HUT61810A - Apparatus, process and laboratory plates for laboratory tests - Google Patents

Abstract

Laboratory apparatus (110) comprises an incubator (112) formed with a housing (114) substantially enclosing an interior space. The incubator maintains in the interior space prescribed conditions appropriate to survival and reproduction of at least one predetermined collection of living cells. A liquid handling station (134) is provided, and a robot (136) transfers to the liquid handling station (134) cells from the incubator (112) and chemicals that when mixed with the cells to form a mixture that is then stored under the prescribed conditions produce a detectable change in the cells. The liquid handling station (134) includes nozzles for mixing the chemicals with the cells to form a mixture, and the robot transfers the mixture to the incubator (112) for incubation therein. The robot transfers the cells after their incubation to a luminometer (162) that produces a detection output signal in response to the detectable change in the cells.

Description

FIELD OF THE INVENTION The present invention relates to apparatus, a method and a laboratory plate for performing laboratory test tests. The proposed apparatus is provided with an incubator which provides the living and reproductive conditions of at least one set of cells of a predetermined nature. In carrying out the method of the present invention, the living and growth conditions of a cell sample containing at least one set of living cells are generated in an incubator and the cell sample is assayed under the conditions of a given assay. The laboratory plate useful in the apparatus and useful in carrying out the proposed process is provided with recesses of tissue culture compatible material to receive the cell cultures to be tested and adapted to transmit detectable signals from the cell cultures.

The present invention provides novel and highly efficient automated laboratory tests that can reliably, at high speed and at low cost, analyze the effects of a large number of chemicals on a variety of materials. Among the various substances, first and foremost, living cells should be mentioned. The invention further proposes an aid that can facilitate the necessary data collection tasks in conjunction with a tissue culture compatible structure and in particular an automated apparatus implementing the method of the invention.

The effects of different chemicals on different substances need to be determined in many ways, and it is especially important to determine how chemicals affect living cells. In general, it is necessary to determine what effect a given chemical has on, for example, the growth of a protein and reproduction and production of the samples by classifying the cells in a particular strain and sorting them based on the results obtained. This can, of course, be accomplished by manual means, whereby cell cultures are prepared, chemical solutions prepared in various dilutions are added and the results observed.

In practice, manual solutions are not as monotonous and labor-intensive as they are slow, in particular the almost infinite number of possible combinations of cell cultures from different strains and the chemicals to be tested. In addition, the procedure requires multiple treatments of cell cultures and chemicals, since cells, which should be kept for as long as possible in order to maximize the reliability of the information obtained from the assays, should be incubated under strictly controlled conditions. Many of these should be diluted just prior to the start of the test and made multiple dilutions to be tested.

Many attempts have been made in the technical field to develop highly automated laboratory apparatus which enables the testing, treatment and processing of the results of various materials. For example, a robot system with interchangeable transport arms is described in U.S. Pat. No. 4,488,241. U.S. Pat. Zymark Corporation (Hopkinton, Massachussetts) • «

Ih.

Maz Under the brand name II, it markets laboratory robots that fulfill many of the features of this patent. The Swiss TECAN AG (Landhaus, Holgass), under the trade mark RSP 5052, markets programmable equipment for the preparation of chemicals of various dilutions and thus for testing. Other sophisticated equipment has become known, the most important of which are on the market:

1) ML 1000 light meter equipment from Dynatech (Chantilly, Virginia);

2) EL-403 plate washer equipment from Bio-Tek (Winooski, Vermont);

3) AE 240 scale machine from Mettler, Hightstown, New Jersey;

4) Dynatech four-position microplate laboratory shaker; and

5) a Pro / Pette fibronectin coating device and disc carrier manufactured by Perkin Elmer Cetus (Norwalk, Connecticut).

The patent literature describes several solutions for laboratory test equipment. Thus, U.S. Pat. No. 4,812,392 discloses U.S. Pat. US Patent No. 4,123,125 describes a cell culture incubator equipped with a photoelectric device for sensing. A pipette provides the nutrients needed to maintain cell culture. To maintain or control the temperature of the incubator, see U.S. Patent No. 4,495,892. U.S. Pat. U.S. Pat. No. 4,336,329; US Patent Specification for Moisture Control and Control ··

solution.

Closest to the subject matter of the present invention is US-A 4,812,392. U.S. Pat. No. 5,123,011, which discloses an arrangement for assaying growth of individual cell cultures in individual wells. The aim is to set the optimum growth rate on individual samples. The disadvantage of this is that the tests take a long time, analyzing a larger number of samples with this layout is extremely time consuming and very labor intensive.

Known devices can be connected to a known computer by carrying out obvious measures, which are capable of processing signals from the light meter or other sensor unit and thereby generate a corresponding measurement result.

The above-described and commercially available devices may also be utilized in addition to the present invention. The same can be said for other similar equipment not listed and solutions to be developed in the future if they are capable of performing the functions described below.

The sophisticated laboratory equipment described and otherwise known, whether commercially available or not, has significantly contributed to improving the efficiency and repeatability of various laboratory test assays. Whatever the connection between these devices is, the efficiency is not improved because no automated connection can be made between the various devices. The elements essentially perform their original functions, and the laboratory staff must provide functional links between the equipment and the equipment must be supervised.

Laboratory disks developed for laboratory tests are also well known, and many types are used by laboratories. Most laboratory plates are typically formed with a large number of wells in which solutions, suspensions, solutions, suspensions, their components, and possible reaction products can be tested individually with different substances, including cell cultures. A standard laboratory plate is made of a plastic material that is hydrophilic in structure, can contain living cells and survive, and normally has eight rows and twelve columns with a total of ninety-six wells.

The basic variants of laboratory disks are tissue culture compatible, although they effectively separate the contents of the recesses, but do not provide sealing of signal pathways and thus do not always provide reliable, fast data collection. For example, the beam of light used to illuminate an indentation may pass through the entire laboratory plate, enter an additional indentation, and thus the metering device associated with this second indentation may already detect a false measurement result in which the first indentation measurement is also present. This can, of course, be a source of error, in which case the contents of the two recesses cannot be reliably identified.

In view of the above, there is a need for a laboratory test · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · possessiar were simple

- 7 equipment, procedures and laboratory plates for conducting tests.

It is an object of the present invention to better meet this need.

Our task is to develop a laboratory test equipment that allows reliable analysis of a large number of samples at low cost and the ability to contact a large number of samples with different dilutions of a variety of chemicals. We have also set ourselves the task of creating a laboratory disk that is particularly well-suited for working with an automated data acquisition laboratory tester, providing the basis for reliable and fast data collection.

As a further task, we set out the following.

Laboratory equipment shall be provided which shall consist of automated components and the connection or transition between two components shall be automated.

The laboratory equipment to be created must be able to operate unattended for a long period of time, meaning that it can free laboratory staff from meticulous manual work and allow them to work creatively and with high levels of skill.

The laboratory equipment to be developed must be capable of performing the test procedures with high reliability and reproducibility; this applies to the preparation of test materials, the conduct of test tests, and the use of data.

- 8 to collect.

The laboratory plate to be created should be tissue culture compatible and allow for the separation of a large number of cell samples and the effects of the applied samples should reflect the results of one cell sample only, ie the test signal obtained in one sample should not be affected.

It is also a requirement of the laboratory plate to be created that it contains, apart from tissue culture compatibility, recesses spaced apart from one another, ensuring that the conditions of the signal used for testing between recesses are present, so that each recess can be generated separately. information from the recess should not distort.

As a solution to this problem, we have developed equipment, procedures, and laboratory disks that provide the optimum conditions for efficient automated testing of laboratory tests.

The apparatus of the present invention is provided with an incubator for providing living and reproductive conditions to at least one set of cells of a predetermined type of living cells, while the incubator is provided with a housing defining housing, wherein the internal space within the incubator accommodates the cell set. they form compartments and the compartments contain laboratory plates with recesses for receiving the living cells arranged in chemicals in containers for the recesses a liquid treatment station with nozzles for dispensing, a laboratory means for receiving sensing means for receiving and determining changes in living cells, and a transport unit fitted to the incubator, wherein the transport unit is disks are formed by first and second robots for transferring discs from the incubator to the incubator, from the liquid treatment station and from the liquid treatment station, and from the chemical disks, preferably chemicals in the containers, and from the disks to the sensing device and the sensing device.

An embodiment of the apparatus of the present invention in which the incubator has a temperature sensing device and a first regulator and / or humidity sensing device in the compartments and / or a humidity sensing device in the compartments and about 100% air in the compartments is particularly suitable for meeting the conditions normally required for an incubator. a second regulator for adjusting and holding the relative humidity.

A particularly advantageous embodiment of the apparatus of the present invention is that the first regulator is provided with an infrared lamp which illuminates at least a portion of the internal space of the incubator, which is mounted in a temperature control circuit, while the second regulator is provided with and an arrangement comprising an adjusting and holding unit for controlling the relative humidity of the compartments by extracting wet air from the compartments, wherein 4 outlets and inlets are provided. they are coaxially arranged with each other, and a sensor device for measuring the relative humidity of the air is connected to the inlet (s) and to the outlet.

The automation of laboratory testing is facilitated by the preferred embodiment of the apparatus according to the invention, wherein the transport unit is provided with a work space which overlaps at least partially the incubator compartments and the incubator is provided with a rotary support table leading to the compartments.

The multi-chemical test assay is facilitated by a preferred embodiment of the apparatus according to the invention, wherein the liquid treatment station is adapted to prepare various dilutions of the chemicals and to dispense one of the various dilutions into one of the wells of the laboratory plate. For this purpose, the proposed apparatus is preferably provided with nozzles forming a first nozzle system for delivering chemicals and solvents to the recesses of the master plate (170) and a second nozzle system for delivering a solution of chemicals and solvents to the recesses of the laboratory plate.

The determination of the measurement results obtained by the test assays is facilitated by the embodiment of the apparatus according to the invention, wherein the detecting means for detecting changes in living cells is adapted to detect emitted light and comprises at least one photometric unit with a photomultiplier.

Testing of a large number of samples is facilitated by the purpose of the apparatus according to the invention.

- 11 ·· «· • · **« V · »

9, wherein the sensing means is a unit for moving a laboratory plate and a sensing means, in particular a photomultiplier, relative to one another. and equipped with a detector for detecting depressions in the laboratory plate, in particular a photomultiplier.

For the operation of the apparatus according to the invention, it is preferable for the first controller for maintaining the temperature to comprise an electrically heated plate mounted on the surface of the incubator.

If the effects of the test chemicals and other materials are to be recorded with the aid of an excipient, an embodiment of the apparatus of the present invention comprising an additional dosing unit for delivering additional chemicals to the laboratory plate recesses and a delivery unit for the laboratory plates to the auxiliary dosing unit and is designed for transporting the auxiliary feed unit.

The highly preferred embodiment of the apparatus according to the invention, which is provided with a washing machine for removing chemicals from the recesses, and whereby the transport unit is adapted to transport the laboratory discs to and from the washing equipment, makes it possible to meet the requirements of numerous test tests.

It is also an object of the present invention to provide a novel method for solving the life and reproduction conditions of a cell sample containing at least one set of living cells in an incubator and requiring a specific assay.

In accordance with the present invention, it is essential that a plurality of cell samples are placed in the incubator, treated with a plurality of chemical samples in each sample, and that conditions are created for the effect of the chemicals, the treated cell samples are incubated in the incubator. the incubation conditions necessary for the development of changes in them are detected, and any changes in the cell samples are detected and the average test time is up to 2.5 s per cell sample.

If the applied chemicals interfere with the sensing or their presence is undesirable after a while, the method of the present invention is preferably performed by removing the pre-added chemicals from the cell samples and adding additional chemicals to the cell samples before detecting the changes. we create conditions that allow its effect to develop.

A laboratory plate provided with tissue culture compatible recesses for receiving cell cultures to be tested and transmitting perceptible signals from cell cultures which are useful in transmitting the cell cultures of the present invention is useful in operating both the apparatus of the present invention and the process outlined above. and made of non-propagation material on the lower level of the recesses, allowing individual examination of the recesses.

Most of the test assays meet the requirements of the laboratory plate according to the invention for its particularly favorable

- 13 vectors, which are suitable for optical examination and consist of opaque material in the directions between the recesses.

In general, the requirements of the test assays are satisfied by the preferred embodiment of the laboratory disc according to the invention, which is suitably designed for optical examination and consists of a light opaque material.

To facilitate the use of the apparatus described above, the laboratory plate of the present invention is preferably formed with recesses arranged in a rectangular grid, preferably with ninety-six recesses arranged in the usual manner, i.e. eight rows and twelve columns.

For performing more complex assays, it is preferred that the laboratory plate according to the invention is formed with recesses having an open surface at the top for washing the cell culture and a filter with apertures at the bottom for retaining the cell culture elements in the event of washing.

The invention will now be exemplified by way of example only. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS and embodiments thereof, with reference to the accompanying drawing. In the drawing it is

Figure 1 is a schematic top view of a laboratory test apparatus according to the invention showing the most important components,

Figure 2 is a side view of the fluid handling station of the apparatus of Figure 1, a

Figure 3 illustrates the most important steps of the process of the invention

• · · · Outline flowchart, a

Figure 4 is a top plan view of a master plate for operating the apparatus of Figure 1 and an ampoule drawer arranged in the master plate for receiving thirty-two samples;

Figure 5 is a plan view of six laboratory disks in a plate holder designed to operate the apparatus of Figure 1,

Figure 6 is a plan view showing details of the design of the master plate of Figure 4, a

Figure 7 is a top view of one of the six laboratory plates containing cell samples shown in Figure 5,

Figure 8 is a flowchart of a program for determining the movement of one arm of the fluid treatment station of Figure 2,

Figure 9 is a flowchart of one of the subroutines of the program represented by the flowchart of Figure 8, a

Figure 10 is a flow chart of a program for determining the movement of another arm of the fluid treatment station of Figure 2, while

Figure 11 is a flowchart of one of the subroutines of the program represented by the flowchart of Figure 10.

In accordance with the present invention, a laboratory apparatus 110 is provided for performing laboratory test tests (Figure 1). This, when mounted on a suitable support such as a table 111, constitutes the apparatus of the invention. The laboratory apparatus 110 is provided with an incubator 112 in which

a proper house is constructed and this defines the interior space. The interior is divided as follows, but it is important to provide living and reproducing conditions for the living cells of at least one cell culture. The housing is formed, for example, as a compartment 114, which is divided into compartments 116, 118, 120 and 122, for example four. The compartments 116, 118, 120 and 122 respectively define interior spaces 124, 126, 128 and 130. Each of the compartments 116, 118, 120, 122 has shelves or brackets for holding ten brackets and housing a set of six laboratory plates in each bracket. In the compartments 116, 118, 120 and 122, the support members are located one above the other. Within the incubator 112, each of the compartments 116, 118, 120, and 122 provides the living and reproduction conditions required by the invention. This means, for example, a temperature of about 37 ° C and a relative humidity of almost 100%, and the carbon dioxide content of the air is at a certain level, generally 5 vol. The incubator 112 also has a rotary support table 132 located below the socket 114 so that each of the compartments 116, 118, 120 and 122 can be rotated in a desired position.

An important part of the laboratory apparatus 110 is a treatment unit, such as a liquid treatment station 134. It is also important that it has 136 transport units, which are preferably provided with 138 first and 140 second robots. The transport unit 136, and in particular the second robot 140, is used for transporting specimens, in particular cells, to be tested for exposure to various chemicals, i.e., from the 112 incubator to the test specimen 4 · · · · · · · · · · · · · · · · · · ♦ ···· * · ··

- Takes 16 tats to 134 fluid handling stations. The transport unit 136, and in particular the first robot 138, serves to supply the liquid treatment station 134 with the necessary chemicals. The chemicals are mixed with the cell samples and the resulting samples are maintained in incubator 112 for a sufficient period of time, allowing various changes to occur. The liquid treatment station 134 is provided with a first arm 164 and a second arm 166 connected to a laboratory shaker 184, the first arm 164 having nozzles 164A and 164B, and the second arm 166 having nozzles 174, 176, 178 and 180. Underneath the first arm 164, 172 ampoule drawers and 170 master plates can be placed, while underneath the second arm 166, six laboratory plates containing test substances can be placed. Liquid treatment station 134 includes washing devices 213 and 237.

An important part of the apparatus of the present invention is a component capable of tracking changes in cells due to chemicals and generating an output signal that reflects the changes. For example, if photons are formed as a result of changes in cells, the unit following the changes is preferably formed by 162 photometers (Figure 1). After incubation of the cells, the cell plates containing the cell cultures are maintained in the 112 incubator. This usually takes about 6 hours. After this, the transport unit 136, and in particular the second robot 140, moves the cells to the photometry unit 162 together with the laboratory plate. For example, the photometric unit 162 includes a photomultiplier, and a component that allows the photomultiplier to perform in the laboratory.

- Scanning through recesses formed in 17 discs and optically scanning their contents. The laboratory plate is specifically designed as described below, so that the signals from the indentations are perceptible for each indentation, and are not disturbed by signals from other indentations or other effects.

A preferred embodiment of the liquid treatment station 134 is shown in FIG. In this, 142 mixers serve to mix chemicals and cell cultures to form a mixture to be tested in more detail below. This is transported by the second robot 140 to the incubator 112, where incubation, as mentioned, occurs. The incubator 112 should maintain the desired flow rate of warm, moist air, preferably maintaining the volume ratio of carbon dioxide. The control is provided by a feedback loop 146, which travels to the interior spaces 124, 126, 128, 130, to supply high humidity air through the inlet 148 and to remove the moist air from the internal spaces through the outlet 150. The inlets 148 and the outlet (s) 150 are coaxially arranged, their common axis being the axis of rotation of the rotary table 132. The turntable 132 is generally made of metal or other material exhibiting good thermal conductivity, illuminated from below by an infrared lamp 144, or connected to another source, such as an electrical source, to maintain the desired temperature within the incubator 112, e.g. 37 ° C.

Transport unit 136 is provided with working spaces 152 and 154. Work space 152 provides space for the first robot 138, • * • «

J

18 ~ while the second robot 140 fits into the working space 154. As mentioned above, the socket 114 of the incubator 112 has compartments 116, 118, 120 and 122, and the rotation of the rotary table 132 allows the socket 114 to be rotated so that one of the compartments 116, 118, 120 and 122 is always in the working space 154. 140 second robot workspace. Thus, the second robot 140 is capable of removing or repositioning the desired objects from the respective compartments 116, 118, 120, 122, the timing and execution of which are described below.

Figure 3 summarizes the most important steps of operation of the apparatus according to the invention and shown in Figure 1. Before the measurements, initial steps A and B are performed first. In step A, the microtitering laboratory plates or plates with 96 wells, the structure of which is described below, are subjected to pre-treatment and sterilization when tissue culture is received. Pre-treatment and sterilization are not usually carried out in the testing laboratory and are provided by the supplier of the laboratory plates or sheets. The invention is an example. In the implementation of the project we used the sheets provided by Dynatech. The laboratory plates are then coated with a fibronectin coating in step C, for example a Pro / Pette plate coating from Cetus. Finally, in step D, the test substance is applied to each of the wells 96 in a microtiter with a culture medium. For example, Cetus's Pro / Pette sheet coating equipment can be used. During the preparation process, we usually fill up one drawer with six such lab disks, that is to say, · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Six laboratory plates should be prepared and placed manually in the 112 incubator. The incubator 112 is filled at intervals, for example at sixteen hour intervals, and can thus be adapted to examine a large number of samples. Typically, approximately 20,000 cells are placed in a well, providing a good basis for generating signals of the desired intensity, with ninety-six wells per lab plate, six drawers per well, and ten drawers per compartment, and the 112 incubator itself has four Contains 118, 120, 122 compartments. With the proposed apparatus, samples can be handled so that readings can be made at average intervals of about 2.5 seconds. This average interval also takes into account waiting and development times, so that the unit can run unattended on a single charge and provides the ability to read about twenty-three thousand wells after charging.

Independently of steps A, C and D, step B requires the preparation, storage, recording and encoding of the chemicals. This operation may otherwise be assisted by an automated barcode device. The execution of steps A, B, C, D represents a non-automated phase of operation of the apparatus according to the invention.

After the above steps, the measurements are performed in F, G, H and I automated basic steps. These can be summarized as follows:

In step F, the liquid treatment station 134 delivers chemicals of the same composition to each of the laboratory plates in the drawer, that is, six laboratory plates. All Labs * ·

- 20 ratium plates contain cells of different strains whose characteristics are essentially known. Cells are transferred to each laboratory plate by filling ninety-six wells. Thus, six cell types can be treated and observed simultaneously. Prior to this, in step E, the first robot 138 provides the fluid handling station 134 with a set of thirty-two ampoules and an empty master plate containing ninety-six recesses and supported in a master drawer. The ampoules contain a predetermined amount of the chemical and their solutions in DMSO or Molecusol. These various dilutions, each of which are three dilutions, are prepared by the first arms 164 of the liquid treatment station 134 (Fig. 2) from each chemical using the master plate 170 as described below. The second arm 166 then delivers the chemical solutions from the master plate 170 also to the six laboratory plates, which are properly arranged, in step F. The second robot 140 is then responsible for delivering the six laboratory plate holding means to the incubator 112, which also belongs to step F. After incubation in step G, which is typically carried out for six hours and is carried out in incubator 112 at 37 ° C in a water atmosphere saturated with steam of about 5% carbon dioxide, the laboratory plates are removed from incubator 112 by step 140, if necessary, the chemical is washed and the developing chemical is added thereto, and in step I, they reach the photometric unit 162, as shown in Figure 1, whereby a bioluminescent signal is measured and then, with the second robot 140, the laboratory plates from the incubator 112

- 21 removed.

In the steps listed above, the following operations are performed. First, in step E, after weighing, for example, on a Mettler scale, the chemicals are transferred to ampoules and diluted therein with solutions containing, for example, dimethylsulfoxide (DMSO) or the Molecusol brand containing inclusion molecules. The ampoules containing the determined amount of chemicals dissolved in the required amount are then delivered to the liquid treatment station 134 by the first robot 138.

In the next step, the second robot 140 pulls the holding device containing the six laboratory plates from the incubator 112 (Fig. 1). Each laboratory plate (Figure 5) contains ninety-six wells in eight rows and twelve columns. The second robot 140 places the holding device on a tray holder 156 (Fig. 1), removes the covers from each laboratory plate and delivers the open-labeled laboratory plates to the liquid handling station 134 on the holding device. Meanwhile, the first robot 138 shipped the ampoule tray and master plate at the same time as mentioned above and added the chemicals to the cell samples on the laboratory plates at the liquid treatment station 134. The second robot 140 then moves the laboratory plates to the tray holder 156, repositiones the covers onto the laboratory plates (which have since been stored in the lid holder 158) and delivers the tray with the laboratory plates to the incubator 112.

As shown in Figure 3, step G is followed by 9 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ♦

The essence of the invention is that the cells in the laboratory plates are provided with the desired living and growth conditions in the incubator 112, for example, for six hours in a high humidity atmosphere of 37 ° C and 5% by volume of carbon dioxide.

In step H, the second robot 140 then moves the six-tray tray holder from the incubator to the tray holder 156, removes the covers of the lab disks, removes the removed covers via the slide 158, and delivers the lab disks to the tray washer 161. The plate washer 161 washes the chemicals from the laboratory plate and the second robot 140 takes the laboratory plates to the reagent dispenser 165, where one or more chemicals are introduced into the wells to initiate changes in the cell samples to detect changes in the first chemical addition. it becomes easier. For example, the second chemical may be a compound or composition that is capable of disrupting cell membranes and thereby providing access to cellular contents. In this case, the compound or composition is selected to elicit a reaction beyond the rupture of the cell membrane that produces a signal which is readily detectable by the available means.

The second robot 140 then in step I delivers the laboratory disks to a test apparatus selected according to the signal to be generated. For example, if a change in light is to be expected as a result of the changes, the second robot 140 is connected to a 162 metering units that are connected to the second robot.

- Receives 23 laboratory discs. To this, a rotary table 163 is assigned, on which the second robot 140 places a laboratory plate and is thus suitably spatially adjustable and can be examined from different directions with the selected apparatus. For example, the photometric unit 162 measures a bioluminescent signal that is transmitted to a computer and processed. The contents of the laboratory plate are examined with the photometric unit 162, after each indentation has been scanned, the second robot 140 removes the laboratory plate from the rotary table 163 and delivers it to the slide 160.

Steps F, G, H and I described above are described in more detail with reference to Figures 4, 5, 6 and 7. In Figure 4.

Fig. 6A shows a master plate holder 168 having a master plate 170 with ninety-six recesses and a vial holder 172 provided with thirty-two vials. These units can also be found in Figure 2. In the vial container 172, the thirty-two vials are arranged in the appropriate order, denoted by numbers 1, 2, 3, 4, 32 in FIG. The ampoules contain the appropriate chemicals.

At the fluid treatment station 134, both the first arms 164 and the second arms 166 can be independently moved in three directions (X, Y, Z) of space. The first arm 164 can access each of the vials in the vial drawer 172 and each position of the master plate with ninety-six recesses. The nozzles 164A and 164B formed in the first arm 164 are located relatively close to each other and are thus operable to operate simultaneously with a single recess or vial, so that they can be positioned over position 24 of the master plate 170 or vial holder 172. The nozzles 164A and 164B can be used independently or simultaneously to aspirate or dispense material. During operation, each of the nozzles 164A and 164B is guided to the appropriate location of the container 172 and the sample is aspirated with one of the nozzles, for example 164A. Make this space 1 position of the 172 ampoule drawers.

In the next step, the first lever 164 is moved so that the nozzles 164A and 164B are positioned above the selected position of the master plate 170, optionally with a recess receiving a sample (Fig. 6). Here, the first solution of sample 1 is formed. The first sample drawn from the ampoule is introduced into the selected recess using nozzle 164A, while the nozzle 164B supplies the same solvent, resulting in a first solution of the first sample. The first lever 164 then moves the nozzles 164A and 164B over their designated positions to form the second solution of sample 1, whereupon the nozzle 164B supplies the required amount of solvent. In the next step, the first lever 164 is replaced with the third solution of sample 1, and the nozzle 164B again supplies the solvent.

4, the sample 1 in the vial 1 at position 1 of the vial holder 172 is moved by the first arm 164 to the lower right position of the master plate 170 of Fig. 6, indicated by a black field. The second and third solutions of sample 1 in the same figure are represented by an increasingly lighter gray color.

Similarly, the sample 2 at position 2 of the vial container 172 is transferred to the indentations in the arrangement shown in Figure 6, whereby the first solution of sample 2 is prepared to the left of the first solution of sample 1. Similarly, a second solution of sample 2 is prepared with the second solution and a third solution of sample 2 with the third solution. In the same way, the sample is transferred from the vial holder 172 from position 3 to the further position of the master plate 170 where the first solution is to the left of the first solution of sample 2. Similarly, the contents of the vial drawer 172 from position 4 are positioned to the left of the position of the master plate 170 containing the first solution of sample 3. As in Sample 1, the second solution contains the second solution and the third solution the third solution.

After filling four depressions on the right side of the bottom row of the master plate 170 in Figure 6 with four different patterns on the right side of each of the two further rows, the second dilution 166 of the fluid treatment station 134 is moved to the first position. the nozzles 174, 176, 178 and 180 are respectively above the first solution of sample 1, the first solution of sample 2, the first solution of sample 3 and the first solution of sample 4. The nozzles thoroughly mix the materials in the recesses by first sucking and then blowing them back, and this operation is repeated several times as desired, for example three times. The second arm 166 then delivers the same amounts of carefully homogenized first solutions of the different samples to the recesses receiving the second solution of the samples 1,2,3,4,4 through nozzles 174, 176, 178, 180. Subsequently, the second arm 166 moves at the fluid treatment station 134 over the laboratory plate of Figure 7, where it fills sixteen recesses from the first solution through the nozzles 174, 176, 178 and 180, • * ♦ • ·

26 namely the first solution of sample 1, the first solution of sample 2, the first solution of sample 3 and the first solution of sample 4. Each of the nozzles 174, 176, 178 and 180 thus draws in and out a different sample.

The laboratory plate shown in Figure 7 corresponds to one of the six laboratory plates shown in Figure 5, and the second arm 166 fills sixteen recesses in each of the six laboratory plates as described above.

The second lever 166 is then guided back over the master plate 170 such that the nozzles 174, 176, 178, 180 have recesses containing the second solution of sample 1, the second solution of sample 2, the second solution of sample 3, the second solution of sample 4. above. So far, these depressions contained only solvent. The nozzles 174, 176, 178, 180 are then fed the first solutions of samples 1, 2, 3, 4 into the recesses and, if necessary, homogenizing the mixtures by multiple suction and dispensing. This produces the second solution of the selected composition.

In the next step, the second arm 166 draws equal amounts of the second solutions, moves over the indentations of the laboratory plate of Figure 7, and then replenishes sixteen wells with the second solutions of the first four samples. Likewise, these second solutions are added to the appropriate wells of the six laboratory plates in the respective holding device. Thus, as shown in Figure 7, further wells of each of the six laboratory plates are filled with the second solutions of the samples 1,2,3,4.

Next, the second arm 166 in the mes27 of Fig. 6 · ··· · Returns above the field plate, feeds the second solutions of samples 1, 2, 3, 4 through the nozzles 174, 176, 178, 180 into the third solutions of samples 1, 2, 3, 4, and then dilutes the samples. These solutions are carefully homogenized by repeatedly aspirating and blowing, and then, as described above, added to the appropriate wells of the laboratory plate of Figure 7, sixteen wells. Repeat the same procedure for the other five laboratory plates.

The above procedure is repeated until the wells are filled in each of the laboratory plates as shown in Figure 7. The laboratory plate shown in Figure 7 contains eight samples at three different dilutions, and each sample dilution is four times present in each of the six laboratory plates. The amount of solvent is chosen relative to the volume of the sample, of course, considering the nutrient initially added, and therefore one solution differs from the other by a concentration factor of about ten, thereby demonstrating the effect of the selected chemicals on the test cells over a wide range. Concentrations are plotted multiple times on each laboratory plate to provide statistically significant data with a much greater degree of reliability than if one were to be inferred from a single dilution.

The above description essentially covers all important steps of the process of the invention, although for the sake of simplicity some obvious operations are not mentioned. so, for example, rinsing is not a separate step to prevent the co-presence of chemicals, nor is it specified shaking to improve homogeneity.

8, 9, 10 and 11 are flow charts for operating the first lever 164 and the second lever 166, i.e., the liquid treatment station 134, including the rinsing and shaking operations.

8, the operation of the first lever 164 in step 186 is enabled. Then, in step 188, the upload configuration is set up. In step 190, the presence of the vial holder 172 containing the thirty-two ampoules and the master plate 170 on the master plate holder 168 (Fig. 4) is required and verified. This is followed by step 192 when awaiting an enable signal for the transport unit 136 to be operational. The transport unit 136 generates this signal in the manner set by the manufacturer. After receiving the required standby signal, the wait signal generated in step 190 is canceled in step 194. A suitable subroutine is then performed in step 196 to process the thirty-two samples. The subroutine is described with reference to Figure 9. In the next step 198, the program waits for the second lever 166 to operate, details of which are shown in Figures 10 and 11. Then, in 200 steps, we check that the program is complete. If so, in step 202, operation is stopped. If not, from step 190, the process is restarted, steps 192, 194, 198, and 200, and subroutine 196 is repeated.

The structure of the subroutine to be performed in step 196 is shown in Figure 9.

• · · · · ····· "·· ·« · «♦ ·« · · · ·· "·· <" shows. This begins with step 204, whereby the first arm 164 processes the preselected one of the thirty-two patterns of the vial drawer 172, that is, transfers it to the master plate 170 as discussed above. Then, in step 206, a solvent is added to the sample to be tested and the mixture is homogenized. In the next step 208, the solvent is added to two further depressions as described above. If four samples have not been processed in this way, this is checked in step 210, and the program moves to step 212, while once the four samples have been processed, step 214 follows which enables the second lever 166 to operate. The second lever 166 is connected to the program at step 212. In this step, the program stops the second lever 166, and then ensures that the first lever 164 enters the nozzles 164A and 164B into the washer 213 where it controls their rinsing. In step 216, the program checks whether the preparation of the thirty-two samples selected for testing is complete. If so, the subroutine is completed, followed by step 218 of the base program of FIG. In contrast, in the preparation process, the subroutine jumps from step 216 to step 204 and repeats the steps listed.

Figure 10 shows a basic program for operating the second arm 166, also performed at the fluid treatment station 134. Operation is enabled in 220 steps. Subsequently, in step 222, the program provides the processing of the samples and their transfer to the laboratory plates. This is done by executing the subroutine shown in Figure 11. After executing the subroutine, in 224 steps, the program examines whether to enter it.

- 30 completed or not. If so, it stops operation in step 226, and returns the subroutine to return to step 222 if no signal to finish the program is received.

Figure 11 shows details of step 222 of Figure 10. The subroutine shown here begins with step 228 whereby the nozzles 174, 176, 178, 180 of the second arm 166 homogenize the material of the four samples on the master plate 170 and absorb a given amount of the sample. In step 230, the presence of the three solutions to be prepared is checked. When the solutions are complete, the program continues with step 232, or, if not, the program first proceeds to step 234, in which a given amount of the same material is introduced into the next indentation, and then step 232 follows. In the latter, the program ensures that the nozzles 174, 176, 178 and 180 of the second arm 166 dispense the diluted samples into the appropriate recesses of the laboratory plates. Then, in step 236, the program provides for moving the second lever 166 to the washer 237 and controls the washing of the nozzles 174, 176, 178, 180 there. Subsequently, in step 238, the laboratory plates are shaken with the laboratory shaker 184 for 15 seconds while the nozzles 174, 176, 178, 180 are cleaned by operating the washing machine 237. In step 240, we check that all wells of the laboratory disks have been filled. If the result of the check is to determine that all the wells in the lab disks have material, the program will require the next step, 242, to replace the lab disks. Subsequently, in 244 steps, the second arm 166 waits for an enabling signal to be used.

136 transport units. If in the subsequent step 246 it is determined that the processing of the selected thirty-two samples has not been completed, the program returns to step 228 and repeats the previous steps. However, if as a result of step 246 the completion of the processing of the thirty-two samples can be determined, the program returns in step 248 to the basic program of Figure 10.

Tissue culture-compatible versions of conventional laboratory discs are made of light-transparent or translucent plastic. If a cell sample in the recess of such a conventional material laboratory plate generates a bioluminescent signal, this may penetrate another recess through the translucent plastic. Therefore, a photomultiplier unit capable of scanning multiple depressions simultaneously detects not only the signal characteristic of the pattern in this other depression, but also the light signal coming from the aforementioned or possibly further adjacent depressions. This is because the individual recesses are not well insulated from one another and, as a result, the measurement results in false information about the material in the recess. This problem can be avoided by making a laboratory plate of a signal insulating material according to the invention. One way to do this, for example, is to make a laboratory plate of opaque material, especially plastic, which can be coated with both fibronectin and cells. However, it is not necessary to make the whole of the laboratory plate from this opaque material. The only condition is that each of the recesses has a stone.

- The signal must not extend, ie it must not penetrate from one recess to another. Accordingly, an opaque lattice support can also be used to create drawers separated by opaque, non-light-illuminated zones. Such a lattice structure may also consist of elements of a vertical arrangement, wherein the recesses receiving the sample may be themselves made of a conventionally used material, such as a transparent or transparent plastic.

If the cells can adhere to the bottom of the prepared wells in the laboratory plate, the wells can be rinsed without the risk of leaching the cells until auxiliary chemicals, such as detergents that break cell membranes or other cellular reactants, have been added to the wells. However, if the cells are unable to cling to the lower level of the depressions and are in suspension, they cannot be rinsed without risk of loss. Therefore, according to the invention, the lower levels of the laboratory plate in each well are supplemented with filtering material having pores smaller than the cell size, so that cells in the wells can be rinsed with fluid from above and below. In this case, the filter structure prevents cell removal during the rinsing process. Filters can be made from the material of a laboratory plate or other material such as plastic.

Thus, in accordance with the present invention, we propose an innovative and highly efficient automated laboratory test apparatus that can be run at a low cost at a high rate. 4 ··································· ··

- 33 reliable measurements on a very large number of samples of the substances to be tested. For example, the effects of various chemicals on living cells can be controlled simultaneously and in large numbers. According to the present invention, we also propose a laboratory plate which, by its novel and highly efficient structure, is not only compatible with tissue culture, but particularly fulfills the requirements of automatic cell culture assays.

Numerous embodiments of the apparatus, process, and laboratory disk described above can be provided, particularly in light of the documents defining the state of the art. so per chemical! The number of solutions, the number of laboratory plates placed on the supports, the number of compartments in the incubator, and the number and nature of the various subassemblies forming the transport unit 136 are broadly variable, essentially not limiting the effect of the various cell cultures and chemicals. The use of the transport unit 136 can greatly facilitate the cooperation between the incubator 112 and the liquid treatment station 134, the incubator and the light meter 162, the incubator 112 and the liquid treatment station 134 and the light meter 162. The proposed laboratory plate may be wholly or partially opaque, i.e. partially opaque or translucent, the essential being that opacity ensures that signal paths from one recess to another are cut off.

The apparatus, process and laboratory disk of the present invention can be provided in a wide variety of shapes as described above.

* · · ♦ · · <· ···· * · * «··» · ··

- 34 lakes created. To accomplish these, the foregoing description and the appended claims provide clear guidance to those skilled in the art.

Claims (22)

An apparatus for conducting laboratory tests comprising an incubator (112) for providing living and reproductive conditions to an individual set of cells of at least one predetermined living cell, characterized in that the incubator (112) has internal spaces (124, 126, 128, 130). ) provided with a defining socket (114), wherein the internal spaces (124, 126, 128, 130) in the incubator (112) form compartments (116, 118, 120, 122) for receiving the cell set and the compartments (116, 118, 120) , 122) disposed laboratory plates with recesses for receiving living cells, liquid treatment stations (134) provided with nozzles (164A, 164B, 174, 176, 178, 180) for dispensing chemicals contained in the recesses, and receiving them for laboratory discs. a detecting means for detecting changes in living cells, and supplying the incubator (112) with a unit (136) is provided, wherein the transport unit (136) transports the laboratory plates from the incubator (112) and into the incubator (112), from the fluid treatment station (134) and the liquid treatment station (134), and preferably comprising first and second robots (138, 140) for transferring chemicals, preferably in tanks, and laboratory disks to the sensing device and to the sensing device. Apparatus according to claim 1, characterized in that the incubator (112) comprises a temperature sensing device and a fv · ····························································································· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Apparatus according to claim 1 or 2, characterized in that at least a portion of the interior spaces (124, 126, 128, 130) of the incubator (112) is provided with an irradiating infrared lamp (144) which is mounted in a temperature control circuit. Apparatus according to any one of claims 1 to 3, characterized in that the incubator (112) comprises a humidity sensing device and a second regulator for adjusting and holding approximately 100% relative humidity of air in the compartments (116, 118, 120, 122). . Apparatus according to any one of claims 1 to 4, characterized in that the incubator (112) is provided with an outlet (150) for blowing wet air into the compartments (116, 118, 120, 122) and from the compartments (116, 118, 120, 122) comprising an adjusting and holding regulator for controlling relative humidity formed by wet air extraction inlets (148), wherein the outlet (150) and the inlets (148) are coaxially arranged with each other, and with the inlet (s) (148) and a sensor device for measuring the relative humidity of the air is connected to the outlet (150). Apparatus according to any one of claims 1 to 5, characterized in that the transport unit (136) has a working space (152, 154) at least partially overlapping the compartments (116, 118, 120, 122) of the incubator (112). an incubator (112) is provided with a rotary support table (132) for receiving the compartments (116, 118, 120, 122) into the work space (152, 154). * · · · · · ·············································································• 7. Apparatus according to any one of claims 1 to 4, characterized in that the liquid treatment station (134) is adapted to prepare solutions of various dilutions of chemicals and to add one of each of the solutions of different dilutions to one of the wells of the laboratory plate. Apparatus according to any one of claims 1 to 7, characterized in that there are nozzles (164A, 164B) forming a first nozzle system for delivering chemicals and solvents to the recesses of the master plate (170). 9. Apparatus according to any one of claims 1 to 4, characterized in that it is provided with nozzles (174, 176, 178, 180) forming a second nozzle system for delivering a solution of chemicals and solvents to the recesses of the laboratory plate. Apparatus according to any one of claims 1 to 9, characterized in that the detecting means for detecting changes in living cells is adapted to detect light emitted and comprises at least one photometric unit (162) having a photomultiplier. Apparatus according to any one of claims 1 to 10, characterized in that the sensing means is provided with a detector, in particular a photomultiplier, for comparing the laboratory plate and the sensing means, in particular the photomultiplier relative to one another and the increment of the laboratory plate. Apparatus according to any one of claims 2 to 11, characterized in that the first controller for maintaining the temperature comprises an electrically heated plate mounted on the surface of the incubator (112). Apparatus according to any one of claims 1 to 12, characterized in that it comprises an auxiliary dosing unit for supplying additional chemicals to the wells of the laboratory plate and a transport unit (136) for the laboratory dosing units to and from the auxiliary dosing unit. is suitable for transporting. Apparatus according to any one of claims 1 to 13, characterized in that it comprises a washing device (213, 237) for removing chemicals from the recesses, and a transport unit (136) for washing the laboratory plates (213, 237), and adapted for transporting the washing device (213, 237). 15. A method of performing laboratory assays, wherein the living and growth conditions of a cell sample containing at least one set of living cells are created in an incubator and the cell sample is assayed under the conditions of a given assay, comprising placing a plurality of cell samples in the incubator. treating a plurality of cell samples with the chemicals applied to each sample and creating conditions for the chemical to exert its effect; An average test time of 2.5 s is provided. 16. The method of claim 15, wherein, prior to detecting changes, the pre-added chemicals are removed from the cell samples and additional chemicals are added to the cell samples, and conditions are created to allow the effects of the additional chemicals on the cell samples to develop. 17. Laboratory plate for carrying out laboratory test tests, which is provided with recesses of tissue culture compatible material receiving cell cultures to be tested and adapted for transmitting detectable signals from cell cultures, characterized in that is made of an active material. 18. A laboratory plate according to claim 17, characterized in that it is suitable for optical examination and consists of opaque material in the directions between the recesses. Laboratory plate according to claim 17, characterized in that it is suitable for optical examination and consists of opaque material. Laboratory plate according to any one of claims 17 to 19, characterized in that the recesses are arranged in a rectangular grid. Laboratory plate according to any one of claims 17 to 19, characterized in that it comprises ninety-six recesses arranged in eight rows and twelve columns. Laboratory plate according to any one of claims 17 to 21, characterized in that it is provided with openings from the top which allow the cell culture to be flushed and with filtering apertures formed at the bottom to retain the cell culture tour in case of flushing.