 Thank you for the invitation, Hamels, to speak here. So issues within vivo models, cell and tissue culture. Let's not make the same mistake we make in animal experiments. And the main driver for this talk, and we have heard it before, is the reproducibility crisis that Mona Baker already published in 2016. I'm quite happy that reproducibility was discussed already this morning, because it's still a great problem. Also, in vivo studies, in laboratory animal science, we knew already long before 2016 that when working with animals, we have reproducibility problems. And I will very briefly discuss two of those issues. The first one is feed composition. And the way we feed animals. So already in, oh, I have to go back. Yeah, already in 1985, Anton Bain has showed that what you feed the animals has an effect on their physiology and will affect the results of your study. And it's not only what you give the animals, but also how you feed them. Because at limited feeding, what is normal with rats and mice, gives obese mice, they live shorter. And they're not as healthy as animals that get intermittent feeding. So we are actually studying non-healthy mice, but the logic mice. So how can we relate the results to, for instance, in preclinical studies to human situations? So feed is very important, and the environment we keep the animals. The other one, and we have heard that before, is substrains. We also have substrains in animals. And the nicest example is the black-six mice. People refer always, well, I do my experiments on black-six mice. But some of them are not aware that there are several black-six strains. So there is a black-six strain from Jackson. And there is a black-six strain from the NIH. And they originally got the same mice, but because of genetic drifting, they showed a different phenotype over the time. And we still see people, I'm an assessor of animal protocols, that use different strains, so either the Jackson or the one of NIH. And they still want to compare those, and they're not really comparable. So in animals, substrains, you get very quickly a substrate. So as this article show, in as little as three generations, so there's really fast, you are already getting a genetic drifting and a new substrate. So you have to regularly back cross to a bona fide black-six line to maintain your original characteristics. Okay, now we go to the in vitro studies. Because when Mona Baker released her study, I started to realize that actually we have the same problems with in vitro studies. I mean, I'm not a vitro person, scientist, and I always claim that in vitro studies are much better than animal studies, also with regard to reproducibility. But actually we have a lot of issues ourselves. And we had a very nice example of that, I have to mention, it is also not only important this reproducing results, but also reproducing quality of products or production consistency. So when you use the cell lines, for instance, for the production of vaccines, etc., it's very important that you use always a nice cell of which you know what it actually is. So in 2021, there was an internal laboratory study published. And they studied the variability of A5 for nine cells. So there were two laboratories, one in Switzerland, one in the UK. They followed the same SOPs. They got the cells from the same supplier, ATCC, and they got fetal bovine serum from the same supplier, Sigma. So those are the two issues I'm going to focus on. And these are the results, just the basic cell properties. Cell viability was different, number of cells per square centimeter was different, and the trans-epidereal electrical resistance of the cells of the two cultures were different, significantly different. And then they started to investigate what's the problem behind that, what is causing this difference. And the first culprit they found was FBS. And they found out that they didn't get the same FBS, they got FBS from different lots. And so they say that FBS can strongly affect the outcome. And in the results they refer to our 2010 paper, where we discussed that batch to batch variability related to variations in the concentration of serum components because it's a biological product. So it can't always have the same composition. In addition to the unknown exact composition of FBS, we still don't know what's the exact composition. Can also ultimately lead to experimental variability and limit inter-laboratory reproducibility. And that led the authors to conclude that if you use FBS in different studies, it will be very hard to get the same results because it will be very hard to get FBS in all the different laboratories from the same lot. So it's very likely you get FBS from different lots, and it's very likely that you get different results. So they suggest that we should go to alternative approaches, and not involving field bovine serum. So animal free serum preferably. And that there is a batch to batch difference was already known at the time that the first paper was published showing that FBS was a nice growth medium for the cells. Because Puck who invented it in the same paper already observed that there was variability of performance when serum was collected in winter or in spring and compared to the other seasons. And we now know that there are a lot of difference not only to the different seasons, but also to the areas where we raised the hurts. And in addition, and that's important nowadays, we have when we produce pharmaceuticals, he also noted that there are toxic factors in serum. And that's for instance nowadays one of the reasons why EMA here in Europe and EPA don't allow serum in the medium when you are going to produce pharmaceuticals with in vitro methods. Okay, serum provides proteins, vitamins, hormones. Shea force protection, attachment factors and trace elements. And it's very important for its growth factors, so almost all cells grow. With serum, it has a limited number of antibodies and therefore it's regarded as a universal medium. So that's why it's so popular still at the moment. But there are scientific problems. So the composition of FBS is unknown, so you don't know what you add to your cells and what is influencing the compounds that you might be testing. We have seen that qualitative and quantitative variation between different serum batches may contain different amounts of endotoxins, hemoglobin and other adverse factors and may be contaminated with viruses, bacteria, fungi, microplasma and prions. And that lead to reproducibility problems in experiments and safety of the products. In addition, there are animal welfare issues. So there are problems with the transportation of the pregnant cows, which is a problem nowadays in Europe because they have restricted the transportation of pregnant cows. But also the withdrawal of blood from the fetus is a problem. But we cannot always get the guarantee from the slaughterhouse that the fetuses are already dead at the moment they do the heart puncture to collect the blood. So the conclusions when considering supplementing cell and tissue culture with animal serum actually do not, unless principle, should be applied. Preferentially the medium should not contain any animal derived component unless it was proved to be an absolute requirement. So how do you replace fetal bovine serum? Well, one of the solutions is go to the FCS-free database. We have established the FCS-free database together with the annual free research UK. And if you go into the FCS-free database, we have 286 different cell types there at the moment, so it's still rather small for which we have 651 different medium. The reason that it is small is that only until recently people haven't developed CM3 media since the last five to ten years people start to be developing those CM3 media. And it involves a lot of time before you have CM3 medium. So you go, the FCS-free database is composed of commercially available products, that was the original basis. And now also of literature based records. So modifications of commercially available products and formulations. The problem we have with commercially available products is that you still don't know what's in there. It could be still and will base products in the commercially in the media. And also they change the formulations without informing the customers. This is what the database looks like. You can here fill in the cell line that you're interested in. Then you get, if you have the cell line CHO, you get all the CHO cell lines here. And when you press on one of the records. Here's the CHO, all the CHO results. When you press on one of the records, you get more information on that particular CM3 media and a link to the source, either the journal or the commercial supplier of it. Also, if there is not a CM3 medium available for your cells, you may go to the reference and review paper part of the website. And there we have a lot of papers that helps you show different strategies to develop CM3 medium for your own cell. But also, and that's really important, some people tend to forget that, the adaptation procedure. I mean, if you have been able to obtain CM3 medium for your cells, don't put your cells directly in the CM3 medium, they will die. They need to get accustomed, adapted to the new medium. And that's a very careful procedure to do that. And that's described in, for instance, very well described in the bottom paper. So conclusions, preferentially, the medium should not contain any animal derived component, unless it was proved to be an absolute requirement. And that brings us also to another very popular supplement, a medium in which we grow, for instance, organoids, and that's matrigel, because matrigel is also animal derived. And because it's animal derived, we also have batch to batch differences. But again, a lot of people claim that they use organoids and the other things we, now let's first claim what is matrigel. It's a basement membrane matrix produced by Corning Life Sciences. And it's used for 3D culture of organized cultures, stem cell growth and differentiation, angiogenesis essays, tumor essays, and a lot more essays. But it's animal derived, and it may have different compositions. And may contain also xenobiotic contaminants and growth factors. So the origin is the Engelbreth-Holm-Swan-Sacoma, and that is maintained and grown in mice. In the base of the tail, they inject the sacoma and there it is grown. For every five milliliters of matrigel, one mouse is killed. So you see already when you claim that you are using organoids to replace animal experiments, and you do it in matrigel. You're not replacing anything, you're still using animals. And there is a lot of suffering involved, because the tumor can be bloody or contains a lot of pus, and they inject the matrigel. At a low temperature and injecting a low temperature in a warm animal is causing suffering, so there's also suffering involved. And the tumor size is a problem, so they have to be very alert on the size of the tumor and also, again, as it says here, there could be pus involved. So an infection of the animal. So both for FBS and matrigel, it holds that also in vitro methods may involve animals and animal suffering. The production of the substance may involve animal suffering again. Many animals are killed for these substances. And there are batch to batch differences, and therefore, with respect to results reproducibility issues. And preferably in vitro methods are xenofree and animal component free. And chemically defined, that would be the best situation. Okay, so far, so much for the environment in which we grow the cells. Now about the cells themselves, and there will be some repetition of what you have heard this morning. Because another culprit that caused that difference in results between the two laboratories was, they obtained the different batch of cells. A different lot of cells, they were not informed by that. They had to go back to the ATCC to find out that they obtained a different lot. And again, different batches of cells potentially introduce experimental variability because the exact differences between the batches is not disclosed by the provider. And one of the causes, and we have heard that before is genetic drifting. And Cuevado found that that is prevalent in almost cell lines with a medium of 4.5% to 6.1% of the total genomes. So ATCC also recommends to passing the cell line for no more than two months of continual culturing. Because after the two months, the cells may have diverted so much away from the original cell that it is not really representative anymore of the original cell. So that's what was used also concluded. They no longer represent reliable models of the original source material. So you should not overpassage your cells. And then the one that has already a lot of attention this morning is the cell that you're working with, really, your cell. And then we refer to the cell of Sauer's database, where we have the problematic cell lines and we have seen this already. It's already in the menu. And when you go to the, you get a whole list of the problematic cell lines where you can check of your cell, whether your cell is problematic. And that is really flagged, clearly flagged in the cell of Sauer's database. And the cell contamination, and Amanda has already showed that, is not new, it is already known for a very long time. So in 1962, Brent already showed that several human, monkey, rabbit, swine, calf, and whatever. Tissues are actually either mouse or human species. And we heard the name Gardler this morning. Also already, and he showed in 1960, that four cell lines, three stated of human origin and one of rabbit origin, were actually mouse origin. A probably consisting of L cells, so the contamination. We have that already for a long time. And also, Gardler showed that different cell lines were not the cell lines that were supposed to be, they were actually the cervical cancer cell line, Hila, and that's, well, we know, that's a very problematic cell line that affects a lot of other cell lines. So go to the cell of Sauer's database. So what are the cost of unauthenticated cell lines? Between 18 and 36 of cell lines may be contaminated or misidentified at the moment, at the moment of 2018 when News published his results. So that's quite a lot, between 80% and 36%. So that really shows what others have already argued this morning, that you have to have your cells regularly checked, either by DSMZ, ATCC, and whatever organization that provide these services. Then one thing I would like to draw attention that has been mentioned this morning also, that there are gender differences between cells. You may recall that in 2014, NIH introduced a new policy where they required that there is a balance between the sexes when you do cell culture and animal studies. Because men and women are different, and that's well known. So here you see an example of 2015 where, as the air studies were done, of 855 annotated female cells, actually 331 were males. The other way around is a little bit less of 600 annotated males, only 10 were females. So also here in cell we misidentify the sex of the cells. And sex of the cells is different, two examples. There are gender-specific differences in expression in human lympho, blastoid cell lines, and we do have the gender of cells really matter when screening for novel anti-cancer drugs. So when you do some pre-screening, you really need to be aware what the sex is of your cell, otherwise you may come up with not very reliable or translatable results. So in conclusion, over subculturing cells leads to genetic drifting. There's a problem with misidentification of cells and sex of gender of cells matter. And there are a lot more issues with in vitro methods, which I can't discuss now. Thomas Hartung already discussed all these issues in 2007, so there are a lot more issues related to in vitro methods that we should be aware of. And luckily enough, like with animal studies, nowadays we have guidelines that helps us identifying those issues and improving our studies. So those are the different good cell culture practices guidance and toward good in vitro reporting standards. So ECFOM produced good cell culture practice, two different reports for just ordinary cell culture, but later on they also produced good cell culture practices for stem cells and stem cell derived models. And good cell culture practice for human primaries, stem cell derived, and organoid models as micro physiological systems. And actually for me, the best and the most extensive guidance report was published in 2018 by the OECD, where they not only focus on the actual experiment, but also all the conditions around the in vitro studies, like the roles and responsibility of in vitro method developers, quality consideration, the facilities, strategies to avoid cross-contamination, requirements for all the materials and reagents, and so on and so on. Oh, staff training and development also very important. So it's really interesting to read this extensive paper and see whether you can learn something from that. Because most of the time we are so stuck with our routine practices that we forget that there are also other issues that we have to take care of. And then the last warning in vitro reporting standards. So now Barozova found out that the two different laboratories had different strains for their studies. They now reported not only what cell they used, they also report that they have different lots and they reported the different numbers. So that in case you want to repeat their studies, and you've got different results, you can tell that you might have a problem with the lot you obtained from the ATCC. So we are pushing that with animal experiments, but we have to push that again, and it was said this morning already. And Anita has said that also, the materials and methods have to be meticulously written down, because otherwise we have a reproducibility problem. Okay, so in overview, we have a problem with medium, particular FCS and matric gel, cell identification, genetic drift, the general sex of the cells. So also in vitro methods face comparable issues as animal experiments, so we can learn from them as well. And performance of good and well-informed research could avoid these problems. And very important there is experimental design and reporting on the experimental mental design and knowing the ins and outs of your model. A regularly updated good cell practice, culture practice could facilitate best in vitro practices and detailed reporting contributes to reusability. And I hope then that in a couple of years, we can answer this question with a no. And with that, I thank you for your attention. Thank you very much. And I think it's very, very important for all people working in labs that all this consideration. I was thinking about something because in the lab and I could notice that also a very important aspect is the time when you are doing your experiment, especially when you are working on metabolism, for instance, the cells are submitted to circadian clock. And do you think that this is also something that we should consider? And we should maybe also give this kind of information in the papers. Yeah, yeah. We have seen, well, I didn't have time to discuss that, but in a much longer presentation, I had that as an example and I showed you their papers where they showed different results dependent on the time of the day because indeed cells do have a circadian written. Yeah. Thank you for the talk. It was really interesting and informative. Do you think it would be a good step towards having FCS alternatives if providers like ECAC, ATCC, when you buy a cell line instead of giving the cell and the media standard one with the FPS, they give you a steady alternative immediately how to grow the cells. Because a pushback that we have all the time is, oh, I've gathered all my data with this condition and they won't be any more reproducible. Yeah. Which I understand, but if you start from scratch and you go to ATCC. Absolutely. When you start a new cell line, I think it's an absolute must to start it with CM3 medium. Yeah. It will be good to have when you buy them that the first option is actually the serum free. Yeah, yeah. And then not the serum plus. And you know the problem is that some people say, well, when I move away from fetal calf serum, my cells get different properties. And then my question is, well, maybe those are the properties of the original in vivo cell because I think fetal calf serum introduces a lot of artifacts that are not related to original in vivo cell. Absolutely. And also the other thing that I personally believe that should be an overall completely of the culturing condition in general, because for example, the amount of glucose in the media is a lot. I mean, we have diabetic cells most of the time. Yeah. It's true because 3.5 grams liters is a lot. And maybe considering a hypoxic condition instead of a normoxia and maybe CO2 content. So I think it would be nice to have this starting, this topic starting on all to create a real better reproducible system as a baseline for animal studies. Yeah. Thank you. One question, I mean, in your list of programs you didn't have time to talk, so was zombies, can you in one or two sentences? Well, that has to do with the traditional way of 2D culture in which we change the medium one or two times a week. So the cells are not stimulated, they're just sitting there, the zombies. And the biggest problem then arises when you change the medium. They suddenly get pushed again and then again they turn into zombies because they have to wait for another couple of days before the medium is refreshed and before they get all the nudges then. And they're flowing around in their own waste material which also make them zombies. Thank you very much. Sorry, so where's the... I'm sorry, yeah. I have a question. So I wonder how people made serum-free medium. How they make it? So I mean, recently some research group create tissue culture medium based on the data of RNA sequencing data. So meaning that we can optimize tissue culture medium based on the proteome or transcriptome data. So do you think we need to improve the serum-free culture medium? Sure. And do you know somebody who... Well, go to the page I showed you. There are several papers that show different ways to get to serum-free medium for your particular cell type. It's a tedious process, I have to say. The other thing you can do, Stina Ortson, I think it's last year or the year before but I can give you the reference, produce a serum-free medium and according to her result, several different cell types already grew in that medium because for the ones who are not familiar with serum-free medium, up until now the new medium you develop was really particular for each cell type. Each cell type needs its own serum-free medium. So it was a challenge now to find a universal serum-free medium and it seems that Stina Ortson has found a serum-free medium. So that's worth trying. It's a gift to full formulation in a paper so you can easily use that as a basis for your cells. Thank you.