Frontiers of Biomedical Engineering

BENG 100 - Lecture 5 - Cell Culture Engineering

Chapter 1. Applications of Gene Transfer [00:00:00]

Professor Mark Saltzman: There were a few things I didn't get to finish last time in talking about particularly gene transfer in mammals. I want to finish with that and then start on the topic for this week which is an introduction to cellular physiology. In particular, cell culture technology or how you culture cells outside the body.

This is the slide that I left on last time and the idea here was to use plasmids, which I talked about last Thursday, to introduce genes into animals. Here, the plasmid is directly injected into a cell. The cell is a fertilized egg, so you take a fertilized egg and you directly inject plasma DNA into the pronucleus, into one of the pronucleus--pronuclei. This solves a major problem which I'm going to talk about just at the beginning of class here in a slide or two, is 'how to get DNA into cells?

We talked last week, or the week before, about the plasma membrane and something about the chemistry of the plasma membrane: a lipid bilayer that's stable in water because it's arranged in such a way that the hydrophilic parts of the lipid molecules are exposed to both the outer and the inner surface of the cell. There's lipid chains in between so cell membranes are lipid rich layers that can exist in water. Because of that they're barriers, they separate the inside of the cell from the outside of a cell, and only certain kinds of molecules can pass through a cell membrane. It has to be small, and it has to be lipid soluble in order for it to pass through a cell membrane.

Well, plasmid DNA is neither of those. It's very water soluble and it's a big molecule, a very big molecule; it can't naturally get inside cells on its own. We talked about this a little bit in section last week, when you eat vegetables or meat, you're eating a lot of DNA but that doesn't enter the cells of your body because of the barrier properties of cell membranes. To get genes and gene transfer vectors into cells is a problem. Here, the problem is solved by injecting it directly into the cell, shown here, and then that's one issue with gene delivery. The second issue is that the gene vector, in this case the plasmid, has to be compatible with the cells that you're trying to express the gene in. that means that the cells have to be able to replicate the DNA on the plasmids, in order to make many more copies of it. It has to have an origin of replication, which we talked about last time, which is compatible with the cells.

It also has to have a promoter for the gene that the cells can recognize. In this case the promoter, which is a sequence of DNA that is positioned right in front of the gene of interest, the promoter is the betagalactoglobulin promoter in this case, and betagalactoglobulin is a milk protein. You're taking advantage of a promoter system, or a gene activation system, that this species knows about because adult animals can make milk. When they make milk they have signals for turning on the betagalactoglobulin promoter and expressing the gene of interest. Both of those things have to be correct. You have to be able to get the DNA into the cell, it has to be incorporated into a vector of some sort, and the vector has to be compatible with the species and the cells that you've provided the DNA to.

In this case, if everything goes right, you inject the DNA into the pronucleus, you implant this fertilized egg into a foster mother, the mother gives birth to offspring, it develops and when it develops this foreign DNA is in the offspring. When this animal makes milk, it makes all the normal milk proteins, but it also activates your gene of interest as well. So you could collect your gene of interest in the milk. Does that make sense? This concept of introducing foreign genes, genes that are made of recombinant DNA into animals using this kind of technique is widely used in biomedical research. One of the ways that it's used is to take the fertilized egg from an animal, a mouse, usually it's a mouse and inject in a vector that contains a gene that is involved in human disease of some sort.

One of the big problems with studying human diseases is that you can study how they occur in people but there's only a limited amount that you can learn from people. If animals get a similar disease then you can learn about that disease progression in much more detail in the animal. But, unfortunately, there aren't good animal models for many of the diseases that we'd like to study. Alzheimer's disease is a good one. Millions of humans affected by Alzheimer's disease, other species don't get Alzheimer's disease. You can produce animals that have a disease that's similar in some ways to Alzheimer's disease by taking the genes that are involved in Alzheimer's and introducing them into mice, for example. You do that by expressing the genes in the mice in the way that I described on the last slide and it's shown in a little bit more detail on this slide. Those animals that you produce are called transgenic animals because they're expressing, usually at high levels, a transgene or a gene that's not normally present in their species.

Chapter 2. Gene Therapy [00:06:41]

How would you accomplish this kind of - how would you accomplish gene transfer in adult humans? Those two examples I just gave you, one had to directly inject a gene containing vector into the pronucleus of a fertilized egg and you don't have that opportunity in adults. You already have an adult organism and you'd like to get the gene transferred. Before we talk about how to do it, and we don't have perfect ways to do it yet but I'll describe some of the ways that are used, I want to talk just briefly on this slide about what the goals for gene therapy might be.

There's a variety of different ways to think about using gene therapy and they're illustrated in this picture. One way is to replace a gene that's defective in a disease. Usually this is a disease that's caused by a defect in a single gene. We talked about sickle cell anemia last time, that's an example. Another example is cystic fibrosis. Cystic fibrosis is a disease that affects many organ systems, but particularly the lung. You get cystic fibrosis because one protein that's made by your lung epithelium is not expressed properly. You're able to make the protein but the protein doesn't function properly. You could potentially treat cystic fibrosis by providing the correct gene and providing it to all the cells of the lung epithelium.

Because of this, because cystic fibrosis is a disease that's caused by a single gene defect, there's been great interest in trying to treat cystic fibrosis with gene therapy. The idea would be to try to give, usually in the lungs--people have thought about this because that's where the main manifestations of the disease occur--and introduce a gene vector into the lungs, perhaps by having a patient inhale it or by somehow instilling into the lungs, in such a way that these gene vectors get taken up by lung cells and the gene gets expressed throughout the lung. This might be a plasmid. It might be a plasmid that contains a promoter that works in lung cells and that has the cystic fibrosis gene.

Well, now, how would you get the gene into the cells? Well, one way you can get it is by mixing the gene with lipids, with lipid molecules. Special lipids that do this are called cationic lipids. They're lipids but they also have a charged portion, a positively charged portion which interacts with DNA. So you form DNA lipid complexes and because the DNA is complexed with lipids it's more soluble in membranes and more likely to enter cells. That's one way to do it and I'll talk about it in other ways in just a minute. You get the idea here, is to deliver the gene that you're interested in directly to the tissue where it's being used and in this case, the idea is to introduce a gene that's missing.

Now, in other cases you might want to introduce a gene that's not even normally present in people. You're doing that because you want to treat disease in a different way. One way that that's done is by introducing genes into cancer cells, genes that aren't normally expressed in any mammalian cell, but that will cause the cancer cell to die. This example here, the idea is to introduce a gene into tumor cells of a tumor. It's a gene that allows you to deliver a drug that's not active but the gene causes that active - that inactive drug to be converted into an active form that kills the cell. This is called a suicide gene. Basically, you're introducing this gene into cells that when you give the right inactive drug that will cause the cell to make chemicals that kill it. Does that make sense?

This is a way of introducing cytotoxicity or cell killing ability into a cell, and that's been used to treat many kinds of cancers, particularly cancers of the brain. The problem is the same, how do I get the gene that I want into only the cells that I want? You can see that it would be problem here if the gene that you were trying to give to the cancer cells also went to normal cells--that would be a problem. This is a common problem in gene therapy. You would like to get the gene that you're interested in expressed in some population of cells within the body but not in other cells. So, making gene therapy specific is a key problem in making it work.

These are two examples I just gave you of introducing genes that affect the life of a cell, either by making it express a protein that it's not making properly that's important to its life, or by intentionally killing a cell by having it express a fatal gene. The other examples on the bottom of the slide here are introducing genes that affect neighboring tissues. So one way to do this might be to - if you're trying to treat a heart that has disease and usually it's disease in one of the coronary vessels or the blood vessels that serve the heart, that provide blood to the heart. Coronary artery disease is one of the major causes of death in western countries. It occurs because blood flow gets reduced in that blood vessel and so the heart muscle, which is actively beating all the time, needs large quantities of oxygen provided by blood can't get the blood through the vessel.

Here, a concept is to introduce a gene that causes the process of new blood vessel growth. If you could introduce that gene you'd make the chemical, in this case it's a protein called vascular endothelial growth factor. The name of the molecule isn't important, but it's a molecule that stimulates the heart to produce new blood vessels around it. This is introducing a gene, it's a natural gene but it's not - it's a natural gene that's found in humans and is actually expressed all over your body in different levels, but you're concentrating it or over expressing it in one particular region of tissue in order to have a particular effect. You're taking a normal gene and you're over expressing it or expressing it more abundantly than normal in one particular spot of the body to have an effect.

One last example here is that you might want to put a gene in that makes a protein in a tissue that doesn't ordinarily make it so that that tissue can make large quantities of the protein and serve sort of as a source of that protein as a drug. The example might be insulin. Your pancreas normally makes insulin. Diabetics whose pancreas does not function properly, so don't make enough insulin, don't have enough insulin present for normal glucose metabolism so they can't handle sugars properly. What if you injected the gene for insulin into their muscle so that their muscle cells in one particular location started making insulin? Then that insulin would accumulate in the muscle, enter the blood, and circulate all over the body and so you'd turn the muscle into an insulin making tissue. Those are some example of different strategies for using gene therapy in people.

Chapter 3. Potentials and Limits of Hijacking Viruses [00:14:45]

Well, I talked about the problem of using plasmids in gene therapy, that it's very difficult to get them into cells. There are other vectors that are very efficient at getting DNA into cells. Viruses, in particular, are very effective at delivering their viral DNA into cells. If you catch a cold you get exposed to someone who has a virus that's causing an upper respiratory infection, they cough near you that virus enters your respiratory system, it infects cells of your respiratory system and those viruses replicate. They basically make many, many copies of their DNA and they make all of their viral proteins and they assemble into new viral units.

Well, what if you could hijack that virus to carry genes that you want? You could do that by using the techniques we described with restriction enzymes and ligases to cut and paste the viral genome so that it contains the gene of interest to you. You make a recombinant virus that has all the normal components of the virus but also has a gene that you want. Now, all you have to do is introduce that virus into an organism and the virus will do what it naturally does, which is infect some cells. Now, the other nice thing about viruses is not only are they very efficient but most viruses are specific for certain kinds of cells. The kinds of viruses that we get when we have colds are very good at infecting the respiratory system. If you get a flu that affects your intestinal system then those viruses are infecting gut cells. There's other viruses that infect cells of the brain, of the kidney, of the liver. Hepatitis is a virus that specifically affects the liver, so viruses are often specific to certain kinds of cells. That is also an advantage of using them as gene therapy vehicles because they will only infect cells that they are - that they have an affinity for or that they're prone to affect.

This picture here, we're going to come back to this later in two weeks when we start talking about vaccines, and we're going to talk about how you make vaccines for viruses and so in doing that we're going to think about the life cycle of a virus. This is a life cycle of a particular class of viruses called retroviruses. You know about retroviruses, the most famous and important one at this time is HIV, which is a retrovirus. This shows the greatly magnified compared to the size of a cell because viruses are much, much smaller than cells. They might be 100 nanometers or so in diameter, whereas, a cell is ten microns or so in diameter. These very small particles are able to specifically recognize certain kinds of cells. They enter the cell after recognizing, they fall apart inside the cell, they reproduce their DNA, or their genetic material using host mechanisms. They, in some cases, they integrate their DNA into the host chromosome, that's what retroviruses do and they make viral proteins. Because they've made many copies of their genome and they've made all the proteins that are necessary to - for assembly of a new virus, then this cell can make many new viruses. That's another advantage of using viruses as a gene therapy vector. They can reproduce and so you only need to give a few of them in order to make many, many inside the body.

Why isn't that the solution to gene therapy? If you can genetically engineer viruses like this and viruses have all those good properties as gene therapy vehicles that I described. Why isn't this a solution? Well, there are problems with viruses as well. Retroviruses, for example, can only insert - can only transduce and express their DNA in cells that are dividing. So if it's not a cell that's going through division you can't use a retrovirus to express the protein. They integrate their DNA into the host genome and you might not want to make that kind of a change in the cell. These are some problems with retro viruses.

Because of this people have sought other kinds of viruses to use as vectors and one that's commonly used is an adenovirus. Adenoviruses are viruses that give upper respiratory infections, they cause colds. They can transduce non-dividing cells so that's a good part. They can insert genes into non-dividing cells and cause those genes to be expressed. A problem with adenoviruses is that your immune system recognizes them, and if your immune system recognizes the virus then it can attack the virus and eliminate it from the body. Now, normally that's a good thing because if you're out in the world and you get infected with adenoviruses you recover from it, your immune system can get rid of it. But if you're trying to use that virus as a gene delivery vector and your body gets rid of it then the therapy has failed, it's ended. People have tried to produce viruses that are different, adenoviruses for example, that your immune system can't recognize. Because of this, because of the natural properties of viruses which sometimes are unwanted like in retroviruses, and the natural properties of your immune system which can cause you to reject viruses, we've had great problems in making viruses that are both safe and effective for gene therapy in people.

I talked about just not using viruses but using plasmid DNA together with lipids and that's another strategy for gene therapy. We'll talk later about other kinds of vehicles when we talk about new methods for delivery of drugs. That's what I wanted to say in finishing up last week's topic of DNA technology. What I want to do for the rest of the time today is talk about - a little bit about cell physiology but I'm not going to say too much for a couple of reasons. I think most of you know something about the sort of architecture and normal structure of a cell and the components within it and the chapter, Chapter 5, has a good description of that for those of you want a refresher. We'll talk a little bit about the principles of cellular physiology and then I'll move next time to talking about how you culture cells outside of the body and what that's useful for.

Chapter 4. Bacterial and Human Cell Physiology [00:22:31]

I said this sentence in one of the first two classes, 'all cells are the same', and you know that's true. We talk about cells as being kind of the fundamental unit of humans and other multi-cellular organisms. Why do we talk about them as the fundamental units? Because cells on their own are alive, they can do the things that we associate with life, they replicate, they reproduce, they metabolize, they can move, they can grow, and they can use nutrients and make wastes, and they work cooperatively in order to perform some function.

This is a picture that I showed you before, but basically tells you something that you already know, that humans are made up of organ systems, organs and organ systems, those organs are composed of tissues. What I didn't mention before was the four major types of tissues which make up the organs of our body: muscle tissues, and the function of muscle tissues is to do work, to contract in order to do work, nervous tissue and the function of nervous tissue is to communicate using electrical impulses primarily to communicate, epithelial tissues which form linings in the body so your skin is an epithelial tissue, the lining of your gut is an epithelial tissue, the linings of all the glands in your body like the pancreas are epithelial tissues, and connective tissues which are responsible for holding all these things together into one unit.

All cells are the same and I say that because any cell within you or within me is the same in many respects. It has a cell membrane. If I looked inside it has the same kinds of components inside, it has the same DNA inside the nucleus. Let's take a step back and ask are all kinds of cells really the same and start by talking about two cells that I know you know are quite different. Cells that I took from a human and cells that are in a bacterium.

What are the differences between a bacterial cell and a human cell? What are some of the differences between a bacterial cell and a human cell?

Student: [inaudible]

Professor Mark Saltzman: The bacterium doesn't have a nucleus, and in fact, they don't have very well formed organelles in general. Human cells have well-described organelles inside of them. You took biology in high school, you learned the names of these organelles and their principle functions, the nucleus, the endoplasmic reticulum, the Golgi apparatus, mitochondria, lysosomes. Those things are all reviewed in the chapter in the book. I'm not going to talk about them in detail but please look at that to remind yourself of those kinds of specialized sub-cellular units which are present in cells in humans but not present in bacteria.

Why aren't they present in bacteria? Why don't bacteria need a distinct nucleus and that's necessary in a human cell? What else is different about human cells and bacteria other than these formed organelles?

Student: [inaudible]

Professor Mark Saltzman: Human cells don't have a cell wall but most bacteria do. There are different kinds of cell walls in bacteria but they have a rigid cell wall and our cells don't have a rigid cell wall, we have a fluid lipid like cell wall. Why is that the case? Well, you could think about the cell wall of a bacterium as its skeleton, it's what protects it from mechanical forces that are out in the world. We have other ways of protecting; many of our cells are inside our body protected by specialized cells of our skeletal system. They don't need that kind of mechanical strength.

Another important difference between human cells and bacterial cells is their size. Bacterial cells are small, one to two microns in diameter and this shows a picture of a common bacterium called E. coli. This is a bacterium that's normally present in humans; it colonizes all of our intestinal systems. It's a common bacterium and most strains of E. coli are not harmful to people, in fact, they provide us with some beneficial properties. A human cell shown here on the opposite side has these formed organelles that I talked about a minute ago, mitochondria, Golgi, endoplasmic reticulum, nucleus. It's encased within a membrane, a cell membrane which we talked about the structure of. It's much larger than a bacterium, it's ten to 30 microns let's say instead of one to two microns, so almost ten times bigger.

One of the reasons why human cells have compartments is because their functions are segregated into regions. The nucleus contains all the DNA, and so the early stages of gene expression, transcription, happened inside the nucleus where all the DNA is. One of the reasons why you would want to segregate things into certain regions is that molecules have to move in order to accomplish these things; molecules have to move from one place to another.

In general, inside of a cell molecules move by a process called diffusion. They move from regions of high concentration to regions of low concentration by this natural flow down a concentration gradient. Movement down a concentration gradient occurs fairly rapidly over short distances like a micron or so, like the size of a bacterium. Diffusion mixes things very well, but as the size gets bigger, diffusion becomes very slow. You're familiar with this, if I had a jar of acetone or some kind of a volatile liquid, let's say I have perfume. I have a jar of perfume and I sit it here, if the room was perfectly still the molecules of perfume would diffuse out of this jar and they'd start moving through the room and those of you in the first row would smell it within a few minutes, sometime later you'd smell it one row back, sometime later - and the farther away you get the longer it takes.

Because of the physics of diffusion it doesn't go linearly with distance. That is, it doesn't take one second to go the first, and then two seconds to go to the second, and three seconds to go to third. It varies with the square of distance so it goes - in order to go twice as far it takes four times as long. These details aren't important, it is described in your book, but the thing that is important to know is that diffusion occurs fast over short distances, but because of the physics of diffusion it occurs slowly over longer distances. Because of that you can't count on molecules moving rapidly over the size of a cell that's ten to 30 microns in diameter. Because of that, in order to efficient function, then you group all the like activities together. That's one way of thinking why animal cells have cells from animals and humans have sub-cellular compartments.

Another thing that's different about human cells and animal - human cells and bacterial cells is that many bacterial cells can swim and can exist in suspension, that means just suspended in a fluid. In fact, that's their preferred state is suspended in a fluid where nutrients are widely available to them, and where they can swim from one spot to another in order to get the nutrients they need, or in order to run away from toxic compounds. If you're a bacterium your whole life is about swimming to nutrients and running away from things that would kill you. Human cells, most human cells, don't function very well in suspension. If you take cells from my skin and disperse them and we'll talk about ways to do that next time, and you try to maintain them in a beaker. Even if that beaker had all the nutrients that skin cells needed they wouldn't survive very well in solution, they tend to need to be stuck to a surface in order to survive.

That's what this picture on the bottom shows you here. It's a picture of cells actually growing and moving in cell culture. This is a cell from connective tissue called a fibroblast. Instead of being round like this cartoon of a cell when you have the cell in culture, in a plastic or a glass dish, it tends to stick to the dish and spread out so it looks much more like a pancake, a funny shaped pancake, but a pancake than a sphere. This property of cells in culture is called anchorage dependence - that the cell needs to be anchored to a surface in order to function properly. That's another difference between human cells and bacteria.

Now, what does that have to do with the life of a cell inside the body? It's - this picture on the bottom shows the cell stuck to plastic. Certainly that's not what it's like when it's inside the body. Well, it's true that it's not stuck to plastic but it is stuck to other materials The other materials that it's stuck to include other cells that surround it and this complex matrix which surrounds all of the cells in our body called extracellular matrix and I'll get to that in just a few moments. In some senses most of the cells in our body are stuck to a surface. They're stuck to a surface that's provided by the environment that's around them, they're not swimming free in solution.

Chapter 5. Cellular Division [00:33:44]

Well, you know that cells in our body can reproduce. Most of you have probably at some point or another learned about the cell cycle, or the process of a cell going from being a single cell to being two cells. That's shown schematically here, let me talk about the bottom diagram first, which shows the process of mitosis. Mitosis is an orderly sequence of events by which one parent cell becomes two daughter cells. Several things happen during mitosis, first, when a cell enters the process of mitosis it already has twice the DNA that it needs. So before a cell enters mitosis it has to have synthesized its DNA so it has two copies of all of its chromosomes.

What happens during mitosis is that the cell is able to separate this double set of chromosomes into two sets and that happens in a way that's shown in this cartoon here. It separates those duplicated chromosomes to two sides of the cell, physically separates them, and the cell itself pinches off to form two new cells. If this happens correctly, each of the new cells has exactly the same composition as the parent cell.

Mitosis is one event that happens during this larger event called the cell cycle. It's usually shown like a clock is shown here, where time 0, which is at the top here is right at the end of mitosis where you have two new cells that are formed. If I followed one of them it is in a phase that's called G1 first and this is a resting phase before it enters the process of synthesizing new DNA in preparation for another round of mitosis. G2 is a phase that indicates the gap between when DNA synthesis occurs and when the next mitotic event occurs. If cells were actively proliferating, that is you had a population of cells that are actively proliferating, they would be going through continuous rounds of the cell cycle. New cells are formed, they wait a bit, they synthesize DNA, they wait a bit, they divide, and on, and on, and on.

Well, not all of the cells in your body are dividing at any one time. Not all the cells are dividing; in fact, cells in your brain don't proliferate at all. Some small subsets do but most of the cells in your brain don't proliferate and that's because they get trapped at one portion of the cell cycle. They get arrested in the G1 phase, for example, and they never progress through to the S phase where they synthesize DNA. Cells can stop in their lifetime at some point in the cell cycle and persist for long periods of time.

This process of cell proliferation or cell division is, of course, very important during development of an embryo. During development of the embryo it goes from being a single cell, the fertilized egg, to being a multi-cellular organism with billions of cells in it. Those billions of cells come from successive rounds of cell division. Now, I said before that all cells are the same. When I talked about mitosis, I talked about cells making perfect copies of each other, so each parent cell becoming two daughter cells that are the same. So if that was all that was happening then an embryo would just be many, many cells that are all identical and you know that's not the case. Cells start to change and they change in complex ways so that eventually when a fetus is born it has some cells that make up its brain, some that make up the heart, some that make up the liver and the skin, and all those cells are different in important ways.

Chapter 6. Cell Differentiation [00:38:41]

I'm going to talk next week, or next time on Thursday, about the processes that lead to differences between cells. The general word for that process of making differences between cells is differentiation. When cells become more like cells of the mature brain they're said to differentiate into brain cells. When they become more like the liver, they differentiate into liver cells. So we're going to talk about that process more next time. What I want you to remember now is that abundant cell proliferation takes place during development. All of the cells that are reproduced during development are the same in certain ways, they contain the same DNA, they're all progeny of the same fertilized egg, but differences are acquired within specific cell populations during the process of development.

Some of those differences in cells are obvious if you look at the cells in an adult organism. In fact, some cells are very different from the cells that are proliferating early in development. One example of a very different cell is a red cell or red blood cell, these are the cells that give blood it's red color. They make up about 45% to 50% of the volume of blood is red cells. They're red because they contain a special protein called hemoglobin which is very concentrated inside the cell. Hemoglobin, as you know, is an oxygen carrier and so red blood cells concentrate in bags of hemoglobin which circulate in your blood and they carry oxygen. Well, it was not very hard for me to draw this red blood cell here, it's just an oval, and one reason I draw it like an oval, is that that's basically what it looks like if you look at it. One of the most obvious things about a red blood cell is it doesn't have nucleus, it has no nucleus. Red blood cells are terminally differentiated cells. They have become as different as they can be. In fact, they're no longer capable of reproducing; all they can do is exist as red blood cells and then die. So, they've lost one of the essential characteristics of these cells that are the same that I talked about earlier. That's just an example of a cell that in its very mature or differentiated form is quite different from other cells inside the body.

On the other side of this diagram I show a picture of a neuron. This is just an example of what a neuron might look like. Here's the nucleus in the middle of the cell body and what - if you looked at a picture of a variety of different cells that were isolated from adult organisms you would recognize this immediately as a neuron or something that's like a neuron because of the many small thin projections that come out of the body of the cell. Neurons have these projections because one important property, or one important function of neurons, is to communicate with each other. The way that they communicate with each other is by physically touching each other, and the way that they connect across distances is by having these process that extend from one place to another. Like many cells in neurons, the shape of the cell is intimately related to its function within the organism. That's obvious when you look at neurons and it will be obvious when we look at other kinds of cells as well.

Because of this, often a biologist can discriminate between different kinds of cells that they see in section of a tissue for example, merely by its shape and the characteristics that it has. One could easily, for example, differentiate between a red cell and a neuron, but a skilled biologist who's looked at many, many different cells could differentiate between different types of neurons. Maybe by the different branching patterns that these processes had, or how many processes were on a different kind of neuron, you'd be able to tell something about its function and probably something about where it came from. You might be able to tell just by looking at it what was - if there was a disease what was wrong with it.

In between these two pictures I show a picture of a fibroblast. Fibroblasts are specialized cells that exist in connective tissues. The connective tissue is one of those four main types of tissue, they're supporting material - supporting tissues that surround other cells in the body and give the cell - and give the body much of its mechanical property. One of the important functions of a fibroblast is to allow for healing of wounds. This shows a fibroblast that is very stretched out. Remember when I showed you that picture of cells attached to plastic a few minutes ago; I said those were fibroblasts that were growing on a culture dish.

If I looked within the skin, one of the lower layers of your skin, the connective tissue layer, you'd find many fibroblasts there and they're just waiting in a more rounded shape than this, just waiting to do their job. Their job begins if you happen to cut yourself, for example, or you get a wound. When you get a wound several things start to happen. First, hopefully your blood clots and so that stops you from dying from loss of blood there, but your body tries to heal this wound that's created. One of the first steps in that healing is that fibroblasts like this cell crawl into the space that's created by the wound and they grab a hold of both sides of the wound and contract and try to pull it together. To do that they have to be able to stretch out and they have to be able to pull. If you look at this picture of this fibroblast you can imagine that it's firmly stuck up here. It's firmly adherent to the surface here, it's firmly adherent at the back here, and in between its trying to pull these ends closer together, you can imagine that that's what it's doing, and in fact, that's one of the functions of fibroblasts. This is another example of how the shape of a cell can tell you something about its function, how you can discriminate between different cells by looking at how they look in their native environment.

Humans are collections of cells, all cells are the same in important ways, but cells acquire differences during development, and so we are collections of billions of cells. What holds us together so that when I leave the room most of my cells go with me? What holds all these cells that are doing different things together? Well, one thing that holds cells together is that they're able to adhere to each other. That cells that form tissues often have junctions that hold them together so that they don't fall apart and they exist as a solid piece of tissue.

This diagram is supposed to make you think about the intestinal tract, this tube that runs through our bodies and allows us to acquire nutrients from the environment. This tube, this hollow tube, which food passes through is made up of many, many epithelial cells which directly abut one another and would be a flat sheet except that it's rolled up into a tube. It's this tube-like structure, which is made of many, many epithelial cells all directly adjacent to each other, is what separates the outside world from our internal bodies. We take bits of the outside world like food and we swallow them. One can imagine that when they're inside this tube here, they're still really outside the body. If it passed directly through the tube and nothing happened to it, it would come out to the outside world again. There's a continuous path from the outside world right through the intestine. That food only becomes part of us if it gets absorbed through the intestinal wall. That is, if it can be absorbed through this layer of epithelium. What happens in the intestine is that food gets broken down into constituent molecules, some of those molecules are absorbed into our bodies. They become part of us, and the rest get - we get rid of and they remain part of the outside world.

It's these cells that are sitting at the surface here that are responsible for determining what becomes part of us and what stays outside. One of the ways they can do this is if there's no space in between these cells. This cell can only be an effective barrier if there's no way to get past it. Cells of the epithelium are actually welded to one another through these junctions called tight junctions. These junctions, these red dots here are actually made up of proteins, some are synthesized by one cell, some are synthesized by each other, and they lock together to form a very tight barrier, to form a tight junction between the two cells. Because of that, if I eat something that's rich in glucose, so it has lots of sugar molecules, the sugar molecules can't pass between the cells. That's how tight the junctions are, not even a small molecule like glucose can pass between them.

In order for glucose to enter our body it has to go through the cells. In order for it to get inside the cells and go through them there have to be transporters which specifically allow glucose to pass into the cell and then out into our body. There's a couple of features of cellular physiology which are shown here. One is that cells form tissues that are mechanically intact, they have mechanical integrity because they can adhere to one another. The other is that this kind of adherence gives the tissue a property that's useful, in this case the property is it can serve as a barrier to nutrients from entering our body.

The other reason why cells stay together is that they're surrounded by a matrix that's called extracellular matrix. That matrix has properties that vary in different locations in the body, but basically it's a highly hydrated or water-rich gel. You know what Jell-o is? Everybody knows what Jell-o is, right? It's usually colored because there's food dye in it, but it wouldn't be colored in its natural state. Jell-o is just a hydrated solution of collagen and it contains a lot of water. A piece of Jell-o has probably 99% water in it, but it has a shape. If you make Jell-o and you make it right, then once it's set or gelled, it has mechanical properties. If you made it in a cup and you tried to pour it out it would still have the shape of the cup when it came out. Because of this my mother used to make Jell-o that had fruit suspended in it, and the fruit doesn't sink to the bottom like you'd expect it to sink through water, but it stays suspended inside. Anybody's mother make Jell-o with fruit in it? That's how cells exist in extracellular matrix. Extracellular matrix in most tissues is abundant in collagen and other proteins like collagen. This collagen is highly hydrated, it forms a gel that has water-like properties but it also has solid properties.

We'll talk about exactly what a gel is later when we talk about biomechanics and cells are suspended within it. The extracellular matrix or the collagen gel gives the tissue integrity, and in part, determines its mechanical properties.

Student: [inaudible]

Professor Mark Saltzman: Where does the collagen come from? The collagen is excreted by cells themselves, so the matrix is made by cells. In fact, fibroblasts, which I talked about before, are very good producers of collagen, they produce the collagen matrix within which they live. Some cells digest collagen. What extracellular matrix you have in any particular tissue is there because there's a balance between it being produced by one kind of cell and digested by another, and you're in this sort of state of dynamic equilibrium. Okay, I'm going to stop there and we'll continue on Thursday.

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