 Okay. Thanks, Amos, very much for inviting me to this stimulating symposium. And also, thanks for developing cellosaurus, which I think is a brilliant contribution to the use of animal cell lines in research. And thanks to you folks for staying to the end. Okay, so what I want to argue today is that cell lines have two sets of properties. Anti-factum properties, or before the fact, and then post-factum properties after the fact. And then I'm going to use those to organize cell lines. And these ideas are presented in this paper in last year with me, Dr. Lucy Lee, who's here, and Georgina Dowd, who's in New Zealand. Okay, what are animals? Animals are multicellular eukaryotic organisms or metazoans in the kingdom, Animalia. And you can divide them in different ways. So you can have, one way of looking at it, you can have five lineages. So you have perifera or sponges, nedarians such as jellyfish and corals, tenophoras, comb jellies, and placoza that are sometimes referred to as flat animals. And together they belong into the non-bilateral phyla. And then the fifth lineage is the bilateria, which there's about 30 phyla. And they have in common is they all go through that stage where they have three germ layers in their development that sets the adult body pattern. And I'll just mention, for examples, a bilaterian phyla, nematodes, mollusks, arthropods, and chordates. And here's where we've been most of the day, way down there. Okay, so you've probably waited all day to find out what the heck are cell lines. So here's my definition. So my definition is a population of multicellular, of cells from a multicellular organism that can be propagated in vitro. And really I'm asking them to have only two properties. One is that they proliferate in vitro, and so I have to sub-cultivate them or passage them into new vessels. And then the second criteria is that I have to be able to cryopreserve them, and that includes recovering them from the liquid nitrogen doer. So those are what cell lines are in my eyes. So what animal groups have cell lines being developed from? And of course you can go to cellosaurus and you can figure this out. But there's two cell lines from the non-bilaterian phyla. And this first one is from the sponge, and I don't know if it's in cellosaurus. I was so hoping I could make a contribution and say, point this out, this is only from a few months ago. And in cellosaurus is one from Naderian, from a coral, and then cell lines from three or four bilaterian phyla, nematodes to cell lines, mollus. There are cell lines listed, but I question them. Arthopods, there are clearly many cell lines from ticks and even an enormous number from insects. And then for the chordates, six of the seven vertebrate subclasses are represented in cell lines. Okay, so I argue that the discipline of animal cell lines is complex. I think from everything I've heard today it is indeed complex. And the minutiae can be overwhelming. I suggest in 2017 propose three new terms. I like making terms. So in vitro-matics is the discipline that describes cell line development. In vitro-omics is for cell line use, and in vitromes is for grouping of cell lines around themes, which is going to be most of my talk. So I'll just go through those three terms because I'm so proud of them. So in vitro-matics is the science and history of establishing, characterizing, engineering, storing, and distributing animal cell lines. And that, I think, has three branches. The scientific or technological. So that's about really, we haven't talked too much about that today. You know, all the business, you know, of, you know, what particular media, how I could hold the pipette, what trypsin to use. So that, of course, is what drives, you know, hundreds and thousands of papers. And then there's in vitro-matics, which is documenting, storing, and distributing cell lines. And this is where cellosaurus is such a terrific contribution. And then cell line repositories that, any repository that helps curate and distribute cell lines would be informatics. And then historical, we heard a great history of C.H.O. Like how many remembered that Theodore Puck was the origin of C.H.O.? I forgot that. Okay, and so I just have one, you know, you have HeLa's history. This one is for WI-38. So that's my breakdown of in vitro-matics. Now we go to in vitro-omics. The applications are uses of cell lines. And I've given four broad categories. Cell lines as general experimental tools to acquire knowledge. That can be basic knowledge, applied knowledge, whatever. Cell lines as models of some in vivo process. So for example, tumors or say myogenesis. And cell lines of sensors of something like a toxicant or viruses. And then as we've heard just now, cell lines as factors. So proteins such as monoclonal antibodies, viruses for vaccines, cells for transplantation as cell tissues or organs. And I almost forgot my lovely wife would want me to point out cell lines to eat. So, you know, the in vitro meat is part of cell lines now. Okay, so that's, and then we come to in vitro-omics. So I'm defining this as a collection or grouping of cell lines around the theme. And the broadest theme would be all the animal cell lines that have been reported in the literature. And so I would call that the animal in vitro, or another way of saying it, it's the cellosaurus in vitro. And I'm proposing there are two general classes of determiners or properties for these cell lines that I'm going to use to subdivide the animal in vitro. So these are the anti-factim or post-factim properties can be used to subdivide the animal in vitro into innumerable specific in vitro. So it's sort of like I'm a Canadian collecting hockey cards and you have a whole bunch of teams and you can put them into the Canadians or the Toronto Maple Leafs, the two most famous teams in the world. No, I'm not sure. Okay, so now what the heck are these anti-factim or before the fact and post-factim after the fact properties? So I'm saying that anti-factim properties are set before the birth of the cell line. The birth of the cell line, I'm saying, you might want to disagree with me, is the first sub-cultivation of the primary cell culture. So that's my definition of when a cell line begins. So the anti-factim properties are all those set before the birth. The identifiers can be the scientists, the sample from which you started the primary culture and of course the species from which the sample was taken. And then the post-factim properties develop after the birth of the cell line. So properties that appear during development characterization and the use of the cell line. And the cases we've heard today, they're continuing to appear, even cell lines that have been around for a long time. Okay, so a simple example of using anti-factim and post-factim properties. So the fish in vitro would be all the cell lines that have been reported from fish. And then the epithelium in vitro would be all the animal cell lines with epithelial-like shape. And then you can combine them, so anti-factim, post-factim properties, so that you have, say, the fish epithelial in vitro, all the fish cell lines with an epithelial-like shape. Okay, so now why construct these in vitro? And I would say to show where new cell lines should be developed, to reveal large patterns and new information and hopefully new research questions. And to allow the more effective use of cell lines in experiments, which I think is sort of being a theme of the talks today. So the use, and so I will illustrate the use of anti-factim and post-factim properties with the fish in vitro, which is approximately 900 cell lines. I can't keep up with cellosaurus. I know there's a very accurate figure, but... Okay, so the anti-factim properties of fish cell lines. So remember, these are set at the point of primary culture. So I have them in three categories. The properties of the laboratory of scientists. So for example, there's so many Indian scientists publishing on fish cell lines. So I would say the fastest growing fish in vitro is the Indian fish in vitro. So we won't say anything more about that. Properties of the sample taken from the fish, properties of the fish species from which the sample was taken. So I will talk further about those two sets of properties. So for the properties of the sample used to initiate the cell line, we can consider location. So the geographical origin, for example, of the fish from which the sample was taken to start the cell line. And the life cycle stage, the fish from which the sample was taken. And then the anatomical site from which the post embryonic sample was taken to initiate the cell line. So I'll just briefly look at location. So origin of the fish from which the sample was taken. And I use, say... This was my chance to show you where Waterloo is in Canada. It's there just to the west of Toronto. Great lakes all around this. So all the fish that have come from the great lakes, sampled from the great lakes and being used as a source of cell lines, I would say is the great lake fish in vitro. But you could do it other ways. You could have it political so that all the fish cell lines from Switzerland that were developed from fish in Switzerland, from Swiss fish would be the Swiss fish in vitro. And then you could have consider whether the fish were wild fish. So a wild fish in vitro or whether they're from a hatchery or whether they're from a fish farm. So that is the origin of the fish. And then there's the life cycle stage of the fish. So we'll just use the simple one of, say, salmon. So we have embryo, larvae, juvenile, adult and spawning fish. And so cell lines have been developed from all of these. And the smallest is from spawning fish, only two, maybe three cell lines. And the largest is from juvenile and adult fish. Next, we can have the anatomical site of the sample. So nearly all fish sources have been, fish organs have been a source of cell lines. But the number is a little hard to say, because some people consider, say, a pectoral fin as an organ. And others just say fin. And sometimes it's not clear what fin they're talking about in the origin of the cell line. So I'm going to say there's approximately 20 organ in vitro. And the largest is if you consider that the cell line was from fin in general. So the fin in vitro has got approximately 133 cell lines. And then, of course, you can consider whether it was from a normal tissue or organ or whether it was from a tumor. And nearly all the cell lines are described as normal, as most of us are not pathologists when we grab that liver from the fish. But to us, it seems normal. But there are at least two cell lines from hepatomas, RTH149 from rainbow trout and PLHC from hepatomas. And that's because they could experimentally induce hepatomas in the fish and then grab the hepatoma and start the cell line. So most organs have then been the source of cell lines. And then now I can go to the properties of the species used to initiate the cell line. And I'm going to divide this in three different ways. One is the significance of the species to humans. So you could have the aquaculture in vitro. So all the cell lines from species subject to aquaculture or the endangered fish in vitro. So people often develop cell lines because they say, hey, this fish is endangered. And wouldn't it be great to have some resource around to study it further. But I won't say anything more about those. What I want to talk about are the taxonomy of the species and the biology or natural history of the species. So taxonomy. So what the public refers to as fish is about 35,000 species. And we can divide those into the jawless fish, such as lampraise, no cell lines. So that leaves us with the jawed fish. And the jawed fish can be divided into the cartilaginous fish, which there might be two or three. So certainly two or three cell lines reported. And then the bony fish. Now the bony fish can be subdivided into the low-fin fish, such as those siloed cats lurking down, I don't know off the coast of South Africa. And then there are no cell lines. And then that leaves us the ray-fin fish, which can be subdivided into the cladistia, no cell lines. And then that leaves us the next class, the octeride, which we can subdivide into the conterstee, which is the sturgeon, approximately 10 cell lines. And then that leads us to the whole stea, or the gar, which is just like seven species in the world. And there's one cell line. And then that then leaves us with the teleos, so 34,000 species, approximately, of teleos. So they represent about 96% of fish. And they constitute approximately 850 cell lines from approximately 590 species. So that would be the teleos in vitro. So overall, about 2% of fish are represented in cell lines. And then we could take this further, I'll just very quickly say so, teleos can be divided in 70 orders, approximately 500 families in 3,000 genera, and then of course, 34,000 species. And I'll just use the, just say with the salmon, there's approximately 220 species with cell lines from approximately 10 species. And the largest is with the rainbow trout in vitro, with about 74 cell lines. But of those 70 orders, about half of them have, and an example is stomiforms, which is dragonfishes and their allies, has about 433 species, but no cell, but no cell lines. So many, many orders have no cell lines derived from them. Okay, so now, and this is actually the one that I wanna emphasize is the natural history of the species. So here I'm gonna give it, read a quote. Organisms can supply structures into which a vast range of biological data can be fitted. Natural history is the principal source of information about organisms living under natural conditions. So what I wanna stress is even though all fish live in water, their natural history is tremendously variable and complex, like how they swim, what they eat, how they have sex. It is just fabulous actually in a way. And so I'm just gonna use habitat as one example. So we can have cell lines from marine versus freshwater fish. So there's slightly more freshwater species than marine species, and there used to be most of the cell lines were reported from freshwater fish, but now there's more and more cell lines being developed from marine species. It could be from another habitat criteria is demersal versus pelagic. So demersal means that the post larval stages live at or near the bottom, and then pelagic, they live in the water column. And so cell lines have been developed from both demersal and pelagic fish. So this is just to now illustrate we're changing gears. So we've been talking all there on the left, and hey, we're gonna move to the right. And I'm just gonna very quickly go over this. And so for most of my career, I've been concerned with the things on the right. So postfactum properties are likely limitless, but often have variable certainty and are more difficult to classify. And so I've done it this way. Properties that appear as the cell lines develop, properties identified deliberately through cell line characterization, and then properties that arise from the use of the cell line, say in experiments or as a regulatory agent, and then properties related to cell line informatics availability and storage. So I've summarized this in this slide, and I just want to say, so for development, the two obvious criteria is what is the shape of the cell line and how does the cell line grow? Does it grow, you know, require a solid substrate? Can it grow in suspension or does it grow in some matrix? So those are things that you notice, you know, early in your development of the cell line. And then the characterization, say in fish, is rather poor for the most part. So we do things like cytoskeletal proteins, but you could have functional lineage markers. And then of course what is really needed in fish cell lines is all those omic profiles. So transcriptome or epigenome. And then a final characterization is the stemness or potential for multi-cellular organization. So those are development and characterization, then are two post-bactin properties. And then the others arise, can arise through use. So using the cell lines tells you a lot about them. So the reason fish cell lines were developed was for the relationship to viruses. So the relationship to pathogens. So you can describe cell lines that are susceptible to a particular virus, say viral hemorrhagic septicemia virus is a very famous fish virus called VHSV. So all the cell lines that supported the replication of that virus, I would describe as the support of viral and vitro, or the support of VHSV in vitro. And then I have say response to contaminants. A favorite class of industrial contaminants are the dioxin and dioxin-like compounds who have this quality of activating their own hydrocarbon receptor and triggering a signal transduction pathway and a readout of genes. So only some cell lines respond. And so I would call that say the dioxin responsive in vitro. And then there would be cell lines used in regulation. And then we come to storage and availability. And so I would just like to say so. So when I, in the 2017 paper, I said there was three types of storage and distribution situations. There was the curated in vitro. So those were cell lines where I went to Willie and said, Willie, give me that perfect cell line that I know that you've characterized. And it has no microplasm and it's just beautiful. And so that's the curated in vitro. But my fish colleagues are cheap because fish grants are not really great in size. So what we have is the informally shared in vitro. So people want, could you just pass me a flask or two? And so that has gone on. I would say, well, we know it's gone because we're asked so many times to give us the cell line. And it's kind of hard to say no. But of course, I always stress we don't have the quality assurance that Willie would have. And then finally, and now here I'm going to take Jan to task here, is we have the zombie in vitro. Those are those cell lines that have been reported in the literature. And we don't know, are they in a liquid nitrogen doer or are they not? Like somebody developed them in the, say, 60s or 70s and their lab is being closed. We don't know if they exist, but they might rise up. So that's a zombie. What you were describing is a cell in G naught. That's not a zombie. Yeah. Okay. So, so finally, what I'm, what I'm going to do is I just have two more slides. And what I'm also going to do is because I told Amos that I was going to do this, is that I'm going to wander up the lecture hall, make sure no one's sleeping. And just say that you can also, and by the way, that's how I always scared undergraduates too. And the longer I lectured, the older I got, the more worried they got. But what I was going to say is that the ocean can be divided by depth. So I'm up here in the epipelagic zone. So the top 200 meters. And so that's where all the fish cell lines have been developed from. They've been developed from fish in the epipelagic zone. So as we go down, of course, where's less light. And of course, the atmospheric pressure is starting to get enormous. So when we'll get to the epipelagic, you know, we have six, or greater than 6,000 meters, no light, 750 times atmospheric pressure. And there's approximately 10 species living there. So there are no in vitromes for those species. So I think there'll probably have to be tremendous developments in in vitromatics, i.e. special technologies to be able, well, to retrieve those fish samples and to grow them, you know, in your lab. But I think the in vitro omics, i.e. the cell line use, could be very interesting. So the question is, will the exceptional anti-factum properties, i.e. being in the epipelagic, yield exceptional post-factum properties? Will we find that their cytoskeletal structure is different in some way or regulated in a unique way? Will their vulnerability to UV damage be different? Will they be susceptible to the same viruses that are bugging the epipelagic fish? So on that note, I would I would just say, so considering anti-factum properties, together with post-factum properties should allow cell lines to better contribute to understanding fish, and that cellosaurus might be able to help connect a fish cell line to the natural history of the fish. Thanks for listening. So thank you, we're open to questions. And everyone is awake. I mean, you didn't have a chance to sleep now. Is there a fish with very few chromosomes? You know, there is. So there is some mud, you know, and someone did start a cell line in the late 80s for that purpose. Yeah. But I can't remember how many, like I think it's how many does Chinese hamster have? I think it's down there in that range. So that could be used in, yeah. Are the fish that live really deep delicious? And if so, is that a good reason to get cell lines? Yeah. Yeah. Wouldn't that be something to say that you had had, you know, fish and chips from a fish? Yeah. Yeah. From 6,000 meters.