Hi, my name is Cecillia Lasser and I'm from the University of Gothenburg in Sweden and welcome to this lecture about differential centrifugation. During this first module of differentials centrifugation, we will start with going through what is a particle sedimentation coefficient? And then we will cover the principle of differential centrifugation and how these two are used to isolate extracellular vesicles. We will also talk about some of the problems that occurs during differential centrifugation. Such as, poor separation between different types size particle which can lead to low purity and low recovery. And then we will go through the properties of different type of rotors. And what to consider when choosing a rotor for your centrifugation in isolation of the vesicles that you are interested in. In the second module which is the next lecture, I will explain what clearing factor is. And how this together with the sedimentation coefficient affects time needed to pellet vesicles. We will also look at the calculations you need when you're changing from one rotor to another. Because rotors are different and they have different properties. And lastly, we will go through some of the things that can affect the vesicle yield and the purity during differential centrifugation. But let's start with module one. So first, sedimentation coefficients. This tells you how a particle will behave during centrifugation. And it's a value that it's depending on the mass of the particle and the shape of the particle. But it also depends on the media that the particle is moving through. And here we can see that sedimentation coefficients for some common biological materials. And what we see is that the bigger particles such as organelles have a higher sedimentation coefficient. And they tend therefore to sediment faster. While smaller particle such as soluble proteins and nucleic acids have low sedimentation coefficients. And therefore, it takes longer time to pellet these particles. The larger the s value is, the faster the particle is separated out from the supernatant into the pellet. So centrifugation separates particles of different sedimentation coefficients and that's the principle behind the differential centrifugation. The largest particles in the sample travel to the bottom of the tube first. The pellet can be discarded or saved depending on, if this is vesicle of your interest or not. The supernatant is then used for further centrifugation. And by using an increasingly higher force and/or longer round times smaller vesicles can be isolated. Differential centrifugation is the sequential pelleting of particles with decreasing sedimentation coefficients. So, differential centrifugation has been the golden standard for isolating extracellular vesicles since the early 80s. The exact speed and time for each step may vary, but this is the general outline that we will go through here. So, the conditional media or body fluids, depending on which starting material you have, are first centrifuged at 300 x g to pellet and remove cells. And this is important because if you don't remove the cells before you go to the higher centrifugation speeds, the cells might burst and these cell debri. Intercellular vesicles can contaminate your samples. So always start with a 300g to pellet the cells. So this supernatant is then used in the next step. And approximately 2 000 x g is then used to pellet the largest extracellular vesicles which are the apoptotic bodies. Microvesicles are smaller than apoptotic bodies and therefore be pellet in the next step at a G-force somewhere between 10,000 and 20,000 x g. This supernatant can then further be used to isolate the smallest extracellular vesicles, the exosomes. And here you usually need a g-force greater than 100,000 x g. It's important to perform all the steps even if you're just interested in the exosomes, because if you have not pre-cleared large vesicles from the media, it will be pelleted together with your exosomes at 100,000 x g, sorry or they will burst. And might contaminate your samples due to that. So, if we look a bit out to separation and the recovery, what are the problems that we might find? Because when isolating EVs with differential centrifugation, using the right speed and the right time, is a balance between optimizing the yield but still have good purity. So, if too low speed or too low time is used, it can lead to poor separation and low purity. See, if we go through these different problems step by step, we can see if you have too-low speed or too-short time during the pre-cleaning step. The larger vesicles, so that would be the red vesicles here, will remain in the supernatant and will then be pelleted together with the smaller vesicle isolated in the next step, resulting in a mixture of EV cell populations. So for example, micro-vesicles contaminating the exosome pellet. If your microvesicles significations step was not efficient enough. So then you will have a contamination on larger vesicles. But, if you instead have too low speed, or too short centrifugation time in the step of the vesicle you're interested in, you can instead have Insufficient EV isolation. So this low speed or too short centrifugation time during the isolation step of EV of interest will instead lead to poor recovery and low yield. Poor separation can also occur if too high speed or too long centrifugation time is needed. As used during the isolation of vesicle of interest, so it's not just that okay, so I had a bit of problem of poor separation and low yield. I just increased my time and speed. That is not [LAUGH] the solution because if you have a too high speed or too long, the smaller vesicles will be palette in a mixture with larger vesicles instead. So in this example, exosomes might contaminate your micro-vesicle pellet. Too high speed and/or too low centrifugation time can also lead to poor recovery and low yield in the final step if it has been applied during the pre-cleaning step. Then the smaller vesicles would have been pelleted already in the earlier steps and the material will be lost. Therefore choosing an appropriate speed and time is of high importance. Okay, so what about the different rotors that we can use during differential centrifugation? There are several different rotors available and the two most commonly used for isolation of extracellular vesicles are fixed angle rotors and swinging bucket rotors. In a fixed angle angle rotor, the tubes are held at a static angle in the tube cavities. Therefore the pellet of vesicles will be located on the side of the tube facing outwards If swinging bucket rotors, the sample tubes are loaded into individual buckets that hang vertical while the rotor is at rest. When the rotor begins to rotate the buckets swing out to horizontal position, therefore, the pellet vesicles will be located at the bottom of the tube. So, there are three factors determine the type of rotor required. Which type of centrifugation is going to be performed, differential or gradient centrifugation? As you need to consider the desired g force that you need but also the sample volume So fixed angle rotors are especially useful for pelleting particles by differential centrifugation. And this is because the tube angle, as we can see here to the left. The tube angle shortens the migration path length compared to swinging swinger bucket. And it results in that you can reduce the run times because it's very efficient in pelleting particles. Swinging bucket rotors are particularly useful when samples are to be resolved in a downside [INAUDIBLE]. The longer path length that we can see on the right here permits better separation of individual particles tied from a mixture. However, this rotor is relatively inefficient for pelleting, which we will talk more about in the next lecture. Generally speaking, the size of the rotor is inversely proportional to its maximum speed capability. That is, the larger the rotor is, the more sample volume it can take, the lower the maximum speed is. So if we look at the fixed angle type 45 Ti rotor for example, it can almost take twice as much as sample volume as Type 70 Ti rotor, but its maximum speed is much lower. The same can be seen for, The swinging bucket 32 Ti rotor, that can fit almost three times as much sample volume as the swinging bucket 41 Ti rotor. But its maximum speed is lower. So this is really something you need to consider if you have large volumes of sample, maybe you need to chose the Type 45 Ti rotor or the swinging 32 Ti rotor, but if you're are needed therefore higher G force in these rotors can take maybe you need to choose other rotors instead. To summarize module one of differentual centrifugation. Simplified, it can be said that smaller EVs have a lower sedimentation coefficients and therefore need higher speed, longer time to be pelleted. The principle of differential centrifugation is based on this. The sequential pelleting of particles with decreasing sedimentation coefficients. Changing speed and time will affect the separation and yield. It should be done with care. And when choosing a rotor, consider what type of centrifugation is it that you want. Don't just choose the rotor that happen to be standing next to the centrifuge or the rotor that somebody else is using because they might have a different aim with their centrifugation. Because, if you remember what I said, a shorter migration path length will give you a more efficient pelleting. And, therefore, the centrifugation time can be reduced, while a longer migration path length will give you a better separation of different types of particles. If furthermore, there might be speed and volume limitations that you need to consider when choosing a rotor. So this is the end of the Module 1. And in the second module of differential centrifugation, we will go through the clearing factor and how it's together with the sedimentation coefficient, affects the time needed to pelleted vesicles. And here we will start look at the calculation needed when you're changing from one rotor to another because maybe you get a protocol for somebody who only have a certain type of rotors and the rotors you had have ten or different and these are things that you need to consider when you Start with your own protocol. And then lastly, in the next session, we will go through some of the things that can affect the vesicle yield and the purity such as viscosity and so on. And so I hope to see you at the next lecture.
