Techniques in Mechanobiology: Microfabrication | Cell Shape Control & Traction Force Microscopy

[00:00:01][Music] hello and welcome to today's lecture of introduction to mechanobiology so what the last few lectures started discussing about some of the tools which are relevant for mekin Aboriginal studies in that regard I started off by discussing about hydrogels wherein you can tune the stiffness of these hydrogen's by changing the cross linker or monomer concentration and gave examples of how different kind of hydrogen's can be generated and by different techniques okay so for characterizing the stiffness of cells or gels in that regard I introduced AFM or atomic force microscopy and showed how AFM can be used for proving the properties of hydrogel cells and in an tissues and in that regard I also gave a few instances of how geometry of the cells are boundary conditions might influence the properties of cells or what you are measuring so for example if a gel is very thin okay if a gel is very thin then cell responses on soft thin gel mimic that on thick and stiff substrates okay so while using the s and I discussed how you can measure cortical stiffness of cells okay and in the last class I introduced the techniques decided how you can go about quantifying contractility of cells it and there is for most adherent cell types there is a very close relationship between the two in that if you increase contractility your cortical stiffness or stiffness of cells is going to increase okay you have a linear correlation however a more direct approach of measuring contractility in that regard I introduced three techniques traction force microscopy trypsin by addition and laser ablation while laser ablation gives us information about contribution of single stress fiber to contact the social contact ility trypsin di addition gives us a whole cell contractility measure and traction force gives us forces it bank measures forces exerted by cells so even in both these techniques you measure something called time constant of relaxation okay so this is an indirect measure of contractility while traction force microscopy gives us forces exerted by cells at distinct condition okay now if you talk about contractility contractility is dictated by cytoskeletal organization okay and when I say cytoskeletal organization I am talking about myosin motors okay in non muscle cells you have two or three different isoforms of myosin - - a - B - C D is localized in specially distinct manner okay cytoskeletal organization is also dictated by actin binding proteins by actin binding proteins these would include cross-linking acting cross-linking proteins like philomene like philomene alpha actinin forming etcetera okay but these are all internal controls or internal molecules which will alter cytoskeletal organization but in the context of stem cell differentiation has said that fairy shape is one of the important regulators okay so the question I want to raise today is how do you go about controlling cell shape so if you place cells in culture most of the times the cells will have shapes like this so this might be a standard shape of a fibroblast or this might be another cell so but this you cannot control so these kind of shapes cannot be controlled in culture okay instead the shape is of course very dynamic in nature so how do you go about controlling cell shape in a controlled manner okay and in that regard I want to talk about this technique of lithography so photolithography is the process used in micro fabrication which are to selectively remove part of a thin film or the bulk of a substrate okay so in this technique light is used to transfer a geometric pattern from a photo mask to a light-sensitive chemical this chemical is called photoresist okay so you then have a series of chemical treatments so then you have various chemical treatments okay which either engrave the exposure pattern into or enable deposition of a new material in the desired pattern upon the material under the photoresistor so how do you go about doing this so you use typically a cleanroom so your process workflow is you take a substrate this might be silicon or glass any substrate you coat it uniformly okay with your photoresist so this it is important to have a uniform coating and to achieve this uniform coating you use something called a spin coater it's the idea of a skin spin coater is very simple if this is your substrate okay this is mounted on a motor which rotates it you put a drop so this is your spin coater okay you put a drop of photoresist and you spirited a given rpm okay depending on the omega how fast you're spinning it you might get a very thin layer okay when your Omega is high or a much thicker layer when Omega is low okay so after you have done your photoresist you do a step called soft bake and then using this material you use something called a mask aligner and you shine light onto your material on substrate through a photo mask so this is your spin coated substrate you have your mask aligner and here is your mask okay so the mask has selective zones okay through which light might be able to pass and selective zones which blocks of the light okay so as a consequence so if this is your photoresist in this zone is getting an exposed to the light these two zones are getting exposed to the light and these free zones are not getting any light exposure okay so after you do this okay after doing this you go ahead and do the next step after photo mask you do again a developer you do what is called a develop okay where you put this in a development solution and then finally you do a hard wait which is an optional step okay so in this context there are two different types of photoresist which are possible okay one is the positive photoresist and other is the negative photoresist okay so if this is your substrate okay and imagine okay so you have this is your entire photoresist of which you have exposed this zone to light this is light exposure so if this was a positive photoresist if you develop the material then eventually you will have the following thing on the substrate okay so this is an example of positive photoresist okay and example is Shipley one eight one three is an example of positive photoresist so in this case the polymer or the photoresist is more soluble after light exposure in contrast you can have the other situation okay this is called negative photoresist so the polymer example is su 8 the polymer is less the liver after being exposed okay so in order to have positive and negative photoresist you also we have two different types of photo mask so as I said that onto your substrate okay this is my substrate and this is the uniform photoresist which is being exposed so this gets passed and then this is your light come this object is called the photo mask okay so this is an opaque plate with holes that allow light to pass okay so for a positive photomask so if you go back to the example earlier if this is we are positive photomask then your eventual product at the end of it okay your light is going to pass through this okay so this is a positive photomask and in case of your negative photomask you only have the centered zone and the surroundings will allow light to pass ok so again this is your substrate is a substrate so light will pass through here through here but it won't pass through the middle really get structure in this case it gets stuck in this two corners okay so you can have photomasks of different material example is chrome glass or even transparencies now so this is broadly lithography what is soft lithography the soft lithography is what is used for biology applications so this it refers to family of techniques okay for fabricating structures using so you want to fabricate subjects using glass tumors okay so this technique is cheap and it is well suited for bio applications also is also applications in plastic electronics yes and you can reach resolution up to order 10 nanometers so for soft lithography is the region that we use is PDMS so this was PDMS was also used for making some of the gels hydrogels but in this case PDMS is used for making the must not the you know the patterns okay PDMS is full form is polydimethylsiloxane so once again advantages you can tune the stiffness okay and it conforms to surfaces or large areas okay so after you make something from PDMS you know you can functional eyes something to PDMS using plasma oxidization so where you expose this to oxygen plasma and using this you can have PDMS stuck to glass or PDMS sticking to other PDMS the problem is this process is not reversible okay because covalent bonds are made so how do we go about making a PDMS material okay so what you do you begin with the same thing you have using lithography what you have achieved is you have transferred a given topography of your photoresist on the substrate so this is a photoresist so what you do you first dump PDMS on top of this if you dump PDMS so this is initially a viscous fluid initially a viscous fluid and after you dumped it what you do is you cure it so curing it on a hot plate or an oven it at 60 degree Celsius so what you will have is this material will then polymerize okay once it has polymerized you can actually peel the PDMS so what your with your left with is something like this this is what with your refrigerants okay now once you have done this so of course what you can tweak is these geometrical features okay however you would intuitively expect for example if I am making this kind of a pattern okay but these are very tall these features you might have defects where these actually collapse onto each other so this is an example of a defect okay and this is called as pairing in addition on top of the substrate let us say a busier substrate okay so after you have done the PDMS what you want to do is on a new substrate you want to invert your PDMS okay after coating it on a given with a given ligand okay but in this case you might have the PDMS might sag so you might you might have this kind of a geometry which is so this is a sagging issue okay so using PDMS people have optimized the composition of the PDMS that the use and like this you can use this very well you can use this for getting micro pillars okay so in other words if I were to draw it in a 2d fashion this is what you will have similarly you will have this kind of patterns on a 2d plane so if this was true then you can create a cell on top of this and you can have cells which so if this was ligand coated so the top if you had coated it with some museum ligand then the cell can actually spread and then exert so you will have a cell assemble on this and exert forces which are inward direction okay which are directed inward so as of course so if I were to look at the same thing on from the top what I will get is so let's say I just write down the I am drawing the center point interview okay you have this 4x4 grid well no cells are there so you can design in such a way this D is known this D prime is also known and the diameter of each of these pellets Phi is known okay so when you put the cell you can track the position so let's say if this for the original conditions so if the cell you are exerting contractile forces then the tops would be displaced inward if you can have this and for each of these pillar you can find out what is the displacement Delta D its Delta Delta X is the displacement for each pillar okay so because you know the pillar displacement and if you know the stiffness material of this materials then you can I exactly determine the force at each pillar so the advantage of this technique is first of all you can tune the stiffness of the pillars by changing the pillar length okay so if the pillar is L and let's say it's stiffness is K if you have 2l okay if you have L prime by L prime is greater than L then K prime is going to be less than K so if L prime is greater than L simply because if you make the pillar tall and tall it becomes more compliant so it is easy to deform it yes so this is one of the big advantages of using micro pillars because you don't need linear elasticity theory to solve the inverse problem for finding out the forces exerted by cells okay here once you know the displacement you can exactly map out the force okay so this is one example where microfabrication is really used the other example of controlling cell shapes is using micro contact printing so in micro contact printing what you do is let's say this is your PDMS mold this is a PDMS mold you incubate it with some you see I'm putting of your choice okay so you increment it with your ECM protein of choice let it fit for some time and then what you do you take a substrate ejected in glass you invert the PDMS so after doing this you let it air dry okay so as a consequence if you let it air dry only these points okay all these points will be coated with your UCM protein but when you stamp it okay when you stamp it then what you will be left with is the ECM protein only advanced extra protrusions will get transferred okay so at this point your ECM protein is getting transferred and after you have done this okay you can block the remaining zones okay with BSA or pranic or any other male or pig any non-adherent material so if you look at it in cub view what you will have is as follows okay so let's assume so geometry is were circles and the remaining zones okay so red is non-adherent and green is adherent okay so that is how you can control cell shape but what you have to optimize is to make sure that your coating of tirana corp eggs is uniform if this does not work then your micro contact ability won't work okay so and instead of coating just your ECM proteins you can also micro print DNA on it or any other metal on it so you are not limited to type of thing in which you are with which you're working yes so I just discussed one last study so what was shown is the link the first one of the first studies what it showed was the linkage between cells fading and apoptosis okay so what was done was to make these islands of different sizes okay so the surface area keeps on increasing you plate cells on this and you track what is the percentage apoptosis okay so x-axis is your spread area okay so this area a so what was found was the apoptosis was very high okay when you are spread area was less but it dropped off as soon as your area kept increasing so this suggested that you need an optimal amount of size okay for a cell to survive and as they were dropping a pop to assist if you plot the percentage proliferation so you would see a corresponding increase in the proliferation rate okay so this was the first study to demonstrate the relationship between idle size and Islands and apoptosis okay and what it was also shown so because the increasing spreading area is associated with increase in focal addition area so they did generated different areas where you have one circular eye in other kids this same area you distribute across multiple points at equal spacing so that summation of area is family and in this case what is happening is the overall cell spread area so if a cell spreads like this this cell spreading area is much greater than this area and what it was found that it is the cell spreading area which regulates survival and proliferation yes so with that I stopped my lectures here and to summarize I have discussed how you can make use of geography or microfiber education for tuning generating structures like micro pillars for your special force microscopy studies or using micro contact printing to generate islands of different sizes and shapes and study cell behaviors beat spreading survival proliferation differentiation so on and so forth thank you for your attention [Music]