Patent Application
20080293157
The disclosed invention relates generally to a test device and more particularly to a test device configured to provide high throughput cell-culture studies on biomaterials.
Known multi-well microplates, (e.g., 96-well plate, 384-well plate, 1536-well plate, and others in such a series of increasingly miniaturized well-sizes) are used for high-throughput studies for cell-culture and bioassays, as well as non-biological testing, such as residuals release and durability of materials. Such high-throughput testing approaches, however, have not been used for evaluation of biomaterials. Their application in cell-culture studies is more common for studying soluble molecules and drug-candidates (e.g., in the pharmaceutical industry). Typically, testing of biomaterials includes the use of individual sample coupons. For example, round 1 cm or 1.5 cm discs or coupons are constructed with a particular biomaterial and fitted within a multi-well microplate test assembly. Such test assemblies typically are each separately placed into wells of a cell-culture plate, presenting inherent disadvantages. For example, the individual coupons can be difficult to handle and have increased incidence of contamination and/or damage due to handling. Such damage and/or contamination can result in more variability and inaccuracies in test results. Moreover, artifacts can be introduced in the experimentation, such as cultured cells crawling under the samples from the edges, as well as other edge-effects (e.g., circular samples having different surface finishes and properties at their edge due to their processing).
Other problems associated with these small coupons are higher costs due to the inability to recycle damaged coupons, and an inefficient low-throughput process, as a coupon represents a single replicate of a biomaterial sample. In addition, the set-up time for testing with coupons is typically long, as each coupon is typically secured by a medical-grade silicon gasket and placed individually into a well on a microplate. The result can be long turn-around times per assay, and fewer replicates per assay. Moreover, high-throughput analytical methods cannot be effectively leveraged to complete efficient end-point analysis with the coupon or individual samples. Another consideration is that automation and robotic methods can be difficult to apply in this scenario. Because of these problems, and due to variation that can be generated between users, test standardization is also difficult. Typically, the statistical strength of the testing is also reduced since the number of replicates that can be accommodated is reduced.
Thus, a need exists for an apparatus and method for testing biomaterials that provides high-throughput capabilities, improved productivity, and improved accuracy of results.
In one embodiment, a kit includes a base and a specimen removably couplable to the base. A top plate defines a plurality of apertures and is removably couplable to the base such that at least two of the apertures are associated with a specimen and each aperture defines a well with that specimen. Each well is configured to receive a sample material therein in contact with the specimen. A method includes disposing a specimen within a recessed portion of a test apparatus and coupling a top plate of the test apparatus to a base of the test apparatus such that a sealing engagement is formed between the top plate and the specimen. The top plate defines a plurality of apertures, each of at least two of the apertures collectively with the specimen defines a well. A sample material can be disposed within at least one well.
The apparatuses and methods described herein can be used to test the effects of various sample materials on one or more specimens, such as a specimen constructed with a biomaterial. The apparatuses and methods provide for high-throughput testing and improved performance and accuracy over known test apparatuses and methods. For example, in some embodiments, the release of residuals, biological response and durability of variations on a single material or multiple materials can be tested within a single test and using multiple replicates. The specimen can be optionally coated with, for example, a polymer material, and the effects of various sample materials on the polymer material can be evaluated during a single test cycle. The apparatuses and methods described herein can be used for evaluation of both biomaterials (e.g., biocompatible materials) and material evaluation from non-biological perspectives. For example, the apparatuses and methods can be used to test metals, polymer-coated metals, compressed polymers and other materials used in medical device development. The apparatuses and methods can also be used to test the effects of sample materials on specimens constructed for example, with a mesh, fabric or film, or specimens including a liquid or therapeutic agent. A fixture is also described herein that can be used during a specimen coating procedure, such as spray-coating a polymer material onto a metal specimen.
As stated above, the apparatuses and methods described herein can be used for non-biological testing of materials (e.g. drug-release kinetics from polymers). Moreover, the invention can be applied to studying materials with a combinatorial approach (i.e. with a systematic variation in material samples along the testing-space on a high-throughput layout). Also, the apparatus is amenable to adapt to robotics, automation and high-throughput analytical methods.
In one embodiment, a kit includes a base and a specimen removably couplable to the base. A top plate defines multiple apertures and is removably couplable to the base such that at least two of the apertures are associated with a specimen, and each of the apertures can define a well with that specimen. Each well is configured to receive a sample material therein in contact with the specimen. A method according to an embodiment of the invention includes disposing a specimen within a recessed portion of a test apparatus and coupling a top plate of the test apparatus to a base of the test apparatus such that a sealing engagement is formed between the top plate and the specimen. The top plate defines a plurality of apertures; each of at least two of the apertures collectively with the specimen defines a well. A test material can be disposed within at least one well.
The term “specimen” is used herein to mean an item having a surface formed of a material to be evaluated. A specimen can take one of a variety of different shapes, sizes and forms. A specimen can also have a variety of different types of surfaces to be tested. For example a specimen can have a planar surface, a curved surface, a textured or roughened surface, etc. A specimen can be a variety of different shapes, such as, for example, oval, round, rectangular, elliptical, semi-circular, or diamond shaped. For example, a specimen can be an elongated strip of material. A specimen can also be formed with one or more of a variety of different materials as will be described in more detail below.
The term “sample material” (also referred to herein as “test material”) is used herein to mean a substance, material, component, dye, cell, biological material, non-biological material, plant matter, etc. that can be disposed within a well of a test apparatus adjacent to and/or in contact with a surface of a specimen. In some embodiments, the sample material is a biological material and the effects of the sample material on the specimen can be tested. In other embodiments, the sample material is a non-biological material, such as a buffer solution, that is used to evaluate the rate of elution of an agent, such as a pharmaceutical, from a polymer that is coated on a specimen.
The term “biomaterial” is used herein to mean a biocompatible material used for the construction of medical devices or material being developed for use in the construction of medical devices.
The test apparatus 20 also includes one or more specimens 34 that can be removably received within a recessed portion defined by a frame 56 that is disposed between the base 22 and the top plate 28. The recessed portion of the frame 56 includes a surface countersunk from an upper face 26 of the frame 56. The specimens 34 can be disposed on the frame 56 such that an upper surface 36 of the specimen 34 is substantially flush with, lower than, or extending above the upper face 26 of the frame 56. In some embodiments, the test apparatus 20 does not include a separate frame 56. In such an embodiment, the base 22 can define a recessed portion having a surface countersunk from the upper face 24 of the base 22 configured to receive one or more specimens 34.
When the top plate 28 is coupled to the base 22, an upper surface 36 of the specimen 34 is associated with one or more apertures of the top plate 28 and defines a well with each aperture (not shown in
The lower face 32 of the top plate 28 can optionally be formed with a material to provide a sealing fit between the top plate 28 and the specimen 22. Thus, each of the wells defined by the apertures and the specimen 34 will be sealed at the lower face 32 of the top plate 28. In other embodiments, the test apparatus 20 can include a gasket 40 to provide a sealing fit between the top plate 28 (and the apertures on the top plate 28) and the upper surface 36 of the specimens 34. The gasket 40 can be removably disposed between the frame 56 and the top plate 28 (e.g., on the upper face 26 of the frame 56). The gasket 40 defines openings (not shown in
The specimens 34 can be formed with various materials and compositions, such as, for example, biomaterials such as metals (e.g., stainless steel, titanium), polymers (e.g., compressed polymers), polymer-coated metals, polymer-coated plastics, and other materials used in the development of medical devices. The specimens 34 can be substantially planar and rigid. In alternative embodiments, the specimens 34 can be flexible and include, for example, films of various compositions, such as films that are sprayed, molded or cast. The specimens 34 can also be formed with meshes or fabrics, glass, plant matter or minerals, or with various biological materials such as, for example, bone, tissue, cartilage, and cell sheets. The specimens 34 can also include a material such as a liquid film, or a material saturated or including a therapeutic agent. The specimens 34 can have a smooth or a polished surface to be tested, or alternatively have a treated surface, such as a surface that has been, for example, sanded, plasma treated, or bead blasted.
When assembled, the test apparatus 20 can be used to evaluate the effects of various sample or test materials on one or more specimens 34. For example, one or more sample materials 42 can be disposed within one or more of the wells such that the sample materials 42 are adjacent to or in contact with a specimen 34 associated with the well(s). The sample material(s) 42 can include, for example, fluids, cells, plant matter, etc. Other materials can be disposed within a well such as, for example, a dye that can be used to test the integrity of the seal between the specimen 34 and the top plate 28. After the sample material 42 is disposed within the wells, a cover 44 can optionally be placed over the top plate 28 to seal the wells at the top face 30 of the top plate 28. The wells can be covered or sealed collectively, or individually. In some embodiments, a cover 44 can be in the form of a film. In other embodiments, the cover 44 can be a rigid or flexible lid.
After the sample material(s) 42 has been disposed within the wells, the sample material(s) 42 can then be incubated for a selected period of time (“test cycle”) and the effects of the sample material(s) 42 on the specimen 34 can then be evaluated after the designated test cycle has ended. The test apparatus 20 can be used to test the effects of biological materials (e.g., cell-culture, cell-growth, cell-interaction, bioassays) and non-biological materials on both biomaterials and non-biomaterials.
In some embodiments, the test apparatus 20 can be used to test the kinetics of drug release (“KDR”). For example, the rate of release of a drug (e.g., a therapeutic agent) disposed, embedded, saturated, etc. within a coating (e.g., a polymer coated on a surface of a specimen) on a specimen, or from a polymer specimen, can be tested and evaluated. For example, a stainless steel specimen can have a polymer coating disposed on its surface and a therapeutic agent can be disposed within the polymer. A sample material in the form of, for example, a liquid, or other buffer solution, can be disposed in one or more wells. The buffer can be, for example, a phosphate buffered saline (“pbs”). As the polymer breaks-down (e.g., deteriorates, degrades, etc.) and the therapeutic agent is eluted from the polymer, the eluted portions will be disposed within the buffer solution. The buffer solution can be tested and analyzed to determine the rate of release of the therapeutic agent from the polymer. The test apparatus 20 can also be heated, cooled, or agitated to induce drug release from the specimen. Samples can be taken from the well, or sensors can be used to analyze the well contents and monitor the drug release from the specimen. The rate of degradation of the coating or material on the specimen (e.g., the polymer) can also be tested.
Thus, a test apparatus according to the invention can be used for a variety of different test applications. For example, a test apparatus can be used for testing the effects of biological and non-biological materials on a specimen, testing drug-elution from a polymer specimen or a polymer coated specimen, or testing the specimen itself, for example, the mechanical properties of a polymer specimen, testing for leachables, biocompatibility of a specimen, etc. The test apparatus and methods described herein provide a high-throughput and efficient test process, which can also be used to leverage subsequent testing.
The test apparatus 20 can be used to test multiple different specimens 34 within a single test cycle. In addition, because a single specimen 34 is associated with multiple wells, multiple different sample materials can be tested on a single specimen 34. The ability to test and compare the effects of multiple sample materials on a single specimen 34 reduces the variability and inaccuracies that often occur when each sample material is tested on a different single test coupon. In addition, the specimens 34 are larger in size than a typical test coupon, and therefore, easier to handle during insertion and removal of the specimens 34 to and from the test apparatus 20. This easier handling enables a user to remove the specimens 34 after the test cycle so that the specimens 34 can be further evaluated, for example, on a device such as a spectrophotometer.
As described above, some specimens may be coated with for example a polymer. The configuration of the specimens 34 allow them to be easily coated with a polymer, or other material, prior to testing the specimen 34 in test apparatus 20. In some embodiments, a fixture 46 can be used to hold the specimens 34 during a coating process. The use of a fixture 46 further improves the process of coating the specimens 34, and reduces damage and contamination that can occur when coating smaller coupons.
In some embodiments, the test apparatus 20 can be included in a kit that includes a reader tray 46. The reader tray 46 can define a recessed portion similar to the frame 56 such that the specimens 34 can be placed within the reader tray 46 after the test cycle. In some embodiments, the reader tray can be configured to receive the frame 56 therein, with the specimens disposed within the recessed portion of the frame 56. The reader tray 46 provides a clean and efficient method for transporting the specimens 34 from, for example, the test apparatus 20 to a spectrophotometer or other device for further evaluation.
The top plate 28, the base 22, the frame 56, and the gasket 40 can each be formed with various materials, preferably materials that can be sterilized for repeated use, such as stainless steel and silicone rubber. The top plate 28 can be configured to meet the ANSI standard for well plates (e.g., ANSI/SBS 1-2004, 2-2004, 3-2004, 4-2004), which will allow the test apparatus 20 to be used with existing automated testing equipment. The various components of the test apparatus 20 are removably coupled together, allowing for the test apparatus 20 to be disassembled for washing and sterilizing, and reassembled as desired.
Having described above various general principles, several example embodiments are now described. These embodiments are only examples, and many other configurations of test apparatus 20 and its various components are contemplated, and will be apparent to the artisan in view of the general principles described above and the exemplary embodiments.
The base 122 includes an upper face 124 (
A gasket 140 can be disposed between the frame 156 and the top plate 128, as shown in
In this embodiment, the coupling mechanism by which the top plate 128 is removably coupled to the base 122 includes a set of pins 152 (
As shown in
As stated above, in some embodiments it may be desirable to apply a coating or layer of material to a surface of a specimen prior to placing the specimen within the base 122.
Also as stated above, in some embodiments, a reader tray can be provided. A reader tray 148 can define a frame 170 similar to the frame 156 configured to receive the specimens 134 therein, as shown in
In this embodiment, the base 222 defines a recessed portion 261 having a countersunk surface 263 and that is configured to receive specimens 234, as shown in
In some embodiments, a test apparatus according to the invention can be used to test layered materials. For example, a specimen can be coated with a layer of material, and then a shim or gasket, a mesh, fabric and/or a second coated specimen. These layers of various specimens, films, meshes, plant/animal matter, fabrics, spaces and liquids can be used to simulate material and drug interactions with cells in a lab environment.
A test apparatus 520, illustrated in
As stated above, the specimens can be a variety of different shapes and sizes.
In some embodiments, prior to coupling the top plate to the base, a gasket can be disposed between the base and the top plate, at 90. The gasket can provide a sealing fit between the top plate and the specimen. The top plate defines a plurality of apertures. Each aperture from the plurality of apertures collectively with a specimen defines a well. A sample or test material is disposed within at least one of the wells at 92. The sample material can be, for example, a biological material, a non-biological material, a fluid, a dye, a cell, etc. At 94, the sample material is incubated for a predetermined time period. At 96 the top plate can be removed from the base, and at 98 the specimen can be removed from the test apparatus. At 100, the specimen can optionally be placed in a reader tray after being removed from the test apparatus. The reader tray can then be placed on or within another instrument, such as a photospectrograph, at 102.
While various embodiments of the invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the invention should not be limited by any of the above-described embodiments, but should be defined only in accordance with the following claims and their equivalents.
The previous description of the embodiments is provided to enable any person skilled in the art to make or use the invention. While the invention has been particularly shown and described with reference to embodiments thereof, it will be understood by those skilled in art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
For example, the various features of a test apparatus 20 (120, 220, 320, 420, 520) may include other configurations, shapes and materials not specifically illustrated, while still remaining within the scope of the invention. Thus, a test apparatus can include various combinations and/or sub-combinations of the various features and/or components described in the various embodiments herein. Also, any number of different layers of substrates, sample materials, plates, tissue, meshes, films, coatings, etc. can be tested with a test apparatus described herein.
