HEK293 细胞更为敏感。培养箱即使设定在 37°C，再加上摇床产生的热量，也会导致培养瓶中的温度超过 CHO 细胞的最适温度。
ExpiCHO-S 细胞培养瓶中培养基的最适温度应为 36.5°C 左右。如果怀疑温度过高，则应调低培养箱温度，使培养瓶中达到最佳培养温度。此时，最好重新解冻一管细胞，而不是尝试使经历过高温条件的细胞恢复正常。而且，任何规模的常规传代和蛋白表达工作均推荐使用无挡板培养瓶。
在解冻后的 1–2 次传代时间内，ExpiCHO-S 细胞生长的倍增时间应为 18 小时左右。培养瓶中应该仅有少量的细胞聚集，只有达到较高细胞密度 (即：约 6 x 106 个细胞/mL) 时才会有可见的小细胞团块。如果开始培养时细胞密度为 0.2–0.3 x 106 个细胞/mL 或0.1–0.2 x 106 个细胞/mL，则其分别应在 3 天或 4 天内达到约 4–6 x 106 个细胞/mL 的活细胞密度。如果细胞生长状况不在上述正常范围内，则应进一步优化细胞培养条件。
收到使用干冰运输的细胞后，最好立即将细胞解冻，或者将冻存管放入液氮中储存约 72 小时，使细胞适应环境，然后再解冻；不要将细胞存放在 –80°C 冰箱。将细胞解冻并转移到装有预热培养基的通气、无挡板摇瓶(non-baffled flask) 中，然后将其置于摇床平台上在 37°C、8%CO2 的条件下孵育，其中轨道直径 25 mm 的摇床速度设为 120 ± 5 rpm，轨道直径 19 mm 的摇床速度设为 125 ± 5rpm。细胞解冻时应具有高存活率，解冻后应快速恢复，在 1–2 次传代时间内达到其正常的 18 小时的倍增时间。
若细胞在1-2次传代后形态上存在严重的结团或连成线状，或者未能恢复至正常的生长周期，可能是在运输或收货时存在问题，则不应该再使用。
有的。请访问 ExpiCHO 产品网页 thermofisher.com/expicho，上面有一段简短视频，准确地介绍了我们如何在实验室中使用 ExpiCHO 系统进行表达。这是帮助您上手使用或者解决疑难问题的绝佳资源。
To inquire about a Commercial Production or Service License, please email: outlicensing@thermofisher.com.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
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
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.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
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)
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
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We recommend using the pcDNA3.4 TOPO TA vector (Cat. No. A14697) for optimal expression. This vector contains the native, full-length cytomegalovirus (CMV) promoter and a woodchuck posttranscriptional regulatory element (WPRE) downstream of the cloning site. Other CMV-based vectors, however, have been shown to express similarly to the pcDNA3.4 TOPO TA vector, thus any CMV-driven vector is a good choice for the ExpiCHO system.
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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.