WO2004019039A2

WO2004019039A2 - Method and kit for performing functional tests on biological cells immobilized on an array of scavenger molecules

Info

Publication number: WO2004019039A2
Application number: PCT/EP2003/007440
Authority: WO
Inventors: Hugo Hämmerle; Thomas Joos; Markus Templin; Hansjürgen VOLKMER; Kerstin Ragnitz; Susanne Stumpf; Cornelia Kuschel
Original assignee: NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen
Priority date: 2002-08-05
Filing date: 2003-07-09
Publication date: 2004-03-04
Also published as: AU2003246668A1; DE10236101A1; CA2494558A1; EP1527344A2; WO2004019039A3

Abstract

Disclosed is a method for performing functional tests on test cells, comprising the following steps: a) an array of separate measuring points is supplied, scavenger molecules with which the test cells can bond being immobilized at each of said measuring points; b) a mixture of test cells and reference particles which can bond with the scavenger molecules and can be distinguished from the test cells is created; c) the mixture obtained in step b) is contacted with the array such that test cells and reference particles can bond at each measuring point; and d) the ratio of interesting bonded test cells to bonded reference particles is determined for at least some of the measuring points. In another method, at least one measuring point is split into several measuring areas in the array along with the associated scavenger molecules, said measuring areas being arranged at different locations in the array. In yet another method, the array is moved after being contacted with the test cells and, optionally, the reference particles.

Description

Method and kit for performing functional tests on biological cells

The present invention relates to a method and a kit for carrying out functional tests on biological cells, in which an array of measuring points is used, with at least one capture molecule or binding partner for the biological cells to be tested being immobilized at each measuring point. In the context of the present invention, “functional test” means, for example, but not exclusively or in a restrictive manner, such experiments, tests or measurements in which certain properties or features of the cells or the change in these properties or features as a function of a treatment the cells and / or the type of capture molecules and / or the addition of substances are recorded or evaluated.

The treatment of the cells is e.g. radiation with high-energy radiation, such as is used in radiotherapy. The addition of substances includes e.g. in the context of pharmaceutical screening, the administration of pharmaceutical preparations whose effect on the cells is examined, for example, in the course of a dose-finding study, or the addition of antibodies which are screened against cell surface receptors. The choice of the type of capture molecules affects e.g. Components of extracellular matrix molecules for simulating the natural microenvironment of the cells in order to test their reaction in vitro to radiation, stimulation by ligands and / or added pharmaceuticals under conditions of the natural microenvironment.

In the context of the present invention, “biological cells to be tested” include, for example, but not exclusively or in a restrictive manner, primary animal, in particular human cells, plant or bacterial cells, cell lines, genetically modified cells, cells made of biopsy material, healthy un-degenerate cells, in particular tumor cells , Cells from peripheral blood etc. understood. These cells are referred to below as test cells. The "properties and characteristics" of the test cells include, but are not limited to, their proliferation ability, their viability, the pattern of their cell surface molecules, their ability to exchange signals or to interact with other cells, a possible pathological condition, a genetic degeneration, their genetic profile, their expression profile, their ability to bind to certain substances.

These methods are carried out with arrays of measuring points in order to be able to read out different functions of cells in parallel with just a few cells. An advantage of such arrays is that, in contrast to microtiter plates, the same ambient conditions prevail for all measuring points.

Carrier plates with arrays which are suitable for carrying out such methods and examples of such functional tests can be found, for example, in WO 02/02226 by the applicant or WO 00/39580.

The carrier plates are usually Glass or plastic platelets which have a functionalized, for example aldehyde-activated surface, on which capture molecules or binding partners for the test cells are immobilized at mutually separated measuring points. The surface between the measuring points is blocked to prevent non-specific binding of test cells or other substances.

The measuring points have a diameter of 200 to 800 µm and a center distance of, for example, 500 μm, so that 100 measuring points can be accommodated on an area of 1 x 1 cm. For example, different capture molecules are immobilized on the measuring points. A solution with test cells is then placed on the carrier plate, which is then incubated for a certain period of time before the test cells that are not immobilized on capture molecules are washed off again. The bound test cells are then recorded optically in a spatially resolved manner in order to determine to which capture molecules the test cells have bound. The optical detection can take place, for example, using bright field microscopy or fluorescence measurements, but other measurement principles can also be used, as described, for example, in WO 02/02226 or WO 00/39580.

Measurements carried out by the inventors of the present application with such carrier plates have now shown that the measurement signals between different measuring points within a carrier plate and between measuring points of different carrier plates fluctuate greatly, although the catcher molecules, the test cells and the test batch were identical. This variability between the measuring points on a carrier plate and between different carrier plates means that the measurement results can often not be compared with one another with sufficient reliability.

This problem can also be found in the publication Belov et al .: "Immunophenotyping of Leuke ias Using a Cluster of Differentiation Antibody Microarray", in Cancer Research (2001) 61, 4483-4489.

This publication describes a method in which more than 50 CD antigens are detected on leukocytes. To do this, an array of different, at different Measuring points immobilized antibodies against the respective CD antigens are used, to which a suspension of test cells is placed, the test cells only binding to those measuring points at which antibodies are immobilized, for which they express the corresponding CD antigens. The bound test cells are recorded optically with spatial resolution. The resulting pattern of measuring points occupied with test cells represents the patient's phenotype from which the test cells originate.

The antibody array covers a total of 60 measuring points on an area of 0.72 cm x 0.4 cm, to which 5nl antibody solution was added. The diameter of a measuring point is approx. 400 μm. 100 μl of a cell suspension with a concentration of 10 ⁷ test cells / ml were added to the array. The authors report that around 600 test cells were bound at this concentration per measuring point, while around 100 test cells are bound at 10 ^δ test cells / ml.

In other words, only approx. 0.1 to 1% of the test cells in the suspension can also bind to the measuring points. With such a small proportion of actually binding test cells, however, according to the inventors of the present application, there is the problem that the measurement results are not reliable enough for statistical reasons, in particular if the measurement results for different measurement points are not only qualitative, as in the case of the typing of the immunophenotype in of the publication mentioned, but also to be compared quantitatively, as in the functional tests mentioned at the beginning. The problem becomes even more serious if a few cells are available, i.e. not 10 ⁷ cells / ml but e.g. only 10 ⁵ cells / ml, as is the case with many test batches.

What is also striking about the fluorescence recordings of test results shown in this publication is that the measuring points vary greatly in size and sometimes are very unevenly populated with test cells. According to the knowledge of the inventors of the present application, however, the method described in this publication leads to a falsification of the measurement results because of these fluctuations, so that the functional tests mentioned at the outset cannot be carried out with sufficient accuracy and reproducibility.

Against this background, the present invention has for its object to provide a method of the type mentioned, in which the reliability and reproducibility between the measurement results of measurement points in an array and in different arrays is so great that a reliable comparison of the measurement results and the creation a reliable gradation of the measurement results is possible.

According to the invention, this object is achieved on the one hand by a method for carrying out functional tests on test cells, with the steps:

a) providing an array of mutually separate measuring points, at each of which capture molecules are immobilized, to which the test cells can bind,

b) Create a mixture of the test cells and reference particles, which have the capacity, to the capture molecules bind cool, and which are distinguishable from the test cells,

c) contacting the mixture from step b) with the array so that test cells and reference particles can bind to each measuring point, and

d) determining the ratio of bound test cells of interest to bound reference particles for at least some of the measurement points.

The object underlying the invention is completely achieved in this way.

The inventors of the present application have recognized that the reproducibility and reliability of the measurement results depend on the varying size of the measurement points and the lack of homogeneity of the immobilized capture molecules, and that the use of the reference particles makes it possible to influence the different sizes of the surfaces of the Calculate measuring points and other inhomogeneities, for example in the local concentration of the capture molecules, from the measurement results. The inventors are therefore not taking the path that is actually available on the basis of the inventors' findings to minimize the variability by means of more complex production processes which lead to more uniform areas of the measuring points and a uniform local concentration of the capture molecules, but instead use reference particles. In other words, the number of test cells bound per measuring point and thus the respective measuring signal in the known methods according to the knowledge of the inventors of the present application depends not only on the binding properties between the test cells and the capture molecules but also on the density of the capture molecules per measurement point the size of the area of the measuring points and the homogeneity of the concentration of the capture molecules.

The existing variability in the size of the areas of the measuring points and the inhomogeneity of the capture molecules applied makes it possible because of the reference particles to determine a gradation in the binding of the test cells to the various capture molecules with sufficient accuracy and reliability.

The quotient of the measurement signal for the test cells and the measurement signal for the reference particles at a specific measurement point can be taken as a measure for the binding of the test cells to the capture molecules of this measurement point. The measurement signal for the reference particles is, so to speak, a measure of the number of capture molecules in a measurement point.

In this context, “bound test cells of interest” are understood to mean test cells that have deposited from the suspension on measuring points and adhere there. The ratio of bound test cells to bound reference particles then serves to compare the adhesion behavior of the test cells to different capture molecules to be able to. On the other hand, it is also possible to investigate the apoptosis rate or the viability of test cells which are incubated after adhesion to the capture molecules with the addition of certain substances or under treatment, for example by irradiation. After the incubation, the still vital or the dying or dead test cells are then recorded and standardized for each measuring point by means of the number of bound reference particles, so that, for example, the influence of different capture molecules on the apoptosis rate can be determined.

In addition to the adhesion or the apoptosis rate, other properties of the test cells or changes in these properties can also be examined in this way as part of functional tests. It is important that the number of "interested" test cells on one measuring point is standardized, so to speak, by the number of reference cells that are also bound and is therefore comparable with the number of test cells of interest on other measuring points. The test cells of interest are, for example, all bonded test cells in the context of adhesion tests, the apoptosis rate, viability, the ability to bind to antibodies, exchange signals, etc. in the context of other functional tests.

Artificial spheres, for example latex spheres, can be used as reference particles, which carry surface molecules that enable a binding to the catcher molecules that is comparable to that of the surface cells of the test cells. These balls are inexpensive and easy to manufacture and can be stored for a long time; they are known from other applications in the prior art and have a size that can correspond to that of the test cells. Another advantage of the Ku geln is that their binding behavior to the capture molecules is not influenced by subsequently added substances or, for example, radioactive or UV radiation, so that after mixing and, if necessary, immobilizing test cells and spheres, the influence of this measure on the test cells and, for example, theirs Binding or apoptosis rate can be tracked without affecting the binding of the balls, so that the reference is retained. ^•

On the other hand, it is also possible to use biological reference cells as reference particles which can be distinguished from the test cells in terms of measurement technology, preferably optically, but which bind to capture molecules like the test cells. The reference cells can be untreated test cells, which can be distinguished by measurement technology from the test cells to be examined and treated before mixing.

However, it is also provided that both artificial spheres and biological reference cells are present in the reference particles. This makes it possible to correct several parameters using internal references, so to speak.

The test cells and the reference particles are preferably mixed with one another in a ratio of 1: 1, so that they are the same. Probability to hit the catcher molecules.

In one exemplary embodiment, the test cells can be distinguished from the reference particles by measurement technology, preferably optically, in that the test cells are labeled with a marker other than the reference particles, preferably a fluorescent marker or a genetic marker. It is advantageous here that the array can be read out with two different excitation waves and / or emission filters, with successively or simultaneously spatially resolved optical signals being recorded, from which the ratio of the test cell of interest to the reference particle can then be calculated.

It is also possible to label the test cells with a genetic marker such as a reporter gene such as GFP (green fluorescent protein). If the reference particles are reference cells, the reference cells can also be genetically labeled instead of or different from the test cells.

In a further exemplary embodiment, the test cells can be distinguished from the reference particles by measurement technology in that the test cells are provided with a different radioactive marker than the reference particles.

It is also envisaged to use radioactive and optical markings together, that is to say test cells and reference cells, to radioactively and optically mark, or to use them in a mixed manner, for example to optically mark the test cells and the reference particles radioactively.

The reference cells are preferably genetically different from the test cells in such a way that they do not, or are known to react differently to substances and / or radiation whose effect on the test cells is to be investigated. The advantage here is that after the test and reference cells have been mixed, this mixture can be treated in the manner indicated without impairing the binding to the capture molecules or other properties of the reference cells examined in the course of the functional tests. In other words, while radiation can, for example, lead to a certain percentage of the test cells being killed or its binding properties to the catcher molecules being changed such that it binds poorly or better to the catcher molecules, the binding of the reference cells to the catcher molecules remains unchanged and is therefore a measure of the number of capture molecules per measuring point.

On the other hand, it is also possible to divide a mixture of test cells and reference cells into two arrays and to irradiate only one array after or during the incubation or to bring it into contact with a test substance. The untreated array then serves as a reference for the effect on test cells and on reference cells.

It is also possible to leave the reference cells untreated and to mix them with the test cells only after the treatment.

Two or more types of reference particles can also be mixed with the test cells, the different types of reference particles being distinguishable from one another and from the test cells, for example by three different "colors". It is thus possible to use so-called “beads” as the first type of reference particles, which are not influenced by the treatment and serve as a reference between different arrays, and as second type of reference particles to use reference cells that are used to calculate the variation within an array.

Alternatively or additionally, a reference measuring point can be provided on each array, to which only reference particles bind. This can be used as a reference between different arrays, the reference measuring point being isolated from the other measuring points, so that only reference particles are applied to it.

According to the invention, on the other hand, the object on which the invention is based is achieved by a method for carrying out functional tests on test cells, with the steps:

a) Providing an array of mutually separate measuring points, at each of which different capture molecules are immobilized, to which the test cells can bind, a measuring point with its assigned capture molecules being divided over several measuring surfaces in the array, which are arranged at different locations in the array .

b) contacting a suspension of test cells with the array so that test cells can bind at each measuring point,

c) location-resolved detection of measurement data representing the number of test cells of interest on the measurement surfaces, d) Calculation of a measurement value for at least one measurement point from the measurement data of the measurement areas assigned to it.

In this way too, the object underlying the invention is completely achieved.

The inventors of the present application have recognized that the uneven deposition of test cells on the measuring points, as can be seen from the publication by Belov et al., Loc. Cit., Is not solely due to the inhomogeneity of the measuring points, but also due to the fact that the Test cells in the suspension are not distributed homogeneously, but preferentially deposit at certain points on the array. However, this means that the local number of test cells on the array is not the same everywhere. In other words, for example, despite a strong bond between test cells and capture molecules, a weaker measurement signal can result for a specific measuring point than for a measuring point at which the binding is weaker because the local concentration of the test cells is lower than at the first measuring point the second measurement point.

According to the inventors of the present application, the distribution of a (logical) measurement point over a number of measurement areas at different locations in the array, however, averages the statistical probability of adhesion for different measurement points, that is to say averaged over the assigned measurement areas. If the individual measuring surfaces become so small that, in the statistical sense, it is no longer possible for a sufficient number of test cells to bind in order to generate a reliable measurement signal, the reference particles and measures discussed in detail above can also be used here, which leads to a synergistic effect.

Ideally, based ^'on the number of test cells in the suspension so large areas realized for a measuring point, that at least capable of binding than 50%, ideally close to 100% of the test cells from the supernatant on the measurement points more. For this purpose, according to the invention, a specific ratio between the area (F) of a measuring point and the number (N) of test cells in the suspension is required, which depends on the adhesive area (H) of the respective cells and is selected as follows:

F ≥ a x N x H; a = 0 .5, preferably approx. 1 . 0

Adhesive area is understood here to mean the size of the area with which a cell lies on the substrate. This is typically H = 1 μm ² for bacteria and H> 100 μm ² for animal cells. Approx. 800 bacteria or 8 animal cells can be deposited on a measuring area of F = 800 μm ² , so that in this case (F = 800 μm ² ) in the suspension applied to the array at most N = 800 bacteria or N = 8 animal cells should be included.

In order to obtain a higher measurement signal, according to the invention either the area F of a measurement point is chosen larger or several measurement areas are combined to form a logical measurement point with a total area F. Under these conditions, almost all test cells can bind from the overhang without interfering with each other. This choice of the ratio between the number and type of test cells and the size of the surface of a measuring point, which determines the adhesive surface, is in itself new and inventive and, together with one or both of the above-mentioned measures, namely the reference particles and / or the distributed measuring surfaces, leads to one synergistic effect. When using reference particles, the above ratio should be applied to the sum of the test cells and reference particles present in the suspension as follows:

F> a x (NT x HT + NR x HR)

where NT and HT denote the number and the adhesive surface of the test cells and NR and HR the number and adhesive surface of the reference particles and a is a factor of 0.5, preferably approximately 1.0.

For a given array with known areas of the measuring points, the amount of test cells and possibly reference particles in the suspension / mixture to be applied to the array must be selected in such a way, according to the knowledge of the inventors of the present application, that the above ratio is maintained in order to To achieve a situation in which almost all test cells / reference particles can bind to a measuring point, so that there is a competition between the measuring points around the cells. This enables quantitative evaluations that would be falsified with a higher number of test cells. This ratio is advantageous, for example, if a few cells are available, as in tumor biopsies, or if the "homing" of stem cells is to be investigated. A preference of the test cells for certain catcher molecules can only be determined quantitatively in the low numbers of test cells used according to the invention. This also applies if a subpopulation is to be examined separately in mixed cell populations.

In this connection, the inventors of the present application were able to show that with larger amounts of test cells they also bind to substrates for which they have no proven preference.

In general, it is still preferred if the array is moved after contacting with the suspension / mixture, i.e. during the incubation with the test cells and possibly the reference particles, for example on a vibrator or a cradle or by means of a pump, for example via microfluidic flow to reduce the local concentration differences in the test cells and possibly reference particles. As a result of the movement, new test cells or reference particles are also repeatedly brought to the measurement points, so that a larger number of them have the chance to bind to capture molecules. In contrast to protein arrays, where the law of mass action applies and this shaking would only have limited advantages, shaking surprisingly increases the number of bound test cells and possibly reference particles, as the inventors of the present application were able to show. Against this background, in a method mentioned at the outset, this measure is also new and inventive in itself, but is preferably used together with one or more of the above-mentioned measures.

Overall, it is possible that the array is either applied to a carrier plate, as is the case in WO 00/39580 and WO 02/02226 mentioned at the outset, or that it is a logical array of individual balls which are loaded with capture molecules and in the usual way, for example can be distinguished from one another by color markings. A measuring point then corresponds to either one sphere or several spheres, each of which represents a measuring surface in the above sense.

If balls are used as an array, in the simplest case they are added to the solution / mixture and incubated, for example on a shaker, with gentle agitation.

In certain applications, the test cells are subjected to a treatment before or after contacting the array, which treatment is selected from the group: irradiation with high-energy radiation, for example UV light or radioactive radiation, contact with test substances such as pharmaceutical active ingredients, etc. Cells, chemotherapy drugs, components of extracellular matrix proteins, antibodies, lectins and other biopolymers.

Different capture molecules are immobilized at the measuring points, which are preferably selected from the group: protein such as, for example, components of extracellular matrix proteins, receptors. Ligands, poly-lysine, peptides from laminin sequences, Control peptides, peptidomimetics, antibodies, lectins, antigens, allergens.

The invention further relates to a kit with an array of mutually separate measuring points, at which capture molecules are immobilized, to which test cells can bind, and with reference particles, which bind to the capture molecules.

The reference particles are preferably the reference particles described in more detail above, with further capture molecules preferably being immobilized at the measuring points, which are preferably selected from the group: protein such as, for example, components of extracellular matrix proteins, receptors. Ligands, poly-lysine, peptides from laminin sequences, control peptides, peptidomimetics, antibodies, lectins, antigens, allergens, nucleic acids, nucleotides.

The array is either applied to a carrier plate or it is a logical array of individual balls that are loaded with catcher molecules, further preferably at least one measuring point with its associated catcher molecules being divided over several measuring surfaces in the array, which are located at different locations in the array are arranged.

Further advantages and features emerge from the description below and from the attached figures.

It goes without saying that the features mentioned above and to be explained below not only in the respectively specified combination but also in other combinations or in Can be used alone without leaving the scope of the present invention.

Exemplary embodiments of the invention are shown in the figures and are explained in more detail in the description below. Show it:

1 shows a schematic example of an array of measuring points arranged on a carrier plate, the measuring points being distributed over different measuring surfaces;

FIG. 2 shows a schematic side view of the carrier plate from FIG. 1;

3 pictures of test cells and reference cells bound to a measuring point of the array from FIG. 1, with A) the bright field picture for test cells and reference cells, B) a fluorescence picture for the test cells and the reference cells, C) a fluorescence picture for the test cells and D) is a fluorescence image for the reference cells;

4 shows different fluorescence images which show the binding of test cells to different measuring points which were not moved (A, C and E) and moved (B, D and F) during the incubation;

Fig. 5 is a diagram showing the binding of test cells to different capture molecules depending on the shows the number of cells used in the applied suspension; and

Fig. 6 is a diagram showing the settlement of a measurement area depending on the number of cells used in the applied suspension with and without movement.

Example I: Carrier plate with an array of measuring points

In FIG. 1, 10 denotes a rectangular support plate made of glass or plastic, on which some measuring surfaces 11 are arranged in an array, to which biological cells, not shown in FIG. 1 - hereinafter: test cells - or reference particles can bind. Areas 12 of the carrier plate 10 are provided between the measuring surfaces 11, to which test cells and reference particles cannot bind.

The measuring surfaces have a diameter of 500 μm and an edge distance of 250 μm, so that there is a center distance of 750 μm. In this way, 96 measuring surfaces 11 can be accommodated on a carrier plate 10 with an edge length of 6 mm × 9 mm.

As can be seen in the basic side view of FIG. 2, the carrier plate can be designed as the base plate of a cell culture vessel. The carrier plate 10 bears on the section 17 a functionalized surface 18 on which capture molecules 19, 21, 22 are immobilized in the measuring points 11. The catcher molecules 19, 21, 22 generally differ from measuring surface 11 to measuring surface 11, it being possible for different measuring surfaces 11 to be combined to form a logical measuring point. The measuring surfaces 11 of a logical measuring point carry identical catcher molecules 19, 21 or 22 and are statistically distributed over the carrier plate 10.

Test cells 23 can bind to the catcher molecules 19, 21 and 22, and their binding behavior to the catcher molecules 19, 21, 22 or their reaction to co-stimulation by catcher molecules 19 and test substances or to a treatment such as radiation should be examined.

In the areas 12 between the measurement areas 11, the functionalized surface 18 is blocked by molecules 24, so that the test cells 23 can only be bound in the area of the measurement areas 11.

A reference particle is indicated at 25 in FIG. 2, which binds to the capture molecules 22. Biological cells or plastic spheres are suitable as reference particles 25, which carry molecules on their surface with which they bind to the capture molecules 19, 21, 22. The reference particles 25 are provided as an internal reference in order to be able to calculate out fluctuations in the size of the measurement areas 11 or the number of capture molecules in the measurement area both within an array and between different arrays.

Example 1 of the aforementioned WO describes how such a carrier plate 10 with measuring surfaces 11 can be produced 02/02226, the disclosure of which is hereby expressly incorporated by reference.

Example II: Incubation of a Mixture of Test Cells and Reference Cells on an Array with Measuring Points Distributed on Different Measuring Surfaces

For this experiment, test and reference cells are marked with different membrane dyes.

Hu AO SMC or GLZ are used as test cells, which are labeled with the Vibrant Dil Red Fluorescent Cell Linker Kit (V22885Y MoBiTec) according to the manufacturer's instructions.

PC12 serve as reference cells, which are marked with the Vibrant DiO Green Fluorescent Cell Linker Kit (V22886Y MoBiTec) according to the manufacturer's instructions.

To display test and reference cells in one image, both cell types were stained blue with the Vibrant Cell Labeling Solution DAPI.

Various matrix proteins were immobilized as capture molecules in the measurement areas.

The test and reference cells were mixed in a ratio of 1: 1, the mixture was added to the carrier plate with the array, and the carrier plate was then incubated for 4 hours in an incubator at 37 ° C. FIG. 3 shows, by way of example, for a measuring point with laminin from the human placenta as a capture molecule, that both test cells (GLZ) and reference cells (PC 12) bind to the measuring point, but the reference cells in a significantly smaller number than the test cells. It can be seen that the measuring point is evenly populated with capture molecules.

3A shows a bright field measurement for both cells, FIG. 3B shows a fluorescence image of the measuring point with a blue filter for test and reference cells after DAPI staining, FIG. 3C shows a fluorescence image with a red filter that is permeable to the emission of the test cells, and FIG. 3D shows one Corresponding image with a green filter permeable to the emission of the reference cells.

In the table below, the total number and percentage of the bound cells is given as an example for some measuring points, each with laminin (human placenta) as a capture molecule for a mixed suspension of test and reference cells, 100,000 cells of each type being found for PC12 with GLZ an array was given and for PC12 with hu AO SMC 35,000 cells of each type.

Colonization of laminin measuring areas by test cells (glioma cell line GLZ) and reference cells (neuronal cell line PC12)

Colonization of laminin measuring surfaces by test cells (smooth muscle cells huAO SMC) and reference cells (neuronal cell line PC12)

Table: Adhesion of test and reference cells; the number of cells bound per measuring point and the respective relative proportion of the populated area are indicated Although there are extreme deviations in the number of bound cells between individual measuring points, the percentage of bound test cells is approximately constant within the range of fluctuation common for biological measurements. For example, while only 53 cells at measurement point 1 and even 569 at measurement point 4 had a total of bound cells, 64% and 75% of the bound cells were test cells (GLZ). The ratio for measurement points 2 and 3 was also in this range, at 70% and 75%, respectively.

This constant ratio is changed by the fact that PC 12 binds to the catcher molecules more poorly in comparison with GLZ than in comparison with hu AO SMC, but it also remains sufficiently constant here with 9% to 17%.

In other words, the ratio between the two respective cell types is approximately constant if the ratio F> N x H is maintained, regardless of the number of cells per measurement area and regardless of whether the two cell types compete for a capture molecule. This makes it possible to use reference cells to calculate the variation between measuring points.

The percentage or the ratio between bound test and reference cells is therefore a more reliable measure of the binding of the test cells to the individual measuring points than the absolute number of bound test cells.

Example III: Incubation with and without shaking

Test cells (PC 12) were in a concentration of 0.5 to 50 x 10 ^s cells / ml on measuring surfaces of the area 280,000 μm ² with different capture molecules, namely collagen I, collagen II and collagen III, incubated for 4 h each, the carrier plates being shaken during the incubation in one case and left to rest in the other case. For shaking, the carrier plate was moved manually at intervals of 10 min to mix the cell suspension over the arrays.

In FIG. 4, the upper line shows the binding to collagen I, the middle one the binding to collagen II and the lower one the binding to collagen III. Incubation without shaking is shown on the left for A, C and E and incubation with shaking on the right for _^ B, D and E.

It can be seen from the recordings that the shaking leads to a significantly increased and also significantly more uniform binding of the test cells to the capture molecules.

FIG. 5 shows the number of cells per measuring surface as a function of the number of cells used and the binding of the test cells to the three capture molecules mentioned in FIG. 5.

6 shows the number of cells per measuring surface as a function of the number of cells used and the binding to the laminin substrate and the influence of the substrate movement.

It can be seen that the shaking ensures that a measuring area is populated with cells at most. A "saturation" of the measuring surfaces already occurs at a concentration of 20 x 10 ⁵ cells / ml, which can be seen from the fact that the number of bound cells does not increase any further from this concentration. It can also be seen in the left branches of the curves that at low concentrations, that is to say with a lower number of cells per measurement point, there is virtually no binding to thrombospondin, but rather that almost all cells bind to laminin or collagen I. This is also to be expected, because PC12 binds to thrombospondin considerably less than to the other capture molecules. Only through a high number of cells in the supernatant, the effect of competition is weakened and the ^cells' also bind to thrombospondin.

Example IV: Determination of the sensitivity of cells in their natural microenvironment

An example of how the method according to the invention can be used is the investigation of how tumor and normal tissues react to radiation or the addition of toxic substances, depending on their natural microenvironment. It is known that cells whose radiation sensitivity is tested in a cell culture without the addition of extracellular matrix components (ECM) are more radiation-sensitive than parallel cultures which have been cultivated on ECM. This means that the composition of the extracellular matrix is of crucial importance for the cell-specific reactivity of both tumor and normal tissue. Furthermore, the combination effect of radiation and chemotherapy can be further optimized in the natural microenvironment.

These experiments are carried out using the ECM as capture molecules. The test cells are placed in a mixed suspension with reference cells on an array of different capture molecules and the number of bound test cells as well of the bound reference cells is determined and from this the normalized number of test cells per measuring point is calculated. The test cells are then treated with staurospondin and incubated. After a certain incubation period, the number of dead test cells per measurement point is determined and the apoptosis rate is determined from this number and from the normalized number determined before the incubation.

Experiments with staurosporin-induced apoptosis in PC12 cells showed that these cells are significantly better protected from the damaging effects of staurosporin by collagen IV and laminin, i.e. their natural substrates, than by PLL (poly L lysine), which is probably not a protective effect has. The apoptosis rate of test cells on lamin was increased by a factor of 4 after staurospondin treatment, while test cells that grew on PLL showed a factor of 15 increase. This difference could only be determined by using the reference cells.

Claims

claims

1. Procedure for performing functional tests on test cells, with the steps:

b) creating a mixture of the test cells and of reference particles which have the ability to bind to the capture molecules and which are distinguishable from the test cells,

2. The method according to claim 1, characterized in that in step b) test cells and reference particles are mixed in a ratio of 1: 1.

3. The method according to claim 1 or 2, characterized in that the reference particles comprise artificial balls (beads), for example. Latex balls, which carry surface molecules that allow a comparable binding to the capture molecules as the surface molecules of the test cells.

4. The method according to any one of claims 1 to 3, characterized in that the reference particles comprise biological reference cells which can be distinguished from the test cells in terms of measurement technology, preferably optically, but which bind to capture molecules like the test cells.

5. The method according to claim 4, characterized in that the reference cells are preferably genetically different from the test cells in such a way that they do not react or are known to react differently to substances and / or radiation whose effect on the test cells is known than the test cells.

6. The method according to any one of claims 1 to 5, characterized in that the test cells from the reference particles are optically distinguishable in that the test cells are marked with a different marker, preferably a fluorescent marker or a genetic marker than the reference particles.

7. The method according to any one of claims 1 to 6, characterized in that the test cells from the reference particles can be distinguished by measurement technology in that the test cells are provided with a different radioactive marker than the reference particles.

8. The method according to any one of claims 1 to 7, characterized in that at least one measuring point with its associated capture molecules is divided into several measuring surfaces in the array, which are arranged at different points in the array.

9. The method according to claim 8, characterized in that a measurement value for the test cells and the reference particles is calculated for the at least one measurement point from measurement values that are determined for the assigned measurement areas.

10. .Procedure according to one of claims 1 to 9, characterized in that there is a ratio between the area F of a measuring point and the number NT of the test cells and the number of NR reference particles in the mixture, that of the adhesive area HT of the test cells and the adhesive area HR depends on the reference particle and is selected as follows:

F> a x (NT x HT + NR x HR), a = 0.5, preferably about 1.0

11. Procedure for performing functional tests on test cells, with the steps:

a) Providing an array of mutually separate measuring points, at each of which different capture molecules are immobilized, to which the test cells can bind, with at least one measuring point with its assigned capture molecules being divided over several measuring surfaces in the array, which are arranged at different locations in the array .

b) contacting a suspension of test cells with the array so that test cells can bind at each measuring point, c) location-resolved detection of measurement data representing the number of test cells of interest on the measurement surfaces,

d) Calculation of a measurement value for at least one measurement point from the measurement data of the measurement areas assigned to it.

12. The method according to claim 11, characterized in that between F, the sum of the measurement areas of a measurement point and N, the number of test cells in the suspension, there is a ratio which depends on the adhesive area H of the test cells and is selected as follows:

F ≥ a xN x H, a = 0.5, preferably about 1.0.

13. A method for performing functional tests on test cells, comprising the steps:

a) providing an array of mutually separate measuring points, at each of which capture molecules are immobilized, to which the test cells can bind, and

b) Contacting a suspension of test cells with the array so that test cells can bind to each measuring point, with a relationship between the area F of a measuring point and the number N of test cells in the suspension that depends on the adhesive area H of the test cells and how is selected as follows: F ≥ ax N x H, a = 0.5, preferably approx. 1 . 0.

14. The method according to any one of claims 1 to 13, characterized in that the array is moved after contacting the test cells and possibly the reference particles, for example on a vibrator or a cradle.

15. Procedure for performing functional tests on test lines, comprising the steps:

b) contacting a suspension of test cells with the array so that test cells can bind at each measuring point, and

c) Incubating the array with the suspension, the array being moved, preferably on a vibrator or on a cradle.

16. The method according to any one of claims 1 to 15, characterized in that the array is either applied to a carrier plate or that it is a logical array of individual balls that are loaded with capture molecules.

17. The method according to any one of claims 1 to 16, characterized in that the test cells are subjected to a treatment before or after contacting the array, which treatment is selected from the group: irradiation with high-energy radiation, for example UV light or radioactive radiation, contact with test substances such as pharmaceutical agents, other cells, chemotherapeutic agents, components of extracellular matrix proteins, antibodies, lectins or other biopolymers.

18. The method according to any one of claims 1 to 17, characterized in that different capture molecules are immobilized at the measuring points, which are preferably selected from the group: protein such as components of extracellular matrix proteins, receptors. Ligands, poly-lysine, peptides from laminin sequences, control peptides, peptidomimetics, antibodies, lectins, antigens, allergens.

19. Kit with an array of separate measuring points, at which capture molecules are immobilized, can bind to the test cells, and with reference particles, which bind to the capture molecules.

20. Kit according to claim 19, characterized in that the reference particles are the reference particles from one of claims 3 to 7.

21. Kit according to claim 19 or 20, characterized in that different capture molecules are immobilized at the measuring points, which are preferably selected from the group: protein such as, for example, components of extracellular mat- rix proteins, receptors. Ligands, poly-lysine, peptides from laminin sequences, control peptides, peptidomimetics, antibodies, lectins, antigens, allergens.

22. Kit according to one of claims 19 to 21, characterized in that the array is either applied to a carrier plate, or that it is a logical array of individual balls which are loaded with capture molecules.

23. Kit according to one of claims 19 to 22, characterized in that at least one measuring point with its associated catcher molecules is divided into several measuring surfaces in the array, which are arranged at different points in the array.