Patent Application
20080227656
The present invention relates to the identification and analysis of biocompatible materials and structures, in particular materials and structures that affect a cellular function such as direct cellular growth, expansion, differentiation, dedifferentiation, mineralization, intra- or intercellular organization, etc.
Physiologically relevant cell culture models for high-throughput screening constitute a major bottleneck for the improved treatment of many human diseases. Some physiological and patho-physiological conditions can be reproduced in 3-dimensional cell and organ cultures, but they are not optimal for quantitative readout in a high throughput setting. Many 2-dimensional culture systems for specialised cell types depend upon co-cultivation with a layer of feeder cells, which has the major drawback of inconvenience and irreproducibility. The immobilisation, isolation, expansion, differentiation and culture of living cells on a substrate is thus highly desirable for a variety of biotechnological assays, in particular for cell replacement and the screening and validation of candidate drugs. Microchips to which cells can be surface attached would facilitate high throughput screening of many candidate drugs simultaneously. The ability of a candidate drug to modulate biochemical or physiological pathways implicated in a given disease state could be examined using microchips on which cells are selectively immobilised or attached.
US 2004/0053334A1 patent application describes a substrate comprising a first layer including a heater, and a second temperature-responsive layer that exists in two states, whereby at temperatures below its ‘lower temperature solution state (LCTS)’ it exhibits a non-cell binding state, while when heated above its LCTS it exhibits a cell-binding state.
Surgical treatment often requires the introduction of implants, for example prostheses, and implantable devises, where the need to overcome a body's own rejection mechanisms and to support tissue regeneration presents a formidable challenge. The clinical success of an implant can thus depend of the cellular behaviour in the immediate vicinity of the interface between an implant and the host tissue. A key element in the progress in this field relies on the identification and use of biocompatible materials for the fabrication of these devises.
Chesmel and Black (1995) J Biomedical Materials Res. 29:1089-1099, describe a system for in vitro examination of the response of cells to changes in the chemistry and morphology of polystyrene tissue culture surfaces. The system provides 9 model biomaterial surfaces formed by polystyrene replicas (with different styrene monomer content) caste on micromachined silicon wafers, with radial surface grooves of varying depth (none; 0.5-, or 0.5-μM). Turner et al (1997) J Vac Sci Technol B15: 2848-2854 describe the effect of textured silicon surfaces on the attachment of astrocytes from the central nervous system, employing silicon substrates with two surface textures consisting of a silicon grass and a wet-etched surface that are organised in alternating rows.
In the ongoing search for improved biocompatible materials and structures there remains a need for a high-throughput screening tool to identify structures and surfaces that are optimal for a specific type of cell or tissue.
These and other needs are addressed by providing a screening tool comprising a biosurface structure array (BSSA) comprising a number of tester areas whereby each area has a surface topology nano- and/or micrometer scale features.
The present invention is based on the recognition that cellular functions like direct cellular growth, expansion, differentiation, dedifferentiation, mineralization, intra- or intercellular organization, etc, are strongly influenced by the cell's microenvironment. While not wishing to be bound by theory, it is thought that proliferation and differentiation of a specific cell type in vitro and in vivo may depend on the provision of a suitable structure, e.g. a completely artificial structure with no resemblance to structures observable in nature, or structures whose surface properties mimic those of the natural environment of the desired type of cell or tissue, to which the cell can attach. In particular the invention recognises that the 2- and 3-dimensional architecture, or topology, of surfaces in the microenvironment of a cell, is a critical factor in determining cell growth and differentiation. There are a myriad of different microenvironments in which different cell types proliferate and differentiate in vivo, and hence the specific microenvironment that will foster the growth of any specific cell type is difficult and time consuming to determine.
Hence there is a need for a high-throughput screening tool to test different microenvironments for cell cultivation, and in particular to test structures having different surface topology. The manufacture of suitable structures, e.g. artificial structure or structures that mimic surface architectures observed in nature, requires techniques capable of defining features that have micrometer scale or nanometer scale dimensions. The present invention exploits the tools presently developed within nanotechnology, which allow the design and construction of structures whose surface architecture may have a lateral feature size as small as approximately 5 nm. In particular, the use of these techniques allows the manufacture of a vast array of different surface topologies each of which is precisely defined, whereby any selected topology can be precisely reproduced in later relevant applications.
The invention provides a biosurface structure array comprising a plurality of tester areas on a single structure, such as a wafer. In order to maximise the number of tester areas per unit area of the wafer, and thereby increase its efficiency as a screening tool, it is desirable to minimise the dimensions of each tester area. Based on the recognition that an individual cell in the body sees its surrounding tissue architecture at the level of micro- and nanostructures, embodiments of the present invention provide a reproduction of such micro- and nanopatterns on a 2-D platform for screening purposes. The identification of a surface structure well suited for obtaining a 2-D layer of a defined type of cells or eventually a structure that best supports the uptake of drugs into the cells will be based upon high-throughput screening of wafers with surface structure arrays (or gradients).
According to one aspect, a key determinant of the minimal required dimensions of a tester area, are the number of cells required in order to attain a statistically significant screening assay, and that may be accommodated per tester area. It has turned out that a statistically significant screening result is achieved when each of the tester areas has an area of at least 0.0625 mm2, wherein one linear dimension is at least 250 μm, corresponding to 5 cells of diameter 50 μm each, thereby avoiding that edge effects at the border of tester areas have a statistically significant influence. In particular, the attachment of cells located near/at the perimeter of the tester area is influenced by the difference in affinity of such cells to the respective topological structures of the tester area and the adjacent tester area. Hence, the attachment of the cells to either one of the adjacent areas is influenced by the migration of cells from one tester area to the other. By suitably selecting the lower size limit of each tester area, such edge effects are prevented from having a statistically significant influence on the screening results. The ratio of cross-sectional cell size to surface area of a tester area should preferably be no less than 1/25, allowing a tester area to accommodate an array of 5×5 cells, i.e. 3×3 cells in the bulk and one row of cells along the perimeter.
According to another aspect, a biosurface structure array for screening cells comprises a plurality of tester areas, wherein each tester area has a surface to be exposed to said cells, wherein the surface has a predetermined periodic topographical structure. When the topographical structure is periodic along one or more lateral directions, e.g. one or more directions parallel with respective edges of the tester area, the structure has a high degree of uniformity across the tester area, thereby increasing the reproducibility of the screening results. Furthermore, periodic structures may be defined/classified by a small number of parameters, thereby allowing a systematic comparison, and a high degree of reproducibility. In some embodiments a cross section of a tester area along one of said lateral directions may be described by a periodic function.
Embodiments of a biosurface structure array include a substrate layer, e.g. in the form of a wafer, and, optionally, a surface layer.
When the substrate layer is silicon, e.g. in the form of a silicon wafer, existing manufacturing techniques for generating a patterned surface structure may be employed. Other suitable base materials for the preparation of the biosurface structure array include any semiconductor (doped or not-doped), a single metal, an alloy, a ceramic, a polymer, a co-polymers, a composite, a drug delivery system, e.g. a polymer with bioactive molecules, other bioactive compounds or any combination thereof.
In embodiments of the invention, the surface layer comprises a material that supports the appropriate response from the cells to be cultured. More specifically, the material needs a suitable degree of biocompatibility.
Examples of surface layers include a metallic surface deposit, e.g. tantalum, titanium, Ti—Al—V alloys, gold, chromium, metal oxides, semiconductor oxides, metal nitrides, semiconductor nitrides, polymers, biopolymers, or other alloys.
In some embodiments, the biosurface structure array comprises additional components such as one or more bioactive compound, e.g. a polymer comprising a polypeptide, deposited on a surface layer of said array, adsorbed bioactive polymers or compounds, and/or the like. For example, said polymer may be selected from the group consisting of an antibody, antigen, glycoprotein, lipoprotein, DNA, RNA, polysaccharide, lipid, organic compound, inorganic compound
A surface of the wafer is divided into a plurality of tester areas, i.e. sub-areas of the total surface area of the wafer. The surface of each tester area includes a regular nano- or micrometer scale structure along one dimension or two dimensions within the surface plane of a tester area. Examples of structures with nano- or micrometer scale features in one dimension include features that extend over the entire length of the tester area in a longitudinal direction and have nano- or micrometer scale dimensions in a lateral direction. Examples of two dimensional structures include structures including features having nano- or micrometer scale dimensions in two directions within the plane defined by the surface of the tester area.
The topographical structure is regular, i.e. the structure comprises features that are arranged in a predetermined ordered, non-stochastic pattern, thereby ensuring the reproducibility of the screening results. It is a further advantage that regular topographical structures of micro and/or nanometer scale can be reproducibly produced by known production techniques, such as photolithography, e-beam lithography, hot embossing, nano-imprint lithography, microcontact printing, focussed ion beam etching, etc. Other examples of production steps that may be involved in the production of the biosurface array include chemical etching, laser ablation, plasma spray coating, abrasive blasting, engraving, scratching, micro-machining, etc.
When the topographical structure is periodic along one or more lateral directions, e.g. one or more directions parallel with respective edges of the tester area, the structure has a high degree of uniformity across the tester area, thereby increasing the reproducibility of the screening results. Furthermore, periodic structures may be defined/classified by a small number of parameters, thereby allowing a systematic comparison. In some embodiments a cross section of a tester area along one of said lateral directions may be described by a periodic function.
The term nanometer scale as used herein is intended to refer to a length scale in the range of between about 1 nm and about 100 nm, in particular between about 1 nm and about 100 nm. The term micrometer scale as used herein is intended to refer to a length scale in the range of between about 1 μm and about 1000 μm.
In embodiments of the invention the lateral dimensions of the topographical features in at least one lateral direction are within the interval 1-105 nm, e.g. 1-2 nm, 2-4 nm, 4-10 nm, 10-100 nm, 100-1000 nm, 1-2 μm, 2-4 μm, 4-10 μm, 10-100 μm.
In some embodiments, the scale of the features varies from tester area to tester area, e.g. some tester areas have features on a nanometer scale, i.e. between 1-1000 nm, while other tester areas have features at micrometer scale, i.e. between 1-1000 μm. Similarly, each tester area may comprise features at different scales, e.g. a structure with a small period along one direction and a larger period, e.g. by a factor of 10, 100, or 1000 larger, in another direction. In some embodiments a structure may even have features at different scales along the same direction, e.g. features at a small scale superimposed features at a larger scale. For example, a wafer with tester areas having regular nanometer or micrometer scale structures as described herein may subsequently be processed so as to superimpose an irregular/random structure on the surface of some or all of the tester areas, e.g. a so-called silicon grass structure as described by Turner et al. (1997) (ibid.)
The depth/height of the features, i.e. their linear dimension in a direction projecting out of the surface of the tester area may likewise be on the nano- or micrometer scale, i.e. the structures may have heights/depths in the range 1 nm-1000 μm. In some embodiments the structures have a depth/height of greater than 0.6 μm, preferably between 1.2 μm and 1000.0 μm, and more preferably at least 1.2 μm, 2.0 μm, 5.0 μm, 10 μm, 20 μm, 50 μm, 100 μm or 1000 μm.
In some embodiments, the structures may further include patterns along the direction projecting out of the surface. For example, the protruding surfaces may be structured by a chemical or other process, e.g. a chemical etching process.
In some embodiments, the period of the periodic structure is smaller than the linear dimension of the tester area, e.g. the linear dimension of the tester area is an integer multiple of the period of the structure.
In embodiments of the invention, the surface structure comprises individual features, such as protrusions, pillars, ridges, channels, recesses, holes, etc. of predetermined lateral dimension and height/depth. Alternatively, a feature of the topological structure may be defined as a period of the periodic structure. Hence, the lateral dimensions of a feature of a periodic structure may be defined as the period of the periodic shape/function, i.e. the length of the shortest interval over which the structure repeats its shape.
The lateral cross section of each of the features may have any shape with a suitably defined circumference and/or geometrical shape, such as square, rectangular, hexagonal, polygonal, round, circular, etc., or a combination thereof. The top and/or side surfaces of the features may be substantially flat, curved and/or structured, e.g. modified by a chemical or other process. Different features may have the same or different shapes.
In embodiments of the invention, the smallest lateral/vertical dimension of the structure is smaller or at least comparable to the linear dimension of the organisms/cells to be exposed to the surface, such that the organisms experience a structure at a scale smaller or at least comparable to their own size.
The wafer on which the tester areas are located may have any suitable size, e.g. a 2, 4, 6, 8, 12, 14, or 16 inch wafer, or an even smaller or larger wafer, thereby providing biosurface structure arrays of different sizes and allowing parallel screening of both small and large diversities of different structures.
The tester areas may have any geometrical shape, such as circular or polygonal, e.g. hexagonal. Nevertheless, rectangular tester areas, in particular square tester areas, are convenient and provide an efficient utilisation of the wafer area. Square tester areas further provide a small boundary relative to the area of the tester area and, at the same time, an efficient utilisation of the wafer area. Hence, a large effective area for screening is provided while limiting the boundary effects at the edges of the tester areas.
The tester areas may have the same or different dimensions along each lateral direction. In embodiments of the invention, the dimension of the tester areas in each direction is at least 0.25 mm, preferably at least 0.3 mm, more preferably at least 0.5 mm, most preferably at least 1 mm, e.g. in the interval between 0.25 mm and 25 mm, preferably between 0.3 mm and 20 mm, more preferably between 0.5 mm and 10 mm, most preferably between 1 mm and 5 mm, e.g. the tester areas may be squares with dimension of about 2 mm×2 mm, 5 mm×5 mm, 10×10 mm, 0.25×0.25 mm, or the like.
In one embodiment each tester area has dimensions of between about 2 mm×2 mm to about 10 mm×10 mm providing between about 60 to about 1500 tester areas on a standard 4 inch wafer. It is an advantage that a screening of cells with a size of approximately 50 μm×50 μm can be conducted with a high statistical significance by providing tester areas of 2 mm×2 mm, where potentially circa 1600 cells can be accommodated per tester area.
When the surface area is at least 0.09 mm2, and wherein each linear dimension is at least 300 μm, at least about 16 cells of dimension 50 μm×50 μm can be accommodated in the bulk of the tester area, surrounded by at least one row of cells along each edge of the tester area. Hence, the tester area is sufficiently large to provide a uniform microenvironment for 16 cells without border effects. When the surface area is at least 0.1225 mm2, and wherein each linear dimension is at least 350 μm, at least about 9 cells of dimension 50 μm×50 μm can be accommodated in the bulk of the tester area, surrounded by at least two rows of cells along each edge of the tester area, thereby further reducing the influence of edge effects. Alternatively, when the surface area is at least 0.1225 mm2, and wherein each linear dimension is at least 350 μm, at least about 25 cells of dimension 50 μm×50 μm can be accommodated in the bulk of the tester area, surrounded by at least one row of cells along each edge of the tester area, thereby increasing the number of cells available for analysis.
Embodiments of the present invention can be implemented in different ways, including the biosurface structure array described above and in the following, a method of screening for a biocompatible surface structure, further product means, and one or more uses thereof, each yielding one or more of the benefits and advantages described in connection with the first-mentioned aspect, and each having one or more embodiments corresponding to the embodiments described in connection with the first-mentioned aspect and/or disclosed in the dependent claims.
In particular, the invention further relates to a kit comprising a biosurface structure array described herein. Such a kit may be used for one or more of the following: A cell attachment assay, a cell differentiation assay, a cell spreading assay, a cell proliferation assay.
A kit comprising a BSSA suitable for screening one or more cellular effects of the surface structures on the wafer may comprise: One or more BSSA wafers, and optionally may include standard cell media, sterile culture containers, or other standard culture items, standard cells, cells labelled with fluorescent dyes, and/or the like. Preferably, the size of the tester areas on the one or more BSSAs is selected such that each tester area can accommodate at least 25 of the cells dimension 50 μm or of the cells included in the kit, preferably at least 50 cells, more preferably at least 100 cells, most preferably at least 1000 cells.
For example, such a kit is suitable for measuring the ratio between attachment of two cell types. Accordingly in this example, the kit may include or be used to screen cells labelled with e.g. the fluorescent proteins EYFP and ECFP. The fluorescent proteins can be expressed in the cells using standard gene transfer techniques. To facilitate the monitoring of the cell numbers of the two different cell types the fluorescent proteins can further be localized to the nucleus.
The results of screening with a BSSA wafer may be analysed by a variety of methods including an optical inspection by means of a microscope. When the microscope is placed in an incubator capable of controlling humidity, CO2, temperature and/or other parameters, a controlled environment is provided for the cell cultures on the BSSA. An embodiment of a device suitable for automatic or at least semi-automatic acquisition of screening results comprises a microscope positioned in an incubator, a support for receiving a BSSA wafer, an image recording device, and a control unit for controlling the position of the wafer relative to the optical axis. The control unit is further adapted to control the microscope to acquire one or more images of each tester area. The control unit further comprises an image processing unit adapted to receive and the recorded images and to perform an image analysis of the received images. For example, the image analysis may include the detection of cells of different fluorescent colours and the calculation of the ratio of the number of cells of different fluorescent colours. Alternatively or additionally, the image analysis process may include a measurement of the total area of a tester area covered by cells with a given fluorescent colour, or similar image analysis steps.
The invention further relates to a method of screening for a biocompatible surface structure, comprising
Using such BSSA technology enables screening of a large number of structures in one single experiment by either a single or a mixed cell culture. The biosurface structure array described herein may be used for assaying a large variety of screening parameters localized to the individual tester areas on the BSSA wafer including: mineralization, differentiation markers on the cells, cell number (enhanced or decreased), gene induction in reporter cells, bio fouling, attachment and expansion of a specific cell type, etc.
Accordingly, in some embodiments, the method further comprises comparing the frequency of attachment of said cells or organisms to any one tester area of said biosurface structure array, thereby determining differences of attachment of cells to the respective surface structure of the different test cells. In another embodiment, the method further comprises comparing spreading of said cells or organisms on any one tester area of said biosurface structure array. In yet another embodiment, the method further comprises comparing differentiation of cells or organisms attached to any one tester area of said biosurface structure array.
In some embodiments, the method further comprises incubating (a) under conditions suitable for the maintenance and/or growth of said cells or organisms.
The biosurface structure array described herein may be used for detecting/testing different microenvironments for cultivation of a large variety of cells or organisms, including cells, such as animal cells, plant cells, mammalian cells, yeast cells, eukaryotic or prokaryotic cells or organisms, etc.
When each linear dimension of the tester area is large enough to accommodate a row of at least 5 cells, preferably at least 10 cells, more preferably at least 20 cells, the tester area is large enough to accommodate a sufficiently large number of cells in the bulk area, i.e. removed from the edge of the tester area, to provide a screening parameter result of sufficient statistical relevance.
The invention further relates to a biocompatible material having a surface, wherein at least a part of said surface is characterized by a topology comprising nanometer scale or micrometer scale features, the surface being obtained/obtainable by the method described above and in the following.
The invention further relates to a wafer comprising a biosurface structure array described herein, and use of such a wafer in an in vitro cell bioassay. The invention further relates to a kit comprising such a wafer for performing an in vitro cell bioassay.
The invention will be explained more fully below in connection with embodiments and with reference to the drawings, in which:
The BSSA wafer 100 comprises 60 tester areas. A number of tester areas 2 are left “blank”, i.e. they have not been processed to have a structured surface. Consequently, the surfaces of the tester areas 2 are substantially flat. Consequently a control experiment is inherently included in each parallel screening test with the BSSA screening tool. The remaining tester areas, designated S1, S2 . . . , S54, comprise respective structured surfaces as described herein. The tester areas are squares of dimension 10 mm×10 mm.
It is noted that the
A biosurface structure array described herein can be manufactured by a number of production techniques. Examples of procedures for the manufacture of a BSSA include one or more of the following techniques which are known as such in the art:
Furthermore, the manufacture of a BSSA may include further modification processes for modifying the surface structures of the tester areas, thereby allowing an even larger range of microenvironments to be screened. Examples of such techniques include but are not limited to topographical modifications directly applicable on 3D, such as various blasting techniques, e.g. sandblasting, microblasting and glassblasting. Further examples of such modification processes include chemical modifications, such as processes resulting in one or more gradients in one or more directions across a wafer, e.g. by protein adsorption, peptide adsorption, chemical deposition, chemical etching, and/or electrochemical deposition. For example, a chemical gradient may be produced by gradually submerging a wafer into a suitable solution. Consequently, on a single wafer, a number of different structures may be screened and, if tester areas with the same structure are repeated along the chemical gradient, each structure is screened under varying chemical conditions. Consequently, a highly parallel screening of different microenvironments is facilitated.
A single-sided polished silicon wafer (4 inch) with a thickness of 525±25 μm provided a substratum for the manufacture of a biocompatible material. The wafer was an n-type wafer with a resistivity of 1-20 ohm cm. A micrometer-sized pattern was printed onto the polished side of the silicon wafer by standard photolithography and reactive ion etching in a SF6/02 discharge according to the following protocol:
Each specific lateral structure is repeated 4 times. In
Hence, the line structure of
It is understood that the preparation method described above may also be applied to wafers with other forms and sizes of tester areas as well as other types of structures. The same production process may be used for a variety of different wafers, where the layout of the tester areas and the particular surface structures are determined by the mask through which the wafer is exposed.
Each wafer was placed in a P15 dish (NUNC, Biotech line) and washed with 70% ethanol and then PBS (6.8 g NaCl, 0.43 g KH2PO4, 0.978 g Na2HPO4*2H2O in 1 liter double distilled water ˜pH 7.4). The wafer was seeded with cells of a MC3T3-E1 murine osteoblastic cell line (Sudo, H et al. 1983, J Cell Bio/96(1):191-98), at a concentration of 20,000 cells/cm2. The cells were cultured for 4 days in plain medium (alpha-minimal essential medium [a-MEM], 10% fetal calf serum [FCS], 100 U/ml penicillin, and 100 microgram/ml streptomycin (supplied by Gibco, Invitrogen). The cells were maintained in a humidified incubator (5% CO2/95% air atmosphere at 37° C.), and subsequently 50 μg/ml ascorbic acid (Wako Chemicals, DE) and 10 mM β-glycerophosphate (Sigma-Aldrich, DK) were included in the growth medium. The cells were cultured for 3 weeks, with a change of growth medium twice a week.
Examples of methods of demonstrating the biocompatible properties of materials include cell attachment assays as described above, a cell spreading assay, a cell motility assay, a differential cell attachment assay, a cell proliferation assay, a gene induction assay, etc.
For example, in a cell proliferation assay cells may be labelled with fluorescent proteins and cell proliferation determined by scanning the BSSA wafer several times for a period lasting up to several days. When the wafer is placed in an automated analysis device including a microscope in an incubator as described herein, the device can perform the scanning in an automated fashion.
Hence, statistically relevant results may be achieved while reducing sources of error caused by gradient effects due to cells close to an edge of a tester area being influenced both by the structure of the tester area and the surrounding structure, e.g. the structure of an adjacent tester area. Larger tester areas can accommodate even more cells, thereby providing more cells for analysis, e.g. 5×5, 10×10, 20×20 cells, 40×40 cells, or even more cells, thereby providing improved statistics. Furthermore, large tester areas allow disregarding of 2, 3, 4 or more rows of cells along the edges, thereby reducing the influence of border effects.
As mentioned above, the BSSA described herein can be used in a screening assay for the differential attachment of any two or more different cell types. In one example of such a differential cell attachment (DCA) assay, one cell line is genetically modified to stably express the enhanced yellow fluorescent protein (EYFP) gene whereas the other either expresses (enhanced cyan fluorescent protein (ECFP) or no fluorescent protein at all. The two cell lines are seeded in various proportions on the BSSA wafer. After one to seven days in cell culture the proportion of the two cell types are calculated by fluorescence microscopy. By comparing with the reference tester areas the structures can be examined for advantages in attachment, proliferation etc., as a higher percentage of the relevant cell type is attached to the surface. For example, by use of 4 inch BSSA wafer it is possible to screen approximately 1500 squares with a size of 0.2 mm×0.2 mm, each comprising a different surface structure. Such a square can accommodate several thousand cells, making the quantification of the DCA effect statistically highly relevant.
The control unit may be implemented by hardware comprising several distinct elements, by means of a suitably configured and programmed computer, e.g. a personal computer, or as a combination thereof.
The microscope and the software controlling the microscope and performing the image analysis is adapted to automatically process wafers of one or more different sizes, e.g. wafers up to at least 6000 tester squares of a size of 2×2 mm. For each tester square one to several pictures are obtained. The microscope, the sample holder, and the control unit are further adapted to process wafers of different sizes, e.g. 4 inch wafers, 6 inch wafers, 8 inch wafers, and/or 12 inch wafers, in an automated fashion.
A kit for screening for insulin producing cells comprises different BSSA wafers, and may further include standard cell culture media and sterile containers as well as various primary cells generated from an insulin Knock-in mouse. By using such cells, surfaces can be assayed for their capability to differentiate e.g. mesenchymal stem cells into beta-cells (insulin producing cells), it can be used to assay for preferential attachment and expansion of beta-cells etc. For human primary cells, cells can e.g. be fixed and stained with antibodies for the relevant genes. The above kit may be used in connection with a microscope described herein. Alternatively or additionally, the kit may comprise such a microscope and/or a software package with control software and/or analysis software for automatic or semiautomatic screening of the wafers.
A BSSA described herein may be used for the screening of biocompatible surfaces in connection with a number of diseases:
For example, any structure that can direct a cell type to the formation of dopaminergenic neurons are of interest as a direct treatment of Parkinson disease as well as they are of potential interest for a variety of other nerve related diseases like Alzheimer. Cells maintained in such a differentiation state further are of great interest, because they can be utilized for screening as well as initial drug testing purposes. The efficiency of cell type attachment to the different surface structures can be measured using immunostaining techniques using commercially available antibodies that recognise cell type specific protein markers, e.g. synapsin I (neurons), GFAP (astrocytes) and PLP (oligodendrocytes), Glia with different coloured fluorescent tags and identify the different cell types by double immunostaining and fluorescence microscopic technologies. The presence of different cell types can be measured at different time intervals which also allows an identification of surface micro- and/or nanometer scale structures that support expansion of neuronal populations.
DAergic neurons can be identified by immunostaining using antibodies specific for tyrosine hydroxylase (TH) and the dopamine transporter (DAT). The ability of the surface structures to support the presence and differentiation of DAergic neuron relative to other cell types, e.g. other neuronal types and astrocytes, can be measured.
Furthermore, any structure that facilitates expansion of Beta-cells or induces differentiation of stem (mesenchymal) cells into insulin producing beta-cells will be of interest in diabetes. Such cells can be used to treat the growing proportion of the population that develops type I diabetes due to lifestyle and/or old age. Insulin producing cells grown on such a surface may be used for drug testing/validation assays.
Structures that enable and/or facilitate the isolation, expansion, priming or differentiation of cells from bone marrow aspirates for bone forming capabilities can be identified by using BSSA. Cells grown on such structures can be used in conjunction with orthopaedic surgery. Primary cells grown on such structures can be used for various drug testing applications.
It is further desirable in a variety of medical applications to be able to transfect primary cells without the use of viral transduction. The BSSA described herein can be applied to screen for nano- and/or micrometer scale structures that either alone or in conjunction with different coatings (e.g. PLA, PLG, PAGA, and poly(D,L-lactide-co-glycolide) (PLGA) polymers) will increase the uptake of DNA in various cell types. For example, the reporter system may consist of a mammalian DNA expression vector encoding enhanced green fluorescent protein (EGFP). This plasmid is applied to the BSSA and structures of interest are identified by fluorescent microscopy.
Such structures will be of major interest in siRNA technologies as well as gene transduction of primary cells for transient expression of an effector gene e.g. BMP-2 in bone forming cells used in orthopaedic research
Embryonic fibroblasts are large stretched cells with long and thick stress fibres (actin filament bundles). Furthermore, embryonic fibroblasts are motile cells. As can be seen from
MC3T3 are smaller cells with thinner stress fibres. Furthermore, MC3T3 are less motile cells. As can be seen from
All stainings in
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2005 00610 | Apr 2005 | DK | national |
| PA 2005 00981 | Jul 2005 | DK | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DK06/00216 | 4/25/2006 | WO | 00 | 5/7/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60675096 | Apr 2005 | US | |
| 60700306 | Jul 2005 | US |
