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Growth of Mammalian Cells at Liquid Interfaces

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Growth of Mammalian Cells at Liquid Interfaces
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This is a general comment to Ivar Giaever’s remarkable set of 11 recorded lectures on biophysics 1976-2004. Giaever has so far (2014) participated in no less than 16 Lindau Meetings, starting in 1976, when he received his first invitation to lecture at the Lindau physics meeting. But it wasn’t until the 2008 meeting, after more than 30 years, that he finally disclosed what he actually received the 1973 Nobel Prize in Physics for, the discovery of tunnelling in superconductors. In year 2000, he did not give a lecture, but sat in panel discussion, and in 2012 he gave a critical talk on global warming, which for a long time has been on top of the list of most viewed Mediatheque videos. But at all the other 13 meetings, he lectured on his activities in biophysics and how these led him into starting a high tech business in the US. It is fascinating to listen to the 11 existing sound recordings, starting with 1976 and following through all the way to 2004. Giaever is smart enough to having realized that the most important part of the audience, the young scientists, change from year to year, so some parts of the lectures (including jokes) appear over and over again. But as time goes, he makes progress in his biophysics research and this leads to important developments and inventions. The starting point in all lectures is the possibility to study biological phenomena in the laboratory using methods from physics. With his background in electrical engineering, it is not surprising that he in particular has used techniques from optics and from the measurement of very small electromagnetic fields. The first two lectures mainly concern proteins on surfaces, but already in the last ten minutes of the second talk, Giaever describes his ideas about working with cells on surfaces. The rest of the talks all concern his studies of the properties of living cells on surfaces. The cells are grown and kept in what is called a Petri dish, a cylindrical shallow glass or plastic container. By inserting a very small electrode made of a suitable metal (e.g., gold) at the bottom of the dish and another above, electronic characteristics of a single cell can be measured. This can be both static and time-dependent properties. A question that has been at the centre of Giaever’s interest has been to develop an objective method to measure the difference between cancer cells and normal cells. Such a method would be an important contribution, since the usual method to distinguish cancer cells from normal cells is by observing their growth pattern in an optical microscope, a highly subjective method where mistakes can be made and have been made. Another question, which Giaever has addressed, concerns what kind of surfaces cancer cells stick to. This can be important to know, because many cancers spread from the original tumour and cancer cells wander to other places in the body and form new growths in places where they stick (metastasis). When he began his activities in biophysics, Giaever worked at General Electric, but after leaving this company in 1988, he accepted a position as Professor at the Rensselear Polytechnic Institute. Together with a colleague he also started a company to develop and market a sensor for cells in tissue cultures. This apparatus is now being produced and marketed (www.biophysics.com). Some of Giaever’s lectures focus on the problems encountered when trying to start a small highly technological enterprise. His account and reflections are interesting and in parts very amusing. Some in the young audience certainly could profit from following in his footsteps, in particular from following this advice: If you don’t get funded for your research, start a profitable business to make your own funding! Anders Bárány
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Transkript: Englisch(automatisch erzeugt)
Ladies and gentlemen, first, I'd like to apologize for not being able to give this talk in German. But I'd also like to say that somebody this morning said the German science was not up to date. Last night, when I sat down to prepare my talk, to my surprise, I already found a summary on my talk
in the folder I got from the organizing committee. So I think they're very efficient. And I would like to have given a talk in German because my talk sounded wonderfully scientifically when it's expressed in German. But I have to give it in broken American. And I'll try to do that as best as I can.
Since this is a physics meeting, you probably expect me to talk about quarks, magnetic monopoles, or something like that. But I'm not going to do that because I have gotten interested in biology. So this is a talk about biophysics. And the problems in biology may not
be of such a fundamental nature as the problem Professor Musbauer talked about. But they are really very fascinating to me. And they're very easy to identify. For example, as I stand here speaking to you, hopefully you will remember tomorrow some of the things I said. The reason you remember is that by speaking to you,
I am changing something in your brains. I mean planning in memory in your brain. Somebody will say, I permanently damage your brain, but I hope I don't do that. Now, amazingly enough, no one knows what memory is.
It could be that you make some protein molecules. It could be you make some new neuron connections. It could mean that you make some DNA or something like that. But the fact is that nobody knows. So you see, biology is full of very fundamental problems like that.
Now, I'm not gonna tackle any problem that difficult. I am going to talk about cells. And one fundamental thing in biology is that all living things are made of cells. The simplest living thing is a bacteria. They are single-celled organisms.
I don't know if you've ever thought about this, but a bacteria never die. If you have a bacteria here, give it a little sugar. In half an hour, the bacteria has grown old and fat. Then cleverly, it divides. You have two new young bacteria
and the process starts all over again. Now, people are also made of cells, but of course, you know that we have a finite lifetime. The reason or one reason maybe for that is that the cells in people are organized as a cooperative effect. And if some cells go haywire, all the cells die.
Now, it is possible to grow cells from people very much similar to how you grow a bacteria. And this work is called tissue culture work. And that's what I'm gonna talk about now. If I could have my first slide, you
will see that this experiment is on a quite different scale than what Professor Musbauer talked about. We have no help from the Swiss Army.
If you take some human cells, you can put them in a plastic dish. And now what you do, you put in a plastic dish a liquid medium. And in the liquid medium is everything you think a cell will like you put in there, like salt, sugar, amino acid, everything you think the cell would think would be good.
And then after you place the cells there, if I can have the next slide, you put the cell in an incubator. This is my co-worker here, Charlie Keyes, who are actually teaching me all he knows about cells. And you put the cells in an incubator, which basically is an oven kept at 37 degrees. And it also gives some carbon dioxide
to keep the pH of the tissue culture medium at the constant level. So really, these are the kind of experiments you can do in your kitchen. Now, if you have given the cell the right thing to eat, what will happen is they will grow and divide. And on the next slide, we see that here is a plastic dish looked from the top,
and it's completely confluent with cells. What you do then, you take a proteolytic enzyme, and you can tear these cells off the surface. You can replant them into new plastic dishes, or many more, if you will, and you can grow them up again.
And now you get two dishes full of cells, and you can continue your experiment. Now, you might think that you can go on forever like that. But it turns out that when you deal with human cells, you can only do the experiment about 50 times. After 50 times, the cell refuses to divide anymore.
Now, this is not an absolute truth, but this is what people now believe in biology, and this is referred to as the Hayflick limit. The reason for the magical number of 50 is that if you start with a human fertilized egg, the egg will divide about 50 times to make a human being. Now, if I took a mouse cell and grew this way,
that cell will divide about 20 times before it refused to divide anymore. And again, you see, this is sort of an interesting thing. Amazingly enough, and maybe ironically enough, if you take cancer cells, they will continue to divide. They will go on forever.
So it seems to me there's some sort of irony in this that the cancer really causes people to die, but the cancer cells will go on and live forever, but the normal cells do not. Now, if I have the next slide, we're gonna look at this thing in a little more detail.
Here is the solid substrate, and what I didn't say is that the medium you feed the cells are what people call optimized. They put everything in there you can think of to make the cell to grow, but the cells do not grow. What you have to do in addition, you have to put about 10% blood into the medium.
Actually, you don't put blood in. You put cerumen, which is blood without the cells and some clotting factors. So you see, it's not a very well-defined science because nobody knows what's in the blood which the cells need to grow, but when you grow human cells, fortunately, you don't need human blood.
You can use calf blood or something like that. Now, in blood, there are proteins, and the first thing that will happen is the protein absorb in a layer on the plastic substrate. Then the cells will come and settle down on top of the protein layer,
and then if you have fed them correctly, they will crawl out and flatten out on the protein layer like so, and they actually pull on this protein layer, and this pull is what I am really very much interested in, and then if they like it, they will again round up,
and now they will divide. You make two cells, and now these cells, again, will go on about their business, and it's this division which, with human cells, only will happen about 50 times. Now, if I can see the next slide, please. Here is a cover of science.
These are two different kinds of cells. These are normal cells growing down here, and they grow sort of in an orderly fashion, and these are cancer cells growing up here, and they are growing more in a helter-skelter kind of fashion. Cancer cells are not subjected to the ordinary
discipline, I would say, that normal cells have. On the next slide, shows there's more in detail, and again, this is not an absolute truth. It's almost truth. Normal cells will grow neatly and cover the substrate in a monolayer. Cancer cells tend to grow helter-skelter
and on top of each other, and so on, and there are really no, there are differences between normal cells and cancer cells, but nobody really know what the differences are, so when you look at cells in tissue culture, you say, well, these cells look cancerous,
and these cells look normal. If you're unfortunate to have a biopsy in a hospital, the doctor take the cells out, he looks at them, and he says, these are cancerous or these are normal, but it's a subjective judgment. There are no objective way of telling a cancer cell from a normal cell, and of course,
this is one reason I am and other people deal with tissue culture work, because what you ideally would like, you would like to take cancer cells and normal cells, put them in some solution, and have the cancer cell turn blue and a normal cell turn red. If you could do that, you will be a very famous man.
Now, on the next slide, shows you what we have started doing. Actually, I said I was very much interested in finding out how hard the cell would pull on the substrate, and what you do then, you start making the substrate thinner and thinner and hope that the cells will buckle your substrate. It turns out that we made the substrate so thin,
we didn't really need it anymore, and what we started doing is to grow cells on an interface between two liquid. Fluorocarbon is a heavy oil, and you put that in a test tube first, then you introduce the cells with the proteins. The protein layer will form on the surface of the fluorocarbon,
and now the cells will go down and grow on this liquid-liquid interface. Actually, they grow on the protein layer on the fluorocarbon. And on the next slide, shows you how one of these experiments looked like. Here is the tissue culture fluid up here.
It looks red, not because it's blood, but because it has a dye indicator indicating what the pH is, and here is the fluorocarbon here on the bottom, and here is the interface between the two. Actually, we don't do it in cylinders like this anymore. On the next slide, shows you how we do it.
We get little plastic dishes which all biologists use as a matrix of these little wells, and here we have a whole set of experiments, then, in the middle, which is like 26 experiments or 24 experiments at the same time. And let me now show you how these cells look like. The next slide shows you some mouse cells
growing on a fluorocarbon liquid, and these cells grow in an epithelial-like fashion. This is one cell here, this is one cell there, and here, what you see, they grow together almost like flagstones, if you will. And biology is a very descriptive science,
and when people grow cells in tissue culture, by and large, they distinguish only between two kinds of cells, epithelial cells and fibroblasts. If epithelial cells look like flagstones, fibroblasts tend to be elongated. This particular cell, on this particular oil, look like epithelial cells.
I'll show you now how it looked like on fluorocarbon oil, which is slightly different from this, and that's the next slide. Here, now, it looks more like a fibroblast, even though it's the same cell, but the substrate it grows on tend to influence how the cell would look. And here, also, we have some large,
what we call lakes, a whole, clear area where there are no cells. The reason for that is that when the cells settle down on the surface, they will grow out, but they will pull on the surface, and suddenly, the protein layer they grow on will break and open up, and you form these lake-like regions.
And this is a very interesting thing to us, because, again, we are interested in finding out how hard the cell pulls on the surface. If I look at the next slide, shows you how cancer cells grows on these surfaces. This is a cell called SV2 cells,
and you see that rather than making lakes, the cancer cells tend to make little warts, and you have all little warts all over the surface. Again, the reason for that is that the layer the cancer cell grow on is not strong enough, and when the cancer cell divide and multiply, they grow out, but they pull on the protein layer,
and the protein layer collapses inward, and the cancer cells pull much harder on this protein layer than the other cells did. So we were very excited about this, Charlie Keyes and I, and we discovered that you can grow cells in liquid-liquid interfaces, because in tissue culture work, it's sort of like an axiom that cell has to grow
on a solid substrate, and these are not solid substrates. So what we did, we did a whole big computer study to try to find out, I mean, anybody had done anything like this before, and the computer come out and said, no, nobody had done anything like this before.
But there's an old thing in physics, which I knew from before, saying if you do something new, it's either well known, or it's wrong, or it's both. And so happened in this case, too, if I can have the next slide. Oh, no, I was, bore myself. What we do with the cancer cells to show that the protein layer breaks
is that you can solidify the protein layer first. And you can take the protein layer, and you can, say, put formaldehyde on it, which would make the protein layer very solid. And now when you grow cells on it, the protein layer is unable to break. And this is shown here. Rather than the previous slide you see now,
the cells do not break the protein layer. Now, if I have the next slide. This is a tissue culture proceedings. Actually, you can't see that. I can't see it myself, even. But it was published in India, held in 1962.
And it turns out a man named Rosenberg had done more or less the same thing, growing cells and liquid interfaces. And it was a big disappointment of us, of course, to finding out that somebody had done it before we did, but that's the way science always is. The only consolation for it was published by a man, it's a publishing company called Dr. Junk.
But this paper is not junk, unfortunately. It's the right stuff. And we can't really understand why people haven't taken this up. But the reason must be this was done about 20 years ago. And at that time, it was so very difficult to grow cells. So people, it's more difficult to grow cells in a liquid-liquid interface than a liquid-solid interface.
So people probably get discouraged by growing the cells that particular way. Anyway, Rosenberg deserves all sorts of credit for having done this work first. Now, there are many advantages growing cells on these liquid-liquid interfaces. And if I can have the next slide.
And I'll show you one. When you're going to harvest cells, here you have the cell growing on a solid surface. Here is the protein layer the cells grow on. And nobody actually knows how the cell attaches to this protein layer. Now, if you want to harvest these cells from the solid surface, you have to get them off.
And the only way you can get them off is to expose the cell to a proteolytic enzyme. And I drew that drawing myself. I don't know what a video game the Pac-Man is popular in Germany, but it's a craze in the United States. And these little enzyme is like the Pac-Man guys. They come along, and they really barber the cell.
Cells have all sorts of proteins referred to as antigens on the surface. And the proteolytic enzyme will barber the cell. The cell doesn't like that a bit. It will round off, and now you can get it off the surface. But you have to recognize you have done severe damage to the cell.
And maybe a lot of people, when I started out saying a cell will only divide 50 times, maybe in that process the cell can't take. Maybe this 50 time limit is not because you can only let it divide 50 times. It may be because each time you have to damage the cell and start anew.
And this, of course, is severe trauma for the cell. If you deal with cells and liquid interfaces, you don't have to do that at all because the cells grow in a protein layer. If you want to have it up, you can just take it up with a pipette. And the cell just come off with no problem whatsoever. And the next slide shows you, this
is one of these cancer warts, which we took off the liquid interface and put on a solid plastic dish. And here you see the cells are happily crawling out from this particular wart and over the plastic dish. So they're all alive and fine. And you do not have to damage the surface of the cell. Another reason that is very important is that a lot of people like to look
for differences between cancer cells and normal cells. And they inject the cells into animals to make antibody to this protein on the cell surface. If you have to damage the cell first, of course, you will not get the right kind of antibody. Now, I work for a general electric company.
And what I really try to do many times is doing applied science. And a lot of people think I ought to be ashamed of that. But I'm not. I really enjoy it. And one thing that led to another in this particular work is that they're important to grow cells in large quantities.
And the problem is that cell has to grow on a surface. So then you need large surface areas. Some smart people about 15 years ago or so found out that you didn't really have to have large. You don't have to use large surface areas. You can grow cells on little beads. You can make plastic beads, glass beads, what have you.
And then, of course, each bead has a large surface area. They can fit them in in a small volume. And the cells turn out to grow very happily in these small volumes. The reason you want to grow a large number of cells is that the cells will manufacture things which you need. For example, you could grow human insulin
in a tissue culture. Interferon is a substance which is very popular today. You can grow that in a tissue culture. Or you could take these cells, infect with a virus. Then you harvest the virus and make vaccine. And this is how most vaccines are made. At least, this is how the polio vaccine used to be made.
So there is great advantage if you can grow cells in large quantities and small volume in an economical fashion. And we got the idea that you could grow cells also on an emulsion. And this is illustrated in the next slide. Here is a, actually, this slide is upside down. It doesn't make any difference. Here is the liquid surface.
And if you imagine the gravity, it goes that way. Here you have the tissue culture medium. And now you see this looks white. The reason it looks white, it's full of little spheres of fluorocarbon. And the fluorocarbon make a stable emulsion because they have a protein layer on the surface.
And so therefore, you have now a very stable emulsion. Now, if you infuse this particular thing with cells, it turns out that the cells happily will grow on that emulsion. And this is shown on the next slide. Here is the picture of the emulsion. And you see, if you look at this large sphere here,
this cell happily grow. There's lots and lots and lots of cells here. And if you look at, actually, it's not very evident in this slide. But to be very successful growing cells, you need a certain size of the sphere. It turns out that the cells are approximately, say, 100 micron in size. And you can grow cells successfully
on spheres, say, 300 to 400 to 500 microns. If you make the spheres too small, the cells don't really like to grow on them because the curvature is too big for the cells to be very happy. So you have to hit the happy medium with the size of the cells and so on.
And this offered the opportunity then to grow cells in an emulsion on a large scale. And that the cells indeed grow is shown on the next slide. Here is the cell number. And here is time in hours, 20, 40, 60, 80, and so on.
And here is the cell number. This is 10 to the fourth. This is 10 to the fifth cells. And you inject a certain number of cells. Then after 20 hours, when you check, you have actually fewer cells left. And the reason for that is that the shock was starting over a fairly large number of cells die.
But after that, the cells like their new environment. And each 20 hours or so, the number goes up. And the cells grow very happily. And so this is a different way then of growing large numbers of cells. And the great advantage it has, we think, is the way the cell can be harvested.
And this is shown on the next slide. That was backwards. Here is the cells, or here is the emulsion. And here are cells growing on the emulsion in the tissue culture fluid. And now if you take this thing and you centrifuge it to spin it, what will happen is that the forces will destroy the emulsion.
And the fluorocarbon, again, is heavier than the tissue culture fluid will be at the bottom. The tissue culture fluid is the lightest. And the cells are in immediate density. And you can pick them off in the middle. And this makes it very, very easy to harvest a large number of cells.
Now unfortunately, even though we like this method very well, I have to be honest and say that we can't grow human fibroblasts very well in this emulsion yet. But we are working on that. And the attack we take is that you can change, you can stabilize this emulsion by putting various kinds
of protein on the sphere surface. And by changing the protein, we find that some cells like some kind of protein better than other kind of protein, and now we are playing around with this kind of boundary condition to see if it can grow human fibroblasts very easily
in this particular fashion. That's enough for the slides. In United States, and I don't know how this is in Germany, after a half hour or so of entertainment, you have to go and sit through some advertising. And believe it or not, you have had your entertainment,
and so now we're gonna sit through some advertising. And the advertising I want to do, I want to speak on behalf of Professor Rabi. I and Professor Rabi had planned to travel together to come to this meeting, and we called each other a few times, and unfortunately, Professor Rabi got sick.
He ended up in the hospital, and he couldn't make it. And I talked to him two days before I left, and if I can quote Professor Rabi, he said, gosh, I will miss so being there. And he was very touched, but he said the doctor could not let him come. And I'm very sorry that he couldn't make it,
so I promise to give every one of you his greeting, which I do, and I will like to write his name and address down, in case any of you would like to send him a letter, I'm sure that would cheer him up.
This is Professor I.I. Rabi, 450, Riverside. I can't do well up in that pen. Well, I'd do well up there, I guess. Riverside, New York, New York, 10027.
It doesn't read very well, but at least I can see it from up here, and I will end up by saying, as they do in television in the United States, hurry, hurry.
Thank you very much.