EP1159443A1 - Procede pour tester des principes actifs in vitro, dispositif approprie et son utilisation - Google Patents

Procede pour tester des principes actifs in vitro, dispositif approprie et son utilisation

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Publication number
EP1159443A1
EP1159443A1 EP00907670A EP00907670A EP1159443A1 EP 1159443 A1 EP1159443 A1 EP 1159443A1 EP 00907670 A EP00907670 A EP 00907670A EP 00907670 A EP00907670 A EP 00907670A EP 1159443 A1 EP1159443 A1 EP 1159443A1
Authority
EP
European Patent Office
Prior art keywords
cell culture
membrane
cell
active substance
container
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00907670A
Other languages
German (de)
English (en)
Inventor
Herma GLÖCKNER
Horst-Dieter Lemke
Christoph Meyer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Acordis Industrial Fibers GmbH
Original Assignee
Acordis Industrial Fibers GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Acordis Industrial Fibers GmbH filed Critical Acordis Industrial Fibers GmbH
Publication of EP1159443A1 publication Critical patent/EP1159443A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types

Definitions

  • the invention relates to a method for testing active substances on cells in vitro, a device and the use thereof.
  • Animal models for the screening of unknown cytostatics are based on the fact that, per treatment of the recipient animal, human tumor cell samples in Hollow thread devices are encapsulated and implanted in the recipient animal, as described in WO 94/25074 and US 5676 924.
  • this procedure has its limits, since animal models are naturally difficult to automate and involve a lot of effort and costs.
  • the idea of animal welfare is also increasingly opposed to testing in animal models.
  • test results on test animals which are mostly rodents, can be transferred to humans.
  • Metabolic differences between humans and animal species play an important role here, which lead to different pharmacokinetics in the animal model.
  • pharmacokinetics means the dynamic process in which an active ingredient with different kinetics is absorbed into the organism, i.e. absorbed, distributed, metabolized, i.e. implemented and eliminated. These processes cannot be quantified independently of each other in vivo because they overlap in time and are interdependent.
  • Possinger, Springer Verlag, (1996) therefore use mathematical models that describe the distribution of a drug in fictitious body spaces or compartments.
  • the form of administration in particular also has an effect on the pharmacokinetics of the substance.
  • the pharmacokinetics of an active ingredient is usually determined in vivo by measuring the serum concentration as a function of time.
  • in vitro models for testing substances is also widespread.
  • the cultivation of human primary cells in monolayer cultures for patient-specific testing is preferred in order to achieve a high sample throughput.
  • Such a procedure is described, for example, by GJL Kaspers et al. in Blood (1997), Volume 90, No. 7, pages 2723-29 and by R. Pieters in Blood (1990), Volume 76, No. 11, Pages 76, 2327-36.
  • the disadvantage of the known in vitro methods is that the microtiter plates used there are three-dimensional Do not allow cells to grow. Solid tumors e.g. For example, different subpopulations are likely to develop due to gradients in pH and nutrient supply. These gradients cause regional variations in cell vitality, metabolism and sensitivity to treatment with cytostatic agents.
  • in vitro pharmacokinetics which take place in the human organism have not hitherto been able to be achieved in vitro, i.e. cannot be modeled by appropriate in vitro pharmacokinetics.
  • in vitro pharmacokinetics This is to be understood to mean that the concentration of active substance in the environment of the target cell changes as a function of time in vitro in the same way as in the in vivo environment of the target cell, such as, for example, in the case of leukemia in serum, irrespective of the means by which this target is achieved.
  • the primary criteria are the curve shape and the absolute value of the active ingredient concentration, while the time axis can be gathered or stretched compared to the in vivo situation.
  • a third disadvantage of all known in vitro methods for drug testing is that no combination therapy can be simulated. This is of crucial importance because today only one cytostatic agent, i.e. monotherapy, is used as an exception, most of the therapies are combination therapies in which a chronological sequence of different cytostatics is used, as from the Compendium Internistic Oncology, publisher HJ . Schmoll, K. Höffgen, K. Possinger, Springer Verlag, (1996). With the standard methods in vitro it is not yet possible, as in Organism, once an active substance has been introduced into the assay, is removed again before the next active substance is administered.
  • the object of the present invention is to provide an in vitro method for testing active substances on cells and a device which can be used for the said method, whereby the aforementioned disadvantages are at least substantially reduced and in particular as an approximation the in vivo pharmacokinetics is made possible by corresponding in vitro pharmacokinetics and a combination therapy with different active substances can be simulated.
  • a method for in vitro testing of active substances on cells comprising at least the steps a) making available a cell culture container with an interior and an inner wall and with a first and second membrane system arranged in the interior, between the membrane systems and the inner wall a cell culture space is formed in the interior, b) presenting a cell culture and a cell culture medium in the cell culture space, c) feeding a liquid nutrient medium into the cell culture space and removing metabolic products by means of the first membrane system, d) supplying at least one gaseous medium into the cell culture space by means of the second membrane system, e) metering at least one active substance into the cell culture space, the metering taking place in accordance with a set active substance concentration-time curve and f) monitoring the cell vitality.
  • the active substances to be tested are understood to be substances whose effect on the cells to be examined is not known or is not sufficiently known before the test. This can also be gaseous active ingredients, such as Act on respiratory toxins or other possibly toxic gases. Among the substances defined above, cytostatics, antibiotics, cytokines, growth factors or antiviral agents are preferably used. In addition, basically all substances as defined above can be tested with regard to their effect on cells if these substances can be introduced into the cell culture area in dissolved form.
  • cell cultures can be tested which preferably consist of primary cells or cell lines, with tumor cell lines being particularly preferably used.
  • the volume of the cell culture space is preferably at least 0.3 ml and at most 2.0 ml, particularly preferably between 0.5 ml and 1.5 ml.
  • the first membrane system located in the interior of the cell culture container consists of at least one semipermeable membrane which is suitable for supplying a liquid nutrient medium, ie allows continuous mass transport through the membrane wall due to diffusive or convective transport mechanisms.
  • a liquid nutrient medium ie allows continuous mass transport through the membrane wall due to diffusive or convective transport mechanisms.
  • dialysis membranes such as Cuprophan ® or hydrophilic microporous membranes such as microporous polyethersulfone membranes can be used.
  • the second membrane system located in the interior of the cell culture vessel consists of at least one gas transfer membrane, in particular a Oxygenationsmembran as it is for example available as Oxyphan ® commercially.
  • the membranes of the first and second membrane systems are preferably hollow fiber membranes.
  • a cell culture container in which the first and second membrane systems consist of hollow fibers which are arranged in layers in a layer above one another in the interior.
  • the cell culture is introduced when using a cell culture container with a lid by adjusting the cell density in the cell culture medium, opening the lid of the cell culture container, pipetting in the desired volume of the cell suspension into the interior of the cell culture container and closing the cell culture container with the lid.
  • the cell culture can be introduced via an opening in the side wall of the interior, which e.g. is provided with a connection for a syringe or the like located on the outside of the cell culture container.
  • the cell culture can be introduced via at least one septum, via which access into the interior of the cell culture container is possible.
  • the cells can be in the form of suspension cells or cells in need of attachment, suspension cells generally being cells which are derived from the blood. and cells that need to be attached are usually cells that originate from the body tissue.
  • the latter can either be placed as undivided pieces of tissue directly into the interior of the cell culture container or the pieces of tissue are first separated and then placed like suspension cells.
  • the interior of the cell culture container is advantageously provided with a surface to which the cells preferentially adhere. This surface preferably consists of protein-coated polycarbonate or of additional textile material made of polyester which is introduced into the interior of the cell culture container.
  • the cell culture space preferably contains at least 1 ⁇ 10 5 cells per ml cell culture space, particularly preferably at least 1 • 10 6 cells per ml cell culture space.
  • the cell density in the cell culture space is more than 5 ⁇ 10 7 cells per ml cell culture space.
  • suspension cells with a cell density of at least 1 ⁇ 10 5 cells per ml cell culture space are used. This enables the cell densities to be approximated as in the blood.
  • the method according to the invention is preferably carried out at a cell density of at least 1 ⁇ 10 5 cells per ml of cell culture space in order to approach the cell densities of the body tissue after appropriate cell growth.
  • Each cell advantageously has an average distance of 0 ⁇ m to 600 ⁇ m to the closest membrane of the first and second membrane systems.
  • the cells are evenly supplied with nutrient medium and gas.
  • the metabolic products are removed again uniformly, so that overall a condition is simulated in the cell culture that is similar to the in vivo conditions.
  • Media can be used as liquid nutrient medium or as cell culture medium, as are usually used for supplying cells with nutrients or for culturing cells.
  • RPMI 1640 is preferred.
  • a nutrient medium containing RPM1 1640 and fetal calf serum is used.
  • a gaseous medium is preferably used as the gaseous medium, which has an oxygen partial pressure pO 2 of 0 to 160 mmHg and a carbon dioxide partial pressure pCO 2 of 0 to 115 mmHg.
  • the cell culture medium contains a bicarbonate buffer and the pCO 2 in the gaseous medium supplied is adjusted so that the pH of the cell culture medium is between 6.8 and 7.8.
  • the desired composition of the gaseous medium can be set by carrying out the method according to the invention in a room in which this composition prevails.
  • the gaseous medium is fed into the second membrane system via a sterile filter.
  • the second membrane system can also advantageously be used to remove gaseous metabolic products if it is operated in cross-flow mode and is connected to a gas supply line and a gas discharge line.
  • individual active substances and / or combinations of several active substances are metered in at different times.
  • a single active ingredient can be administered over a period of time in several successive doses, or different individual active ingredients can be added to the cell culture medium with a time delay.
  • a sequence of active substance combinations can also be metered in with a time delay, the active substance combinations remaining the same or being able to vary in their composition.
  • individual active substances and combinations of active substances of the type described above can also be metered in with a time delay.
  • the active ingredient can be supplied directly or via the first membrane system, or in the case that it is a gaseous active ingredient, via the second Membrane system in the cell culture room.
  • the active substance is supplied via the first membrane system, the active substance is fed into the culture medium stream and enters the cell culture space with the culture medium via the membranes of the first membrane system.
  • a gaseous active substance is supplied via the second membrane system, it is fed into the gaseous medium and enters the cell culture space with the gaseous medium via the membranes of the second membrane system.
  • the course of the active ingredient concentration over time can be set, for example, by the permeability of the first or second membrane system, by the duration of the addition of active ingredient and by the active ingredient concentration.
  • the procedures described above thus allow the simulation of a wide variety of pharmacokinetics of individual active substances and / or active substance combinations.
  • the active substance addition can take the form of a ramp in the form of a continuous infusion or a peak in the manner of an intravenous administration.
  • the cell culture container is kept at a temperature suitable for culturing the cells, preferably at 37 ° C.
  • monitoring cell vitality is understood to mean monitoring metabolic activity, proliferation, apoptosis or, in general, cell death.
  • a cell vitality dye is used to monitor the cell vitality.
  • Alamar Blue ® is particularly preferred. This substance can be supplied to the cell culture room via the nutrient medium and the first membrane system. Alamar Blue ® penetrates the cell membranes into the interior of the cell, where it is converted into a fluorescent dye by cell metabolism. Therefore, the amount of fluorescent dye so formed can be used as a measure of cell vitality and with with a fluorescence sensor either on-line or after sampling in the nutrient medium. It is particularly advantageous that sampling in the cell culture room is not necessary, but can be carried out in the liquid stream that leaves the cell culture container. A further advantage is that the cell vitality dye can not only be supplied via the first membrane system, but can also be completely removed again, as a result of which any interference in the cell culture by the dye can be corrected and reversed.
  • At least one sensor can be used to monitor the cell vitality, which provides information about the state of the cells.
  • These are preferably miniaturized sensors for determining proliferation, vitality, apoptosis or in general cell death. It is particularly preferably a sensor for fluorescence.
  • Suitable sensors can be used for process monitoring in the method according to the invention. Sensors for monitoring the temperature, pH, partial pressure of oxygen pO 2 , or carbon dioxide pCO 2 , glucose or lactate are preferably used. In a particularly preferred embodiment, the sensors are integrated into the interior of the cell culture container as microsensors, as a result of which there is no disruptive influence of the sensors on the cell culture. One or more of the sensors mentioned can also be used in the cell culture room or in the nutrient medium and the sensor signals can be tracked online. For example, pH, p0 2 , glucose and lactate can be measured simultaneously.
  • a device which comprises a cell culture container with an interior suitable for receiving a cell culture in a cell culture medium, at least first means for feeding in the interior a nutrient medium and second means for supplying at least one gaseous medium are arranged, the means each having a supply side and a discharge side, and wherein a cell culture space is formed between said means and the inner wall of the interior, and wherein the first means with their supply side via a
  • the nutrient medium metering unit is in fluid communication with at least one nutrient medium container, and the second means are in fluid communication with their supply side via a gas metering unit with at least one gas storage container, and the device is characterized in that the cell culture space has a volume of at most 5 ml and at least 0.1 ml has that the device further contains means for supplying at least one active substance into the cell culture space and means for specifying an active substance concentration-time curve in the cell culture space.
  • the discharge means are in fluid connection with their discharge side with a waste container.
  • the discharge means are in fluid connection with their discharge side via a recirculation line with the at least one culture medium container.
  • a device that can isolate or detect certain metabolic products can be installed in the line between the discharge side of the first means and the waste container.
  • the first means located in the interior of the cell culture container preferably consist of at least one membrane suitable for the supply of liquid nutrient media.
  • the at least one membrane of the first means preferably consists of a semipermeable membrane which is suitable for supplying a liquid nutrient medium, ie which permits continuous mass transport through the membrane wall due to diffusive or convective transport mechanisms.
  • a semipermeable membrane which is suitable for supplying a liquid nutrient medium, ie which permits continuous mass transport through the membrane wall due to diffusive or convective transport mechanisms.
  • it is a nanofiltration, ultrafiltration or microfiltration membrane.
  • dialysis membranes such as Cuprophan ® or hydrophilic microporous membranes such as microporous polyethersulfone membranes can be used.
  • the second means located in the interior of the cell culture container preferably consist of at least one membrane suitable for gas exchange.
  • the at least one membrane of the second means preferably consist of a Oxygenationsmembran, more preferably of at least one Oxyphan ® membrane.
  • the second means are in fluid communication with a gas storage container.
  • the gas storage container from the gas space e.g. of an incubator and the gas space and the second means are in contact via a gas-permeable sterile filter.
  • the second means are in fluid communication with at least one gas storage container under gas pressure via at least one gas metering unit. In this way, different concentrations of different gas components can be set in a simple manner.
  • the membranes of the first and second means are designed as hollow fibers.
  • the hollow fibers are particularly preferably arranged in a plurality of layers in the interior in layers.
  • the maximum distance between the hollow fibers forming the respective means is between 50 ⁇ m and 600 ⁇ m.
  • the cell culture container has a bottom and a lid which delimit the interior, lie opposite one another and consist of a transparent material. The transparency of the bottom and lid allows microscopic observation of the cells during the drug test.
  • a heating system suitable for thermostating the cell culture container to 37 ° C, e.g. in the form of a heating foil, which allows sufficient transparency for, for example, microscopic observation of the cell culture.
  • the cell culture room preferably has a volume of 0.3 ml to 3.0 ml.
  • the advantage of this miniaturization is the particularly low consumption of cells, active substances, liquid and gaseous media.
  • the means for supplying active substance preferred in the device according to the invention consist of at least one active substance storage container, at least one active substance dosing unit and a line system which connects the at least one active substance storage container via a respective active substance dosing unit either directly or via the first means to the cell culture space of the cell culture container.
  • the means for specifying an active ingredient concentration time course in the cell culture space consist in the permeabilities of the membranes of the first means.
  • the device according to the invention comprises a means for monitoring cell vitality.
  • a particularly preferred embodiment of the device according to the invention contains, as a means for monitoring cell vitality, at least one sensor which is suitable for providing information about the state of the cell culture.
  • At least one sensor which is suitable for providing information about the state of the cell culture.
  • sensors for determining proliferation, vitality, apoptosis or in general cell death. It is particularly preferably a sensor for fluorescence.
  • the device according to the invention can contain sensors suitable for process monitoring, preferably sensors for temperature, pH, partial pressure of oxygen pO 2 , or carbon dioxide pCO 2 , glucose or lactate, which are attached in the interior of the cell culture container.
  • the sensors mentioned can be arranged individually or in combination in the interior of the cell culture container.
  • the object on which the present invention is based is further achieved by a modular active substance testing system which comprises at least 2 of the devices according to the invention.
  • the modular active substance testing system preferably comprises 6, 24 or 96 of the devices according to the invention, which are suitably arranged to form a modular structure.
  • the object on which the present invention is based is achieved by using the device according to the invention or the active substance testing system according to the invention for in vitro testing of the effect of active substances on cells.
  • the device according to the invention or the modular active substance testing system according to the invention can be used excellently to determine the influence of pharmacokinetics on the vitality of cells.
  • FIG. 1 flow diagram of a device according to the invention
  • FIG. 2a cross section of a modular active substance testing system with devices according to the invention
  • Figure 2b cross section of a modular drug testing system with three superposed levels with devices according to the invention
  • Figure 3 Top view of a modular drug testing system comprising six devices according to the invention.
  • FIG. 4a three active substance concentration-time profiles referred to as profiles 1 to 3.
  • Figure 4b Representation of the vital cells in four different cell culture containers as presented (inoculum) and as harvested (cell harvest).
  • FIG. 1 shows a culture medium container 4 which is fluid-connected via a line 14 and a culture medium metering unit 3 to the supply side of the first agent contained in the interior 2 of the cell culture container 1.
  • the discharge side of the first means located in the interior 2 of the cell culture container 1 is fluidly connected to a waste container 10 via the line 14.
  • a device 12 which can isolate or detect certain metabolic products.
  • the recirculation line 11 enables the liquid flowing from the discharge side of the first agent located in the interior 2 of the cell culture container 1 to be returned to the nutrient medium container 4.
  • An active substance storage container 7 is connected via a line system 9, an active substance metering unit 8 and a line 9b to the supply side of the Interior 2 of the cell culture container 1 located first means fluid-connected.
  • an active ingredient reaches the interior 2 of the cell culture container 1 via the first means.
  • the switching element 9c and the line 9a enable a direct fluid connection between an active substance storage container 7 and the interior 2 of the cell culture container 1.
  • At least one gas storage container 6 is fluidly connected via a gas metering unit 5 to the supply side of the second means located in the interior 2 of the cell culture container 1, the discharge side of which is connected to a gas discharge line 6a.
  • FIG. 2a shows in cross section a modular active substance testing system which is fixed in a holder 13.
  • a nutrient media container 4 is fluidly connected to a nutrient medium line 14 via a nutrient medium metering unit 3 designed here as a peristaltic pump, with the supply side of the first means 1a in the interior of the cell culture container 1, the connection between the nutrient medium metering unit 3 and the supply side of the first means 1a being realized by the lines 14 and 9b .
  • the discharge side of the first means 1a in the interior of the cell culture container 1 is fluidly connected to a waste container 10 via a line 14.
  • the discharge sides of several of the devices according to the invention can also be connected to a common waste container.
  • the cell culture container 1 is placed on a base plate 15 and fastened thereon with a locking mechanism.
  • a heating foil which is suitable for tempering the cell culture container and the medium to the required temperature, preferably to 37 ° C., shortly before it enters the cell culture container.
  • the heating foil can also be integrated into the bottom of the cell culture container 1, as long as this integration permits sufficient transparency of the cell culture container bottom, which is advantageous for visual or microscopic observation of the cell culture.
  • devices 16 which are suitable for taking a sample and can be designed, for example, as a septum.
  • the active substance storage container 7 is connected via the line system 9, the active substance metering unit 8 and the line 9b to the supply side of the first means 1a located in the cell culture container 1.
  • the liquids carrying lines are preferably silicone hoses with an inner diameter of 1 mm.
  • FIG. 2b shows in cross section a modular active substance testing system with three levels arranged one above the other, each in a holder 13, which contains devices according to the invention.
  • FIG. 2b illustrates that a large number of these devices can be provided by means of the modular arrangement of the devices according to the invention, as a result of which a large number of in vitro active substance tests can be carried out simultaneously.
  • FIG. 3 shows a top view of a modular active substance testing system in a holder 13, which fixes six devices according to the invention. Five of these devices are identical to the devices described in Figure 2a.
  • the sixth device shown at the bottom in FIG. 3, shows an arrangement 17 suitable for combination therapy, the three active substance storage containers 18a, 18b and 18c of which are fluid-connected to the supply side of the first means via separate active substance pumps 19a, 19b and 19c. Together with the active substance storage container 7 and the active substance metering unit 8, a combination therapy with four active substances can thus be carried out in the cell culture container of the lowest device according to the invention from FIG.
  • the devices of the modular drug testing system close nutrient media, cell cultures, drugs and waste solutions to the outside world. All parts of the device that come into contact with active substance are preferably designed as single-use articles. Therefore, each device of the modular system can be removed individually from said system by means of the locking mechanism already mentioned, without the operating personnel coming into contact with the partially highly toxic active ingredients. All parts that come into contact with the nutrient medium, active ingredient and cell culture must be sterilizable. Sterile operation of the device over 10 days has been demonstrated.
  • the modular arrangement of the devices allows a very large number of drug tests with different pharmacokinetics and drug combinations, to which the possibility of the modular system also contributes, to choose the number and occupancy of the individual devices in a variety of ways.
  • reference devices without the addition of active ingredient can be operated in parallel with devices with active ingredients.
  • the modular structure of the system from individual devices also has the advantage that the individual devices can be manipulated individually.
  • the same manipulation can also be carried out in different channels (multiple measurements).
  • a modular active substance testing system consisting of 6 devices weighs less than 10 kg and can easily be carried by one person. Because of the small dimensions, for example, a modular active substance testing system consisting of 24 devices can also be operated in a commercially available CO 2 incubator.
  • a personal computer is preferably used for system control, sample identification, data acquisition and data evaluation, on the screen of which the measured values currently present can be followed.
  • a data comparison between individual channels is also possible.
  • the evaluation software is just as capable of trend analysis as it is of analyzing the difference between reference and drug channels. The results can be evaluated and saved in relation to the patient.
  • the following example shows how the modular drug testing system can be used to measure the influence of pharmacokinetics on the vitality of cells.
  • the leukaemic cell line CCRF CEM is placed in a density of 1 »10 7 per ml cell culture medium (RPM1 1640 and 10 vol.% Fetal calf serum based on RPMI1640) and in a volume of 300 ⁇ l in four cell culture containers of a modular active substance test system according to the invention from 4 devices according to the invention .
  • the modular active substance testing system is enclosed in an incubator, in which a temperature of 37 ° C and a gaseous medium consisting of 5% CO 2 , 74% N 2 and 21% O 2 is present.
  • the gaseous medium just mentioned is supplied diffusively via sterile filters into the second membrane systems designed as Oxyphan ® in the interior of cell culture containers 1 to 4.
  • RPMI 1640 and 10% by volume fetal calf serum based on RPM11640 are used as the nutrient medium.
  • the nutrient medium at a flow rate of 7 ml / min is recirculated, whereby the leukemic cell lines as on the Cuprophan ® - are powered formed hollow fibers membranes of the first membrane system with nutrient medium.
  • the Nährmedienzucht is interrupted and the cytostatic idarubicin in three different active ingredient concentration-time comparison runs at a flow rate of the nutrient medium of 0.2 ml per minute over the Cuprophan ® membranes in the respective cell culture container of the devices of the invention as hereinafter dosed as described:
  • the active ingredient concentration-time profiles are designated as profiles 1 to 3 in FIG. 4a).
  • Profile 1 A solution of 0.20 ug per ml idarubicin the aforementioned cell culture medium in a period of 75 minutes through the Cuprophan ® - directed hollow fibers of the cell culture vessel.
  • Profile 2 A solution of 0.50 ug per ml idarubicin the aforementioned cell culture medium in a period of 20 minutes by the Cuprophan ® - directed hollow fibers of the cell culture vessel. 2 Then a solution of 0.25 ug per ml idarubicin is the above-mentioned cell culture medium in a period of 20 minutes by the Cuprophan ® - directed hollow fibers of the cell culture vessel. 2 Profile 3: A solution of 1, 00 ug idarubicin per ml of the above cell culture medium, in a period of 15 minutes by the Cuprophan ® - passed the cell culture container 3 hollow fibers.
  • No idarubicin is metered into the interior of the cell culture container 4.
  • This cell culture container serves as a control.
  • the active substance was added in such a way that the same area under the respective curve (area under curve, AUC) resulted for all of the active substance concentration-time profiles described above.
  • the cell culture container is rinsed for 1 hour with fresh nutrient medium (RPMI 1640 and 10% by volume fetal calf serum based on RPMI 1640) by passing the nutrient medium through the membranes of the first membrane system at a flow rate of 0.2 ml / min and the liquid stream emerging from the membranes is directed into the respective waste container.
  • fresh nutrient medium RPMI 1640 and 10% by volume fetal calf serum based on RPMI 1640
  • the nutrient medium recirculation is then resumed at a flow rate of 7 ml / min. After 72 hours, the nutrient medium used so far is replaced by the same but fresh nutrient medium. After 96 hours, the cells are harvested from the four cell culture containers and the number of vital cells and, as vitality, the percentage of the number of harvested vital cells in the total number of harvested cells determined according to the following relationship:
  • FIG. 4b behind “Inoculum”, the number of vital cells presented in the cell culture containers 1 to 4 is shown. Since the cells were placed in a volume of 300 ⁇ l and in a cell density of 1 ⁇ 10 7 per ml cell culture medium, the number is presented vital cells in the cell culture containers 1 to 4 30 • 10 5. The number of vital cells obtained after the cell harvest is plotted behind “cell harvest” in FIG. 4b. It can be seen that the profile of the active ingredient concentration over time reduced the number of vital cells the most.
  • the vitalities of the cells from the cell culture containers 1 to 4 were determined with the following result:

Abstract

L'invention concerne un procédé permettant de tester in vitro des principes actifs sur des cellules, qui comprend comme étapes au moins le fait de mettre à disposition un récipient pour cultures cellulaires comportant une chambre intérieure et une paroi intérieure, ainsi qu'un premier et un second système membranaire disposés dans la chambre intérieure. Une chambre de culture cellulaire est formée entre les systèmes membranaires et la paroi intérieure de la chambre intérieure. Il est prévu de placer une culture cellulaire et un milieu de culture cellulaire dans la chambre de culture cellulaire, d'introduire un milieu nutritif liquide dans la chambre de culture cellulaire et d'évacuer des métabolites à l'aide du premier système membranaire. Il est également prévu d'introduire au moins un milieu gazeux dans la chambre de culture cellulaire à l'aide du second système membranaire, d'acheminer au moins un principe actif dans la chambre de culture cellulaire, cet apport s'effectuant d'après une courbe temps-concentration en principe actif ajustée et de surveiller la vitalité cellulaire. L'invention concerne en outre un dispositif approprié et l'utilisation dudit dispositif pour tester l'effet de l'idarubicine sur la ligne cellulaire leucémique CCRF CEM.
EP00907670A 1999-03-09 2000-03-08 Procede pour tester des principes actifs in vitro, dispositif approprie et son utilisation Withdrawn EP1159443A1 (fr)

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DE19910540 1999-03-09
DE19910540 1999-03-09
PCT/EP2000/002011 WO2000053797A1 (fr) 1999-03-09 2000-03-08 Procede pour tester des principes actifs in vitro, dispositif approprie et son utilisation

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US6429013B1 (en) 1999-08-19 2002-08-06 Artecel Science, Inc. Use of adipose tissue-derived stromal cells for chondrocyte differentiation and cartilage repair
NL1017973C2 (nl) * 2000-05-10 2002-11-08 Tristem Trading Cyprus Ltd Inrichting.
EP1357922B1 (fr) * 2001-02-07 2011-05-25 McComb Foundation Inc. Technique de preparation de suspension cellulaire et utilisation
AUPR298901A0 (en) 2001-02-07 2001-03-08 McComb Foundation, Inc., The Cell suspension preparation technique and device
CN104471052B (zh) * 2012-11-13 2017-06-13 海马生物科学公司 用于基于控制介质流动的三维组织测量的装置和方法
AU2013205148B2 (en) 2013-03-14 2014-10-30 AVITA Medical Americas, LLC Systems and methods for tissue processing and preparation of cell suspension therefrom

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US4661458A (en) * 1983-08-31 1987-04-28 Cell Environmental Systems, Ltd. Cell culture system
US4748124A (en) * 1984-10-30 1988-05-31 E. I. Du Pont De Nemours And Company Compartmentalized cell-culture device and method
JPH0292270A (ja) * 1988-09-30 1990-04-03 Terumo Corp 細胞培養装置
US4937196A (en) * 1989-08-18 1990-06-26 Brunswick Corporation Membrane bioreactor system
FR2771421B1 (fr) * 1997-11-27 2001-05-04 Bertin & Cie Dispositif d'amplification de cellules hematopoietiques et ses applications
DE19810901C1 (de) * 1998-03-13 1999-06-17 Ascalon Gesellscchaft Fuer Inn Bioreaktor

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