[Music] [Music] welcome back to the course on cell culture technology today we are into the third class of the first week so the first two class I made an humble attempt to kind of request you to think beyond cell culture as just a culture technique and I told you that at every point of it there is a philosophy there is a philosophizing in subject instead of blindly following certain things and there is simple chemistry physics and biology involved in it so 2d the next two classes what two two three whatever you know whatever it takes us we'll talk about the biology of the cells visibles the in vivo and the in vitro conditions because what should I understand the biology of the cells it will help us to recreate a situation which is similar to that of inside the body ok so we are into week one and today's hour plus three okay so today we will talk about the biology of the cell to be taken into account to grow the cells outside the body okay so this is the key point what we'll be dealing next few classes okay so in order to address this point first of all we have to understand under what conditions are cells go inside the body so again let's take the example of one of the previous examples where you dealt with okay so say for example if take the same example again and let's take the example of the cardiac cells cardiac or the heart okay so these cells which are growing out here the cardiac myocyte which i've already explained earlier and here are the gap junctions okay these cells out here have the first parameter they need oxygen and they give out carbon dioxide and they need oxygen so there is something called they have a mechanism of gaseous exchange inside the system very very first thing you need it because in the absence of oxygen in its absence which is an anaerobic situation these cells will die okay so they need sufficient oxygen and not only that these carbon dioxide which is produced here has to be sent outside the system otherwise this will make the environment acidic because co2 plus h2 we know that it will perform carbonic acid okay so that is something one has to keep in mind so there has to be a proper gaseous exchange which should take place without that gaseous exchange these cells are not going to grow so first thing what one has to keep in mind if you are taking all this tissue out here and you should be able to provide the right milieu of gaseous exchange okay and we will come to that how the gaseous exchange how this menu is being maintained in a system outside the system okay parameter one next parameter is this we have to figure out if you remember that in one of the lectures give a pause I say if these cells adhere to the surface so there are two kind of cells cells which are not here of course there are other parts of what we have we are adhering self-adhering cells and non adhering cells which cells are we culturing so talking about non adhering self adhering so you could see these are all adhere to each other right in our hand now talking about the non adhering cells blood cells they are circulating in your body and non adhering right they do not adhere if they are there then will collapse right because they have to carry oxygen so are we talking about RBC's red blood cells or we are talking about WBC's white blood cells are we talking about of course our descendants all the lymphocytes leukocytes macrophages are we trying to culture these and if you're trying to culture them do you want them to address or we want them to you know in a suspension to move around what are we culturing this is a very critical point so think of it the hard tissue which is growing out here it is exposed to blood and that blood contains lot of non adhering cells moving through and those non hydrating cells while they are moving through say for example is a non attaining cell once again let me change occurs non adding cells it is rolling you know it is moving through but what will it is moving through it may secrete out certain things right in a real life we do not know what it is secreting and that what it is secreting to its surrounding may influences the another cell which is which is an earing cell sitting out underneath these pink colors are the adhering cells which are sitting underneath and top of that this red cells is moving through by secreting certain XYZ compound XYZ chemical and this is influencing some of its biology we really do not know that so first we have two digits to decide are we culturing adhering or non is reddening at this point I will stick to their adhering cells we talked about that earring we are not taking into account the non adhering so that's why I give a pause in somewhere in my first or second lecture that you know are we talking about what I did not introduce that complexity so here I thought that I bring it back so if we are talking about adhering cells so the cells the way it works is something like this say for example I have a cell like this okay here I have a nucleus to the DNA sitting there hear all sorts of organelle mitochondria and the plasmic reticulum er nucleus mt stands for mitochondria likewise we have the plasma membrane PM what these cells have lot of you know membrane proteins which are sitting there now if these cells have to adhere to a substrate or adhere to each other they secrete specific cementing materials and by virtue of which these blue arrows coming out are telling or telling you about the cementing materials these cementing materials helps it to adhere to another cell in its vicinity of its type or another type or whatever G is the Golgi apparatus likewise okay so again these two cells then form the colony so say for example this leads to secrete another set of cousin it invites the third one to form they are slowly forming a colony so in order for them to adhere these compounds which are shown in blue out here this forms a matrix so if you could remove all the cells what you will be seeing behind they will be left behind will be something like this those blue color arrows what I am trying to show and this matrix is called in technical terms it is called extra-cellular because it is extra it is not inside the cells right it is that extra extra outside this extra this is not part of the cell extra cellular matrix it forms a matrix like this here is a matrix and this extracellular matrix are mostly proteins and part of carbohydrates and there are metal ions and several things which are involved in it this extracellular matrix is one critical feature in our body which ensures the cells remain adhered to their specific locations because this particular aspect is very critical when we'll be talking later about cancerous cells okay so this extracellular matrix is a kind of you can call it the signature of the colony of cells what do I mean by the colony of cells now let me come back to the previous diagram so this this is the I named it as a hard colony okay okay now out here where I have the brain this is the brain colony okay now I have this stomach and everything this is the say stomach colony then the series of things so each one of them are colonies and within colonies there are sub colonies how is it so say for example if I break open the heart within it say the structure of the heart is something like this where it has four chambers like this you know now within it these are the two upper chambers depending on which angle you are looking at it from which side LS stand for left R stand for right use system proper chamber Elsa stand for lower chamber your chamber so within this colony there are multiple colonies and each one of these they have different cell types which are involved in it ventricular cells so the lower ones are the ventricular cells which are covering this part ventricles the lower two chambers are called ventricles these are called Oracle's and they have different size types with mild variations so there are different colonies and different colonies leads to different extra cellular matrix system which I introduced in the P in short this is called and biological jargon they call it ECM extracellular matrix and nothing but identification of that location these cells grows here under these these conditions so it's a very classic signature of accessory matrix now if I have to coming back here if I have to grow these cells outside we take out these cells the first thing would have to mimic is you have these cells out here I should have the right set of ECM extracellular matrix so if I have to grow these cells outside on a dish then it's the prime prerequisite is if I have a dish like this but this should have the right set of extra cellular matrix so I should be able to coat this I'm just putting blue because since I showed you the extracellular matrix in blue I'm just putting it with blue should have the right set of extracellular matrix proteins out here now one can since I'm talking about extracellular matrix protein so if one has a hypothesis saying that the say for example I mark this extracellular matrix as say I call this as blue this blue is showing the accessory matrix okay and I called it as B now if I give you a hypothetical situation if I say if in the extracellular matrix B you add compound P which I'm representing by say compound P something like this and if you grow these cardiac myocytes out here on top of this then it will change the properties of cardiac myocyte so they will not adhere to each other so in that situation you will only see the cells sitting there they will not move they will not migrate and form the structure what I showed you then if they have to form they should show something like this like if you remember when I showed you this structure they should show the electro mechanical activity and all this thing they should come close to each other remember this a structure which I showed you in probably in the first or second lecture so they will not be coming close to each other now Here I am telling you that you can actually test such things if you say this P will actually prevent their migration and say for example P is a some form of a toxin which binds to XSL or a matrix and prevent the cardiac myocyte to come close to each other so how to test it here is an example so you see if you know the b6 then you can answer in finite number of questions what you have to understand the basic very right because as I'll go through you see you know you just cultured the Dacian many people as a matter of fact because of lot of phenome budding graduate students you know they don't even understand the significance they buy coated dishes to culture things without even realizing that there is a tremendous chemistry and biology involved in it it is not something like you know just mix something mix match something something is going to happen it doesn't work like that there's a logic and one has to because it's a very tough process I mean one has to go through that whole grill to understand that what is the significance of having these kind of under standing or say for example we start predicting that okay these are ECM which are natural these are natural easy which consists of as I have mentioned of proteins and / carbohydrates now I have a synthetic molecule a synthetic analog which exactly mimics a natural analog big discovery and we'll talk about it there are synthetic analogs which exactly behaves the same way as their biological counterpart how to discover it if you do not know the basics and within scientific and within this one can have synthetic synthetic things which may be inorganic counterpart may be an organic molecule but in order to decipher it one has to understand the biology and chemistry behind it because I told you there is another very interesting thing which is related to this extracellular matrix protein that is coming back to this picture now when you talk about carcinomas or cancer cells so what are really cancer cells are cancer cells are originating from your own body these cells for example we talk about a colony okay let me draw it something say for example you consider your body as colony it says say a blue colony red colony green colony - a green colony orange colony of pink colony fade blue color so this is good applicant and each one of these colonies are different you know cells which governs them right these are the different cells and this whole system lives in harmony without you know creating ruckus okay one minute okay there you are right and they are connected with each other with blood vessels and you know they cross talk with each other system which is functioning in complete harmony with it with Excel and they're happy-go-lucky family now say for example in this colony say for example one of the kid become rogue this is that rope kit and this rope kid decided that I am going to create problem what this will do it will travel it will come out of it so now think of it these colonies identification of these colonies are their ECM there is a different ECM here except cellular matrix there is a different ECM here there is a different ECM here ECM is their barcode you remember the barcode whenever you buy some material you see the barcode it is a barcode of that cell which decides which part of the body it is going to grow but here this rogue fellow decided that it will deceive the barcode and it will go anywhere and it will is going to grow it will invade me it may invade this part and you know it will grow like this because it is now more of a suspended cell it can grow anywhere and sit it does not have anymore the barcode to grow you have you know kicked out its barcode it goes here and Internet create trouble it may go here and create trouble whatever whatever it decides it is a rogue fellow so all of a sudden harmonious system goes for a different swing and that's precisely what happens in a cancer cell a cell which decides that hey I do not want to become part of this colony how I cannot become part of the colony I shut off my barcoding and I cheat this colony the red Colin if I start with the red colony I cheat the read colony vessel I moved out from that colony I went to the dark green colony or a light green colony or to the pink colony and I spread myself because I do not have any barcode now I can grow anywhere and as much as I can grow this is precisely what happens and that so I am Telling You understanding this part of the biology of understanding this extracellular matrix extra again I am reiterating this point because try to understand the signs behind it extracellular matrix is so so so so very important so please as the next generation upcoming scientists do not follow things blindly do you think this is always a chemistry behind it without that biology does not exist there has to be a basic fundamental chemistry behind it so please think over it I have seen people buying dishes coated with some exercise it accessible images and they grow cells there surely is it something will grow I mean who is stopping them from growing life expresses itself there is a thumb rule but then that's not the way to do the science one has to understand the basic concepts what is happening if I change it so you should be able to play with the cells you know if I change this extrasolar metric this is already there if I use this this is what is going to happen how the cell behavior changes as a matter of fact within the same extrasolar matrix with slight difference their physical parameter changes so this is that part where when our self become broke when the cell becomes cancer because this extracellular matrix signature or the barcode is compromised so for those who are from outside biology you can call it as a compromised ECM barcode this is how we should remember it the barcode has been compromised when the barcode is compromised they can cheat anybody and they do cheat they go to some other part of the body and then create records so this is how it works this is the second aspect what one has to understand that what the ECM does in the next class will follow further with a biology of these cells which will help you because based on this so we have talked about today about the gaseous exchange in how we mimic it we haven't talked about the mimicking but how we have to mimic that condition second we talked about the ECM right we will follow it up in the next class thank you and thanks for your patience [Music] [Music] you [Music]