Embarking on the daunting task of processing large volumes of conditioned cell culture media or other samples for EV isolation? Avoid the arduousness of ultracentrifugation – a time-consuming process that often yields subpar EV isolates – and forge a better path. Our suggested mode of metaphorical transport? Large-scale size exclusion chromatography (SEC) with our large qEV columns. This article highlights our largest ‘off-the-shelf’ SEC column, the qEV100. Below we will guide you through the critical considerations for large-scale EV isolation, demonstrating why the qEV100 (or possibly one of our even larger custom columns) is the clear winner over ultracentrifugation. Additionally, we'll show you the data which demonstrates how optimising the qEV100 can get the best out of your starting sample.
How does the qEV100 compare to ultracentrifugation for practicality?
Time is valuable, so it is unsurprising that protocol length is an important factor for large-scale EV isolation. Whilst ultracentrifugation protocols often take a full day to execute, the qEV100 collection procedure takes 15 minutes. All in all, with column priming and cleaning, the whole qEV100 protocol takes less than hour to complete. When it comes to time then, there is no contest.
Verdict: Clear qEV100 win for speed of protocol
Here the two methods are pretty evenly tied. qEV100 columns are clearly far cheaper to start using, but have the ongoing cost of buying new columns. Conversely, an ultracentrifuge and everything you need to run it come at a huge initial financial outlay. If this was all there was to it, eventually ultracentrifugation would be cheaper, but the staff time taken to run these day-long protocols and the expensive regular services that the ultracentrifuges require make it dead even.
Aside from maintaining accuracy, the regular servicing of ultracentrifuges is imperative for safety reasons. If an ultracentrifuge is unbalanced or a component fails, ultracentrifuges can be extremely dangerous due to the high g force required for EV isolation. Whole laboratories have been destroyed by ultracentrifuge accidents and in 1992 a man was killed when an ultracentrifuge exploded.
Using a qEV100 column is immeasurably safer.
Verdict: Clear win for the qEV100
Considering the ''Time'' and ''Cost'' sections above...
Verdict: Clear win for the qEV100
How does the qEV100 compare to ultracentrifugation for isolate quality?
EV Yield and Purity
The two big metrics used to assess EV isolation success are yield and purity. Data from the literature suggests that when isolating from larger volumes (e.g., when using cell culture media or biofluids like milk or urine), the yield of EVs is no different when comparing qEV to ultracentrifugation. Purity, however, is different between the two. qEV column isolation results in purer EV isolates (p=0.036), meaning that using qEV100 columns maximise purity without compromising yield. This data was gathered on Legacy qEV column data. With the new Gen 2 qEV columns showing improved purity and the new 20 nm series showing improved yield, you can pick the qEV column that suits your purity and yield requirements.
Verdict: Clear qEV100 win
One of the biggest reasons to choose qEV columns over ultracentrifugation is the gentle nature of SEC, which results in greater maintenance of EV functionality as compared with UC-isolated EVs.7,8 This is likely in part due to the lack of extreme centrifugal forces such as those used in ultracentrifugation.
Verdict: Clear qEV100 win
Which factors impact upon qEV100 performance?
Whilst the qEV100 was nominally created for a 100 mL loading volume, this isn’t a ‘one-size-fits-all’ rule. For highly concentrated cell culture media, the point of maximum loading volume may be lower. This is why we recommend that you determine the maximum sample loading volume for your sample type. Theoretically, the yield in EVs should double when the sample volume added to the column is doubled. As you can see in Figure 2A, this occurs when doubling the loading of plasma from 25 mL to 50 mL, and between 50 mL and 100 mL (p<0.01). A similar picture is seen with purity (Figure 2B) where increasing the loading volume results in greater purity (p<0.05).
Next we sought to show that sample characteristics matter when it comes to choosing the optimal sample loading volume. Here we took 50 mL of plasma and diluted it in another 50 mL of PBS to get a final volume of 100 mL before loading it onto the qEV100. As you can see in Figure 3, there is no difference in the EV yield between the neat or diluted sample. However, there is increased purity for the diluted samples (Figure 3b; p<0.05), demonstrating that 100 mL of a more dilute sample is optimal for the qEV100 than 50 mL of a more concentrated sample. As such, if you have a highly concentrated sample, or have a sample that is less than 100 mL, you may wish to trial dilutions to determine whether you can improve yield and/or purity.
As we wrap up this exploration of large-scale EV isolation, it is evident that the qEV100 column sets a new standard in the field. Its swift processing time, combined with high-quality EV isolation and enhanced safety, positions the qEV100 as the optimal choice over the traditional ultracentrifugation. The qEV100 column is designed for a nominal loading volume of 100 mL, but this is not a fixed limit as the optimal volume can be lower for highly concentrated samples. It's recommended to determine the best sample volume for your specific sample type. The qEV100 gives you the flexibility and the opportunity to optimise EV isolation to suit your sample, meaning that you will get the best EV isolate every time.
Whether you want more information on our larger qEV columns or you are interested in learning about our customised large-scale EV isolation columns, our team is here to help.