Abstract:
Systems and methods are providing for performing high-throughput, programmable, multiplexed assays of biological, chemical or biochemical systems. Preferably, a micro-pallet includes a small flat surface designed for single adherent cells to plate, a cell plating region designed to protect the cells, and shaping designed to enable or improve flow-through operation. The micro-pallet is preferably patterned in a readily identifiable manner and sized to accommodate a single cell to which it is comparable in size. Each cell thus has its own mobile surface. The cell can be transported from place to place and be directed into a system similar to a flow cytometer. Since, since the surface itself may be tagged (e.g., a bar code), multiple cells of different origin and history may be placed into the same experiment allowing multiplexed experiments to be performed.

Description:
RELATED APPLICATION  
       [0001]     This application claims the benefit of U.S. provisional application Ser. No. 60/564529 filed Apr. 21, 2004, which application is incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The present invention relates to microfabricated devices for single and multiple cell analysis and, more particularly, to devices and methods for performing automated, programmable, high-throughput analysis of adherent cells.  
       BACKGROUND OF THE INVENTION  
       [0003]     Adherent cell assays have typically been performed on slides, in flasks, or in petri dishes. Cells are inspected by microscopy whereby the experimentalist seeks out each cell and images each independently for fluorescence or other indicating measurables of the experiment. Alternatively, the experimentalist may look at the effects of the entire colony of cells as a whole by measuring a global quantity, such as overall fluorescence or absorbance. The methods are not conducive to high throughput analysis, or multiplexed analysis.  
         [0004]     Tagged (“bar-coded”) microdevices have been developed for the purpose of performing multiplexed assays. Most devices are not suited to carry cells and they do not offer any added functionality other than bar coding. One system currently marketed includes a small metal device that has a bar code pattern etched on it. This enables multiple assays to be performed simultaneously. At the end of the assay, the bar coded “beads” fall to the bottom of the container and they are read by an imaging system. Another system includes beads that can be identified by their adsorption of two dyes in differing ratios, thus enabling bead identification by the use of two photodetectors. This system works in a flow-through manner, but tends not to be able to be used with cells. At least two systems are currently marketed to enhance single cell analysis. One system allows flow-through analysis of single cells by encapsulating the cell in a spherical gel matrix, then flowing the gel balls though the analysis system (e.g., a flow cytometer). Another system provides moderately sized glass plates (0.5 mm×0.35 mm) with color codes on the side to enable cells to plate and be read by imaging. It tends not to be able to be the method used in a flow-through system, nor can the current method of manufacturing enable further miniaturization.  
         [0005]     Thus, it is desirable to provide devices and methods that facilitate performing automated, programmable, high-throughput analysis of adherent cells.  
       SUMMARY  
       [0006]     The systems and methods described herein provide for performing high-throughput, programmable, multiplexed assays of biological, chemical or biochemical systems. In one embodiment, a micro-pallet is provided that includes a small flat surface designed for single adherent cells to plate, a cell plating region designed to protect the cells, and shaping designed to enable or improve flow-through operation. The micro-pallets are preferably patterned in such a manner so that they can be readily identified using standard optical equipment.  
         [0007]     The micro-pallets are preferable sized to accommodate a single cell and are comparable in size to the cell itself. In this manner, each cell has its own mobile surface. The cell can thus be transported from place to place and be directed into a system similar to a flow cytometer. Furthermore, since the surface itself can be tagged (e.g., a bar code), multiple cells of different origin and history can be placed into the same experiment allowing multiplexed experiments to be performed. The micro-pallet enables adherent cells to be used in flow-through analysis systems such as lab-on-a-chip system.  
         [0008]     In another embodiment, the micro-pallets are patterned in an array on a plate.  
         [0009]     Other systems, methods, features and advantages of the invention will be or will become apparent to one with skill in the art upon examination of the following figures and description. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0010]      FIG. 1A  is a plan view of a micro-pallet of the present invention. The pallet is preferably small enough to carry a single cell.  
         [0011]      FIG. 1B  is a perspective view of an alternative embodiment of a micro-pallet of the present invention.  
         [0012]      FIGS. 2A and 2B  are schematic diagrams of exemplary manufacturing processes for micro micro-pallets shown in  FIGS. 1A and 1B .  
         [0013]      FIG. 3  is a perspective view of an electrical poration and loading system that utilizes the micro-pallets shown in  FIGS. 1A and 1B .  
         [0014]      FIG. 4  is a perspective view of an optical readout system for use with the micro-pallets shown in  FIGS. 1A and 1B .  
         [0015]      FIG. 5  is a plan view of flow system having a mechanical means for directing detected cells to different channels.  
         [0016]      FIG. 6A  is a micro-pallet plate having an array of micro-pallets.  
         [0017]      FIG. 6B  is a schematic of an imaging system in which the micro-pallet plate is moved through at high speed and a selected plates are released.  
         [0018]      FIG. 7  is a schematic of a high content screening and cell selection system utilizing a micro-pallet cassette comprising an array of micro-pallets. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0019]     The systems and methods described herein provide for performing automated, programmable, high-throughput analysis of adherent cells. In a preferred embodiment, microscopic structures such as cell pallets, are manufactured using micromachining techniques. The structures or micro-pallets, which preferably range in size from about ten microns to about one millimeter, and preferably less than five millimeters in any given dimension, are patterned in such a manner so that they can be readily identified using standard optical equipment and designed to carry chemical, biological or living samples through a fluidic system. Preferably, the cell pallets are surface treated to enable adherent cells to plate against them and designed to provide a portable scaffolding for the adherent cells—carrying the cells to different locations through an experimental system such as a flow-through system. For example, the pallets can be run past electrodes for cell wall poration or an optical system designed to detect scattering or fluorescence in a manner similar to a flow cytometer. Typically, flow cytometer cannot be performed on adherent cells because they cannot live in free solution as they require a surface to grow on. The pallets provide the surface to grow on and are constructed to protect the cells from electrical, mechanical and shearing forces and to provide alignment of the pallet in a flow stream, i.e., the pallets can utilize the flow stream to align the cells appropriately.  
         [0020]     Additional functions are added by building tagging systems (e.g., bar codes) on the pallets, by adding optical components (e.g., apertures, lenses), or by adding magnetic material to the pallets to allow magnetic sensing or actuation of the pallet. Electrical contacts can be included if desired to facilitate electrical poration of cell walls.  
         [0021]     In preferred embodiments, the pallet or micro-pallet is a small, flat piece of material designed to carry molecules, cells, or tissue in a fluid medium. Surface treatments can be provided to affect the adsorption of molecules, cells, or tissue on to the micro-pallet, and produce distinct regions that have biological properties of importance to the cells, such as regions that promote or discourage cell adhesion. The micro-pallet can have markings that vary between opaque and transparent that can encode information about the micro-pallet (e.g., “bar code”). In addition, the micro-pallet can be shaped in a manner that also encodes information about the micro-pallet and/or affect its movement through fluid.  
         [0022]     The micro-pallet can be used in a system designed to move the pallet from at least a first location to a second location to (a) perform a step in an assay protocol, or (b) to enhance the ability to detect or image the micro-pallet. External mechanisms, including but not limited to electromagnetic fields, optical fields, moving surfaces, centrifugal forces, vibration, fluid flow and the like, can be used to move the micro-pallet through the system. The micro-pallet can also be used in a system designed to capture the micro-pallets in fixed locations for the purpose of knowing its location to ease assaying, detection or imaging. The fixed locations can be in array, linear, or any form suitable for the assaying instrument.  
         [0023]     Turning in detail to the figures, an embodiment of the micro-pallet is illustrated in  FIG. 1A . As depicted, the micro-pallet  10  includes a body  12  preferably formed from metal, glass, polymer or the like with a cell plating region  14  prepared to facilitate cell growth on the micro-pallet  10 . The plating region  14  is preferably prepared using a suitable surface coating (e.g., hydrophilic) and can be recessed to protect the cell from harm as the micro-pallet  10  is moved through an experimental system.  
         [0024]     Another embodiment of a micro-pallet is illustrated in  FIG. 2 . As depicted, the micro-pallet  20  includes a body  22  preferably formed from metal, glass, polymer or the like. A cell plating region  24 , prepared using a suitable surface coating (e.g., hydrophilic) to facilitate cell growth, is preferably formed in a recess  28  in the body  22  of the micro-pallet  20 . The wall  29  of the recess  28  tends to protect the cell(s) from harm as the micro-pallet  20  is moved through an experimental system.  
         [0025]     The shape of the pallet  20  is preferably optimized to enable it to line up in a flow stream. As such, the pallet can be long and thin and can include fins, rudders, or other devices to achieve a desired alignment in a flow stream.  
         [0026]     The pallet  20  is preferably made to be opaque, whether through the use of an opaque material or the application of an opaque coating (e.g. thin film of metal). Small openings  26  in the opaque material of the body  22  can be used for identification purposes in the form of a recognizable pattern or, as depicted, a bar code. The bar code  26  can be read by an imaging device or by directing the flow of the pallet  20  past an optical system that can detect the transparent regions of the pallet  20 . Alternatively, the pallet  20  can be tagged using conductive patterns, magnetic patterns, or the like.  
         [0027]     In addition, the opaque material is preferably patterned to leave a transparent region, which forms an optical aperture, directly below the cell or cell plating region  24  of the body  22  The optical aperture enables imaging of the cell and can reduce optical background.  
         [0028]     The pallet  20  can also contain magnetic components to enable electromagnetic sensing and/or actuation of the pallet  20 . For example, a pallet  20  containing magnetic components can be moved or directed through a flow system into different channels or reservoirs by application of an external magnet.  
         [0029]     The micro-pallets  10  and  20  can be manufactured using several methods common to micromachining. In one embodiment, shown in  FIG. 2A , the micro-pallets  10  and  20  are manufactured in a process  100  comprising the following steps: (A) a photosensitive polymer  104  is applied to a surface of a substrate  102  formed of glass or the like; (B) the polymer  104  is then photo-patterned  108  through a mask  106  comprising features of interest on the micro-pallets; (C) the polymer  104  is developed, removing unwanted polymer; and (D) the supporting substrate  102  is stripped away leaving freestanding micro-pallets in the polymer material.  
         [0030]     In another embodiment, shown in  FIG. 2B , the micro-pallets  10  and  20  are manufactured in a process  110  comprising the following step: (A) a structural material  114  is placed over a sacrificial layer of material  113  on a substrate  112  formed of glass or the like; (B) the structural material  114  is etched or cut through to the sacrificial layer  113  using such methods as wet or dry etching, laser ablation, die-stamp cutting, or the like; (C) additional materials (if desired) can be added to the surface of the cut material  114 ; and (D) the supporting substrate  112  and sacrificial material  113  are stripped away leaving freestanding micro-pallets in the structural material  114 .  
         [0031]     The small cell pallets  20  and  10  allow adherent cells to be readily stored, transported, and manipulated in a flow-through system. Cells can be visualized one at a time using an imaging system, or they may be detected using optical, electrical, or electromagnetic systems that are configured to detect the cells.  
         [0032]     Exemplary embodiments of flow-through (lab-on-a-chip) systems that utilize the micro-pallets  20  and  10  are illustrated in  FIGS. 3, 4  and  5 . Turning to  FIG. 3 , an electrical poration and cell loading flow through system  30  is illustrated. The system  30 , as depicted, is preferably formed in a polymer chip  31  and comprises first and second inlet channels  32  and  34  and an outlet channel  38 . A pair of electrodes  36  are positioned along the outlet channel  38  just beyond the junction region  35  of the first and second inlet channels  32  and  34 . In this system  30 , two or more flow streams flowing through the first and second channels  32  and  34  are brought together at the junction region  35 . One of the flow streams flowing through the second channel  34  is preferably a buffer containing a substance intended to be loaded into the cells (e.g., DNA, enzymes, or fluorescent molecules). Another of the flow streams flowing through the first channel  32  contains or transports the micro-pallets  10  and adherent cells plated thereon. The loading reagent comes into close proximity to the micro-pallets  10  as they flow through the channel junction  35 . In the embodiment shown, the channels are about 0.5 mm wide and the micro-pallets or carriers  10  are about 0.1 mm wide.  
         [0033]     The micro-pallets  10  pass by the two electrodes  26  that carry current (preferably alternating current, to avoid electrolysis). Passing so close to the electrodes  26  tends to ensure that the cells are electrically porated by electrical breakdown of their cell membranes. Following poration, the cells are rapidly loaded with the loading reagent because the loading substance tends to be in very high concentration in regions very close to the porated cells. Serpentine geometries or external magnetic fields can be used to induce mixing and, thus, improve reagent loading.  
         [0034]     High speed readout of the cells and pallets may be performed with a suitably designed optical system  130  such as that shown in  FIG. 4 . In this embodiment, cell pallets  10  are directed into a single file through injection into a laminar flow stream and flow through a channel  132  formed in a polymer chip  131 . The injection system (not shown) may include the use of a laminar flow sheath to pinch the cell pallets  10  into alignment. The cell pallets  10  can be aligned through the use of shear forces in a laminar flow stream, or through the use of external magnetic fields (if the cell pallets have magnetic elements). A microscopic imaging system  136  is positioned over the channel  132  adjacent the outlet  138  of the channel  132  to image the cells as they flow past. Imaging preferably occurs by high speed flash and photography, triggered by light scatter of beads. Alternatively, imaging can occur by continuous readout of a linear detector array (scanner mode). Other readout systems, including optical systems, electrical systems, and chemical systems can be used.  
         [0035]     Other operations, such as directing the micro-pallets  10  into different channels  237  and  239  or reservoir regions, based on preprogramming, or based on detection criteria can be performed, as shown in  FIG. 5 , to provide automated, programmable sample preparation and cell sorting capabilities. Micro-pallets  10  can be moved using fluid flow, electric fields, magnetic fields or mechanical means. As depicted in  FIG. 5 , micro-pallets  10  flowing through a channel  238  are directed to different channels. Micro-pallets  10  carrying cells having a first detection criteria  11  are directed to a first channel  237  via mechanical means such as a movable gate  233 . Likewise, micro-pallets  10  carrying cells having a second detection criteria  13  are directed to a second channel  239 .  
         [0036]     Turning to  FIGS. 6A and 6B , a micro-machined micro-pallet plate  40  is shown. The pallet plate  40  preferably includes a pre-set array of releasable pallets  44  for cell culturing that are releasably positioned atop of a plate  42  formed of glass or the like. The pallets  44  are treated to promote cell growth at the center  45  of the pallets  44 . The pallets  44  are preferably indexed, e.g., bar coded, so that their positions are known in advance of use of the pallet plate  40 . Cells are allowed to grow on the pallets  42 .  
         [0037]     As shown in  FIG. 6B , the pallet plate  40 , immersed in a buffer solution  47 , is moved through an imaging system at a high speed over an imaging device  48 . Selected pallets  44 A can be released from the surface by a laser pulse  49  and the like.  
         [0038]     As shown in  FIG. 7 , a disposable cassette  140  comprising a substrate or plate  142  formed of glass or the like and a cover  146  can include an array of micro-pallets  144 —e.g., providing 500,000 (50×50 microns) pallet sites—positioned on the plate  142 . The cassette  140  can be used with a microscope attachment  50  for imaging, fluorescent analysis, sorting, and the like. Analysis software provided on a CPU  60  can be used for high content screening and cell selection. A pallet extractor can be used to extract a selected pallet from the cassette  140 .  
         [0039]     While the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the appended claims.