WO1999060094A2

WO1999060094A2 - Method for measuring the interaction of chemicals with cells

Info

Publication number: WO1999060094A2
Application number: PCT/DE1999/001443
Authority: WO
Inventors: Thomas Noll; Heinz MÜHLENSIEPEN; Markus Cremer; Manfred Papaspyrou; Karl-Josef Langen; Ralf Engels; Richard Reinartz; Kurt Hamacher; Manfred Biselli; Markus Holschbach
Original assignee: Forschungszentrum Jülich GmbH
Priority date: 1998-05-20
Filing date: 1999-05-12
Publication date: 1999-11-25
Also published as: EP1078039A2; DE19822827A1; WO1999060094A3; JP2002515589A

Abstract

The invention relates to a method for determining the interaction of chemicals with cells in a reactor, e.g. in a fixed-bed reactor or in a fluid-bed reactor. To this end, a signal is measured which is produced by one of the components affected during the interaction. The signal is suited for being detected by a detection system situated outside of the reactor. The component which produces the signal can be the chemical itself, in which case, it can concern the analysis of the pharmacokinetic characteristics of the chemical. An additional possibility relates to the analysis of an influence of the chemical on processes within the cell. A device for carrying out the inventive method comprises a reactor for accommodating cells, a circulation system, control units for monitoring the composition of the medium, a system for gassing the medium, an entry for the chemical and at least one detection system for detecting the interaction.

Description

Procedure for performing a measurement of the interaction of

Chemicals with cells.

The invention relates to a method for carrying out a measurement of the interaction of chemicals with cells, the cells being contained in a reactor. The invention also relates to an apparatus for performing the method and its use.

Interactions between chemicals and cells are often measured in biological research. The focus of such investigations can vary. On the one hand, such tests are carried out to determine whether chemicals have a certain effect on the cells. This can be a pharmacological activity if the aim of the tests is to find new active substances, but it can also be a toxic property. Both types of measurements will be carried out as part of a substance screening, where it always depends on a certain ratio of biological effectiveness compared to toxicity.

Another application is the detection of the pharmacokinetic properties of a chemical. The pharmacokinetic properties of chemicals must also be investigated in the course of product development if the product is intended to be used on humans or animals. As part of the study of pharmacokinetic properties, the absorption of the chemical, its distribution in the body, its metabolism and its excretion are recorded. What can be excellently examined in the cell system is the absorption of the chemical into certain cells, its metabolism in these cells and their excretion by these cells.

The biological activity of chemicals is ultimately recorded in animals. However, a great advantage of tests in cell systems are their better standardization, their simplicity and their lower costs. Tests on animals are not only made more difficult by legal regulations, they are always expensive and complex and the resistance in the population to such tests is constantly increasing to.

For this reason, it is welcome and necessary to be able to test the properties of chemicals in a test procedure without consuming animals screen. Even if such test methods will never completely replace animal testing, their great advantage is already due to the fact that they are used as a preliminary screening in the first phase of the

Development of chemicals will make many animal experiments unnecessary.

Attempts to take measurements of the interaction of chemicals with

Cells are known in the art.

P 195 37 033.3 describes how measurements are carried out on cells in a reactor to determine pharmacokinetic and toxicokinetic properties. A fluidized bed reactor is used as the reactor and is bubbled without bubbles. The cells are attached to carrier bodies. With this procedure it is fundamentally possible to solve a number of questions. The method is characterized by good applicability. A disadvantage of the known method, however, was the fact that it was intended according to this method to carry out an off-line acquisition of the data, i.e. it was necessary to regularly take samples from the reactor so that the effects of the chemical on the cells were recorded.

Constant sampling of data to collect data is problematic in some respects. Withdrawal of material from the reactor leads to a change in the system; if frequent removal is required, this change can even be significant. In addition, this type of detection leads to a certain delay in the procedure. If it is necessary to make changes in the system based on the recorded measured values, for example by adding the chemical, this change can only be made after the respective measured value has been determined. This sluggishness is fundamentally disadvantageous. The fact that measurements have to be carried out at all is also undesirable. Measurements tie up material and personnel and are undesirable for the establishment of procedures in routine screening.

It was the object of the present invention to provide a method which did not have the disadvantages of the prior art and is easier to fit into a screening system by means of a simple acquisition of measurement data. It was a further object of the present invention to provide a device with which the method according to the invention can be carried out.

The first-mentioned object is achieved by a method with the characterizing features of the main claim.

The process relates generally to the interaction of a chemical with cells. The method is therefore expressly not restricted to a specific interaction.

It is therefore possible within the scope of the method according to the invention to detect an effect of the cells on the chemical. This therefore includes an absorption of the chemical into the cells, a conversion of the chemical in the cell and an excretion of the modified or unchanged chemical by the cells. This effect of the cell on the chemical affects processes that can generally be subsumed under pharmacokinetic studies.

On the other hand, the system offers the possibility of examining the effect of the chemical on the cells of the test system. Such interaction is often observed as a deterioration in the condition of the cells. A direct lethal effect of the chemical on the cells will usually be regarded as a toxic effect, unless the cells in the test system are downright target cells for a desired attack by the chemical, such as for tumor cells, in which case the lethal or toxic effect is desired is. However, it should be pointed out that this type of interaction also includes influencing biochemical processes within the cell, the biochemical processes being able to represent effects at the level of protein synthesis, DNA synthesis or RNA synthesis.

The process is carried out in a reactor. A reactor is the general name for a vessel that contains cells. Depending on the arrangement of the cells in the reactor, this is called differently. The process can be carried out with a fluidized bed reactor (also known as a fluidized bed reactor) or with a fixed bed reactor. These options will be specified later in the description. Other arrangements can also be considered. The reactor is not limited to a particular format. A reactor with a total volume of about 50 ml is preferred, but other dimensions are also possible.

If the reactor is a fluidized bed reactor, the process according to the invention can be carried out very well with the reactor described in P 195 37 033.3. Such a preferred reactor has a total volume of about 50 ml. The reactor is tapered at the lower end and the diameter of this end corresponds to the diameter of a line which is assigned to this end. The reactor tube has a diameter of approx. 10-20 mm with an opening angle of approx. 25 °.

There are also several options for the exact arrangement of the cells in the reactor. The cells can be used as cell culture, being freely available in a suitable liquid medium. The biological specialist literature provides the specialist with sufficient information on how to proceed with regard to the establishment of a cell culture.

However, an embodiment in which the cells are not freely available but have settled on carrier particles and they are thus immobilized on these carrier particles as solid particles has proven particularly useful. This embodiment, which e.g. P 195 37 033.3 describes leads to surprisingly high cell densities. If a reactor with a volume of 50 ml is used, it is preferred to work with a carrier amount of 20 to 25 ml bulk volume. 1 to 5 g of cells can be immobilized on this amount of carrier material. The process can be very high

Lead cell density: cell densities between 5 * 10 ⁷ and 10 ⁸ cells are achieved per ml carrier. The density achieved with this method is much closer to the natural situation than the previous in vitro methods.

The method is not limited to specific cells. The selection will primarily depend on the suitability for use in the method, because not all cell types are necessarily suitable for use in a free cell culture, and likewise not, according to the preferred embodiment, are suitable for settling on support material.

The method can be carried out very well with animal or human liver or kidney cells, a selection which is particularly useful in the context of pharmacokinetic Investigations is extremely useful. Use with tumor cells is also preferred, especially in the context of investigations that focus on screening for anti-carcinogenic

Straighten connections. In general, it can be said that cells with which the method according to the invention cannot be carried out are currently. are not known.

The solid particles are not bound to any particular embodiment. With regard to these solid particles, too, there is any variation of possibilities. The only condition for carrying out the method according to the invention is the requirement that the solid particles are not soluble in the selected liquid medium. Otherwise, the material of the particles and their exact geometry should only be used for their purpose in the process. Within the scope of the invention it will certainly be a preferred variant to start from more or less spherical particles. These offer a relatively large surface area, can be stacked perfectly into a pack and still allow an undisturbed flow of liquid medium.

Of course, the possibility of carrying out the method by means of a fixed bed or a fluidized bed affects the free-floating cells as well as the cells adhering to solid particles.

As will be discussed later in the context of the exemplary embodiment, it is a preferred embodiment of the method according to the invention to use it in the cultivation of human body cells. In such experiments, certain particles have proven to be extremely useful. Porous vertebral bodies with a diameter of 200 - 1500 μm and a density of at least 1.1 kg / 1, the pores of which are filled with medium, are particularly useful.

First, favorable experiences were made with Siran® balls (from Schott, Mainz). Such Siran® balls are porous glass substrates with about 50% porosity and a grain size of 200 - 1500 μm, preferably 400 - 700 μm. The glass slides can be pretreated with a diluted gelatin solution before use in the process. In experiments with human or animal cells, the Siran® balls have repeatedly shown excellent properties in terms of adhesion and stability. Another variant which is also preferred in the context of investigations with

Cell material is used as collagen microcarriers. These carriers are also available in different sizes and designs. Collagen microcarriers show similarly good results

Properties in the experiment have a special property compared to the glass balls, which can give them a decisive advantage as required. In fact, when using particles with attached components, it is often the case that it is desired to recover these components at the end of the reaction, with the aim of reuse or subsequent processing. In such cultivations, the grown cells represent the purpose of the experiment and it is therefore inevitable that they are separated from the particles, e.g. to loosen the Siran® balls or the collagen microcarriers. It is precisely then that the advantage of the latter particles becomes apparent because the collagen microcarriers can simply be transferred to a system containing a collagenase, with the result that the carriers dissolve and the cells which are not attacked by the enzyme can be isolated from the solution.

The process according to the invention is carried out in a liquid medium. The conditions in the reactor, such as the composition of the culture medium, the temperature, the type of fumigation, etc., must be determined and optimized individually for each cell type. The optimized reaction conditions are preferably to be regulated automatically in the process according to the invention.

It is a special feature of the method according to the invention that one of the components involved in the interaction between the chemical and the cells is a component producing a signal. Such a component is directly or indirectly affected in the interaction between the chemical and the cells.

The component can thus be the chemical itself, for example if the focus of the investigation is to determine whether the chemical is influenced by the cells at all. Information will often be requested as to whether the chemical is absorbed by the cell. The knowledge that a certain chemical is not absorbed by certain cells can be of considerable importance. Recording a time course of the intracellular concentration of a chemical can also provide important information regarding the effectiveness or side effects of chemicals. However, the component can just as well not be the chemical itself, but another component if, for example, the focus of the investigation is to determine whether certain intracellular processes are changed in the presence of the chemical. For example, it can be investigated whether the chemical causes a change in the protein synthesis in general, or the synthesis of a specific protein or the synthesis of the genetic material or the division of the cell. When it comes to an assay for protein synthesis, the component involved may be a particular amino acid or two amino acids, but if it is an assay for a change in DNA or RNA synthesis, it may become one of the components of DNA or RNA can be used as the affected component. If another process is examined again, other components may also be examined. It therefore depends on the question during the investigation which component becomes the subject of the investigation.

The component concerned, which is the subject of the investigation according to the method according to the invention, must be a component producing a signal. This produced signal enables the investigator to determine whether there is an interaction between the chemical and the cells and how this interaction works.

The signal produced by the affected component can be any signal. The only requirement is that such a signal is produced and that it can be detected by a detection system outside the reactor. Such a signal can be, for example, radioactive radiation if a radiotracer is used to carry out the method. Another possibility relates to fluorescent radiation if a fluorescent component is used when carrying out the method. In the context of studies which deal with the influence of the cell on the chemical, that is to say studies with a pharmacokinetic background, the chemical can be offered as an affected component labeled with a radioactive isotope. The radiation produced by the radioactive isotope can be detected by an appropriate radioactivity detector outside the reactor. A different radioactive label can be used. For the selection of the relevant marking in the examination, one can choose from the available offer. The Selection will of course be affected by which labels are available or usable for the intended study. However, there are other practical considerations. Short-lived radioactive compounds have been found in the

Carrying out the tests according to the method according to the invention has clear advantages, because with such compounds it is possible with considerably less effort to carry out separate tests in the same reactor at relatively short intervals. In terms of cleaning, disposal and residual radiation, it is therefore very advantageous to use such short-lived emitters. As such short-lived emitters, for example, 1 _unc } 18Q come in

Consideration. In contrast, e.g. Studies with ^ C represent clear disadvantages.

However, it will not be a fundamentally new problem for the person skilled in the art to consider which radiotracers are best suited for his question. The use of radioactivity in biological research is a routine procedure that is used in many experiments.

It can also be considered to use two different labeled compounds in one test. This option can be useful if an investigation is to determine the direct effect of a chemical on an intracellular process and its pharmacokinetics. In such an investigation, there is thus a labeled chemical and possibly a labeled precursor for the intracellular process.

It is a further feature of the method according to the invention that the signal is detected with a detection system outside the reactor. Within the scope of the description of the device according to the invention, the embodiment of the detection system will be discussed in more detail. The execution will of course be determined primarily by the signal produced. For measurements on components with a radioactive isotope, the detection system will include a system that is suitable for the detection of the radioactive radiation, while a device for the detection of fluorescence beams is selected for the detection of fluorescence.

It is another preferred feature of the present invention that the signal produced in the reactor by one of the affected components, which is outside the Reactor is detected by a detection system, immediately fed to a computer and processed further. Such a preferred embodiment has many advantages. First of all, it is necessary to prepare appropriate software that enables the processing of the data. Once this requirement has been met, the method according to the invention can run fully automatically without the need for manual actions and corresponding binding of operating personnel. With such an embodiment in particular, the great advantage of the method according to the invention becomes clear. The regular taking of samples in the prior art method, the need to further process the taken samples outside the reactor depending on the course of the reaction, and finally to further process the data obtained, lead to a complex and costly procedure. This effort does not apply to the method according to the invention. Where the great expense in the method according to the prior art may have led to the fact that this known method is only used occasionally in very specific examinations, the method according to the invention readily offers the possibility of using the method in a routine screening. For example, it is a great advantage of the method according to the invention that when the chemical is in the test system, the need to carry out such a measurement by adding an indicator is eliminated. This addition of an indicator was described in the prior art method and was undesirable if only because it could never be completely ruled out that there was an interaction between the chemical and the indicator. In addition, it could only be assumed that the behavior of the two compounds takes place in the same way in the process. Particularly when the chemical is taken up and metabolized by the cells, the assumption that the indicator and the chemical behave in the same way is doubtful.

The procedure is usually carried out with cells. However, it should be expressly pointed out that an application without cells is also possible. A simple example is an examination which can be carried out to check the specificity of monoclonal antibodies. For this purpose, the monoclonal antibodies can be bound to solid particles, the above-described ones in turn being preferably usable, in order to subsequently carry out L-examinations with marked substrates in order to test the binding of these substrates. The depletion of the marked The substrate or its enrichment in the area of the fluidized bed or fixed bed allows the

Specificity of binding and determination of binding kinetics.

Investigations into the influence of the antibody-substrate complex on test substances and temperature or pH changes can be carried out in the same way.

Another possibility of use in a cell-free system is also described further below in the description of some preferred uses of the method according to the invention.

The invention also relates to a device with the features of claim 6. The device was invented with the focus on being used in the method according to the invention. A device is thus provided which is to be used for measuring the interaction of chemicals with cells.

The device has a reactor for taking up the cells, or according to the preferred embodiment of the method, for taking up the cells immobilized on solid particles.

The reactor can be designed differently and is adapted to the process. A fluidized bed is often provided in the process according to the invention and the reactor accordingly represents a fluidized bed reactor. In a fluidized bed reactor, liquid medium is usually fed to a reactor part containing the cells at such a flow rate that the cells cannot settle on the one hand, but continuously from the liquid flow be whirled up without being carried along.

Such fluidized bed reactors are described, for example, in P 195 28 871.8. Such a fluidized bed reactor can have, for example, a cylindrical reactor tube and a conically tapered end. In the case of a reactor with a total volume of, for example, 50 ml, the reactor tube can have a diameter of 10-20 mm, while the tapered end has a diameter which corresponds to that of an intended circulation system. The fluidized bed reactor can have a membrane or filter in the tapered lower end in order to prevent cells from getting into the circulation system counter to the flow of the liquid medium, but this is not absolutely necessary since the experience with such fluidized bed reactors has shown that the cells with the correct setting of the

Do not leave the area of the cylindrical reactor tube.

The reactor can also be designed as a fixed bed reactor. The reactor can then basically also have a format as described above in relation to an embodiment as a fluidized bed reactor. The fixed bed reactor can thus also have a cylindrical reactor tube and a tapered end. In the case of a design as a fixed bed reactor, the liquid medium will preferably flow from above through the fixed bed, which of course requires the arrangement of a filter, a membrane or a sieve in the reactor tube or in the section of the tapered end.

Other types of reactors can also be considered. Any spatial arrangement which is suitable for accommodating cells can be considered as a reactor in the device according to the invention.

The reactor will normally be made of glass. Other materials can also be considered, but it is not easy to imagine what advantages a certain other embodiment would have for the device according to the invention if it were made in a different material. Glass is cheap, stable, easy to clean, transparent and permeable to the signals which have to be detected according to the method according to the invention.

The device according to the invention must also have a circulation system. It must be provided to stir or swirl the liquid medium in the device so that an effective homogenization of the liquid medium can be carried out in the system.

The circulation system can be of any design as long as it ensures an intensive and thorough stirring process. It is a preferred embodiment of the device according to the invention that the device has a circulation system which comprises a first opening at the lower end of the reactor and a further opening at the upper end of the reactor, a line being arranged between the two openings. With this arrangement, the liquid medium is constantly in a kind of circular movement pumped round, a circulating pump preferably being arranged in the line for this round pumping.

The dimension of the openings in the reactor and the format of the line is not an essential feature of the device according to the invention. If the reactor according to the preferred embodiment described above is a fluidized bed reactor which consists of a cylindrical reactor tube with a tapered lower end, the line is connected to the tapered lower end. The diameter of the tapered end is usually less than 7.5 mm and with such a diameter, a circulation hose can be used in a relatively simple setup. In this preferred embodiment, the second opening, which preferably has the same diameter, is arranged in the upper region of the cylindrical reactor tube, so that an identical hose can be used for the line between the two openings.

The device must have control elements for monitoring the composition of the medium. Reproducible and comparable studies require constant conditions. Consistency in the composition of the liquid medium is important for working with cell systems because this is the only way to ensure that the system is sufficiently vital. Parameters such as concentration of the medium's ingredients, concentration of nutrients, pH, osmolarity and oxygen concentration can be monitored with appropriate sensors. The control bodies such as a pH probe, a pO2 electrode or flow injection analysis for substrate and metabolite analysis can be arranged in the reactor and in the circulation system. Ideally, the control elements are built into an automated control system, which also provides that any adjustment of the composition of the liquid medium that may be required is also controlled automatically. Such an adjustment can be carried out as the supply of any ingredients or nutrients (dissolved or undissolved), as the supply of fresh medium or an intensification of the fumigation.

As already indicated in the penultimate paragraph, the device according to the invention has an arrangement for gassing the medium. In most studies, this kind of fumigation means an arrangement that specifically supplies oxygen. For most biological systems, a certain minimum oxygen pressure is a decent one Course of the intracellular processes is an absolute requirement. That is why a well-functioning arrangement for fumigation is particularly important.

It does not depend on a particular execution of the arrangement for fumigation. In the prior art according to P 195 37 033.3, which has already been cited several times, an arrangement for gassing is provided which comprises a silicone membrane, via which oxygen reaches the liquid medium. The device according to the prior art preferably provided a coaxial arrangement in the reactor tube for this silicone membrane. However, such a coaxial arrangement has disadvantages in the device according to the invention because, as will be described in more detail below, the device comprises a detection system and the arrangement of the silicone membrane in the reactor tube interferes with a particularly preferred embodiment of the detection system. It is therefore more favorable not to arrange the gas supply device in the reactor tube, but rather at some point in the circulation system.

The device according to the invention must have a point for metering the chemical. It can be provided that only one point is provided for metering, but it is also possible to provide two different positions for metering, one of the two points can be used for an acute injection, the other for metering which is carried out as an infusion becomes.

It is an essential feature of the present invention that the device according to the invention has a detection system for detecting the signal produced by the interaction of the chemical with the cell. In the description of the method according to the invention it has already been shown that the method can be carried out equally with different signals or even in a single test run with different signals. It is obvious that the detection system has to be adapted to the respective signal. However, in the basic description of the device according to the invention, it is of no particular importance for which signals the detection system is intended to be detected. Since the device according to the invention detects the interaction of a chemical with

Purpose cells, the arrangement of the detection system in the area of the reactor where the

Of course, cells are usually the most sensible.

When carrying out an experiment with a chemical that itself produces the signal, positioning the detection system in the area of the cells will make it possible to determine whether the chemical is accumulating in the area of the cells and a time course of the signal intensity will become clear. If the signal is not from the chemical itself, but e.g. is produced by a component of the cell, the influence of which is to be studied, the detection of the signal in the area of the cells provides information on the extent to which the chemical effects an increased uptake or discharge of this component over time.

Although a device with only one detection system thus makes sense for different questions, a further more preferred embodiment with two detection systems will lead to a better result. In this more preferred embodiment, the first detection system will be located in the area of the cells, as has already been described above for a device with only one detection system. However, the second detection system should be positioned at a location in the device where there are no cells. Such a preferred embodiment allows a much more meaningful statement about whether a certain signal level in the area of the cells really comes about through an interaction with the cells or rather represents a general change in the entire system, which ultimately only results from a signal at the level containing the cells liquid medium comes about. The second detection system will make it possible to detect interactions between chemicals and cells with much greater accuracy and sensitivity. The detection of the signal intensity in the circulating medium and in the fluidized bed allows the continuous determination of the amount of chemicals absorbed by the cells.

This description of the device according to the invention makes it clear why an embodiment of the device which provides a coaxial position of the gas membrane for the oxygenation of the medium is a disadvantage in connection with the detection of the signal in the region of the reactor. The arrangement of the gas membrane within the Reactor must inevitably lead to disturbances in the measurement process. Not only can

Interfering with the presence of the membrane, in addition, the arrangement displaces the medium and accordingly causes the signal to weaken.

According to a first embodiment, it is provided that the detection system comprises a detector. According to a further more preferred embodiment, the detection system should comprise two separate detectors, which are arranged, for example, on opposite sides of the reactor (coincidence measurement). This more preferred embodiment is equally preferred in a single detection system and in a double detection system.

According to further, even more preferred embodiments, further detection systems are provided in the device according to the invention. A first further arrangement is provided in the area of the access of the chemical, provided that the detected signal is produced by the chemical itself. The advantage of this arrangement relates to the possibility of carrying out a targeted and precise metering of the chemical into the liquid medium, because the increase in the signal in the system as a whole is detected by means of the direct detection of the signal in the inlet area.

According to yet another, likewise preferred embodiment, a further detector is provided in the area of the circulation system. With a detection system arranged at this position, a further refinement of the monitoring of the activity in the system is possible.

The detectors are each connected via a main amplifier and a window discriminator (for setting to the radionuclide used) to a counter card in a computer which displays and / or stores the data with a corresponding program.

According to a particularly preferred embodiment, the detection system consists of two or more measuring heads (these can be gamma radiation detectors, for example, depending on the radiation detected), an associated standard readout electronics and a data acquisition system (computer). Due to the different radiation energy which is to be detected in the fluidized bed reactor, a high dynamic range must be ensured.

There are various options for the execution of the measuring head, e.g. the

Use of semiconductor detectors or scintillation detectors. Both types of detectors are suitable for single and coincidence measurements and must be carefully shielded with a lead sheath. The ionizing radiation can be detected directly (semiconductors) or indirectly (scintillation). The scintillator can be coupled to the photomultiplier either directly or via optical fibers. In order to increase the sensitivity of the measuring arrangement, the detectors can be arranged around the reactor.

When carrying out the process with radio tracers, it is necessary to shield the reactor with a lead shield to protect the operating personnel. The detectors are collimated to the respective field of view by lead shielding with a suitable geometry or, in the case of positron emitters, optionally by coincidence measurements.

The aim of the invention is basically based on the fact that the investigation can be carried out completely on the closed system and that it is not necessary to take samples from the device. This goal can also be achieved if the method according to the invention is carried out as described above. However, examinations may be intended where removal is required. Such removal is e.g. required if the examination provides information about the identity of the metabolites produced by the cells, about a binding to certain cell organelles, about damage to the cells, and similar information that can only be carried out by a subsequent examination outside the device. In such cases, the method according to the invention provides that, in addition to the automatically recorded and on-line evaluations, samples are taken at certain times, which are processed for evaluation outside the device.

If it is necessary to take samples from the device, a removal point is provided in the device. The method according to the invention is fundamentally suitable for use in tests which aim to investigate the pharmacokinetic properties of chemicals. The method is also suitable for carrying out studies on

Assessment of the toxic properties of chemicals.

Another use of the method according to the invention relates to the measurement of the porosity in the reactor.

Porosity of a fluidized or fixed bed means the proportion of the liquid medium in the area of the bed, which is composed of the intermediate grain volume and the accessible cavities inside the carrier.

In the cell-free reactor, the porosity is thus the quotient of the signal intensity measured in the area of the bed and the intensity measured in the carrier-free circulation. It is a prerequisite for such a measurement that the labeled substance does not adhere to the carriers, because this would lead to an accumulation in the area of the bed and thus lead to an incorrect result.

In the case of a fluidized bed, the increase in the volume flow in the circulation of the fluidized bed reactor leads to an expansion of the fluidized bed and thus to an increase in the porosity.

The porosity of a fluidized or fixed bed can be determined by means of continuous data acquisition. The porosity is of particular interest for procedural questions.

A very specific question can arise if, in the course of an investigation with a catalyst immobilized on solid supports, it is desired to measure whether the immobilized catalyst is bleeding (being flushed down by the supports). This question is an examination which is carried out in the absence of cells. The carrier which contains the carrier as a fixed bed or as a fluidized bed is flowed through by a liquid phase comprising the substrate. The porosity of the bed can be measured by regularly injecting a signal producing compound, eg a radiotracer. If the catalyst is lost, a change in the porosity is determined. Description of the invention with reference to illustrations.

Figure 1 shows schematically an experimental device with a fluidized bed reactor. Figure 2 schematically represents an experimental device with a fluidized bed reactor. Figure 3 shows the result of an investigation with ¹⁸ FDG in the device according to the invention.

Figure 4 shows the result of an examination with * ⁸ FDG on human glioma cells. Figure 5 shows the result of a porosity measurement.

A device according to the invention is shown schematically in FIG. The device has a reactor 1, which consists of a cylindrical part 2 and a tapering end 3. The reactor 1 is surrounded by a heating jacket 4. A circulation system 6 is assigned to the reactor. The liquid medium present in the reactor is withdrawn from the reactor by means of a peristaltic Umlau 5 and fed to it again via the circulation system 6. In its simplest design, the circulation system 6 consists of a hose which is connected to the reactor 1 at positions 8 and 9. At position 7, liquid medium can be removed from the reactor. Control elements 10 are assigned to the circulation system and enable the composition of the medium to be continuously monitored. The control members 10 always include an oxygen probe, but can otherwise comprise any measuring devices. Fresh liquid medium can be supplied, specifically via the line 11. The silicone membrane for gassing the liquid medium is integrated in an arrangement 12 in the circulation system 6 in order to avoid a possible disturbance of the measuring processes in the area of the reactor 1. Addition of chemicals or substances producing a signal takes place via a line 15. The arrangement shown in FIG. 1 comprises a common supply line 15 which can be used as an infusion for an acute injection such as for adding the chemical or the signal-producing substance. If an acute injection is carried out, with which process an acute injection is practically to be imitated on humans or animals, the injection is carried out by means of a syringe 16 through a membrane 17 into the part 18 of the line. If an infusion is provided, this is carried out, for example, by means of an infusion pump 27 in part 19 of the line. The positions of the feed line 11, the control elements 10, the gassing arrangement 12 and the feed line 15 are all assigned to the circulation system, but their exact position does not necessarily have to be be as shown in the schematic figure, other positions can also be considered for carrying out the method according to the invention, the

Order of arrangement in the circulation system can be varied. The device according to the invention has detectors for detecting the signal produced when the cells interact with the chemical. FIG. 1 shows a device which comprises two detectors 20 and 21, the detector 20 being associated with the upper region of the reactor 1 and the detector 21 with the lower region of the reactor 1. Another detector 22 is assigned to the part of the circulation system which connects directly to the tapered end of the reactor. A further detector 23 is located on the feed line 15. The detectors can be optimized for certain types of beams or fluorescence signals, they can consist of an excitation light source and an optical separation unit in addition to the detector, or they can be designed as a combination of different detection systems. The reactor 1 is closed with a lid 24 which is connected to the reactor with a screw cap. The lid 24 has devices for taking samples, which are not shown here. This removal can be done sterile with a sterile bench.

When carrying out the method according to the invention, the reactor 1 is filled with a liquid medium which is pumped by the peristaltic circulation pump 5 through the circulation system 6. If the examination which is to be carried out relates to a reaction in a fluidized bed, the liquid medium is conveyed by the peristaltic pump 5 in the direction of the arrow 25. It speaks for itself that in the case of an orientation as a fixed bed, funding is provided in the opposite direction. The reactor has in the liquid medium the solid particles 26 which are constantly stirred up by the constant inflow of the liquid medium, but without leaving the lower region of the reactor 1. The on-line detection is carried out by the detector 22 as a control for compliance with the test conditions, by the detector 21 as an indicator for the activity absorbed by the cells and by the detector 20 as a detector for the activity remaining in the medium. According to the invention, a labeled test liquid is used for the calibration of the system, ie for the quantitative detection of the activity absorbed in the cells. By comparing the measurement data from the detectors 20 and 21, it allows the determination of the marked amount of substance which has accumulated on the cells or is taken up and metabolized by the cells. FIG. 2 shows another possible embodiment of a device according to the invention. In contrast to the device shown in FIG. 1, the feed line 15 is provided at a different position in the circulation system. The execution of the detection system is even more important. In this embodiment, all of the detectors 20, 21, 22 and 23 are not provided as individual detectors but as a duplicate. Such an embodiment allows a significantly more precise detection of the signal intensity.

Description of the invention using exemplary embodiments.

Example 1.

Studies on the uptake of [18F] -labeled fluorodeoxyglucose (18FDG) on human glioma cells (86HG39) in a fluidized bed reactor.

Human brain tumor cells (86HG39) were immobilized on open - pore borosilicate glass substrates (Siran®, Schott, Mainz) in a spinal fluid reactor and up to a cell density of approx

1 * 10 ⁸ cells cultivated per gram of carrier. Commercial LMDM with 10% calf serum was used as the medium. The fluidized bed reactor was two

Scintillation detectors equipped, one at the level of the fluidized bed (corresponding to detector 21 in Figure 1) and one above in the area of the cell-free circulation (corresponding to detector 20 in Figure 1).

At the point in time shown in FIG. 3, the fluidized bed reactor was charged with [^ ⁸ F] -labeled fluorodeoxyglucose (^ ⁸ FDG) in the form of a step function.

1 ⁸ FDG is used in the clinic to investigate glucose metabolism and to diagnose tumors. The compound is absorbed into the cells but, unlike natural glucose, is not metabolized. The signals in the fluidized bed and in cell-free circulation are shown in FIG. 3. The intracellular accumulation of the radiotracer in the immobilized cells results directly from the difference between the signal in the fluidized bed and in the cell-free circulation (shown in FIG. 4). The radioactive decay and the glass content in the fluidized bed have already been taken into account by the measurement data acquisition.

At various times, carrier samples with immobilized cells were taken from the fluidized bed reactor (using a carrier sampling system integrated in the cover 24 shown in FIG. 1) in order to determine the intracellular accumulation of the radiotracer off-line and to compare it with the signal obtained on-line.

FIG. 4 shows the result of the comparison of the off-line and on-line determinations. It is obvious that the course of the curve, which documents the on-line binding of the radiotracer to the particles, corresponds perfectly with the result of the off-line determination.

Example 2.

In a fluidized bed reactor there was a fluidized bed consisting of open-pore borosilicate glass substrates (Siran®, Schott, Mainz) with a diameter of 500-560 μm. Deionized water was used as the circulating medium in the test. The reactor was equipped with two scintillation detectors, one at the level of the fluidized bed (corresponding to detector 21 in FIG. 1) and one above in the area of the cell-free circulation (corresponding to detector 20 in FIG. 1).

At time t = 0, an aqueous [18F] -labeled fluoride solution was injected into the system (by injection at position 18 in Figure 1). After about 5 minutes, the activity in the reactor had mixed uniformly.

The quotient formed from activity in the fluidized bed and the activity in circulation corresponds to the current porosity of the fluidized bed, this value is usually represented as a percentage.

Different volume flows were then set for the circulation by means of the circulation pump (circulation pump 5 in FIG. 1). Depending on the volume flow, the expansion of the fluidized bed changed and thus also the porosity, which was measured on-line as shown above by forming the quotient of the measured activities in the area of the fluidized bed and the circulation. FIG. 5 shows the porosity determined in this way as a function of the circulating volume flow.

Claims

Expectations.

1. A method for carrying out the measurement of an interaction of a chemical with cells contained in a reactor, characterized in that one of the components involved in the interaction between the chemical and the cells represents a signal-producing component, which signal is for detection by a Detection system outside the reactor is suitable.

2. The method according to claim 1, characterized in that the chemical is detected in the reactor by means of a detectable signal.

3. The method according to claim 1, characterized in that the chemical is detectable by detecting the radioactivity.

4. The method according to any one of claims 1 to 3, characterized in that the cells are immobilized on solid particles.

5. The method according to any one of claims 1 to 4, characterized in that the detectable signal is fed to a computer and processed on-line.

6. Device for performing the method according to one of claims 1 to 5 for

Measurement of the interaction of chemicals with cells in a liquid medium, comprising a reactor for taking up the cells, a circulation system,

Control elements for monitoring the composition of the medium, an arrangement for gassing the medium and at least one input for the chemical, characterized in that at least one detection system for detecting the interaction is arranged in the device.

7. The device according to claim 6, characterized in that the circulation system comprises an opening arranged at the lower end of the reactor, which is connected by means of a line to an opening arranged at the upper end of the reactor.

8. The device according to claim 6 or 7, characterized in that a circulation pump is arranged in the line.

9. Device according to one of claims 6 to 8, characterized in that the reactor is a fluidized bed reactor.

10.Device according to one of claims 6 to 9, characterized in that the

Arrangement for fumigation of the medium comprises a fumigation membrane for bubble-free fumigation.

11.The device according to claim 10, characterized in that the arrangement is arranged in the line according to claim 7.

12.Device according to one of claims 6 to 11, characterized in that a

Arrangement for taking samples is arranged.

13.Device according to one of claims 6 to 12, characterized in that the

Device comprises a detection system in the area of the cells and a further detection system in the area of the cell-free supernatant.

14.Device according to claim 13, characterized in that a further detection system is assigned to the line according to claim 7.

15.Device according to one of claims 13 or 14, characterized in that a further detection system is assigned to the input for the chemical.

16. Use of the method according to one of claims 1 to 4 for measuring pharmacinetic properties of chemicals.

7. Use of the method according to one of claims 1 to 4 for the detection of pharmacological or toxic properties of chemicals.

Use of the method according to one of claims 1 to 4 for measuring the porosity in a fluidized bed or a fixed bed.