The presently disclosed subject matter relates to techniques for culturing tissue, including by using a microfabricated 3D cell culture system.
The architecture of tissues can directly impact cellular and multicellular function. Within the in vivo microenvironment, cells adhere to the surrounding extracellular matrix (ECM), which has compositional, mechanical and topographic properties that are tissue specific. Cells can also be exposed to complex spatial distributions of soluble factors and can engage in physical interactions with neighboring cells. When cells are removed from this setting and cultured in vitro, typically on tissue culture polystyrene, they can rapidly lose their native phenotype. Cellular functions such as proliferation, migration and differentiation can be drastically altered in vitro. Certain in vitro studies have offered limited insight into the mechanisms governing biological processes.
One alternative to culturing cells in vitro is to observe them in vivo. When used in conjunction with genetic knockout methods and intravital microscopy methods, animal models such as zebrafish, drosophila and transgenic mice can be very informative, particularly in studies of organism development. However, in vivo assays generally can be more expensive, lower throughput, and harder to manipulate than in vitro assays. As such, they are often not employed as extensively in more commercial-driven applications such as drug discovery.
In addition to conventional in vitro cell culture and animal models, in vitro organotypic cultures can recapitulate some of the elements of in vivo tissue architecture. For example, breast cancer can be modeled in vitro by culturing a mass of epithelial cells within Matrigel, a mouse-derived mixture of ECM proteins. The cells can subsequently organize into hollow spherical structures known as mammary acini. As another example, the granulation tissue that forms during wound healing can be modeled as a polymerized matrix of collagen I in which fibroblasts can be embedded. The fibroblasts can subsequently contract the collagen matrix, yielding a dermal equivalent. One limitation of these organotypic models is that they are essentially self-assembled processes within a microenvironment whose composition is still poorly defined. To partially address some of these limitations, bioengineering approaches can be used, particularly in a 2D environment. For example, synthetic ECMs with tunable stiffness, adhesiveness and bioactivity can be engineered. Microfabrication-based approaches can be used to specify adhesive topography, modulate substrate stiffness, generate gradients soluble factor, and constrain cell-cell interactions.
One aspect of these culture models is that nearly all tissues have fluid-filled tubes that are lined with cells and surrounded by interstitial extracellular matrix containing a mixture of cells. This tissue architecture is true of glandular tissues such as pancreas (epithelialized tubes are filled with digestive enzymes), breast (lactiferous ducts carry milk), liver (bile ducts carry bile), brain (ventricles contain cerebrospinal fluid), intestines (transports food) and kidney (renal tubules carry waste filtrate). It is also true of all tissues fed by the circulatory system, as blood passes through an endothelial cell-lined vascular tree that permeates nearly all tissues in the body of all species with a closed circulatory system (including humans).
Accordingly, there remains a desire for improved in vitro environments for 3D cell culture.
The presently disclosed subject matter relates to devices for culturing tissue, particularly to systems and methods of a microfabricated 3D cell culture system including fluidic and matrix compartments.
According to some embodiments, a device for 3D cell culture includes a substrate having at least one interior chamber, at least one opening providing access to the at least one chamber for introduction of an extracellular matrix into the chamber, and at least one channel through the extracellular matrix. In some embodiments, the device for 3D cell culture can include at least one reservoir in fluid communication with the at least one channel.
In one embodiment, the device for 3D cell culture can include a plurality of channels through the extracellular matrix. Each channel can be connected, in fluid communication, to at least one reservoir for introduction of liquids, including but not limited to culture media, biological fluids (e.g., blood or its components, urine, milk, mucus, gastrointestinal fluids, or bile), and environmental fluids (e.g., seawater or lake water) to the channel. In one embodiment, each channel can be connected, in fluid communication, to two reservoirs.
In another embodiment, a method of fabricating a device for 3D cell culture includes providing a top master mold for fabricating a top layer. The mold can have raised portions defining at least one opening for introduction of extracellular matrix and at least one reservoir for introduction of media. A top layer can be cast from the master mold. A bottom master mold can be provided for fabricating a bottom layer. The bottom master mold can have raised portions defining an interior chamber and reservoirs for introduction of media. A bottom layer can be cast from the bottom master mold. The top and bottom layers can be treated to adhere the layers together.
In one embodiment, the top and bottom layers can form a microfabricated gap between the layers for introduction of an acupuncture needle into the internal chamber. The extracellular matrix can then be introduced, form a gel, and the needle can be removed.
In other embodiments, methods of using the device disclosed herein are provided. In one embodiment, the device can be used for introducing endothelial cells into a first channel and angiogenic factors into a second channel, creating a gradient of angiogenic factors extending across the extracellular matrix. In other embodiments, tumor cells can be introduced into either channel or the extracellular matrix while the first channel is endothelialized. In yet other embodiments, immune cells and fibroblasts can be introduced into either the channels or the extracellular matrix.
The disclosed subject matter is generally directed to techniques for culturing tissue in a microfabricated 3D cell culture system. Reference will now be made in detail to embodiments of the disclosed subject matter, examples of which are illustrated in the accompanying drawings.
Throughout this specification, the term “tubular compartment(s),” “channel,” “sink channel,” and “source channel” shall mean a cavity through an extracellular matrix. The terms “tube,” “tubular compartment,” and “channel” shall be synonymous with each other unless the context indicates otherwise. It shall be appreciated that a “tubular compartment” is not required to have a circular or elliptical cross section, but could instead take on other shapes.
Likewise, the term “interstitial” region shall mean the region of the extracellular matrix adjacent to or between one or more channels. The term “extracellular matrix” or “ECM” can be used to refer to any material that provides structural support for the channels. The ECM can have a synthetic composition (e.g., polyethylene glycol, poly(lactic-co-glycolic acid)) or be derived from natural components such as proteins (e.g., collagen, fibrin) or polysaccharides (e.g., alginate, agarose, dextran), or living cells themselves and their byproducts.
In one aspect of the disclosed subject matter, a device for 3D cell culture includes a substrate having at least one interior chamber. The substrate includes an opening that provides access to the interior chamber(s) for introduction of an extracellular matrix. The extracellular matrix has at least one channel defined therethrough.
In one embodiment, a device for 3D cell culture comprises one or more tubular channels that are encased within extracellular matrix representing the interstitial region. In some embodiments, the channels can be connected to fluidic reservoirs. The tubular channels can be individually addressable compartments that can be seeded separately with any cell type and perfused with any type of fluid. For example, for a device having two channels, both compartments could be lined with different cells and perfused with different media. The surrounding ECM can be comprised of any type of native or synthetic ECM or filled with any cell type. Moreover, this device can be re-configured in a number of ways, including altering the interactions, number, geometry, and orientation of tubular compartments. In certain embodiments, for example, the tubular channels can be interconnected.
As disclosed herein, the tubular compartments can contain more than one concentric layer of different ECMs or other biocompatible and biodegradable coating materials that can be seeded with different cells. For example, and not limitation, the tubular compartments can contain an endothelium, intima layer, media layer (e.g., where smooth muscle cells are located), and aventitia layer associated with a blood vessel.
The techniques described herein can provide for improved in vitro environments for 3D cell culture not limited to a particular cell type. For example, an exemplary device in accordance with the disclosed subject matter can provide an environment for 3D cell culture with unconstrained cell migration (e.g., cell migration can occur within an extracellular matrix without constraint by pillars, posts, or other structures). In another embodiment, an exemplary device in accordance with the disclosed subject matter can provide tubular channels suspended in the extracellular matrix. That is, for example, the tubular channels can be completely lined with extracellular matrix and cells seeded in the channel need not be in contact with any physical surfaces (glass, PDMS, etc) of the substrate. In this manner, cells seeded into the channels can form lumens therein, as opposed to merely coating part of the interior surface of the channel. Cell migration into the extracellular matrix can be unimpeded by any physical surface, and therefore, unconstrained in any direction. For example, the direction of cell migration can be observed in response to a gradient of soluble factors established in the extracellular matrix.
For purposes of illustration, and not limitation,
While
It will be appreciated that in place of the acupuncture needles, other suitable techniques may be employed to create channels. For example, a sacrificial filament may be implanted in the ECM which can be subsequently dissolved. Alternatively, a suture or string can be placed in the ECM which can subsequently be removed to create a channel. 3D printing of biocompatible and biodegradable materials can also be employed to generate channels and network of channels. Non-limiting and exemplary methods to create channels in the ECM are known in the art, e.g., as disclosed in Chrobak K M, et al., Formation of perfused, functional microvascular tubes in vitro, Microvascular Research, 2006. 71(3):185-196 and Golden A P, et al., Fabrication of microfluidic hydrogels using molded gelatin as a sacrificial element, Lab on a Chip, 2007, 7:720-725.
After the needles are removed, vacuum grease 170 can be used to seal off the PDMS gaps 140. Endothelial cells 180 can be seeded into the channel to form a monolayer. The substrate can be placed on a rocker or ported with syringe pump 190 to enable flow through channels 150, as depicted in
In one embodiment, for example, gravity-driven flow can be obtained by porting the ends of the channels with vertical fluid reservoirs where the fluid levels are at different heights and tunable. Fluid reservoirs can be connected to syringes to maintain differential fluid heights. Gravity-drive flow can additionally be generated by placing the substrate on a rocker. The rocker can be a piece of laboratory equipment comprising a platform that tilts back and forth on one axis or orbits around an axis. In some embodiments, the platform can tilt up to 30 degrees away from the horizontal plane in both directions. The substrate can be positioned on the rocker such that tilting causes the reservoirs connected to a given channel to be at different vertical planes. The fluids contained in the reservoirs are thus at different heights, resulting in a hydrostatic pressure difference which can drive flow through the channel. Thus, the rocker can cause fluid flow to occur in both directions in an oscillatory manner. A rocker used in conjunction with one embodiment of the presently disclosed subject matter, depicted in
In other embodiments, a syringe pump 190 can be used, and can achieve arterial and supraphysiological flow rates. The reservoirs can be plugged with silicone tubing. The tubing can be attached to a syringe on a syringe pump 190 which pushes fluid through the tubing. In some embodiments, physiologic and supraphysiologic flow rates can be achieved. Mucus can flow at 2-5 mm/min, which can be equivalent to approximately 7 nL/s in the device configuration of one embodiment of the disclosed subject matter, disclosed in
In other embodiments, a channel can be lined with contractile cells such as cardiac muscle cells or smooth muscle cells that can respond to stimulant(s) to generate contraction forces to constrict and dilate the channel thereby driving fluid flow through the channel.
In one exemplary embodiment, an exemplary device can be used to generate an organotypic model of angiogenesis. Two tubular compartments 150, approximately 400 μm in diameter and spaced approximately 1 mm apart, can be encased in collagen I gel. One of the tubular compartments 150b can be lined with endothelial cells and perfused with basal cell culture medium. The other tubular compartment 150a can be perfused with cell culture medium containing soluble angiogenic factors. The resultant diffusion of media into the interstitial collagen gel from both channels can create a stable angiogenic gradient, which stimulates matrix degradation and invasion, multicellular sprouting of endothelial cells, and formation of vessel lumens from the endothelialized cylindrical compartment in the direction of the source of the angiogenic factors.
Furthermore, for purpose of example and not limitation,
In another embodiment, the device disclosed above can be scaled up into a multi-well format, e.g., for use in high-throughput assays. This arrangement can be used, for example, to screen the effects of specific growth factors, cytokines, small molecules, ECMs, and non-endothelial cells (e.g., fibroblasts, immune cells) on angiogenesis or other cellular processes. As depicted in
One of ordinary skill in the art will recognize that a variety of other configurations are contemplated within the spirit and scope of the presently disclosed subject matter. For example, and with reference to
In another aspect of the presently disclosed subject matter, the device can be used in a method for culturing cells. In some embodiments, the device can be used to identify the mechanisms of action of test compounds and materials. Well known genetic tools such as RNA interference and genetic knockout can be applied to cells cultured in the device to identify gene targets for modulating angiogenesis or other cellular processes.
In other embodiments, the device can also be used as a diagnostic test for personalized medicine. For example, cancer cells extracted in biopsies can be cultured within a tubular or interstitial compartment to determine whether they attract angiogenic sprouting from an adjacent endothelialized tube. Moreover, and with reference to
In other embodiments, the device can be used to study any cellular process guided by gradients of soluble factors. For example, with reference to
In another example, with reference to
In yet another example, with reference to
In yet other embodiments, the device is used to investigate the effect of heterotypic cell interactions on cell function. For example, with reference to
In one embodiment, as depicted in
In another embodiment, as depicted in
In yet other embodiments, with reference to
In one embodiment, with reference to
As depicted in
The disclosed subject matter provides for the mimicking of in vivo microenvironment for a plurality of types of cell-based assays, including, but not limited to: angiogenesis, inflammation, tumorigenesis, tubulogenesis, proliferation, migration, differentiation, and signaling assays. The disclosed subject matter also provides for the evaluation of cellular migration, endothelial cell sprout migrations, anastomosis, vessel lumen formation, immune cell trafficking, and tumor mass migration. The disclosed subject matter has broad applicability in drug discovery, tissue engineering and basic cell biology.
In addition to endothelial sprout migrations, anastomosis and vessel lumen formation, the disclosed subject matter can also have application in immune cell trafficking (i.e., extravasation) and tumor cell migration (as seen in metastasis). In these embodiments, endothelium can be absent. For example, in one embodiment, only immune cells (for example, neutrophils) can be cultured in the channels while endothelial cells can be omitted. The movement of immune cells between the tubular and interstitial compartments can be observed.
Moreover, the presently disclosed subject matter has can be applied to models of different types of vasculature—lymphatic vessels, veins, arteries, and capillaries all contain endothelial cells but these endothelial cells can express different markers. For example, endothelial cells from microvasculature (capillaries) can respond differently to angiogenic factors than venous endothelial cells. Thus, tissue-specific endothelial cells can be used in the substrate to generate more physiologically relevant models of blood vessels and lymphatic vessels. Furthermore, other cell types, notably smooth muscle cells, can form part of the arterial vasculature. For example, a thick smooth muscle wall layer surrounding the endothelium. The substrate disclosed herein can incorporate this additional complexity.
In some embodiments, smooth muscle cells can be cultured inside the tubular compartment, followed by the addition of endothelial cells to create a multi-layer vessel analog. In another embodiment, brain cells may be seeded initially, followed by endothelial cells serving to model the blood-brain barrier. Additionally, the disclosed subject matter can be applied to models of non-vascular tissues disclosed herein. Other glandular tissues such as pancreas (epithelialized tubes which are filled with digestive enzymes), breast (lactiferous ducts that carry milk), liver (bile ducts that carry bile), brain (ventricles that contain cerebrospinal fluid), intestines (that transports food) and kidney (renal tubules that carry waste filtrate) can be modeled. For example, in one embodiment, kidney epithelial cells can be seeded into the tubular compartment to form an artificial renal tubule. Solutions containing metabolites can be flowed into the tubular compartment to assess the re-absorption function of the artificial renal tubule. The artificial renal tubule can be treated with pharmacological toxins which can impair the ability of the cells to re-absorb metabolites from the filtrate solutions.
With reference to
The present application is further described by means of the examples, presented below. The use of such examples is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, this application is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification. The invention is to be understood by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.
Examination the processes of angiogenic invasion and sprouting from an existing vessel in accordance with the disclosed subject matter will be described, with reference to
In a first example, the impact of various pro-angiogenic factors on directed invasion and sprouting from the parent vessel is examined. Six common factors associated with angiogenesis in the literature were selected: basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), sphingosine-1-phosphate (S1P), and phorbol 12-myristate 13-acetate (PMA). After these factors were added individually to the non-endothelialized source channel, phase-contrast and confocal microscopy were used to assess the organization and development of EC invasion over four days. In connection with this example, for purpose of illustration and not limitation, VEGF, MCP-1, HGF or bFGF alone did not induce significant invasion (e.g., single cell invasion or collective cell invasion quantified by length of invasion and/or density of invasion) into the matrix, while S1P and PMA resulted in substantial directed invasion. This invasion was oriented directly toward the source channel, despite the fact that cell migration from the endothelium was not artificially constrained in any direction by the device used in connection with this example (
In connection with this example, for purpose of illustration and not limitation, S1P and PMA stimulated different modes of cell migration. S1P drove chemotactic migration primarily of single cells from the endothelialized channel, whereas PMA triggered collective cell migration that manifested itself in the form of sparse, long, multi-cellular sprouts into the matrix (FIG. 11Ci,ii). Progressively more complex combinations of the six factors yielded more substantial multicellular sprout-like structures, especially in the case of two distinct combinations that drove robust sprouting—HGF, bFGF, MCP-1, VEGF, and S1P (HFMVS) and MCP-1, VEGF, PMA, and S1P (MVPS). HFMVS-guided invasion exhibited numerous sprout-like structures that extended hundreds of micrometers from the endothelialized parent vessel as well as large numbers of solitary cells migrating into the matrix (FIG. 11Ciii,iv). The MVPS cocktail induced an even greater multicellular sprouting response with less single cell migration (FIG. 11Cv). In both cases, the sprouts continued to invade toward the source channel as long as the gradient was maintained.
When the tips of these sprouts reached the source channel (typically after one week), they breached into the source channel, forming new microvessels connecting the two parallel channels (
In a second example, changes in cellular organization during early stages of invasion were examined, in connection with the MVPS cocktail. Prior to stimulation, cells in the endothelialized channel exhibited the expected apical-basal polarity as demonstrated by the localization of CD34 apical marker podocalyxin to the luminal face. On the basolateral side of the endothelium laminin deposition was observed. Upon stimulation, occasional single ECs began invading into the matrix and extending filopodia-like protrusions in the direction of the angiogenic gradient (
As the sprouts continued to invade and extend into the matrix, they became longer, contained progressively more cells, and began to branch (
Upon formation of neovessels spanning the two channels, non-perfused filopodial protrusions disappeared (FIG. 12Hi). The neovessels were lumenized end-to-end (FIG. 12Hii, iii), and cells were aligned with flow as in the parent vessel, demonstrated by actin stress fiber alignment (FIG. 12Hiv). Additionally, deposition of laminin around the neovessels (
In a third example, the physiological response of sprouts to agents known to perturb the angiogenic process was examined. First, a VEGF receptor 2 (VEGFR2) inhibitor Semaxanib was added with the HFMVS angiogenic cocktail. If added from the outset, the inhibitor abrogated sprout initiation (
In a fourth example, the morphogenetic responses to anti-angiogenic factors was examined with reference to the effects of perturbing S1P signaling, which acts as a strong chemoattractant through a G-protein coupled receptor (S1PR) and is known to regulate angiogenesis. Exposing cells to the S1PR inhibitor Fingolimod resulted in abrogation of sprout initiation when introduced at Day 0, and inhibited further sprout extension when given at Day 3 (
In connection with the examples described herein, an exemplary device in accordance with the disclosed subject matter was fabricated from two patterned layers of poly(dimethylsiloxane) (PDMS; Sylgard 184; Dow-Corning) bonded to each other and sealed against a glass substrate (e.g., as depicted in
Cells were cultured and seeded in accordance with the disclosed subject matter into the device described in connection with the first example. Human umbilical vein endothelial cells (HUVECs) (Lonza) and human microvascular endothelial cells (HMVECs) (Lonza) were cultured in EGM-2 and EGM-2MV, respectively. While all experiments shown were conducted with HUVECs, HMVECs also sprouted in response to angiogenic cocktails. ECs were concentrated at 107 cells/mL and seeded into one of the two channels. The device was inverted to allow ECs to adhere to the top surface of the channel for 10 min, and then flipped upright to allow cells to adhere to the bottom surface of the channel for another 10 min. Cells that adhered in the fluid reservoirs were scraped off with a pipette tip, and unattached cells in the channel were thoroughly flushed out with phosphate-buffered saline (PBS). Media was immediately added thereafter and the devices were placed on a platform rocker (BenchRocker, BR2000). Cells were cultured in channels for 1-2 days before angiogenic factors were introduced.
For immunofluorescence staining, cells in the devices were fixed in situ with 3.7% formaldehyde for 45 min. For CD31 labeling, cells were permeabilized with 0.1% Triton-X for 30 minutes, blocked in 3% BSA overnight at 4° C., washed 3 times with PBS and incubated with mouse monoclonal antibody against human CD31 (1:200, Dako). For laminin and podocalyxin labeling, samples were blocked with 3% BSA overnight at 4° C., washed 3 times with PBS and incubated with either rabbit polyclonal antibody against laminin (1:100, Chemicon) or goat polyclonal anti-human podocalyxin (1:100, R&D) overnight at 4° C. Before secondary antibody incubation, the devices were washed overnight with PBS at 4° C. All secondary antibodies (Invitrogen) were used at 1:500 dilution. Cell nuclei were labeled with DAPI (1:500, Sigma). F-actin was labeled with Alexa Fluor 488-conjugated Phalloidin (1:100, Sigma).
Brightfield images of sprouts, as described herein below, were acquired with a Nikon TE200 epifluorescence microscope (Nikon Instruments, Inc.) using 10× objective. Confocal immunofluorescence images were acquired with either 10× air objective or LD C-Apochromat 40x, 1.1 numerical aperture (N.A.) water immersion objective attached to either an Axiovert 200M inverted microscope (Zeiss) equipped with an CSU10 spinning disk confocal scan head (Yokogawa Electric Corporation), and an Evolve EMCCD camera (Photometrics) or an Olympus IX 81 microscope (Olympus America, Inc.) equipped with an CSU-X1 spinning disk confocal scan head (Yokogawa Electric Corporation), and an Andor iXon3 897 EMCCD camera (Andor Technology). ImageJ was used to merge channels, perform z-projection for all confocal stacks, and generate longitudinal and transverse cross-sections.
In connection with screening, the endothelialized parent vessel was perfused with culture media while the source channel was perfused with media enriched with angiogenic factors. Angiogenic factors include vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), hepatocyte growth factor (HGF), and basic fibroblast growth factor (bFGF), all purchased from R&D Systems. Sphingosine-1-phosphate (S1P) and phorbol myristate acetate (PMA) were purchased from Cayman Chemical and Sigma, respectively. VEGF, MCP-1, bFGF, HGF, and PMA were all used at 75 ng/mL while S1P was used at 500 nM. Inhibitors targeting VEGFR2 (10 μM Semaxanib, Cayman Chemical) or S1P receptors (100 nM Fingolimod, Selleck Chemicals) were administered into both channels. MMP inhibitor (0.6 μM Marimastat, Tocris Bioscience) was administered into the source channel. Media in both channels were refreshed daily.
After neovessels bridged the two preformed channels in the device, a solution of CellTracker CM-DiI (Invitrogen) was delivered into the parent vessel to label cells in situ. Fluorescent beads (Polysciences) of 3 μm diameter were suspended in PBS and perfused into the parent vessel at a flow rate of 5 μL/min. Images were acquired at 40 frames/sec using an Eclipse TE2000 and an Evolve EMCCD camera.
Custom MATLAB code was written to measure the individual distances from the leading protrusions of tip cells to the wall of the parent vessel. Tip cells were additionally quantified as either attached to stalk cells extending from the endothelialized channel or as isolated single cells (FIG. S1). Sprouting metrics were quantified for the screening experiment (N=2 samples per condition), the VEGFR2 and S1P inhibitor experiment (N=5 samples per condition), and the MMPs inhibitor experiment (N=3 samples per condition).
Projections from z-resolved confocal stacks, which were taken with a 25× objective, Axiovert 200M inverted microscope (Zeiss), and spinning disk confocal scan head, were used to analyze filopodia length and number. A custom MATLAB code was used to determine the distance from the tips of filopodia to the center of cell nuclei and count the number of filopodia. The number and length of filopodia were averaged over the number of cells across 3 samples per condition.
Sample populations were compared using unpaired, two-tailed Student's t-test. P<0.05 was the threshold for statistical significance. Data points on the graphs represent mean values and error bars depict SEM.
Although the disclosed subject matter has been described in connection with particular embodiments thereof, it is to be understood that such embodiments are susceptible of modification and variations without departing from the inventive concept disclosed. All such modifications and variations, therefore, are intended to be included within the spirit and scope of the appended claims.
This application is a U.S. continuation of PCT/U.S. Ser. No. 13/024,450, filed Feb. 1, 2013, which claims priority to U.S. Provisional Application Ser. No. 61/594,084, filed Feb. 2, 2012, and both of which are incorporated herein by reference in their entireties.
This invention was made with government support under Grant No. EB00262, awarded by the National Institute of Health. The government has certain rights in the invention.
Number | Date | Country | |
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61594084 | Feb 2012 | US |
Number | Date | Country | |
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Parent | PCT/US13/24450 | Feb 2013 | US |
Child | 14334408 | US |