WO2003056335A2

WO2003056335A2 - Micro-recess array for size-dependent sorting or separation of cells suspended in a flowing medium and method for analysing the functional activity of individual cells

Info

Publication number: WO2003056335A2
Application number: PCT/EP2002/014762
Authority: WO
Inventors: Dirk Lassner; Holm Uhlig; Johann Michael KÖHLER; Günter Mayer
Original assignee: Institut für Physikalische Hochtechnologie e.V.
Priority date: 2001-12-31
Filing date: 2002-12-27
Publication date: 2003-07-10
Also published as: AU2002361220A1; AU2002361220A8; WO2003056335A3

Abstract

The invention relates to a micro-recess array for size-dependent sorting or separation of cells suspended in a flowing medium in individual reaction chambers, and to a method for analysing the functional activity of individual cells which are separated by said micro-recess array. The micro-recess array is characterised in that it comprises a cell array made of a microstructured solid phase with a plurality of recesses (10).

Description

Micro-well array for size-dependent sorting or separation of cells suspended in a flowing liquid and method for examining the functional activity of single cells

description

The invention relates to a method for examining the functional activity of single cells on milαrostructured solid phase surfaces and a microwell array used therefor. The invention is used in medical diagnostics, immunology and biotechnology.

A number of techniques are available with which the detection of secretion products from single cells not organized in tissues or single cells isolated from tissues can be carried out. Techniques are listed below that have been developed to type and / or sort cells according to their functional activity and the production of secretion products. Methods are also given for arranging biological molecules and cells in high density in a suitable pattern on solid phases, which forms the basis of the solution shown below.

The analysis using FACS (fluorescence activated cell sorting) allows the detection and sorting of cells essentially according to surface markers. The detection of the functional activity of cells in the sense of measuring intracellular products is only possible using standard techniques after permeabilization and fixation of the cells (Prussin 1997, Assemissen et al. 2001). A large number of cells can be analyzed in a short time using this technique.

Miltenyi technology can be seen as a method for cell separation based on the criteria for the production of cell secretion products (eg cytokines). It allows the measurement of secretion products from single cells using bifunctional antibodies, which on the one hand bind to the cell surface and on the other hand bind secretion products such as cytokines. The secretion products bound on the cell surface can They can then be detected using suitable labeling of the secretion products in sandwich technology and assigned to the producing cell (Manz et al. 1995, Assenmacher et al. 1998, Brosterhus et al. 1999). The technique allows both the detection of secretion products and the sorting of cells according to their secretion products using magnetic or FACS sorting. No permeabilization and fixation of the cells is required.

The analysis of cell secretion products by means of in-situ detection can also be carried out microscopically after cytocentrifugation with immunophenotyping. Here, the secretion products can be detected as with the FACS. Fixation and / or permeabilization of the cells is necessary. The evaluation is carried out using microscopy.

The ELI spot technique (enzyme-linked immuno-spot) for the analysis of cell secretion products (in particular antibodies produced by B cells and by B and T cells, cytokines produced by antigen presenting cells) allows the characterization of cells on the basis of their secretion. Cells are cultivated in culture chambers with special membranes. The coating of the membranes with ligands against cell secretion products allows the binding of secretion products, which can then be detected using sandwich technology. However, due to the dyeing techniques used (typically peroxidase and / or alkaline phosphatase), only one or two-color markings are possible. This technology is primarily based on up to 96-well plate systems with special membrane systems as the plate base. Due to the plate systems used up to now, only a relatively low throughput of cells is possible and a rather complex analysis is necessary, since the cells arrange themselves in a random pattern on the membrane.

Detection of cytokines can also be carried out if the cells are embedded in a matrix which prevents the diffusion of the secretion products and which, on the other hand, permits subsequent detection steps to detect the secretion products. A sorting of cell After the detection procedure has been carried out, the cells can be removed from the matrix (Turcanu and Williams 2001)

Array systems are increasingly used to analyze ligand-ligand interactions using high-throughput methods using miniaturized, defined arrangement of defined molecules or cells. A wide variety of miniaturized systems for molecular interactions have so far been described (gene experiment arrays for an overview, see Deyholos and Galbraith 2001, protein arrays (Mac Beath and Schreiber 2000), antibody arrays (de Wildt et al 2000). Cell spot arrays have been described for measuring DNA expression (Ziauddin and Sabatini 2001), but not for single cell analysis and not for the detection of cell secretion products on living cells. In immunology, array systems from peptide libraries are used to measure the epitope properties of T cells (Hiernstra et al. 2000, Toby et 2001. However, these systems are not comparable to the single cell characterization system presented in the present invention, microelectrode arrays using semiconductor technology were used to analyze the electrical activity of cells (Israel et al. 1984, Connolly et al. 1990, Bhattacharjee 2000 Microstructured Ob Surfaces of other authors for positioning cells by coating cell adhesion molecules on solid phases allow cells to be positioned, but not in the precision pattern shown below (Chen et al. 1998) and differ fundamentally from the sorting principle described below.

Disadvantages of the techniques listed:

In the detection of intracellular products, permeabilization of the cell membrane and cell fixation is required, which affects the activity of the cells and is incompatible with the viability of the cells in many cases. The measurement of time-dependent, sequential processes is for an entire cell population, but not for Single cells possible.

The previous techniques allow a high throughput analysis, but the sensitivity of the method is dependent on the capacity of the anchor molecules dependent on the cell surface, which limits the binding capacity and detection limit of the system. An analysis of time-dependent sequential processes is also only possible for the total population, not for single cells, when using FACS technology. An evaluation of cells marked in this way by means of microscopy in principle also allows sequential processes to be studied, but is limited by the low throughput in the methods available hitherto.

The analysis of rather low cell counts and the need for permeabilization of the membrane are major disadvantages of this technique, which is why it has not become established for the screening of cells outside of defined scientific questions.

The ELI spot is typically carried out after the cells have been washed off the membrane. The enzyme reactions lead to an insoluble reaction product, which is deposited on the membrane. A broader immune typing of the cells with different markers is not possible with the existing systems. Due to the staining method used, only one or two-point markings are used. The unstructured arrangement of the cells on the membrane complicates the evaluation of the assay. The result of this is that the assay can only be meaningfully evaluated with a relatively low cell density. The recovery rate is also very limited.

The Turcanu and Williams test requires the cells to be embedded in a matrix, which requires additional work steps and makes later sorting or later analysis more difficult. A direct structured arrangement of the cells is also not possible in this test.

Due to the existing systems (cf. gene experiment arrays and protein arrays) an arrangement of single cells in a fixed pattern and the detection of the secretion products of these cells have not been described so far. A structured arrangement of cells using automatic spotlights is in principle conceivable, but very complex and very expensive when using living cells in the routine laboratory. intensive. The cells are often physically injured or destroyed. Furthermore, the Milαroarray principle is based on the application of liquid drops on a solid surface. This amount cannot be reduced to a minimum (at least 100 nl) and therefore the spotting of single cells is to be regarded as a coincidence.

The disadvantages of the various systems result on the one hand from the need to manipulate the cell membrane, and on the other hand to change the environment of the cells in such a way that the secreted product can be detected. Furthermore, a structured arrangement of individual cells is difficult due to the membranes used, which complicates an evaluation of the system and, consequently, necessarily reduces the density of the cells to be examined. The difficulty in arranging living individual cells on surfaces using blot techniques lies in the complicated technical solution of the spot process, which must consist of cell detection and the actual spot process using high-precision mechanics.

By means of the desired solution, single cells not organized in tissues or single cells isolated from tissues should be typified according to their functional activity and the production of secretion products. The process of cell typing itself should affect the vitality of the cells as little as possible. A solution is to be found which allows an arrangement of cells to be characterized in a suitable pattern, on the one hand to enable a high cell density and on the other hand to facilitate the evaluation of the marking. The cells should not change their position in the course of the test in order to allow the measurement of time-dependent processes. The cells should be able to be characterized on the basis of their surface markers, cellular proteins, their genetic material (DNA, RNA) as well as their secretion products and metabolic activity.

A structured arrangement of individual cells (which should be spatially separated from one another) on a structured solid phase would, on the one hand, permit high-throughput analysis, on the other hand, the detection of the secretion products on the solid phase due to immobilization improve, but allow the surface to be separated from other cells. A multi-channel analysis is possible when using fluorescence labeling.

The object is achieved by the microwell array for size-dependent sorting or separation of cells suspended in a flowing liquid and the associated method for examining the functional activity of single cell cells with the features mentioned in claims 1 and 18.

The microwell array for size-dependent sorting or separation of cells suspended in a flowing liquid is distinguished by the fact that the microwell array comprises a cell array consisting of a microslrucrured solid phase with a large number of wells. The solid phase preferably consists at least partially of silicon, glass or plastic. The cells are arranged in the wells by sedimentation due to the gravity and shear forces in the liquid flow, but can also be done by other aids such as magnetic beads, application of an electric field or by optical manipulations (e.g. optical tweezers). The cells position themselves in the corresponding zones on the solid phase in such a way that cells with a smaller to maximally the same size can be positioned in a well of a certain size.

In a preferred embodiment of the invention, a surface covering the solid phase of the cell array is provided, which consists in particular of microscopic glass. In this way, a later optical evaluation of the cell sorting, for example by fluorescence microscopy, can be carried out particularly simply and reliably. A distance between an upper edge of the depressions and the glass plate is preferably 10 μm to 500 μm. This can prevent turbulence in the liquid flow.

Furthermore, the densest possible arrangement of the depressions on the solid phase is preferably predetermined, so that a very large number of cells can be separated at the same time and then examined. This can be achieved in particular by are arranged offset to one another and / or a size of the depressions is adapted to the cell sizes to be examined. In the latter case, it is preferred that the size of the depressions is only slightly larger, in particular 0.5 μm to 5 μm, larger than the cell size. A cell depth also preferably corresponds to the cell size. A density of the depressions on the solid phase is further preferably up to 1,000,000 depressions per cm ² . The measures mentioned, individually or in combination, contribute to increasing the density of isolated cells for the subsequent examination.

It is further preferred that the solid phase is coated with antibodies or antigens. This makes it particularly easy to carry out tests on the individual cells. It goes without saying that the wells on the cell array can carry different antibodies and / or antigens depending on the question of the examination. In particular, several antibodies and / or antigens can be bound in one well or regions with different antibodies and / or antigens can also be specified on the cell array.

It is further preferred that the solid phase is coated with a mixture of proteins and antibodies. The protein can in particular be albumin, in particular the albumin coating applied directly to the solid phase subsequently carrying antibodies covalently bound via succinididyl ester. Alternatively, the solid phase can have a coating made of polylysine with subsequently attached antibodies. The modifications mentioned have proven to be particularly practical in the production of coated solid phases and are also suitable as a standard system for investigations. If the solid phase is made of silicon, depending on the intended application, the silicon surface can be treated in such a way that its charge and hydrophobicity change. In this respect, the system can be variably adapted to the respective requirements.

When examining the functional activity of single cell cells with a microwell array, the cells are separated in a first step and then examined in a subsequent step the functional activity of the individual cells. The orderly separation of a large number of individual cells is an essential aspect of the method and only favors the success of the subsequent investigations. The separation can take place by sorting according to cell size and / or typing according to cell type. It can also be supported with the help of latex particles, magnetic beads or optical tweezers. The measures allow for the first time the parallel investigation of a large number of single cells with regard to their functional activity.

It has proven to be advantageous to load the microwell array with a cell suspension of high density, in particular of approximately 2 × 10 ⁶ cells per 100 μl. Only 10 to 20 μl of such a cell suspension are preferably taken up in the microwell array. This means that very small amounts of sample can be processed.

After loading, the microwell array is rinsed to remove non-positioned cells. A liquid flow during rinsing is preferably 0.01 to 0.3 ml per second. A rinse quantity of 1 to 2 ml is preferably applied per rinse cycle. Pulse-like loading and rinsing has proven to be particularly effective. The above-mentioned measures for sample preparation have proven to be particularly suitable for the standardization of the process.

Accordingly, the present invention proposes to form network-like depressions in the possible size of the individual cells to be examined (silicon micro-well array) on silicon, plastic or glass surfaces by means of etching or embossing techniques. The depressions serve to accommodate and thus to arrange individual cells. If a suspension / solution with single cells is applied to the array, the cells accumulate both on the non-structured surface and in the depressions. Cells that are not arranged in depressions can be washed off the surface by applying a liquid stream. The cells placed secrete certain metabolic products or change their environment through certain biochemical processes. A coating of the surface of the cell array with antibodies or other ligands of cell secretion products allow the cell secretion products to bind or influence the activity of the cells. The secreted substances or the microenvironments modified by the cells can be made visible by detection reactions with (eg fluorescence) labeled ligands (eg antibody sandwich technology).

The functional characterization of single cells (e.g. B cells, T cells, antigen presenting cells, endocrine cells, tumor cells) using the microwell array shown is made possible by the possibility of multi-channel anaryysis for different secretion products and surface markers.

The array allows the defined arrangement of very large cell numbers that do not change their positions in the course of the test phase in a simple manner. It is therefore possible, on the one hand, to characterize the cells on the basis of surface markers, on the other hand to detect and measure the secretion products of these cells in their immediate surroundings, which requires very little manipulation of the cells to be characterized, on the other hand can be carried out in various detection steps and then in the respective well / respectively cell can be assigned. Using the method described for determining the secretion products of a cell, time-dependent characterization of individual cells and detection of the secretion products is possible without the alteration of the cell membrane of these cells.

The advantages of the proposed solution are therefore once again:

1. Simultaneous arrangement of cells in an increased cell density (up to 1 million cells / cm ² ) compared to conventional cell spot methods.

2. An orderly immobilization of cells is possible and thus also a high detection rate by optical methods (eg microscopy / laser scanning microscopy, immunofluorescence scanning method) after various types of treatment (rinsing and washing steps). 3. Due to the spatial separation of the individual cells, locally separate examinations of a large number of different cells (lymphocytes, macrophages, circulating tumor cells, cell lines) can be carried out.

4. Time-dependent processes can be carried out and analyzed sequentially on one and the same cell (real-time analysis). The increase in fluorescence signals with stable fluorescent markers and the possibility of analyzing bleaching effects with non-stable fluorescent markers can be used to measure time-dependent processes.

5. A parallel examination of cell surface markers and detection of their secretion products is possible.

6. Quantifiability of the secretion products and assignment to the secreting single cell.

7. Investigation of the selα: ion function under different environmental conditions (medium, temperature, cell toxins, hormones, cytokines) is given by the possibility of changing the cell media with a constant cell position.

8. In contrast to the ELI spot technique, other suitable detection systems (conventional fluorescence microscopy, laser scanning microscopy), but also luminescence markings can be carried out due to the use of fluorescent markers. The use of chemoluminescence would make the test described highly sensitive.

9. The high number of cells in the analysis on the cell array allows a good statistical evaluation of the cell population examined.

10. The immobilization of the cells in the fluid flow can be combined by a variety of other methods (latex particles, magnetic beads, optical tweezers). 11. The examination of living toes allows the subsequent use of the cells in a subsequent experiment (for example after magnetic sorting), or after detachment of the cells using stamping techniques.

12. Due to the rigid arrangement of the wells in the solid phase, the described functional test can be carried out sequentially several times in succession and also with different cells (multiple use of the structured solid phases is possible).

13. The technique is suitable for the characterization of single cell cells in activity, toxicity and carcinogenicity tests using high-throughput screening.

A method for the functional characterization of single cells that are not organized in tissues or isolated from tissues and an associated device have been created. For this purpose, microstructured solid phases (eg silicon, glass, plastic), on which milvalue depressions are applied, are used. Individual cells can be arranged in a suitable pattern in the microwells in a very high cell density (for example 150,000 per cm ² ). Ligands against secretion products of the individual cells can be applied to such a cell array (microweU irnmuno chip), which allow detection of the secretion products of individual cells. The characterization of the individual cells should be carried out by analyzing their secretion products, but can be expanded by ZeUtyping using surface analysis or intra-cellular markers. It is possible to separate cells after analysis.

A method and a device are available for the separation of parti- / cells from large populations of nikrostnikturierte solid phases and the subsequent molecular typing of the toes, such as genetic material or surface or toe proteins.

For effective separation and cloning of toes, living single cells from large cell populations can be isolated, based on specific properties are examined and selected after typing for subsequent processes.

Likewise, for locally separated, sequential examination of separated toes, such as the secretion of cytokines and chemokines on single cells on co-structured solid phases, after separation from large cell populations.

The method is also used to detect the secretion of isotype-specific or antigen-specific antibodies on isolated B cells using a MikroweUarray.

It is possible to use the microwave array for the separation and later screening of isolated hybridomas for the production of antigen-specific antibodies.

The method is suitable for the detection of secreted enzymes on single cells, e.g. secreted proteases which are tied up by tumor cells, the enzyme activity of which can be demonstrated by converting specific, immobilized substrates on the surface of the microcavity.

It is possible to use the microcavities for the detection of surface-based enzyme-mediated and / or immune-mediated cell reactions, also for the interaction between different cells. To do this, the cavities are first loaded with a ZeU type. Then other cells are flushed in and double loading of the cavity is achieved. This means that cell counter actions can be measured spatially separately.

A method for the detection of proliferation of individual cells has been created. After the separation of individual cells in micro-cavities, the proliferation of cells can be investigated in such a way that the cells leave the cavities after proliferation due to cell growth and this can either be detected on the surface or can be detected using DNA markers. The surface can be coated with stimulating, proliferation-promoting factors and the effect of these substances can be examined on isolated cells.

The method can be used for the selection of cells if specific cells are promoted or inhibited with respect to vitality or proliferation by surface coating, or are bound and thus selectively influenced.

The separation of individual cells from large populations for later analysis of the genetic material. Like DNA of the RNA is possible.

The invention soU be explained below using an exemplary embodiment and with reference to drawings. Show it:

Figure 1 is a schematic representation of a depression in the solid phase with a coating of a ligand;

Figure 2 shows the recess of Figure 1 with a few ZeUe and secretion products;

Figure 3 shows the well of Figure 2 after washing off unbound secretion products;

Figure 4 shows the recess of Figure 3 after marking with markers;

5 shows the well of FIG. 4 after removal of the cells and treatment with labeled ligands and

Figure 6 shows the well of Figure 4 after treatment with labeled ligands.

The separation and investigation of cells by means of the milα "structured solid phase, from which only one depression 10 was selected in each case for the illustration in FIGS. 1 to 6, can be carried out as follows: Coating of the solid phase

The solid phase made of silicon, as indicated schematically here, is coated in the area of the recess 10 with antibodies 12 against secretion products (e.g. cytokine assay) or with antigens for the detection of a secretion product to be measured (FIG. 1). Due to the surface properties of silicon, antibodies against secretion products such as cytokines can be directly adsorbed, on the one hand, and antibodies 12 can be coated together in a mixture with other proteins such as albumin. The direct coating with albumin and the subsequent covalent binding of the antibodies via succinimidyl ester has also proven to be a sensible option.

Furthermore, protein A can be bound to the silicon surfaces, to which antibodies can then be bound. Experiments have shown that an optimal solution must be found in order to achieve a high ligand density on the one hand, the ligand should retain its binding activity and the unspecific binding of cells should be as low as possible. Thus, the direct adsorption of antibodies allows a high level of binding, but the bound antibodies prevent a significant proportion of their binding activity (penaturation). After coating the surface with polylysine, a good binding density of antibodies is achieved which have good functional activity; the surface coated in this way also has a very high binding capacity for cells.

Blocking the solid phase

The further surface of the ZeUarray is blocked with a protein such as albumin that is irrelevant for the test in order to block the remaining binding sites on the silicon surface for proteins and to minimize the adhesion for ZeU surface proteins.

Loading with ZeUen

The microwell arrays are made with a zeus suspension of about

Load 2 ^• 10 ⁶ cells per 100 μl. 10-20 μl are sufficient for the actual coating. The volumes must be adjusted depending on the dead volume of the system used. The arrangement of the follows through the gravity in the fluid flow, but can also be done by other aids such as magnetic beads, application of a magnetic field or optical manipulations (eg optical tweezers) or reinforced by these methods. The ZeUen position themselves according to their size in the prepared wells of a similar size. The aim is to achieve the highest possible cell density, with the highest possible number of microwells being filled with just one cell. This ZeU density is achieved both by a high ZeU density in the ZeUsuspension (see above), by the densest possible arrangement of the micro-wells, by the arrangement of the Mifao wells in specific, sometimes offset patterns themselves, by a size adapted to the ZeU size (only slightly (0 , 5-5 μm) larger micro-depressions than the ZeU size), by the shape of the micro-recess (ZeUtiefe should correspond to the ZeU size), by the surface properties (charge, hydrophobicity) of the surface. The application of a magnetic field or the use of optical methods can secure or support the holding or placing of the cells in the wells.

Rinsing of not positioned cenuts Not positioned in the wells are removed after the application of a suitably strong liquid flow. Washing steps should on the one hand avoid washing out the cells located in the depressions due to swirling, and on the other hand damage to the cells by a massive flow of liquid. Flow rates of 0.3-0.01 ml per second have proven to be sensible. The distance between the upper edge of the depressions and a microscopic surface, for example quartz glass, is of essential importance for avoiding turbulence and thus washing out the cells. At distances from 500 μm very strong turbulence occurs, at distances below 10 μm the liquid flow with ZeUen is severely impaired. Multiple, pulse-like loading and washing cycles increase the density and effectiveness of the loading. The washing steps are also effective in pulse-like cycles. A total of 1 to 2 ml of washing volume is sufficient for pulsed washing. Enriching the washing solution with cellular medium or protein (albumin 0.1-1%) increases the vitality of the cells during the test. Incubate the cells

The ZeUe 18 is incubated with a culture medium on the ZeU array in order to maintain the vitality of the cell 18 and a secretion of metabolic products 14, 16, e.g. To cause cytokines in the cytokine assay or antibodies in B lymphocytes in the environment (FIG. 2). The metabolic products 14, 16 are bound or modified (e.g. in the case of enzymes) in another way by the immunized antibodies 12 or possibly antigens or change the initial coating of the surfaces. Incubation should take place for 1-72 hours in order to be able to detect the metabolic products 14, 16 on the one hand, and on the other hand to avoid adulteration caused by ZeUtod and innovations. Certain ZeU types such as macrophages tend to actively change their position, which affects the result, other ZeU types such as lymphocytes maintain their position for hours.

Rinsing the solid phase

The entire chamber contents are rinsed several times with washing solutions in order to rinse off unbound metabolic products 14, 16 (FIG. 3). The secretion products 14 to be detected bind to the antibody 12 bound to the solid phase surface. The washing conditions are to be selected as described above. A variation of the rinsing flow and the distance between the upper edge of the microwave and the chamber surface can be used to rinse unfixed cells if necessary.

Incubation of markers

The ZeU array is incubated with color-labeled antibodies 20 which are directed against surface antigens of the ZeUe 18. The antigen-positive cell 18 is stained by direct or indirect labeling. The concentration of the antibodies 20, 22 depends on the dilutions customary for fluorescence microscopy. If necessary, the surface marking of the ZeUe 18 can also be carried out before application to the ZeU array. After permeability of the cell membrane, the cells 18 can also be labeled by antibodies 22 against cytosolic or other antibodies or other dyes (for example DAPI or propidium iodide for cells / nucleic acids). Vital dyes can be used Detected to estimate the vitality of the ZeUen for each individual ZeUe during the test. Due to the small volumes required to coat the well array (10-20 μl), small amounts of reagents are required.

The entire chamber contents are rinsed several times with washing solutions in order to rinse off the excess antibodies 20, 22.

Examination The examination is followed by a microscopy of the ZeU array for different fluorescence (green e.g. FITC, red e.g. Texas red, blue e.g. DAPI, white e.g. photoluminescence). A laser scanning microscope is suitable because this measuring method has a high throughput rate with a high sensitivity for the ZeUmarker. On the other hand, a number of reflection and scattering effects occur within the micro-recess, which can be used for the evaluation and which can increase the sensitivity. A non-confocal measurement mode is also sensible because a large part of the recess surface that can be used for the measurement (4/5) is arranged vertically, so that it almost only leads to reflection and stray light, which would then almost be masked out with confocal measurement. With a 50-fold microscope magnification, around 14,000 wells can be analyzed simultaneously with a 10 μm microwe size.

When washing the array, there is the possibility of removing the removed and possibly marked cells 18. In FIG. 5, a metabolic product 14 is detected without and in FIG. 6 in the presence of cells 18. To detach cell populations held by magnetic beads, this may have to be done Magnetic field can be changed. Changes in flow rates can be used as described. In order to hide signals from the cell marking and to use the respective channel several times for further measurements, the fluorescent dye can be bleached with briefly high-dose UV light. In the example shown in FIGS. 4, 5 and 6, there was a deposit of specific metabolic products 14. In the example of FIGS. 5 and 6, the secretion products 14 are detected by incubating the ZeUarray with further fluorescence-labeled ligands 24 against the metabolic product 14, for example other antibodies which have a different epitope than the primary antibodies used to bind the metabolic product 14 become. On the one hand, monoclonal antibodies are possible that recognize different epitopes, or the use of a monoclonal antibody for binding and a polyclonal for detection.

After detection, the ZeUarray is washed to remove excess antibodies.

Further microscopy steps for the detection of other secretion products can follow.

This is followed by an analysis of the signal intensities of the respective microscopy steps and assignment of the surface antigen negative / positive cells to the signals of the corresponding secretion products measured. The combination of the different measurements in the different color channels allows a characterization of the functional activities of the individual cells. The secretion products can be detected by multi-color fluorescence without removing the cells.

Further microscopy steps for the detection of background signals and for time-dependent analysis are possible. After the scanning process, zeUs can be removed from the well array using special stamps and further analyzes carried out.

A specific example is described below: coating the surface with 100 μl lmg / ml Ghadin solution for four hours, washing with PBS, 1% bovine serum albumin, blocking with 10% bovine serum albumin solution for one hour. Wash with PBS / l% bovine serum, load the wells (16 μm) with a B-lymphocyte hybridoma that produces IgG antibodies to gliadin. ed. Wash excess ZeUen, incubation of the ZeUen for twelve hours in RPMI1640 (additions 10% FCS, penicillin, streptomycin, mercaptoethanol). Permeabilize the ZeUen, incubation with DAPI (0.001 mg / ml PBS) for the detection of wells loaded with ZeUen for ten minutes, washing, first microscopy step. Multiple extensive washing steps to wash the toes out of the wells. Incubation with anti-IgG fluorescein-labeled immunoglobulin for one hour (DAKO anti-mouse IgG, polyclonal, 1:50). Washing step. Second microscopy step for the detection of bound anti-Ghadin immunoglobulin-anti-mouse-IgG complexes. AUe washing steps with PBS, 0.1% bovine serum albumin, repeated pulses of approximately 300 μl washing solution over five minutes.

Further microscopy steps for the detection of background signals and for time-dependent analysis are possible. After the respective scanning process, zeUs can be removed from the surface using special stamps and sent for further analysis. The characterization of the individual cells on the basis of their secretion products can, however, be expanded by the cell typing by means of surface analysis and / or by means of intracellular markers. The separation of cells after the analysis can be carried out by using magnetic beads or other physical principles such as stencil techniques or laser-controlled deflection ("optical tweezers") of cells.

Bezugszeichenhste

10 _ wells

12 - Antibodies

14; 16 - Metabolic products

18 - cells

20 - color-marked antibodies

22 - Antibodies to cytoscopic or other anti-antibodies

24 - ligands

Claims

claims

1. Micro-well array for size-dependent sorting or separation of cells suspended in a flowing liquid, characterized in that the micro-well array comprises a cell array of a milαOstructured solid phase with a plurality of wells (10).

2. Micro-well array according to claim 1, characterized in that a surface covering the solid phase of the ZeUarray, in particular made of microscopic glass, is present.

3. microwell array according to claim 2, characterized in that a distance between an upper edge of the recesses (10) and the surface is 10 microns to 500 microns.

4. microwell array according to one or more of claims 1 to 3, characterized in that the densest possible arrangement of the wells (10) is predetermined on the solid phase.

5. Micro deepening array according to claim 4, characterized in that the depressions (10) are arranged offset from one another.

6. microwell array according to claim 4 or 5, characterized in that a size of the wells (10) is adapted to a ZeU size.

7. Micro-well array according to claim 6, characterized in that the size of the wells (10) is only slightly larger, in particular 0.5 μm to 5 μm, larger than the size of the cell.

8. microwell array according to one or more of claims 4 to 7, characterized in that a ZeUtiefe corresponds to the ZeU size.

, Microwell array according to claim 8, characterized in that the ZeUtiefe is predetermined so that two cells can be accommodated (double loading).

IO micro-well array according to one or more of claims 4 to 9, characterized in that a density of the wells (10) is up to 1,000,000 per cm ² .

11. microwell array according to one or more of claims 1 to 10, characterized in that the solid phase made of silicon,

There is glass or plastic.

12. Micro-well array according to one or more of claims 1 to 11, characterized in that the solid phase is coated with antibodies (12) or antigens.

13.M ^ overtiefungsarray according to one or more of claims 1 to 11, characterized in that the solid phase is coated with a mixture of proteins and Antiköφern (12).

14.Mikroveιtiefungsarray according to claim 13, characterized in that the protein is albumin.

15. Micro-well array according to claim 14, characterized in that the solid phase bears a direct coating of albumin with antibodies (12) subsequently covalently linked via succinimidyl ester.

16.Milα: overtiefungsarray according to claim 13, characterized in that the solid phase carries a coating of polylysine with subsequently attached antibodies (12).

π.MϊlαOvertiefarray according to claim 11, characterized in that the solid phase is made of silicon and the surface of silicon is treated in such a way that its charge and hydrophobicity has changed.

18. A method for examining the functional activity of tools with a multi-well array according to one or more of claims 1 to 17, characterized in that in a first step a separation of the tools (18) and a subsequent step an investigation of the functional activity of the isolated ZeUen (18) takes place.

19. The method according to claim 18, characterized in that a sorting by ZeU size takes place during the separation.

20. The method according to claim 18 or 19, characterized in that a typing according to ZeUtyp takes place during the separation.

21. The method according to one or more of claims 18 to 21, characterized in that the separation is supported by means of latex particles, magnetic beads or by means of optical tweezers.

22. The method according to one or more of claims 18 to 22, characterized in that the microwell array is loaded with a cell suspension of high density.

23. The method according to claim 22, characterized in that the microwell array is loaded with a cell suspension having a density of approximately 2 × 10 ⁶ cells per 100 μl.

24. The method according to claim 23, characterized in that the microwell array is loaded with 10 to 20 μl of the zeus suspension.

25. The method according to one or more of claims 18 to 24, characterized in that the microwell array is rinsed after loading to remove non-positioned cells.

26. The method according to claim 25, characterized in that a liquid flow during rinsing is 0.01 to 0.3 ml per second.

27. The method according to claim 25, characterized in that a rinse volume per rinse cycle is 1 to 2 ml.

28. The method according to claim 25, characterized in that the loading and rinsing takes place in pulsed fashion.

29. The method according to one or more of claims 18 to 28, characterized in that the examination is carried out sequentially.

30. The method according to one or more of claims 18 to 29, characterized in that the examination is carried out by means of fluorescence spectroscopy.

31. The method according to one or more of claims 18 to 30, characterized in that locally separate tests are carried out.

32. The method according to one or more of claims 18 to 31, characterized in that a separation of the cells (18) takes place after the end of the examination.

33. The method according to claim 18, characterized in that the

Elimination of metabolic products (14) of the cells (18) is determined locally defined.

34. Method according to claims 18 and 33, characterized in that an assignment to each individual cell (18) takes place.

35. The method according to claim 18, characterized in that the influence of the entire ZeUe (18) on the environment is measured.

36. The method according to claim 18, characterized in that the

Metabolic activity of the ZeUen is measured.

37. The method according to claim 18, characterized in that comparative measurements of two cells are carried out.

38. The method according to claims 18 to 37, characterized in that the cells (18) are incubated for hours in nutrient solution.