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

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Video 1: Dynamic State of Tissues
So, today we will be talking about Tissue Dynamics.We have been talking about cell culture and looked at how cells initially had to be harvested,isolated and cultured, and even differentiated.I hope the assignment gave you an idea of what differentiation is and how complicateddirected differentiation can be.Because achieving differentiation is not too difficult actually.If you are talking about just differentiation, then all you do is expose them to some stresscondition and it will just differentiate to some random cells.And mostly it will just differentiate to something which is very prevalent like a fibroblastor something.But if you want some specific directed differentiation, then it is quite complicated and it can actuallybe quite tricky ok.So, having discussed some of those things, let us move on to tissue dynamics.So, tissues actually can exist in three dynamic states, tissues are actually not static theycan change in their state, status.And do you know what the dynamic states are? you might have studied it in cell biologyI hope.So, it is homeostatic which is when it is normal steady-state functioning.So, this tissue is doing what it is suppose to do and it is just existing in the system.So, why do you think understanding this tissue homeostasis is important from a tissue engineeringperspective?Generally, we prefer the tissues in this condition.Ok So, we ultimately the tissue has to exist in this condition after implantation right.So, it is important to understand what its role is, what exactly it is doing, how thecells should behave and so on, so that we can try to achieve it in our tissues.The next step is tissue repair.So, which is where any wounded tissue actually displays healing process.And why do you think understanding this is important from a tissue engineering perspective?A implant could get damage.Implant could get damage that is one thing, but you would actually be creating a woundduring implantation you, many a times right.So, if you there are things like in situ forming hydrogels or injectable hydrogels, those thingsare not exactly cause wounds, but in many cases you are going to cause wounds and thehealing can actually influence how the tissue in integrates with your engineered tissue.So, it is important to understand this process as well.The last one is the formation or development ok.So, here it is basically the initial formation of the tissue, the original tissue being formedwhich can be understood by studying developmental biology and morphogenesis.Why do you think this is important, not just how to grow the tissue, it like a how to createthe tissue right.So, see how they will migrate and so on.So, how they will behave in general is unknown, but see, with respect to tissue formationwhatever you studied, whatever you did in the assignment for differentiation that comesfrom developmental biology.It does not, so people do not just say I will throw these growth factors and see what happensright.You try to understand how the tissue actually develops when an embryo developing into awhole organ, whole organism.And during that process they try to understand what are all the signalling pathways whichare involved in the tissue development, what are the molecules which are involved and tryto use these molecules to direct the differentiation.So, understanding this can will help you to create tissues especially if we are goingto be using stem cells and we are going to direct differentiation ok.So, these are the actually the three dynamic state.So, it kind of falls with whatever you had guessed.So, if you are going to talk about tissue homeostasis, tissue can actually, some tissueswould be in their homeostatic state for a long period, whereas some tissues are quiteprolific and they keep getting replenish regularly.So, do you, can you guess what are the most prolific tissues in your body?Skin.Skin is one of the most prolific tissues, yes.Anything else?Liver.Liver can regenerate, but it is not that it keeps replenishing itself again and again.Mucosal layer.Mucosal layer, the intestine layer is one which actually gets replenished regularly.Anything else, what would you think the most prolific neither of those is?Blood cells, bloods.Not exactly blood cells, but.white blood cells.Bone marrow.So, bone marrow is one of the most dynamic tissues in your body.So, it might actually get replaced very quickly.And the second most prolific tissue is the villi of the small intestine, which is themucosal layer which is present on the small intestine.So, here the cellular content can actually turnover every 5 days.And third most prolific is the skin.And so the net proliferation rate can actually vary based on the region of your body, andthe turnover is in the order of a few weeks, so that is homeostasis.And see every tissue has its own homeostasis.Will not get into individual homeostasis because it is different for the type of tissue youare going to work with.So, however, the repair process is more uniform right.So, it is something which is common for any tissue which you are talking about.So, when a tissue is injured, the healing process that usually varies with age starts.So, there are two types of healing.You have the fetal wound healing which is rapid and leads to restoration of scarlesstissue, and you have postnatal healing which is slower and it also leads to scarring.It is that is why many of us would not have like scars from when we were very little right,unless it was a real bad gash you had, you are probably not going to have scars fromwhatever injuries we face when you are 1 year old and all of us go through that right.And, but none of us have the scars because the healing process is actually changes asyou age.And if you are in your teens or your later young adulthood whatever wound you have create,this scars are going to be much more lasting.So, the postnatal tissue healing is slower and it leads to scarring.So, wound healing follows a sequence of events, it is actually a very sequential process,it is very well controlled and that actually make sure that the damage tissue gets repairedto form a healthy tissue.This is a YouTube video I would like you to watch, it is about a 4 minute video it explainsthe healing process with good animation it is better to learn from this than to for tome to explain it.So, just have a look at the process.Hemostasis occurs right after initial injury.At the time of injury, epinephrine a chemical that constricts peripheral blood vessels isreleased in an attempt to minimize bleeding into the soft tissues.The key cell responsible for this function is the platelet which causes the body to forma clot to prevent further bleeding.There is an increased aggregation of platelets to enable the wounded vessel to complete theclotting process.Platelets also release key cytokines such as platelet derived growth factor that callin cells to participate in later phases of healing.The objective of the hemostasis phase of wound healing is to control bleeding.Following hemostasis the inflammatory phase begins.The local signs and symptoms that occur during the inflammatory phase are swelling, increasedfluid, perfusion of blood, redness release of epinephrine estimate heat histamine responseand pain.The inflammatory phase is characterized by our host of cells leukocytes and macrophagesinfiltrating the wound sites.Bleeding is controlled by hemostasis.Any bacteria that is present is destroyed by leukocytes, particularly the polymorphonuclearneutrophil.About 4 days after the injury, macrophages work to destroy bacteria, cleansing the woundof cellular debris.Macrophages, replace the leukocytes and produce a host of cytokines and growth factors.These act as chemo attractants to other cells needed for tissue repair.Macrophages also convert macromolecules into the amino acids and sugars necessary for woundhealing.It is thought that the macrophage also attracts contractual cells to the wound to encouragewound contraction, vasodilation with resultant edema warmth and ruber are the result of factorssecreted from the macrophage and other leukocytes present at the wound site in response to theinflammatory process.The objectives of the inflammatory phase at wound healing to clean debris and bacteriaand prevent infection.Scar tissue formation is characterized by three distinct phases, granulation, contractionepithelialization.Click on one of the phases to learn more.In an open wound, granulation tissue is generated producing red beefy shiny tissue with a granularappearance.This tissue consists of fibroblasts, capillaries and neutrophils as this type of tissue proliferate.Fibroblasts stimulate the production of collagen which gives tissue its tensile strength andstructure.As the, as the wound site fills with granulation tissue gives margins contract or pull togetherdecreasing the size of the wound.The extent of contraction is dependent upon the mobility of the surrounding tissue.During the epithelialisation, the final step of this phase cells migrate from the woundmargins, divide and ultimately touch one another sealing the wound.Epithelialization can only occur in the presence of viable vascular tissue.Epithelial cells will not migrate across a dry surface or necrotic tissue.During the maturation phase, the collagen fibers reorganize, remodel and mature gainingtensile strength.Collagen fibers, proteoglycans and fibronectin are rearranged and redistributed.The scar becomes less cellular and gains tensile strength.However, this tissue will always be at risk for breakdown, because its tensile strengthis less than that of uninjured skin.Collagen synthesis begins with the fibroblasts which secretes procollagen.Growth of the procollagen fibres is a complicated process.Macrophage and platelets are key growth factors in the development of procollagen.Procollagen fibers mature into collagen fibril.The fibrils then link together into a very strong rope like collagen fiber.There are about 10,000 fibrils interconnect within a single collagen fiber.Research is currently underway to determine the chemical details associated with collagensynthesis.So, as you saw, the general thing is, it is quite logical right first stop the blood,and then there is an inflammation phase which actually is a phase where you feel the painand so on, and then you have granulation proliferation phase where there is granulation tissue formationand collagen deposition and so on.And then finally, epithelialization and the healing process.So, this actually make sure that any tissue can be healed.So, in impart conditions one of these phases can get prolonged.So, for example, in diabetic wound healing the inflammation phases actually prolongedthat is why it ends up becoming a chronic wound rather than it healing it, healing withina period of time.So, trying to treat these aspects can help you.So, even if you are going to develop tissue engineered an engineered tissue, you mightactually have to consider having some growth factors which will help in the healing process,so that there will be a better host integration and so on ok.In case of fetal wound healing the process is much faster and it is quite effective andthere is no scar formation.The overall steps which are the overall four steps which are involved are similar to thatof adult wound healing.However, the individual mechanisms are different.What happens is in fetus, the gap in the epidermis is closed by contraction of a rapidly assembledactin purse-string.So, basically it is like a zipping up of the wound.Whereas, in case of adult wound healing the epithelial cells actually crawl over the exposedsubstratum and then close the defect.So, this is a slower process to do that.The inflammatory response in case of fetal wound healing is actually minimal.So, the inflammatory phase is quite short and the process goes on quickly.The collagen matrix has a basket-weave form compared to the bundles which you see in adults,so that is one of the reasons you have a lesser scaring when it comes to fetal wound healing.So, the composition of the ECM is also different, you have collagen three and hyaluronic acidin fetal wound healing, whereas, it is primarily fibronectin in adults.So, those are the differences in fetal wound healing versus adult wound healing.

Video 2: Cellular Fate Processes
So, whenever we talk about tissues, the tissues can actually, tissues are basically made ofcells right.So, the tissue dynamics depends on what the cells are doing.So, there are five cellular processes which actually regulate tissue dynamics.So, can you again guess what those five could be, what could the cells be doing?Growth function.Grow.Sorry, what was it, differentiation yes.Proliferation.Proliferation or growth ok.Where is cell death?death.So, we will just call.So, those are the biological aspects of cell death, but we have cell proliferation or growth,you have cell death.You said cell differentiation.What else would a cell do when it is present in a tissue?So, it basically can divide, differentiate, adhere, migrate or die ok.And based on what it does, the tissue dynamics will change.And you are going to have cell populations which are go doing different things in thesame tissue at varying stages right.So, there will always be some cells which are adhere, some which are migrating, somewhich are divide dividing and some dying and so on ok.So, we will go through quickly about fundamentals related to division and death because thatis something most of us would be familiar with because you would have studied at eitheras part of cell biology when it comes to mammalian cells or as part of microbiology and biochemistrywhen you are talking about bacterial cells.So, the principals are still the same.So, we will just quickly go through them.So, cell division basically, we talk about mitotic cell division, so meiotic is not reallyrelevant for tissue engineering, so that is just formation of gametes which has nothingto do with creation of tissues at least from this perspective.So, we will in a mitotic cell cycle you actually have four phases.So, you have the G1 phase which is where the cells are actually present and doing theirjob, and this can have a variable time.And you have the S-phase, which takes about 8 hours which is a period in which the DNAsynthesis happens and here the chromosome duplication and everything is done.And then finally, and then you have the G2 phase which takes about 2 to 3 hours.So, in the G2 phase you have the cells which have the duplicated chromosome, they havetwo sets of chromosomes present.And it moves to the M-phase which is where the mitosis or cell division actually happens,the chromosome are separated, and the cells are divided to form two different cells andtwo daughter cells.And the time taken for S, G2 and M phases together is about 12 hours; and S, G2 andM is 0th order kinetics, it does not depend on the number of cells which is present, itis going to just depend on the process itself right.So, this is so although overall cell division can be depended on the cell number.This particular phases is not depended on the cell number, it is dependent on the processeswhich have to take place for the cell division to happen ok.So, this is what cell division is I am pretty sure that you have studied this in cell biology.And so when we talk about these phases what happens is the cell moves from one phase toanother in a unidirectional fashion which is controlled by check points right.So, it cannot, after it duplicates its chromosomes, it cannot say no I will go back to G1, andit is not possible.It has to go to the M phase or it has to die those are the only options right, so thatis because you have check point.So, do you know what are the check points?After G1.After G1 ok.After S, after S. After S ok.basically after S it checks that the DNA is here synthesised properly.Ok.After G2 it checks about their organelles.Ok.And after G1 its check, all it like raw materials for some reason available.Whether this cell is ready for division ok.So, G1 check point is basically to verify of the cell is large enough whether the environmentis favourable, whether the cell already has any error in its DNA.So, if it is already DNA with some error, it sorry, cell with some DNA error it shouldnot be on a get divided.So, G2 cell again it verifies whether the cell has become is big enough for accommodatingall these and whether the duplication of DNA has actually happened, and whether this isfully complete and you have two proper sets of DNA.And then the final check point is your M check point or metaphase checkpoint, where you verifyif all the chromosomes are attached to the mitotic spindle, whether all the organellesare in proper shape.And if make sure that this happens before the division happens.These are the three check points to ensure that the cell division happens properly.And when one of these check points fail, you end up with damaged cells which can end upbeing cancerous also ok.So, when you are talk about cell division, this again comes back to simple growth rate,and growth kinetics.So, cell division can be modelled and growth rate is basically directly proportional tothe number of cells present in the cycle at a given point of time.So, you have dX dt equals mu X, where mu is the specific growth rate and dX dt is thegrowth rate ok.So, dX dt equals mu X and you have X.So this can be solved to get X equals X 0, e power mu t and growth rate mu is would beequal to ln 2 by td, where td is the doubling time.So, I hope everybody can derive that you should have done it anyways.So, the doubling time for most of the cells which is present in a humans is about 24 hours.So, basically the cells grow very slowly, mammalian cells are not like E.coli right.What is the doubling time for E.coli?It is 20 minutes, almost 30 minutes.20 minutes.Right.It is roughly 20 minutes.So, it doubles very very rapidly.Whereas, mammalian cells are going to grow at a very slow rate, because of this we haveone advantage with mammalian cell culture.Can you guess what it would be, compare to bacterial cell culture?One of the major limitations in bacterial cell culture is not a big problem in mammaliancell culture.They have to keep regenerating.So, however, with respect to mammalian cells, it is not like the cells can keep dividingto get to very large numbers; in bacterial cell, you start with the very small inoculumand it can multiply for hours and you would be able to get very large numbers providedyou maintain the nutrient concentration and so on.It is here that is not really possible because the even if you have nutrients supply thereis only limited space for the cells to grow.So, there can always be limitations with respect to how much cell population can be achieved.So, because of that the equation actually can get little trickier when you are modellingcell division which would become instead of dX dt equals mu X, it will be dX dt equalsmu X times 1 minus X by Xmax.So, which gives you an upper limit where Xmax is the maximum cell density achievable forthe conditions you have.So, growth rate, the specific growth rate mu can actually be a function of many variables,it could be dependent on oxygen growth factors, nutrients supply, oxygen concentration andso on.In many cases is it not like depended on one limiting substrate.So, if it is limit based on, influenced on one limiting substrate, then you can use Monodmodel to model it.However, in many cases there might be multiple thinks which are limiting the rate at whichthe mammalian cells have growing.So, this is the Monods model.And Monods model is the equation similar to enzyme kinetics.So, growth rate with respect to mammalians cells are does not always have to be likefirst order like what we started with and growth rate can also be 0th order and youmight have to model it appropriately ok.So, the whatever mathematical description we have is completely phenomenological, andyou would need experiments to quantify these coefficients.And in general the doubling time of the human cells can range anywhere from 12 hours to30 hours.So, 12 hours would be for progenitor cells and as this as you have cells which are lessproliferative you will have increased doubling time.There can also be cells with much larger doubling time, but those are exceptions.So, in general, the maximum achievable cell density is somewhere between 3 to 5 lakhscells per square centimetre, so that is dependent on the size of each of these cells when theyare adhere to the surface.The cell density can reach up to 10 million cells per cubic centimetre if the reactorsystem is very well designed.So, that is why you have the spinneret flasks and things where you can actually have veryhigh concentration of cells which are growing.So, in some cases, the cell division can only be modelled if we describe the status of thecells itself, because see not all cells are going to divide right.See even when you do bacterial cell culture, what do you do, you make sure that everythingstarts of at log phase, you make sure cells in log phase are the one you are studyingfor understanding growth kinetics right.But in mammalian cells people do that as well people can actually stop cell cycle, arrestit at one phase and then start them proliferating, so that all of them are uniform or you couldalso have things where they are in different phases.So, if you have to account for that, then the model would also have to account for that.Then you end up with the partial differential equation which would take the cell densitychanging with respect to time and cell density also varying with respect to the cell thecell cycle status.So, a is the cell cycle status.So, if you have X as t of a, t and a then a is basically the variable which describesthe cell cycle status.Where a equal 0 would be a newborn cell and a equals 1 would be the cell which is rightabout completing mitosis.So, it can range anywhere in between depending on which phase the cell is in.So, cells can also be lost from the cell cycle.So, this if you are going to have that, then you have to account for this balance takinginto account alpha of a into X, where alpha of a could be the rate of loss of cells inthat particular state.So, this is not exactly cell death, this is more of weeding of cells from that particularstate.See you could also have cell death; cells can die for different reasons tells, cellsdie from tissue damage which is necrosis.Cells also undergo programmed cell death which is called apoptosis.So, apoptosis is actually a major part of tissue development.So, apoptosis itself was first discovered in 1842, but it was rediscovered in 1972.People did not really bothered to follow it up after that and then they realised it wasa crucial part of tissue developmentThere are different phases in apoptosis you have the induction phase.So, this depends on the specific cell death in specific death inducing signals which aresend to the cells, and then you have the effector phase, where a central executioner is activatedand the cell basically commits suicide.And you have the degradation phase where the biochemical and morphological changes arehappening as the cell is getting degraded.So, the cell death process can also be mathematically modelled in a similar way to death cell division.It is except that the rate will be negative compared to the positive of cell divisionok.This gives us the brief introduction on the couple of processes.There were also other cellular processes which we will talk about.We will talk in detail about cell differentiation, and also about cell adhesion and migrationok.We will talk about how these can also be mathematically modelled ok.Thank you.