Episode 1 | Wound-Infection-On-Chip | Back to the Future | Virtual Lab
[Music] foreign [Music] and I'm a science Communicator at the center for predictive human model systems or as we like to call it cphos in this series we're going to take you to some of the coolest labs in India where we're going to learn about Advanced new tools and Technologies being used in the world of biology [Music] [Applause] you've all probably heard yourself while playing at some point you usually just put on a Band-Aid and forget about the wound right but after a few days when you remove the bandage the wound has magically gotten all better have you ever wondered how our body does this our skin is the largest organ of our body it protects and shields us from the outer environment the skin is made up of three layers the epidermis the dermis and the hypodermis when these three layers break due to an injury it results in a wound in humans wound healing involves a bunch of different factors like immune cells which release biochemicals to build new tissue and inflammatory cells which prevent infection and help the healing process some wounds which take a very long time to heal are known as chronic or non-healing wounds now these wounds often develop infections which cause a delay in wound healing and in severe cases this can lead to amputation or even death infections are mostly due to the formation of microbial biofilms a biofilm is a thick slimy layer found on top of chronic wounds this layer is actually an extracellular Matrix which is produced by structured communities of my groups this biofilm induces a long-term inflammatory response prolonged wound healing and can resist antibiotic treatment scientists have studied wounds so far by using animal models the most preferred model organisms are rats and mice because of their small size and availability but the problem is rodent skin is really different from human skin scientists turn to using pigs instead which are physiologically similar to humans but genetically we're quite different from pigs and studying wounds in animals doesn't really make sense if we want to learn about human wounds right and of course we can't study them directly in humans so what do we do well that's what you're going to learn about today scientists have built model systems using human cells which mimic a wound one example of these systems is a wound on a chip which incorporates not just wound but also skin cells microbial biofilms biochemical factors and biophysical forces we are going to learn about this cool model today so let's get into it hello everyone um Welcome to our group I'm Dr Karishma kaushik and my research group is based at savitri by fule Pune University so my background is that I'm a physician scientist and clinical microbiologist and that sparked my scientific interest in human relevant infection States and we do so with we study these human relevant infection states with a difference we study them in the lab under in vitro conditions that we try to make closely mimic the the actual clinical infection micro environment so one of our Focus areas is studying wound infections so we have built a range of different models and tools to study wound biofilms under in vitro conditions in the lab and I'm excited to show you what we do so a wound infection on a chip platform is to mimic clinical wound infection state so our device consists of a space where we can grow cells that are found in the wound bed we add microbes to these cells because that's how biofilms look in when they are present in wounds this is grown in the presence of a wound milieu which is similar in composition to wound fluid and all of this is under a biophysical Force known as shear stress which is also something the wound is normally subject to so together this is our wound infection on a chip platform working with Dr Karishma kaushik at the human relevant infection biology lab in our lab we have developed wound infection on a chip model system where we have managed to mimic the environment of a chronic wound infection before starting working in an animal tissue culture facility we have to take care of something called as good lab practices the first thing no wrist watches or anything on your body secondly you have to wear masks and lab coat and gloves to work in a hood foreign since we're going to be working with cells we need a sterile environment to prevent any contamination the very first step is to disinfect the biosafety cabinet or cell culture Hood we do this by using 70 ethanol to sterilize our hands arms and the inside surface of the cell culture Hood after that we spray each item that goes inside the hood and wipe it clean and by each item we mean every single thing from tubes and pipettes to pens tape and parafil finally we close the glass of the hood and turn on the UV switch for a minimum of 30 minutes this UV irradiation kills any living microbes that might be inside the hood and make sure everything is sterile using computer-aided software and 3D printing we have designed and developed wound infection on a chip model now this Farm size device is made of a biocompatible polymer consisting of centrally placed transparent chamber where the biological interaction happens it also consists of an inlet and an outlet where the flow of fluid using a peristaltic pump happens now we're going to begin our cell culture work the very first thing is to take ourselves out of the incubator and view them under the microscope to see how they look a human keratinocyte and fibroblast cells or in simple words skin and connective cells now that we know our cells are nice and healthy it's time to see how many of them we have we do this by counting the cells the very first step is to remove the growth media from the T25 flask where the cells are currently growing nizam is using a pipette gun and a 10 mL pipette to speed up this process the old media that is removed from the flask will be discarded the cells that we're using today are adherent meaning that they stick to each other and the surface of the flask in order to detach them they must undergo a process called trypsinization foreign is added to these cells and then off they go into the incubator for about two minutes so that the trypsin gets activated foreign detach we can see them floating around in the trypsin under the microscope now we bring the cells back into the hood and add growth media to neutralize the effect of trypsin the next step is to collect all of our cells and transfer them into a 15 mL Falcon tube to be centrifuged centrifugation is a process where we spin ourselves at a very high speed so that they all settle at the bottom of the tube before we start we have to make sure that the volume in the balance tube matches the volume in our tube after that we put the tubes into the centrifuge and set an appropriate speed and time once the cells are done being spun we can see a small white pellet at the bottom of the tube after this we'll gently aspirate the old media from the tube making sure not to disturb the cell pellet at the bottom into this goes fresh growth media in which we gently resuspend the cells and mix it all together now it's finally time to count a small volume about 10 microliters of the cell suspension is taken and mixed with 10 microliters of a Dying agent called tripin glue in an ependorf tube from this mixture we aspirate 10 microliters and add it to this glass chamber called the hemocytometer where all the action happens the cells will be distributed throughout the three by three millimeter chamber and we count the cells in the four corner squares which are one by one millimeters in width the required number of cells are taken from each flask and co-cultured together on a glass surface which is fixed inside the central chamber these cells are left to grow for 72 hours and become confluent after which they are fixed with four percent piriformaldehyde this creates the first layer of our wound simulation the skin foreign cells are growing let's prepare the next layer the microbes which will form a biofilm nizam is creating streaks of two microorganisms staphylococcus aureus and pseudomonas erogenosa on this agar plate so that they can multiply and grow once the streaks have formed he will transfer them to a vessel containing lb broth and let them grow for a while foreign after the cells have been left to grow overnight nizam quantifies them so he knows exactly how much to add to our wound infection on chip device after diluting the cells in lb broth the microbes are added on top of the skin cells we added previously these microbes are left to incubate for two hours so that they can settle and form biofilms [Music] it's finally time to get ready for the big show now that we have our wound bed consisting of skin cells and microbes ready we're going to implement the remaining factors of the wound micro environment biophysical forces nizam is connecting tubes to the inlet and Outlet channels of the device these tubes are connected to a reservoir tube which contains a fluid that makes up the wound milieu this wound fluid is a special lab made solution which contains several important components like proteins inflammatory cytokines and growth factors which contribute to and perpetuate the wound state [Music] [Music] a peristaltic pump is used to transport liquids in tubes by a series of rollers that squeeze it against the walls of the pump housing which can be seen here this is similar to peristaltic movement which can be found in human bodies the speed of flow of liquid and the force it creates mimics the physiological levels of shear stress that is seen in a wound as the solution flows through these tubes and into the device it mimics the wound fluid which is found in a chronic wound Karishma explains that it is a common observation in clinical cases to see pseudomonas out competes to phyliccoccus over time pseudomonas is represented by the red Aggregates which overgrows to phyliccoccus which can be seen as the Green Dots okay now that we have done confucal microscopy of our device we have the raw data but it needs to be processed and so we'll be beginning with the downstream processing we are using biofilm queue to do this it will convert the raw files into a compatible format for image analysis and then once the images are ready we will render it and calculate parameters such as bioflim height mean fluorescence intensity and then we could plot them in this image we have the 3D rendering of the host cell scaffold which we have stained with tapi the dappy stains the nucleus and hence we can see individual nuclei colored in blue at this time point of 4 hours we can see clear intact nuclear but as we move across the time point of 8 24 and 48 Hours the nuclear slowly starts to disintegrate and by the end of the 48 hours we are not able to see any nuclear and the diet P has spread throughout [Music] hi I'm Dr Joey Shepard from the University of Sheffield in the UK I am a microbiologist and tissue engineer So speaking from physiological cellular and molecular standpoints humans we humans are very complex beings so to study biological interactions in humans either healthy or in disease States animal models have been a Mainstay of biomedical research for many years now so animal models like mice and rats have been quite useful and they provided us quite a lot of information because they do share several biological processes with humans however humans are different species we're not 70 kilogram mice and most of us anyway are not quite as furry so these many differences between animal and human biological mechanisms have been increasingly investigated over the years the most common way of Performing investigations with human cells in the past and indeed currently has been using single cell types grown in dishes or flasks and again while this is useful and has provided lots of basic information it doesn't really represent the complex 3D multicellular environment in a whole human very well so now biological research has moved from single cell simple cell cultures in Petri dishes and flasks to growing very much more complex 3D multicellular organ models such as muscle and bone and liver and many others and in our Labs at Sheffield University we developed models of human skin the study infection and working together with Dr kaushik's lab who developed an even more realistic way to model human infection we've put our two Labs work together and we've come up with a small model of infected skin that behaves very much like a real clinical situation so it's very useful tool and that's great but so we can work on many tests in a shorter time we can now as well create in vitro organometer chip model systems so very small and this will allow us to do lots of tests in a much shorter time so we're now capable of creating organ systems in chips the size of microns and these current trends are very exciting to say the least and we're very very happy to be a part of this exciting research the main reason we are trying to build wound infection on a chip model system is to test pre-clinical drugs which are used to fight infection in non-healing wounds we do this by incorporating a variety of factors in our model some examples include human skin cells microbes and external biophysical Force such as shear stress and lab developed wound fluid using this platform we can test different kinds of antimicrobial agents like novel antibiotics traditional remedies enzymatic dressings and many more today we covered one type of an organon chip device but scientists across India and the world are creating many exciting variations like lungs heart kidney and liver on chip models just to name a few and that's not all scientists are also creating organon chip models for diseases like cancer there is so much potential lying in these tiny microfluidic devices and the research landscape in India is adapting to make full use of it well that brings us to the end of today's Virtual Lab we hope your neurons are firing and there will be a lot of questions for us to answer in our upcoming quiz and discussion Circle to learn more make sure you check out your Virtual Lab workbooks tune in next time and follow us to a new lab where we explore how scientists are creating many versions of human organs right in their Labs see you soon [Music]
2023-02-12 20:55
