Patent Application
20160033491
The present invention relates to dehydrated solution microarrays. In particular, the present invention relates to arrayed multiphase assay systems and uses thereof.
The identification of the entire genome sequences of many species, including humans, has set the stage for rapid advancements in the field of functional genomics. Information generated from functional analysis of genes will, in the long run, have major benefits for the prevention, diagnosis and management of many diseases which have been difficult to control. Given the large volume of data from the genome of complex organisms, functional genetic studies demand high-throughput methods to rapidly elucidate the function of many genes in parallel. Cell-based microarray systems are simple and low-cost, yet powerful tools that allow large-scale manipulation of genes in cells and analysis of corresponding downstream phenotypes. Currently, these arrays are realized by using either microwell plates that spatially segregate reagents using physical walls or solid substrates (glass or polystyrene) “printed” in certain spots with reagents suspended in a gel material. The printing method, also known as reverse transfection, offers higher density and simplified fluid handling once the reagents are printed and several groups have shown its potential for high-throughput studies of gene function. Nevertheless, this technique is inflexible in timing of delivery and removal of reagents, which limits the possibility of exposure of cells to a biochemical for a desired time period, and addition of certain components is required to stabilize transfection reagents. Most importantly, the printed gel that immobilizes reagents on the surface, by necessity, becomes the substrate to which cells attach and grow. This is a major concern for phenotypic assays since the influence of interactions between cells and their ECM on gene expression patterns of cells is ignored.
New methods are needed for cellular arrays to allow for parallel analysis of multiple genes in one assay.
The present invention relates to dehydrated solution microarrays. In particular, the present invention relates to arrayed multiphase assay systems and uses thereof.
For example, in some embodiments, the present invention provides a system, comprising: a) a first polymer solution (e.g., comprising a first polymer); b) a second polymer solution (e.g., comprising a second polymer), wherein the second solution is more dense that the first solution, and wherein the first and second solutions form an aqueous two-phase system when mixed, and wherein at least one of the first and second polymer solutions are dehydrated; and c) a solid or semi-solid support. In some embodiments, the first polymer solution is dehydrated, while in others the second polymer solution is dehydrated, and in further embodiments, both the first and second polymer solutions are dehydrated. In some embodiments, just the dehydrated polymer (or polymers) is affixed to the support. In some embodiments, the polymer in solution (e.g., not dehydrated) is mixed with the dehydrated polymer solution to rehydrate the dehydrated polymer solution and form the two-phase solution. In some embodiments, the first and second polymer solutions are rehydrated by an aqueous solution. In some embodiments, the system further comprises reagents for performing an assay (e.g., an immunoassay or a nucleic acid detection assay). In some embodiments, reagents for performing the assay are delivered in one or more of the hydrated polymer solutions, the dehydrated polymer solution, or an aqueous solution used to rehydrate polymer(s). In some embodiments, the aqueous solution comprises cells. In some embodiments, the first polymer is polyethylene glycol and the second polymer is dextran. In some embodiments, the first or second solutions comprise two or more polymers (e.g., DEX-methylcellulose, DEX-polyvinyl alcohol, PEG-DEX sulfate, polyvinyl alcohol-DEX sulfate, hydroxypropyldextran-DEX, or DEX sulfate-methylcellulose).
In further embodiments, the present invention provides a method comprising a) contacting a solid or semi-solid support with a first solution comprising a first polymer (e.g., a dehydrated polymer) to form an arrayed support; b) contacting a portion of the arrayed support with a second solution comprising a second polymer (e.g., a dehydrated or hydrated polymer solution) wherein the first and second solutions form an aqueous two-phase system when mixed. In some embodiments, the first polymer solution is dehydrated, while in others the second polymer solution is dehydrated, and in further embodiments, both the first and second polymer solutions are dehydrated. In some embodiments, just the dehydrated polymer solution(s) are affixed to the support. In some embodiments, the support is for example, plastics, metal, glass, paper, fabric, hydrogels, foam, surfaces of sensors, electrodes, cantilevers, microfluidic device surfaces, inside capillaries, on medical devices, etc. In some embodiments, the polymer in solution (e.g., not dehydrated) is mixed with the dehydrated polymer solution to rehydrate the dehydrated polymer solution and form the two-phase solution. In some embodiments, the first and second polymers are rehydrated by an aqueous solution. In some embodiments, the method further comprises delivering reagents for performing an assay (e.g., an immunoassay or a nucleic acid detection assay). In some embodiments, reagents for performing the assay are delivered in one or more of the hydrated polymer solution, the dehydrated polymer, or an aqueous solution used to rehydrate polymer(s). In some embodiments, one phase of the ATPS is dehydrated and the other is in solution. In other embodiments, multiple phases are dehydrated together. In some embodiments, the aqueous solution comprises cells. In some embodiments, the first polymer is polyethylene glycol and the second polymer is dextran. In some embodiments, the first or second solutions comprise two or more polymers (e.g., DEX-methylcellulose, DEX-polyvinyl alcohol, PEG-DEX sulfate, polyvinyl alcohol-DEX sulfate, hydroxypropyldextran-DEX, or DEX sulfate-methylcellulose). In some embodiments, the dehydrated polymers are lyophilized or dried. In some embodiments, the aqueous solution is a bodily fluid (e.g., blood, urine, saliva or serum). In some embodiments, the aqueous solution comprises cells. In some embodiments, the cells are excluded from at least a portion of the aqueous two phase solution. In some embodiments, the cells form patterns in multi well plates (e.g., exclusion zones). In some embodiments, the cells are used in an assay (e.g., a cell migration assay).
Additional embodiments of the present invention provide a method, comprising a) arraying a plurality of first solutions comprising i) a first polymer; and ii) reagents for performing an immunoassay on a solid or semi-solid support; b) dehydrating the solution to form dehydrated spots on the support; c) contacting the dehydrated spots with a second solution comprising a second polymer and a test sample under conditions such that the second solution rehydrates the dehydrated spots and forms an aqueous two phase solution. In some embodiments, the method further comprises the step of identifying the presence of one or more antigens in the test sample. In some embodiments, the plurality of first solutions each comprise an antibody specific for a different antigen.
Additional embodiments are described herein.
To facilitate an understanding of the present invention, a number of terms and phrases are defined below:
The term “sample” in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture. On the other hand, it is meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin.
Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bears, fish, lagamorphs, and rodents.
Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
As used herein, the term “cell” refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
As used herein, the term “cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
The term “transfection” as used herein refers to the introduction of foreign DNA into eukaryotic cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
The term “stable transfection” or “stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term “stable transfectant” refers to a cell that has stably integrated foreign DNA into the genomic DNA.
The term “transient transfection” or “transiently transfected” refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell. The foreign DNA persists in the nucleus of the transfected cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes. The term “transient transfectant” refers to cells that have taken up foreign DNA but have failed to integrate this DNA.
As used herein, the term “selectable marker” refers to the use of a gene that encodes an enzymatic activity that confers the ability to grow in medium lacking what would otherwise be an essential nutrient (e.g. the HIS3 gene in yeast cells); in addition, a selectable marker may confer resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be “dominant”; a dominant selectable marker encodes an enzymatic activity that can be detected in any eukaryotic cell line. Examples of dominant selectable markers include the bacterial aminoglycoside 3′ phosphotransferase gene (also referred to as the neo gene) that confers resistance to the drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase (hyg) gene that confers resistance to the antibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) that confers the ability to grow in the presence of mycophenolic acid. Other selectable markers are not dominant in that their use must be in conjunction with a cell line that lacks the relevant enzyme activity. Examples of non-dominant selectable markers include the thymidine kinase (tk) gene that is used in conjunction with tk-cell lines, the CAD gene that is used in conjunction with CAD-deficient cells and the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene that is used in conjunction with hprt-cell lines. A review of the use of selectable markers in mammalian cell lines is provided in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp. 16.9-16.15.
As used, the term “eukaryote” refers to organisms distinguishable from “prokaryotes.” It is intended that the term encompass all organisms with cells that exhibit the usual characteristics of eukaryotes, such as the presence of a true nucleus bounded by a nuclear membrane, within which lie the chromosomes, the presence of membrane-bound organelles, and other characteristics commonly observed in eukaryotic organisms. Thus, the term includes, but is not limited to such organisms as fungi, protozoa, and animals (e.g., humans).
As used herein, the term “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
The terms “test compound” and “candidate compound” refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function. Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. In some embodiments of the present invention, test compounds include antisense, siRNA or shRNA compounds.
The present invention relates to solution microarrays. In particular, the present invention relates to an aqueous two-phase system for solution microarrays and uses thereof. In some embodiments, the present invention provides compositions and methods for addressing cells and reagents to one of the multiple phases of an aqueous multiphase cell culture system. In some embodiments, the system and methods are used to deliver reagents (e.g., nucleic acids in a transfection complex or viruses) to arrays of cells. The solution based arrays allow for precise and accurate delivery of reagents to only those locations desired and not to others. Although the compositions and methods described herein are illustrated with the use of printing on cells, the present invention is not limited to printing on cells. The “cell” surface may be replaced by surfaces other than cells and work in a similar manner. In some embodiments, the aqueous two-phase system is dehydrated on the microarray, providing additional stability and functionality.
In some embodiments, the present invention provides multiphase (e.g., two) solution based microarrays. The present invention is not limited to particular components of the microarray. In some embodiments, microarrays comprise aqueous polymers. Preferred polymers are those that form an aqueous two phase system (ATPS) at a wide range of temperatures (See, e.g., WO 2010/027590 herein incorporated by reference in its entirety). Any system that selectively partitions larger molecules (e.g., cells) or smaller molecules (e.g., nucleic acids or viruses) may be utilized. Examples of suitable polymers include, but are not limited to, polyethylene glycol (PEG), dextran (DEX), and combinations of other polymers such as DEX-methylcellulose, DEX-polyvinyl alcohol, PEG-DEX sulfate, polyvinyl alcohol-DEX sulfate, hydroxypropyldextran-DEX, and DEX sulfate-methylcellulose.
In other embodiments, ATPS that exhibit variable phase separation with temperature are utilized. In some embodiments, such systems utilize low molecular weight polymers.
In some embodiments, the first layer (e.g., PEG containing media) is dispensed onto cells (e.g., a confluent layer of cultured cells). In some embodiments, genetic material to be transferred to the array is placed in the wells of a multi well (e.g., 1536 well) plate. In some embodiments, the molecule of interest (e.g., genetic material) is in a solution containing the second component of the ATPS (e.g., DEX). In some embodiments, a transport component (e.g., an array of slot pins) is then used to transfer the molecule of interest onto the cell array. For example, in some embodiments, a multiplex dispenser that allows different materials to be added to different spots on the array is utilized. In some embodiments, the dispenser is a plurality of pins or other dispensing components affixed to a single transport component. In some embodiments, the transport component is automated.
In some embodiments, the component comprising the molecule of interest is denser that the first component and thus displaces the first material and contacts the cells. In some embodiments, cells are then transfected with the genetic material.
Embodiments of the present invention provide ATPS that comprise one or more dehydrated components. Experiments conducted during the course of development of embodiments of the present invention describe a facile, reliable, and scalable method to spatially pattern, dry, and rehydrate droplets of two incompatible aqueous polymer solutions.
A variety of multiplexed assays have been reported in the literature and a number of systems have even been commercialized (LUMINEX®, Mesoscale, Aushon, etc.). These multiplexed assays, however, have not been adapted successfully to clinical settings, even in well-equipped hospitals of developed countries. The primary reason is the multiplicity of challenges associated with assay validation. One limitation of existing sandwich assays (so-called ELISA and related sandwich immunoassays that are required for clinically appropriate specific and sensitive detection of many important biomarkers) is that although the primary antibody can be arrayed separately, the secondary antibody is introduced as a cocktail. The difficulty of validation increases exponentially as the number of assays to be multiplexed increases. If any one of the reagents or antibody becomes defective, the entire assay is jeopardized. This reduces reliability of the entire assay. Any replacement of reagents, even with extreme attention to the integrity of the reagents, requires revalidation of the entire combination and a separate “standard curve”. This greatly reduces versatility and increases cost. What is needed to enable efficient and robust point-of-care diagnoses, therefore, is improved multiplexed biomarker analysis technology where sandwich immunoassays can be efficiently multiplexed without “reagent crosstalk” problems.
In some embodiments, the present invention provides dehydrated aqueous multiphase (e.g., two) systems. In some embodiments, one phase of the system is dehydrated. In some embodiments, the system is rehydrated with a solution of the other phase to reconstitute the aqueous two-phase system.
In some embodiments, all phases of an aqueous two phase system are dehydrated. In such embodiments, the system may be rehydrated using any number of suitable solutions.
Examples include, but are not limited to, water or aqueous solution that does not include either of the components of the aqueous two phase system, samples (e.g., blood, urine, serum, semen, saliva or components thereof).
The present invention is not limited to a particular method of dehydrating ATPS components. Examples include, but are not limited to, drying or lyophilizing.
In some embodiments, the dehydrated phase (or phases) is patterned or arrayed on a solid surface. In some embodiments, the support or solid surface is, for example, plastics, metal, glass, paper, fabric, hydrogels, foam, surfaces of sensors, electrodes, cantilevers, microfluidic device surfaces, inside capillaries, or medical devices.
In some embodiments, the dehydrated phase (or phases) comprises reagents that partition selectively to that phase and remain in that phase when rehydrated. In some embodiments, the reagent is an immunoassay reagent such as bead proximity assay reagents. In some embodiments, the reagent is a nucleic acid reagent for transfection, gene knockdown, or molecular beacon type detection assay reagent.
In some embodiments, the rehydrating solution contains cells. In some embodiments, cells in the rehydrating solution are excluded from the pre-patterned aqueous two phase system to form patterns of cell attachment (e.g., on a solid support such as a multi-well plate). In some embodiments, the patterned cells are used for assays (e.g., cell migration assays or migration inhibitor assays).
Additional uses for dehydrated ATPS are described herein.
The solution based microarrays of the present invention find use in a variety of applications. Any application that requires manipulation of cells, reagents or surfaces in an array format is amenable to the methods of the present invention.
In some embodiments, the compositions and methods of the present invention find use in transfection methods. For example, in some embodiments, cells to be transfected are placed under the first solution. The genetic material is placed in the second solution and selectively delivered to cells. The present invention is not limited to a particular type of genetic material. Examples include, but are not limited to, DNA, virus, phage, R A (e.g., antisense, shR A or siRNA) or DNA encoding antisense, shRNA or siRNAs. Using such methods, it is possible to deliver multiple different types of genetic material to the same array of cells.
In some embodiments, cells are transfected using ultrasound. For example, in some embodiments, DNA is delivered to specific cells using the ATPS systems described herein and ultrasound contrast agent bubbles are used to introduce genetic material into cells via ultrasound.
In other embodiments, additional transfection systems and reagents are included (e.g., lipid based transfection systems, electroporation systems, etc.).
In some embodiments, the compositions and methods of embodiments of the present invention are used in cell migration assays. In some embodiments, the migration assay is based on movement of cells from a confluent monolayer into a circular cell-excluded area within the monolayer. In some embodiments, an aqueous two-phase system is utilized to create cell-exclusion patterning (see, e.g., Example 4). In some embodiments, cell-exclusion patterning is used in cell migration (e.g., drug screening and research applications). For example, in some embodiments, cells are treated with test compounds and their migration is assayed.
In other embodiments, the compositions and methods of the present invention find use in drug screening applications. For example, in some embodiments, cells to be screened (e.g., cancer or disease cells) are placed under the first solution. Candidate compounds are then placed in the second solution and selectively delivered to cells. In some embodiments, candidate compounds are therapeutic nucleic acids (e.g., siRNA, antisense or DNA encoding therapeutic RNAs). Using such methods, it is possible to deliver multiple different candidate compounds to the same array of cells.
In other embodiments, cells are contacted with additional molecules of interest including, but not limited to, cell signaling molecules (e.g., cytokines), growth factors, and proteins, and the effect of the molecule on the cell is assayed.
In still further embodiments, the present invention provides systems and methods for detecting cell-cell and cell-tissue interactions. For example, in some embodiments, a substrate immersed in a first solution is printed with cells suspended in a second solution, allowing localized delivery. The cellular array can then be contacted with a second cell or cell type (e.g., to study cell-cell interactions) or a tissue (e.g., for use in tissue engineering or research).
Following manipulation of cells, altered (e.g., transfected) cells may be detected using any suitable read out method. In some embodiments, the read out is an immunofluorescence method where antibodies to protein expressed from a transfected cell is contacted with the transfected array. Fluorescence is detected using any suitable method (e.g., a microscope or a fluorimeter).
In other embodiments, a different detection method including, but not limited to, fluorescence in situ hybridization, reporter assay (e.g., with fluorescence, chemical or chemiluminescence readout) or other detection method is utilized.
In some embodiments, cell based array methods and read out is performed in a high throughput manner. In some embodiments, high throughput methods are automated.
In some embodiments, the present invention provides compositions and methods for assaying cell migration. Cell migration is a fundamental process during events such as cancer metastasis and the failure of diabetic wound healing. Understanding molecular mechanisms responsible for dysregulated cell migration greatly benefits from high throughput technologies that allow screening libraries of small molecules or siRNAs to identify potential therapeutic compounds. To date, the majority of in vitro cell migration studies rely on the conventional wound healing and Boyden chamber assays. These approaches are limited in throughput and implementation in formats smaller than 96-well plates. An Oris cell migration assay has also been developed that utilizes a physical barrier resting on the surface to generate individual islands of naked surface surrounded by a cell monolayer in wells of a 96-well plate. Migration of cells into the empty space is studied after removal of the barrier. The technique is cumbersome due to the use of various components the need for several wash and incubation steps. More importantly, the physical contact of the barrier with the surface can be damaging to underlying soft extracellular matrix hydrogels. Therefore, new approaches that enable cell migration studies in higher throughputs are required.
Accordingly, in some embodiments, the present invention provides compositions and methods for assaying cell migration. In some embodiments, the assays utilize ATPS comprising one or more dehydrated components.
In some embodiments, the compositions and methods of embodiments of the present invention find use in immunoassays. In some embodiments, the present invention provides multiplexed immunoassays where the sample solution is simultaneously exposed to multiple analyte assay droplets where the primary and secondary antibody reagents for each analyte are segregated into the different droplets with no crosstalk of reagents between the assay droplets. In some embodiments, aqueous two phase systems where immunoassay reagents (both primary and secondary antibodies) are localized into microarrayed dehydrated droplets of one aqueous phase and subsequently the droplets are exposed to sample contained in the second aqueous phase are used. The aqueous two phase system and reagents are designed to allow biomarkers to diffuse from the sample-containing aqueous phase into the assay droplet phase, but the immunoassay reagents does not diffuse out of the assay droplets. These solution microarray assays enable flexible adaptation to analyze different panels of biomarkers such as, for example, antigens, antibodies, cytokines, interleukins, and drugs, to diagnose pathogens, infection, inflammation, compliance, and other point-of-care needs.
An example of immunoassays that can be performed within, for example, dehydrated ATPS droplets include, but are not limited to, proximity bead based assay reagents (e.g., AlphaLISA, PerkinElmer) pre-arrayed and dried in a microarray format with first and second polymers (e.g., dextran (DEX) and polyethylene glycol (PEG)). Body fluid sample is wicked over the dehydrated array of individual immunoassays. In one step, the body fluid sample is exposed to all the arrayed spots where each droplet has immunoassays for different biomarkers. Each spot becomes hydrated and forms distinct phase-separated droplets. Engineered sandwich immunoassay reagent beads against various biomarkers stably partition within each first polymer phase and do not diffuse out into the second polymer phase. Thus, there is no cross-talk of antibodies between different assay droplets. On the other hand, immune system biomarkers (proteins) diffuse freely from one phase to another allowing interaction with the immunoassay reagent beads confined within each phase droplet. Once the biomarker brings the primary antibody-bead and secondary antibody-bead into close proximity, a chemiluminescent or other signal is produced. This bead proximity assay requires no washing or other fluidic manipulation. The simplicity of procedures plus the ability to self-contain each sandwich immunoassay in separate solution droplets with no cross-talk between antibodies, gives the technology reliability, flexibility, and scalability.
In some embodiments, the reagent in the first polymer is a molecular beacon or other nucleic acid detection reagent and multiplexed DNA detection is performed. In other embodiments, the reagent is a proximity scintillation reagent or a fluorescent polarization immunoassay reagent. In some embodiments, the reagent storage stability is enhanced because dried forms are more stable than solutions and because sugars, including dextran, often enhance stability of dehydrated forms of reagents.
The present invention further provides systems and kits comprising the solution arrays described herein. In some embodiments, systems and kits comprise cells (e.g., in a multiwell solid support), multiple solutions for forming arrays, transport components (e.g., robotics), and components for read out of signal from altered cells, including analysis software. In some embodiments, kits further comprise additional component useful, necessary, or sufficient for performing and analyzing the results of the methods described herein (e.g., including, but not limited to, buffers, nucleic acids, candidate drug compounds).
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
This example describes a method to spatially pattern, dry, and rehydrate droplets of two incompatible aqueous polymer solutions. A polymeric aqueous two phase system (ATPS) with 2.5% (w/w) polyethylene glycol) 35,000 (PEG) and 3.2% (w/w) or higher dextran 500 (DEX) as the phase forming polymers were used. In the ATPS solution, PEG always forms the upper phase whereas DEX always forms the lower phase.
Aqueous solutions are hydrophilic; thus, when deposited onto hydrophilic substrates (e.g. glass), the solutions “spread” out over the surface and the contact angle measured is less than 90°. If deposited onto a hydrophobic surface, however, aqueous solutions bead onto the surface or exhibit poor wettability and the contact angle measured exceeds 90°. The contact angle quantifies the angle at which the solid substrate intersects the liquid droplet/vapor interface.
To generate a spatially patterned microarray of DEX droplets, 0.2 μL-1.0 μL DEX phase was dispensed onto poly(dimethylsiloxane) (PDMS), polystyrene, or glass substrates. DEX droplets were allowed to evaporate at room temperature and the length of time required for complete evaporation depends upon the initial droplet volume. After evaporation, dried discrete films of DEX remain adhered to the surface. Addition of an aqueous PEG solution to the dried DEX films causes the DEX droplets to rehydrate as illustrated in
To generate discrete islands of ATPS droplets, the first PEG solution is dispensed onto poly(dimethylsiloxane) (PDMS), polystyrene, or glass substrates. The appropriate DEX phase subsequently is introduced into the PEG phase using a dispensing tool (
This example describes a system that provides a cell migration assay in a high density format such as a 384-well microplate format in a single step without the need for multiple washing and incubation steps. The system is based on the use of a cell culture compatible aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and Dextran (DEX) as the phase forming polymers (
This method was used to generate a monolayer of cell surrounding a circular area defined by the size of the rehydrated DEX droplet. Suspension of cells is mixed with the PEG phase and added to the culture dish containing a DEX film. Cells mainly adhere to the surface around the rehydrated DEX drop and the interfacial tension between the PEG phase and the aqueous DEX phase prevents cells from penetrating into the droplet. After incubation for 30 min, culture medium is added to increase the volume to working volume. This dilutes out the polymer content of the culture system and results in a single aqueous phase. Migration of cells into the available empty space can then be monitored.
This Example describes an exemplary immunoassay system.
All reagents were prepared in a darkly lit room/Cell culture hood, wrapped in foil and placed in desk drawer for incubation period.
Next, the ATPS mixture was pipeted into a well of an Opti-Plate 384. The microplate was placed into a PHERAStar Microplate reader for an hour to cause PEG and DEX to equilibrate and form two-phase system. The AlphaLISA signal is temperature sensitive the microplate was brought to temperature of plate reader. Readings are then taken. It was observed that readings could be taken well into the low μg/mL or femtomolar range of TNFα (
This ATPS immunoassay microarray has advantages over conventional assays even if not performed with dehydrated ATPS in that the amounts of reagents used can be small, and multiple microscale singleplex assays can be performed in parallel without significant crosstalk of reagents as occurs in conventional multiplexed immunoassays.
Stock solutions of 20% (w/w) PEG (MW:35,000, Fluka) and 20% (w/w) DEX (MW:500,000 Pharmacosmos) were prepared in dH20. In 10 ml corneals, 19 ATPSs of differing compositions ranging from 0.56% (w/w) PEG-19% (w/w) DEX to 16% (w/w) PEG-0.94% (w/w) DEX were prepared using the stock phase solutions. The weight of each conical with the ATPS was recorded. Each ATPS was titrated drop-wise with dH20 until a single phase was obtained. The conical was centrifuged at 2000 g for 3 min to ensure formation of a single phase. The final weight of the conical with the one-phase system was recorded and used to calculate the weight of diluent added for one-phase formation. The binodal was determined through plotting the final composition of each system. A relationship derived by Merchuk et. al was fitted to the data using a non-linear least squares regression (Merchuk et al, J. Chromat. B 1998, 711, 285).
6.4%) (w/w) solution of the DEX 500K phase was prepared in culture media and kept at 4° C. until use. The tip magazine of an automated liquid handler (CyBi-Well, CyBio) was loaded with 96 sterile 25 μL pipette tips (CyBio). 75 μL of the DEX solution was pipetted into each well of a 96-well microplate (Corning), which was then placed on the stage of the liquid handler. To load pipette tips with the DEX solution, the stage was slowly raised until the pipette tips were slightly inside the DEX solution. 10 μL of the DEX solution was aspirated into the tips at a rate of 5 μL/s. The loaded tips were brought within 200 μm of the bottom of the wells of an empty microplate and 0.8 μL of the DEX solution was dispensed onto the surface at a rate of 0.5 μL/s to form circular droplets. The microplate was kept in a culture hood for at least 24 hrs to allow the droplets to dry. Images of the dried droplets were captured using a bright-field microscope (Nikon, TS-200). The Feret diameters of the droplets were determined using the “Analyze Particles” function of ImageJ (NIH). Droplets whose Feret diameters were not within ˜1% of the desired mean diameter were excluded from use in the assay. The entire printing and drying process was performed in a sterile culture hood.
Cell Culture
Three different human cancer cell lines were used: MDA-MB-231 breast cancer cells (ATCC) were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Invitrogen), 1% glutamine (Invitrogen), and 1% antibiotic. A549 lung cancer cells (ATCC) were cultured using F-12K medium (ATCC) supplemented with 10% HI-FBS and 1% antibiotic. PC-3 prostate cancer cells were maintained in DMEM supplemented with 10% HI-FBS and 1% antibiotic. Cells were maintained in a cell culture incubator at 37° C. with 5% CO2 and >95% humidity. When at desired confluence, cells were washed with PBS and Hank's-based enzyme-free cell dissociation buffer was added to the flask. Cells were incubated for 30 min and then collected and suspended in complete growth medium. After centrifuging down for 5 min at 4° C. and 1000 rpm, cells were resuspended in appropriate volume of the culture medium to give a density of 2×106 cells/ml.
Cell-exclusion patterning using the two-phase media 5.0%) (w/w) solution of the PEG 35K phase was prepared in culture media and kept at 4° C. until use. This solution was added to the cell suspension at a 1:1 (v/v) ratio to give a final PEG phase concentration of >2.5%. Prior to this step, cell suspension density was adjusted by addition of culture media to result in a final density of 4.5×104 MDA-MB-231 cells, 4.0×104 A549 cells, and 4.5×104 PC-3 cells after addition of the PEG-containing media. These cell densities were selected from preliminary experiments that evaluated the number of cells required to form a confluent monolayer per unit surface area. 80 μL of the resulting solution was added to each well of a 96-well plate, which was incubated at 37° C. with 5% CO2 and 95% humidity overnight (12 hrs). Then, the two-phase media was washed out and replaced with regular culture media. This was set as the time zero of the migration experiment.
Treatment of Cells with Blebbistatin and Anti-Cancer Drugs
After allowing MDA-MB-231 cells to spread and form a confluent monolayer around the cell excluded gaps, they were treated with (±)-blebbistatin at different concentrations in the range 5-200 μM or anti-cancer drugs paclitaxel, colchicine, and nocodazole in the concentration range of 1-1000 nM. Cells were incubated for 18 hrs in the presence of blebbistatin, washed with PBS once, and regular culture medium was added. Incubation continued for another 12 hrs before imaging. To study the effect of anti-cancer drugs, cells were incubated with each drug for 2 hrs, washed with PBS, and then supplied with culture medium. Imaging was performed after 30 hrs of incubation. Experiments were set in 30 replicates. All reagents were purchased from Sigma.
After migration experiments were complete, cells were stained with a 5 μM Calcein AM and the plates were incubated at 37° C. for 30 min. The dye solution was gently removed and culture medium was added to wells.
MDA-MB-231 cells were stained for actin filaments. First, cells were fixed in a 4.0% paraformaldehyde (Sigma) solution for 10 min. After washing cells with PBS, they were permeabilized with 0.1% Triton X-100 (Sigma) solution in PBS for 5 min. To reduce nonspecific binding, cells were treated with 1.0% BSA as the blocking reagent for 30 min. Cells were incubated with Alexa Fluor 594-conjugated Phalloidin at a concentration of 165 nM for 20 min. Finally, cells were washed three times with PBS before imaging using an inverted fluorescence microscope (Nikon, TE300). The resulting images were pseudocolored in Photoshop 10.0 CS3 (Adobe).
The migration assay is based on movement of cells from a confluent monolayer into a circular cell-excluded area within the monolayer. To develop this assay, an aqueous two-phase system was utilized. A droplet of the denser aqueous phase, DEX, printed on a tissue culture dish is allowed to dehydrate and form a solid disk (
The importance of interfacial tension for this cell patterning procedure was demonstrated by comparing the efficacy of two biphasic systems with a similar PEG concentration of 2.5% but different DEX concentrations of 3.2% and 6.4%. The interfacial tension of an ATPS is determined by the concentration of the phase forming polymers. A phase diagram of this polymer pair was used to determine the initial and final compositions of both systems (
Migration of MDA-MB-231 cells into the empty circular spot did not start until cells had completely spread and covered small intercellular spaces. Monitoring cells over time post printing showed that tight association of cells and the initiation of this process takes about 12 hrs. This is consistent with the finding that a threshold cell density is needed before the cellular gap closure process starts (Rosen et al, Proc. Natl. Acad. Sci. USA 1980, 77, 4760). At this point, cells present at the free edge experience pressure from the monolayer side. The presence of the circular cell-excluded discontinuity within the epithelium creates a net force on the boundary cells to move in the path of least resistance into the free available space until it is fully occupied by migrating cells (
The cell migration technology was adapted to a high throughput format by printing a single DEX droplet in each well of a 96-microwell plate. Subsequent addition of cells suspended in the PEG phase generated parallel migration assays. A major challenge with the use of high-content cell migration platforms is image analysis and quantification of a large number of experiments. Cell migration was quantified by calculating the percentage of closure of the cell-excluded area over a given time period before the gap is completely closed, i.e. % Area closure=[(Al−A2)/Al]×100, where Al and A2 denote the area of cell-excluded region at the beginning and endpoint of an experiment.
To determine Al and A2, cells were stained with a fluorescent dye and imaged at a magnification of 2.8×. In order to perform unbiased measurements of area on a large number of images, an automatic protocol to process images and measure the void area within each image not occupied by migrating cells was developed. Using the scripting language of ImageJ (Abramoff et al, Biophoton. Inter. 2004, 11, 36), each image was loaded and its background was removed. Then all empty regions within the image were determined using thresholding. Subsequently, a set of despeckling, dilating and eroding steps was used to remove artifacts of thresholding and further correct the images by eliminating regions and objects that were too small compared to the size of a cell. This resulted in a single open area in the middle of the image with a precise contour of the edge formed by the migrating cells. To account for the small number of cells already inside this area, they were subtracted them from the total area. Overlaying the resulting image with the original unprocessed image confirms correct identification of the border of migrating cells and the accuracy of this image processing procedure. This procedure was performed automatically for all images resulting in the corresponding area data. After ensuring that the size of cell-excluded area was consistent throughout a plate for a given printed DEX droplet diameter, only cells at the end time point of the experiment to were stained and imaged, in order to save user time.
Migration of MDA-MB-231 cells was studied in microwell plates containing cell-excluded spots with three different diameters of 1.6 mm, 0.8 mm, and 0.5 mm. Cell seeding density was adjusted to give equal number of cells per unit area, i.e. ˜1400 cells/mm2, in all three conditions. Imaging was carried out every 4-6 hrs until the initial gap was completely occupied by migrating cells. A gap size-dependent closure rate of cell-excluded areas was observed, with the highest rate of cell migration into the smallest gap and the slowest rate into the largest gap (
The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, the basic mechanism of cell motility is similar in many cases and involves polarization of cell body accompanied by continuous reorganization of actin cytoskeleton, integrin-mediated adhesion to the underlying substrate, formation of membrane protrusions at the cell front, traction of cell body through actomyosin-mediated contraction, and disassembly of adhesion sites at the rear of the cell to enable the cell translocate. As a test of the platform to assess cell migration, cells were treated with a potent inhibitor of machinery of cellular motility, blebbistatin. This compound interferes with rigid actomyosin cross-bridging and abates cell motility (Kovacs et al., J. Biol. Chem. 2004, 279, 35557; Watanabe et al, Am. J. Physiol. Cell Physiol. 2010, 298, CI 118). MDA-MB-231 cells were treated with six different concentrations of blebbistatin ranging from 5-200 μM for 18 hrs. Cells showed a dose-dependent response to blebbistatin and their migration capacity decreased significantly with increasing concentration of the inhibitor, especially at concentrations greater than 50 μM (
The effect of three different compounds on migration of MDAMB-231 cells was screened. Paclitaxel, colchicine, and nocodazole, each of which interferes with cell proliferation and normal dynamic function of microtubules were utilized. Paclitaxel is a potent cancer chemotherapeutic agent that stabilizes microtubules and prevents their depolymerization to tubulin monomers (Schiff et al, Proc. Natl. Acad. Sci. USA 1980, 77, 1561; Yvon et al, Mol. Biol. Cell 1999, 10, 947). Colchicine and nocodazole, on the other hand, inhibit polymerization of tubulin monomers to microtubules (Cronstein et al, Clin. Invest. 1995, 96, 994; Mythreye et al, Proc. Natl. Acad. Sci. USA 2009, 106, 8221). Drugs were tested at four different orders of magnitude of concentration in the range of 1-1000 nM. Cells were treated with compounds for a 2 hr period because it has been indicated that these agents can be effective even at short treatment times. Paclitaxel showed potency for inhibiting cell motility within the concentration range of 10-100 nM (
To demonstrate generality of the method for use with different cell types, two other cancer cell lines were tested in addition to the breast cancer cells (MDA-MB-231): A549 lung cancer cells and PC3 prostate cancer cells. The results in
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in electrical engineering, optics, physics, and molecular biology or related fields are intended to be within the scope of the following claims
This application is a Continuation of U.S. patent application Ser. No. 13/635,697, filed Dec. 9, 2011, which is incorporated herein by reference and which is a U.S. National Stage entry of International Application PCT/US2011/028941, filed Mar. 18, 2011, which claims priority to U.S. Provisional Application 61/315,183, filed Mar. 18, 2010, which is incorporated herein by reference.
This invention was made with government support under HL084370 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61315183 | Mar 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13635697 | Nov 2012 | US |
| Child | 14879566 | US |
