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Module 1: Signaling, Bioreactors and Challenges in Tissue Engineering

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Video 1: Cell Migration in Tissue Engineering
So, Cell Migration plays a crucial role in developmental biology and in Tissue Engineering.So, we will try to understand what cell migration is, why cells migrate, what are the rolesof the cell migration and so on, ok.So, this is in general very important process for both unicellular and multicellular organisms.For unicellular organisms, it is usually for finding food.So, the cells migrate, they usually swim through a fluid, try to find if there is nutrientswhich they can use, right.So, this or they can also crawl on a surface as an amoeba would do.Multicellular organism also does cell migration, but for more complicated process, it is notjust for finding food.It could be for tumor invasion metastasis, embryogenesis, angiogenesis or immune responsesand so on.So, if you remember when we looked at wound healing; so, the aspect which we talked aboutwas migration of inflammatory cells, phagocytes, macrophages and so on to see how they actuallycome to the site and so on.So, migration plays a crucial role in some of these aspects.So, the speed and the pattern of migration is actually determined by the type of celland also the chemicals present in the environment.So, some of these molecules can act as triggers or signals for directing cell migration.So, the fundamental mechanism for forming structures within embryo is cell migration,right.So, think about it, you only have one cell to start with, its starts dividing; if thecell just keep dividing, you just have a blob of cells, right.You are not going to get defined shapes.So, what is going to happen is these cells are going to produce matrices and they aregoing to migrate to form different tissues, right, So, different organs and tissues andso, because of this during embryogenesis cell migration is very crucial.And, it is also very very well controlled and that is why you end up with two eyes onthe place where they are and nose on where it is right.So, it is a very regulated process and this ensures that cell migration is controlled.So, this requires cell migration to be controlled.So, development of tissue structure and cell migration are interdependent, right.So, only when you have the cells migrating to a particular area, the tissues can startdeveloping.So, people have used magnetically labeled oligodendrocyte progenitors and monitor themusing MRI to show that the cells can actually cover a region of greater than 8 millimeterin size during the first 10 days when you inject it to spinal cord.So, when you are talking about tissue engineering application, cell migration is can be veryimportant.Because, if cell migration is going to be there, you might have host cells coming tothat tissue construct and integrating very nicely with the cells.And, also in case of angiogenesis, cell migration is required for vascularization to happen.However, it is not desirable if cell migration can cause side effect such as loss of functionat the transplantation site.So, basically if you have a cell seeded scaffold and these cells are just going to migrateaway then you would have a problem, right.So, you would want to ensure that the cell migration is optimized.So, can you think of a way you would minimize this type of loss of cells from a cell seededscaffold?In if they adhere properly.If they adhere more strongly, if they prefer to adhere to the scaffold compared to whatis present surrounding it, then they will probably not migrate away, right.So, optimizing the scaffold for cell addition can actually help in controlling cell migration.So, these are some of the cells which migrate very commonly and these are the roles whenit comes to the migration.So, neutrophils migrate for phagocytosis, lymphocytes for destruction of infected cells,macrophages in for antigen presentation and, in response to antigen presentation and endothelialcells to create angiogenesis.Epidermal cells and fibroblast during the process of wound healing, tumor cells duringmetastasis they will migrate.And neurons and axons during the development and regeneration of nervous tissues and embryoniccells migrate during embryogenesis, right.So, if you are looking to study migration of cells.How would you go about designing an experiment to study cell migration?Culture cells.So, no migration does not always happen through the blood vessels.It is not that the cells which are adhere to a surface actually entered into the bloodvessel and they are carried to someplace.It can happen during metastasis in some cases, but its migration.Migration requires some.For it to move so, it cannot it has to come from somewhere to the blood vessel, if itis even doing do that, right.Cell migration would have to happen, can happen on the surface or as well right.And, see if any time cells are proliferating, they also have to migrate right otherwiseeverything will just be forming a blog in that.So, what you basically do is you will fluorescent label the cells and these cells will be trackedusing time lapse microscopy or conventional microscopy.Confocal can be used, but confocal is usually more complicated.You can actually use it, if you tag it with fluorescence you can use simple microscopytechniques.And, what you would do is you will see where the cells you seeded are and where it actuallyis looked at ok; how far it has gone.You can use sectioning to see how far it has gone and so on, ok.So, what is the commonly used fluorescence molecules for these studies?Can methylene blue be used?Not really.What is the one common fluorescence molecule which you know for biological studies?Something of fluorescein.Fluorescein diacetate; fluorescein diacetate is to stain live cells, but yeah you can,you may be able to use it.But, the most common molecule which is used is GFPs.GFPs.Ok.So, lymphocytes and other phagocytic cells can also be tracked using MRI because, theywill ingest these superpara superparamagnetic particles.So, these are phagocytic cells, they gobble up stuff, right.So, if you put an nanoparticle there, it will eat it up and you can track them using MRIor other microscopy techniques.So, when we are talking about cell migration experiments, what you look at is to introducesome kind of genetic markers to the cells that can actually be tracked.So, time lapse techniques can permit quantitative analysis of individual cell movements.And, you were, you might have seen these videos in some publications, they usually have thesetime lapse videos.I do not know if you have.None of you might have taken my immunology right.Did anybody take immunology here?Ok.So, you did ok.So, I am pretty sure Dr. Vani would have shown that video where a neutrophil runs behinda bacteria.So, a neutrophil basically, that is it is a time lapse video actually, it is not thatit is going at that pace it is about a 32, 30 seconds to 60 second video.And, you would be able to see a neutrophil chasing a bacteria to eat it.And so, you can look up such videos, these are actually time lapse videos which weretaken over a long period of time.And, you would, many a times you would actually see a clock which is running much faster thanthe actual time to show you what is the real time frame.So, the most common migration assays which are used actually expose cells to very simplesystems compared to in vivo setup, right.So, in vivo the tissue is much more complex whereas, a migration assay, if you are goingto culture it on a plate and look at the migration it is going to be very simple.So, can you think of a common limitation which you would experience with such systems?While doing it on a plate can be of 2 dimensional versus 3D as you are putting down.Yeah, that is usually the major problem, right.So, you, most of the cell culture which is done is done in the 2D set up, like only whenpeople work on tissues, they start looking at 3D setups.So, that is important because when you have a 3D environment that the migration directionsalso increased.So, you might have to observe things from different angles.So, when you are talking about 3D environment for migration basically what you do is similarto what Karan was alluding to.So, you basically have a collagen gel, on which the cells are seeded and you followthe cell infiltration into the gel by following the leading front of the cell population.So, not all cells are going to migrate at the same rate.So, you might have a lead cell which is what you would track to see what is the max, whatis, how it is actually moving.So, this gives important information about cell migration; however, this experimentalsystem would have an important limitation.Can you think of any two limitations?Is this experiment for all kinds of cells you?Yeah.Any migrating cells any cell migration.Because, of its 3D motion the cell goes out of the focus.Not necessarily, you can those are I am not talking about observational problems; I amtalking about a design problem in the experiment.The scaffolds design will impede how it moves, migration.But, that is what is supposed to happen right; the ECM is going to have its own.Design.Barrier.So, ok; so, I will tell you.So, one thing is it actually is a very long process.So, it can take days for it to infiltrate and monitoring it and maintaining the correctenvironment, making sure the cells do not die are all quite big challenges when it comesto performing these experiments.And, the other issue is more of a proper design problem which is the leading front distanceonly gives information about the fastest moving cell, it is not the average cell populationwhich you are looking at.So, whenever you are looking to collect data you are trying to get something representativeof the sample.This will not be truly representative of the sample; this will only represent the fastestcell.Sir, how are these fastest cells chosen likely?No, because that is the leading edge you would be able to observe right; when you observeit in a microscope, you will just be seeing the leading front as its moving.Ok.That is all.So, see if you were to have a fluorescence tagged thing and you are observing in a microscope;you just look at the far the cell from the starting point that is all you are lookingfor.So, this is how a cell which is migrating would look like.So, what you see here is the front region which is called the lamellipodium or the leadingedge and the rear region is the uropodium which is seen in the back.So, what happens during this time is, the cells can actually cells actually sorry.what happens during this time is.the cells which are adhered basically start moving and they go and adhere to some othersurface.And, then the; then they get detached from the previous site, ok.So, once they have done that, they have actually moved one position.So, the periods when the cell is moving forward in a straight line without turning is calledas the persistence time.So, when you are talking about a cell migrating, the cells are not going to always move inrandom directions, right.So, they will continue in one direction for a while and then they may change directionsas well.So, this time frame in which it actually moves in one direction is called the persistencetime and this persistence time can be altered based on having chemoattractants and so on.So, if you have a chemoattractant then the cells are going to try and migrate towardsthat attractant and in that case the persistence time will be longer.Otherwise if it is going to be uniform distribution of the chemoattractant, the persistence timewill be much shorter, ok.The cell movement itself is initiated by active membrane protrusion in the lamellipodial region.So, in case of fibroblasts, the leading edge of the cell protrudes and retracts in a cyclicfashion.So, basically the peak velocity it can reach is about 50 to 60 nanometers per second andit averages a net speed of 5.5 nanometers a second; so, that is the rate at which theyusually move.So, during this cell movement, new cell addition sites have to be formed right, only then thecells can leave the previous adhesion site and go to the new adhesion site.So, these are some of the cells, their persistence time, speeds and their motility coefficients.So, what you can see is, some of the cells which are very motile would have lesser persistencetime like your neutrophils or macrophages have lesser persistence times.So, especially neutrophils have significantly lesser persistence times because, they justhave to figure things out in that region where they are, right.So, they need to, they cannot just keep moving in one direction whereas, if you were to lookat endothelial cells they are actually not as migratory as these cells.So, they will have a much higher persistence time and whereas, if you are looking at speeds;obviously, the cells with lesser persistence times end up having higher speeds and so on.So, this is the process which describes cell migration.So, this is a repetitive process of lamellipodial searching for a new adhesion site.So, the new front edge which is coming or forming will basically have to go and adhereto a new surface.And, once it is adhered to a new surface the uropodial region has to get detached and itmoves to the new site.For this to happen, what would have to happen is the cell would have to strongly adhereto the new site.So, the initial step is for the cell to stretch to figure out what is the right place to adhere.And, once it adheres there, there is now a tension in the cell right, this is becausethe cell is stretched.So, this tension could cause it to either move forward or move backward, depending onwhichever site has the higher adhesion properties, higher affinity.So, if the cells have higher affinity to the new site, it will move forward; if it hasmuch higher affinity to the old site it will move backwards.So, this is something which you can even simply test as well, right.So, like I do not know what are these jelly kind of clay which is called like now theyare.Slime.Slime yeah.So, you can play with that and you will be able to emulate something like this.So, if you stick it strongly at one site and you stretch it out and place it on anothersite; it will either come to this site or go to the other site based on how strong youradhesion is, ok.So, let us look at some of the different adhesion properties of the surfaces and the how thatwould affect your cell migration.So, what would happen to the cell migration speed, if your substrate has very high adhesiveproperties?The initial site or the?The surface in general has very high adhesion.So, because it is not like, I am not going to look at one cell right.If I am preparing a scaffold with very good adhesive, cell adhesion properties, then howdo you, how do I expect the migration speeds to be?If it is high it will be slow.It will be slow because it is already adhere strongly, it might not want to go to anothersite ok.What if it is very low?Then the it will this migration.That depends, I mean its.Easier for it to migrate, but that does not necessarily mean through want.I might be faster.If it is not adhered to the site, it means that it does not really like the site right.So, it might want to migrate as well.It is not getting the right grip to pull its.So, that would be the problem.So, in the first case what will happen is it will not be able to detach itself fromthe first site; whereas, in the second case it will not have a position where it can actuallyattach and move.So, in both cases the migration rate will be very low.So, when it is medium that is when you would have high migration rate and as I have alwayssaid biology always has some optimal values, right.So, high, low are never good, something in between is where you are looking for and thesepredictions have actually been confirmed experimentally as well.

Video 2: Patterns of Cell Migration
So, when you are talking about cell migration there are two major types of migration.You have a random migration.So, this is observed for, when you look at it for a sufficiently long period of time.So, what would happen is if you are going to observe, see we looked at the persistencetimes, right.So, we said that the persistence time for endothelial cells is 300 minutes based onthe table.So, if I have; if my period of observation is only 150 minutes, then I am going to thinkthat the cells are moving only in one direction right.And, if I am going to observe only one cell as well, like smaller number of cells thatis going to happen.If I were to observe a very large number of cells for 3000 minutes, then what is goingto happen is different cells are going to keep moving in different directions.So, it will look clear that it is a random migration which is happening, assuming thereis no signal which is actually directing the migration.Directed migrations happen when it comes to cells actually responding to their environment.So, the rate of migration will depend on the composition of the local environment; whetherthe receptors are present and what soluble factors or surface bound factors are present.So, if the ligands are present in the on the surface, then the cells will migrate towardsthat and so on.So, if the environment contains uniform concentration then the migration becomes random right.So, if you have one region which is rich in these ligands, then you might have a directedmigration.So, if spatial variations are present, the cells are actually capable of detecting thesegradients and changing their migration pattern to towards these attractants.So, this process of moving in response to some gradient is called as taxis and it iscritical for regulation of any biological event that involves cell movement.Because, otherwise if cell movement is always going to be random then you would have verylittle control over tissue development or anything right.So, it, this directed migration ensures that this is happening ok.So, in case of the random migration I had also put some term called Brownian motion.So, what is Brownian motion?Movement of particles in random direction.Movement of particles in?Random direction, I think.Ok.Why would it happen?It is.Kinetic energy.It has its own energy kinetic and internal energies which makes it move and as it collides,they change directions.So, that is why it is random motion.So, that is why it is Brownian motion ok.So, if you observe random migration of cells for a long period of time and you track thecells, it will look like it is a Brownian motion because it will be quite random.So, basically Brownian motion is a solute particle moves as the results of collisionsand their own energies.So, the momentum is transferred from the, from one particle to the another during thecollisions, particle move with the velocity; I do not know whether v prime has actuallymoved.So, they can move with the velocity of the v dash until the collision causes that tochange directions and then move with the new velocity.Let me just fix this, ok.So, they can move with a particular velocity until some collision causes it to change directions.So, otherwise it will keep persisting in that.That is your persistence time right.So, only when there is a collision that is going to be change in direction so, that willaffect your speed as well velocity as well.So, either could be momentum which is lost or momentum gained based on the velocitiesof these two molecule, velocities and mass of these two particles which are colliding.So, collisions occur from all directions with equal probability, that is what causes thiskind of a random motion.If it is going to be in one particular direction, then you are going to have, you are not goingto have a random moment, right.So, that in case of Brownian motion, the time between these collisions will depend on thedensity of the molecules right.So, if there is going to be a higher concentration of the solute molecule, there is going tobe higher chance of collision and so on.So, the average time between collisions can be estimated from the average distance theparticle travels between collisions, which is called as the mean free path and the averagespeed at which these are moving.So, the movement of a collection of Brownian particles is typically characterized by therate of increase in the mean-squared displacement over a period of observation.So, usually this timeframe for observation is much larger than the mean free path interval.So, similarly if you were to draw analogy to our system, your period of observationhas to be much larger than the persistence time.If you are a; so, that is why I was saying, if you are looking at 300 minutes and haveobserved it only for 150 minutes it is not going to work.So, the persistence time has to be much smaller than the period of observation for you tounderstand random migration.So, this behavior can be characterized by a diffusion coefficient D which can be givenby this Stoke-Einstein equation, ok.So, we talked about how random migration can look like Brownian motion and what Brownianmotion is.But, are the mechanism similar?Random migration is different.Ok.So what would be the difference?I do not think there could be collision between, Sir.Yeah.So, it is not that the momentum transfer is happening causing the change in directions,but change in directions is happening because of adhesive properties and other things, right.And, the other thing is cells do not, this change in direction is not instantaneous.In case of molecules colliding, you are going to keep up, keep coming boom and then go atdifferent ways, it is not this way.The cell migration is going to, cell change in direction is going to be very very slowok.So, and it is also because it is not a transfer of momentum, but it is a sequence of significantnumber of events which include attachment to a new surface, a contraction of the intracellularfiber system and detachment from the previous surface.So, dead or fixed cells can also move because of Brownian motion.So, they will not migrate on the surface.So, those would just be particles right, those will be particles in a suspension.So, neutrophils can actually migrate through a collagen gel 20 times faster than you wouldexpect them to diffuse in water.So, although collagen is a much much more viscous system than water; showing you that,it is not that, it is a going to be dependent on the momentums it, but it has mechanismsto make sure it reaches things faster, right.So, neutrophils usually are trying to attack stuff when they have to migrate through thecollagen matrix so, they move very very fast in that environment.So, although cell migration is similar to Brownian motion when you observe it for along period of time.The striking difference would be that the cells can actually move in the same directionat a nearly constant speed, when you observe it for relatively short intervals.So, the change in direction can occur randomly and this is completely stochastic in nature.And it depends on the membrane adhesions and cytoskeletal contraction events.But, substantial changes in direction occur only after small random perturbations havebeen accumulated.So, it is not like the cell which is being moving this way, suddenly starts changingit in directions and moving the other way.It will then move slightly slightly to another site another site, another site, before itcompletely changes directions, right.So, in spite of these differences you can actually use mathematical studies based onBrownian motion for understanding cell migration, for random cell migration, ok.So, in our department there is a Professor Murugan, I do not know if you any of you havemet him or not, but he actually works on, not exactly on cell migration but, he workson aspects of protein migrations and protein folding using Brownian motion and so on.So, if you are interested in understanding these topics he might be a person who mightbe able to help you with that, ok.So, when you are talking about directed migration that is actually spatial variations of factorswhich regulates cell migration and this leads to cells detecting these gradients and changingtheir migration pattern.So, this process is called as taxis.So, there are different types of taxis which you would have heard off.The topotaxis is enhanced turning towards the stimulus.Orthotaxis is increased cell speed when the cell is already oriented towards the stimulusand you have klinotaxis which is decreased turning when the cell is oriented towardsthe stimulus, right.So, these are the four ways that cells can respond in the presence of a stimuli, ok.And, based on what stimuli actually is causing the migration, the process can be labeledas chemotaxis, haptotaxis or phototaxis, right.So, chemotaxis is when you have dissolved chemicals, some chemicals which are actingas a signaling molecules and cell adhesion would cause haptotaxis and light could bethe trigger for phototaxis.Certain agents can cause chemokinesis and which is a change in the kinetics of cellsrandom migration that does not require a gradient, but it will depend on the total concentrationof an agent itself.So, this is in response to a particular concentration the cells will move and again we use the sameterms like orthokinesis and klinokinesis.And, orthokinesis is variations in cell speed versus klinokinesis is variations in cellpersistence time.So, the pattern for the random migration of cells as we said can be similar to Brownianmotion if you observe it for a long period of time; however, if you, if you look at itonly for short periods of time it will not be.So, this, you need to make sure that there is significant directional changes so, thatyou can actually look at it as random migration, as Brownian motion, ok.So, however, cell migration is more, is actually very good example of something called a persistentrandom walk, ok.So, what happens is at short observation times, the cells seem to be persisting towards onedirection and for long observation times the cells are migrating at in random, right.So, that is what is a persistent random walk, it represents both sites, ok.

Video 3: Random walk and Chemotaxis
So, in the simplest random walk, what happens is that a particle is constrained to movealong a one, along a one-dimensional axis and in a time interval tau the particle canmove a distance of delta depending on the speed in which it is moving right.So, that is quite logical so, at the end of this time period the particle will changeits direction.So, this is the persistence time.So, at the end of the persistence time, it will change directions and go into somethingelse.So, the particle can actually go into left or right.So, in an unbiased random walk there is actually equal probability that it will go to eitherleft or right, you understand so here.So, I am walking towards a certain direction say.So, from here let us say I am walking like this and I, if 3 steps are my persistenceto walk and then I can turn left or right and I can actually, either way is fine.So, I do not really have a preference and that would be a unbiased random walk.So, at each step, a new direction is chosen randomly and with the particles history ofthe moment having no impact that is quite logical, right.So, if you are to think about the cell, if once it gets detached from the previous site;the previous site has no role to play in where the cells are moving to, towards.So, if many particles move based on these simple rules at the end of the first timeinterval tau, half the particles will be at minus delta and half the particles will beat plus delta, right.So, if both have exactly equal, if all cells have exactly equal probability of moving leftor right, then half would have moved left, half would have moved, right.So, they would have move to so, center is 0 it would have moved to minus delta, anotherwould have moved to plus delta.So, the position of each particle after n time intervals can be given as x of n equalsx of n minus 1 plus or minus delta, right.So, that would basically mean at 1; at n minus 1 this is my position, it could either bethis or this, right.So, those are the positions I can be in after one step.So, from this equation you can actually calculate the mean particle position using this; so,basically you have just divided it by the number of like particles; like summation ofthe, all the positions divided by the number of particles, ok.So now, you need to simplify this equation.So, if you were to simplify this equation can you think of what would happen to thesecond term?Does it not get cancel.Why?Because, that is plus delta minus delta plus delta minus.Yeah.Delta dealt cancels.So, half will be plus half will be minus so, it goes to 0, ok.So, this happens only in an unbiased random walk.So, if both are, if there are biased then you cannot do the simplification.So, basically what happens is x of n equals x of n minus 1.So, what does this mean?You mean position of the cells remains the same.Yeah, it is independent of time; the position of the particle is actually not dependentdirectly on time right.So, all the average position has not changed, the particles are actually spread across theaxis, right, it is not that the cells are not migrating.For every cell which is moving to the right, there is another cell moving to the left.So, its mean distance from its origin is different they have increased, but if you were to addall cells that would even out.ok.So, the mean squared displacement will give you the extent of spread that is why you lookat this squared value rather than just the actual value because that will give you a0, ok.So, as the means squared distance is 0 at times 0, the equation over here indicatesthat the mean squared distance increases with the number of steps, right that is logical,right.So, as you have more steps, you would have more distance covered and more spread.And, with that we will come to the conclusion of all the fundamentals related to tissue engineering