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Module 1: Cell source, Isolation, Growth, and Differentiation

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Video 1: Cell Size and Density Based Isolation
So, in the previous class we were talking about cell culture and we were looking atdifferent aspects of cell culture which are required for Tissue Engineering.And we talked about isolation techniques, we were talking about different ways cellscan actually based on different things, cells can be isolated.So, we actually spoke about cellular characteristics such as, surface charge and adhesion whichcould be used for like we were talking about how surface charge and adhesion can actually,be used for separation and isolation of cells.So, we will move on to the other aspects, so we will talk about how size and densityof cells can actually be used.And we can also talk about cell morphology and physiology which can be used for cellseparation and also surface markers, which could be surface antigens and so on, whichcan be used for separation of cells.So, we will first talk about that size and density-based isolation.So, this is actually a crude way of separating, but it is quite effective for certain typesof cells; and it is being done extensively.So, this relies on the size of the cell and the density of the cell and techniques like,simple centrifugation or filtration are the are used actually make sure that the cellsare separated right.So, this is frequently used to separate specific cell types from peripheral blood or bone marrow.So, when you have this kind of liquid source, you can just spin it down and you would endup with the pellets in the bottom or there will be different layers.And you can get different cell sources in the different layers, so that is what thisis used for, this is very commonly done in even in clinical settings and so on.So, gradient centrifugation or density gradient centrifugation is the most frequently useddensity-based separation method.So, here what is done is a sample is centrifuged using a suitable gradient medium at an appropriatespeed, till the different cell types are actually fractionated into the different layer or phasesdepending on their densities.So, this medium will basically contain molecules of; will contain things of different densitywhich will all align itself and the cells will also align themselves with that particulardensity.The sedimentation rate of the cells will depend on the cell size and density, the medium densityand viscosity and also the centrifugal force which is used.So, with this, with respect to centrifugation processes, like the rate at which, the centrifugationrate which is used is going to have a, speed which is used will have a significant rolein how the cells are isolated.Because, you will actually experience a lot of shear stress when you are having this highcentrifugation speeds.So, that is one of the reasons people use this for separating cells in peripheral bloodand so on, which can withstand the shear rates.If you have a source of cell or like a cell population, which cannot withstand the shearrates, then you would not want to go something like centrifugation yeah.So, this is usually used as the initial step to enrich certain cell populations beforesome more sophisticated techniques like FACS and so on.Let us say you would not want to use this as the only thing to separate unless you arejust trying to isolate platelets and so on.So if you take blood and you perform centrifugation you can easily get platelets, so that likeplatelet rich plasma can actually be easily obtained.But the does not mean that it will be only platelets you have got.Let us say if you want something which is very specific, if you want only one specifictype of cell; then it becomes more crucial to actually have something which is more sophisticateddown the line.So, the medium which is used is actually made up of the gradients that encompass the densitiesof all the possible cell types which are present in the mixture which you are going to be using.During the centrifugation process each cell type will actually start sedimenting, butit will not go down to the bottom.It will actually reach a some kind of an equilibrium at the gradient, where the density is sameas the density of the cell right.So, this you would have even seen with multiple layers of different liquids rights so, itis not everything is gonna go to the bottom rights, so it is not going to push to thebottom.So, at equal densities the sedimentation rate will be 0 and at this point cells will settleand that is where they will stay that it will not go, it will not move further even if youcontinue with the centrifugation process.So, this point is called the isopycnic point and you try to do this to ensure that thecells are separated.If the cells are really heavy the much denser than anything in the medium, they will startto form pellets in the bottom.And these pellets can actually be removed very easily right, people will take out thepellets and throw it away if that is not needed and or use it for further studies.So, the lightest ones and the dead cells will actually float on the top of the gradient.So, you would have some amount of cell damage in this kind of a technique, you are goingto have anyways some shear and there is going to be some damage to cells.So, the dead cells which are lysed, will actually we will very light and they will end up floatingon the surface.So, you can actually use two different types of gradient medium, the gradient medium couldeither be discontinuous or continuous.So, discontinuous medium is basically distinct bands can actually be obtained.So, this is made of distinct bands with different densities and what happens is, you would havethese densities which will help in fractionating the blood cell population.In the continuous gradient then you are going to have a smoothly increasing density, sowe are from the top you would have lighter density and the bottom would be the heaviestthing.But it is not going to be clear regions; you are going to have a smooth gradient.And this kind of a gradient medium is more complex to create; how wide range of densityis available when you use this which means the cells can actually have a better chanceof isopycnic banding.When you are using discontinuous gradient, you need to have enough number of bands forthe cells to actually reach the isopycnic point, otherwise the cells are going to floatsomewhere in between and you are not going to isolate them effectively.So continuous gradient is better for isolation, but it is more complex to create.So, what should be the property of a gradient medium?So, a gradient medium should first of all have the sufficient density range for thecells which you are using.So, that isopycnic banding will happen right.So, if you are going to have cells of different densities, all the range should actually betaken care off; otherwise, the cells might just pellet out or it might just start floatingon the top or it get mixed up in the regions in the interface regions which is not a goodthing.So, the gradient medium should also have pH and osmolarity and ionic strengths similarto the physiological condition.So, why are these important, what can happen if say for a example osmolarity is different.Cells will not maintain the integrity.Yeah.The cells can actually lyse ok, cells can either shrink or swell basically kill thecell.And this should ideally have the gradient medium should ideally have low viscosity becauseonce it becomes highly viscous the it becomes very difficult for the cells to move around.And you have to give higher shear for it to separate which will cause damage to cells,this medium should also be non toxic it cannot kill the cells which you are trying to separate.It should not be able to penetrate into the biological membranes because, if its startsgoing in then it is going to damage the cells and like maintaining the osmolarity can helpin that aspect.So, it should be able to form continuous and discontinuous gradients depending on whatyou are trying to do.And, it should also be easily removable from the isolated cells because, whatever cellsyou are isolating then should not have a problem of actually separating it from the gradientmedium.It should not just get adhere to the gradient medium or should not become too complex toactually separate them.So, there should not be any effect on downstream assay or culture procedure which are goingto be taken care of which are going to be done in the future rights.So, whatever cells you are isolating it should not just for fun that you are doing that right.So, these cells are going to be used for something.So, if you are going to be using it for something then whatever procedure you are using forisolation cannot affect the cellular properties for the downstream processes.So, the application should not get affected because of the gradient medium.So, we need to make sure that those aspects are also taken care of.So, the most common application for density gradient centrifugation would be fractionationof blood so or exclusion of dead cells.So, if you just want to have only the live cells and then you can easily take out thedead cells on the top.And a specialized gradient media can actually be prepared for isolation of cells like fromliver, pancreas, lungs, testes and intestines not this is not common medium common gradientmedium which is used for blood; yeah.It is not use for blood there is no gradient medium which is used for blood?No, gradient medium can be used for blood as well, but without gradient medium alsoyou can actually have a density-based separation in blood.So, you just take blood and you spin it and you are going to get different layers.So, it has anybody done those experiments like that; we do not have clinical biochemistrykind of lab, but if we did you would have done that a clearances are usually a painto get the ethical clearances for just drawing blood that would be a problem that is whywe do not do this experiments.But you guys should do this a part of biofest or something.So, there people would not ask you for ethical clearances and you can just draw blood fromsomebody and show some of these experiments right ok.So, the advantage of some of this technique is its actually very simple and cost effective,it can actually be scaled up or down you can have 50 ml centrifuges, where you do it oryou can have 1.5 ml centrifuges where you do this fractionation and it is reasonablyscalable.And the yield of cells obtained especially for blood samples is quite high, it is notlike you are going to lose the cells during this process right, so, the yield is reasonablyhigh.However, the limitation is the purity of different cell fractions is not very good and it isusually low, it can be time consuming and low through put because you would have toeach sample can be processed individually only right.So, you cannot actually process multiple samples simultaneously it is actually quite challenging.So, you would; you can have motors with multiple samples, but you still will be able to doonly whatever the motor can actually that sorry the router can actually deal with.So, if you are going to use 15 ml falcon tubes, you can only load like 8.So, you can only do that many together or you need multiple centrifuges to work with.So, filtration is another simple process where cells are separated based on size.So, cells which are smaller than the pore size of a filtration device will pass throughand the larger cells are trapped.So, there can be two things two ways of filtration one is a passive filtration and the otheris an active filtration.A passive filtration is a process where only gravity or capillary action is used for separationand active filtration uses some kind of a motorized vacuum pump.So, the applications is this is filtration is actually almost exclusively used in cancerbiology specifically for isolation of circulating tumor cells, which are much larger than healthiercells.So, it is easily to isolate them from the healthy cells.So, the advantages it is very simple and easy to reproduce, it is quite high throughputbecause you can just keep having a flow system continuous flow system where you can processmultiple samples.And the filters which are containing the captured cells can I we directly used for downstreamassays because you are not actually changing the cell in anyway right, you are not addingany media or anything to the cells.So, the cell property would not have changed you take the cells and you can process itfor any downstream application without any issues.But the limitation is there is very poor specificity and purity.So, any cell which is large is going to get entrapped in that.So, if you have a macrophage which is very large it will come along with tumor cellsyou cannot prevent that.Surface phenotypes can actually be lost because of this if especially you are gonna use somethinglike an active filtration process, where you have a motorized vacuum pump which is being used.

Video 2: Cell Morphology, Physiology and Surface Markers
So, other than density and size, morphology and physiology of the cell can also be usedfor isolation of the cells.So, what people do is selective culture, so where a specific cell type can actually beselected from an unwanted population, over an unwanted population, like culturing itin a medium which will provide advantage to the type of cell which we are looking at.This could be done by adding specific growth factors or cytokines, which will help thatparticular cell grow while other cells do not really used that, use those sources nutrientsin the media.So, what you do is, you create a selective growth medium, which could either be an antibioticresistance medium, which is usually used for isolation of like engineered cells.So, you could actually have cells, which are transfected and have an antibiotic resistancegene, and then you can use that anti biotic every other cell will die and only the cellswith that anti biotic resistance will survive.This is a very common technique used in molecular biology to ensure that you take out only thetransfected the cells right and then you can use that for whatever studies you are goingto be working with.Metabolic or biosynthetic enzymes can also be used if there are cells which specificallyexpress certain enzymes then you can actually create media which would; so, which can beused only by the cells which produce these enzymes.If the cells are not producing the enzyme, they will not be able to use the media andthey will just die out.So, one common example for that is the hypoxanthine, guanine, phosphoryl.HGPRT.HGPRT so, phosphoribosyl transferase.So, this enzyme production this is a gene which actually can produce expresses certaintypes of enzymes and this can take up the HAT medium.So, if the cells do not produce this gene, they cannot take up HAT medium and they willjust die.So, the advantage of this selective media isolation is it is again reasonably simpleall you are doing is culturing the cell you are just using a different media.And it is quite reproducible, because you are looking at the property of the cell whichis unique to the cell type which you are going to be working with.It will give adequate cell yield because there can still be transfected cells or cells withthe desired types which can die and you will not able to recover them.The limitation is there is going to be risk of contamination because you are looking atthe culturing process which is usually a much longer process than centrifugation or a filtrationprocess right.Cell culture is going to take one days or three days or seven days depending upon atype of the cell you are working with.So, there can also be an emergence of spontaneous resistant clones that do not carry the geneof interest.So, you can always have be isolating some of the cells, which are actually not transfectedthey just have some mutation which actually creates the resistance which you are usingfor screening.There is also a high cost associated with this process because, any time you work withcell culture it is going to be very expensive.Now, it is also time consuming and significant amount of labor is required.Another factor which is used for separation of cells is, cell surface markers.So, these are used for very specific separation.So, antibodies are raised against cell surface antigens and they are used to target the cellswhich are expressing those antigens.So, these techniques became common after the discovery of the CD markers; so, CD is a clusterof differentiation markers, with these are surface receptors which are involved in signalingand adhesion.So, when this was identified people realized ok there are unique markers on cells whichcan be used for isolation of cells.These unique CD profiles can be used for a defining and separating different cell populations.So, people actually use CD markers to identify cell populations as well.So, after isolation also even if you have to confirm that, this is a particular celltype then CD markers are very commonly used right.So, these can be used to isolate rare cell cells because, that particular cell type mightactually have a combination of CD markers which can easily be used for specificallytargeting them and isolating them.This can also differentiate between control and treated cells.So, one process which where these kind of surface markers are used for separation isfluorescence activated cell sorting or the FACS.So, this is a specialized type of flow cytometry this was invented by Bonner and Herzen berg.And the populations are stained with different fluorophores tagged antibody antibodies, whichcan then be separated on the basis of the fluorescent signals which are generated bythese cells.So, these are used exclusively for positive selection and isolation of cells it.So, we talked about positive selection and negative selection and so, on earlier.So, this is used for positive selection.So, FACS actually has two major aspects when it comes to instrumentation.One is fluidics aspect.So, the fluidics basically has to allow the cells to flow in a stream and ideally in astream of one cell right, so that it should be optimized in a way there is one cell willpasses those these things.And the optics have to be there for it to identify the cells detect the cells.So, the FACS basically has a fluorescent dye, which is coupled to an antibody of interest,the cells are labeled with this antibody and a laser beam is used to capture the fluorescenceof these cells.So, the drops which contain the cells will form positive or negative electrical chargeand a strong electrical field is finally applied for the cells to be separated based on thecharge.So, this is the simple procedure which is followed for cell separation using FACS.The advantages is, it is used sorry, the application is used extensively in hematopoiesis research,it is used for isolation of different type of cancer cells, enrichment of transfectedcells and so on.The advantages is it is highly sensitive and it is high through put procedure, it can sortmultiple cell populations based on just the immuno-phenotype.It can separate cells based not only on surface markers, but also on things like cell size,granularity, cell cycle status, intracellular cytokine expression, metabolic status andso on.So, it can be used for separating cells based on so many factors which gives it is a hugeadvantage.So, the limitation is the sorting process itself is low and recovery of the for mostrecovery of the cell recovery is about 50 to 70 percent and the operation and maintenanceof these machines are quite expensive.And this requires highly skilled and trained personnel who can actually operate this so,that makes it very difficult to actually have for individual labs and so on.In our department there are a couple of these so, they think two are there in the commonequipment facility.And I think one is, one or two is present with Professor Mahalingam.So, there are facilities is there, but yeah it is quite maintaining it is quite painfuland operational costs are also quite high.Other surfer marker-based techniques could be magnetic activated cell sorting, insteadof FACS its MACS and panning.So, in case of MACS what is done is, cells are made into be,, made magnetic by attachingsome labels, label antibiotic antibodies which will have some of these magnetic beads coupledto them.And the magnetic cells are separated from the non magnetic cells using a strong magneticfield.So, magnetic beads can be avoided from for implantation, if you are actually if you donot want these beads actually detach them using some antiserums and so on or you canalso use also use negative selection.You basically just do not tag the cells you want tag other cell then you will get cellswithout any of these tags.So, panning is a technique where you it is a simpler techniques all you are do is youcoat antibodies to culture plates and only the desired cells are adhere and other cellsare easily washed away, when they do not adhere strongly it can just easily just wash it awayok.So, whatever cells you are isolating would also have to be expanded.So, tissue engineering usually requires large numbers of cells.And for cell expansion there are different things can be done.A Monolayer on a culture dish is what is commonly done which 2D cell culture so, this is donefor anchorage dependent cells.You can also have suspension cultures; suspension cultures are usually done for your using semisolidculture media.And you have culture on feeder cells; I think i mentioned this earlier as well.So, what is done in feeder cells are grown on grown as a monolayer on this culture platesand then, the growth is arrested.On top of the feeder cells you culture the cells which you want ok.So, growth medium you use has to be designed to supply all the nutrients.So, this would not be like what you have for a bacterial culture, but it still has thesame principles.You still need some carbon source, some source for nitrogen some proteins, peptides, serumwhich would be basically contain different growth factors and other nutrients.So, serum which is usually used is fetal bovine serum or it you can also use from horses.So, you can use it is called fetal calf serum, which can be either from bovine source orfrom a horses right.So, you would be using peptides and hormones and other nutrients such as minerals and metabolitesmight have to be added.So, this growth medium is usually complex medium, define media is almost never used.So, you would have some kind of a complex medium.So, you would have things like DMEM which are, which have different things combinedand with serum without serum and so on, depending on how you want the cells to grow.So, dedifferentiation of cells is one of the factors which needs to be accounted for whenyou are talking about in vitro expansion.So, several cells types can actually change their phenotype towards fibroblast like cellsbecause, while they are growing in these kinds of culture environments.This depends on the passages and the method used for expansion; the nutrients which youprovide, the conditions you provide will direct them into different lineages.So, it can go into transdifferentiate into fibroblast like cells which is not a goodthing, you would want the specific cell types right.So, there have been studies which have shown that chondrocytes can differentiates to formsomething like a myocytes myogenic cells.So, when you actually have low passage cells this kind of trans differentiation or de differentiationis usually lesser, but as the passages increased you would have some of these problems ok.So, the culture conditions would can favor cell populations that respond to the cellculture, that respond more rapidly and because of that these particular cell types will probablymove towards another cell types ok.