Multi-well micropatterning by ablation
The present invention is drawn to the generation of micropatterns of biomolecules and cells on standard laboratory materials through selective ablation of a physisorbed biomolecule with oxygen plasma. In certain embodiments, oxygen plasma is able to ablate selectively physisorbed layers of biomolecules (e.g., type-I collagen, fibronectin, laminin, and Matrigel) along complex non-linear paths which are difficult or impossible to pattern using alternative methods. In addition, certain embodiments of the present invention relate to the micropatterning of multiple cell types on curved surfaces, multiwell plates, and flat bottom flasks. The invention also features kits for use with the subject methods.
This application claims the benefit of U.S. Provisional Application No. 60/851,101, filed Oct. 12, 2006, which application is incorporated herein by reference in its entirety.
GOVERNMENT SUPPORTThe U.S. government may have certain rights in this invention, pursuant to the Ruth L. Kirschstein National Research Service Award (NRSA), grant no. # 1F32DK072601.
BACKGROUND OF THE INVENTIONThe growth and function of anchorage dependent cells is closely tied to local microenvironmental cues surrounding the cell including the nature of the underlying substrate, the degree of cell-cell contact (both homotypic and heterotypic), paracrine signaling, and physical forces. Mooney, D.; Hansen, L.; Vacanti, J.; Langer, R; Farmer, S.; Ingber, D. J Cell Physiol 1992, 151, 497-505; Reid, L. M.; Fiorino, A. S.; Sigal, S. H.; Brill, S.; Holst, P. A. Hepatology 1992, 15, 1198-1203; Nelson, C. M.; Chen, C. S. Febs Letters 2002, 514, 238-242; Bhatia, S, N.; Balis, U. J.; Yarmush, M. L.; Toner, M. Faseb J 1999, 13, 1883-1900; Tan, J. L.; Tien, J.; Pirone, D. M.; Gray, D. S.; Bhadriraju, K.; Chen, C. S. Proceedings of the National Academy of Sciences of the United States of America 2003, 100, 1484-1489; Galbraith, C. G.; Sheetz, M. P. Current Opinion in Cell Biology 1998, 10, 566-571. Cell adhesion to culture materials can be modulated through adsorption of extra cellular matrix (ECM) components which interact with the cell through various integrin signaling pathways. Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428; Flaim, C. J.; Chien, S.; Bhatia S. N. Nature Methods 2005, 2, 119-125. In addition, the degree of cell-cell interaction can influence their fate and function through both contact-mediated and soluble signals from neighboring cells. Schwartz, M. A.; Ginsberg, M. H. Nature Cell Biology 2002, 4, E65-E68. Traditional tools to address this microenvironmental parameter space are limited to bulk manipulations of the culture conditions. Kan, P.; Miyoshi, H.; Yanagi, K; Ohshima, N. Asaio J 1998, 44, M441-444. Adsorbing biomolecules such as ECM components to the substrate can modulate cell-matrix interactions. Cell-cell interactions are probed through seeding densities (higher densities increase homotypic interactions and lower densities reduce homotypic interactions), or co-cultivation with other cell types at various ratios to alter the homotypic and heterotypic interface. Hamaguchi, K.; Utsunomiya, N.; Takaki, R.; Yoshimatsu, H.; Sakata, T. Experimental Biology and Medicine 2003, 228, 1227-1233. While these techniques have yielded valuable experimental data and insight, engineering microenvironmental cues through micropatterning in high throughput biological formats enables precise experimentation not currently available using traditional techniques. Montesano, R; Mouron, P.; Amherdt, M.; Orci, L. Journal of Cell Biology 1983, 97, 935-939.
Recently, various methods to control cell-matrix and cell-cell interactions through protein and cellular micropatterning have been demonstrated. Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. PNAS 2006, 103, 2480-2487. Some examples include microcontact printing, microfluidic patterning, photolithographic patterning, stencil patterning, and ink-jet printing. Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696-698; Chiu D. T.; Jeon, N. L.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proceedings of the National Academy of Science 2000, 97, 2408-2413; Bhatia, S. N.; Yarmush, M. L.; Toner, M. J Biomed Mater Res 1997, 34, 189-199; Folch, A; Jo, B. H.; Hurtado, 0.; Beebe, D. J.; Toner, M. Journal of Biomedical Materials Research 2000, 52, 346-353; Pardo, L,; Wilson, W. C.; Boland, T. Langmuir 2003, 19, 1462-1466. However, these techniques often require specific substrates (gold for microcontact printing or ink jet printing), are limited to simple geometries (microfluidic and stencil patterning) and flat surfaces (glass or silicon for photolithography), and cannot be utilized in high-throughput platforms such as multi-well plates.
Precise engineering of cellular microenvironments is an exciting new addition to the biologist's toolkit; however, the fabrication complexity of many techniques impedes their implementation in standard biological labs. Accordingly, a need exists for a method for etching of physisorbed biomolecules utilizing inexpensive, easily accessible off-the-shelf cell culture materials including multi-well plates, flasks, and bottles, which method would empower biological investigations through streamlined micropatterning.
SUMMARY OF THE INVENTIONMicropatterning biomolecules and cells allows precise experimentation investigating the role of the microenvironment on cellular fate and function; however, the fabrication complexity of many micropatterning techniques limits the practicality of integration with high throughput formats. Successful large scale biological investigations require simple procedures and standard materials in order to integrate seamlessly into the process flow of the biological research lab. By merging micropatterning with standard lab materials, large scale experimentation can exploit the benefits of micropatterning without increasing experimental complexity.
One aspect of the invention described herein relates to techniques which can be used to generate micropatterns of biomolecules and cells on standard laboratory materials through selective ablation of a physisorbed biomolecule with oxygen plasma. In certain embodiments, oxygen plasma is able to ablate selectively physisorbed layers of biomolecules (e.g., type-I collagen, fibronectin, laminin, and Matrigel) along complex non-linear paths which are difficult or impossible to pattern using alternative methods. In addition, certain embodiments of the present invention relate to the micropatterning of multiple cell types on curved surfaces, multiwell plates, and flat bottom flasks. Importantly, the techniques described herein integrate seamlessly into many biological protocols through adapting to commonly used materials.
Merging micropatterning techniques with high-throughput biological experimentation facilitates biological discovery through enabling precise control over microenvironmental cues not attainable through traditional techniques. Therefore, an object of the present invention is to distill micropatterning into the simplest form to facilitate broad use in standard research labs. Transitioning new technologies, such as micropatterning, into standard research labs is facilitated through the use of materials and platforms common to these fields. It follows that a method which incorporates micropatterning with the ubiquitous plastic multi-well plate would reduce the hurdle to implement such a technique in biological experimentation.
Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
In this specification and the appended claims, the singular forms “a,” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.
All publications mentioned herein are incorporated herein by reference for the purpose of describing and the components that are described in the publications which might be used in connection with the presently described invention.
As summarized above, one embodiment of the invention relates to the use of an etch mask to micropattern molecules in a multi-well plate. Once an etch mask is fabricated, generating the micropatterned multi-well plate is accomplished in two simple steps: first molecules (e.g., biomolecules) are physisorbed to the surface followed by selective ablation (e.g., with oxygen plasma).
In certain embodiments, an elastomeric etch mask can be used. Two of the advantages of using an elastomeric etch mask are that it easily conforms to the substrate and it protects the physisorbed molecules from ablation. In certain embodiments, the etch mask can be formed of PDMS, rubber, chrome, or plastic. For example,
Certain embodiment of the present invention require that the material which is etched can physisorb from solution and can withstand air drying. In certain embodiments, the material used is a biomaterial. In addition, in certain embodiments of the invention the concentrations of solutions used should be above 25 μg/mL as some physisorbed material may adhere to the etch mask upon removal. Due to this, etch masks should be dedicated to specific biomolecules to reduce the risk of cross-contamination. However, the adhering of biomolecules to the etch mask can be exploited to generate patterns without the use of oxygen plasma, by using the etch mask of the invention as a flexible mask.
In certain aspects, a biomolecule is adsorbed onto the surface of a substrate. The biomolecule may be a peptide, polypeptide, nucleic acid, nucleic acid binding partners, proteins, receptors, antibodies, enzymes, carbohydrates, oligosaccharides, polysaccharids, cells, cell aggregates, cell components, lipids, arrays of ligands, non-protein ligands, liposomes, and microorganisms, such as bacteria or viruses.
Any particular cell may be micropatterned according to the subject invention so long as the specific cell type requires an extracellular matrix or at least some other protein or peptide derived from the matrix for attachment such as hepatocytes or endothelial cells. Additional examples of various cell types include but are not limited to cells derived from other organs such as kidney, muscle, pancreas, epithelium cells, tissue/skin cells, intestinal cells etc. or stem-cell derived cells such as hepatocyte-like cells derived from embryonic stem cells.
For example,
In certain embodiments, more than one type of cell is micropatterned according to the subject invention. In this aspect, the subject methods would be employed to micropattern at least two different types of cells.
In other aspects, different coatings may be used with different pattern configurations. For example, one large post of a multi-well mold can have a different pattern configuration (i.e., one well can have 100 micron islands and another well can have 500 micron islands). As such, each well may have a different coating of a biomolecule (or other material). For example, hepatic tissue may be coated with one particular pattern on fibronectin and a different pattern on collagen or one well may have patterned hepatic tissue and another well may have kidney cells with a different pattern. In certain aspects, the coating may range from 10 nm to 1 mm.
In another aspect of the invention, the coatings do not necessarily have to be a biomolecule. For example, a virus may be patterned onto the substrate such as lentivirus. As such, when cells attach to lentivirus spots, the cells get transfected with the nucleotides the lentivirus is carrying for over-expression or knockdown of RNA and proteins. Therefore, this type of platform may be used for screening lentiviral libraries in a high-throughput format (e.g., 384 well plate). In addition this platform may also be used to deliver drugs or other agents via the micropatterned microparticles to cells. As such, the subject invention may be further employed to pattern any material, biology or non-biology related so long as the material can be protected by the posts in select regions, while the remaining regions are susceptible to plasma.
Further, in another embodiment of the invention, the etch masks of the invention can be used, as shown in
In certain embodiments, the material of the invention is etched with a gas plasma. Gas plasmas, the fourth state of matter, consist of a mixture of electrons, ions, radicals, and photons and can be created by the application of RF power to a gas under vacuum. RF power excites free electrons to gain sufficient kinetic energy to causing the collision with other molecules to generate ions and radicals. These reactive radical species oxidize and ablate the adsorbed molecules (e.g., a carbon based biomolecule) and this oxidation can pattern through application of a etch mask (e,g., a PDMS etch mask). Previously, oxygen plasma micropatterning has been demonstrated for patterning biomolecules on glass substrates with selective ablation through PDMS stencils. Tourovskaia, A.; Barber, T.; Wickes, B. T.; Hirdes, D.; Grin, B.; Castner, D. G.; Healy, K. E.; Folch, A. 2003, 19, 4754-4764. Remarkably, herein it is disclosed that the plasma can penetrate more than 10 cm along non-linear paths so that the surface to be patterned does not need to be directly exposed to the oxygen plasma; this fact greatly expands the functionality of oxygen plasma based micropatterning. The technique is adaptable to any oxygen plasma system; however, the etching rate will vary with power and configuration. Importantly, this aspect of the invention enables micropatterning on standard biological materials, such as multi-well plates and flat bottom flasks, which would not be attainable through other techniques.
As such, the gas plasma used in the subject invention may be oxygen plasma, nitrogen plasma, hydrogen plasma, argon plasma, or halogen plasma. In a particular embodiment, the gas plasma is oxygen plasma.
In certain aspects, the material to be coated may be patterned by other methods. For example, the material, e.g., biomolecule, may be patterned onto the substrate by stamping techniques or pin-spotting techniques. For example, a microtechnology-based process utilizing elastomeric stencils may be employed to miniaturize and characterize human liver tissue in an industry-standard multiwell format. This approach incorporates ‘soft lithography,’ a set of techniques utilizing reusable, elastomeric, polymer (Polydimethylsiloxane, PDMS) molds of microfabricated structures to overcome limitations of photolithography. A process uses PDMS stencils consisting of 300 μm thick membranes with through-holes at the bottom of each well in a 24-well mold. In order to micropattern all wells simultaneously, the assembly was sealed against a polystyrene plate. Collagen-I was adsorbed to exposed polystyrene, the stencil was removed, and a 24-well PDMS ‘blank’ was applied. Co-cultures were ‘micropatterned’ by selective adhesion of primary hepatocytes to collagen-coated domains, which were then surrounded by supportive murine 3T3-J2 fibroblasts.
The surfaces of polystyrene multi-well plates contain more irregularities compared with the atomically uniform standard micropatterning substrate of silicon. Hence, the inventive etch masks of the invention are preferably fabricated out of elastomeric material (e.g., PDMS) in order to facilitate compression assisted sealing over the large area (8×12 cm) of the multi-well plate. In certain embodiments of the invention, the entire multi-well plate can be micropatterned with a single etch mask, which greatly reduces the time required to pattern as all 96 wells are masked in one step. In certain embodiments, each pillar of the etch mask can have a different micropattern (as shown in
A custom clamp can be used to compress uniformly the etch mask to the surface of the multi-well plate as shown in
Micropatterns of fluorsescently labeled type-I collagen, fibronectin, laminin, and Matrigel are shown in
The power of this technique lies in the adaptability and simplicity of the method. To further demonstrate, the interior surface of flat-bottom tissue culture flasks and bottles were micropatterned with type-I collagen and seeded with primary hepatocytes as shown in
The resolution and performance of this technique were explored through the quantification of etching distances along straight microchannel etch masks as shown in
The resolution of the technique can be limited by the resolution of the etch mask. In certain embodiments, patterns were generated down to 20 μm, the limit of the high resolution transparency photomasks used far SU8 mold fabrication; however, smaller features could be fabricated through the use of a high resolution chrome photomask. However, smaller features often require longer etching times, and increasing the etching time can cause the temperature of the substrate to increase. For example,
The oxygen plasma ablates any exposed surface within the barrel of the plasma asher and can generate complex patterns, such as spirals and non-linear paths as shown in
This invention has broad potential applications as an in vitro model of cellular systems. Micropatterning of biomolecules can be designed to engineer precisely cellular position, shape, and interactions with extra cellular matrix. For example, the invention contemplates a hepatocyte-fibroblast micropatterned co-culture system as an in vitro model of the liver, as shown in
Furthermore, micropatterning in multiwell plates will facilitate high throughput biological experiments not possible with traditional techniques. Current micropatterning techniques are primarily in the proof-of-concept stage and are not easily adaptable to large scale investigations. This invention overcomes these limitations and enables large scale experimentation. To date, there have been no demonstrations of micropatterning inside standard multiwell plates. This fact is primarily due to most micropatterning procedures either require solvents that would degrade standard tissue culture plates, extremely flat surfaces (Si wafers or plasma cleaned glass slides), or specialized substrates (gold substrate for self-assembled monolayers). The methods described herein are robust and require no specialized surfaces. Importantly, due to the deep penetration properties of plasma, virtually any surface morphology can be patterned in one step.
Selected Methods of the InventionOne aspect of the invention relates to a method of forming a micropatterned substrate, comprising the steps of: adsorbing molecules onto a surface of a substrate, thereby forming a coated surface of the substrate; compressing a micropatterned etch mask onto the coated surface of said substrate; and exposing the compressed micropatterned etch mask and coated surface of the substrate to a gas plasma for a period of time, thereby ablating the exposed surfaces of the substrate.
In certain embodiments, the present invention relates to the aforementioned method, further comprising rinsing and drying said coated surface after the adsorbing step.
In certain embodiments, the present invention relates to the aforementioned method, wherein said exposing step is carried out in a plasma asher.
In certain embodiments, the present invention relates to the aforementioned method, wherein the micropatterned etch mask is one solid elastomeric piece.
In certain embodiments, the present invention relates to the aforementioned method, wherein the micropatterned etch mask comprises a plurality of pillars.
In certain embodiments, the present invention relates to the aforementioned method, wherein the micropatterned etch mask comprises chrome or elastomeric poly(dimethylsiloxane) or rubber or plastic.
In certain embodiments, the present invention relates to the aforementioned method, wherein the micropatterned etch mask comprises elastomeric poly(dimethylsiloxane).
In certain embodiments, the present invention relates to the aforementioned method, wherein the micropatterned etch mask comprises an about 50 μm to about 1 mm thick piece of elastomeric poly(dimethylsiloxane).
In certain embodiments, the present invention relates to the aforementioned method, wherein said substrate surface is ceramic, metal, glass, or plastic.
In certain embodiments, the present invention relates to the aforementioned method, wherein said substrate surface is plastic.
In certain embodiments, the present invention relates to the aforementioned method, wherein said substrate comprises fluoropolymers, fluorinated ethylene propylene, polyvinylidine, polydimethylsiloxane, polystyrene, polycarbonate, and polyvinyl chloride, fused silica, polysilicon, or single silicon crystals.
In certain embodiments, the present invention relates to the aforementioned method, wherein said substrate is a tissue culture flask, a tissue culture bottle, or a cell culture multiwell plate.
In certain embodiments, the present invention relates to the aforementioned method, wherein said substrate is a 24-well, 96-well, or 384-well cell culture plate.
In certain embodiments, the present invention relates to the aforementioned methods wherein said molecules are biomolecules.
In certain embodiments, the present invention relates to the aforementioned method, wherein said molecules are biomolecules; and said biomolecules are selected from the group consisting of peptides, polypeptides, nucleic acids, nucleic acid binding partners, proteins, receptors, antibodies, enzymes, carbohydrates, oligosaccharides, polysaccharides, cells, cell aggregates, cell components, lipids, arrays of ligands, non-protein ligands, liposomes, and microorganisms.
In certain embodiments, the present invention relates to the aforementioned method, wherein said molecules are hyaluronic acid, collagen, fibronectin, lamanin, or matrigel.
In certain embodiments, the present invention relates to the aforementioned method, wherein the thickness of the coating on the surface is in the range from about 100 nm to about 200 nm.
In certain embodiments, the present invention relates to the aforementioned method, wherein the thickness of the coating on the surface is about 150 nm.
In certain embodiments, the present invention relates to the aforementioned method, wherein the gas plasma is an oxygen plasma, nitrogen plasma, hydrogen plasma, argon plasma or halogen plasma.
In certain embodiments, the present invention relates to the aforementioned method, wherein the gas plasma is an oxygen gas plasma.
In certain embodiments, the present invention relates to the aforementioned method, wherein said time is in the range from about 5 seconds to about 1000 seconds.
In certain embodiments, the present invention relates to the aforementioned method, wherein said time is about 5 seconds, 10 seconds, 20 seconds, 40 seconds, 60 seconds or 120 seconds.
In certain embodiments, the present invention relates to the aforementioned method, wherein the substrate is a 96-well plate; and the molecules comprise collagen, fibronectin, lamanin, or matrigel.
In certain embodiments, the present invention relates to the aforementioned method, wherein the substrate is a tissue culture flask; and the molecules comprise collagen, fibronectin, lamanin, or matrigel.
In certain embodiments, the present invention relates to the aforementioned method, wherein the substrate is a tissue culture bottle; and the molecules comprise collagen, fibronectin, lamanin, or matrigel.
In certain embodiments, the present invention relates to the aforementioned method, wherein the substrate is a 96-well cell culture plate; the molecules comprise collagen; and the time is about 1000 seconds,
In certain embodiments, the present invention relates to the aforementioned method, wherein the substrate is a 96-well cell culture plate, and the micropattern is generated within all 96 wells simultaneously.
In certain embodiments, the present invention relates to the aforementioned method,
wherein the substrate is a 384-well cell culture plate; the molecules comprise collagen; and the time is about 1000 seconds,
In certain embodiments, the present invention relates to the aforementioned method, wherein the substrate is a 384-well cell culture plate, and the micropattern is generated within all 384 wells simultaneously.
In certain embodiments, the present invention relates to the aforementioned method, further comprising the steps of: removing the micropatterned etch mask; and contacting said micropatterned substrate with cells.
In certain embodiments, the present invention relates to the aforementioned method, wherein said cells are hepatocytes.
In certain embodiments, the present invention relates to the aforementioned method, wherein said cells are human or rat hepatocytes.
Selected Multi-Well Cell Culture Plates of the InventionAnother aspect of the invention relates to a multi-well cell culture plate, wherein each cell is micropatterned with a material.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein said multi-well cell culture plate is plastic.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein said multi-well cell culture plate is a 24-well, 96-well, or a 384-well cell culture plate.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein said material is a biomolecule.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein said material is a biomolecule; and said biomolecule is selected from the group consisting of peptides, polypeptides, nucleic acids, nucleic acid binding partners, proteins, receptors, antibodies, enzymes, carbohydrates, oligosaccharides, polysaccharides, cells, cell aggregates, cell components, lipids, arrays of ligands, non-protein ligands, liposomes, and microorganisms.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein said material is hyaluronic acid, collagen, fibronectin, lamanin, or matrigel.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein the thickness of the material on the multi-well cell culture plate is in the range from about 100 nm to about 200 nm.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein the thickness of the material on the multi-well cell culture plate is about 150 nm.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein the multi-well cell culture plate is a 96-well plate; and the molecules comprises collagen, fibronectin, lamanin, or matrigel.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein each cell of the multi-well cell culture plate has the same micropattern.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein not all of the cells of the multi-well cell culture plate has the same micropattern.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, further comprising cells.
In certain embodiments, the present invention relates to the aforementioned multi-well plate, wherein said cells are hepatocytes.
Selected Etch Masks of the InventionAnother aspect of the invention relates to a micropatterned etch mask comprising a plurality of pillars.
In certain embodiments, the present invention relates to the aforementioned micropatterned etch mask, wherein the micropatterned etch mask is one solid elastomeric piece.
In certain embodiments, the present invention relates to the aforementioned micropatterned etch mask, wherein the micropatterned etch mask comprises chrome or elastomeric poly(dimethylsiloxane) or rubber or plastic.
In certain embodiments, the present invention relates to the aforementioned micropatterned etch mask, wherein the micropatterned etch mask comprises elastomeric poly(dimethylsiloxane).
In certain embodiments, the present invention relates to the aforementioned micropatterned etch mask, wherein the micropatterned etch mask comprises an about 50 μm to about 1 mm thick piece of elastomeric poly(dimethylsiloxane).
KitsFinally, kits for use with high throughput biological experiments and/or for in vitro models of cellular systems are provided. The subject kits at least include the cell culture plates and etch masks of the subject invention. The kits may further include one or more additional components necessary for carrying out the biological experiments or creating the in vitro models, such as sample preparation reagents, buffers, labels, and the like.
In addition to above mentioned components, the subject kits typically further include instructions for using the components of the kit to practice the subject methods with the subject devices. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
EXEMPLIFICATIONThe invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. Note that these studies employed a 4″×6″ 100 W barrel asher (March Plasmod, Concord, Calif.) as the oxygen plasma system.
Example 1 Fabrication of the 96-Well Etch MaskA mold was machined with the same center to center spacing as a standard 96-well plate and was used to generate the support structure for the 96-well etch mask. This support structure consisted of an array of 96 pillars spaced evenly to allow nesting in a standard 96-well plate. The support structure is molded out of poly(dimethylsiloxane) (PDMS) (sylgard 184, Dow Corning, Midland, Mich.) and prepared using standard techniques. Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Analytical Chemistry 1998, 70, 4974. In a separate step, mold masters for the micropatterns were fabricated with SU8 photoresist (Microchem, Newton, Mass.) using a high resolution transparency photomask. The molds were fabricated to have 50 μm thick features. These mold masters were coated with a layer of PDMS 2 mm thick The PDMS was peeled from the mold and circles were punched out with a cork borer and glued to the pillars of the PDMS support structure using PDMS as an adhesive. This resulted in a single etch mask with 96 micropatterned pillars able to nest inside a standard 96-well plate and conform to the bottom of the plate under slight compression.
Example 2 Patterning Protein and Cells in 96-Well PlatesBiomolecules were physisorbed to each well of a standard multi-well plate (solutions of type-I collagen, fibronectin, Matrigel and laminin at 50 μg/mL in water. Note that 1×PBS may be used instead of water. The multi-well plates were incubated for 1 hour at 37° C. followed by rinsing with water and allowed to air dry. This results in an adsorbed thickness of approximately 150 nm. Gurdak, E.; Dupont-Gillain, C. C.; Booth, J.; Roberts, C. J.; Rouxhet, P. G. Langmuir 2005, 21, 10684-10692. In some cases, micropatterned proteins were fluorescently labeled via incubation (1 hour at room temperature) with Alexa Fluor® 488 carboxylic acid, succinimidyl ester (Invitrogen, Carlsbad, Calif.) dissolved in phosphate buffered saline (PBS) at 20 μg/mL. A single etch mask was inserted into the multi-well plate and compressed in a custom clamp consisting of two blocks of polycarbonate flanking the masked multi-well plate and compression was applied by tightening screws joining the two blocks as Shown in
Primary rat hepatocytes were seeded into the multi-well plate isolated and purified by a modified procedure of Seglen. Seglen, P. O. Methods Cell Biol 1976, 13, 29-83. Briefly, 2-3 month old adult female Lewis rats (Charles River Laboratories, Wilmington, Mass.) weighing 180-200 g were anesthetized prior to in situ perfusion of the portal vein. Following a two-step perfusion of Krebs Ringer Buffer and collagenase, dissociated cells were passed through nylon mesh and purified on a Percoll gradient.
Example 3 Ablation QuantificationA PDMS etch mask containing dead-end microchannels with channel widths of 50, 100, 150, and 200 μm repeated for three channel heights of 25, 50, and 75 μm (12 channels total) was used to quantify the etching rate. The mask was placed onto p60 petri dishes physisorbed with type-I collagen and exposed to oxygen plasma for several time points (5, 10, 20, 40, and 60 seconds) with each time point on a separate dish. Primary rat hepatocytes were then seeded onto the patterned dishes and cultured for 24 hours. The hepatocytes specifically attached to regions with type-I collagen, hence plasma ablated distances could be directly correlated to hepatocyte attachment as hepatocytes will not attach to adsorbed collagen substrates from solutions less than 0.5 μ/mL. The distances were measured using Metamorph (Universal Imaging, Sunnyvale, Calif.) and plotted as a function of time.
Example 4 Micropatterning Two Cell TypesPrimary hepatocytes and a supportive murine fibroblast were co-cultivated by first micropatterning adhesive type-I collagen microdomains on the substrate of a 96-well plate. Hepatocytes were seeded at a density of 0.5×106 cells/mL in serum free media and allowed to spread on the collagen domains. Unattached hepatocytes were rinsed off and fibroblasts were seeded in media containing serum at 0.5×106 cells/mL. Components of the serum adsorbed to the regions of the substrate not containing type-I collagen domains (or hepatocytes) and fibroblasts attached and spread.
Example 5 Flask and Bottle MicropatterningType-I collagen solution (50 μg/mL) was physisorbed to the surface of the flask or bottle for 1 hour at room temperature, followed by rinsing and drying. A 1 mm thick PDMS etch mask was inserted into the flask or bottle and pressed against the walls. This was placed inside the plasma asher and exposed to oxygen plasma at 100 W for 2 minutes. Following plasma ablation, primary rat hepatocytes were seeded and cultured overnight. The cells were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 0.5 mg/mL which is cleaved into a visible product in viable cells for imaging.
Example 6 A Miniaturized, Multiwell Human Liver Tissue ModelThe system is comprised of primary human hepatocytes organized in micropatterned colonies surrounded by supportive stromal cells. Model utility is demonstrated via gene expression profilings, phase I/II metabolism, canalicular transport, secretion of liver-specific products, and susceptibility to hepatotoxins.
Elastomeric stencils were used to miniaturize and characterize human liver tissue in an industry-standard multiwell format. In order to micropattern all wells simultaneously, the assembly was sealed against a polystyrene plate. Collagen-I was adsorbed to exposed polystyrene, the stencil was removed, and a 24-well PDMS ‘blank’ was applied. Co-cultures were ‘micropatterned’ by selective adhesion of primary hepatocytes to collagen-coated domains, which were then surrounded by supportive murine 3T3-J2 fibroblasts.
The diameter of through-holes in the stencils determined the size of collagen-coated domains and thereby the balance of homotypic (hepatocyte/hepatocyte) and heterotypic (hepatocyte/fibroblast) interactions in the microscale tissue. We varied collagen island diameter over several orders-of-magnitude and observed that hepatocyte clustering consistently improved liver-specific functions when compared to unorganized cultures. Furthermore, hepatocyte functions were maximal for the configuration containing ˜500 μm islands with ˜1200 μm center-to-center spacing. These findings are consistent with our rodent data in that 3T3 fibroblasts were able to stabilize hepatocyte functions across both species; however, human hepatocytes were more dependent on homotypic interactions than rat hepatocytes. Thus, the microscale human liver tissue developed and characterized herein represents 24-well plates with each well containing ˜10,000 hepatocytes organized in 37 colonies of 500 μm diameter and surrounded by 3T3-J2 fibroblasts (micropatterned co-cultures), for a total of 888 repeating hepatic microstructures per plate. The microscale architecture remained stable for several weeks in culture which enabled microscopic tracking of individual hepatocyte islands for weeks.
In order to qualitatively assess the stability of micropatterned co-cultures, hepatocyte morphology was monitored over time and found to be maintained for the duration of the cultures, typically 4-6 weeks (
In order to assess the utility of micropatterned co-cultures for drug metabolism studies, cytochrome P450 (CYP450) activity, phase II conjugation, and canalicular transport was characterized. CYP450 activity in micropatterned co-cultures was assessed over several weeks using both fluorometric substrates well-suited for high-throughput screening and isoenzyme-specific probe substrates requiring chromatographic separation of metabolites. Activities of several major CYP450 isoenzymes (CYP2A6, 2B6, 3A4) were retained well (>50% of fresh hepatocyte levels) for several weeks in un-induced micropatterned co-cultures (
In order to obtain a more global perspective, the microscale tissues were gene expression profiled over the span of several weeks. Prior to extraction of hepatocyte RNA, fibroblasts were removed via selective trypsinization (˜95% purity, see supplemental methods online). The ability to obtain purified hepatocyte RNA from micropatterned co-cultures is enhanced by clustering via micropatterning and is advantageous for genome-wide analyses (e.g. toxicogenomics). Gene expression profiles of hepatocytes in this platform were compared to gene expression in several models, which included: a) all cell types of the human liver immediately after tissue disruption but prior to hepatocyte purification (universal mixture of all cells or UMIX); b) freshly isolated pure hepatocytes in suspension (day 0) widely used as starting point controls for in vitro studies; and c) unorganized pure hepatocyte monolayers 1 week after plating as a model of deteriorating functions. In this study, global scatter plots were utilized coupled with linear regression analysis to compare gene expression in the aforementioned liver models.
Overall, these expression profiling experiments showed that hepatocytes in micropatterned co-cultures were stable for 4-6 weeks as indicated by high levels of expression of liver-specific genes (relative to fresh controls and pure hepatocytes) relevant for evaluating drug metabolism and toxicity in vitro (i.e. Phase I, II, III, nuclear receptors, liver-enriched transcription factors). Specifically, global gene expression analysis revealed a positive correlation (R2 between 0.7-0.8) between expression intensities in hepatocytes purified from micropatterned co-cultures (up to 6 weeks) and intensities in fresh controls, which included all cell types of the liver as well as purified fresh hepatocytes in suspension (
In addition to Phase I and II drug metabolism genes, the gene expression levels of several other classes of genes relevant in evaluating drug disposition were also analyzed, which included: nuclear receptors (NR) that can modulate the expression of drug metabolism enzymes following exposure of hepatocytes to endogenous and exogenous stimuli; liver-enriched transcription factors (LETF) which regulate broad classes of hepatic functions; and influx and efflux transporters. Several important genes were found from these classes which were expressed at statistically significant levels in micropatterned co-cultures (up to 6 weeks) (
In order to assess the utility of micropatterned co-cultures for toxicity screening, the acute and chronic toxicity of model hepatotoxins was quantified. Compounds were characterized by their TC50, defined as the concentration which produced 50% reduction in mitochondrial activity after an acute (24 hours) exposure (
The induction and inhibition of drug metabolism enzymes (i.e. CYP450) typically underlie drug-drug interactions which can lead to serious pharmacological and/or toxicological consequences in the clinic. The induction of CYP450 activity in micropatterned co-cultures was demonstrated using prototypic clinical inducers and both fluorometric and conventional CYP450 substrates (
Modulation of CYP450 activities depends on both the dose and time of exposure to compounds. In
An advantageous feature of our platform is its modular design in that various liver or non-liver derived stromal cells can be used to surround hepatocyte colonies to form microscale tissues. We chose 3T3 (sub-clone J2) fibroblasts because of their ready availability, ease of propagation, lack of liver-specific gene expression (albumin, P450 activity), and evidence showing that this cell line can induce high levels of liver-specific functions. Nonetheless, to demonstrate versatility of our platform, we co-cultivated micropatterned human hepatocytes with the non-parenchymal fraction of the human liver and also observed stabilization of hepatic functions, though not to similar levels or duration as in co-cultures with 3T3 fibroblasts (data not shown).
We demonstrated that micropatterned clusters of pure human hepatocytes outperformed their randomly distributed counterparts by several-fold, consistent with reports that confluent hepatocyte cultures retain liver-specific functions better than sparse cultures, partly through cadherin interactions. Subsequent introduction of non-parenchymal cells further enhanced hepatocyte functions and longevity of the liver tissues. Thus, our optimized micropatterned co-cultures use an order-of-magnitude fewer hepatocytes (10K vs. 200K) and maintain phenotypic functions for a longer time than conventional pure cultures (weeks vs. days) in similar multiwell formats. Furthermore, we explored many hepatocyte sources. In our platform, we observed induction of liver-specific functions in fresh hepatocytes across donors of different age groups, genders and medical histories. Nonetheless, due to limited availability of fresh cells, we demonstrated successful incorporation of cryopreserved human hepatocytes into micropatterned co-cultures (
Hepatocyte culture models typically employed in the pharmaceutical industry rely on manipulating the extracellular microenvironment of hepatocytes with Matrigel and/or collagen (i.e. Matrigel overlay, collagen gel sandwich culture). When utilized with near confluent (80-100%) monolayers of hepatocytes, these models allow better retention of hepatocyte cytoarchitecture (i.e. bile canaliculi, tight junctions) and activity of specific CYP450 enzymes for a few more days (˜1 week) than that seen in monolayers on rigid collagen. However, studies have shown that cell-cell contacts (homotypic and heterotypic), more than the extracellular matrix configuration or composition, play a critical role in maintaining phenotypic functions of primary human hepatocytes in vitro. Primary rat hepatocytes, on the other hand, functionally respond better to extracellular matrices of different compositions and topologies. Our own studies with primary human hepatocytes confirmed the data reported in the literature (
Several other in vitro models of the liver that utilize ‘3D’ tissues (aggregate-based) and/or continuous perfusion have been proposed in the literature. Many of these strategies were initially developed for applications in cell-based therapies (i.e. tissue engineering) where the challenges are often around scale-up; however, a few have been scaled-down for utility in drug screening applications. 3D architecture is indeed likely to be critical to therapeutic applications of liver tissue; however, it can introduce challenges when applied to high-throughput applications that stem from limited in situ observation of cells with conventional microscopic techniques and nutrient transport limitations. Nutrient transport limitations can be overcome by the inclusion of convective transport in the form of flowing medium. Nonetheless, the inclusion of a flow circuit for each well in a high-throughput screening format introduces complexities in liquid handling (pumps rather than liquid dispensers) and larger fluid volumes that require larger quantities of novel compounds. As a result, static 2D monolayer systems (confluent monolayers, collagen sandwich or Matrigel overlay) are currently widely favored in industrial settings. In this study, we show that the phenotypic functions of primary human hepatocytes can be maintained remarkably well in a static, 2D platform. These micropatterned co-cultures can be mapped seamlessly to existing laboratory protocols including robotic fluid handling, in situ microscopy, and colorimetric/fluorescent plate-reader assays.
In conclusion, microtechnology was utilized to fabricate and miniaturize human liver tissue that can be used for high-content cell-based assays in preclinical phases of drug development. We anticipate that the adoption of microscale liver tissues for ADME/Tox screening will be facilitated by the long-term retention of human hepatocyte functions as well as the ability to seamlessly integrate the multi-well, 2D tissues into the existing technological infrastructure. Such a platform has potential to reduce development costs, increase likelihood of clinical success, and reduce the risk for patient exposure to unsafe drugs. In the future, in vitro tissue models with precisely defined microenvironments may find utility in many other arenas that could benefit from realistic models of human tissue biology.
Micropatterning of CollagenElastomeric Polydimethylsiloxane (PDMS) stencil devices, consisting of thick-membranes (˜3000m) with through-holes (500 μm with 1200 μm center-to-center spacing) at the bottom of each well of a 24-well mold were manufactured by Surface Logix, Inc (Brighton, Mass.). Stencil devices were first sealed (via gentle pressing) to tissue culture treated polystyrene omnitrays (Nunc, Rochester, N.Y.), then each well was incubated with a solution of type-I collagen in water (100 μg/mL) for 1 hour at 37° C. Purification of collagen from rat-tail tendons was previously described. The excess collagen solution in each well was aspirated, the stencil was removed and a PDMS “blank” (24-well mold without stencil membranes) was applied. Collagen-patterned polystyrene was stored dry at 4° C. for up to 4 weeks. In some cases, micropatterned collagen was fluorescently labeled via incubation (1 hour at room temperature) with Alexa Fluorg 488 carboxylic acid, succinimidyl ester (Invitrogen, Carlsbad, Calif.) dissolved in phosphate buffered saline (PBS) at 20 μg/mL. For experiments in Supplemental
Primary rat hepatocytes were isolated from 2-3-month old adult female Lewis rats (Charles River Laboratories, Wilmington, Mass.) weighing 180-200 g. Detailed procedures for rat hepatocyte isolation and purification were previously described79. Routinely, 200-300 million cells were isolated with 85%-95% viability and >99% purity. Hepatocyte culture medium consisted of Dulbecco's Modified Eagle's medium (DMEM) with high glucose, 10% (v/v) fetal bovine serum, 0.5 U/mL insulin, 7 ng/mL glucagon, 7.5 μg/mL hydrocortisone, and 1% (v/v) penicillin-streptomycin. Primary human hepatocytes were purchased in suspension from vendors permitted to sell products derived from human organs procured in the United States of America by federally designated Organ Procurement Organizations. Hepatocyte vendors included: In Vitro Technologies (now part of Celsis, Baltimore, Md.), Cambrex Biosciences (now part of Lonza, Walkersville, Md.), BD-Gentest (Woburn, Mass.), ADMET Technologies (Durham, N.C.), CellzDirect (Pittsboro, N.C.) and Tissue Transformation Technologies (now part of BD-Gentest, Edison, N.J.). All work was done with the approval of COUHES (Committee on use of human experimental subjects). Upon receipt, human hepatocytes were pelleted via centrifugation at 50×g for 5 min (4° C.). The supernatant was discarded, cells were re-suspended in hepatocyte culture medium, and viability was assessed using trypan blue exclusion (typically 70-90%). Liver-derived nonparenchymal cells, as judged by their size (<10 μm diameter) and morphology (non-polygonal), were consistently found to be less than 1% in these preparations.
Hepatocyte-Fibroblast Co-CulturesIn order to create micropatterned co-cultures, hepatocytes were seeded on collagen-patterned substrates, resulting in a hepatocyte pattern due to selective cell adhesion. The cells were washed with medium 2 hours later to remove unattached cells (˜10,000 adherent hepatocytes in 37 collagen-coated islands) and incubated in hepatocyte medium overnight. 3T3-J2 fibroblasts were seeded (30,000 total) in fibroblast medium 12-24 hours later to create co-cultures. Fibroblast to hepatocyte ratio was estimated via a hemocytometer to be 4 to 1 once the fibroblasts reached confluency in cocultures and their growth was contact-inhibited. Culture medium was replaced to hepatocyte medium 24 hours after fibroblast seeding and subsequently replaced daily (300 μL per well in 24-well format). For cultures, hepatocytes were seeded on substrates (glass or polystyrene) with a uniform coating of collagen. In some cases, hepatocytes were fluorescently labeled via incubation (1 hour at 37° C.) with Calcein-AM (Invitrogen) dissolved in culture medium at 5 μg/mL. Fibroblasts were fluorescently labeled with CellTracker (Orange CMTMR, Invitrogen) as per manufacturer's instructions.
Biochemical AssaysSpent medium was stored at −20° C. Urea concentration was assayed using a calorimetric endpoint assay utilizing diacetylmonoxime with acid and heat (Stanbio Labs, Boeme, Tex.). Albumin content was measured using enzyme linked immunosorbent assays (MP Biomedicals, Irvine, Calif.) with horseradish peroxidase detection and 3,3′,5,5″-tetramethylbenzidine (TMB, Fitzgerald Industries, Concord, Mass.) as a substrate.
Cytochrome-P450 InductionStock solutions of prototypic CYP450 inducers (Sigma) were made in Dimethylsulfoxide (DMSO), except for Phenobarbital, which was dissolved in water. Cultures were treated with inducers (Rifampin, B3-Naphthoflavone, Dexamethasone and Omeprazole at 50 μM each, and Phenobarbital at 1 mM) dissolved in hepatocyte culture medium for 3-4 days. Control cultures were treated with vehicle (DMSO) alone for calculations of fold induction. To enable comparisons across inducers, DMSO levels were kept constant at 0.1% (v/v) for all conditions.
Toxicity AssaysCultures were incubated with various concentrations of compounds dissolved in culture medium for 24 hours (acute toxicity) or extended time periods (chronic toxicity, 1-9 days). Cell viability was subsequently measured via the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma) assay, which involves cleavage of the tetrazolium ring by mitochondrial dehydrogenase enzymes to form a purple precipitate. MTT was added to cells in DMEM without phenol red at a concentration of 0.5 mg/mL. After an incubation time of 1 hour, the purple precipitate was dissolved in a 1:1 solution of DMSO and Isopropanol. The absorbance of the solution was measured at 570 nm (SpectraMax spectrophotometer, Molecular Devices, Sunnyvale, Calif.).
Statistical AnalysisExperiments were repeated at least 2-3 times with duplicate or triplicate samples for each condition. Data from representative experiments is presented, whereas similar trends were seen in multiple trials. All error bars represent standard error of the mean.
Example 7 A Long-Term Model of the Rat Liver for Evaluating Drug Disposition Long-Term Morphological and Functional Stability of Co-CulturesPreviously, rat hepatocytes were arranged in collagen-coated circular islands of prescribed dimensions and surrounded by 3T3-J2 fibroblasts to create micropatterned co-cultures. We found that co-cultures with a larger initial heterotypic interface (i.e. single hepatocyte islands surrounded by fibroblasts) had highest levels of liver-specific functions as compared to other configurations. Furthermore, hepatocytes in smaller patterns (<250 μm) intermingled significantly to dissipate the pattern, whereas larger islands (>450 μm) assumed a relatively stable conformation for several weeks. In this study, liver-specific functions in micropatterned and rat co-cultures were characterized over a period of several weeks. The pattern geometry with ˜500 μm hepatocyte islands (˜1200 μm center-to-center spacing) was selected since it provided high functional capacity along with retention of pattern fidelity for the duration of the co-cultures. The stability of the miniaturized rat liver tissues was assessed using both qualitative and quantitative criteria. Hepatocyte morphology in icropatterned co-cultures was found to be stable for over 2 months (
Next, liver-specific functions were characterized in micropatterned pure hepatocyte cultures and co-cultures and compared them to their randomly distributed counterparts (random cultures and co-cultures). Consistent with our previous studies, we found that both albumin secretion (
Resorufin-derivatives, ethoxy-resorufin (ER) and methoxy-resorufin (MR) was utilized in order to demonstrate activity of cytochrome-P450 (CYP450) phase-I enzymes in co-cultures of primary rat hepatocytes and 3T3-J2 murine embryonic fibroblasts. ER and MR dealkylation into fluorescent resorufin is mediated by CYP1A1 and CYP1A2 enzymes, respectively. Low levels of baseline (untreated) ER and MR were observed in metabolism in co-cultures. Therefore, in order to improve the signal-to-noise ratio, levels of CYP1A was ‘induced’ by pre-incubating co-cultures with 3 μM 3-Methylcholanthrene (3-MC) for 3 days prior to assessment of substrate metabolism. 3-MC is a known inducer of CYP1A1 and CYP1A2 expression in hepatocytes via the ligand-activated nuclear receptor AHR (aryl hydrocarbon receptor) [303]. In
Besides albumin secretion, urea synthesis and Phase I and II activities, another marker of liver-specific function is the formation of functional bile canaliculi between hepatocytes. In
Modulation of CYP450 enzyme levels by pharmaceutical compounds is an important parameter in the occurrence of clinical drug-drug interactions. Cultures of primary hepatocytes from different species (i.e. human, rodent) are widely utilized to evaluate CYP450 induction and inhibition by drugs. In order to demonstrate that CYP450 enzymes can be induced in co-cultures of rat hepatocytes and 3T3-J2 fibroblasts, the prototypic CYP1A inducer, 3-Methylcholanthrene (3-MC) was utilized. Co-cultures were incubated with 3-MC for 72 hours before assessment of CYP1A1 and CYP1A2 activities via dealkylation of ethoxy-resorufin and methoxy-resorufin, respectively (
Drug-drug interactions due to the induction or inhibition of CYP450 enzymes can lead to serious toxicological consequences. In order to demonstrate such effects, a well-established in vivo model was utilized in which the toxicity of Acetaminophen (APAP, analgesic found in many over-the-counter medications including Tylenol) is enhanced upon induction of CYP3A. We first pre-treated co-cultures with increasing doses of dexamethasone (DEX) for 2 days to induce CYP3A levels. Then, co-cultures were incubated with a 5 mM dose of APAP for 24 hours, followed by assessment of viability via the MTT assay (see Methods). Data showed a substantial increase in APAPmediated toxicity in DEX-treated co-cultures over untreated controls (
Toxicity of pharmaceuticals due to chronic exposure is clinically relevant. Since hepatocyte culture models utilized in the pharmaceutical industry lose viability and phenotypic functions within a few days, toxicity due to repeated drug exposures over days or weeks cannot be evaluated. Since co-cultures remain functional for several weeks, dose- and time-dependent toxicity of four known Hepatotoxins was investigated, including: Acetaminophen, ethapyrilene, Pyrilamine and Troglitazone. Co-cultures were incubated with varying doses of toxins dissolved in culture medium over several days. Mitochondrial activity (MTT assay, see methods) was evaluated at different time points to assess viability in co-cultures. Minimal toxicity (i.e. 90-100% viability relative to untreated controls) was seen in co-cultures that had been treated for 24 hours with Acetaminophen (APAP) doses ranging between 3 and 30 mM. However, following 6 days of repeated exposure, the viability in co-cultures ranged from 77% for 3 mM APAP to 19% for 30 mM (
Next, the chronic toxicity of pyrilamine was evaluated in rat co-cultures. As with APAP and methapyrilene, we observed dose- and time-dependent toxicity in pyrilaminetreated co-cultures. Specifically, the TC50 value dropped from greater than 1 mM after 1 day of treatment to ˜0.2 mM following 7 days of exposure (
Pure hepatocyte monolayers rapidly lose phenotypic functions and thus represent an ‘unstable’ model of the liver. On the other hand,
Significant variations in hepatocellular functions (i.e. CYP450 enzymes) across different species have been implicated in the inability of animal models to adequately predict adverse outcomes in clinical trials involving humans. Nonetheless, studies in live animals are required by the FDA since they provide valuable in vivo data in a preclinical setting. Therefore, the selection of an animal species (i.e. rat, mouse, monkey, guinea pig) in which the liver-specific metabolism and toxicity of a particular candidate drug are similar to that seen in humans is crucial for clinical success. In vitro human and animal models that closely resemble in-vivo functionality may find use in such a selection process. Here, to demonstrate utility in drug development, the metabolism of specific Phase I and II enzyme substrates were compared in micropatterned co-cultures utilizing either primary human or rat hepatocytes (
Co-Cultivation of Hepatocytes with Liver-derived Nonparenchymal Cells
In this study, non-liver-derived 3T3 murine embryonic fibroblasts were utilized to stabilize phenotypic functions of rat hepatocytes in vitro. The ‘co-culture effect’, however, has been reported for nonparenchymal cells from both within and outside the liver. In this study, the 3T3 model was functionally compared with co-cultures that contained liver-derived primary nonparenchymal cells. Therefore, primary rat hepatocytes were co-cultivated with the whole non-parenchymal fraction of the rat liver (NP-fraction). In
Regardless of any functional improvements, micropatterned co-cultures offer several advantages over randomly distributed ones, which include: precise control over homotypic and heterotypic interactions towards consistent modulation of phenotypic functions; and, imaging and tracking of individual hepatocyte islands over time to monitor cellular responses to specific stimuli. It should be noted though that in this work, a few studies demonstrating utility of co-cultures in drug development were carried out with 1-2 week old randomly distributed co-cultures (high functioning) partly due to the ease with which such co-cultures can be created as compared to micropatterned ones.
Nonparenchymal Cell Culture3T3-J2 murine embryonic fibroblasts were acquired and cultured as described in chapter 2 of this dissertation. In some cases, fibroblasts were growth-arrested by incubation with 10 μg/mL Mitomycin C (Sigma, St. Louis, Mo.) in culture media for 2 hours at 37° C. The nonparenchymal fraction of the rat liver was recovered via centrifugation following digestion of the liver with collagenase. Briefly, a suspension with different types of liver cells (i.e. hepatocytes, sinusoidal endothelial cells, and kupffer.
6Hepatocyte-Nonparenchymal Co-CulturesPrimary rat hepatocytes were isolated and purified. For studies comparing species-specific responses, primary human hepatocytes were purchased in suspension from commercial vendors and cultured. Co-cultured hepatocyte-fibroblast co-cultures were generated. Briefly, collagen-coated polystyrene plates (24-well format) were seeded with hepatocytes (100,000 cells per well) in hepatocyte culture medium (500 μL per well). For co-culture experiments, 3T3 fibroblasts (1:1 ratio with hepatocytes) were seeded in their respective medium 12-24 hours after initiation of adherent hepatocyte cultures. The culture medium was replaced to hepatocyte culture medium the day after fibroblast cell seeding and subsequently replaced daily. Briefly, elastomeric polydimethylsiloxane (PDMS) stencil devices, consisting of thin membranes (˜300 μm) with through-holes (500 μm with 1200 μm center-to-center spacing) at the bottom of each well of a 24-well mold were used to create collagenous domains on tissue culture polystyrene. Hepatocytes were seeded in serum-free hepatocyte culture medium on collagen-patterned substrates, resulting in a hepatocyte micropattern due to selective cell adhesion. The cells were washed with media 2 hours later to remove unattached cells and incubated with serumsupplemented hepatocyte media overnight. Growth-arrested 3T3 fibroblasts were seeded onto hepatocytes to create co-cultures as described above; however, a 3:1 fibroblast to hepatocyte ratio was used due to lack of fibroblast proliferation. In some cases, liver derived nonparenchymal cells (10:1 nonparenchymal to hepatocyte ratio) were used instead of fibroblasts to create co-cultures.
Hepatocellular Function AssaysSpent media was stored at −20° C. Urea concentration was assayed using a colorimetric endpoint assay utilizing diacetylmonoxime with acid and heat (Stanbio Labs, Boerne, Tex.). Albumin content was measured using enzyme linked immunosorbent assays (MP Biomedicals, Irvine, Calif.) with horseradish peroxidase detection and 3,3′,5,5″-tetramethylbenzidine (TMB, Fitzgerald Industries, Concord, Mass.) as substrate. For some experiments, cultures were treated with 3 μM 3-Methylcholanthrene (Sigma) dissolved in hepatocyte culture medium for 3 consecutive days to induce cytochrome-P450 1A (CYP1A) enzyme levels. Control cultures were treated with solvent alone (Dimethylsulfoxide, DMSO) to measure baseline enzyme activity. CYP1A1 activity was assessed via dealkylation of ethoxy-resorufin (ER, Sigma) into fluorescent resorufin, while methoxy-resorufin (MR, Sigma) was used as a substrate for CYP1A2. Briefly, cultures were incubated with 5 μM substrate dissolved in DMEM without phenol red for 30-60 min. Resorufin fluorescence (excitation/emission: 530/590 nm) in collected supernatants was quantified by means of a fluorescence micro-plate reader (Molecular Devices, Sunnyvale, Calif.). Protocols in chapter 5 were followed to evaluate the Phase I-mediated hydroxylation of coumarin, dealkylation of 7-methoxy-4-rifluoromethylcoumarin (MFC) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC), and phase II-mediated conjugation of 7-Hydroxycoumarin (7-HC) in rat co-cultures.
Staining of Functional Bile CanaliculiCo-cultures were washed three times with phenol-red free DMEM, incubated with 6 μg/mL CFDA (5-and-6-carboxyfluorescein diacetate, mixed isomers—purchased from Invitrogen, Carlsbad, Calif.) for 10 minutes, and washed three times again prior to examination with fluorescence microscopy (excitation/emission:495/520 nm). Specimens were observed and recorded using a Nikon Diaphot microscope equipped with a SPOT digital camera (SPOT Diagnostic Equipment, Sterling Heights, Mich.), and MetaMorph Image Analysis System (Universal Imaging, Westchester, Pa.) for digital image acquisition.
Acute and Chronic Toxicity StudiesAll chemicals were purchased from Sigma. In order to evaluate the acute toxicity of compounds, cultures were incubated with various concentrations of compounds dissolved in culture medium for 24 hours. For chronic studies, culture media with fresh Hepatotoxins was added every 2 days. Cell viability at different time points was subsequently measured via the MTT assay.
Drug-Drug Interaction StudiesAll chemicals were purchased from Sigma. Co-cultures were first treated with dexamethasone (1-10 μM) or ethanol (2% vol/vol) dissolved in culture medium for 2 days to induce CYP3A levels. Next, co-cultures were incubated for 24 hours with either fresh media or fresh media supplemented with one or combinations of the following compounds: Acetaminophen (5 mM in culture medium), Caffeine (5 mM), and Troleandomycin (TAO, 100 EM). TAO was specifically used to inhibit CYP3A enzymes in co-cultures. Following the 24 hour incubation period, viability was assessed using the MTT assay.
INCORPORATION BY REFERENCEAll publications, U.S. patents and U.S. published patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. U.S. patent application publication 2001/0023073 is expressly incorporated by reference.
EQUIVALENTSWhile several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
Claims
1. A method of forming a micropatterned substrate, comprising the steps of:
- adsorbing molecules onto a surface of a substrate, thereby forming a coated surface of the substrate;
- compressing a micropatterned etch mask onto the coated surface of said substrate; and
- exposing the compressed micropatterned etch mask and coated surface of the substrate to a gas plasma for a period of time, thereby ablating the exposed surfaces of the substrate.
2. The method of claim 1, further comprising rinsing and drying said coated surface after the adsorbing step.
3. The method of claim 1, wherein said exposing step is carried out in a plasma asher.
4. The method of claim 1, wherein the micropatterned etch mask is one solid elastomericpiece.
5. The method of claim 1, wherein the micropatterned etch mask comprises a plurality of pillars.
6. The method of claim 1, wherein the micropatterned etch mask comprises chrome or elastomeric poly(dimethylsiloxane) or rubber or plastic.
7. The method of claim 1, wherein the adsorbed molecules are different.
8. The method of claim 7, wherein the different molecules each have a different pattern.
9. The method of claim 1, wherein the micropatterned etch mask comprises plastic.
10. The method of claim 1, wherein the micropatterned etch mask comprises an about 50 μm to about 1 mm thick piece of plastic.
11. The method of claim 1, wherein said substrate surface is ceramic, metal, glass, or plastic.
12. The method of claim 1, wherein said substrate comprises fluoropolymers, fluorinatedethylene propylene, polyvinylidene, polydimethylsiloxane, polystyrene, polycarbonate, and polyvinyl chloride, fused silica, polysilicon, or single silicon crystals.
13. The method of claim 1, wherein said substrate is a tissue culture flask, a tissue culture bottle, or a cell culture multiwell plate.
14. The method of claim 1, wherein said substrate is a 24-well or a 96-well or a 384-well cell culture plate.
15. The method of claim 1, wherein said molecules are biomolecules.
16. The method of claim 1, wherein said molecules are biomolecules; and said biomolecules are selected from the group consisting of peptides, polypeptides, nucleic acids, nucleic acid binding partners, proteins, receptors, antibodies, enzymes, carbohydrates, oligosaccharides, polysaccharides, cells, cell aggregates, cell components, lipids, arrays of ligands, non-protein ligands, liposomes, and microorganisms.
17. The method of claim 1, wherein said molecules are hyaluronic acid, collagen, fibronectin, lamanin, or matrigel.
18. The method of claim 1, further comprising the steps of:
- removing the micropatterned etch mask; and
- contacting said micropatterned substrate with cells.
19. The method of claim 3, wherein said cells are hepatocytes, endothelial cells, kidney, muscle, pancreas, epithelium cells, tissue/skin cells, intestinal cells or stem-cell derived cells.
20. The method of claim 19, wherein said cells are rat or human cells.
21. A multi-well cell culture plate wherein each cell is micropatterned with a material, wherein said plate is prepared by a process comprising the steps of:
- adsorbing molecules onto a surface of a substrate, thereby forming a coated surface of the substrate;
- compressing a micropatterned etch mask onto the coated surface of said substrate; and
- exposing the compressed micropatterned etch mask and coated surface of the substrate to a gas plasma for a period of time, thereby ablating the exposed surfaces of the substrate.
22. The multi-well plate of claim 21, wherein said multi-well cell culture plate is a 24-well or a 96-well or a 384-well cell culture plate.
23. The multi-well plate of claim 21, wherein said material is a biomolecule; and said biomolecule is selected from the group consisting of peptides, polypeptides, nucleic acids, nucleic acid binding partners, proteins, receptors, antibodies, enzymes, carbohydrates, oligosaccharides, polysaccharides, cells, cell aggregates, cell components, lipids, arrays of ligands, non-protein ligands, liposomes, and microorganisms.
24. The multi-well plate of claim 23, wherein said material is not a biomolecules.
25. The multi-well plate of claim 24, wherein said cells are human or rat hepatocytes.
Type: Application
Filed: Oct 12, 2007
Publication Date: Sep 11, 2008
Inventors: David T. Eddington (Wheaton, IL), Sangeeta N. Bhatia (Lexington, MA)
Application Number: 11/974,341
International Classification: C12M 1/22 (20060101); B44C 1/22 (20060101);
