WO2011023363A1

WO2011023363A1 - Method and device for cell sample diagnosis

Info

Publication number: WO2011023363A1
Application number: PCT/EP2010/005174
Authority: WO
Inventors: Sebastian Koball
Original assignee: Universität Rostock
Priority date: 2009-08-25
Filing date: 2010-08-24
Publication date: 2011-03-03
Also published as: DE102009038520B4; DE102009038520A1

Abstract

The invention relates to a method for cell sample diagnosis on a cell sample or cell culture. In addition, the invention relates to a computer program product, a data storage medium with a computer program product stored or written thereon according to the invention, and a device for cell sample diagnosis on a cell sample or cell culture. The method according to the invention is characterized in that a) at least one extract from the cell sample or cell culture is captured by video microscopy over a pre-definable measuring time period, b) at least one cell (1) from the cell sample or cell culture is individualized in the captured video signal over at least part of the measuring time period, c) at least one property (21 - 23) of the at least one individualized cell (1) with the change over time thereof is captured, d) the property (21 - 23) of the individualized cell (1), captured with the change over time thereof, is compared with at least one comparative value with respect to the registered property (21 - 23), and e) from the agreement or deviation of the captured property (21 - 23) of the individualized cell (1) with or from the reference value, the functional state, state of vitality and/or state of damage of the cell and the change thereof over time is/are determined.

Description

Method and device for cell sample diagnostics Description

The invention relates to a method for cell sample diagnostics on a cell sample or cell culture and to a device for cell sample diagnostics on a cell sample or cell culture. The present invention finds particular application in the study of the effects of new drugs, potentially toxic and other substrates on living cells, particularly human or animal cells. This represents a major area of responsibility for the compatibility tests of new drugs. Such studies are first carried out on cell cultures isolated and cultivated in the laboratory, in which the compatibility of such substances is tested before they take place on humans or on the animals themselves. Corresponding studies, i. Methods for cell sample diagnostics on cell samples or cell cultures are known.

In known methods for cell sample diagnostics are cultivated Incubated cells are incubated with the substance to be examined and it is observed, which effects the substances have on the vitality of the cells. The study examines the behavior of cells after the addition of test reagents, which allow to assess the vitality, the metabolism or individual functions of the cells as a whole. Instead of or in addition to the addition of analytes, physical or physical stimuli such as the application of alternating electromagnetic fields, light intensity or temperature control, and the like may be used, either for the entire cell sample or cell culture or locally limited.

In a known class of procedures for Zeilprobendiagnostik5 by adding dyes living and dead cells are differentiated from each other. These colorants or changes in hues are usually measured in the cell suspension as a whole. However, individual cells can also be examined by fluorescence optics or microscopy. Dead cells can also be detected by light microscopy in some cases even without color additives.

Metabolic performance of cells is typically measured by the addition of substrates that are transposed by the cells. Metabolic performance in this case is measured indirectly by the extent of reaction of the added substrates. Concentration changes of test substances in the whole cell suspension provide information about the activity of the cells. In addition, measurements of electrical potentials on cell surfaces, which are indirect, are possible for specific questions

Provide information about the state of the cells in the culture. With the exception of the light microscopic diagnosis of dead cells, all methods used hitherto by metrology are based on the addition of test substances or the introduction of test devices into the cell culture, in addition to the introduction of the active substances or pollutants to be investigated. These test substances, mostly dyes or radioactively labeled test substances, are themselves involved in the test environment and are often themselves toxic, so that they can considerably alter the test results determined. The test results are therefore not based solely on the reaction of the cells to the investigating active or pollutants. It is often unknown to what extent the application of the investigational reagents changes the actual interpretation of the results. Furthermore, in known methods usually only a single test with a cell sample is possible at the same time. However, long-term observations with repeated application of test substances and dyes are often not possible. With known test methods, in particular in the measurement of

Color changes in the entire cell culture or the dissolved supernatant, the spatial resolution is often not sufficient to assess cell damage in detail. Many test methods are based on measuring the release of intracellular enzymes and proteins. This release takes place with the dissolution of the cell membrane, ie at a very late time, when the cell is already completely dead and the integrity of the cell wall is abolished.

When cell culture mixtures are used, an assignment of substances released in cell death to the individual cell types - A -

due to the lack of spatial resolution of these tests not possible. As a result, only statements about the entire cell culture are possible. Some differentiation is via dyeing methods for

Detection of cell death possible in principle with a corresponding spatial resolution under the microscope. However, different types of cells often show different follicular behavior, so that a reliable interpretation of the results is not always possible.

In contrast, the invention has for its object to provide a method and an apparatus that allows a Zeilprobendiagnostik on a cell sample or cell culture, with which a fast, accurate and damage-specific and cell-type specific spatially resolved analysis is possible.

This object is achieved by a method for cell sample diagnostics on a cell sample or cell culture, which is developed by a) at least a section of the cell sample or cell culture over a predetermined measurement period is detected by videomicroscopy, b) at least one cell of the cell sample or cell culture in c) at least one property of the at least one individualized cell is detected with its temporal change, d) the property of the individualized cell detected with its time change is compared with at least one comparison value with respect to the detected property; and e) the conformity or deviation of the detected property of the individualized cell with or from the comparison value of the function, vitality and / or Damage state of the cell and its temporal change is determined.

The method according to the invention is based on the observation that animal and human cells behave and move as their environment changes according to specific behavioral patterns. In addition to cell death characteristic changes in the cell shape are specific for specific damage patterns

To detect changes in the movements or movement or behavior patterns of the cells. These changes in behavioral patterns are captured by the video signal and evaluated for their temporal change or variability. In individual properties these temporal changes and the

Dynamics of these changes are very characteristic and significant, so that a small number of individualized cells is already sufficient, may already be a single individualized cell.

For the method according to the invention, animal or human cells or other cells are kept on a glass plate, in a Petri dish or in a special solution, for example a special nutrient solution. Suitable sample carriers are, for example, bone substitutes or other carrier materials which are used, for example, for biocompatibility tests. Typical cell types studied in this way are, for example Connective tissue cells, liver cells, peritoneal cells, blood cells or nerve cells, wherein the applicability of the method according to the invention is not limited to these cell types. The method according to the invention is based on the optical detection of cells in at least one section of the cell sample or cell culture, which may be a culture of single cells or a cell carpet or a multi-dimensional reconstruction of cell clusters or cells on carrier materials. The detection takes place, for example, in the optically visible range

Grayscale analyzes or color shades. In addition to light microscopy, phase contrast microscopy, fluorescence microscopy and laser scanning microscopy are also suitable for use with the method according to the invention. In particular, laser scanning microscopy offers the advantageous possibility of evaluating three-dimensional images.

The evaluation of the video signal can be done on-line during the recording or off-line based on the completed video recording.

The process according to the invention can be carried out alone or compared with conventional processes, which are often carried out in parallel. It allows the u. a. also directly comparing the effects of different drug concentrations and evaluating the cell's responses to it.

A property encompassed by the invention is preferably a cell shape, a cell circumference, a cell area, a number or size of cell spurs, a ratio of sizes of cell spurs, a cell nucleus location in the cytoplasm, a cell nuclear to plasma size ratio, a cell surface to cell ratio a movement pattern with a direction of movement and a movement speed or a number of cell divisions or cell mergers. Upon contact with irritants, active substances or pollutants, these variables are characteristically variable.

In an advantageous embodiment, a detected property is a movement pattern with changes in location, wherein interactions between neighboring cells are detected, in particular between different cells of the same type and / or between cells of different types and / or species, or wherein the ratio of

Self-movement of at least one individualized cell is detected to cell collective movements. For example, a characteristic response to a local stimulus is movement of the cell away from a damage stimulus or to an attractant. Even more complicated reaction patterns occur in the change of location of cells or the change in their interactions, in which two or more of the above properties change in a certain way. The parameters mentioned are typical parameters of the cell appearance and of the movement and activity pattern of the cells, which react to the addition of substances or physical stimuli. Thus, a cell that is damaged may either expand or contract, the nucleus may change its shape or size, the activity of the foothills and the number and

Extent of shoots may decrease or increase, the number of cell contacts, displacement of neighboring cells, penetration of cell aggregates or neighboring cells, cell mergers, rejections, cell divisions or resolutions may increase or decrease, damaged individualized cells may move against cell collective movements, etc. There is one Variety of possible changes of these parameters, which are characteristic for the respective cell type and for the respective damage pattern.

The analysis of the individualized cell or cells according to the invention is based on a, in particular dynamic, measurement of the

Cell shape and its changes. For this purpose, it is provided in a preferred embodiment of the method according to the invention that the videomicroscopic recognition of properties of the individualized cell on the basis of individual recordings of the video signal, in particular automatically happens, wherein for each individual recording of the video signal one or more of the following steps are performed cumulatively: a) detection the cell wall and in particular calculation of the area content of the cell, and in particular determination of the position of the cell in the detected section, b) detection of the cell nucleus and the nuclear boundary and in particular calculation of the area of the cell nucleus, the nuclear-plasma relation and / or the orientation of the nucleus in the

Cell, c) detecting cell organelles and / or inclusions and determining their position relative to the cell nucleus and the cell wall.

Cumulative execution in this context means that at least the process step a), or the process steps a) and b) or the process steps a), b) and c) are performed.

The steps mentioned relate to the detection of the geometric relationships of the cell in the individual image of the video signal, whose changes is examined in the sequence of individual images. Thus, the detection of the cell wall at the same time also allows the determination of Zellausläufern and the calculation of a suitably calculated Zellausläufer index, especially in relation to the surface of the cell. From the obtained geometric data of the cell and the

Nuclei and the cell organelles can be derived essential properties.

The detection of the cell wall or of the cell nucleus also includes, in particular, the detection of the location at which the cell or the nucleus is located, so that an absolute movement of the entire cell or of the cell nucleus can also be tracked from one single exposure to the next. Preferably, a detected property is a time, a period of time, a frequency, or a frequency in which or at which a detected property changes. This makes the dynamics of the changes particularly clear. The dynamics of the changes in the detected properties form an important part of the method according to the invention.

The comparison values are preferably snapshots of acquired properties or temporal developments of detected properties on the basis of known functional, vitality and / or damage states and / or their changes. Thus, it can be examined whether the extent and / or rapidity of a measured change of detected properties corresponds to a known damage state, or whether the change is within the normal range of vital or healthy cells. The variability of the properties in healthy or damaged cells is taken into account. Preferably, the detection of properties and their comparison with comparative values is carried out for a plurality of individualized cells, in particular forming a cell group, wherein preferably between 5 and 500 cells, more preferably between 10 and 100 cells, are examined. The examination is carried out in particular by sampling. Examination of a large number of cells makes it possible to improve the statistical data base and therefore to improve the informative value of the examination. In particular, in addition to the study of monocultures, the study of culture mixtures with different types of cells can also be carried out, the high spatial resolution with respect to the cell type and the interactions of the individual different cells being particularly advantageous. For this purpose, preferably the cell sample or cell culture is a mixed culture of at least two different cell types or cell types. Also, three to five different cell types can be examined in this way. The inventive method is theoretically not limited to the top. However, the performance of the video-microscopic device used and the computing capacity of the data processing systems used for the analysis place an upper limit on the number of different cells to be investigated simultaneously, and in particular cell types. An investigation based on culture mixtures can in practice also be followed by an examination of initially only one cell type.

Preferably, the videomicroscopic detection takes place while the cell culture is incubated, in particular with a chemical or pharmacological substance and / or under the action of one or more physical stimuli.

Preferably, the comparison value is from a cell thereof Cell type whose functional, vitality and / or damage state is known. Alternatively or additionally, the comparison value from the same cell or a cell of the same cell type originates from the captured segment earlier in the measurement period. The two alternatives can also be mixed with several recorded properties. The first mentioned alternative is particularly advantageous for known cell types for which reference values are known. The latter alternative is advantageous because it also allows for detecting relative changes in the observation of cells during incubation with previously unknown drugs, which may be sufficient for statements to assess the effect of incorporated substances. In addition, the dynamics of previously unknown cells can be investigated, such as the description of the growth of tumor cells of a patient and characterization of the effects of various drugs on these cells. Since these are patient-specific tumor cells, there are usually no comparative data for this particular cell line, but the effects of substances on these cells are important.

Furthermore, preferably positive and / or negative controls of the cell sample or cell culture, in particular simultaneously, are incubated and recorded by video-microscopy and the at least one detected property is also recorded for at least one individualized cell of the control and used as comparison value. This makes a direct comparison between the recorded values and comparison values possible, since the comparison values for the same cell type or the same cell culture mixture are obtained under the same conditions as the recorded values of the cell cultures contaminated with harmful substances or active substances.

In an advantageous development of the method according to the invention The procedure is carried out under standard culture conditions. In particular environmental factors, in particular temperature, carbon dioxide content or light are changed. The technical implementation of the new measurement method is carried out by videomicroscopic recording of cell growth and cell movement under cell culture conditions. The cells to be examined are incubated with the test substance and observed over a period of usually four to twelve hours. The period can be shorter or longer. The cell changes are recorded in a, in particular digital, video film or video signal from a sequence of individual images. This sequence of individual images from the video signal represents the data basis for the following data analysis. Preferably the video-microscopic acquisition is performed with a refresh rate of 0.01 Hz to 1

Hz ₁ in particular between 0.05 Hz to 0.2 Hz, in particular of 0.1 Hz made. This corresponds to a spacing between the individual images of 1 to 100 s, in particular 5 to 20 s, in particular 10 s. These intervals are based on the typical velocities and change times of the cell cultures or

To adapt cell samples, which may vary depending on the cell type. In a few cases, a refresh rate of more than 1 Hz is necessary. The image repetition rate may preferably also be variable, whereby, for example, after the addition of an active substance, it is first recorded with a comparatively high image repetition rate in order to record the immediate reaction in detail, while in the course of the examination period the distances between individual images become greater if the activity of the examined Cells subsides. This achieves a reduction in the amount of data to be processed without affecting the accuracy of the analysis. lysis.

Data analysis relies on a large database of cell movement data, which stores comparative data or reference data obtained during studies under well-defined environmental conditions with known toxic agents. In addition to single-cell transduction data, this database also contains data on cell-cell interactions and interactions with physical structures or stimuli in combination with known drugs. Data for cell cultures with one to three cell types or even more cell types are stored in the database. By computer-aided comparison of the new data with the results from the database and / or a positive or negative sample statements with respect to the effects and toxicity of different drugs are possible. As a rule, the database does not contain any videomicroscopic individual images or image sequences, but only the parameterizations of the abovementioned properties. This leads to a very efficient data reduction.

The method according to the invention requires no additional reagents which would cause a falsification of the test environment and make the interpretation of the measured values difficult. The test system has by the observation of single cells, even a plurality of single cells, a high spatial resolution. Damage patterns can be assigned to individual cells and cell subpopulations and thus allow a higher specificity in the interpretation of the test results. Even studies with more than two or three cell populations are possible. ZeII damage in connection with structural peculiarities are possible, specific reactions to mechanical cell damage can be detected more sensitive than previously. The method according to the invention reacts early to changes in cell behavior and is thus more sensitive than comparable microscopic examination methods. Broad-based screening studies, eg in pharmacological drug discovery, are thus possible with a much lower time factor. The lack of chemical reagents minimizes the running costs of testing. Long-term observations are possible without problems, the effort for the cell quantities to be kept is minimized.

With the method according to the invention not only cell cultures with isolated cells can be examined, but also with cells that are present in cell carpets or cell lawns. Even in cell turf there is a brisk movement, as single, mobile

Cells can penetrate the neighboring cells, i. can squeeze between two adjacent cells. Individualization and tracking of the individual cells from frame to frame of the video signal is also possible in this environment.

The object on which the invention is based is also achieved by a computer program product with program code means, in the execution of which the steps of a method according to the invention described above are executed or can be carried out in a data processing system. Also, the object underlying the invention is achieved by a data carrier with a computer program product stored or written on it. The object underlying the invention is also by a

Device for the delivery of a cell sample or cell culture, comprising a video microscopy device, the is adapted to make, in particular digital, video recording of the incubated cell sample or cell culture, wherein the resolution of the video microscopy device is sufficient to individualize individual cells of the cell sample or cell culture, wherein the device comprises an evaluation device which is formed from the

Video recording at least one cell to customize, at least one property of the individualized cell and their temporal change to capture and compare with a comparison value.

The device according to the invention is preferably set up to carry out the method according to the invention as described above. The evaluation device preferably has access to a database with comparison values, in which comparison data regarding the recorded properties for one or more cell types or cell types with known functional, vitality and / or damage status are stored. In addition, it is preferably additionally stored in the database by which substances or physical substances

Stimuli of the functional, vitality and / or damage state was caused, which correspond to the comparative data. Preferably, the database is supplemented by the evaluation device to the newly acquired data.

This also provides a device with which, according to the invention, the evaluation of cell functions, cell vitality and damage states is possible better than with currently used examination methods and devices. By observing cell movements and changes in shape over time, the method and the device according to the invention avoids the shortcomings of the known methods and devices. obligations. In particular, cells can be observed by means of videomicroscopic observation over variable periods of time and movements or changes in the shape of the cells can be recorded. Individual environmental factors such as temperature, carbon dioxide content or light and any other physical environmental variables can be changed in a controlled manner. Under constant physical environmental factors, the effects of chemical agents to be investigated or specific physical stimuli can be recorded and investigated.

The tests are carried out on the vital cell culture and no additional test reagents are used which could falsify the results by their own action or side effects. The collected data can be collected and evaluated both for single cells and for cell aggregates as well as for the analysis of cell formations changes in shape as a reaction to neighboring cells and their movement behavior can be analyzed. Since the collective analyzes always take place at the level of the individual cell, a particularly high spatial resolution is also given in the collective analyzes. This also applies to the study of mixed cultures.

The invention will be described below without limiting the general inventive idea by means of embodiments with reference to the drawings, reference being expressly made to the drawings with respect to all in the text unspecified details of the invention. Show it:

1 is a schematic representation of a cell,

2 is a schematic diagram of the temporal evolution of detected characteristics with conventional or the method according to the invention and

Fig. 3 is a schematic representation of a device according to the invention.

In the following figures, identical or similar elements or corresponding parts are provided with the same reference numerals, so that a corresponding renewed idea is dispensed with.

In Fig. 1, a cell 1, which has been individualized in the method according to the invention, shown schematically. The cell 1 has a cell wall 2 and a cell nucleus with a cell nucleus boundary 4, as well as organelles 5 and inclusions 6. These components of the cell 1 are recognizable by video-microscopy, in particular via

Grayscale analyzes or color shades. These constituents can also be detected in light, phase contrast fluorescence microscopy or laser scanning microscopy and can be used for the analysis according to the invention. Methods for the automatic recognition of these cell boundaries and cell components in the single image are known.

FIG. 1 already shows a first step of an exemplary further analysis. There are connecting lines 7, 8 between the cell nucleus border 4 and the cell wall 2, which are shown in points 7 ',

7 "or 8 ', 8" touch the cell wall 2 or the cell nucleus 3. These connecting lines 7, 8 represent such connecting lines in which a local maximum or a local minimum of the length of the connecting lines exists from the family of possible connecting lines. In FIG. 1, those connecting lines with a locally maximum length are drawn through or solidly drawn, while the connecting lines are shown with dashed lines of locally minimal length. are. The longest of these connecting lines mark cell spurs 9.

For detecting which connecting lines have a local maximum or minimum length, several different methods are applicable, which can lead to slightly different results in each case. In the temporal pursuit, however, they are essentially equivalent and it is particularly important that each be applied consistently. Thus, the lines can originate from the center of the cell nucleus 3, they can emanate from the irregular nucleus boundary 4, from a circle around the center of the nucleus 3, or it is calculated back from the cell wall 2 to the nucleus boundary 4 and optimized in each case with suitable algorithms.

The connecting lines drawn in FIG. 1 already allow a conclusion to be drawn about the number and extent of cell spurs 9. An alternative method to the illustrated method is approximately in the Fourier analysis of the cell wall 2 and in particular the cell nucleus boundary 4. Other suitable geometric analysis methods are applicable for this purpose. In order to limit the effort of the necessary analysis, it can be provided that only a number of the most significant spurs 9 are determined in the manner described and tracked over the sequence of individual recordings. As a result, the development of the foothills, their evisceration and recovery, traceable. The distribution of the positions and / or significances of the extensions can also be detected. It can also be detected in which areas the cell 1 changes the fastest, ie in which areas are particularly fast forming or retreating.

In Fig. 1 it can also be analyzed where the organelles 5 and 5 inclusions 6 are, where they are in relation to the cell nucleus 3 and, on the basis of the successive individual images, how they move.

A corresponding analysis can be carried out for a large number of individualized cells 1, which can also be of different cell types. Typically, for example, an examined slice contains several hundred cells that are either all examined or sampled, which are then subsequently tracked over the duration of the frame and frame video signal.

For most cell types, the likelihood of cells in the slice being examined remaining in the observed slice throughout the observation period is relatively large. It is possible to select those cells that remain in the clipping, or to adapt the analysis to take into account that some examined cells move out of the clipping and other cells in their place are newly included in the examination.

5

FIG. 2 shows the time profile of a conventional examination variable as well as three indices determined by the method according to the invention over a period of 260 minutes. The study concerns a cell culture incubated in a toxic dialysis solution. Dialysis solutions for peritoneal dialysis, which are optionally provided with lactate as the sole buffer, generally have a pH which does not correspond to the pH value. fit by developing human body cells well. The toxic dialysis solution used is acidic and hyperosmolar.

On the vertical axis, two different units are shown. On the one hand, it is an index which is characteristic of the three properties 21 to 23 detected and determined according to the invention. These indices correspond to arbitrary units. The curve labeled 20 is the time course of the concentration of the enzyme lactate dehydrogenase (LDH), which is measured in the unit IU / I, ie in international units per liter. Lactate dehydrogenase is an enzyme found in all cells that is released upon cell damage, particularly damage to the cell wall, and is a standard test for investigating cell damage. The level of the LDH reading correlates with the number of damaged cells and the intensity of cell damage. The release leads to an increased concentration of lactate dehydrogenase in the medium.

From the diamond-marked LDH curve 20, it can be seen that massive cell damage occurs with increasing time during the incubation of the cells in the toxic dialysis solution, and thus, as a measurable sign for this, an increase in the LDH values. This increase begins significantly after about two hours and experiences near-saturation after about three hours, after which only a slight further increase in the LDH value is detectable. This is explained by the fact that the majority of cell damage has already occurred after about three hours.

The index profiles provided with the reference symbols 21, 22 and 23 are calculated from a plurality of individual variables. The The reference numeral 21 refers to changes in the cell shape and cell total area, ie, the area, perimeter, and foothold of the cells. The index indicated by reference numeral 22 is calculated from several properties relating to movements of the cell as a whole, namely changes in the location of cells, their velocity and the direction and direction of their movements. The index indicated by reference numeral 23 is calculated from movements of the cell in place, namely, changes in the cell surface, the dynamics of foothill formation, and the movement of cell organelles. It can be clearly seen that, even at the beginning of the measurement, the index 23 concerning dynamics of the movement of the cell at the location, ie the changes of the cell surface, the dynamics of the formation of foothills and the movement of cell organelles decreases significantly. After a first steep decrease of this index in the first 30 minutes of the measurement, this falls further, but not so steep.

The index indicated by the reference numeral 22, which indicates the movement of the cell as a whole, is initially flat, while it also decreases significantly after about an hour. After 1 hour in the toxic dialysis solution, the cells thus lose their mobility in the medium, ie the change of location, speed and the directionality of the movement. After about 2 Vz hours, the index 22 drops only slightly. However, after 2 ^Λ A hours, this index 22 moves at a fraction of the level at the beginning of the measurement. The cell movement has therefore essentially come to a standstill. The third index, reference numeral 21, has a flatter course. A first relatively significant change relates to a decrease after about 2 hours, after which the cell shape, the total cell area and also the number of shoots decrease. This decrease continues in the further course of the measurement.

From Fig. 2 it can be seen that on the basis of the indices 21, 22, 23 from the inventive method already much earlier than with the known method of measuring the Lactatdehydro- genase values in the solution, a change in properties of the cells in the toxic dialysis solution is recognizable. It is also much more accurate than previously possible to see what kind of changes occur in the corresponding individualized and examined cells.

FIG. 3 shows by way of example a schematic representation of an embodiment of a device 40 according to the invention. This cell sample diagnostic apparatus 40 includes a cell sample container 41 having a cell sample or cell culture disposed in an incubator 42 together with a video microscope 43 directed to the cell sample container 41. The incubator 42 is connected via a control line 45 and a signal and control line 46 to a data processing system 44. The data processing system 44 includes an evaluation device 47 which receives and evaluates signals from the video microscope 43 via the signal and control line 46 as well as signal and control paths 51, 52. For this purpose, the evaluation device 47 accesses a database 48, characterized by a double arrow, which contains or comprises comparative data on detected properties of one or more cell types investigated. The data processing system 44 also includes a control device 49 with which the incubator 42 and the video microscope can be controlled. Thus, for example, the environmental conditions of the incubator 42 can be changed by the control device 49 and the recording by the video microscope 43 can be controlled.

The evaluation device 47 and the control device 49 can also be in contact with each other, so that the control device 49 can inform the evaluation device 47, for example, that the environmental conditions in the incubator 42 are changed or the image repetition frequency of the video signal is changed.

Furthermore, it is shown that via a signal and control path 53 and a data and control line 54, a display and operating device 50 is controlled outside the data processing system 44, on which analyzes can be performed or on the video data, intermediate analysis data, analysis end data, etc. can be displayed.

It is also shown that the evaluation device 47 can send data or control commands to the database 48. This can be the

Database 48 are extended to recognized comparison data or it may follow a control command, with the particular data from the database 48 are retrieved. Some or all of these mentioned devices can also be combined in a single data processing system or distributed in each case to different data processing systems or other suitable electronic systems which form a network.

All the above features, including the drawings alone to be taken as well as individual features, in combination with disclosed other features are considered alone and in combination as essential to the invention. Embodiments of the invention may be accomplished by individual features or a combination of several features.

LIST OF REFERENCE NUMERALS

1 cell

2 cell wall

3 nucleus

4 cell nucleus border

5 organelle

6 inclusion

7 connecting line with locally maximum length

T point on the cell wall

7 "point on the nucleus

8 connecting line with locally minimal length

8 'point on the cell wall

8 "point on the nucleus

9 cell spurs

20 LDH concentration

21 Index 1

22 Index 2

23 Index 3

40 Device for cell sample diagnostics

41 cell sample containers

42 incubator

43 video microscope

44 data processing system

45 control line

46 signal and control line

47 evaluation device

48 database

49 control device

50 Display and operating device

51 - 53 Signal and control paths

54 data and control line

Claims

Method and device for line sample diagnostics Claims

1. A method for Zeilprobendiagnostik on a cell sample or cell culture, characterized in that a) at least a section of the cell sample or cell culture over a predetermined measurement period is detected videomicroscopy, b) at least one cell (1) of the cell sample or cell culture in the detected video signal over at least one C) at least one property (21-23) of the at least one individualized cell (1) is detected with its temporal change, d) the property (21-23) of the individualized cell (1 ) with at least a comparison value with respect to the detected property

And (e) from the agreement or deviation of the detected property (21-23) of the individualized cell (1) with or from the comparison value of the functional, vitality and / or damaged state of the cell and its temporal Change is determined. A method according to claim 1, characterized in that a detected property (21-23) comprises a cell shape, a cell circumference, a cell area, a number or size of cell spurs, a ratio of sizes of cell spurs, a location of the cell nucleus in the cell plasma, a size ratio of nucleus 5 to plasma, a ratio of cell surface to cell foothills, a motion pattern with a direction of movement and a velocity of movement, or a number of cell divisions or cell fusions. O

3. The method according to claim 1 or 2, characterized in that a detected property (21-23) is a movement pattern with changes in location, whereby interactions between neighboring cells are detected, in particular between different cells of the same type and / or between cells of different types and / or Species, or wherein the ratio of proper movement of the at least one individualized cell (1) is detected to cell collective movements.

4. Method according to one of claims 1 to 3, characterized in that the video-microscopic recognition of properties (21-23) of the individualized cell (1) on the basis of individual images of the video signal, in particular automatically, takes place, wherein for each individual recording of the video signal one or more of the following steps are performed cumulatively:

5 a) Detection of the cell wall (2) and in particular calculation of the surface area of the cell (1), and in particular the position of the cell (1) in the detected section, b) detection of the cell nucleus (3) and the cell nucleus boundary (4) o and in particular Calculation of the area of the

Cell nucleus (3), the nuclear-plasma relation and / or the orientation of the nucleus (3) in the cell (1), c) detecting cell organelles (5) and / or inclusions5 (6) and determining their position relative to the nucleus (3) and the cell wall (2).

A method according to any one of claims 1 to 4, characterized in that a detected property (21 - 23) is a time, o a period of time, a frequency or a frequency in or with which a detected property (21 - 23 ) changes.

6. The method according to any one of claims 1 to 5, characterized in that the comparison values snapshots of he-5 detected properties (21 - 23) or temporal developments of detected properties (21 - 23) due to known functional, vitality and / or Damage states and / or their changes are. O

7. The method according to any one of claims 1 to 6, characterized in that the detection of properties (21 - 23) and their comparison with comparative values for several, in particular an individualized cell (1) forming a cell structure is made, wherein in particular between 5 and 500 cells, in particular between 10 and 100 cells, are examined.

8. The method according to any one of claims 1 to 7, characterized in that the cell sample or cell culture is a mixed culture of at least two different cell types or cell types.

9. The method according to any one of claims 1 to 8, characterized in that the videomicroscopic detection takes place while the cell culture is incubated, in particular with a chemical or pharmacological substance and / or under the action of one or more physical stimuli.

10. The method according to any one of claims 1 to 9, characterized in that the comparison value comes from a cell of the same cell type, whose functional, vitality and / or damage state is known.

11. The method according to any one of claims 1 to 10, characterized in that the comparison value of the same cell (1) or a cell of the same cell type from the detected cut comes at an earlier time within the measurement period.

12. The method according to any one of claims 1 to 11, characterized in that positive and / or negative controls of the cell sample or cell culture, in particular simultaneously, incubated and recorded by videomicroscopy and at least one detected property (21 - 23) for at least an in- individualized cell (1) of the control and used as comparison value.

13. The method according to any one of claims 1 to 12, characterized 5 characterizes that the method is carried out under standard culture conditions, in particular environmental factors, in particular temperature, carbon dioxide content or light are changed. o 14. The method according to any one of claims 1 to 13, characterized in that the videomicroscopic detection with a refresh rate of 0.01 Hz to 1 Hz, in particular between 0.05 Hz to 0.2 Hz, in particular of 0.1 Hz, is made.

5

15. Computer program with program code means, in the execution of which is carried out on a data processing system, a method according to one of claims 1 to 14. 0

16. A data carrier with a stored or written computer program according to claim 15.

17. A device (40) for cell sample diagnostics on a cell sample or cell culture, comprising an incubator (42) for a cell sample or a cell culture, a video microscopy device

(43) adapted to take a, particularly digital, video of the incubated cell sample or cell culture, wherein the resolution of the video microscopy device (43) is sufficient to individualize individual cells (1) of the cell sample or cell culture marked that the

Device (40) comprises an evaluation device (47) which is formed from the video recording at least one cell (1) to individualize, to record at least one property (21 - 23) of the individualized cell (1) as well as its temporal change and to compare it with a comparison value.

18. Device (40) according to claim 17, characterized in that it is arranged to carry out a method according to one of claims 1 to 14.

19. Device (40) according to claim 17 or 18, characterized in that the evaluation device (47) has access to a

Database (48) with comparison values, in which comparison data relating to the recorded properties (21-23) are stored for one or more cell types or cell types with known functional, vitality and / or damage status.

20. Device (40) according to any one of claims 17 to 19, characterized in that in the database (48) is additionally deposited by which substances or physical stimuli of the functional, vitality and / or damage state was caused, the comparison data correspond.