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Module 1: Signaling, Bioreactors and Challenges in Tissue Engineering

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Hi everyone.So, today will be seeing about bioreactors that are used in Tissue Engineering.So, what are bioreactors? and what is its function?So, bioreactors is usually used to maintain controlled environment, to ensure that thebiological reaction can proceed.So, for that intent, you maintain the nutrient and product concentration.And also you get a high degree of control and reproducibility of these products on usingthese bioreactors.Role of bioreactors in tissue engineering too is similar to what it is normally usedfor.So, it is to actually initiate, maintain and direct cell cultures in 3D environment bymaintaining that environment and also the aseptic condition.So, why use bioreactors?So, the aim of tissue engineering has been to get organs or tissues off the shelf whereinYou are able to replace a tissue or organ which is damaged by as you see as you buya prosthetic.The factors which play an important role to making this feasible is the efficacy of theproduct.So, the product has to do what the natural tissue or organ does.Then it has to comply to regulatory standards.And also, you should be able to produce these tissues or organs in a cost-effective manner.So, this is where the bioreactors come in.So, products which are manufactured on the table top in a lab, these might be interestuseful to study about how you can grow this organ in lab ex vivo.But to produce it in large scale you would need something like a bioreactor.In addition, such a controlled environment like a bioreactor ensures that we can studyin detail on how the tissue development happens by controlling all the parameters and changingwhatever we require.So, these are the basic types of bioreactors which are used in tissue engineering.So, the first one is a spinner flask wherein there is low degree agitation which is givento the flask.The second one is the rotating wall vessel wherein the cells remain in suspension andthe bioreactors keep rotating.So, this offers some slight degree of shear stress, which is required for cell lines likeepithelial cells and all.Then hollow fibers.So, this hollow fibers have capillaries which run through them carrying the media.And the media can actually diffuse out outside.And on the outer surface of these capillary tubes there are cells which are seeded onit.So, the spent media is then taken out and can be recycled and circulated again.Direct perfusion so, that is d.So, here what happens is a cell and scaffold contract is placed in the centre of the chamberand the media is perfuse through it.So, that the spent media comes out on the other side and you can re-circulate it orchange the media.Then load cell bioreactors.So, this is when the tissue that you are growing require some sort of mechanical force to growin the right way like tendons or bones.So, you can simultaneously stretch or compress the tissue as they are forming, thereby givingit the physiological conditions required for its development.So, the basics that we look at is, so, what do we need to keep the cells alive.So; obviously, we need a media.And this media needs a carbon source or nitrogen source, oxygen and serum and other thingslike growth factors etcetera.The cells need space to grow and also you need an optimum temperature and pH.So, temperature effects.So, the optimum temperature required by the cells can vary based on the cell type.So, they are divided into three classes based on their required temp temperature.So, psychrophiles which required temperature is below 20 degree Celsius; mesophiles, whichrequire a temperature range of 20 to 50 degree Celsius.And thermophiles, which require high temperature greater than 50 degree Celsius.So, above this optimum temperature growth rate decreases and thermal death results.So, the effect of temperature can be shown by the Arrhenius equation.So, in that this shows the growth rate constant and this shows the death rate constant.And using the death rate and the growth rate constant, we can calculate the rate of cellconcentration with respect to time.So, the activation energy for growth is usually around 10 to 20 kilo cal per mol and of deathis around 60 to 80 kilo cal per mol.So, from this we can see that if you can substitute it, that thermal death is more sensitive totemperature changes.So, having the right temperature range is quite important.Then coming to pH effect.So, pH is basically the H plus concentration, which in turn affects the enzyme activityand the growth rate.So, these are few of the examples of different organisms and their preferable pH range.So, for mammalian tissues its usually around 6.5 to 7.4 pH.The cells are capable of regulating their intracellular pH even when there is a variationin the external pH so, there is a buffering system usually for it.Now, looking at the main functions of a bioreactor in tissue engineering.So, we have a cell source and a scaffold.So, the cells initially need to be seeded on to the scaffold and then we should be capableof maintaining the right environment for the cell proliferation and growth.And there should be some level of physical conditioning which is required.So, it varies depending on the tissue that you are trying to grow.So, it can be fluid driven, or mechanical, or electrical conditioning.And also, all these parameters are continuously monitored and using sensors and they can becontrolled.So, that the preferable main environment is maintained.So, first let us look the cells seeding part.So, traditionally cell seeding was done by dropping the cells onto a scaffold using apipette.So, here you rely on gravity to take the cells into the scaffold.But the disadvantages of this is there would be a uniform, there would be a uniform distributionof the cells into the scaffold.And there may not be a complete penetration of the cells throughout the scaffold and itspoor efficiency and reproducibility is also affected.So, that is why dynamic cells seeding is used mainly in the bioreactors.So, here what is done is.So, here what is done is, the its ensure that the cells end up penetrating throughout thescaffold and they are distributed in a more even fashion.So, these are some other techniques normally used for seeding the cell within the bioreactor.So, a spinner flask uses normal mechanical steering to ensure that the cells are seededcompletely into the scaffold.But the spinner flask has some limitations which are mentioned here.So, we use wavy walled which has sinusoidal pattern.Waves can be created because it has irregular not irregular, a patterned outer layer.So, this structure allows for enhance perfusion of the cells into the scaffold.Then the next one is a rotating wall vessel which we saw before.Amongst the dynamic cells seeding techniques, the most efficient one is perfusion seedingwerein it relies on active perfusion rather than using gravity for seeding the cells ontothe scaffold.So, it can penetrate throughout the scaffold and in a uniform fashion the cells can beseeded.So, it has been actually used in converting porcine heart valve by decellularizing itand recellularizing it with human cells by perfusion.So, the main parameters which determine how the cells are seeded are the cell concentrationthat you use for your seeding.The flow rate of the medium, the flow direction and timing of perfusion pattern.So, currently moreover what is used is a trial and error approach in determining what isthe best parameters that need to be used to achieve the best seeding.Modeling is quite challenging because each cell type can have a lot of variability.Still there are models which can help us achieve maximum seeding density based on the porosityof the matrices.So, maintenance of control culture environment.So, a convective media flow around the construct and also perfusion of this media through thepores can aid in overcoming the diffusional transport limitations.So, perfusion can enhance the formation and deposition of ECM as shown in these examples.And also improve the cell proliferation in blood vessels etcetera.So, computational fluid dynamics can be used to design new perfusion bioreactors and alsoto optimize their operating conditions.But what is required for this mainly is to get to know the underlying mechanisms associatedto perfusion, underline perfusion associated cell proliferation.So, what is important for this to happen is to get a good grasp on the underline mechanisms.So, the challenges in maintaining homeostasis is an abrupt change in concentration of metabolitesand catabolites.So, this happens especially in lab culture environment.The challenge in maintaining homeostasis is the abrupt change in concentration of metabolitesand catabolites.So, this can be overcome in a bioreactor by using a semi-continuous or automatic replenishment.So, thereby there is not a huge and sudden change in the metabolites or the catabolitesduring the reaction.Also, we can use a feedback-controlled addition of fresh media.Physical conditioning of developing tissue, this would be the third aspect that bioreactorsneed to look at.So, physical forces as mentioned in the previous talk do play a important role.Different types of physical forces play a role like hydrodynamic or hydrostatic forces,mechanical or electrical forces.So, the aim of the bioreactor here would be to simulate these in vivo conditions insidethe bioreactor too.So, some of the basic physical conditions in vivo are fluid driven mechanical simulation,wherein shear stress can be induced on the cells by the fluid especially in the heartvalve etcetera.So, also create a differential pressure, we can even combine these two mechanisms especiallyin making vessels and heart valves.In the mechanical condition, wherein you give a tension or compression like in bone, cartilageetcetera.And electrical stimuli, which is used in cardiac, skeletal and muscle constructs.So, the challenges in developing a bioreactor would be that we need a very thorough understandingof what is happening in the in vivo condition.And if we can make a computational model of the cell and tissue development in the bioreactorssetting and the sensing and monitoring techniques could be improved wherein we can get realtime information on how the construct is being developed and what its parameters are likeits morphology etcetera.Thank you.