Welcome to this first lecture in the third module of this course, Collection and processing of cell culture media and body fluids prior to isolation of extracellular vesicles. You will later hear about isolating EVs from body fluids such as urine and plasma, but I will start here by talking about some of the specific technical issues for in vitro research. So my name is Cecilia Lasser, and I'm from the University of Gothenburg, Sweden. So these are the topics that we will be covering in this lecture. So FBS, fetal bovine serum, contains EVs. And as we have that in our cell culture media, we need to deplete them prior to use. We'll talk about the importance of looking at the Viability of your cells that you isolate your vesicles from. And we will talk a bit about that you can Simulate EV release, the problem of Microbial contamination and the importance of Stable cell culture conditions. So first, Fetal Bovine Serum Contains Extracellular Vesicles. You could see it here on the EV, electronmicroscopy picture where we see several vesicles. You could also see that they contain RNA in this Bioanalyzer graph and that they contain proteins. Furthermore the FBS, vesicles have a density between 1.09 and 1.16 and so it's similar in density to the EVs that you will be isolating from your cells. So you need to remove these vesicles prior to starting your cultures otherwise you will be looking at FBS and vesicles. And not necessarily only the vesicles that are released from the cells of interest, so one option would be to use serum free media. However, as we can see here for the human mast cell line, HMC1. That when you culture them without FBS, which is the blue line. The proliferation stops, and the viability drops. So FBS-free conditions can induce a stress response which may lead to the release of vesicles with different composition. Or perhaps your cells will actually not release any vesicles at all, enduring this stressed state. So there are advanced media that can be used for some cells, and this is a media that allows you to culture your cells without supplementing it with FBS. So here we can see that for the HEK293 cells, the advanced media, the cells are perfectly happy within this media compared to media supplemented with FBS. But if we look at the FL3 cells instead we can see that these cells have a drop in the viability if they are cultured in advanced media. So this is something you need to consider, that this medium might work for some cells, but not for others. So, the most common way to tackle this problem is to deplete the FBS by. The FBS EV by Ultracentrifugation, so in this paper from our group, we compared what happened if you centrifuged FBS for one and a half hour or 18 hours. And we did it both with pure FBS, or diluted to 30% in media. And then this FBS was added to media to a final concentration of 10%, and this was used to go through a normal excess on isolation procedure that has been 120,000 x g for 70 minutes. But these media had never been in contact with any cells. So if we would see anything, that would come from the FBS. So here we can see that in the control where we used FBS that had not been ultracentrifuged at all, we will get a lot of RNA in the vesicle pellet. And this is all contributing from FBS. Then we can see for 1.5 hour and 18 hours, that the 18 hours almost 95%, 90 to 95% of the FBS EV RNA while 1.5 hour only remove between 50-60%. So 1.5 hours is not efficient to remove all the FBS EVs when it comes to the RNA. Just a short note that if this EVs do effect the cells that you are culturing or at least some cells, because you can see here that even though the U87. Which is the red line, this cell seems to be perfectly happy in the EV-depleted FBS media, while other cells, such as the green and the gray, which is Hela cells and Hex cells. Might not or the viability will be slightly affected by growing them in EV-depleted FBS. However, what the author saw was that when they induced DNA damage, these cells that were cultured in the EV-depleted FBS. Was equally good at resisting this induced DNA damage where they responded similar to the control cells. And this was also true for the induced endoplasmic reticulum stress. So what about cell death? EVs from dying cells can contaminate your isolated EVs derived from live cells. And it has been suggested that a maximum acceptable cell death of 5% to have a pure EV isolate, and this is from the Whitwer position paper in 2013. And what we have seen is that when we used TRAIL to induce apoptosis in TF-1 cells. We will have more vesicular RNA in all three types of cell populations of EV such as apoptotic bodies, micro vesicles, and exosomes. So cell death will definitely affect your isolated EV population. So the EV release can be stimulated, for example, with PMA and calcium ionophore. And here we can see in the black bars, which is the calcium ionophore stimulate itself, that they release more vesicles as measured by NTA here. Just remember that the stimulated release might lead to a release of different vesicles compared to vesicles that are released during the natural release. And then microbial contamination, so infect the cells, do release EVs of different composition, which has been shown previously. But also, the microbes are quite small, so they can actually contaminate your EV pellet. And mycoplasma, for example, are approximately 300 nanometer. They lack a cell wall and therefore they are not affected by penicillin used in cell culture media. And if you look at the end picture here, you might even agree that they actually looks a bit like vesicles, so it can be hard to detect them in your cell culture. But it's something that you need to be aware of. And I think this might be one of the most common questions that I get. And also I see them in different forums where EVs are discussed. How much EV can you expect to collect from your cells? I would say that the answer is I don't know and that it's very hard to know because all cells are different. For example here we can see that the FL3, which is a human urinary bladder. A cell cancer cell here the yield was 1.8 microgram at 100,000 K, but 100,000 K spin, which is 100,000 g, for the HEK293 cells will you give you more than double that yield. So it's very different for a different cell. And what this paper is showing is also that depends on which type of sub populations of EVs you're interested in. Is it apoptotic bodies? Is it that the microvesicles where the exosomes yield will be different and here the yield is measured as RNA. And then just a final note on stable cell culture conditions and I think you all very well aware of this. So I'm juts going to mentioned it quickly, don't change the settings during your experiments. You don't know how that will affect your cells, if it will release more vesicles or less vesicles or if it just will release different vesicles containing different RNA or protein. So don't change the setting during your experiments. Don't switch media from one company to another even though. It should be the same, you never know. Don't change culture flasks or culture dishes. The plastic might be different and stimulate your cells differently. The seeding density, don't change that. It could be that the cells release less or more vesicles when they're low confluency or high confluency. We don't necessarily know that for every cell type, so it's good to keep that consistent. If you're collecting your vesicles off 24 hours, 48, 72, I think it's good to stick with the same. Because that could also be different, different cells that would be released as the cells become more and more confluence. So the conclusion is that optimize the conditions for each cell type and then don't change the conditions during the project, unless that's the research questions that you have. If this contains EV and that will contaminate your vesicle sample, unless you remove them prior to using your cell cultures. Measure the viability of your cells at the EV collection point to make sure that you have viable cells, happy cells. Confirm mycoplasma free status of your cells. So thank yo for listening to this lecture and I hope you enjoy the rest of the course.