Tissue Engineering
Transcript of Part 2: Microscale Liver Tissue Engineering
00:00:00.00 Hi, my name is Sangeeta Bhatia 00:00:02.08 and today I'm going to be telling you about tissue engineering of the liver. 00:00:06.21 To orient you, this is a schematic of the liver. 00:00:12.24 The liver has over 100 billion cells and performs over 500 functions in your body. 00:00:19.04 300 million people worldwide suffer from liver disease 00:00:23.29 which one can acquire from viral insults like hepatitis 00:00:27.13 or a drug insult like chronic alcohol exposure 00:00:32.05 or even an over the counter drug in high doses. 00:00:34.15 What happens when the liver gets damaged is it forms scar tissue 00:00:38.27 seen here in the middle panel. 00:00:40.23 The scar tissue evolves into this nodular pattern known as cirrhosis. 00:00:46.09 A cirrhotic liver is prone to developing cancer over time. 00:00:51.26 So patients who have liver disease often progress to liver failure and liver cancer. 00:00:58.11 The gold standard for treatment of these patients is whole organ transplantation. 00:01:03.24 However, there are not enough donor organs available for transplanting those in need. 00:01:08.21 Because of this and because the liver provides so many functions that are vital for life 00:01:14.25 many groups have thought about supporting liver function with cell based therapies 00:01:20.24 wherein the cells would be hepatocytes, 00:01:23.24 the cell of liver that performs most of those 500 functions. 00:01:28.06 The idea is that living cells would perform liver function in a variety of hybrid formats. 00:01:35.05 So, here I'll just show you two examples. 00:01:38.03 These are extra-corporeal devices. 00:01:41.00 They are akin to kidney dialysis machines. 00:01:44.12 The blood would run outside the body through this cartridge 00:01:48.06 and the cartridge would house living hepatocytes that would process the patient's blood. 00:01:53.06 The idea is that this device would bridge the patients to transplantation 00:01:57.15 or even support the patient while the liver regenerates 00:02:01.12 because, interestingly, the liver is one of the few tissues that actually can regenerate 00:02:06.03 in certain kinds of injury. 00:02:07.25 Another idea which our group has been pursuing as well as several others 00:02:14.17 is this idea of an implantable, tissue engineered liver. 00:02:17.24 This would one day, we hope, serve as an adjunct or an alternative to liver transplantation. 00:02:24.24 The problem in the field has been that liver cells in these devices 00:02:30.13 do not perform the 500 some odd liver functions that are necessary for patient support. 00:02:36.07 We know from a variety of work in the field that this is because the liver microenvironment, 00:02:42.21 depicted here, is disrupted when the cells are isolated and 00:02:46.24 put in contact with the plastic of the dialysis cartridge or the polymer scaffolds 00:02:52.15 that are going to be implanted in people. 00:02:54.27 So the liver microenvironment, as you can see, is quite complex. 00:02:58.29 Here we see a repeating unit of the liver. 00:03:01.10 This is called the liver acinus and each of these units is about 1mm in diameter. 00:03:07.21 The hepatocytes, the cell of interest that performs most of those 500 functions, 00:03:13.05 are aligned in these cord-like structures. 00:03:16.13 These are actually 3-dimensional structures known as hepatoplates 00:03:19.27 and you can see that along each one of these cords lies a blood vessel. 00:03:24.21 So each hepatocyte is only one or two cell widths from the blood flow 00:03:30.10 and that provides them the ability to very efficiently process the blood; 00:03:36.18 both to metabolize drugs and hormones in the body 00:03:41.26 as well as to produce secreted products for the blood stream. 00:03:46.15 For example, your liver makes most of the clotting proteins 00:03:49.27 important for clotting blood after you cut your finger. 00:03:53.18 In addition, it makes albumin which helps you keep your fluid inside your blood vessels. 00:03:59.18 It metabolizes all the drugs that you take from your doctor. 00:04:05.04 And in addition it regulates energy metabolism. 00:04:10.23 So all of these functions are important and they reside in these hepatocytes 00:04:15.17 that are dependent on this very complex microenvironment to function. 00:04:20.07 When we remove hepatocytes 00:04:22.20 in order to incorporate them into one of these cell based therapies 00:04:25.25 and put them in culture this is what happens to their function. 00:04:29.23 So here I'm showing you data for just one liver specific function 00:04:33.22 and that's the production of albumin 00:04:35.18 which is important for oncotic pressure, the fluid pressure in your blood stream. 00:04:40.27 And you can see that these are hepatocytes here. 00:04:43.12 They've been cultured on collagen coated tissue culture plastic. 00:04:47.01 And after a couple of weeks in culture they're spread out, and they're dying, 00:04:51.20 and all of their liver specific functions dramatically decline. 00:04:55.11 So in our group, what we've been intersted in is 00:04:58.19 seeing whether we could recreate a happy microenvironment 00:05:02.29 for these hepatocytes outside of their native microenvironment 00:05:07.03 in such a way that it could be useful for incorporation into a therapeutic device. 00:05:13.09 Because the environment of the cells in vivo is specified at the 10-100 micron length scale, 00:05:21.05 we have been drawn to technologies that allow us to manipulate 10-100 micron length scale. 00:05:27.08 And in fact, if one looks at the computer revolution, 00:05:31.02 the computer revolution was driven by miniturizartion technologies that have exactly this property. 00:05:37.12 So what I'm showing you here is what we achieved as a community, over 50 years of progress 00:05:43.28 from a single transistor to 100 million transistors 00:05:47.21 through a very simple process known as photolithography. 00:05:51.19 So this is a process by which computer chips are made. 00:05:56.07 And what's done is one takes crystalline silicon and coats it with a light sensitive material, 00:06:01.26 shines light through that light sensitive material and creates a pattern on the surface. 00:06:06.29 Now if you were making a computer chip, one could use that pattern then to make 00:06:11.14 integrated circuits, resistors, transistors and so on. 00:06:15.11 But we and others have been thinking about using these technologies 00:06:19.05 to create cellular microenvironments for the applications of tissue engineering that I mentioned. 00:06:25.08 In this talk, what I'll be telling you about is our work 00:06:29.13 in constructing human tissue microenvironments for the liver, 00:06:34.03 our interest in interrogating these microenvironments in high throughput 00:06:39.25 for, for example, drug development, 00:06:42.05 and furthermore, our interest in using these liver tissues 00:06:46.21 for studying the interaction with pathogens that only normally interact with the human host. 00:06:52.16 So I'm going to start by telling you how we are interested in 00:06:55.28 exploiting these tiny technologies for constructing engineered liver tissues. 00:07:01.14 So the first step that I need to tell you about is 00:07:05.13 how we adapted that same process I mentioned earlier which is called photolithography, 00:07:10.08 where normally one patterns metals on silicon, 00:07:14.17 here to pattern cells on glass. 00:07:18.00 And what we did was we took the same liver cells that I showed you before, 00:07:21.11 now they're color red, and we surrounded them with another cell type 00:07:26.00 So we knew, in the liver, in its native microenvironment, 00:07:28.27 that there's at lest four other cell types that are close to the hepatocytes 00:07:32.24 and so here we've just added one other cell type back 00:07:35.08 and this is a randomly organized co-culture of two different cell types. 00:07:40.18 What we did with photolithographic processing 00:07:43.21 was create what we called a micropatterned co-culture and that's shown here. 00:07:48.10 In this system what we've done is use the same light-based systems 00:07:51.27 to pattern collagen extracellular matrix on a glass coverslip. 00:07:56.06 The hepatocytes (the liver cells of interest) stick on those spots 00:08:00.20 and then we can fill in the spaces with the supportive cells. 00:08:03.23 And in this way we can control the architecture of the tissue in a two-dimensional plane. 00:08:09.23 And we found over the years is that the geometry of this architecture, 00:08:15.17 the control over which cellular neighbors are right next to the hepatocyte, 00:08:20.06 whether they're self (homotypic) or non-self (heterotypic), 00:08:24.22 determines the level of function that one gets. 00:08:27.04 And in particular, for human tissues that one can rescue the phenotype of the cells, 00:08:32.24 those 500 functions of interest, for about 4-6 weeks. 00:08:36.16 In addition, we've been interested in using another version of microfabrication technology known as MEMs. 00:08:43.05 In this process, instead of just making a flat, planar patterned structure, 00:08:47.29 what groups have done over the years is develop etching techniques 00:08:52.08 to etch crystalline silicon like this part that you see here. 00:08:55.13 So this part is first patterned with light 00:08:58.13 and then deep-reactive ion etching is used to etch out the structure that you see there. 00:09:03.23 Using this part, we were able to manipulate our cells at the microscale. 00:09:09.09 So here what we've done...You're looking at two interlocking combs. 00:09:12.17 You seed one cell population on one part and the supportive population on the other part. 00:09:18.01 And what we're able to do is move them together and apart 00:09:22.03 and ask questions about whether they need to signal each other continuously or reciprocally. 00:09:28.09 So, for example, in this experiment, in this movie, what's being done 00:09:33.13 is the cells are being brought together 00:09:35.26 using a simple micropipetman manipulation in a biosafety hood. 00:09:41.15 And you can see that they've been brought together in what we call gap mode, 00:09:45.02 where they're separated by an 80 micron gap but not allowed to touch. 00:09:49.25 Now they've been brought into contact mode where they're allowed to touch. 00:09:55.23 And now they're separated completely. 00:09:57.13 This allows us to study the dynamics of cell interaction using microfabrication. 00:10:02.29 What we've learned as an ensemble in these sets of experiments, is the following. 00:10:08.23 That this big fat cell here is supposed to represent the hepatocyte 00:10:12.15 and this cell is the supportive, stromal cell. In this case it's a fibroblast. 00:10:18.01 What we've learned is that the cells need to touch each other for about a day 00:10:22.16 in order to get the rescue that I described earlier. 00:10:25.29 After the first day, however, they can produce soluble factors that can diffuse over that 80 micron moat. 00:10:35.20 And this soluble factors are completely sufficient to support the hepatocyte. 00:10:39.29 So what's exciting to us about this finding as engineers 00:10:43.15 is it might suggest that after an initial priming period one could support hepatocytes 00:10:49.11 in an implantable tissue or in an artificial liver device 00:10:52.12 without needing to have another living cell taking up space and nutrients. 00:10:57.19 So I've told you about how we used microfabrication 00:11:02.15 to learn about how to stabilize the liver cells 00:11:05.17 but one can also use these light based patterning techniques 00:11:09.22 to build three-dimensional, implantable parts. 00:11:12.06 And in doing this, we were inspired by this rapid prototyping technique known as stereolithography. 00:11:19.04 So stereolithography is a technique whereby one will draw a three-dimensional part 00:11:25.00 using CAD software in the computer and then use it to drive this robot. 00:11:29.20 And the robot works as follows. It drives this stage. 00:11:33.20 The stage can move up or down in a vat of light-sensitive polymer. 00:11:38.19 So when the stage is at the top and one shines a light in a pattern it crosslinks the polymer 00:11:44.03 and then we can make it drop a level and shine a different pattern. 00:11:48.26 And so on and so forth. 00:11:50.07 At the end you build very complicated three-dimensional parts. 00:11:54.06 And in fact, there are programs now to be able to drive these 00:11:58.06 with three-dimensional, patient-specific anatomic data. 00:12:02.04 So one can imagine then taking CAT scan data and driving a robot like this 00:12:08.14 to make a three-dimensional living part. 00:12:10.03 So in order to make a living tissue using a system like this 00:12:14.08 we need to create cells within this three-dimensional light-sensitive system. 00:12:21.29 In order to do that we borrowed a chemistry 00:12:25.17 that was first developed by a scientist named Jeff Hubble, which is described here. 00:12:30.27 This is a polyethylene glycol polymer. So it's a long chain of polyethylene glycol molecules. 00:12:37.13 It's a long chain polymer with reactive end groups. 00:12:41.02 And what one does is simply mix in photo-initiator (a light-sensitive chemical) 00:12:46.29 with cells and when one does this mixing and shines light, 00:12:51.17 you get a photo-crosslinked network with cells imbedded in the polymer structure. 00:12:55.26 So this is like the cells are fruit and the polymer is the jello. 00:13:00.09 So the cells are the fruit in the jello. 00:13:02.20 And the nice thing about this is that one can then change the pattern of light 00:13:07.16 that one shines and create different structures. 00:13:10.22 So in order to get hepatocytes (the liver cells) living in this system, 00:13:15.06 as I mentioned, they're very sensitive to their microenvironment, 00:13:17.23 a graduate student in our group spent about five years discovering how to do that. 00:13:23.06 And in this slide, I'll summarize her work. 00:13:26.19 So what she showed was that if you put liver cells just in this material system, 00:13:31.07 and tracked the albumin secretion, which is one of those liver-specific functions I mentioned earlier, 00:13:36.15 they survive but they don't function like liver cells. 00:13:40.14 If instead you add those supportive cells that I showed earlier, the cells do a little bit better. 00:13:46.19 If one further adds a peptide into this inert polymer, this is an RGD peptide, 00:13:55.00 which mimics the binding site of fibronectin, an extracellular matrix molecule in the liver, 00:14:00.15 as it interacts with the integrins, the receptors on the hepatocytes, 00:14:04.14 one gets even more function. 00:14:06.23 And finally, if one adds a third cell type which exists in the liver, 00:14:10.18 the endothelial cells which are the natural neighbor of the hepatocytes and 00:14:15.14 normally live only 1 micron away from the hepatocyte. 00:14:18.14 If one adds those into the network, 00:14:20.13 now one can get a highly functional liver cell in this photo-encapsulated system. 00:14:25.14 So using this system we've created many, many different PEG-based tissues. 00:14:31.26 And this is just a smattering of the kinds of architectures we've been able to make over the years. 00:14:36.08 In this movie, what you'll see is in this latest version, 00:14:41.01 we're now able to make these microfluidically. 00:14:43.17 So this another type of microfabrication where one can make a microfluidic device out of polymers. 00:14:50.01 Here the polymer is called PDMS, poly-dimethylsiloxane. 00:14:54.14 And what you see entering into this microfluidic nozzle is a mixture of cells and the polymer suspension. 00:15:01.12 As they bud out into these droplets then they are ready to have light shone on them. 00:15:07.26 And then they can be polymerized in these microfluidic droplets. 00:15:11.19 And this an example of cells inside these microtissues. 00:15:16.19 So, we've made progress now on the fabrication of these hepatocyte-based tissues. 00:15:23.27 The next thing we wanted to do was see how they would do in vivo. 00:15:28.16 And that experiment is shown here. 00:15:32.05 So in this experiment what we've done is taken these hepatocytes, these liver cells, 00:15:37.03 and genetically modified them with a virus, a lentivirus, 00:15:41.15 that drives a gene known as the luciferase gene 00:15:46.11 when a particular promoter is active, the albumin promoter that I was mentioning earlier. 00:15:52.15 So if the hepatocytes are happy and functional, they will express luciferase. 00:15:58.21 And here what we've done is implant these tissues in the the interperitonial space, 00:16:04.14 the abdomen of the animal or in the subcutaneous space. 00:16:07.25 So we're looking at different site of implantation. 00:16:10.22 And then we look for light generation by this tissue as a marker of how it's doing over time. 00:16:16.10 You can see in these experiments these tissues survive in the interperitonial space 00:16:21.23 for up to three months by this measure. 00:16:24.26 And if we look in the blood of the animals and look for markers of the human proteins in the mice, 00:16:31.00 we see that the cells are making human albumin in the blood of the animal. 00:16:36.21 And this is a sign that the blood vessels in the mice 00:16:40.22 have been recruited to that construct and they're hooked up. 00:16:44.17 So the construct has been vascularized. 00:16:47.23 OK, so that's as far as we've gotten in terms of fabricating tissues 00:16:54.25 that we hope to implant in patients one day. 00:16:57.03 We have lots of work to do on the scale up of these tissues. 00:17:00.14 The tissues that I've mentioned to you only have about a million cells in them 00:17:04.18 and in order to get a therapeutic effect in patients we probably need a billion cells. 00:17:09.17 So we have lots of ideas now about who to scale up by three orders of magnitude. 00:17:14.23 Hopefully next time I talk to you I'll be telling you about that. 00:17:18.01 What I'd like to now mention in the last two parts of my talk 00:17:21.00 is how one could use these model systems not just for therapeutic applications 00:17:26.15 but for scientific discovery by interrogating them. 00:17:32.01 So, the application that we first became interested in 00:17:36.02 when we realized that we had human microtissues growing in the laboratory was this one. 00:17:41.13 This is the infamous drug discovery pipeline 00:17:45.24 and what you can see here is that typically it takes... 00:17:49.21 one can start with about 10000 chemicals 00:17:52.06 and over the course of about 15 years and close to a billion dollars, 00:17:56.11 one can get one FDA approved drug on the market. 00:18:00.02 What's problematic is that actually even after all of the pre-clinical studies that are done, 00:18:09.00 all of the in vitro screens and the required animal screens, 00:18:12.08 so these are typically rodent models and large animal models, 00:18:15.25 when one enters into Phase I trials, which is when the drug is first exposed to patients, 00:18:20.29 a third of the time they exhibit liver specific toxicity. 00:18:27.11 So they exhibit toxicity to the human liver 00:18:30.22 which was not predicted earlier in the development process. 00:18:34.09 So we were interested in this idea 00:18:38.13 which was whether we could bridge the gap between animal models and clinical trials 00:18:43.25 with some of these technologies we could make engineered human livers in vitro 00:18:48.27 and use hem to study this process. 00:18:51.08 So, what I'm going to tell you about now is a two-dimensional, in vitro human microliver. 00:18:56.06 And one can also do the implantable three-dimensional models that I mentioned earlier 00:19:01.14 to humanize a mouse model and again bridge the gap between mice and clinical trials. 00:19:07.06 So when one discovers new drugs, one often doesn't have very much of the compound. 00:19:14.17 So you'd like to develop a much higher-throughput way 00:19:17.20 of fabricating the tissues than the ways I mentioned in the past. 00:19:20.22 The photolithographic process that I described to you in the beginning of my talk 00:19:24.28 was one wherein we patterned coverslip by coverslip 00:19:28.04 and we created these microarchitectures that would stabilize the cells for about four to six weeks. 00:19:33.27 But that's not a practical way to screen thousands of compounds. 00:19:38.01 So one thing that our group worked on was miniaturizing that technology further. 00:19:42.29 We came up with this. This is a multi-well device. 00:19:46.02 And the idea is that this looks just like a 24-well or 96-well plate 00:19:51.01 This is sort of the workhorse of the field of biology. 00:19:54.15 And this can be fed with fluidic robots. 00:19:57.08 At the bottom of each of these wells is a micro-patterned co-culture 00:20:01.23 of the two cell types I mentioned earlier, human hepatocytes and the supportive fibroblasts. 00:20:07.06 And now they're organized in their optimal size and shape for human cells. 00:20:12.23 The way we do this is we create a soft polymer part 00:20:16.23 that has a stencil at the bottom of each well. 00:20:19.20 When you pour collagen through this stencil you get collagen spots on the underlying surface. 00:20:25.27 And now again, as before, you can seed human hepatocytes 00:20:28.29 and then surround them with those stromal neighbors. 00:20:31.20 And in this way one can reuse this part over and over again 00:20:35.06 and never have to go back to the microfabrication facility 00:20:37.22 and have a sort of high throughput format of the microliver tissues. 00:20:42.06 So if one now looks at the albumin function of these tissues, 00:20:46.08 and this is the curve I showed you earlier where the albumin function is lost very rapidly, 00:20:50.25 one can see that it's rescued and is stable for about four to six weeks. 00:20:55.27 So, the next question then is are these useful for drug screening. 00:21:02.01 So, one important aspect of liver tissue in vivo and in vitro, is the one I already mentioned 00:21:08.27 which is that animal livers and human livers differ in their drug metabolism. 00:21:13.20 There are a couple of reasons for that. 00:21:16.11 The first are a class of enzymes known as the P450 enzymes 00:21:20.13 and they have species specific differences. 00:21:22.25 Furthermore, there's a process known as induction. 00:21:26.23 Induction is the process whereby exposure to a chemical 00:21:31.02 upregulates the P450 metabolism and alters the metabolism of that drug. 00:21:37.27 So, the induction process also varies in a species-specific way. 00:21:43.02 So what we wanted to do is make micropatterned co-cultures out of rat cells and human cells 00:21:49.10 and expose them to inducers that are known to have species-specific differences 00:21:55.08 and ask whether in vitro we could predict these findings 00:21:59.00 that are normally reported at the level of the organism. 00:22:02.21 And you can see here with these two drugs, Omeprazole and beta-naptholflavone, 00:22:07.07 we were able to upregulate the P450 metabolism in the human system 00:22:12.29 and relatively less in the rat system and that recapitulates what we know about the clinical picture. 00:22:19.01 So this was encouraging for the utility for species-specific induction studies. 00:22:25.09 Furthermore, this process of induction 00:22:28.13 is actually the mechanism by which drug-drug interaction occurs. 00:22:33.01 So, many of you may have noticed when you go to the pharmacy and you get prescribed a medicine, 00:22:39.01 it will tell you sometimes not to take the medicine with another drug; 00:22:42.16 that the two drugs might interact. 00:22:44.09 And the drug-drug interaction often happens at the site of the liver. 00:22:48.11 So this is an example of Tylenol (acetaminophen) exposure to the liver. 00:22:54.27 And so what you can see here is that the acetaminophen exposure to the liver is not consequential. 00:23:00.13 These two drugs, one which is an anti-seizure medicine 00:23:04.05 and the other which is a heart medicine, are not consequential to the liver. 00:23:08.07 However, if you give acetaminophen together with the anti-seizure medicine, 00:23:12.10 or with the other one, then you see the emergence of drug-drug interaction. 00:23:16.17 And we know the mechanism of this has been well worked out, 00:23:19.07 that that's recapitulated in this system. 00:23:21.16 So this is promising for the ability to maybe study drug-drug interaction in humans 00:23:27.24 again, in vitro and in high-throughput. 00:23:30.00 And finally, one can then study the hepato-toxicity of drugs with these livers. 00:23:39.26 So, this is an experiment where one calculates the toxic concentration 00:23:44.27 at which 50% of the cells die. That's called the TC50. 00:23:50.18 And one can do this over 24 hours of exposure with a variety of clinic compounds 00:23:55.26 and this is what's typically done now for novel small molecules. 00:23:59.20 And one can rank order these chemicals 00:24:01.13 and we can see that they rank order in the way 00:24:03.16 that they should based on what we know about clinical exposure. 00:24:06.27 What we're more excited about 00:24:09.10 is the potential to do things actually differently than the way people do things now. 00:24:13.12 And that's to do chronic exposure. 00:24:15.18 We know that all of us take our medicines in low dose, multi-day format. 00:24:20.08 And if one looks at the emergence of hepatotoxicity clinically, in clinical trials, 00:24:25.13 it actually usually unfolds on the order of weeks. 00:24:28.24 So, in similar experiments, what we've done is shown that one can do 00:24:34.10 multi-day exposure and score the relative toxicity of compounds over a chronic, lifetime. 00:24:44.15 And that in fact the answers change if you do chronic exposure as compared to an acute exposure. 00:24:49.15 So we're excited about the potential to change the way people do toxicity testing with small molecules. 00:24:55.23 OK, so I've told you about building implantable livers, 00:25:01.15 about building arrays of tiny livers in vitro, 00:25:04.09 and the last thing I'd like to tell you about is how one might learn something 00:25:08.08 about pathogens that infect the normal human liver using a platform like this. 00:25:14.13 So, in fact I've already told you about one class of pathogens that infect the human liver--Hepatitis. 00:25:20.06 So there's Hepatitis A, B, C, D and many more. 00:25:24.00 In particular, we've been working on Hepatitis C for which there's 00:25:26.26 no vaccine and no good, effective drug therapy. 00:25:29.29 Another pathogen that's very interesting for which there are not good in vitro models 00:25:35.07 are the human malarias. 00:25:37.26 So, just to remind you how human malaria work 00:25:43.01 Plasmodium falciparum and Plasmodium vivax are two parasites 00:25:46.28 that are transmitted by a mosquito bite. So typically the mosquito will bite a human. 00:25:53.00 The sporozoite, the parasite, will travel through the bloodstream, infect the liver. 00:25:59.11 When it infects the liver, it will traverse several hepatocytes 00:26:03.01 and pick one in which to set up shop. 00:26:05.03 It sets up shop in that hepatocyte. It will then multiply and grow and undergo morphogenesis. 00:26:13.04 And then burst out into the bloodstream 00:26:15.25 where it can then infect the erythrocyte (the red blood cells). 00:26:19.19 And it's at this stage that one would get the clinical symptoms that one associates with fever. 00:26:24.26 What's interesting and exciting about the opportunity to get drugs to work on the liver stage 00:26:31.08 is that typically 30 sporozoites going into the liver 00:26:35.12 would be amplified to about 300,000 merozoites bursting into the blood. 00:26:40.26 So, if one could kill the parasite at the liver stage of infection and before there were any symptoms, 00:26:47.12 one could prevent the symptomotology and also this amplification in the patients. 00:26:52.11 So, the trouble with studying all of this is that the human malarias 00:26:58.06 don't infect animal models. 00:27:00.28 So for example, they don't infect mice or rats in the laboratory. 00:27:04.00 So, we've been interested in studying our human micro-livers in vitro 00:27:09.17 and seeing whether one can infect them with the human malarias 00:27:12.27 and recapitulate this liver stage of the malaria lifecycle 00:27:16.27 both for drug discovery of new anti-malarials and for vaccine applications. 00:27:23.00 So, in this movie what you see is a typical sporozoite that would be swimming through the bloodstream 00:27:27.23 and you can see what they do when they swim like this 00:27:32.08 is they undergo this gliding mechanism 00:27:35.05 and as I've mentioned, they traverse several hepatocytes on the way 00:27:38.08 before picking the one in which they set up shop. 00:27:40.27 The hepatocytes that they traverse ont he way are wounded. 00:27:46.11 So, the membranes are temporarily breached and they self seal if they survive. 00:27:52.04 So we've recently been infecting our micro-livers 00:27:56.19 those 500 micron colonies of hepatocytes that I mentioned earlier 00:28:00.11 with the human malarias. 00:28:02.03 So, here what we've done is add a fluorescent dextran, a red dye, to the media 00:28:08.23 and as the sporozoite traverses through the cells, it injures the membranes 00:28:15.03 and the cells take up the dye and if the membranes seal, then they stay lit up like this. 00:28:20.05 So, essentially, this is a molecular footprint of the sporozoite traveling 00:28:24.22 through our human micro-livers in culture. 00:28:27.21 And so, we've now done this with both Plasmodium falciparum and Plasmodium vivax 00:28:33.18 and we're very excited to get a glimpse of the elusive stage of Plasmodium vivax 00:28:39.13 that's referred to as the hypnazoite. 00:28:41.21 It's a dormant stage of the parasite that no one's ever seen before in vitro 00:28:45.29 and we're dying to see if we have it growing. 00:28:49.18 So just to summarize, I think that the tiny technologies 00:28:54.12 have been very powerful for the study tissue engineering and tissue microenvironments 00:28:59.18 and I've given you the example of the liver but I think this is broadly true because 00:29:04.16 the microarchitecture of all tissues actually have repeating units on these same length scales. 00:29:09.12 So here are just a list of technologies that are emerging in the bioengineering field 00:29:14.17 from the 100 micron length scale all the way down to the 100 nanometer length scale 00:29:18.15 that are really ripe to be borrowed in this field. 00:29:21.23 00:29:25.14 Just to summarize, then, I've told you how one can use microscale technologies 00:29:29.14 to fabricate liver tissues, to interrogate arrays of liver tissues, 00:29:33.08 and then finally to learn more about pathogens that infect the liver. 00:29:37.11 So that you for watching and I'be like to thank our group for all the hard work 00:29:42.13 and in particular, Megan Shan and Alice Chen who helped me work on this talk. 00:29:46.26
