Session 7: Tissue Engineering

00:00:00.23 Hi, my name is Sangeeta Bhatia. Today I'm going to tell you about tissue engineering.
00:00:05.12 In particular I'll be telling you about engineering tissue replacements for patients.
00:00:09.28 In this seminal paper in 1993 Bob Langer and Dave Vacanti described the concept of tissue engineering.
00:00:19.22 The idea was that one would take a degradable biomaterial, basically a plastic,
00:00:25.25 and mix cells with this degradable biomaterial, culture them together,
00:00:31.00 and create an engineered tissue.
00:00:34.05 These hybrid tissues that are part cell and part material could then be implanted
00:00:40.14 for patients with tissue failure of various kinds.
00:00:43.20 In this same paper they examined the number of procedures per year
00:00:49.20 that were done for patients that had tissue failure of some sort.
00:00:54.24 And you can see that many, many millions of procedures are done
00:00:58.14 for all kinds of medical conditions every year.
00:01:01.07 So, how has this gone?
00:01:04.03 Well, first one of the challenges.
00:01:07.06 If one looks at how tissues are organized in the human body,
00:01:11.04 we see that they're hierarchically organized.
00:01:14.09 So for example, here is an image of the liver and if you zoom in on the liver
00:01:19.14 you can see this fundamental unit of the liver which is known as the liver acinus.
00:01:23.20 This unit is about 1 millimeter in diameter and there are many millions of these unit in the liver.
00:01:30.19 If one zooms in on these further, one can see that within the acinur unit
00:01:36.21 are hepatic cords and these hepatic cords are rows of hepatocytes (liver cells)
00:01:43.05 that are organized in a way to process the blood efficiently as it's flowing through the liver.
00:01:48.25 Finally, if one zooms in all the way to the single cell level,
00:01:53.08 one can see that these hepatocytes, these liver cells, are surrounded by at least 5 other cell types
00:01:59.28 in their home, in their what we call micro-environment.
00:02:03.18 So, these are the challenges of the tissue engineer: to recreate
00:02:08.16 the microenvironment of these cells
00:02:10.24 in a way that there could be many, many of them in healthy microenvironments
00:02:16.07 in tissues that could replace the tissue function of the damaged tissue.
00:02:20.20 So, first you might ask why one would create a bioengineered tissue that's part plastic and part cells?
00:02:30.02 This is a very famous image of one of the first tissue engineered pieces of cartilage
00:02:35.21 in the shape of a human ear in the back of an immune compromised mouse.
00:02:40.19 So why would one do this?
00:02:42.25 Well, tissues engineers would typically do this only if
00:02:46.26 one needed to improve upon one of the following existing medical therapies:
00:02:51.14 if no simple medical device could improve the tissue function.
00:02:57.17 For example, an implantable hip is a piece of metal or a piece of plastic
00:03:02.10 and if that could achieve the tissue function that you were interested in,
00:03:06.08 you wouldn't create a hybrid device.
00:03:08.07 A drug: if one could administer a drug to patient, again, one wouldn't need a hybrid device.
00:03:14.20 A cell that was minimally manipulated: this is the term that
00:03:20.27 has been invented by the United State Federal Drug Administration
00:03:24.05 and it refers to cells that have come out of the body and are re-infused with very minimal manipulation.
00:03:30.14 So if one could minimally manipulate a cell,
00:03:32.28 one wouldn't need to engineer a tissue to improve the tissue function.
00:03:36.27 A surgical reconstruction: if one could simply borrow another part of the patients body
00:03:44.02 for replacement of the tissue that's damaged, surgically, again,
00:03:48.19 one wouldn't need a complex functional replacement.
00:03:52.14 And finally, a transplant: if one could use a cadaveric or donor organ,
00:03:57.09 a whole organ, and replace the tissue function of interest
00:04:00.23 one wouldn't need a bioengineered tissue.
00:04:02.22 But importantly, these criteria are often still not met
00:04:07.17 and one does need to, in fact, turn to tissue engineering.
00:04:10.02 So, there's actually a spectrum of tissues that can be engineered
00:04:15.02 and all of these fall in that category.
00:04:18.19 So engineered tissues include biomaterials and cells in various combinations.
00:04:24.16 And on the first slide I described to you biomaterials that housed cells.
00:04:29.14 So these are plastic, typically, and cells together in a hybrid form.
00:04:34.24 But there are also various incarnations of these engineered tissues.
00:04:40.15 So, for example, there are complex biomaterials that can be implanted and recruit cells.
00:04:46.11 So, upon implantation they don't carry any cells but they can recruit host cells
00:04:51.26 and become hybrid in situ.
00:04:54.11 These are also in the category of what one would call engineered tissues.
00:04:59.04 And finally there are materials that are completely derived from cells.
00:05:04.13 So we know that cells themselves can make scaffolding which is called extracellular matrix.
00:05:09.15 And if one cultures cells in the laboratory, and creates structures that are part cell and part extracellular matrix,
00:05:18.00 those also can count as engineered tissues but they have no synthetic materials within them.
00:05:23.22 So all three of these categories of engineered tissues have been
00:05:29.12 widely experimented on with varying degrees of success
00:05:32.01 and what I'd like to do now is to show you some examples of each of these.
00:05:35.20 So we are going to start with acellular biomaterials.
00:05:38.28 This is an acellular biomaterial which is a very effective engineered tissue.
00:05:46.13 It's called small intestine submucosa.
00:05:50.12 It has no cells whatsoever.
00:05:52.26 So if we look in this bottom image here, what we see if a cross section
00:05:58.04 of the intestine, in this case of a pig.
00:06:01.00 So we see these vila structures of the intestinal lining
00:06:05.28 and that's how the intestine absorbs nutrients,
00:06:08.19 and underneath that structure are these muscular layers.
00:06:12.23 And lying between these two layers is what's called the submucosa.
00:06:17.01 The submucosa looks like this on an electron micrograph.
00:06:22.26 This is a very strong extracellular matrix structure
00:06:26.20 made up primarily of collagen and mixed with lots of other extracellular matrix types of molecules.
00:06:33.17 So what was done originally by Steve Badylak and coworkers and many other groups since,
00:06:38.27 is that one can take porcine small intestine and
00:06:42.25 strip away the cells with various detergent type procedures,
00:06:46.20 and come up with large scaffolds that are acellular, that are very strong mechanically.
00:06:52.25 It turns out that when one implants these at sites where regeneration is required,
00:06:58.02 so for example in tendon and ligament repair,
00:07:01.10 these recruit host cells by inducing proliferation and differentiation of those cells
00:07:07.22 and promote wound healing very effectively.
00:07:10.11 These types of materials have been in millions and millions of patients.
00:07:14.06 Another more complex example is a hybrid example.
00:07:20.13 So I told you before that once can combine biomaterials and cells
00:07:25.11 to make hybrid engineered tissues and this is one of the best successful examples of having done this.
00:07:31.17 So here we see engineered skin.
00:07:34.20 So, on this side we see a cross-sectional micrograph of what human skin actually looks like
00:07:43.02 and we see that there's a dermal layer here and an epidermal layer here.
00:07:47.20 The dermis is composed of connective tissue cells like dermal fibroblasts
00:07:52.20 and the epidermal layer is composed of epithelial cells
00:07:56.18 which undergo what we call stratification as they mature and form this multilayer surface.
00:08:02.12 And way out here is where the skin interfaces with air.
00:08:06.14 So this structure is what's damaged in the case of burns, for example.
00:08:11.07 And tissue engineers sought to create tissue engineered constructs to replace this skin.
00:08:17.20 So, on this side what you see is an engineered version of skin
00:08:21.17 and in this material what's been done is a biomaterial, a collagen gel, an extracellular matrix gel,
00:08:29.25 that has the consistency sort of like jello, has been mixed with dermal fibroblasts.
00:08:35.12 These dermal fibroblasts remodel the collagen and they form very tight mess-work.
00:08:41.19 On top of that mess-work, one can seed those epithelial cells, the epidermal cells
00:08:47.28 and they will stratify and form a multi-layer structure on top of that dermal layer.
00:08:54.25 And this engineered skin can be implanted in the setting of burn or other injured skin
00:09:02.02 and promote wound healing and to great benefit.
00:09:06.11 Another example of a hybrid tissue engineered system is this one.
00:09:11.25 This one was developed originally by Toy Atala and coworkers
00:09:15.00 and in this system instead of using a natural scaffold, like collagen,
00:09:19.19 we're using a synthetic scaffold called polylactic co-glycolic acid.
00:09:25.15 So this is a polymer which I'll describe later in a moment,
00:09:29.03 but for now you can think of it like a plastic
00:09:31.09 and this plastic was cast in the shape of a balloon structure that you see here.
00:09:36.11 On the inside of this balloon, we see that lining cells from the bladder were seeded
00:09:45.03 and on the outside of the balloon muscular cells that are important for bladder function,
00:09:50.04 for the contraction of bladder, were seeded.
00:09:53.03 Together these were cultured in vitro and then implanted in animal experiments and now in humans.
00:09:59.07 And these hybrid tissues that are composed of two cells types and a polymer scaffold
00:10:04.13 together promote better bladder function, better compliance in this setting.
00:10:10.01 Interestingly, in this classic paper, where they first did the experiments in dogs,
00:10:15.23 these hybrid bladders were also re-enervated.
00:10:19.28 So they promoted nerve regeneration in the host
00:10:23.29 and these dogs regained voluntary bladder function.
00:10:27.00 This was unexpected and one of those exciting areas of research that is still unexplained.
00:10:33.16 So I've told you about acellular grafts and hybrid grafts.
00:10:38.25 Now let's talk about completely cellular derived grafts
00:10:42.11 that have no synthetic or natural biomaterials in them.
00:10:45.21 So here's an example of a biomaterial-free engineered tissue.
00:10:50.13 This is blood vessel that's made by Nicola L'Heureux and colleagues
00:10:55.03 and in this system they do what they call sheet-based tissue engineering.
00:10:59.15 The vision is that one would take a sample from a patient, a little biopsy sample,
00:11:04.23 from which one could harvest endothelial cells which line blood vessels
00:11:09.05 and fibroblasts, those same dermal cells that I described earlier, and culture them in flasks.
00:11:15.16 And if one cultures them in flasks...if one cultures the fibroblasts in flasks,
00:11:20.02 one can see that the fibroblasts deposit their own extracellular matrix.
00:11:25.05 They deposit the same types of collagen that you saw in the small intestinal submucosa earlier,
00:11:31.08 but they deposit it on the bottom layer of the flask.
00:11:34.01 And one can manipulate the culture conditions to promote
00:11:37.00 lots and lots of extracellular matrix deposition.
00:11:40.11 Then one can detach the system and it creates a sheet
00:11:44.14 that can be manipulated and rolled on a mandril.
00:11:47.15 This mandril then, now is a living cinnamon roll of cells and extracellular matrix
00:11:56.13 and it's cultured in this living format so that
00:11:59.19 the layers of the cells and the extracellular matrix will fuse into a very strong wall.
00:12:04.28 One can then seed the inside of these tubes
00:12:08.19 with the endothelial cells that were originally obtained
00:12:11.27 and now you have a construct that has endothelial cells on the inside
00:12:15.26 and this nice, strong surrounding structure made of fibroblasts and their extracellular matrix.
00:12:23.23 And these can be implanted in patients.
00:12:27.01 And these are what these tissue engineered blood vessels look like.
00:12:31.02 These blood vessels have no scaffolding material that was introduced from the outside.
00:12:37.24
00:12:40.15 OK, so these are the different flavors of engineered tissues that exist.
00:12:44.12 If one were thinking about designing a bio-engineered tissue to replace a particular organ function,
00:12:50.17 one important thing to note is that, as tissue engineers,
00:12:53.23 we can frequently...we are unable to replace all of the functions of interest.
00:13:00.03 So, for example, if one thinks about the functions of skin that are are being replaced,
00:13:04.03 it's simply the barrier function in the example I showed you.
00:13:07.16 Those pieces of skin have no hair follicles, no sweat glands, no immune cells,
00:13:13.19 and that's a deliberate choice on the part of the tissue engineer
00:13:17.13 to go after the function that's most critical for life.
00:13:20.09 Similarly, if one thinks about pancreatic tissue engineering,
00:13:24.10 one often sees that folks are interested in the implantation of beta cells
00:13:28.24 that will produce insulin in a glucose responsive way for patients with diabetes.
00:13:33.10 There are many other functions of the pancreas
00:13:36.02 that are often excluded there from tissue engineering
00:13:38.28 and again this is a deliberate decision on the part of the tissue engineer.
00:13:42.21 So it's important to keep in mind that one can't do everything, one has to have priorities.
00:13:48.00 OK, well then, once you've made your decision about what functions you're most interested in,
00:13:54.02 what would one then to go further and actually engineer a tissue from scratch.
00:13:58.22 So the next thing to do would be picking some raw ingredients.
00:14:02.22 The raw ingredients that one has to work with are cells.
00:14:06.09 There's actually a variety of cell sources that one can consider.
00:14:10.06 So there are somatic cells and stem cells.
00:14:13.24 Somatic cells are cells of the body that are not germ cells, that is, not eggs or sperm.
00:14:21.08 So they are part of the adult organism.
00:14:23.18 And they can come from the person that you plan to treat. Those are called autologous.
00:14:29.22 They could come from another person. That would be allogeneic, of the same species.
00:14:36.10 Or they could come from an organism that is another species.
00:14:40.14
00:14:40.14 and those would be called xenogeneic.
00:14:42.11 And in tissue engineering one actually sees examples of all of these types of cell sourcing.
00:14:47.18 So, for example, the skin that I showed you earlier was allogeneic.
00:14:52.05 The dermal fibroblasts and the epidermal cells
00:14:55.12 were from a non-self human to create those skin tissues.
00:15:00.21 Another source of cells, which is becoming more and more exciting, is stem cells.
00:15:06.02 One can think about using both adult stem cells or pluripotent stem cells
00:15:10.13 that have been derived from various methods that I'll describe in a moment.
00:15:13.20 So one certainly needs raw ingredients of cells.
00:15:16.29 The other thing one needs is biomaterials.
00:15:20.07 And finally one needs to cultures these together in a way
00:15:23.25 that the cells have sufficient nutrients to remodel those biomaterials and create a tissue.
00:15:30.00 Once one has the right raw ingredients, we have to think about fabrication.
00:15:35.13 How to assemble these structures, how to process these, how to create culture environments
00:15:41.19 how to preserve them so they can get to the clinic
00:15:44.28 and finally how they can then integrate with the host at that site.
00:15:49.03 So in the next few slides I'm going to talk to you about some of the issues
00:15:52.21 that are related to both the raw ingredients and the fabrication of engineered tissues.
00:15:56.27 First, let's talk about cells.
00:15:59.07 So, if one is thinking about somatic cells, culturing primary cells,
00:16:05.12 they can be derived in various ways;
00:16:07.24 from an adult organism, from an embryo, or from an egg.
00:16:11.28 Regardless of the source, what's typically done is these are mechanically dissected
00:16:16.14 in the laboratory and then they can be further processed in one of three ways.
00:16:20.14 If they are just slightly processed and they retain their three-dimensional structure
00:16:27.20 from the in vivo environment, we call that organ culture.
00:16:31.18 These organ cultures are often done at air-water interfaces
00:16:35.04 that mimic the environment of particular tissues in vivo.
00:16:39.17 For example, the skin that I showed you earlier is actually cultured at the air-liquid interface,
00:16:45.03 just like the skin on your body is at an air-liquid interface.
00:16:48.05 One could further dissect these tissues, finely chop them,
00:16:51.28 and culture them in a flask and allow cells to grow out of these structures.
00:16:57.19 That's called explant culture.
00:16:59.11 And that's actually the way that the bladder cells were first derived that I showed you earlier.
00:17:03.26 Finally, and more commonly now, these tissues can be enzymatically digested
00:17:09.22 to create a single cell suspension, a suspension of cells in a liquid.
00:17:14.18 And those can be cultured in flasks and if they are a proliferative cell, if they can grow,
00:17:19.20 once they grow to what we call confluence, once they fill up that flask,
00:17:24.13 one can remove them, put them in a new flask and passage them.
00:17:28.16 That's called passaging--grow them, expand them, over and over again.
00:17:32.00 And that's how you would create the cells for your engineered tissues.
00:17:35.26 One limitation of using somatic cells was first described by a scientist named Hayflick in 1965.
00:17:45.26 And that's known as the Hayflick limit.
00:17:48.02 So, what he observed in his experiments was the following.
00:17:52.04 If one takes fibroblasts, those same fibroblasts that I was describing to you earlier,
00:17:56.11 from patients of different ages (so, fetal fibroblasts all the way up into your 80s)
00:18:03.24 and cultures them and passages them, in the flasks as I showed you and counts doubling time,
00:18:10.04 one sees that the number of cell doublings that one can achieve is less as the patient source is older.
00:18:19.21 So as patients age, their cells are able to double a fewer number of times.
00:18:25.23 And what that implies is that cells have a finite lifetime in culture,
00:18:31.05 and, in fact, in vivo.
00:18:33.00 And we know now that the molecular basis of this is that
00:18:36.00 the ends of chromosomes have something called telomeres, the so called molecular clock,
00:18:40.29 and every time the cell divides, those telomeres get shorter until the cell reach senescence.
00:18:46.18 So, this observation has led to much of the excitement in the field of tissue engineering
00:18:53.03 about stem cells because in fact stem cells don't have this property.
00:18:57.11 They are what we call immortal.
00:18:59.06 They elongate their telomeres every time they divide
00:19:02.21 by expressing an enzyme known as telomerase.
00:19:05.06 They actually are not susceptible to the Hayflick limit
00:19:08.26 and they can grow in perpetuity in culture.
00:19:12.06 So as a cell source they are very exciting resource.
00:19:15.18 Furthermore, stem cells have been identified that are pluripotent.
00:19:23.27 So here's a diagram describing a number of ways that one can make
00:19:27.29 stem cells that can differentiate, that can be encouraged
00:19:33.23 to grow into the various cell types of interest for any tissue of interest.
00:19:38.15 The idea here is that you could have a single cell source.
00:19:41.20 It could be immortal. It could be expanded.
00:19:44.14 And then, it could be used to engineer any tissue of interest.
00:19:47.10 A further idea that's been very exciting in the field
00:19:50.19 is the idea that one could make individualized pluripotent stem cells.
00:19:55.18 This is the idea of patient specific, or personalized medicine.
00:19:59.14 So let me walk you through how this idea goes.
00:20:02.05 There are a variety of ways to make these cells.
00:20:05.03 So, in this system what we see is that you take, again, a fibroblast, a skin fibroblast
00:20:09.19 just like the ones I showed you earlier
00:20:11.17 and they have two copies of DNA. So these are somatic cells.
00:20:18.00 One can take out the nucleus of that cell and put it in an enucleated egg.
00:20:23.21 Those cells then will start to grow into a blastocyst structure shown here.
00:20:29.29 And the blastocyst, which is the early embryo, has a pocket of cells known as the inner cell mass.
00:20:36.14 If one cultures cells from the inner cell mass,
00:20:39.22 one can create so called pluripotent stem cells or embryonic stem cells.
00:20:44.25 Pluripotent stem cells, the embryonic stem cells, will have the same genetic identity
00:20:49.28 as the original patient.
00:20:51.15 Another way to create pluripotent stem cells from that patient
00:20:55.15 would be to take the same somatic cell that we saw earlier
00:20:58.15 and fuse it with an embryonic stem cell.
00:21:01.13 And that process of fusion also generates patient specific pluripotent stem cells.
00:21:07.12 Finally, recently, in a very exciting set of discoveries,
00:21:11.03 Yamanaka and coworkers have defined a way to reprogram somatic cells
00:21:16.26 without going through an ES cell intermediate or an egg intermediate.
00:21:21.29 What they did, in their classic experiments, was show that
00:21:25.18 a certain number of molecules known as transcription factors
00:21:29.06 could be expressed in a somatic cell and that transduced cell would be reprogrammed
00:21:36.09 into a pluripotent stem cell.
00:21:38.02 So, this now offers the potential to create differentiated cells
00:21:43.19 that are genetically matched to patients.
00:21:46.27 It offers the further possibility to make pluripotent stem cells
00:21:50.27 from patients to fix a genetic defect in the laboratory
00:21:55.14 and then to create a tissue out of those genetically altered cells
00:22:00.07 to give back to the patient.
00:22:01.29 So, given the Hayflick limit,
00:22:05.20 the promise of stem cells has been very exciting in the field of tissue engineering.
00:22:10.21 OK, so we've talked about cellular ingredients. Now let's turn to biomaterials.
00:22:17.00 So, one classical tenant of biomaterials in the tissue engineering field
00:22:23.13 is that one wants to match the degradation of the biomaterial
00:22:29.17 with the synthesis of extracellular matrix scaffolding in the host.
00:22:34.12 So, one would not want the biomaterial to degrade too quickly.
00:22:39.01 Otherwise, it wouldn't perform its scaffolding function.
00:22:42.15 On the other hand, one typically would not want the biomaterial to be persistent indefinitely.
00:22:48.21 Because one might incur what's called to foreign body response.
00:22:52.02 So, where does this concept come from?
00:22:54.26 This concept was originally defined by Yannas and coworkers
00:22:58.27 in these classic experiments that were done in the skin.
00:23:03.01 In these experiments, what they did was they made tissue engineered skin
00:23:07.27 that had a variety of degradation rates.
00:23:10.25 And what they looked at was the wound healing time of a full thickness wound,
00:23:16.07 a cut through both the epidermal and dermal layers of the skin in an animal model.
00:23:22.09 And what they found was that if one didn't put any scaffolding in that wound,
00:23:29.19 it would contract and close very quickly and form scar.
00:23:35.02 That was what we were hoping to prevent with the use of the tissue engineered scaffold.
00:23:40.18 If one then implanted tissue engineered scaffold that has a very rapid degradation time,
00:23:46.09 those wound proceed to contract, just as in the control conditions,
00:23:53.02 as if there was no scaffold in the system at all.
00:23:55.04 However, if one further cross-links the material,
00:23:58.24 one can see that one can delay that contraction time as seen here.
00:24:05.18 And then after that, if one makes the material even less degradable,
00:24:09.11 there's really no benefit to that and the wound actually just persists the same amount of time.
00:24:13.25 So these experiments led to the idea that there's an optimal degradation rate
00:24:19.07 for tissue engineered scaffolds
00:24:22.06 and one can actually see that in the SYS experiments, in this bottom graph here,
00:24:27.13 they conceptualized this as follows.
00:24:29.21 So, the degradation time of the material is decreasing
00:24:33.06 and the production of extracellular matrix is increasing.
00:24:38.26 And the SYS actually bridges the gap in those two time scales.
00:24:43.20 So, it's important to match the degradation with the host synthesis.
00:24:47.25 So that's a material property that's important. What are the materials actually?
00:24:53.18 So there are a variety of synthetic scaffolds that one has to choose from
00:24:58.06 and if one looks in the community we see new chemistries coming out on a daily basis.
00:25:03.17 I've just listed for you here some of the classical chemistries
00:25:07.22 that are used for tissue engineering.
00:25:10.07 Many of them are what we call polymers. So, for example, PLGA is polylactic co-glycolic acid.
00:25:17.26 These are macromolecules that have repeating structures.
00:25:22.03 In this case, a lactic acid and glycolic acid.
00:25:25.16 And when they're implanted and they degrade in the body by hydrolysis,
00:25:30.13 by the interaction with water, they degrade into units of lactic acid and glycolic acid,
00:25:36.02 which already occur in your body.
00:25:38.10 So these are actually originally what sutures were made of, biodegradeable stitches.
00:25:45.01 So there are many, many novel, polymeric materials that have been developed.
00:25:50.09 Another very popular one which has the property of being a hydrogel...
00:25:54.14 Again, a hydrogel is a material that absorbs a great amount of water and
00:26:02.02 has a sort of intuitive feel of jello, gelatin.
00:26:04.27 Here we have a polyethylene glycol which is a very popular biomaterial these days
00:26:11.15 and you can see that it looks a lot like a soft contact lens.
00:26:14.05 What's interesting about some of these hydrogel materials
00:26:17.08 is that rather than being pieces of plastic, where cells can be attached to the plastic and they can remodel the plastic,
00:26:26.07 these are materials where cells are imbedded within the plastic.
00:26:29.28 And this material in particular is very interesting because it can be crosslinked with light.
00:26:35.04 So the way the experiment works is
00:26:37.10 you take polyethylene glycol macromers (so long chains of polyethylene glycol).
00:26:43.19 You mix in cells and a chemical that's sensitive to light
00:26:47.22 and when you shine light you get a network of polymer
00:26:51.17 that looks like this contact lens structure with cells imbedded within it.
00:26:55.09 So these are examples of synthetic scaffolds
00:26:58.27 and one can tune these to have different material properties.
00:27:02.08 They can be inert, like polyethylene glycol.
00:27:05.05 You can modify their chemistry to interact specifically with cells.
00:27:09.17 For example, one can put in peptides that can bind to cellular receptors like integrins.
00:27:15.08 They can have different mechanical properties
00:27:17.08 which we know is important for some cells types.
00:27:19.19 And then they can have different mechanisms of degradation.
00:27:22.04 So they don't all degrade just by interactions with water.
00:27:26.00 Some of them can degrade in very specific ways;
00:27:28.07 for example, by interacting with enzymes that the cells are making.
00:27:31.18 Another example of a kind of scaffold that one would use are the so called natural scaffolds.
00:27:38.11 So I already mentioned collagen earlier.
00:27:40.26 Collagen is one of the most popular natural scaffolds.
00:27:43.29 Hyaluronic acid is another natural scaffold.
00:27:46.13 The SYS material that I showed you is a more complex natural scaffold
00:27:51.00 because it contains all the extracellular matrix molecules that were in the submucosa
00:27:55.16 in addition to the growth factors
00:27:59.03 and other bioactive molecules that would be in that natural material.
00:28:03.25 An example of such complex natural scaffolds that is becoming quite popular these days
00:28:11.23 is an idea that was first described by Doris Taylor's group a couple of years ago
00:28:15.27 and these are whole organs that have been de-cellularized
00:28:19.23 in much the same way that the small intestinal submucosa was de-cellularized.
00:28:23.26 So here what you're looking at is a picture of a heart that's being perfused with the detergent.
00:28:30.12 And you can literally see the cells melting away
00:28:33.13 so that you have what they call a ghost heart at the end.
00:28:37.01 This preserves all of the microarchitecture of the organ that I described to you earlier.
00:28:43.10 You can see here under the microscope that it has a fibrolar collagen organization
00:28:49.14 and what she did in these experiments was re-cellularized this de-cellularized heart
00:28:55.29 using that as a scaffold for tissue engineering.
00:28:59.23 So various groups have now gone on to do this in other organ systems including the liver.
00:29:06.07 And what's exciting about this potentially is one can think about reusing organs.
00:29:12.03 Instead of having to just transplant a living organ from a donor into a patient,
00:29:17.11 one can think about using cadaveric organs as complex scaffolding
00:29:22.03 for cells from some of the cell sources that I described earlier.
00:29:26.00 OK, so I've described the raw ingredients; cells and biomaterials.
00:29:31.01 I won't say much about nutrients except for to say that
00:29:33.24 there is a whole science about how to feed cells in culture.
00:29:39.10 And that's pretty well understood at this point.
00:29:42.19 The next thing I'd like to just touch on briefly is how to fabricate these organs;
00:29:47.15 how to assemble them, how to process them before implantation and
00:29:51.17 importantly, how to preserve them so that one can get them to the clinic at the time of need.
00:29:57.11 OK, so I mentioned earlier that tissues are organized hierarchically.
00:30:03.07 One can see here in this diagram that when one starts at the level of the liver,
00:30:07.23 we have this multi-scale organization where the liver acini are 1 millimeter in dimension,
00:30:14.17 the liver cords here are 500 microns long,
00:30:17.22 and the cells here are about 20 microns in diameter.
00:30:21.07 So it's this hierarchy which tissues engineers have to try and grapple with
00:30:26.07 in the sense that cells respond, actually, only locally to their microenvironment.
00:30:31.07 So even though you're trying to build a large structure,
00:30:34.12 if you care about cells performing the functions of interest
00:30:38.22 and here are some functions that one might care about,
00:30:41.04 proliferation, differentiation, apoptosis, migration, or metabolism,
00:30:46.23 If these are the functions of interest for having a healthy tissue,
00:30:49.29 that we know that the cells are actually responding to their local microenvironment
00:30:54.23 to these kinds of cues; soluble factors, interactions with other cells,
00:30:59.17 extracellular matrix interactions, physical forces, and cell shape.
00:31:04.24 So the challenge for the tissue engineer is to maintain the cellular microenvironment
00:31:09.25 that will impact the cell behavior on the sort of 10 to 100 micron length scale
00:31:15.12 but simultaneously be scaling up the structure
00:31:18.06 to be a large enough body of cells to implant to achieve a therapeutic outcome.
00:31:23.17 So a good example of how cells have local microenvironmental interactions
00:31:30.12 that one calls 3-dimensional in the field
00:31:33.09 is cartilage.
00:31:34.09 We see this is cross section of articular cartilage
00:31:37.17 and if one looks at cartilage one sees that the chondrocytes
00:31:40.23 that make the extracellular matrix that makes up cartilage,
00:31:43.26 have different organizational structures.
00:31:46.09 So here we can see that they are in these columnar structures.
00:31:50.06 Whereas, in the superficial layer of cartilage that sits right next to the joint space
00:31:54.25 they actually are flat, elongated and in clusters.
00:31:58.24 So if you were to look down at the cartilage, you would see that they were in clusters.
00:32:02.18 So, it's been known for quite some time now that chondrocytes in culture,
00:32:09.07 when one puts them on plastic of any type for tissue engineering purposes
00:32:12.28 they will actually spread out and lose many of these functions
00:32:17.14 They will start proliferating and stop producing the extracellular matrix
00:32:20.25 that one is trying to recapitulate in a tissue engineered construct.
00:32:24.23 And so, in fact, if one embeds them in a 3-dimensional microenvrionment,
00:32:28.24 where they have this round shape and more favorable mechanical properties,
00:32:32.23 one stimulates them as they are stimulated mechanically in vivo,
00:32:36.18 one can actually improve the quality of the extracellular matrix that they produce
00:32:41.23 and therefore the mechanical strength of the graft.
00:32:44.26 So this is just one example of a tissue where
00:32:47.11 the 3-dimensional organization of the microenvironment,
00:32:50.14 in addition to the chemical composition of the microenvironment is very important.
00:32:54.19 And one will hear a lot about 3D tissue engineering in years to come.
00:32:58.09 One can then think about how to assemble these microenvironments
00:33:03.18 where cells have very precise, local chemical and mechanical cues into larger structures.
00:33:09.12 There's a set of technologies emerging as 3D fabrication technologies
00:33:14.05 that have come out of the rapid prototyping field
00:33:17.23 that are starting to be borrowed in tissue engineering.
00:33:20.10 Some people call this organ printing.
00:33:22.12 This is an example of a couple of parts that were made in a layer by layer fashion
00:33:27.17 known as stereolithography
00:33:29.28 and stereolithography is one example of one of these rapid prototyping technologies.
00:33:34.23 So, in this technology, what's done is
00:33:37.28 a 3-dimensional drawing is drawn on the computer and sent to this robot.
00:33:43.14 And the robot has a movable stage that sits in a vat of light sensitive material.
00:33:50.16 And in every layer of the material, light is shone in a pattern.
00:33:55.21 It polymerizes and the stage drops and the next layer is built and so on and so forth.
00:34:00.28 And using this, one can create complex structures; some that are actually biomaterial structures.
00:34:06.06 Some folks envision that one could make personalized tissue engineered parts
00:34:10.26 using a technology like this because one can envision
00:34:13.09 going from a 3-dimensional medical imaging data like CAT scan data, to tissue engineered parts.
00:34:19.13
00:34:24.02 Another thing that's required is to culture these tissues before they're implanted
00:34:28.18 and these are a couple of examples of the kinds of bioreactors that are designed and used in the field.
00:34:34.06 At the top what you see is a quite well known bioreactor developed by NASA.
00:34:39.06 This is a circulating bioreactor and it never allows the cells to touch the walls of the bioreactor
00:34:47.02 so they're forced to aggregate in 3-dimensional clusters.
00:34:49.29 And this has been shown to promote tissue formation in both cartilage and heart cells.
00:34:55.01 Furthermore, techniques from the computer chip industry
00:35:00.13 are now being borrowed to create micro-reactors.
00:35:03.22 So here what you're looking at is a coverslip and some inlet ports from a microfluidic device.
00:35:09.18 And if one looks in the microscope, inside that coverslip,
00:35:12.19 one can see these little tissue units that have microstructure,
00:35:16.24 that have been fabricated in the computer lab at 100 micron length scale.
00:35:22.06 So they have 3-dimensional microenvironments
00:35:25.01 that have been specified in a scalable fashion for building larger pieces of tissue.
00:35:30.17 So, one can assemble tissues, one can culture these to mature them in the laboratory,
00:35:37.08 and then finally one needs to think about how to preserve them the deliver them to the patients.
00:35:41.16 So, there's a whole set of technologies that have grown up in the field
00:35:47.06 about cryopreservation. I should say that not all tissues are frozen.
00:35:51.18 Many are actually just refrigerated to 4 degrees C
00:35:55.06 and in fact, the skin tissue engineered product that I showed you earlier
00:35:58.02 is sent that way to the clinic.
00:36:00.13 However, in the field of cryopreservation,
00:36:03.15 which most of us agree will be important to really make this technology have it's highest impact.
00:36:11.03 There has been a lot of research on how best to make cells, especially in 3-dimensional structures,
00:36:17.10 survive the insults of cryopreservation.
00:36:19.18 So, the insults of cryopreservation have now been categorized
00:36:23.21 into two classes of insults and those are depicted here.
00:36:27.03 So I need to walk you through what a cell feels as it gets frozen
00:36:31.14 so you can see what the two classes of insults are.
00:36:34.25 So if you have a cell here and it's been cooled just under the seeding point of ice,
00:36:40.03 and one artificially seeds external ice in the environment, two things can happen.
00:36:45.15 If one slowly cools the solution further, you can see that water has time
00:36:52.03 to cross the cell membrane and leave the cell and form ice in the extracellular space.
00:36:57.25 And this causes what people call dehydration injury to that cell.
00:37:03.07 You can see that its shape has shrunken. There is a concentration of the intracellular solutes.
00:37:09.19 And those cells when they are thawed again, exhibit damage from this dehydration process.
00:37:15.13 If instead one takes this solution and rapidly cools it,
00:37:20.03 one actually finds that you can have intracellular ice crystal formation.
00:37:25.02 The formation of intracellular ice crystals, in and of itself, can cause damage to cells.
00:37:30.10 So the process by which cells are susceptible to slow cooling or fast cooling
00:37:35.11 turns out to be cell type dependent and dependent on various additives in the system
00:37:40.21 as well as the 3-dimensional architecture of the tissue.
00:37:44.01 And so this is being worked out now for many, many cell types in the field.
00:37:47.18 OK, so to conclude, I've told you that engineered tissue replacements can
00:37:52.15 be made from combinations of cells and biomaterials to replace a subset of tissue functions.
00:37:58.17 I've told you that the cells can be derived from somatic cells or stem cells.
00:38:03.06 I've told you that the biomaterials in these tissue replacements can be natural or synthetic.
00:38:09.02 I've also told you that tissue structure in the body is hierarchical
00:38:13.18 and therefore the tissue engineer has the challenge of maintaining the cellular microenvironment
00:38:18.27 in a way that can be scaled up to build a tissue that's large enough for therapeutic impact.
00:38:25.03 And finally, I hope you've noticed that the convergence of
00:38:28.20 cell biology, medicine and engineering is really advancing this field.
00:38:33.11 In part two of my seminar, I"ll be telling you about our work on liver tissue engineering
00:38:39.05 both in the progress we're making towards therapeutic goals like the ones I've described,
00:38:44.18 as well as progress that we're making in using these engineered liver tissues
00:38:48.20 to advance scientific discovery.
00:38:51.02

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