Session 7: 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