DE102009038520A1 - Method and device for cell sample diagnostics - Google Patents

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 carrier with a computer program product according to the invention stored or written thereon 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 a section of the cell sample or cell culture is recorded by video microscopy over a predeterminable measurement period, b) at least one cell (1) of the cell sample or cell culture is individualized in the recorded video signal over at least part of the measurement period, c) at least one property (21-23) of the at least one individualized cell (1) is recorded with its change over time, d) the property (21-23) of the individualized cell (1) recorded with its change over time with at least one comparison value the detected property (21-23) is compared 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 damage status of the cell and whose change over time is determined.

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 field of activity of the compatibility tests of new active ingredients. Such studies are first carried out on cell cultures isolated and cultivated in the laboratory, at which the compatibility of such substances is tested before they take place on humans or on the animals themselves. Corresponding investigations, d. H. Methods for cell sample diagnostics on cell samples or cell cultures are known.

In known methods for cell sample diagnostics, cultured cells are incubated with the substance to be investigated and it is observed what effects the substances have on the vitality of the cells. The study investigates the behavior of cells after the addition of test reagents, which allow to assess the vitality, metabolic performance or individual functions of the cells as a whole. Instead of or in addition to the addition of substances to be examined, it is also possible, for example, to use physical or physical stimuli, such as the application of alternating electromagnetic fields, control of light intensity or temperature, and the like, either for the entire cell sample or cell culture or locally ,

In a known class of methods for cell sample diagnostics, living and dead cells are differentiated by the addition of dyes. 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 without color additives in some cases.

The measurement of metabolic performance of cells is usually done by the addition of substrates that are reacted 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 are possible for specific questions that indirectly provide information about the state of the cells in the culture.

With the exception of the light microscopic diagnosis of dead cells, all methods previously used in 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, it is not possible to assign substances released in cell death to the individual cell types because of the lack of spatial resolution of these tests. As a result, only statements about the entire cell culture are possible.

A certain differentiation is possible by dyeing methods for the detection of cell death in principle also with a corresponding spatial resolution under the microscope. However, different cell types often show a different color behavior, so that a secure 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 cell sample diagnosis of a cell sample or cell culture, with which a fast, accurate and damage-specific and cell-type-specific spatially resolved analysis is possible.

• a) wenigstens ein Ausschnitt der Zellprobe oder Zellkultur über einen vorgebbaren Messzeitraum videomikroskopisch erfasst wird,
• b) wenigstens eine Zelle der Zellprobe oder Zellkultur im erfassten Videosignal über wenigstens einen Teil des Messzeitraums individualisiert wird,
• c) wenigstens eine Eigenschaft der wenigstens einen individualisierten Zelle mit ihrer zeitlichen Veränderung erfasst wird,
• d) die mit ihrer zeitlichen Veränderung erfasste Eigenschaft der individualisierten Zelle mit wenigstens einem Vergleichswert bezüglich der erfassten Eigenschaft verglichen wird und
• e) aus der Übereinstimmung oder Abweichung der erfassten Eigenschaft der individualisierten Zelle mit oder von dem Vergleichswert der Funktions-, Vitalitäts- und/oder Schädigungszustand der Zelle und dessen zeitliche Veränderung bestimmt wird.

This object is achieved by a method for cell sample diagnostics on a cell sample or cell culture, which is further developed in that

• a) at least a section of the cell sample or cell culture is recorded videomicroscopically over a predefinable measurement period,
• b) at least one cell of the cell sample or cell culture in the detected video signal is individualized over at least a part of the measurement period,
• 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 temporal change is compared with at least one comparison value with respect to the detected property, and
• e) from the coincidence or deviation of the detected property of the individualized cell with or from the comparison value of the functional, 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 changes in the cell shape characteristic of cell death, specific changes in the movements or movement or behavior patterns of the cells can be detected for specific damage patterns. 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, even a single individualized cell may already be sufficient.

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 which are investigated in this way are, for example, connective tissue cells, liver cells, peritoneal cells, blood cells or nerve cells, the applicability of the method according to the invention not being 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 via gray value analyzes or via 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. Laser scanning microscopy offers, in particular, the advantageous possibility of evaluating three-dimensional images.

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 direct comparison of the effects of different drug concentrations and the evaluation of the reactions of the cell to it.

A property encompassed by the present invention is preferably a cell shape, a cell size, 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 area to cell sprout ratio, a movement pattern with a direction of movement and a movement speed or a number of cell divisions or cell merges. 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, in which 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 where the ratio of self-movement of the at least one individualized cell Cell Collective movements is detected. 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 of their interactions.

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 shape or size, the activity of the foothills and the number and extent of the foothills 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 can increase or decrease, damaged individualized cells can move against cell collective movements, etc. There are a variety of possible changes of these parameters, which are relevant for the respective cell type and for the cell type respective damage pattern are characteristic.

• a) Detektion der Zellwand und insbesondere Berechnung des Flächeninhalts der Zelle, sowie insbesondere Bestimmung der Position der Zelle im erfassten Ausschnitt,
• b) Detektion des Zellkerns und der Zellkerngrenze und insbesondere Berechnung des Flächeninhalts des Zellkerns, der Kern-Plasma-Relation und/oder der Orientierung des Zellkerns in der Zelle,
• c) Detektieren von Zellorganellen und/oder Einschlüssen und Bestimmung ihrer Position relativ zum Zellkern und der Zellwand.

The analysis according to the invention of the individualized cell or cells 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 video-microscopic 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 of the cell wall and in particular calculation of the surface area of the cell, and in particular determination of the position of the cell in the captured section,
• b) detection of the cell nucleus and the cell nucleus boundary and in particular calculation of the surface area of the cell nucleus, the nuclear-plasma relation and / or the orientation of the cell 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 change in the sequence of the individual images is examined. 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 cell nucleus as well as the cell organelles essential properties can be derived.

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 forms 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 characteristics corresponds to a known damage condition, or whether the change is within the normal range of vital 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, particularly preferably between 10 and 100 cells are examined. The investigation is carried out in particular on a random basis. 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.

The comparison value preferably originates from a cell of the same 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 method 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, 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 to be adapted to the typical speeds and change times of the cell cultures or cell samples investigated, 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 can preferably also be variable, whereby, for example, after the addition of an active substance, it is first recorded at a comparatively high image repetition rate in order to record the immediate reaction in detail, while the intervals between individual images increase as the activity of the examined cells decreases in the course of the examination period , This achieves a reduction in the amount of data to be processed without degrading the accuracy of the analysis.

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 movement 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.

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. Cell damage related to structural peculiarities are possible, specific reactions to Mechanical cell damage can be detected more sensitively than before.

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. As in the 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 which are present in cell carpets or cell lawns. Even in cell turf, there is a lively movement, as individual, mobile cells can penetrate the neighboring cells, d. H. 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 underlying the invention is also achieved by a computer program product with program code means, in the execution of which in a data processing system the steps of a method according to the invention described above are executed or can be carried out. 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 solved by a device for cell sample diagnostics on a cell sample or cell culture, which comprises a video microscopy device, which is designed to make a, 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 adapted to individualize from the video recording at least one cell to detect at least one property of the individualized cell and its temporal change and to compare it 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 deposited in the database by which substances or physical stimuli the functional, vitality and / or damage state was caused, to which the comparative data correspond.

Thus, a device is provided with the present invention, the evaluation of cell functions, cell vitality and damage states is better than currently used with conventional examination methods and devices possible. By observing cell movements and changes in shape over time, the method and apparatus of the invention avoids the shortcomings of the known methods and devices. 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 are always carried out 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 a schematic representation of a cell,

2 a schematic diagram of the temporal evolution of detected characteristics with conventional or the inventive method and

3 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 1 is 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 nucleus with a cell nucleus border 4 on, as well as organelles 5 and inclusions 6 , These components of the cell 1 are identifiable by video microscopy, in particular via gray value analyzes or via color shades. These constituents are also recognizable in light, phase contrast fluorescence microscopy or laser scanning microscopy and can be used for the analysis according to the invention. Methods for automatically detecting these cell boundaries and cell constituents in the frame are known.

In 1 a first step of an exemplary further analysis has already been presented. They are connecting lines 7 . 8th between the cell nucleus border 4 and the cell wall 2 shown in points 7 ' . 7 '' respectively. 8th' . 8th'' the cell wall 2 or the nucleus 3 touch. These connecting lines 7 . 8th represent such connecting lines, where from the family of possible connection lines a local maximum or a local minimum of the length of the connecting lines exists. Here are in 1 those connecting lines drawn with a maximum local length or drawn solid, while the connecting lines are shown in dashed lines with locally minimum length. 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. So the lines can be from the center of the nucleus 3 They may come from the irregular nucleus border 4 go out, from a circle around the center of the cell nucleus 3 or it gets off the cell wall 2 on the cell nucleus border 4 calculated back and optimized with suitable algorithms.

In the 1 already drawn conclusions on the number and extent of Zellausläufern 9 to.

An alternative method to that shown is, for example, in the Fourier analysis of the cell wall 2 and in particular the cell nucleus border 4 , Other suitable geometric analysis methods are applicable for this purpose.

In order to limit the effort of the necessary analysis, it may be provided that only a number of the most significant spurs 9 is determined in the manner described and is tracked across 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 changed fastest, ie in which areas spurs form or retreat very quickly.

In 1 can also be analyzed where the organelles 5 and inclusions 6 where they are in relation to the nucleus 3 and, based on the successive frames, how they move.

An appropriate analysis can be used for a variety of individualized cells 1 which may also be of different cell types. Typically, for example, a section of interest includes several hundred cells that are either all examined or sampled, which are then 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.

In 2 the time course of a conventional examination size and three indices determined by the method according to the invention over a period of 260 minutes is shown. 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 only buffer, usually have a pH that does not correspond to the pH adjusted by human body cells develop 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 that is suitable for the three properties detected and determined according to the invention 21 to 23 is characteristic. These indices correspond to arbitrary units.

At the with 20 The curve referred to 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 rhombus 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 there is only a slight further increase in LDH 20 is detectable. This is explained by the fact that the majority of cell damage has already occurred after about three hours.

The with the reference numerals 21 . 22 and 23 provided index profiles are calculated from each of several individual sizes. The with the reference number 21 The indicated line refers to changes in the cell shape and total cell area, ie, the area, perimeter, and foothold of the cells.

The one with the reference number 22 provided index is calculated from several properties that affect movements of the cell as a whole, namely changes in the location of cells, their speed and the directionality and direction of their movements.

The one with the reference number 23 characterized index is calculated from movements of the cell in its place, namely changes in the cell surface, the dynamics of the formation of foothills and the movement of cell organelles.

It is clear that already at the beginning of the measurement the index 23 on cell dynamics dynamics of the cell, that is, cell surface changes, the dynamics of foothill formation, and the movement of cell organelles decrease 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 one with the reference number 22 indicated index, 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 ½ hours the index falls 22 only a little bit off. However, this index is moving 22 after 2 ½ hours 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 number 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.

Out 2 Thus, it can be seen that the indexes 21 . 22 . 23 from the method according to the invention a change of properties of the cells in the toxic dialysis solution can be recognized much earlier than with the known method of measuring the lactate dehydrogenase values in the solution. It is also much more accurate than previously possible to see what kind of changes occur in the corresponding individualized and examined cells.

In 3 is an example in a schematic representation of an embodiment of a device according to the invention 40 shown. This device 40 for cell sample diagnostics includes a cell sample container 41 with a cell sample or cell culture, in an incubator 42 together with a video microscope 43 is placed on the cell sample container 41 is directed. The incubator 42 is via a control line 45 and a signal and control line 46 with a data processing system 44 connected. The data processing system 44 includes an evaluation device 47 passing through the signal and control line 46 as well as signaling and control paths 51 . 52 Signals from the video microscope 43 receives and evaluates. For this purpose, the evaluation device attacks 47 to a database 48 back, characterized by a double-headed arrow, which contains or comprises comparative data on recorded properties of one or more investigated cell types.

The data processing system 44 also includes a control device 49 with which the incubator 42 and the video microscope can be controlled. For example, the environmental conditions of the incubator 42 through the control device 49 changeable as well as the recording by the video microscope 43 controllable. The evaluation device 47 and the control device 49 can also be in contact with each other, so that the control device 49 for example, the evaluation device 47 can communicate that the environmental conditions in the incubator 42 be changed or that the refresh rate 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 control device 50 outside the data processing system 44 on which analyzes can be performed or on which video data, intermediate analysis data, final analysis data, etc. can be displayed.

It is also shown that the evaluation device 47 Data or control commands to the database 48 can send. This allows the database 48 it can be extended by recognized comparison data or it can be followed by a control command, with which certain data from the database 48 be 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 mentioned features, including the drawings alone to be taken as well as individual features that are disclosed in combination with 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 NUMBERS

1

cell

2

cell wall

3

nucleus

4

Nucleus border

5

organelle

6

inclusion

7

Connection line with locally maximum length

7 '

Point on the cell wall

7 ''

Point on the nucleus

8th

Connecting line with locally minimum length

8th'

Point on the cell wall

8th''

Point on the nucleus

9

cell processes

20

LDH concentration

21

Index 1

22

Index 2

23

Index 3

40

Device for cell sample diagnostics

41

Cell sample container

42

incubator

43

video microscope

44

Data processing system

45

control line

46

Signal and control line

47

evaluation

48

Database

49

control device

50

Display and operating device

51-53

Signal and control paths

54

Data and control line

Claims (20)

A method for cell sample diagnostics on a cell sample or cell culture, characterized in that a) at least a section of the cell sample or cell culture is recorded videomicroscopically over a predeterminable measurement period, b) at least one cell ( 1 ) the cell sample or cell culture in the detected video signal is individualized over at least a part of the measurement period, c) at least one property ( 21 - 23 ) of the at least one individualized cell ( 1 ) is recorded with its temporal change, d) the property recorded with its temporal change ( 21 - 23 ) of the individualized cell ( 1 ) having at least one comparison value with respect to the detected property ( 21 - 23 ) and e) from the agreement or deviation of the recorded property ( 21 - 23 ) of the individualized cell ( 1 ) with or from the comparison value of the functional, vitality and / or damage state of the cell and its temporal change is determined. Method according to claim 1, characterized in that a detected property ( 21 - 23 ) 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 nucleus to plasma size ratio, a cell surface area to cell spur ratio, a motion pattern with a direction of movement and one Movement speed or a number of cell divisions or cell merging is. Method according to claim 1 or 2, characterized in that a detected property ( 21 - 23 ) is a movement pattern with location changes, 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 the at least one individualized cell ( 1 ) is detected to cell collective movements. 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, wherein for each individual recording of the video signal one or more of the following steps are performed cumulatively: a) detection of the cell wall ( 2 ) and in particular calculation of the area of the cell ( 1 ), and in particular the position of the cell ( 1 ) in the captured section, b) detection of the nucleus ( 3 ) and the nuclear boundary ( 4 ) and in particular calculation of the area of the cell nucleus ( 3 ), the nuclear-plasma relation and / or the orientation of the cell nucleus ( 3 ) in the cell ( 1 ), c) detecting cell organelles ( 5 ) and / or inclusions ( 6 ) and determining their position relative to the nucleus ( 3 ) and the cell wall ( 2 ). Method according to one of claims 1 to 4, characterized in that a detected property ( 21 - 23 ) is a time, a period of time, a frequency or a frequency in or with which a detected property ( 21 - 23 ) changes. Method according to one of claims 1 to 5, characterized in that the comparison values snapshots of recorded properties ( 21 - 23 ) or temporal developments of recorded properties ( 21 - 23 ) due to known functional, vitality and / or damage states and / or their changes. Method according to one of claims 1 to 6, characterized in that the detection of properties ( 21 - 23 ) and their comparison with comparative values for a plurality of individualized cells, in particular a cell structure ( 1 ), wherein in particular between 5 and 500 cells, in particular between 10 and 100 cells are examined. Method according to 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. Method according to 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. Method according to 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. Method according to 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 captured region earlier in the measurement period. Method according to 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 video microscopy and the at least one detected property ( 21 - 23 ) also for at least one individualized cell ( 1 ) of the control and used as comparison value. Method according to one of claims 1 to 12, characterized in that the method is carried out under standard culture conditions, in particular environmental factors, in particular temperature, carbon dioxide content or light are changed. Method according to one of claims 1 to 13, characterized in that the video-microscopic 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. Computer program with program code means, in the execution of which on a data processing system a method according to one of claims 1 to 14 is executed. Data carrier with a computer program stored or written thereon according to claim 15. Contraption ( 40 ) for cell sample diagnostics on a cell sample or cell culture comprising an incubator ( 42 ) for a cell sample or cell culture, a video microscopy device ( 43 ) adapted to receive, in particular digital, video recording of the incubated cell sample or cell culture with the resolution of the video microscopy device ( 43 ) is sufficient to separate cells ( 1 ) of the cell sample or cell culture, characterized in that the device ( 40 ) an evaluation device ( 47 ), which is formed, from the video recording at least one cell ( 1 ), at least one property ( 21 - 23 ) of the individualized cell ( 1 ) as well as their temporal change and to compare it with a comparative value. Contraption ( 40 ) according to claim 17, characterized in that it is arranged to carry out a method according to one of claims 1 to 14. Contraption ( 40 ) according to claim 17 or 18, characterized in that the evaluation device ( 47 ) Access to a database ( 48 ) with comparative values, in the comparative 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. Contraption ( 40 ) according to one of claims 17 to 19, characterized in that in the database ( 48 ) is additionally deposited by which substances or physical stimuli the functional, vitality and / or damage state was caused, to which the comparative data correspond.

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