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

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Video 1: Cell Harvest and Retrieval
So, in the last class, we were talking about cell source right.So, we talked about where the cells will be obtained, and what type of cells we can getand so on.Because when you are talking about tissue engineering, cells are the second aspect ofthe triad.And during that time, I also said the cells which we get would also have to be cultured.So, there are different aspects to it.So, we identified what could be the source and what type of cells we could use, but weshould also know how to actually separate these cells, how to actually get these cellsfrom the tissues and how to get the specific cell type from the tissue because a tissueis going to have multiple cell types.So, we need to be able to isolate the specific cell type which we are interested in, so thatis what also crucial.And once you have that we should also look at how the cells would be cultured.And in case we are working with stem cells how the cells would be differentiated right.So, all these aspects are crucial when you are talking about cells.So, the cell culture aspect is what we are going to be talking about.So, we will talk about first harvesting the cells, selecting and isolating the cells,expanding or culturing the cells and differentiating the cells.So, these are different aspects of cell culture.So, harvesting just means that taking cells from a tissue.So, it could be from different tissues.You just take out the tissue of interest and there could just be some simple mechanicaldigestion or some simple treatment to get a mixture of cells from the harvested tissue,that could be used as the cells itself that mixture could be used as the cells itself.So, one example would be just using platelet rich plasma or platelet poor plasma wherenot much of selection or anything is done.There is a little bit of a isolation being done, but it is primarily just harvestingand using it.You could also have selection which could be a lot more specific, where you try to identifyspecific cell types which you are interested in and taking those cells and actually usingthem for tissue engineering applications.It could either be in vitro or it could be directly injected into a person for regenerationof the tissue right.So, these are two options when you are talking about cell sorry cell selection and harvesting.But the cells you get is usually not of enough numbers we did talk about this earlier right.So, we need a lot of cells.And especially if you are going to take it from an autologous source, then you are notgoing to have enough number of cells.So, you would have to culture them.So, usually this is done in vitro, so that you can get enough numbers.So, even if you are going to harvest 10 power 4 or 10 power 5 cells, you might need 10 power8 or 10 power 10 cells.So, you might have to multiply these cells over a period of time to get enough cellsand then use it for regeneration or tissue engineering.So, culturing the cells is a crucial aspect and this is going to be dependent on the typeof cell you are working with right.So, if you are going to use for example, if you are working with hepatocyte, the cultureconditions would be different compared to what you would have for an epithelial cellright.So, you need to know what culture conditions are and how you actually maintain these cultureconditions, what are the growth media required and so on.So, if you are using stem cells, then obviously, the expansion would also have to make surethat the stemness is maintained.So, in the sense that, as it is dividing and multiplying, it should not differentiate,it should only multiply, so that is a crucial aspect.If it starts multiplying you will get enough numbers, then the stem cell has to be differentiatedto get the specific cell type for the application.So, the differentiation process itself will be different.So, you how you direct the differentiation to get the desired cell type is also a challengethat needs to be understood and this will be different for different cell types.So, first thing is harvesting the cells.There are different methods and procedures.So, the method you use would depend on the cell source or the cell type itself.So, depending on the tissue, which you are going to extract, you are going to use differenttechniques right.So, most of these cell harvesting techniques actually come from some type of a diagnosticprocedure.So, there are different diagnostic procedures for studying, for checking the pathology,checking some disease condition.So, here tissues are taken for these studies.So, these techniques are used for cell harvesting as well.So, the there is a high demand for patient safety because these procedures actually exposethe patient towards potential infections and so on.So, you need to make sure that these are done in proper clinical environment and you donot cause any complication to the patient.So, you need to maintain the correct environment, the clean environment for doing this.So, special care for handling storage and transportation of these tissues is also required.Because depending on the tissue type you might have to store it under different conditionsright.So, you might have to store it in liquid nitrogen or you might have to treat it as soon as,you process it immediately and so on.So, on depending or you might have to heparinized it if you are going to using blood or citratedblood might be required.So, depending on what tissue you are working with and how you are going to use it in thefuture, you would have to handle it and store it appropriately.So, one of the most common sources for cells in tissue engineering is bone marrow aspirates,because these contain the mesenchymal stem cells.So, these are usually harvested under local anesthesia.People are given local anesthesia and it is taken the bone marrow aspirates are takenfrom the upper part of the hip which is the iliac crest I mentioned this earlier as well.So, this is the iliac crest.So, you have the you have the hip bone.So, what you see here this, these are the iliac crests.So, here the bone is sorry the bone contains the bone marrow, and you can actually getthe bone marrow aspirate from which you can get this mesenchymal stem cells.These can also be isolated from the sternum.Sternum is what is in your ribcage.So, the ribcage is actually connected by something called as sternum, so that is what this tissueis this bone is.And tibia is a other bone in your legs.So, you actually you can harvest stem cells mesenchymal stem cells and bone marrow aspiratesfrom these tissues ok.So, iliac crest is the most common tissue from where you try to harvest it.So, what you do is there is something called an aspirate needle.So, these are large needles maybe 16 gauge or something.And these are penetrated into the skin and to the and it also penetrates the outer partof the bone which is the hard bone and reaches the softer central part which contains thebone marrow.So, this is a painful procedure right, so that is why you have to have a local anesthesia.So, we are actually causing a puncture through the skin through the muscle tissue into thebone.So, it is going to be quite painful.And from this softer part of this bone where the bone marrow is present, the marrow issucked out using a heparinised syringe ok.So, why do you think we use heparinise syringe?What is the role of heparin?To prevent blood clot.So, these aspirates can actually clot very rapidly.So, to prevent that you need to use a heparinised syringe ok.So, these aspirates can will contain hematopoietic cells, adipocytes, endothelial progenitorcells and osteoprogenitor cells as well.So, if more than 2 ml is aspirated, then peripheral blood can actually dilute the mesenchymalstem cells.So, it is important to control how much you are aspirating and then whatever you are gettingis a now a mixture of cells and you might have to separate cells to get the specificcell which you are looking for.So, another technique which is used is tissue biopsies.So, tissues from any in almost any organ can be biopsied.So, you basically just take a piece of the tissue, cut a piece of the tissue and useit for your cell isolation.So, there are different techniques for this.A scrape is basically cells are removed from the surface of a tissue, you just take a crave,scrape of the tissue.Or you can have something called a punch biopsy where a punch which is round shaped is usedto cut and remove a disk of tissue.So, this is like what you would see in a punch hole right.So, you create a small hole small punch, and this disk shaped tissue can be harvested andthat can be processed further to get the desired tissues sorry desired cells.You can also have needle biopsy.So, here a needle is used to remove a sample.So, usually this is for liquid samples.So, it could be for the lymph, for the blood and so on.You have stereotactic biopsy.Here stereotactic system which uses the 3D coordinates is use to identify a small targetregion and specifically remove tissues.So, these things are used in places where you need to have a lot more control.For example, if you are going to take brain tissue and so on.So, you would not want to damage the organ.So, you need to have precise control over which part of the tissue you are actuallyharvesting.So, for that reason, you actually use something like stereotactic biopsy.So, what are the reasons for harvesting brain tissues?You might want to work with brain cells right.So, they and there could also be reasons like diagnostics for tumour and so on.So, there are different reasons where you do biopsies.A colposcopic biopsy is where colposcope is used, this is a close focused telescope thatallows the doctor to actually look at the cervix to look at in detail.So, this is usually done for again diagnostic, diagnosing cancer cervical cancer.Endoscopic biopsy is where an endoscope is used to collect the sample.An endoscope is basically a long thin lighted optical instrument, which is used to get deepinside the body and it can also examine and operate on organs ok.So, you might have seen endoscopy done to the nose or to the mouth, those are very commonlydone ok.So, this is just a long tube which is inserted into your body.And so, these are some of the different techniques which are available readily for performingbiopsies.So, what I am showing here is the punch biopsy.So, this is one of the simplest things done, usually this is done for tissues like skinwhich are easily cut open.So, what you do is under local anesthetic, you take this punch tool which is sharp knifewhich is round in shape, and the punch is placed over the area which you want to harvestand it is pushed down and rotated to remove a circular piece of the skin.The skin sample is then lifted with the forceps or a needle and this can be cut from the tissuefor before it is further processed.So, biopsies for internal organs, however, cannot be done with very a such a simple andcrude way.You would need more of a fluoroscopic control you would either combine it with endoscopeor an X-ray machine, so that you actually can keep track of where you are going andwhat tissue you are removing.So, the tissue, the biopsies you got or the aspirates you got basically are a mixtureof cells right.So, you might have to process them further before you can use them for tissue engineeringapplications.Bone marrow aspirates are basically suspensions.So, cell retrieval can actually be done from these suspensions.Whereas, tissue biopsies have extra cellular matrix attached with it right.The cells are adhered to the ECM and you have actually taken out the tissue.So, now, you need to make sure that the ECM is removed before you start working with thecells ok.If you are only wanting the cells, you do not need the ECM.So, this is the opposite of decellularization.You need to remove the ECM without actually damaging the cells.So, the first step is usually mechanical.So, what do you do is you could either have a vortexing with digestion buffer, or justdicing with scalpel and so on.So, those are simple techniques to try and remove as much of the ECM as possible.The disrupted biopsies could then be implanted or further processed or it could be placedin culture for expansion as well.So, what does do you think you can do for further processing.So, the first step is just mechanical right.So, after that if you want to remove ECM, what do you think you can do?Collagenase digestion.Collagenase or something.So, where you actually use some protease some enzyme which could actually disrupt the ECM,and release the cells from these ECM right, so that would be one way to do this.And trypsin could actually just dislodge the cells from the ECM.Instead of destroying the ECM, trypsinization actually make sure the cells do not adhereto the surface they just come out of the ECM right.So, there are different techniques which you can use ok.So, there can also be calcium dependent molecules being targeted which are what the which areused for cell adhesion.So, you can use chelating agents that will actually target these calcium dependent celladhesion molecules and help in the release of cells from the ECM.So, basically cell retrieval process can be of two or three different steps.The first step is mechanical homogenization, where you can use sonication, manual pulverizationor high-speed blending, this is usually done for soft tissues.And enzymatic digestion is done for soft and cartilaginous tissues, where you use thingslike collagenase, proteinase k and so on.Acid digestion is done for osseous and fibrous tissues.So, instead of using enzymatic digestion when you have hard tissues, you can use acid digestionso which is usually done using HCl.
Video 2: Cell Isolation
So, the harvested cells, the cells which you have harvested actually contain mixture ofcell types right, because you have not done anything to specifically attract one celltype.Whatever you have done till now, you have just done mechanical characterisation mechanicalisolation or enzymatic digestion which will just retrieve all the cells that are presentin the tissue.This would mean there is a mixture of cells.But now you might want to use only one specific cell type.So, if you want hepatocytes alone, then you would have to make sure that hepatocytes areseparated.So, there are many techniques which can be used to enrich these cells.So, how you do this is dependent on three major factors.It is based on adhesion properties of the cell or the morphology of the cell which wouldinclude the density and size of the cell or it could be antibody binding and so on, sothe physiology of the cells and so on.So, you based on the distinct characteristics which are unique to a cell, you try to separateit from the other cells ok.So, the procedure can be either positive selection or negative selection.What I mean by that is, in positive selection, you try to isolate the target cell type fromthe entire population.So, you might have like 8 or 10 cell types and you try to target one cell type and takethat out of the mixture.Whereas the other thing is negative selection where you deplete all other cell types andonly the target cells remain, both approaches are possible.Both of them have their own advantages and disadvantages.In positive cell types, advantage is there is a high purity, because you are targetedone specific cell type you know for sure only that cell type is going to come.There is going to be a unique property which you identify and you are going to use thatto separate it, so which means it is going to be highly pure.It is not going have any issues with purity.Also, the cells which you get can be further processed again and again for purificationright, because you are having one only one type of cells.And even if a few other things are present with it, if something if one of the techniquesyou use is not very specific to one cell type and it can actually attract two cell types,then what happens is you now again have a mixture and you can still further processit.But the disadvantage is you have actually used some antibody or a labelling agent thatwill attract the cell type of interest.So, your cell might actually have this, attach to it and that could interfere in the furtherprocessing.So, if you are going to culture them or if you are going to use it in vivo, this couldactually be a problem.Yeah Vasundra.Sir, Have they tried using same media or something?So, people do use that.So, we will talk about that as well, so that is also possible people use selective mediato so just like how you do it for transfected cells, but that is primarily done for transtransfected cells, without transfection the cells might not have that level of selectivity.So, in case of transfection, you can have something like that antibiotic assistanceand then you culture the cells in the presence of the antibiotic, every other cell will dieand only the transfected cell will survive.But if you are talking about cells which are just isolated, it is not very easy to do that.So, negative selection the problem is it is very complex to design a cocktail which willactually remove everything except that cell you are interested in.It because you need to now target so many different cell types, so it is actually adifficult process to do it.Because of this, you will be end up with something which is of lesser purity, you may not beable to get the highly pure mixture.However, the advantage is the cells will not have any of the antibodies or labelling agentsafter you do this ok.So, both these techniques can be used you would have to decide on what is of more interestyou what level of purity or whether the cell should not have any antibodies attached andso on.So, depending on what you are going to do with the cells and how you are going to processthem, you would choose negative or positive selection techniques.So, when you are trying to isolate a cell type from a mixture of cells, you want toidentify unique properties of the cell that of that cell type and try to exploit that.You want to identify something which is specific for that cell type.So, the choice of isolation technique will depend on the cell type itself right.So, what do you think are the parameters that will actually govern the choice of isolationtechnique, what do you think will affect how the cell isolation is done?Cell surface marker.Surface markers is one.Density.Cell density ok.Size and geometrics.Size and shape.Charge.Cells surface charge ok.The type of product we finally want.What do you mean by that?Depending on the final, I guess.You mean the application or the product.So Sir application.So, what you are going to use the cells for ok.Anything else?So Sir pH resistance.So PH resistance is one specific aspect, butin general what I would say so ok.So, depends on the, so amount of stress which a cell can withstand right.So, you might have cells which can withstand certain pH or certain temperature or certainsheer and you can only choose techniques which can handle that.So, for example, if you are going to use something like a centrifugation technique, cells whichcannot withstand sheer cannot be used a separated using centrifugation.You are going to lyse too many cells right.So, those are factors which you have to take it account.So, other thing is ultimately what do you want, now how pure and of the cells you wantor are you focused on yield if you want isolate as many cells as possible right.In some cases, yield is more crucial than purity, you would want to ensure all the cellsof the type are recovered, but it is ok if the purity is only 80 percent.So, you might want to use a technique which is suitable for that.So, again negative or positive selection which will depend on the application we are using.And any other specific requirement which the application might have, also you have to lookat time and cost right.So, you cannot have a process which is too time consuming which is very low through putand it is going to be very expensive, so those things will not be the most attractive options.So, you need to figure out things which are viable for the specific application you arelooking at ok.So, the aspects like surface charge and surface markers would be the cellular characteristicswhich you are looking to use for isolation.So, surface charge and adhesion properties would be important because these determinethe extent of attachment to a surface and can be used to separate adherent cells fromsuspension cells.So, there are cells which are suspension cells as well, mammalian cells which can survivein suspension are there.So, these suspension cells can be separated from adherent cells right.So, what are these cells?Suspension cells.No, I mean mammalian cells which can.Survive under suspense.So, blood cells would be one , all the immune cells are also they can survive a suspensioncells.Cancer cells can actually survive a suspension cells.There are different cell types which can actually, stem cells can actually survive a suspensioncells.So, mesenchymal stem cells are anchorage dependent, but there are other adult stem cells whichare un-progenitor cells which are actually suspension cells, they can actually form somekind of an aggregate and survive as an aggregate.So, we will talk about it ok.So, cell size and density is a parameter which can be used for separation.So, sedimentation, filtration, or density gradient, centrifugation are techniques whichcan be used for separating cells.So, these have been extensively studied and people will use it very commonly.Cell morphology and physiology, basically the shape, histological staining, media selectivegrowth, redox potential and other visual and behavioural properties of the cell can beused for identifying and isolating the cells.People have used many of these techniques for getting specific cell types.So, last is the surface markers, it is the most specific method.So, what you do is there is specific binding of a surface antigen either to the antibodiesor to aptamers to selectively capture cells that have a specific surface phenotype.So, you try to attract cells which are specific or which are of specific interest to you.So, people also work with combination of these techniques.So, instead of just using one method, people try to use combinations there by getting somethingwhich is much more pure right.So, usually combinations would include one of the first three techniques, first threefeatures and the last one ok.So, because the first three aspects are a lot cruder compared to the last one right.The last one where you are trying to use surface markers is very specific and also highly sophisticatedcompared to the other three.Because if you are talking about just surface adhesion, all you are doing is culturing cellsright.So, you are separating adherent cells from non-adherent cells, it is quite simple.And same goes for cell size and density filtration is a very simple process and so as centrifugation.So, all you need is get a suspension of the cells and centrifuge it and you will be ableto get a gradient centrifugation done, and you will be able to separate the cells.So, morphology again those things are not too difficult to do.These are reasonably simple to perform, selective media and things like that are not too difficultto work on work with.Whereas, surface markers can actually be quite tricky and quite tedious, because this isthis requires a lot of information about the cell you are interested in, and designingof proper antibodies and antigens, choosing the correct molecules which can actually adhereto the cell you are talking about, and using very sophisticated equipment like cell sorterswhich can actually use this to separate the cells.So, what people usually do is, perform one of the first three options first and thenperform the last one.So, there way you that way you do a combination, and you get much higher chance of gettinga much higher purity right.So, this is very commonly done for getting specific cell types.
Video 3: Cell Isolation Techniques
So, this is just an overview of all the techniques that are there.We will talk about individual techniques in greater detail.So, the first technique is the plastic adhesion which is based on the surface charge or surfaceadhesion properties.So, this can either be positive or negative selection.The purity is very low, but the yield is quite high.Because if you are trying to separate all you are doing is segregating it has two differentclasses, adherent and non-adherent cells right.So, every non-adherent cell is going to be in the suspension; every adherent cell isgoing to adhere to the surface.So, you are obviously going to get very high yield, but obviously, the purity is goingto low, because you are going to get a mixture of adherent cells and mixture of non-adherentcells.Density gradient centrifugation is based on cell density and this is a positive selectionfor separation.And again the purity which you get would be low, because you are centrifugation is notgoing to make sure it is distinct separations, and again many cells have could similar densitiesas well.So, you are going to have cell populations in certain regions.So, the purity is going to be low.And the yield is going to be quite high, because in that particular region you are going tohave cells all the cells of that particular density.Filtration is based on cell size.It is a positive selection mechanism where you have low purity and the high yield.Again, everything which is larger than the filter pore size is going to be retained,and that is going to be a mixture of cells again right.So, you are not able to get one particular cell type from that.FACS, MACS, aptamer binding or all surface antigen binding techniques which have veryhigh purity and the yield is actually not very high, yield is low to medium.So, that is why you try to use the first technique before this, so that you make sure you collectall of the cells before you actually perform further purification ok.So, if you have to perform the FACS or MACS or something like that, the cells sorter firstthen; obviously, you are not going to have enough to cells to work with.So, there can also be a selective growth media or culture where you use a physiology of thecells.So, usually this is negative selection.And here the purity could be medium to high depending on what types of cells you are workingwith, and what actually is the mechanism you are trying to use.And yield could again be low to medium.So, LCM is actually a laser-based technique which is used for cutting of cells, identifyingcells and isolating cells, and that is a morphology-based technique.And this again uses positive selection, and the purity is high and the yield is low.With RBC rosetting and immune-LCM, you are actually trying to use combinations here.So, a RBC rosetting is the basically using the size and surface antigen, whereas LCMimmune-LCM would be using morphology and surface antigen.These will give you very high purity, because obviously you are using the surface antigensas the second technique.So, you are going to have very high purity.Yield will again be medium to low ok.So, these are the general techniques which have been extensively studied.And we will talk about some of these techniques in greater detail and some of them we willnot discuss.Let us first look at the first criteria which is adhesion-based isolation.So, as I said there are two types of cells adherent cells and the suspension cells.Adherent cells basically require a suitable surface on which they can attach in orderto thrive.Examples would be macrophage, fibroblasts and mesenchymal stem cells.Suspension cells do not require an attachment surface and can occur in suspension in thebody.They are usually found in fluids like blood or lymph right.So, all your blood cells are suspension cells right.They do not have to adhere to something to grow, they will survive in suspension.So, lymphocytes, granulocytes and other lymphocytes granulocytes and other immune cells are allexamples of suspension cells.So, cancer cells which have actually lost the ability to be adherent are also suspensioncells ok.So, some cancer cells due to the mutations lose their ability to be adherent.So, they do not have to be adherent.So, they will be in suspension.So, those cells are also suspension cells.So, this technique has evolved as the cell culture techniques has evolved.So, when cell culture was first done, people are using just Pyrex glass flasks.So, on which the cells would they are trying to culture the cells.But this does not actually work because primary cells do not attach to glass surfaces.Glass surfaces are glass surfaces hydrophilic or hydrophobic?May be hydrophobic.Why do you think it is hydrophobic?Why do you think glass is hydrophobic?Uncoated glass is hydrophilic, it is not hydrophobic ok.So, the glasses you see like what you wear or all the window glasses are, all they arecoated, so that is why you have you see droplet standing there, otherwise water spreads verynicely.So, if you want to perform a contact angle study on a pure glass, the contact angle wouldbe close to 0.So, it is the clean surface would be very hydrophilic ok.So, but the cells do not like very hydrophilic surfaces as well, they do not like a veryhydrophobic or very hydrophilic, they like only something in between ok.So, because of these the primary cells do not adhere to Pyrex glasses and also Pyrexglasses were actually too painful to maintain right.So, you have to actually sterilize them, and you have to clean them, there is lot of cost,it will get broken.So, it is very painful to deal with glass culture plates.So, in the 60s disposable polystyrene-based culture plates were brought in and peoplestarting using them more commonly.So, you cannot always purchase cells right you would have to isolate cells from primarycells, isolate primary cells from tissues.So, when you have to do that, you have to perform different studies.