Patent Application
20140206028
This invention relates generally to the field of in vitro cell culture systems and microelectrode arrays. Specifically, the invention relates to neuronal/microelectrode array hybrid systems.
Neurodegenerative conditions as well as traumatic brain injury (TBI) are characterized by widespread loss of functional, active neurons. Investigations into treatments and therapies for these conditions are reliant upon the existence of accurate systems mimicking the conditions found in the human brain. Widely used systems include rat and mouse disease models mimicking pathological symptoms of the diseases, embryonic neuron cell culture systems including dissociated cell culture and brain slices, and later stage testing on human subjects. Some work has been performed on the refinement of in vitro dissociated neuronal systems to use adult brain tissue rather than embryonic brain tissue. Problems exist with these current in vitro culture systems. Difficulty in limiting damage during the dissociation of adult brain tissue, mitigation of cellular trauma from the release of myelin inhibitory proteins, problems promoting both regeneration and long-term survival of adult brain-derived neurons, and proliferation of previously quiescent terminally differentiated neurons are among the most common problems encountered in creating such an in vitro system.
Adult central nervous system neurons have long been described as post mitotic, with neurons arrested in a G0 phase of the cell cycle. However, this cell cycle arrest in neurons is not permanent, but rather, mature differentiated neurons must constantly regulate themselves to keep from progressing through the cell cycle. In vitro, post mitotic neurons revert to a proliferative state due to the action of the essential factor basic fibroblast growth factor (bFGF).
The factor bFGF also promotes GABA-negative neurons to survive by influencing both glucosyceramide synthesis and the VDCCs. It also up-regulates cdk5 expression, triggering neuronal re-entry into the cell cycle. Cdk5, while generally expressed in neurons in the cdk5/p35 complex, is normally expressed by neurons at levels that exert an influence on neurite outgrowth and migration but not proliferation. Cyclin-dependent kinase inhibitors (CKIs), which include the competitive cdk5/p35 inhibitor roscovitine, normally play an important role in regulatory decisions controlling progression through the cell cycle.
Electrophysiological studies into learning and memory formation in the presence of neurotoxic compounds and under neurodegenerative conditions most commonly rely upon patch-clamp electrophysiology. While this method provides detailed information, it is very labor-intensive, complicated, and has limited long-term capability in relation to non-invasive techniques for measuring electrical activity such as microelectrode arrays (MEAs). MEAs are innovative tools used to perform electrophysiological experiments for the study of both neuronal activity and connectivity in populations of neurons from dissociated cultures. Where patch-clamp electrophysiology can only measure the electrical activity of cells over the short-term (<few hours), MEA systems can measure the same population of neurons and the chronic effect of toxic compounds, drugs, etc on those neurons for extended periods of time (days-months. Uses for these systems include studies into the mechanisms of learning and memory formation and investigations into drug discovery, neurodegenerative diseases, and biosensor applications.
A common limitation of neuronal MEA systems as well as neuroscience research in general has been the reliance upon neurons derived from embryonic tissue. While these are differentiated neurons, they are developmentally immature, with transcriptional profiling indicating two-thirds of genes are only expressed postnatal and >95% of the expressed genes show highly significant changes during postnatal development. In fact, the electrophysiological properties of embryonic or neonatal neurons yield promising in vitro epilepsy models. When evaluating the machinery responsible for synaptic transmission, AMPA channel expression is limited at birth, only increasing post-natally. The gene expression for the NMDA channel subunits NR2A and NR2B was not detected until near birth at E21-22, with expression not peaking until P20 14. In vitro, NR2A/B channels are detected at only very low levels until after 2 weeks in embryonically derived neurons. Gene expression for the axonal sodium transporter subunit 1 begins around P15, then increasing until P30. In vitro, expression patterns for all genes, specifically axonal and synaptic channels responsible for signal transmission, is similar to that seen in vivo, with gene expression showing significant changes over the course of weeks. The usefulness of these developmentally immature neurons for studies of neuronal electrical activity and synaptic transmission has been restricted by the limited expression or lack of expression of certain channels responsible for synaptic transmission in the adult brain. In addition, using these developmentally immature neurons in studies of neurodegenerative diseases or drug discovery can yield results that are difficult to correlate with the function or action of mature neurons in adult brain tissue.
What is needed in the art is a dissociated neuronal cell culture system that can be used with microelectrode arrays, in which the neuronal cells have the functional characteristics of adult neurons for studying the function of neurons, neuronal interactions, aging, neurodegenerative diseases, drug studies, neuroprosthetic devices, neurocomputing, biorobotics, and neuroregeneration, especially in traumatic brain injury.
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, the present invention comprises methods of producing a population of electrically active neurons, comprising contacting adult neural cells with a cdk5 inhibitor to control cell division of the adult neural cells and contacting the adult neural cells with a neurotransmitter, thereby producing a population of electrically active neurons.
In another aspect, the present invention comprises methods of utilizing adult neurons for testing purposes, comprising contacting at least one electrically active adult neuron, or a membrane portion thereof, with a test compound, the at least one electrically active adult neuron having been cultured under conditions where cell division is controlled, and observing for an effect of the test compound on the at least one electrically active adult neuron, or membrane portion thereof.
In another aspect, the present invention comprises microelectrode array systems comprising a microelectrode array having a surface and at least one electrically active adult neuron disposed on a surface thereof, the at least one electrically active adult neuron being derived from adult nervous tissue.
In another aspect, the present invention comprises populations of electrically active neurons, wherein the neurons were produced by contacting adult neural cells with a cdk5 inhibitor to control cell division of the adult neural cells and contacting the adult neural cells with a neurotransmitter under culture conditions to promote electrical function.
In another aspect, the present invention comprises methods of identifying test compounds having a possible effect on an electrically active adult neuron, comprising contacting a microelectrode array system with a test compound candidate, the microelectrode array system comprising a microelectrode array having a surface and at least one electrically active adult neuron disposed on a surface thereof, the at least one electrically active adult neuron cell being derived from adult nervous tissue, and observing for an effect of the test compound on the microelectrode array system, at least one electrically active adult neuron cell, or membrane portion thereof.
In yet another aspect, the present invention comprises microscale fluid handling systems, comprising a microfluidic device; and at least one three dimensional multi-cellular surrogate tissue assembly comprising a population of electrically active neurons, wherein the device is used for initiating, culturing, manipulating, and assaying each tissue assembly of the system.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate (one) several embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.
The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description.
The present invention comprises methods comprising exposing adult neural cells to a cdk5 inhibitor to control cell division of the neural cells. The method further involves encouraging development of electrical function of the neural cells by subjecting the neural cells to a neurotransmitter. Adult neural cells may be electrically functioning prior to exposure to a neurotransmitter.
The present invention comprises compositions of and methods for utilizing electrically active adult neural cells for testing and/or screening purposes. In an aspect, a method can involve exposing at least one electrically active adult neuron, or a membrane portion thereof, to a test compound and observing for an effect of the test compound on the at least one electrically active adult neuron, or membrane portion thereof. A person of ordinary skill in the art can observe the electrophysiology of the at least one electrically active adult neuron, or membrane portion thereof, before and after exposure of the at least one electrically active adult neuron cell, or membrane portion thereof, to a test compound. For example, a person of ordinary skill in the art can observe up-modulation or down-modulation of ion flow across the cell membrane of the at least one electrically active adult neuron cell, or membrane portion thereof, indicating that the test compound modulates electrical activity. Other non-limiting examples of effects that can be observed may include changes in frequency and/or amplitude of action potentials, longevity, robustness (health), or protection against the presence of a toxin or other harmful or disease causing agents. Source material for adult neurons may be normal neural tissue, neural tissue from a known disease state, or neural tissue from a suspected or unknown disease state.
The present invention comprises dissociated neuronal cell culture systems derived from neural tissue to produce a stable population of electrically active neurons. Through the action of regeneration-promoting growth factors, for example, basic Fibroblast Growth Factor (bFGF) and the dose-dependent application of anti-mitotic factors, cdk5 mediated cell cycle progression can be activated or deactivated to promote or control the division of adult, terminally differentiated neurons. The application of cell cycle control compositions to an improved serum-free culture system allows for either the expansion of primary adult neuronal cells under controlled conditions or for the development of a stable population of primary neurons that both morphologically and functionally regenerate without expansion. These neurons may be applied to culture surfaces coated with the chemical substrate N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine (DETA), a surface previously shown to be superior in the promotion of attachment, regeneration and long-term survival of neurons in vitro (D. A. Stenger et al., 1993; J. J. Hickman et al., 1994; M. Ravenscroft et al., 1998; M. Das et al., 2005; D. Edwards et al., 2010; A. E. Schaffner et al., 1995). The Examples show an exemplary system comprising a neuronal cell culture system derived from the hippocampus of adult rats.
As used herein, the term “adult” in the context of describing cells or tissue relates to cells that are terminally differentiated, or tissue containing such cells. The term “adult” in the context of describing a subject (human or nonhuman animal) typically means an animal that has reached reproductive capacity. While adult cells may be obtained from an adult subject, cells and/or tissue may be obtained from a non-adult subject and still be considered adult cells and/or tissue so long as the non-adult subject is post-natal and the adult cells and/or tissue are terminally differentiated.
As used herein, a “subject” is an individual and includes, but is not limited to, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, rat, mouse, or other rodents), a fish, a bird, a reptile or an amphibian. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included. A “patient” is a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.
As used herein, a “neuron” is a “neural cell.” The terms can be used interchangeably.
The present invention comprises methods comprising obtaining adult neural cells (neurons) from a tissue sample and exposing the obtained adult neural cells to a growth factor under suitable conditions to induce cell division of the neural cells. Other neural cells, such as glial cells, support cells, Schwann cells or other cells found in the brain or spinal cord, or cells known to those skilled in the art as derived from neural tissue may be included with neural cells in sampling or in culturing. Induction of cell division typically occurs prior to a step of controlling cell division by exposure to a cdk5 inhibitor. The cells may be maintained in the presence of both a growth factor and a cdk5 inhibitor.
Adult neural cells can be obtained by the dissection and enzymatic digestion of, for example, an adult neural tissue in the presence of a dissecting medium. After cells have been obtained from the tissue sample, they can be separated from each other by mechanical dissociation of the tissue sample. The individual adult neural cells can then be plated (attached) to a substrate in the presence of a plating medium for a desired period of time, for example three days. The cells on the substrate can be maintained from, for example day 3 of the culture, through the end of the culture. Exemplary methods of culturing cells are provided in the Examples herein, and those skilled in the art can determine periods of time for growing and attaching cells, and for exposure to particular media.
The present invention comprises methods for producing a population of electrically active (functional) neurons, comprising a) contacting adult neural cells with a growth factor, b) contacting the neural cells of step a) with a cdk5 inhibitor, and then c) contacting the neural cells of step b) with a neurotransmitter, wherein the adult neural cells develop into a population of electrically active neurons. Non-limiting examples of neurotransmitters include acetylcholine, dopamine, serotonin (5-HT), norepinephrine, epinephrine, GABA, prolactin, nitric oxide, histamine (H1, H2, H3, H4), vasopressin, oxytocin, endocannabinoids, endogenous opioids (Mu-1, Mu-2, kappa, delta, ORL, sigma receptors), glycine, and GHB.
Adult neural cells are cells taken from a sample of adult neural tissue. In an aspect, adult neural tissue is adult tissue from the central nervous system of a subject. Thus, adult neural tissue can be tissue from the brain or spinal cord of a subject. In an aspect, adult neural tissue can be hippocampal tissue from the brain of a subject. The peripheral nervous system may be a source for adult neural tissue.
Contacting adult neural cells with a growth factor under suitable conditions for a period of time induces the cells to divide in order to achieve confluence on a substrate. In some aspects, the cells may reach confluence without a growth factor present. A time for contact by the growth factor may be in a range of from 1 hour to about 2 days, to about 3 days, to about 5 days, to about 10 days or longer, and any time period thereinbetween. A non-limiting example of a growth factor is basic fibroblast growth factor (bFGF). In an aspect, a substrate, for example a glass cover slip, can be treated with N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine (DETA), resulting in a cell adhesive surface. Applying dissociated adult neural cells to a DETA-treated glass cover slip in a suitable plating medium allows neurons to attach with minimal attachment of cellular debris.
After adult electrically active neural cells are attached to a substrate, the cells can be maintained by being in contact with a maintenance medium, which can comprise a growth factor and a cdk5 inhibitor. Non-limiting examples of an inhibitor of cdk5 include butyrolactone. indolinone A and roscovitine.
A population of adult electrically active neurons can be created by dissecting adult neural cells. Shown in the Examples is a population of adult electrically active neurons from rat hippocampal tissue in the presence of a dissecting medium. See Table 1 for the composition of a dissecting medium. After a period of time, for example four to five hours, the adult neural cells can then be dissociated from each other in the presence of a dissociation medium. Approximately one to two hours later, the dissociated cells can then be plated with 500 μl of plating medium onto a DETA-covered substrate, for example a glass cover slip, and then washed with Hibernate A and some fresh plating medium. After about three cell divisions, the plating medium can be totally replaced with maintenance medium and 5 μM Roscovitine. After every four subsequent cell divisions, half of the maintenance medium can be replaced with fresh maintenance medium and 2 μM Roscovitine. The cells can be passaged and cultured under suitable conditions to establish electrical activity. One or more electrically active neurons created by the disclosed methods can then be disposed on a microelectrode array (MEA). The neural cells may be electrically active after regeneration.
Further disclosed is a MEA system, comprising a microelectrode array having a surface and at least one electrically active adult neuron disposed on a surface thereof, wherein the at least one electrically active adult neuron is from adult nervous tissue. In an aspect, the at least one electrically active adult neuron has been exposed to a cdk5 inhibitor, for example Roscovitine. In another aspect, the surface of a disclosed MEA system is treated with PDL (polynucleotide-D-Lysine) and laminin.
The present invention comprises a composition comprising a population of electrically active neurons. Such a composition may be produced by a method comprising a) contacting adult neural cells with a growth factor, b) contacting the neural cells of step a) with a cdk5 inhibitor, and then c) contacting the neural cells of step b) with a neurotransmitter, wherein the adult neural cells develop into a population of electrically active neurons.
The present invention comprises methods of identifying a compound that can affect electrical activity of an electrically active adult neuron, comprising a) contacting a MEA system with a test compound, wherein the MEA system comprises a microelectrode array having a surface and at least one electrically active adult neuron disposed on the surface thereof; and b) detecting a change in electrical activity of the at least one electrically active neuron or membrane portion thereof, wherein the detection of a change in electrical activity of the at least one electrically active neuron or membrane portion thereof identifies a compound that can affect electrical activity of an electrically active adult neuron. In an aspect, the electrically active adult neuron is from adult nervous tissue, for example the brain of a mammal. In another aspect, the change in electrical activity comprises changes in frequency and/or amplitude of action potentials, changes in spontaneous neuronal activity, changes in ion flow, changes in longevity, robustness (health), or protection against the presence of a toxin or other harmful or disease causing agent. Cessation of firing of an action potential by a cell indicates a change in the neural cell.
The present invention comprises an automatic top-off system using a surface tension based valve, which in an example requires neither any moving parts nor electronic control devices. An example top-off system is inexpensive and convenient to miniaturize for microfluidic and/or high-throughput systems. In another aspect, the present invention comprises a liquid handling system to automatically fill wells, e.g., culture wells, to desired levels without operator handling. In an exemplary system, fluids are stored in a separate reservoir, and tubing is specifically allocated to each well. More particularly, a siphon system passes media from a fluid reservoir to individual wells based on surface tension of the receiving well. Liquid is passively added while a seal, such as a valve cap, remains open. After the seal is closed and initial flow is induced, the liquid media are maintained in an aspiration well at a constant level without manual manipulation. Non-limiting example applications include high-throughput cell culture and high-throughput in vitro drug screening. See U.S. Patent Publications 20110086427, 2009/0053749 and 20040259177 for examples of microfluidic devices that can be used in conjunction with the cells of the present invention. Implementation of the cells and such microfluidic devices can be used for drug screening. The present invention may be used in systems or combinations of microfluidic devices or a three dimensional multi-cellular surrogate tissue assembly wherein at least one component of the system or assembly comprises a population of electrically active neurons, wherein the device is used for initiating, culturing, manipulating, and assaying each tissue assembly of the system.
Another technique for drug screening provides for high throughput screening of compounds (see, e.g., PCT Application WO 84/03564) that utilize robotic type systems. See, for example, U.S. Patent Publications 20100297685 and 20100105074. For example, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with the disclosed cells, or portions thereof, of the instant application and washed. Cells are then detected by methods well known in the art, using commercially available machinery and methods, for example, the Automated Assay Optimization (AAO) software platforms (Beckman, USA) that interface with liquid handlers to enable direct statistical analysis that optimizes the assays; modular systems from CRS Robotics Corp. (Burlington, Ontario), liquid handling systems, readers, and incubators, from various companies using POLARA™ (CRS), an open architecture laboratory automation software for a Ultra High Throughput Screening System; 3P (Plug & Play Peripherals) technology, which is designed to allow the user to reconfigure the automation platform by plugging in new instruments (ROBOCON, Vienna, Austria); the Allegro™ system or Staccato™ workstation (Zymark), which enables a wide range of discovery applications, including HTS, ultra HTS, and high-speed plate preparation; MICROLAB Vector software (Hamilton Co., Reno, Nev., USA) for laboratory automation programming and integration; and others.
For any of these machines and methods, the assays measure a response by the target cells that provides detectable evidence that the test compound may be efficacious. For example, the cells' response may include, but is not limited to, a change in morphology, neural processes, or synapse formation. The detectable signal is compared to control cells and the detectable signal identified by subtraction analysis. The relative abundance of the differences between the “targeted” and “untargeted” aliquots are simultaneously compared using a “subtraction” analysis (differential analysis) technique such as differential display, representational difference analysis (RDA), GEM-Gene Expression Microarrays (U.S. Pat. No. 5,545,531), suppressive subtraction hybridization (SSH) and direct sequencing (PCT Application WO 96/17957). The subtraction analysis can include the methods of differential display, representational differential analysis (RDA), suppressive subtraction hybridization (SSB), serial analysis of gene expression (SAGE), gene expression microarray (GEM), nucleic acid chip technology, or direct sequencing.
It is to be understood that this invention is not limited to specific synthetic methods, specific culture conditions, or to particular cells, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Thus, reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material, or composition is directed.
All technical and scientific terms used herein, unless defined herein, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. The techniques employed herein are also those that are known to one of ordinary skill in the art, unless stated otherwise.
As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an electrically active adult neuron” can include one or a plurality of electrically active adult neurons. Reference to “a medium” includes one medium or a mixture of two or more media, and the like.
Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the phrase “optionally contacted before” means that contact may or may not occur before a specified event or step.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
All parameters of the adult culture system were optimized for the support of neuronal attachment, regeneration, and long-term survival in vitro (
Media formulations (Table 1) along with the time- and dose-dependent application of growth factor and transcription factor mediators (
Multiple strategies were investigated to prevent primary terminally differentiated adult hippocampal neurons from returning to the cell cycle and dividing. The ultimately successful strategy for preventing proliferation under cell culture conditions was to add roscovitine, a competitive cdk inhibitor. Roscovitine was introduced into the culture medium at 1, 5, and 10 μM both before the onset of neuronal division (2 DIV) and after the onset of neuronal division (7 DIV) (
Other strategies for mitotic elimination that ultimately proved ineffective in the prevention of neuronal proliferation in vitro were also investigated. First, bFGF was removed from the Plating media and/or the Maintenance media. Neurons exposed to medium lacking bFGF did not divide. Rather than remaining static, however, neuronal populations deprived of bFGF decreased rapidly in vitro, with almost complete loss of neurons evident after two weeks (
Mitotic inhibitors were also investigated to eliminate the division of the cultured neurons. FudR (10 and 50 μM) and ara-C were added to the culture medium on 2 DIV (
Neurons were evaluated for electrical recovery after 6, 13, and 25 DIV using whole-cell patch clamp electrophysiology to evaluate the electrical potential of neurons, defined by the ability to move current into and out of the cell and to fire and propagate action potentials. The recovery of electrical activity in vitro was found to be significantly different between populations of neurons allowed to divide unchecked and neurons where mitotic division was prevented through application of roscovitine. When allowed to mitotically divide unchecked in vitro, neurons did not fully recover electrically for up to 3 weeks in vitro and then only after the neurons reached confluency and were stimulated with 25 μM glutamate for more than 24 hours (D. Edwards et al., 2010). Prevention of neuronal division in vitro through application of roscovitine (5 μM days 2-7 in vitro followed by 2 μM roscovitine after day 7) significantly changed the time-scale required for electrical recovery of neurons in vitro. Glutamate (25 μM) had been applied after 5 DIV. Neurons recovered after 6 DIV and showed the ability to move current into and out of the cell as well as to fire action potentials (
The maturity of neurons cultured in this system was probed. Neurons that were allowed to mitotically proliferate were probed for expression of neurofilament-M, Ki-67, and NeuN. While positive for the neuron-specific structural protein neurofilament-M, none of the neurons was positive for the mature neuronal nuclear marker NeuN. Instead, all neurons were positive for Ki-67, expressed exclusively in dividing neurons active in the cell cycle. After roscovitine was added to the cell culture medium, neurons no longer divided, and the expression pattern changed. Neurons still expressed mature structural proteins (MAP2), but instead of the proliferation marker Ki-67, they expressed the mature nuclear marker NeuN (
The combination of bFGF and roscovitine has allowed for a culture system of terminally differentiated primary adult hippocampal neurons in which the neuronal population can be kept stable or can be expanded. When these neurons were expanded under the basic culture conditions described in Figure and Table 1, i.e. without roscovitine, three distinct phases were observed. In phase 1 (0-3 DIV), mature neurons were able to recover from the trauma of cell culture and regenerated morphologically, as shown by the expression of neurofilamant-M, the 150 kDa structural protein uniquely found in neurons in the brain, and NeuN, a nuclear marker expressed exclusively in mature neurons (R. J. Mullen et al., 1992). The components of the Plating medium (Table 1), specifically growth factors, anti-oxidants, and Neurobasal-A with adjusted osmolarity, were essential for this recovery and regeneration. In phase 2 (3-21 DIV), bFGF in the Maintenance media (Table 1) induced neurons to re-enter the cell cycle through its activity in up regulating cdk5 expression (B.-S. Li et al., 2001; Z. Yan et al., 2002; S. M. Goodyear and M. C. Sharma, 2005; B. Menn et al., 2010). Previously quiescent, terminally differentiated phase bright neurons with long axons and branched dendritic trees first retracted their neurites into the cell soma, divided, and finally re-extended their axons and dendrites a period observed to occur on average 90 minutes from neurite refraction, division, and neurite extension. Neurons that have previously divided repeat this process of cell division and double in number on average every 24-36 hours. During these periods, neurons displayed expression patterns consistent with both neurons and dividing cells, with all cells displaying both neuronal morphology and expression of Ki-67, a nuclear marker for proliferating cells (T. Scholzen and J. Gerdes, 2000), and Nf-M. At the same time, however, these dividing neurons were not expressing NeuN. Based on the pattern of all neurons during phase 1, specifically with all cells expressing mature neuronal markers, as well as the universal expression of proliferation markers throughout phase 2, it was determined that mature neurons were dividing, not the subset of neural progenitors known to exist in the dentate gyms of the hippocampus. If only amplification of the smaller progenitor neuronal population of neurons had been occurring, then only a subset of neurons present would have shown evidence of Ki-67 expression.
In phase 3 (21-90 DIV), neurons reached confluence and stopped proliferating, a point at which neuronal expression of Ki-67 ended and NeuN reappeared. However, a passage of the confluent neurons from one DETA cover slip to multiple DETA cover slips at a lower cell density allowed for the continued proliferation of neurons beyond that seen on one cover slip alone, extending phase 2. During phase 2 neuronal electrical activity, specifically the ability to move current into and out of the cell and to fire action potentials (APs) was limited or non-existent. As the cells moved into phase 3, neurons were stimulated to recover their electrical activity through the introduction of 25 μM glutamate to the Maintenance medium. When cycloheximide (CHX), an inhibitor of protein synthesis that had previously been used to block preconditioning in neurons (F. C. Barone et al., 1998), was used to block all possible changes in protein synthesis induced by incubation of the neurons with glutamate, all improvements in neuronal electrical activity were completely blocked. The introduction of glutamate mediated the cellular transcription activity to mature the previously dividing neurons (D. Edwards et al., 2010).
Glass cover slips (Thomas Scientific 6661F52, 22 mm×22 mm no. 1) were cleaned by acid washing using a 50/50 mixture of concentrated hydrochloric acid and methanol. The cover slips were washed three times, 30 min per wash, and were rinsed in distilled de-ionized water between each washing. The DETA (N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine, United Chemical Technologies Inc., Bristol, Pa., T2910KG) monolayer was formed by the reaction of the cleaned surface with a 0.1% (v/v) mixture of the organosilane in freshly distilled toluene (Fisher T2904) (M. Ravenscroft et al., 1998). The DETA-coated cover slips were heated to just below the boiling point of toluene, rinsed with toluene, reheated to just below the boiling temperature, and then oven dried. The DETA formed a reaction site limited monolayer on the surface of the cover slip (M. Ravenscroft et al., 1998). The DETA cover slips were characterized to authenticate the monolayer formation. First, contact angle measurements were taken using an optical contact angle goniometer (KSV Instruments, Monroe, Conn., Cam 200). The contact angle for the DETA-coated cover slips was 54.2±0.2, which was previously shown to be acceptable for neuronal hippocampal culture (M. Ravenscroft et al., 1998). Second, X-ray Photoelectron Spectroscopy (XPS) (FISONS ESCALab 220i-XL) was used to characterize the elemental and chemical state of the DETA-coated cover slip surfaces. The XPS survey scans as well as high-resolution N 1 s and C 1 s scans, using monochromatic Al Kα excitation, were obtained, similar to previously reported results (M. Ravenscroft et al., 1998; M. Das et al., 2005).
The hippocampi of adult rats (Charles River, age 6-12 months) were dissected and homogenized into small tissue fragments in cold medium (˜4° C.) consisting of Hibernate-A, Glutamax, and antibiotic-antimycotic. The tissue was then digested for 30 minutes at 37° C. in calcium-free Hibernate-A (HA) containing 6 mg papain/12 ml (HA). Following digestion, the tissue was washed three times with cold Hibernate-A media to remove any active enzyme. Next, the tissue was suspended in Dissociation Media (Table 1), formulated for dissociation of rat and mouse adult brain tissue into individual cells and dissociated into individual cells through mechanical dissociation with fire-polished Pasteur pipettes. The dissociated cells were suspended in Plating Media (Table 1), used after tissue dissociation to promote neuronal attachment and regeneration, and then deposited onto DETA-coated glass cover slips for 30-45 minutes. DETA, with its triamine functional group exposed at the surface, strongly attaches to neurons and allows for all non-neuronal debris such as ECM proteins, myelin debris, and cell fragments to be washed from the cover slip surface (D. A. Stenger et al., 1993; J. J. Hickman et al., 1994; M. Das et al., 2005; A. E. Schaffner et al., 1995). Following this washing step, fresh Plating medium was applied and remained for the first 3 DIV. On 3 DIV and every fourth subsequent day, one half of the medium was removed and replaced with Maintenance Media (
bFGF (Invitrogen, 13256-029, 5 ng/ml) triggered neuronal cell proliferation until confluence, a period marked both by rapid neuronal division as well as limited or absent functional electrical activity. After neuronal confluence, 25 μM glutamate ((N-Acetyl-L-glutamic acid, Aldrich, 855642) triggers induction of neuronal electrical recovery (D. Edwards et al., 2010). Control of the division of neurons in this culture system was investigated experimentally as follows: (1) bFGF removal from both the Plating and Maintenance medium. (2) Fluorodeoxyuridine (FudR, Sigma-Aldrich, F-0503) addition at 10 and 50 μM (3) arabinoside-D (ara-C, Sigma, C-6645) plus deoxycytidine addition. (4) Trolox (Sigma, 238813) addition at 40 and 100 μM. (5) Aphidicolin (Aph, Sigma, A0781) addition at 1.5 μM. (6) Roscovitine (Rosc, Sigma, R7772) addition at 1, 5, and 10 μM. All factors were added before the onset of neuronal division (2 DIV), some after the onset of division (after 3 DIV).
Whole-cell, patch-clamp recordings were performed at room temperature. pH was adjusted to 7.3 with HEPES acid and base, ion concentrations with NaCl to approximately 130 mM, and osmolarity of 290-300 mOsm. Cells were visualized on the stage with a Zeiss Axioscope, 2 FS Plus, upright microscope in maintenance culture medium. Patch pipettes (4-8 M′Ω) were filled with intracellular solution (K-gluconate 140 mM, EGTA 1 mM, MgCl2 2 mM, Na2ATP 5 mM, HEPES 10 mM; pH 7.2), osmolarity approximately 260 mOsm. Voltage clamp and current clamp experiments were performed with a Multiclamp 700A (MDS Analytical Devices) amplifier. Signals were filtered at 3 kHz and digitized at 20 kHz with an Axon Digidata 1322A interface. Data recordings and analysis were performed with pClamp software. Whole-cell capacitance and series resistance were compensated electronically. Only cells with access resistance less than 22 M′Ω were analyzed. Inward currents that had the characteristics of fast sodium currents, and outward currents that had the characteristics of potassium currents were measured in voltage clamp mode using voltage steps of 10 mV from a −70 mV holding potential. Action potentials were measured with 1 s depolarizing current injections to elicit action potentials from a −70 mV holding potential. Cells were morphologically selected for electrophysiological characterization. Selected cells were phase bright pyramidal neurons with large branching apical dendrites and small basal dendrites. Cells with this morphology stained positive for anti-neurofilament-M (Nf-M) and/or MAP2. Neurons were electrically characterized after 6, 13, and 25 DIV.
To prepare cells for immunocytochemical characterization, cover slips were rinsed twice with Phosphate Buffered Saline (PBS). Cells were fixed with 4% paraformaldehyde for ten minutes at room temperature, and subsequently rinsed three times with PBS. Cells were permeabilized for five min in PBS with 5 mM Lysine and 0.5% Triton X-100, and were then blocked for two hours by adding 5% normal donkey serum. Anti-Neurofilament-M polyclonal antibody (Nf-M, intracellular filament found in mature neurons, Chemicon, AB 1981, diluted 1:1000), anti-NeuN (nuclear marker in mature neurons, Chemicon, diluted 1:1000), anti-Ki-67 (nuclear marker in dividing cells, Chemicon, diluted 1:1000), anti-NR2A (NMDA channel subunit expressed in the post-synaptic cell membrane, AB1548, diluted 1:75), anti-NR2B (NMDA channel subunit expressed in the post-synaptic cell membrane, MAB5216, diluted 1:150) were added in blocking solution for 12 hr at 4° C. After 3 washes with PBS, fluorescently labeled secondary antibodies (Invitrogen, A1011, A21449, and A11029) were applied for two hours. Vectashield mounting medium (H1000, Vector Laboratories, Burlingame, Calif.) was used to mount the cover slips onto slides. The cover slips were observed with an Ultra VIEW™ LCI confocal imaging system (Perkin Elmer).
Numerical summary results are reported as a mean, plus or minus the sample standard error of the mean (±SEM). Neuronal cell density immediately following plating onto cover slips was normalized to 100 to allow for comparisons of the effect of growth factor removal or mitotic factors intervention. For two-group comparisons, statistical analysis included a two-sample student's t-test on electrophysiological data.
Expansion of a population of mature terminally differentiated neurons derived from the hippocampus of adult rats, passage of that population 4 times, and arresting proliferation in ½ the population after each passage followed by electrical characterization
Method: Mature terminally differentiated neurons were extracted from the hippocampus of adult rats and plated onto DETA cover slips. bFGF in the culture medium caused these neurons to re-enter the cell cycle and proliferate to confluency on the cover slip. The following passaging protocol was used
1. Initial culture—two adult rats plated for 1 hour. Post hour wash to remove myelin debris and addition of Plating medium
2. Cell confluent on cover slip(s) (approximately 14-23 days in vitro)—initial passage
a. Cover slip(s) trypsinized with 0.05% trypsin/EDTA in HBSS
b. Upon initial cell dissociation, trypsin inhibitor added
c. Cells washed off surface, removed to 15 mL flask and spun at 500×g for 5 mins
d. ×2 more washes before replating upon fresh DETA coverslips
½ cover slip(s): proliferation arrested with cdk inhibitor roscovitine followed by electrical characterization through electrophysiology
½ cover slip(s): continued proliferation followed by passaging
3. Subsequent passages. Cells adhere and recover/proliferate to confluency
4. After confluency, neurons dissociated and replated on fresh DETA coverslips and/or microelectrode arrays.
Microelectrode arrays (MEAs) are innovative tools used to perform electrophysiological experiments for the study of synaptic activity and neural connectivity in populations of neurons from dissociated cultures. Uses for such an in-vitro high-throughput test system include investigations into neurodegenerative disease, traumatic brain injury, and stroke. A common limitation of neuronal MEA systems has been the reliance upon neurons derived from embryonic tissue. With MEAs, spontaneous network activity from both embryonic and adult brain tissue was concurrently measured. In addition, introduction of agonists and antagonists that affect maturation of neuronal activity/synaptic transmission established the usefulness of this system for drug discovery and basic research, including neuronal development and regeneration.
Adult neurons: Mature terminally differentiated neurons were extracted from the hippocampus of adult rats and plated onto DETA cover slips. After 4 days the neurons were passaged from these cover slips onto MEAs that had been coated with PDL/laminin (for cell adhesion)
Embryonic neurons: Neurons from the hippocampus of embryonic day 18 rat fetuses were extracted and plated directly on MEAs that had been coated with PDL/laminin.
For each population of neurons, electrical recordings of spontaneous neuronal activity were performed for up to 3 months. In addition, antagonists and agonists were introduced and their effects were measured against baseline electrical activity. 64-channel axion MEAs were used.
Results: Neurons (both embryonic and adult) regenerated on the MEAs and recovered electrically. After two weeks on the MEAs, both populations demonstrated consistently stable electrical activity.
Embryonic hippocampal neurons were cultured. Pregnant rats, 18 days in gestation, obtained from Charles River were euthanized with carbon dioxide and the fetuses were collected in ice-cold Hibernate E (BrainBits)/B27/Glutamaxtm/Antibiotic-antimycotic (Invitrogen) (Dissecting Medium). Each fetus was decapitated, and the whole brain was transferred to fresh ice cold dissecting medium. After isolation, the hippocampi were collected in a fresh tube of dissecting medium. The tissue was enzymatically digested at 37° C. for 10 minutes with papain (Worthington 3119), 12 mg/6 ml Hibernate-E (-calcium) (BrainBits)+0.5 mM Glutamax (Invitrogen). Hippocampal neurons were obtained by triturating the tissue using a fire-polished Pasteur pipette. After centrifugation, the cells were re-suspended in culture medium (Neurobasal/B27/Glutamaxtm/antibiotic-antimycotic) and plated on MEAs. All research was approved by the Institutional Animal Care and Use Committee at the University of Central Florida and conformed to NIH guidelines.
Adult neurons were extracted, dissociated, cultured, and maintained. Briefly, the hippocampi of adult rats (Charles River, age 6-12 months) were dissected and homogenized into small tissue fragments in cold medium (˜4° C.) consisting of Hibernate-A, glutamax, and antibiotic-antimycotic. The tissue was digested for 30 minutes at 37° C. in calcium-free Hibernate-A (HA) containing 6 mg papain/12 ml (HA no calcium). Following digestion, the tissue was washed three times with cold HA media to remove any active enzyme. Next, the tissue was suspended in Dissociation Medium and broken apart into individual cells through mechanical dissociation with fire-polished Pasteur pipettes. The dissociated cells were suspended in Plating medium and then deposited onto DETA (N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine, United Chemical Technologies Inc., Bristol, Pa., T2910KG) coated glass cover slips. After 30-45 minutes, the cover slips were washed with warm HA by gently swirling the medium to remove tissue debris. Following this washing step, fresh Plating medium was applied and remained for the first 3 div. On 3 div the medium was removed and replaced by Maintenance Medium with 5 μM Roscovitine (Rosc, Sigma, R7772). All research was approved by the Institutional Animal Care and Use Committee at the University of Central Florida and conformed to NIH guidelines. After 4 div, the adult hippocampal neurons on the DETA cover slips were passaged to MEAs. Briefly, neurons were dislodged from the DETA with trypsin (0.05% trypsin/EDTA in HBSS, Gibco, 25200). Trypsin inhibitor (trypsin inhibitor, soybean, Gibco, 17075-029) in Dissociation medium at 0.5 mg per ml deactivated the trypsin. The dislodged neurons were collected and spun at 500×g for 5 minutes. The supernatant of deactivated trypsin in HBSS was discarded, and the neuronal cell pellet was suspended in 1 ml Plating medium. Immunocytochemistry and Laser Scanning Confocal Microscopy
To prepare cells for immunocytochemical characterization, cover slips were rinsed twice with Phosphate Buffered Saline (PBS). Cells were fixed with 4% paraformaldehyde for ten minutes at room temperature, and subsequently rinsed three times with PBS. Cells were permeabilized for five minutes with 0.5% Triton X-100 in PBS, and were then blocked for two hours in 5% normal goat serum in PBS. Anti-neurofilament-M (Chemicon, AB5735, 1:500), anti-synaptophysin (Chemicon, MAB368, 1:300), and either anti-NMDAR2A (Chemicon, AB1555P, 1:200), anti-NMDAR2B (Chemicon, AB15557P, 1:200), or anti-glutamate receptor 2/3 (Chemicon, AB1506, 1:50) were added in blocking solution for 12 hr at 4° C. After 3 washes with PBS, fluorescently labeled secondary antibodies (Invitrogen, A11011 (594 nm), A21449 (647 nm), and A11029 (488 nm), 1:200) in blocking buffer were applied for two hours. Vectashield mounting medium with DAPI (H1200, Vector Laboratories, Burlingame, Calif.) was used to mount the cover slips onto slides. Fluorescent images were acquired with the UltraView spinning disc confocal system (PerkinElmer) with AxioObserver.Z1 (Carl Zeiss) stand, and a Plan-Apochromat 40x/1.4 Oil DIC plan-apochromat objective with 26 μm resolution. Z-stack projections of the scanned images were generated and modified within the Volocity image processing program (PerkinElmer).
The MEA chips (Axion Biosystems) provided 64 platinum-black coated gold-electrodes with a diameter of 30 μm, organized in an 8 by 8 array with 200 μm pitch. Clean MEAs were sterilized with 70% alcohol and then incubated with 1 ml poly-L-lysine (100 μg/ml) for 30 minutes. An area just large enough to cover all electrodes was additionally coated with 3 μl laminin (2 μg/ml) over night. Embryonic rat hippocampal neurons were plated directly on MEAs at a density of 500 cells/mm2. Adult rat hippocampal neurons were first cultured on DETA coated cover slips for recovery. While basic fibroblast growth factor (bFGF) was necessary for survival of adult neurons in culture, bFGF caused rat neurons to divide in vitro; administration of Roscovitine at 3 div prevented neuronal mitotic activity. After 4 days on DETA cover slips, the adult neurons were passaged onto MEAs at a density of 500 cells/mm2. Every 2-3 days, half the medium was replaced with fresh Maintenance medium supplemented with 2 μM Rosc. Supplementing the adult neurons with 25 μM glutamate (N-Acetyl-L-glutamic acid, Aldrich, 855642) on the 2 div increased the electrical activity of the adult neurons.
MEAs were incubated with their covers off to allow gas exchange but covered upon removal from the incubator to reduce contamination, media evaporation, and gas exchange while recordings were taken. The head stage of the recording system (Axion Biosystems) was pre-heated to 37° C. before MEAs with adult or embryonic hippocampal cultures were investigated. The activity of neuronal networks was recorded with 25 kHz using the software Axion's Integrated Studio (AxIS). Signal amplitudes 6 times larger than the standard deviation of the base line were detected as action-potential spikes. The spike data was then imported into Matlab 2010b (The MathWorks) for further processing.
Baseline spontaneous activity in adult and embryonic neurons was recorded for 3 minutes on 5 consecutive days per week, starting on 7 div. The synaptic antagonists D-(−)-2-Amino-5-phosphonopentanoic acid (D-APS, 25 μM, Tocris Bioscience, 0106), 6-Cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX, 25 μM, Tocris Bioscience, 1045), and (−)-Bicuculline methobromide (Bicuculline, 50 μM, Tocris Bioscience, 0109) were separately administered to both adult and embryonic neurons on 14 div and at various time points between 30 and 60 div. Three minute recordings were made to quantify the effect from these antagonists on spontaneous activity. If necessary, additional baselines were recorded prior to the administration of antagonists. Each experiment was followed by a complete washout of the antagonist and replacement with fresh medium.
Data analysis was performed off-line using unpublished Matlab functions to evaluate spike files created during each MEA recording. In brief, each 3 minute dataset was processed to extract the following parameters: “active channels” (a number from 0 to 64); “AP frequencies” (reciprocal inter-spike intervals); “Burst” (where at least 10% of the active channels showed more than 1 AP with 1 ms); “Burst duration” (time-interval from the first AP in a burst to the last AP in a burst); “Average burst frequency” (the reciprocal time-difference between two subsequent bursts); “In-burst frequency” (amount of APs within a detected burst divided by the duration of that burst); “Non-burst frequency” (reciprocal inter-spike intervals between APs that were not associated with bursts). In about 25% of the adult cultures and 14% of the embryonic cultures, portions of the neuronal networks detached from the substrate over time and especially after about 6 weeks. In order to accommodate the different numbers of active channels between MEAs, all values were corrected for the amount of active channels on an MEA. For each of the 3 minute recordings, a representative results chart was produced as shown in
Dissociated neuronal cultures from adult and embryonic sources recovered on MEAs and formed networks. Three-minute recordings of spontaneous activity were taken of each MEA, 5 times per week, for up to 70 div. Phase-contrast pictures of the MEAs were taken after each recording for a daily assessment into the condition of the cells as well as verification of physical contact between cells and electrodes
Spontaneous activity of Adult and Embryonic Neurons
Spontaneous firing activity started in both the adult and embryonic neurons between 7-10 div. Movement of MEAs from the incubator to the heated recording stage and the subsequent 3 minute recording period did not significantly affect the pH or temperature of the medium, represented by consistent baseline activity. However, brief increases in baseline neuronal activity were observed as a result of medium changes. Over the more than 2 month culture period, embryonic MEAs (n=6) consistently displayed a higher number of active channels, with an average of 37±8 channels active per MEA versus 15±5 channels in adult MEA cultures (n=9). Action potential (AP) firing frequencies in embryonic cultures with approximately 2-4 Hz were higher as in adult cultures with 1-2 Hz. Spontaneous bursting occurred about one week earlier in adult cultures as opposed to embryonic cultures. The burst development over the first 6 weeks was consistent between the two types of neuronal networks (
Agonist and antagonist were administered to embryonic and adult cultures on MEAs in separate experiments: 25 μM D-AP5 (NMDA channel antagonist), 25 μM CNQX (AMPA channel antagonist), and 50 μM bicuculline (GABAA antagonist). The results were summarized in
D-AP5 (25 μM) caused a significant decrease in the number of active channels in both adult and embryonic MEA cultures. Adult cultures lost a greater percentage of channel activity (90±6% on 14 div, 82±6% on 30-60 div) versus embryonic cultures (65±4% on 14 div, 36±7% on 30-60 div). Changes in the action potential frequency also varied with lower frequencies in adult cultures (76±8% on 14 div, 82±17% on 30-60 div) which were significantly different from the measured effect on embryonic cultures. On 14 div, the firing rate of embryonic neurons increased 90±6%, while in 30-60 day old cultures the firing rate decreased 70±7%. The difference in NMDA channel expression (NR2A and NR2B subunits) in adult and embryonic neurons in vitro likely caused the contrasting reaction to D-AP5 in the two populations of neurons. While NMDA channels were expressed at low levels in embryonic neurons on 14 div, the level of expression was not nearly as high as in adult neurons on 14 div. Because adult neurons expressed a greater number of NMDA channels, their reaction to the competitive NMDA antagonist D-AP5 was much more pronounced.
Addition of CNQX caused the activity of far fewer channels to be lost in both adult and embryonic MEA cultures as compared to D-AP5 (
After MEA measurements were recorded, the antagonists were washed from the neurons with an entire media change. 24 hours after, the activity of the neurons had returned to baseline levels.
Adult neurons cultured from the hippocampus of rats recovered functionally and had the capacity to fire spontaneously on MEAs over 70 div. These studies of neuronal maturation highlight the need for gene and protein expression in study populations of neurons to mirror that in mature adult neurons in vivo. Because embryonic neurons do not express the same machinery responsible for electrical activity or signal propagation in adult neurons, then their response to neurotoxic agents or drug therapies may not be correlative to responses in the mature adult brain.
The disclosed method of culturing adult neurons resulted in a system where NMDA and AMPA channel subunits were expressed throughout the lifespan of the culture. NMDA channel subunits NR2A and NR2B, as well as AMPA channel subunits GluR2/3, were expressed on and after 2 div. This contrasted greatly from neurons derived from embryonic tissue, with delayed NMDA and AMPA channel expression until 14 div and later. Responses of neurons to NMDA and AMPA channel antagonists were found to be significantly different in embryonic neurons as compared to adult neurons, with each antagonist decreasing activity in adult neurons to a greater degree than in embryonic neurons. These results show that embryonic neurons in culture develop a mature profile of ion channel subunits after 3-4 weeks. Therefore, embryonic neurons should not be employed for further experiments until they have fully matured in culture especially in studying neurodegenerative diseases such as Alzheimer's where synaptic protein profiles may play a critical role in the process of synaptic failure.
In comparison to embryonic MEA systems, as disclosed herein, measurements of neuronal activity using adult hippocampal neurons on MEAs, is more applicable to the adult brain. While preparation of these MEAs was slightly more complicated than embryonic neuronal MEAs, the end result yielded a long-term screen methodology that is more correlative to the dynamics of learning and memory formation in the adult brain. Additionally, due to the earlier development of bursts and general maturation, this system can facilitate quicker, more reliable, and more correlative investigations into drug discovery, neurotoxic agents, and neurodegeneration.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/487,251, filed on May 17, 2011, which application is hereby incorporated by this reference in its entirety.
This invention was made with government support under Grant R1 EB005459 awarded by NIH. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/038358 | 5/17/2012 | WO | 00 | 2/28/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61487251 | May 2011 | US |
