Patent Application
20140194321
1. The Field of the Invention
The present invention relates to methods, systems, and computer program products for determining if a compound is a skin sensitizer. More specifically, the present invention relates to using in vitro cells and cell-based assay methods to determine if compounds are skin sensitizers.
2. The Relevant Technology
Allergic contact dermatitis (ACD) is a detrimental health effect that can develop in those exposed to skin sensitizing chemicals and products that contain the chemicals. To decrease the occurrence of this adverse reaction, U.S. Food and Drug Administration (FDA) regulations require that testing be performed to identify chemicals that are responsible for this effect. Products that contain the skin sensitizing chemicals can then be labeled accordingly.
Historically, the use of animals has been required for testing chemicals for the potential for skin sensitization in the U.S. and the E.U. The currently accepted method of testing is the local lymph node assay (LLNA). The LLNA measures cell proliferation in the draining lymph node of a test animal as a measure of skin sensitization. The cell proliferation is measured via radiolabeled cells, following dermal exposure to the compounds during the induction phase of sensitization. Many live animals, typically mice, are required to perform the LLNA for each chemical compound tested.
Recently, a modified version of the LLNA (known as the revised LLNA or rLLNA) was developed that uses a chemical method of measuring cell proliferation and decreases the animals required for the testing of each chemical compound.
Although the LLNA and the rLLNA are able to determine the skin sensitivity of compounds that are tested, both the LLNA and the rLLNA suffer from significant drawbacks. They are time and labor intensive and must use a number of animals to test each chemical compound. And because many consumer chemical compounds must be tested each year for skin sensitivity, a significant number of animals are required. Not only is it expensive to house and maintain the animals, but the chemicals often harm or even disfigure the animals. In addition, despite the LLNA being used as the standard testing method, discrepancies have been found between results of the LLNA and previously used guinea pig tests as well as human data.
In light of the above, providing a simple, efficient, in vitro method of testing for skin sensitizers is of particular relevance to consumer product safety and occupational health.
Various embodiments of the present invention will now be discussed with reference to the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. In the drawings, like numerals designate like elements. Furthermore, multiple instances of an element may each include separate letters appended to the element number. For example two instances of a particular element “20” may be labeled as “20a” and “20b”. In that case, the element label may be used without an appended letter (e.g., “20”) to generally refer to every instance of the element; while the element label will include an appended letter (e.g., “20a”) to refer to a specific instance of the element.
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. It will also be understood that any reference to a first, second, etc. element in the claims or in the detailed description, is not meant to imply numerical sequence, but is meant to distinguish one element from another unless explicitly noted as implying numerical sequence.
In addition, as used in the specification and appended claims, directional terms, such as “top,” “bottom,” “up,” “down,” “upper,” “lower,” “proximal,” “distal,” “horizontal,” “vertical,” and the like are used herein solely to indicate relative directions and are not otherwise intended to limit the scope of the invention or claims.
The basic premise of animal based sensitizing assays is that the assays ultimately produce proliferation in immune cells of the animal, specifically to the draining lymph node from the site of treatment. The LLNA is a simple test that enumerates these proliferating cells. However, the test is costly due to the labor required to perform the assay and house the animals, the disposal of radioactive markers (if used), and the time to analyze the results. Furthermore, the use of live animals for chemical testing is frowned upon by the general public. In light of the above, there is a concerted effort to develop assays that replace animal testing, are high throughput, and do not lose any information in the transition from in vivo to in vitro.
To this end, multiple labs have attempted to devise a method of obtaining similar results to the LLNA via a flow cytometric approach using either primary cells or cell lines. Similar to the LLNA, these assays produce a population-based result. However, instead of using cell numbers, as in the LLNA, the flow cytometric assays analyze the production of particular proteins in the treated cells over a background. While the ultimate outcome of the LLNA is based on cell counts, the LLNA also implicitly identifies that cells travel e.g., from the ear to the lymph node of the mouse that is used for testing. Because flow cytometry methodology lacks a functional aspect, the cytometric assay is simply unable to replicate this aspect of the LLNA.
In contrast, embodiments of the present invention are directed to methods, systems, and computer program products for determining skin sensitivity of compounds using an in vitro assay that replicates the in vivo effects of a skin sensitizer. These effects, which are quantitatively measured in embodiments of the present invention, can include differentiation or maturation with protein production, an increase of cell number/proliferation, and cell trafficking or motility.
Compared to conventional methods (e.g., the LLNA and rLLNA), determination of skin sensitivity according to embodiments of the present invention can be performed in less time, is less labor intensive, and does not require the use of live animals. In addition, unlike in the LLNA and the rLLNA, aspects of the overall skin sensitization pathway can be quantified, and this quantification be done in a cell line based format.
In addition, the collected cell number and morphology data can indicate concentrations at which the chemicals are toxic, allowing adjustments to be made so that determination of the chemical's skin sensitizing potential can be done at lower non-toxic concentrations.
Embodiments of the invention can employ the use of a high-content screening (HCS) system. In a high content screening assay, the processes discussed herein can be more efficiently recapitulated due to the ability to deal with spatial characterization as well as cell enumeration. As a result, information can be quickly and easily obtained for a range of concentrations for a compound.
Thus, embodiments of the present application can apply physical and functional changes over a range of concentrations to a decision tree that predicts sensitivity. Because the LLNA is itself a functional test, embodiments of the present invention provide the best opportunity for an in vitro replacement to the LLNA.
The innovative processes presented herein enable the prediction of skin sensitizing potential for chemical compounds with high specificity and sensitivity (i.e., low false positive and negative rates, respectively).
Although the discussion set forth herein is directed to using the innovative processes for predicting whether a compound is a skin sensitizer, it will be appreciated that the processes can also be used to determine other types of sensitivity. By way of example and not limitation, embodiments of the invention can also be used to determine whether other parts of the body, such as the mouth, throat, stomach, and lungs, among others are sensitive to particular compounds. Sensitivity potential of a compound used in conjunction with non-chemical insults, such as UV-radiation, can also be determined.
Embodiments of the present invention may comprise or utilize a special purpose or general-purpose computer including computer hardware, such as, for example, one or more processors, as discussed in greater detail below. Embodiments within the scope of the present invention also include physical and other computer-readable media for carrying or storing computer-executable instructions and/or data structures. Such computer-readable media can be any available media that can be accessed by a general purpose or special purpose computer system. Computer-readable media that store computer-executable instructions are physical storage media. Computer-readable media that carry computer-executable instructions are transmission media. Thus, by way of example, and not limitation, embodiments of the invention can comprise at least two distinctly different kinds of computer-readable media: computer storage media and transmission media.
Computer storage media includes RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store desired program code means in the form of computer-executable instructions or data structures and which can be accessed by a general purpose or special purpose computer.
A “network” is defined as one or more data links that enable the transport of electronic data between computer systems and/or modules and/or other electronic devices. When information is transferred or provided over a network or another communications connection (either hardwired, wireless, or a combination of hardwired and wireless) to a computer, the computer properly views the connection as a transmission medium. Transmission media can include a network and/or data links which can be used to carry data or desired program code means in the form of computer-executable instructions or data structures and which can be accessed by a general purpose or special purpose computer. Combinations of the above should also be included within the scope of computer-readable media.
Further, upon reaching various computer system components, program code means in the form of computer-executable instructions or data structures can be transferred automatically from transmission media to computer storage media (or vice versa). For example, computer-executable instructions or data structures received over a network or data link can be buffered in RAM within a network interface module (e.g., an “NIC”), and then eventually transferred to computer system RAM and/or to less volatile computer storage media at a computer system. Thus, it should be understood that computer storage media can be included in computer system components that also (or even primarily) utilize transmission media.
Computer-executable instructions comprise, for example, instructions and data which cause a general purpose computer, special purpose computer, or special purpose processing device to perform a certain function or group of functions. The computer executable instructions may be, for example, binaries, intermediate format instructions such as assembly language, or even source code. Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the described features or acts described above. Rather, the described features and acts are disclosed as example forms of implementing the claims.
Those skilled in the art will appreciate that the invention may be practiced in network computing environments with many types of computer system configurations, including, personal computers, desktop computers, laptop computers, message processors, hand-held devices, multi-processor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, mobile telephones, PDAs, pagers, routers, switches, traffic sensors, and the like. The invention may also be practiced in distributed system environments where local and remote computer systems, which are linked (either by hardwired data links, wireless data links, or by a combination of hardwired and wireless data links) through a network, both perform tasks. In a distributed system environment, program modules may be located in both local and remote memory storage devices.
Accordingly, in this specification and in the following claims, a computer system is also defined to include imaging systems (e.g., imaging system 102 in
System 100 can also include an external computing device 112, if desired. External computing device 112 can comprise a general purpose or specialized computer or server or the like, as defined above. External computing device 112 can be used as a controller for the system as well as for performing, by itself or in conjunction with imaging device 104, the analyzing and/or storing of the data obtained by imaging device 104. In some embodiments, external computing device 112 can also display results to the user on user display device 106. External computing device 112 can communicate with imaging device 104 and/or display device 106 directly or through a network, as is known in the art.
In one embodiment of the invention, one or more of the method steps described herein are performed as a software application. However, the present invention is not limited to this embodiment and the method steps can also be performed in firmware, hardware or a combination of firmware, hardware and/or software. Furthermore, the steps of the methods can exist wholly or in part on imaging device 104, external computing device 112, and/or other computing devices.
An operating environment for the devices of the system may comprise or utilize a processing system having one or more microprocessors and system memory. In accordance with the practices of persons skilled in the art of computer programming, the present invention is described below with reference to acts and symbolic representations of operations or instructions that are performed by the processing system, unless indicated otherwise. Such acts and operations or instructions are referred to as being “computer-executed,” “CPU-executed,” or “processor-executed.”
In embodiments of the present invention, skin sensitizing compounds can be identified via the induction of cell surface protein production along with functional chemotaxis assays that can also provide toxicity information for the compounds. Using the data from the biological assays, a prediction can be made as to whether a compound is a sensitizer or a non-sensitizer. In addition, the toxicity of the compound at the tested concentrations can be determined and, if toxic, a recommendation for additional testing of the compound for sensitization at non-toxic doses can be made.
The assay process can consist of several parts. A first part can include a specific manner of treating and fluorescently labeling cells of haemopoetic origin, and quantitatively measuring several imaged cellular properties, functions and/or targets (collectively referred to herein as “targets”) to detect the initiation of skin sensitizing potential. In one embodiment, cell lines derived from dendritic cells are used. A second part can include analyzing and correlating the quantitatively measured data (i.e., the targets) acquired in the first part to determine if the compound of interest is a skin sensitizer.
In many of the examples discussed herein, the cellular targets monitored in the assays are cell gain/loss, specific cell markers, and motility of the cells; however, other cellular properties, functions and/or targets can also be monitored.
In a second step 124 of the exemplary process, the quantitative multiparametric cellular target data acquired in step 122 is analyzed over the range of compound concentrations to determine if the compound of interest is a skin sensitizer.
The exemplary skin sensitization detection and analysis process 120 enables the systematic investigation of the effects a compound has on immune effector cells and accurately predicts therefrom the skin sensitivity of the compound with high specificity and sensitivity (i.e., low false positive and negative rates respectively).
To accomplish step 122, an assay can be performed on immune effector cells treated with various concentrations of a compound of interest, then the responses of those cells can be measured. In some embodiments of the invention, a multi-well microplate can be used.
An example of a multi-well microplate that can be used is well plate 130, shown in
For example,
Wells can also be associated with vehicle control samples (denoted “Vehicle Control” in
The indicators of skin sensitization can be classified in three types of cellular response: i) gross markers, ii) specific markers, and iii) functional outcomes. Any of these can be measured and quantified in the immune effector cells (e.g., U937) treated with compounds in embodiments of the present invention.
Gross markers are used to identify both total and live numbers of cells. Cells can be identified by the fluorescence staining of a major cellular structure, such as by a nuclear or a whole cell dye, and then counted to give the number of cells. This cell number can provide an indication of toxicity of the compound via cell loss. The cell number can be measured via manual counts (albeit labor intensive), flow cytometry, image analysis and plate reader methodologies. In addition to providing cell numbers, imaging and flow cytometry also have the ability to identify the shape of the nucleus or cell body to provide additional toxicity data.
Specific markers are indicative of specific cellular changes, identified by changes in specific cellular targets, induced by the presence of the compound. These specific markers can be cell-type specific and have a role in the differentiation of the cell type that is induced by sensitization. In skin sensitization, markers on U937 cells that are associated with differentiation, e.g., CD54, CD80, CD86, HLA-DR, can be quantified. In many embodiments discussed herein, CD54, an up-regulated marker, is used as the specific marker of U937 that is quantified. However, other cellular molecules can also be detected on the cell surface, either instead of, or in addition to CD54. The measurement of specific markers can also be detected using different detection methods and processes.
Since specific markers are usually cell surface protein molecules that are either translocated from inside the cell or from new protein production, the ability to detect specific markers can follow several forms. For instance, the proteins can be detected by immunofluorescence, by a fluorescent ligand, or with a specialized subclone of the cell type in question that produces a fluorescent variant of the molecule. Automated imaging and analysis or flow cytometry can be used to detect these changes.
Functional outcomes indicate functional responses induced by exposure to the compound. Because the LLNA is a functional test, as discussed above, using functional outcomes helps embodiments of the present invention to replicate the in vivo responses in an in vitro environment. The ability for cells to demonstrate a functional response to treatment of the compound can help to determine how sensitization compounds influence the differentiation of a cell line. Functional outcomes are typically measured using a chemotactic approach, as discussed in more detail below. Chemotaxis is the movement of cells in the direction of a chemical gradient and is useful for measuring many functional outcomes, such as, e.g., cell motility. Virtually any method of measuring movement in response to a compound that induces chemotaxis can be a valid measurement technique.
In step 142, immune effector cells are cultured on a microplate, such as well plate 130. In one embodiment, the area of interest is the skin; therefore the assay can be performed using U937 cells as the immune effector cells. Other types of cells can also be used depending on the body area for which sensitivity is being investigated.
U937 cells are of histocytic origin and mimic skin-resident Langherhans Cells (LCs) which are some of the first immunological cells that come in contact with a skin sensitizer in live beings. In particular, U937 cells mature from a monocytic to an LC-like phenotype after being exposed to sensitizers. U937 cells can be grown and cultured on multi-well microplates in optimal cell growth media,
In step 144, the cultured cells are treated with various concentrations of the compound of interest. Many cellular targets exhibit different responses at different doses of different compounds. For example, a cellular target may show its skin sensitivity response to a particular chemical at an intermediate concentration but may be toxic at a higher concentration and show no response at lower concentrations. To take these conditions into account, many of the embodiments disclosed herein simultaneous monitor responses of multiple cellular targets over a range of compound concentrations.
To do so, each well of cells in the microplate can be treated with a different concentration of the compound so that a range of cellular dose responses for each cellular target is obtained for the compound. For example, in the exemplary plate map 134 of
To take into account sample variations, a plurality of wells can be used for each concentration level of a compound to generate multiple samples for the concentration level. For example, in the exemplary plate map 134 of
In step 146, after being treated with the various concentrations of the compound, the treated cells are stained with specific fluorescent materials, as is known in the art, to detect different cellular targets, such as those discussed above, whose changes are indicative of skin sensitivity. For example, in one embodiment, the cells are stained to help detect the number of cells, the CD54 intensity, and the cell motility. The cell staining procedure can be optimized to ensure the proper staining of different cellular targets. Although specific cellular targets are monitored for the exemplary embodiments discussed herein, other cellular targets can also be monitored for other sensitization assessments by using similar fluorescent probes.
At this point, one of two branches is taken depending on whether a phenotypic approach or a chemotactic approach is required. For a phenotypic approach, which involves no additional chemicals that need to be partially separated from the chemicals in the well, step 148 is next performed; for a chemotactic approach, which involves using additional chemicals partially separated from the chemicals in the well, step 150 is next performed.
In step 148, cellular targets are imaged directly from microplate 130 to acquire images of the stained cells. The fluorescently labeled cells can be detected by manual microscopy or by using a fluorescence imaging system, such as, e.g., imaging system 102 (
As noted above, many functional outcomes, such as, e.g., cell motility, are measured using chemotaxis. To detect and measure these targets, steps 150-154 are followed to obtain the desired data. In step 150, the cells are transferred to a second plate or other device that can allow chemotaxis to occur. For example, after exposure to the compound, cells can be selectively stained with a viability dye to detect live cells. The stained cells can then be transferred to a particular type of microplate that is able to sustain a chemical gradient, such as, e.g., a chemotaxis plate. One embodiment of a chemotaxis plate that can be used with embodiments of the present invention is the Iuvo™ Chemotaxis Assay Plate #6006 manufactured by Bellbrook Labs of Madison, Wis. Of course, other chemotaxis plates can also be used.
In step 152, a chemotactic chemical, such as, e.g., RANTES/CCL5 or SDF-1/CXCL12, can be introduced to the second microplate as a chemokine to induce chemotaxis, and the stained cells can be allowed to migrate in response to the chemokine In many embodiments discussed herein, SDF-1 was used as the chemokine to induce chemotaxis. However, other chemokines can also be used to induce chemotaxis, such as MIP-3beta (CCL19) and CCL21.
As is known in the art, chemotaxis measurements can be performed in several manners. These can include using Boyden chamber based devices, such as, e.g., transwell inserts, that can fit in microwell plates, specifically manufactured microplates designed for chemotaxis that can sustain a chemotactic gradient (e.g., the Iuvo™ Chemotaxis Assay Plate #6006, discussed above), and simple assays where the cells are tracked during introduction of the chemokine.
The chemotaxis devices that subject the cells to a physical separation from the chemokine, may be advantageous, as those devices allow for chemicals that crystallize or fluoresce to be removed from the area of analysis. This enables a more accurate cellular count using automated methods. In many other methods, the presence of fluorescent compounds can make it difficult to identify and accurately count individual cells.
In step 154, once cell response to chemotaxis has occurred, the functional outcome can be imaged in a similar manner to that discussed above with respect to step 148.
In step 156, the acquired images from both phenotypic and chemotactic approaches can be analyzed to obtain and measure relevant data for each cellular target. Once multiple images for each condition have been acquired, different regional areas of each cell can be assigned by image analysis algorithms as are known in the art. For example, to obtain the number of cells, the nuclear region can be masked by DNA staining in the cell and a cytoplasmic area can be masked by the staining of the whole cell with the nuclear area subtracted. Cell number values can be determined by measuring the number of cells in a defined area. The amount of surface antibody staining of cells can be determined by the fluorescence that is associated with a certain region that is a defined distance away from the nuclei of the cell, but still within the area of the cell. Similar processes can be used to obtain data for other cellular targets, as is known in the art.
To accomplish step 124 of
In step 160, the data corresponding to the different compound concentrations is normalized. In one embodiment, measurements generated from the vehicle-treated cell image analysis can be used. Because the vehicle-treated cells have not been treated with any of the compound, the vehicle-treated cells represent a baseline for the cellular targets. As such, a normalization of the data obtained from the compound-treated cells can be determined by subtracting the baseline response of the vehicle-treated cells from the measurements of the responses of the compound-treated cells. Measured values of the compound-treated cells that are below baseline levels can be notated as lacking a response, and can be ignored in further analysis, if desired.
In step 162, a representative value, such as, e.g., the average value, of each of the observed targets of the samples can be calculated for each concentration of the compound. This can be done by calculating a mean value for the measured target at each concentration level, taking into account all of the samples corresponding to the particular concentration level.
For example, mean values for gross markers can be computed for the number of cells using either the number of cell bodies or the number of nuclei detected. For specific markers, mean values can be computed for each of the features (e.g., CD54 (ICAM-1) intensity). To get a relative amount (or intensity) of up-regulated markers (e.g., CD54 (ICAM-1)) per cell, the per-replicate amount of the staining can be divided by the number of cells for the particular replicate. For functional outcomes, mean values can be computed for the number of live cells that demonstrate movement. Other mean values can also be used. In addition, other representative values can also be used for each concentration of the compound, such as a highest measured value, a lowest measured value, or other representative values.
In step 164, a dose response curve is generated for each target. This can be done by plotting on a graph the average value of the target for each concentration.
In step 166, the dose response curves generated in step 164 are qualified to determine a reaction of the cellular targets to the increasing concentrations of the compound. In one embodiment, qualifying the dose response curve can be accomplished by categorizing the curve as “increasing,” “decreasing,” or “flat.”
For example, to determine the curve characterization of a dose response curve, the dose response curve can be compared to a predefined range, such as range 184 in
If the entirety of the curve falls within the predefined range, as is the case with dose response curve 172 of
If the entirety of the curve does not fall within predefined range 184, an evaluation can be made of the relationship between the lowest concentration of the compound and the highest value of the target. If the highest cellular target value occurs at the lowest compound concentration, the curve can be characterized as “decreasing.” For example, in
In contrast, if the lowest cellular target value occurs at the lowest compound concentration the curve can be characterized as “increasing.” For example, in
If the entirety of the curve does not fall within predefined range 184 and the lowest or highest cellular target values does not occur at the highest or lowest compound concentration, it may be unclear how the treated cells are reacting to the compound, and the particular cellular target might need to be discarded. For some cellular targets, these biphasic curves may be indicative of toxicity and more testing may be desired.
In step 170, a decision matrix is applied to the qualified dose response of one or more of the measured parameters to predict skin sensitization. The qualified dose response of any of the measured cellular targets can be used. For example, the cellular targets used in the decision matrix can be selected from the gross markers, specific markers and/or the functional response of the cells. Using the selected properties, a decision matrix can be constructed based upon the expected behavior of the cell type in response to the compound used. The dose response curve behavior (e.g., “increasing,” “flat,” or “decreasing”) of the measured cellular properties can then be used to determine the prediction of sensitizing capability of the compound. In some embodiments, the decision matrix can also be used to determine if the compound is toxic.
Although cell number and CD54 intensity responses are used in decision matrix 194, any of the other measured cellular targets can alternatively be used. For any of the cellular targets, the entry for each position in the decision matrix can be initially determined based upon the expected behavior of the immune effector cell type in response to the compound used. In addition, decision matrices can be designed that use more than two types of cellular targets as variables, again based upon the expected behavior of the immune effector cell type in response to the compound used.
Test data and results are now given. Both known skin sensitizers and known non-skin sensitizers were used for testing. Table 1 lists the compounds used for testing; Table 1a includes the known skin sensitizer compounds used and Table 1b includes the known skin non-sensitizer compounds used. The characterization of each compound as a skin sensitizer or a skin non-sensitizer is based on a previous European Union project, entitled “Sens-it-iv”, that determined which of the compounds were skin sensitizers (European Union, 6th Framework, Novel Testing Strategies for In Vitro Assessment of Allergens LSHB-CT-2005-018681).
An example protocol that was used to culture, treat, and stain the cell cultures during execution of the steps of the inventive methods discussed herein is now given. It is appreciated that the protocol discussed below is exemplary only and that other protocols can also be used.
The protocol is divided into two parts, the first directed to treatment and immunofluorescence of cellular targets that can be obtained using a phenotypic approach and the second directed to treatment and staining of cellular targets that can only be obtained using a chemotactic approach.
In both protocols, 96-well microplates, such as well plate 130 shown in
The compound solutions can be prepared in appropriate solutes. Because of the number of compounds tested per plate and the requisite dilutions, making a master plate of individual wells of the 2× compound solutions that correspond to the plate map is recommended, although not required.
Treatment And Immunofluorescence For Phenotypic Analysis.
For treatment, staining, and immunofluorescence for the phenotypic approach, approximately 10,000 U937 cells can be placed in 100 μl of RPMI-1640 complete media per well in a collagen-I coated 96-well plate and incubated for 16-24 hours at 37° C. in 5% CO2. To reduce variation between wells when using multiple plates, the plates can be spread in the incubator instead of stacked.
The compounds for which skin sensitivity is desired can then be introduced to the immune effector cells. For each concentration level of each compound, 100 μl of the 2× concentrated compound solution can be added to the corresponding wells. Similarly, 100 μl of vehicle solution can be added to the vehicle control wells and the 2× solution of the negative and positive controls can be added to the negative and positive control wells, respectively. The plate can then be incubated for 24 hours at 37° C. in 5% CO2.
After incubation, the plate can be centrifuged for 5 minutes at 125×g. The media can then be aspirated from the wells after which 50 μl of pre-warmed (e.g., to 37° C.) 4% paraformaldehyde can be added to each well. The plate can then be incubated at room temperature for 15 minutes.
After incubation, the plate can be centrifuged for 5 minutes at 125×g. The paraformaldehyde can then be aspirated from the plate and the wells rinsed twice with 1× Phosphate Buffered Saline (PBS).
50 μl of 0.1% Triton X-100 dissolved in 1×PBS can be added to each well. The plate can then be incubated at room temperature for 10 minutes. The plate can be centrifuged for five minutes at 125×g, after which the liquid can be aspirated from the plate.
A primary antibody solution can be prepared as follows: For each 96-well microtiter plate, 6 ml of PBS are added to a 15 ml conical tube. To this, 12 μl of a primary antibody against CD54 (e.g., ICAM-1) are added. The resulting solution in the tube can be briefly vortexed (e.g., for less than 10 seconds).
After aspiration of the liquid, discussed above, 50 μl of the prepared primary antibody solution can be added to each well and the plate can be incubated at room temperature for one hour.
After the primary antibody solution has been added and the plate incubated, the plate can be centrifuged for 5 minutes at 125×g. The primary antibody solution can then be aspirated from the plate, and the wells rinsed twice with 1× Phosphate Buffered Saline (PBS).
A secondary staining solution can be prepared as follows. For each 96-well microtiter plate, 6 ml of PBS can be added to a 15 ml conical tube. To this, 6 μl of a secondary antibody can be added against the primary antibody that recognized CD54, along with 3 μl of Hoechst and 50 μl of the whole cell dye. The resulting solution in the tube can be briefly vortexed (e.g., for less than 10 seconds).
After the primary antibody solution has been aspirated from the plate and the wells rinsed, as discussed above, 50 μl of the secondary staining solution can be added to each well and the plate can be incubated at room temperature for 30 minutes, protected from light (e.g., wrapped in aluminum foil).
The plate can then be centrifuged for 5 minutes at 125×g, after which the liquid can be aspirated from the plate and the wells rinsed twice with 1×PBS.
A final volume of 150 μl of 1×PBS can be added to each well and the plate can be sealed. Image acquisition can then be performed on the plate using an imaging system such as a high-content screening (HCS) instrument using appropriate image analysis software.
Treatment And Staining For Chemotactic Analysis.
For treatment, staining, and immunofluorescence for the chemotactic approach, approximately 30,000 U937 cells can be placed in 100 μl of RPMI-1640 complete media per well in an uncoated 96-well plate and incubated for two hours at 37° C. in 5% CO2. To reduce variation between wells when using multiple plates, the plates can be spread in the incubator instead of stacked.
The compounds for which skin sensitivity is desired can then be introduced to the immune effector cells. For each compound solution, 100 μl of the 2× concentrated compound solution can be added to the corresponding wells. Similarly, 100 μl of vehicle solution can be added to the vehicle control wells and the 2× solution of the negative and positive controls can be added to the negative and positive control wells, respectively. The plate can then be incubated for 24 hours at 37° C. in 5% CO2.
After incubation, 50 μl of a 5× solution of a live cell reagent in 1×PBS can be added to each well, after which the plate can be incubated for 45 minutes at 37° C. in 5% CO2.
The plate containing the treated cells can be carefully removed from the incubator so as not to jostle the cells from their settled state. 150 μl of the media can be removed from each well, and the cells in the remaining 100 μl of media can be resuspended.
A chemotactic solution of SDF-1 can be prepared at a concentration of 100 ng/ml. 200 μl of the chemotactic solution can be added to each well of a 96-well transwell plate with 8 μm pores. The transwell inserts can then be positioned within the wells so that no air bubbles are caught between the membrane of the inserts and the media. 50 μl of the resuspended cells from the original plate can then be added to the top of the transwell inserts.
The transwell plate can then be incubated for 2-6 hours at 37° C. in 5% CO2 until migration has been achieved in the positive control wells. Image acquisition can then be performed using an imaging system such as a high-content screening (HCS) instrument using appropriate image analysis software.
The protocols discussed above were employed in conducting an assay on the twenty four chemical compounds listed in Table 1. The overall results are shown in Table 2.
A 96-well plate similar to well plate 130 shown in
The test protocol was optimized for use with U937 cells. To maintain U937 cells, supplier recommendations were adhered to. Cells were cultured in RPMI-1640 media supplemented with 2 mM glutamine, penicillin-streptomycin, and 10% fetal bovine serum. Cells were fed by replacing 90% of the media when a concentration of one million cells/ml of media was reached. Cells were used at a passage ≦20.
Cells were harvested by centrifugation, and concentrated into complete media so that the density was at one million cells/ml. 100 μl of the cell suspension was added to each well of the 96-well microplate to achieve 10,000 cells/well. Cells were incubated overnight at 37° C. in 5% CO2 before compound treatment and then incubated again for 24 hours.
After the incubation with each of the compounds, the protocol discussed above was used to culture, treat, and stain the cell cultures. The following specific products were used during testing of each compound:
For each compound, cell images were obtained using the Thermo Scientific® ArrayScan® VTI HCS Reader, manufactured by Cellomics Inc. Representative samples 200 of the cell images obtained during the assay are shown in
The data for each compound were normalized and dose response curves generated, as discussed above. The number of cells and the CD54 intensity per cell were used as the cellular targets used for the dose response curves. The dose response curves of each compound were qualified in the manner discussed above, and the decision matrix shown in
Using hexylcinnaldehyde as an example from Table 1, the dose response curves 210 and 212 shown in
As shown in Table 2, the assay yielded an 67% accuracy rate and 100% sensitivity for the skin sensitivity predictions. That is, of the known skin-sensitizers tested, the assay correctly predicted that all of them were skin sensitizers or toxic.
The results did not predict the skin non-sensitizers with as much accuracy. While most of the known skin non-sensitizers were correctly predicted to be skin non-sensitizers, a few of the known skin non-sensitizers were incorrectly predicted to be skin sensitizers. However, all of the chemicals that were predicted to be skin non-sensitizers were indeed skin non-sensitizers. Thus, although a definitive prediction of skin sensitization was not obtained, one of skin non-sensitization was. That is, because all of the chemicals that were predicted to be skin non-sensitizers were indeed skin non-sensitizers, one can deduce that if the outcome of the assay indicates that a compound is a non-sensitizer, then the compound indeed is a non-sensitizer.
This information can be used to reduce the number of compounds required to be animal-tested for skin sensitivity. For example, one can use the assay as a preliminary screening to remove from further testing those compounds that are predicted to be skin non-sensitizers. Additional testing can then be performed on the chemicals that are predicted to be skin sensitizers or toxic. By eliminating the predicted skin non-sensitizers from further testing, a great deal of time, money, and effort can be saved, even if the other chemicals may need to undergo further testing.
Besides being useful as a screening tool, further testing will likely determine further cellular target combinations that may be even more accurate predictors of skin sensitivity than those used in present testing.
Many benefits to skin sensitivity testing over present methods are obtained by using embodiments of the present invention. Some of these include:
Although discussion herein has been directed to determining the sensitivity of skin to tested compounds using the methods presented herein, it is appreciated that other types of sensitivity can also be predicted using the methods. For example, sensitivity of other body tissue or organs to tested compounds can also be predicted. For example, sensitivity of the throat, the stomach, the lungs and associated airways, the eyes, etc. can also be predicted by imaging and analyzing corresponding cells according to the methods presented herein.
Furthermore, sensitivity potential of a compound used in conjunction with non-chemical insults, such as UV-radiation, can also be determined using the methods presented herein.
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
