MICROFLUIDIC DEVICE FOR CELL CULTURE EXPERIMENTS AND USES THEREOF

Description

[0001] According to the invention, a microfluidic device is presented, comprising at least one first culture chamber containing cardiac muscle cells, at least one microfluidic channel, at least one pump (e.g., a micropump), and at least one detector, wherein the detector is configured to detect activity of the cardiac muscle cells contained in the culture chamber. The microfluidic device further comprises at least one control unit configured to control the at least one pump based on activity of the cardiac muscle cells detected by the at least one detector. Further uses of the microfluidic device are proposed.

[0002] Many research groups worldwide are working on the development of artificial heart tissue. New impetus is expected, particularly for animal-free drug testing, as cell-based systems enable patient-centered and individualized drug discovery. Current in-vitro platforms are mostly too simple in design (two-dimensional, no perfusion, lacking a microenvironment) to adequately replicate the physiological behavior of heart tissue. Therefore, there is a need for microfluidic devices, also called microphysiological systems (MPS), that replicate the microenvironment and provide three-dimensional tissue spaces that more closely resemble human tissue. The goal is, on the one hand, to create organ models that are as close as possible to in-vivo models, and on the other hand, to cultivate several such models in a closed circulatory system to enable systemic drug testing (so-called "multi-organ chip").

[0003] Under physiological conditions, cardiac muscle cells begin spontaneous or (electrically) stimulated movement, which can be influenced by the prevailing microenvironment (hypoxia, nutrient deficiency), metabolic products, or the addition of substances. Heart rate, contraction velocity (or relaxation velocity), contraction force, contraction duration, rhythmicity, and maximum deformation are pharmacologically relevant parameters that can be determined, for example, by microscopic observation followed by video analysis. Based on these parameters, the human body possesses a complex regulatory mechanism that is lacking in current microfluidic devices. For example, existing microfluidic devices cannot simulate the fact that, as a self-sustaining organ, the heart's own blood supply decreases when its pumping capacity is reduced (which, in turn, can lead to a further decrease in pumping capacity).

[0004] In the current state of the art, a number of different in vitro cardiac muscle systems are known, differing in size (single cells to macroscopic 3D tissues), origin (e.g., primary rodent cells, cell lines based on stem cells), structure (isotropic or anisotropic), and also in the implementation of perfusion. In most cases, the replication of cardiac muscle focuses on its use as an "electromechanical sensor" to directly determine the effects of drugs or stimuli on the tissues. Only a few studies have actually used the reconstructed cardiac muscle tissue to generate perfusion. Saeed Rismani Yazdi et al.: "Adding the 'heart' to hanging drop networks for microphysiological multi-tissue experiments", LAB ON A CHIP, Vol. 15, No. 21, 1.January 2015 (2015-01-01), pages 4138-4147, discloses microfluidic devices comprising pumps, culture chambers with cardiac muscle cells and detectors, wherein the pumping speed is regulated based on the cardiac muscle activity detected by the detector.

[0005] Due to the low forces generated by the microcardiac muscles, direct generation of perfusion (cardiomyocyte pump) is not practical due to the high fluidic resistance of microchannels.

[0006] The detection of contraction movements or contraction forces in in-vitro cardiac muscle systems is implemented in various ways in the current state of the art. For example, flexible spring bars, 3D-printed strain gauges, and optical analysis methods are used. Furthermore, electrophysiological sensors such as microelectrode arrays (MEAs), patch-clamp electrodes, sharp electrodes, and calcium imaging are employed.

[0007] German patent DE 10 2013 011 768 A1 discloses a circulation system and presents a method for supplying vital fluids to cell cultures in a microfluidic network. In this microfluidic network, a defined supply to several cell culture compartments is achieved via throttles and / or valves. These throttles and / or valves allow, for example, the adjustment and regulation of pH, oxygen partial pressure, carbon dioxide partial pressure, and / or glucose concentration.

[0008] Multilayer microfluidic devices with integrated active components (such as pumps and oxygenators) for the direct cultivation of cardiac muscle cells have also been realized.

[0009] Furthermore, a module with a feedback loop for controlling and adjusting defined gas compositions is known in the prior art.

[0010] Up to now, the MPS systems known in the art using cardiac muscle cells have the disadvantage that they do not, or only inadequately, represent the influence of cardiac muscle cell activity on the fluid flow in the microfluidic device. In other words, the known systems or devices do not allow a specific cardiac muscle cell activity present at a specific time to be translated practically immediately (i.e., without time delay) into a fluid flow in the system corresponding to that activity, as is indeed mediated in vivo by the regulatory mechanism of the cardiovascular system.As a consequence of this disadvantage, the true in-vivo state of a living patient can only be inadequately simulated with currently known microfluidic devices using heart cells, since in a living patient a change in the activity of the heart muscle cells can sometimes have a very strong effect on the fluid flow of the circulatory system and thus on the blood and oxygen supply of individual cells and organs.

[0011] Based on this, the object of the present invention was to provide a microfluidic device with cardiac muscle cells that allows a more accurate and realistic investigation of the influence of certain factors on the activity of cardiac muscle cells and also enables the activity of other biological cells (e.g. liver cells or nerve cells) to be recorded as realistically as possible depending on the changed cardiac muscle cell activity (e.g. under the conditions of an arrhythmia or cardiogenic shock).

[0012] The problem is solved by the microfluidic device having the features of claim 1 and the uses according to claim 15. The dependent claims describe advantageous embodiments.

[0013] According to the invention, a microfluidic device is provided, containing a) at least one first culture chamber containing cardiac muscle cells; b) at least one microfluidic channel; c) at least one pump (e.g. a micropump) for conveying a fluid through the at least one microfluidic channel and the at least one culture chamber; d) at least one detector configured to detect activity of the cardiac muscle cells contained in the culture chamber; wherein the microfluidic device further comprises at least one control unit configured to control the at least one pump based on the activity of the heart muscle cells detected by the at least one detector, wherein the microfluidic device comprises at least one device for influencing the activity of the heart muscle cells contained in the culture chamber, characterized in that the device for influencing the activity of the heart muscle cells contained in the culture chamber comprises an electrode.

[0014] The term "activity of cardiac muscle cells" refers specifically to electrical activity and / or motor activity of cardiac muscle cells (preferably both), whereby the term also includes zero electrical activity (no action potential) and zero motor activity (rest of movement). Motor activity of cardiac muscle cells refers specifically to contractility and / or the movement rate of cardiac muscle cells (preferably both).

[0015] In the device according to the invention, the activity of cardiac muscle cells can be detected and, via a feedback loop to the pump, the amount of fluid delivered (perfusion) can be adjusted according to the activity of the cardiac muscle cells. This measure allows not only a more realistic study of the influence of altered perfusion on the cardiac muscle cells themselves, but also of the influence of the altered perfusion on other biological cells located in further culture chambers within the microfluidic device.

[0016] The feedback loop allows the complex regulatory mechanism of the cardiovascular system of living organisms to be replicated more accurately than with previous systems or devices in the prior art. In other words, the device according to the invention makes it possible to make more realistic and, with regard to the actual in-vivo situation, more relevant statements about the influence of certain factors on the cardiovascular system. Furthermore, the fundamental possibility of circulating fluid in the microfluidic device opens up the possibility of more accurately predicting the risk of delayed secondary diseases, since certain substances or metabolic products can have an effect over a long period.

[0017] Furthermore, the device according to the invention can realistically simulate the effect of altered activity of heart muscle cells on other tissue types (e.g. liver tissue, nerve cells, etc.), which is of crucial importance in particular for systemic testing of (e.g. chemical, biochemical or biological) substances.

[0018] The microfluidic device can be characterized in that the at least one control unit is configured to increase the pump flow rate of the at least one pump when the cardiac muscle cells are highly active, preferably by increasing the pump frequency and / or the pump stroke. Furthermore, the at least one control unit can be configured to decrease the pump flow rate of the at least one pump when the cardiac muscle cells are weakly active, preferably by decreasing the pump frequency and / or the pump stroke.

[0019] The at least one control unit can further be configured to decrease or increase the perfusion of the at least one first culture chamber containing cardiac muscle cells, preferably by opening or closing a short-circuit channel fluidically connected in parallel to the at least one first culture chamber. Furthermore, the at least one control unit can be configured to decrease or increase the perfusion of at least one second culture chamber in the microfluidic device, preferably by opening or closing a short-circuit channel fluidically connected in parallel to the at least one second culture chamber.

[0020] The first culture chamber, at least one of which may contain cardiac muscle cells forming at least one cardiac muscle fiber, wherein the cardiac muscle cells of the at least one cardiac muscle fiber are preferably anisotropically oriented. Furthermore, the culture chambers may contain biological cells other than cardiac muscle cells, preferably cells selected from the group consisting of fibroblasts, endothelial cells, and combinations thereof. In addition, the cardiac muscle cells may contain or be embedded in a hydrogel.

[0021] In a preferred embodiment, the microfluidic device has at least one reservoir containing a nutrient solution for feeding cardiac muscle cells, wherein the reservoir is preferably fluidically connected to the at least one microfluidic channel and the at least one culture chamber, and the at least one pump is particularly preferably configured to deliver the nutrient solution to the at least one culture chamber, in particular via the at least one microfluidic channel.

[0022] The microfluidic device can be configured to increase the outflow of nutrient solution from the reservoir during strong activity of the cardiac muscle cells, preferably by increasing the pumping frequency and / or the pump stroke and / or by pressurizing the reservoir. Furthermore, the microfluidic device can be configured to decrease the outflow of nutrient solution from the reservoir during weak activity of the cardiac muscle cells, preferably by decreasing the pumping frequency and / or the pump stroke and / or by lowering the pressure at the reservoir.

[0023] The microfluidic channel can contain at least one valve and / or at least one throttle. The at least one valve and / or the at least one throttle preferably contains an elastic membrane, which is preferably arranged in at least one wall of the at least one microfluidic channel and particularly preferably contains or consists of plastic, most preferably a plastic selected from the group consisting of thermosets, thermoplastics, elastomers and combinations thereof, in particular a plastic selected from the group consisting of PC, PET, COC, PDMS, TPE and combinations thereof. Furthermore, the elastic membrane can be suitable for being controlled by a pneumatic, thermopneumatic, electromagnetic, electrostatic, magnetic, chemical and / or piezoelectric force, wherein the valve and / or throttle is preferably equipped with at least one actuation channel (e.g.is connected to a pneumatic channel and / or a hydraulic channel) and / or at least one voltage source.

[0024] The at least one control device can be configured to control the at least one valve and / or the at least one throttle based on the activity of the cardiac muscle cells detected by the at least one detector. In this case, the at least one control device is preferably configured to at least partially open the at least one valve and / or the at least one throttle when there is strong activity of the cardiac muscle cells. Furthermore, the at least one control device can be configured to at least partially close the at least one valve and / or the at least one throttle when there is weak activity of the cardiac muscle cells.

[0025] The microfluidic device can include at least one oxygenator for oxygenating or deoxygenating the liquid in the microfluidic device, preferably a gas-permeable membrane and / or a hollow fiber. Furthermore, the microfluidic device can include an O₂ sensor for measuring the O₂ content of the liquid in the microfluidic device, preferably an O₂ sensor selected from the group consisting of optical O₂ sensors, electrochemical O₂ sensors, and combinations thereof.

[0026] The at least one control unit can also be configured to control the oxygenator depending on an O2 content measured via the O2 sensor and / or depending on the activity of the heart muscle cells detected by the at least one detector.

[0027] In a preferred embodiment, the detector includes or consists of an optical detector, preferably an optical detector configured to measure calcium concentration. In the case of an optical detector, the culture chamber of the microfluidic device is at least partially transparent to light of the wavelength of the optical detector (preferably the visible spectrum). For example, the optical detector can include a microscope camera, which is preferably coupled to a real-time evaluation unit, wherein the evaluation unit is preferably configured to determine pharmacologically relevant parameters.

[0028] Furthermore, the detector can include or consist of an electrical detector, preferably a multi-electrode array (MEA). The electrical sensors can perform time-of-flight measurements. A benefit of non-optical detection is the reduced hardware complexity required for image processing. In addition, the detector can include or consist of a mechanical detector, preferably one selected from the group consisting of strain gauges, contact force microscopes, spring beams, and combinations thereof. In a certain sense, the microfluidic device in the case of these detectors can be considered a cell-based (chemo-)(electro-)(opto-)mechanical transducer that responds to changes in the microenvironment, metabolic products, or substance exposure.

[0029] The detector can be configured to send signals about the activity of the cardiac muscle cells contained in the at least one first culture chamber to a data acquisition device, the data acquisition device preferably being configured to record and evaluate the signals as a function of time.

[0030] According to the invention, the microfluidic device includes at least one device for influencing the activity of the cardiac muscle cells contained in the at least one first culture chamber. This allows diseases of the cardiovascular system, such as heart failure, cardiac arrhythmias, etc., to be artificially induced.

[0031] According to the invention, the device for influencing the activity of cardiac muscle cells comprises an electrode, preferably at least one electrode selected from the group consisting of immersion electrodes, planar electrodes, or a combination thereof. In particular, the device comprises or consists of a multi-electrode array. Electrical contact with the cardiac muscle cells via this device offers the possibility of electrically stimulating the cardiac tissue, e.g., to simulate certain conditions such as physical exertion. The advantage of multi-electrode arrays (MEAs) is that they can also serve as electrical sensors, i.e., they can simultaneously stimulate the cardiac muscle cells and record their activity.

[0032] Furthermore, the device for influencing the activity of the cardiac muscle cells contained in the at least one first culture chamber can include at least one actuator, particularly preferably an actuator selected from the group consisting of piezoelectric actuators, electromechanical actuators, pneumatic actuators, hydraulic actuators, surface tension actuators and combinations thereof.

[0033] Furthermore, the device for influencing the activity of the cardiac muscle cells contained in the culture chamber can include a device for supplying or removing gas, preferably a gas exchange membrane, wherein the device for supplying or removing gas preferably has a source of a gas selected from the group consisting of (pure) oxygen, air (e.g. compressed air), nitrogen, carbon dioxide and combinations thereof, in particular a source of a gas selected from the group consisting of air (e.g. compressed air), nitrogen, carbon dioxide and combinations thereof.

[0034] In a particularly preferred embodiment, the microfluidic device includes at least one access point for introducing a substance (e.g., chemical, biological, and / or biochemical). This embodiment is advantageous for pharmaceutical drug testing. Optionally, this access point leads directly into the at least one microfluidic channel, directly into the at least one first culture chamber, and / or directly into the at least one reservoir. The application of one or more chemical, biochemical, or biological substances can thus be carried out directly onto the tissue or indirectly (e.g., through the inlets and outlets of the microfluidic device) via the flowing medium.

[0035] The microfluidic device can include at least one second microfluidic channel, which preferably branches off upstream of the at least one first culture chamber from the at least one first microfluidic channel and opens downstream of the at least one first culture chamber into the at least one first microfluidic channel.

[0036] Furthermore, the microfluidic device can have at least one second culture chamber containing biological cells that differ from cardiac muscle cells. Preferably, these cells are selected from the group consisting of liver cells, kidney cells, nerve cells, adipose tissue, and combinations thereof. The at least one second culture chamber is particularly preferably fluidically connected to the at least one culture chamber, optionally via at least one second microfluidic channel. Upstream of the second culture chamber, a throttle or valve for flow control can be included. The microfluidic device can furthermore have more than two such culture chambers, each of which can have these features. The culture chambers can also each be configured as separate subordinate microfluidic devices connected to a microfluidic channel of the superior microfluidic device, e.g.,are connected via a plug connection.

[0037] The microfluidic device can include at least one second detector configured to detect the activity of the biological cells contained in the second culture chamber. The at least one second detector preferably includes or consists of an optical detector and is particularly preferably configured to send signals about the activity of the biological cells contained in the at least one second culture chamber to a data acquisition device, wherein the data acquisition device is preferably configured to record and evaluate the signals over time.

[0038] In a preferred embodiment, the microfluidic device, preferably the at least one microfluidic channel, which contains at least one first culture chamber and / or at least one reservoir, blood or components of blood, preferably blood cells, particularly preferably cancer cells and / or immune cells, in particular metastatic cancer cells and / or monocytes.

[0039] The at least one control device can include a flow velocity measuring device, preferably a particle image velocimetry device, wherein the microfluidic device is particularly preferably configured to control the at least one pump, preferably all pumps, of the microfluidic device based on a flow velocity measured by the flow velocity measuring device. Furthermore, the microfluidic device can particularly preferably be configured to control at least one valve and / or at least one throttle, preferably all valves and / or all throttles, of the microfluidic device based on a flow velocity measured by the flow velocity measuring device.

[0040] The microfluidic device can contain or consist of plastic. The plastic preferably contains or consists of at least one structured plastic film laminated with at least one further, optionally unstructured, plastic film. Furthermore, the plastic can be selected from the group consisting of thermosets, thermoplastics, elastomers, and combinations thereof, particularly preferably from a plastic selected from the group consisting of PC, PET, COC, PDMS, TPE, and combinations thereof. In addition, the plastic can be at least partially structured by a process selected from the group consisting of laser structuring, cutting plotters, hot stamping, milling, thermoforming, injection molding, soft lithography, 3D printing, and combinations thereof.

[0041] The control device can be configured to adapt the distribution of liquid, solid, and gaseous substances in the microfluidic device to the specific needs of cells or cell groups within the microfluidic device, preferably via selective control of the pump, at least one valve, at least one throttle, and an oxygenator. This selective control is particularly preferably achieved via a mathematical model, which is stored, in particular, in the microfluidic device, preferably in the control device. The mathematical model contains a physical description of the flow and mass transport in the lower- or higher-level microfluidic device, or in the entire microfluidic device.

[0042] The microfluidic device can also include a device for measuring the flow velocity of fluids. This allows the actual flow velocity of the fluid to be monitored. Flow velocity measurement can be performed non-invasively on flowing particles or cells (e.g., blood components) using PIV with an adapted motion-tracking module. However, invasive measurement (e.g., using a thermal anemometer) is also possible.

[0043] Furthermore, the microfluidic device can include a device for measuring the O₂ content in fluids and a gas source for controlling the O₂ content in fluids. This allows for oxygen enrichment of the liquid in the microfluidic device if necessary.

[0044] The microfluidic device can include at least one further microfluidic device that is fluidically connected in parallel or serially to the (first) microfluidic device (e.g., via a single fluidic connection, i.e., a single microfluidic channel). The fluidic connection preferably has at least one fluidic inlet and at least one fluidic outlet (e.g., each in the form of microfluidic channels). A fluid circuit can therefore be formed between the (first) microfluidic device and the further microfluidic device. Here, the (first) microfluidic device can be a superior microfluidic device, and the at least one further fluidic device can be a subordinate microfluidic device. vice versaFurthermore, the microfluidic device according to the invention can itself be divided into, for example, a subordinate part and a superior part. The at least one first culture chamber containing cardiac muscle cells can be contained in a subordinate part of the microfluidic device, and, for example, the at least one pump and / or the at least one detector can be contained in a superior part of the microfluidic device. If the at least one detector is arranged in the superior part, it can be fluidically connected to the subordinate part. In the case of an optical detector, however, this fluidic connection is not necessary. A superior part of the microfluidic device, or a superior, further microfluidic device, can be designed in the form of a layer that contains a subordinate part of the microfluidic device.a subordinate, further microfluidic device is contacted (and . vice versa ) .

[0045] Furthermore, the microfluidic device can have at least two further microfluidic devices that are fluidically connected in parallel or series with the (first) microfluidic device and / or to each other (e.g., each via only a single fluidic connection, i.e., a single microfluidic channel). The fluidic connection preferably has at least one fluidic inlet and at least one fluidic outlet (e.g., each in the form of microfluidic channels). A fluid circuit can therefore be formed between the (first) microfluidic device and the at least two further microfluidic devices. Here, the (first) microfluidic device can be a superior microfluidic device, and the at least two further fluidic devices can each be subordinate microfluidic devices. vice versaFurthermore, at least one, and optionally both, of the at least two further microfluidic devices according to the invention can itself be divided into, for example, a subordinate part and a superior part. Here, at least one further culture chamber can contain cells that are not cardiac muscle cells (e.g., cells selected from the group consisting of liver cells, kidney cells, nerve cells, adipose tissue, and combinations thereof), and these cells can be contained in a subordinate part of the further microfluidic device, and, for example, at least one part different from the further culture chamber can be contained in a superior part of the further microfluidic device. A superior part of the microfluidic device or superior, further microfluidic devices can each be configured in the form of a layer that contains a subordinate part of the microfluidic device or a superior part of the further microfluidic device.subordinate, further microfluidic devices contact (and . vice versa ).

[0046] It is further proposed to use the microfluidic device according to the invention to check whether a certain substance or its metabolites has an effect on the activity of cardiac muscle cells, preferably to check whether the certain substance triggers tachycardia or bradycardia of the cardiac muscle cells or causes arrhythmia of the cardiac muscle cells.

[0047] Furthermore, it is proposed to use the microfluidic device according to the invention to verify whether a mechanical, electrical, and / or gas-induced influence (e.g., via an oxygenator) on the activity of cardiac muscle cells has an effect on the activity of biological cells other than cardiac muscle cells. Preferably, it is verified whether and to what extent this influence has a negative or positive effect on the activity (viability) of the biological cells.

[0048] The following figures are intended to explain the subject matter of the invention in more detail, without limiting it to the specific embodiments shown here. Reference symbol list

[0049] 1.1: Cardiac muscle cells; 1.2: Cells of another organ (e.g., liver cells); 2: Microfluidic channel; 3: Pump (e.g., micropump); 4.X: Detector (e.g., multielectrode array); 4.1: Optical detector (e.g., microscope); 4.2: Electrical detector (e.g., multielectrode array ("MEA")); 4.3: Mechanical detector (e.g., strain gauge); 5: Control unit; 6.X: Data acquisition device (e.g., for recording cardiac muscle cell activity); 6.1: Bradycardia; 6.2: Tachycardia; 7.1: Pump parameter for influencing flow (as a feedback loop signal); 7.2: Flow parameter for influencing flow (as a feedback loop signal); 7.3 Gas composition for the oxygenator (as a signal from the feedback loop); 8: Supply of substance(s); 9: Reservoir; 10: Valve; 11: Throttle; 12: Elastic membrane; 13: Electrical device for influencing the activity of the cardiac muscle cells; 14: Mechanical device for influencing the activity of the cardiac muscle cells; 15: Higher-level microfluidic device; 16.1: First lower-level microfluidic device; 16.2: Second lower-level microfluidic device; 17: Particles (e.g., blood components); 18.X: Structured plastic film; 19: Device for detecting the flow velocity of fluids; 20: Device for detecting the O2 content (e.g., O2 sensor); 21: Oxygenator. 22: Fluidic interface 23. Actuation channel (e.g. channel to a gas source) .

[0050] Figure 1Figure 1 shows an embodiment of a microfluidic device for cell culture experiments according to claim 1. In this embodiment, a microfluidic device is shown which includes a cell culture chamber with cardiac muscle cells 1.1, a microfluidic channel 2, and a pump 3 as a fluidic actuator. For reading the cardiac muscle cell activity, an optical detector 4.1 and / or a multi-electrode array 4.2 are arranged below the cell culture chamber in this embodiment. The signal received by the detectors 4.x is acquired by a data acquisition device 6.x and forwarded to a control unit 5. According to the invention, the received signals are processed in the control unit 5, and the pump parameters 7.1 are adjusted to influence the flow.

[0051] Figure 2Figure 1 shows an embodiment of a microfluidic device for cell culture experiments according to claims 1 and 22. In this embodiment, a microfluidic device is shown which includes at least one microfluidic channel 2 and a pump 3 as a fluidic actuator. Furthermore, several subordinate microfluidic devices are included, each with a cell culture chamber 1.x containing different cell types (cardiac muscle cells 1.1, kidney cells 1.2, etc.). The at least two subordinate microfluidic devices are connected to each other and to the superior microfluidic device via defined fluidic interfaces 22. Fluidic throttles 11 for volume flow regulation can also be arranged upstream of each channel branch. For reading out the cardiac muscle cell activity or physiological parameters of other cell types, an optical detector 4.1 and / or a multi-electrode array 4.1 can be provided in this embodiment.2 and / or a strain gauge 4.3 may be arranged below the cell culture chamber. Furthermore, an O₂ sensor 20 and an oxygenator 21 may be arranged within the microfluidic device. To measure the flow velocity, an optical detector 19 for detecting the movement of circulating particles or cells 17 may be arranged in the microfluidic device. The signal received by the detectors 4.x, 19, and 20 is acquired by a data acquisition device 6.x and forwarded to a control unit 5. According to the invention, the received signals are processed in the control unit 5, and the pump parameters 7.1 for influencing the flow as well as the gas composition for the oxygenator 7.3 are adjusted.

[0052] In this configuration, the cell culture chamber 1.1 further contains an electrode 13 and / or an actuator 14 to influence the activity of the cardiac muscle cells.

[0053] The illustrated microfluidic device can, according to the invention, be used to check whether a specific substance 8 or its metabolites has an influence on the activity of cardiac muscle cells, preferably to check whether the specific substance causes bradycardia 6.1 or tachycardia ( Figure 3 ) 6.2 of the heart muscle cells or causes an arrhythmia of the heart muscle cells can be used.

[0054] Figure 3 shows the microfluidic device made of Figure 2 After adding the substance 8. Based on the changing activity 6.2 of the heart muscle cells due to the addition of the substance, the flow parameters 7.2 and gas composition 7.3 are adjusted accordingly.

[0055] Figure 4Figure 1 shows a microfluidic device according to claims 1 and 22. In this embodiment, it comprises a higher-level microfluidic device 15 comprising a pump 3, at least one microfluidic channel 2, two reservoirs 9, a valve 10, fluidic throttles 11, and an oxygenator 21. The higher-level microfluidic device 15 is connected via defined fluidic interfaces 22 to at least one first lower-level microfluidic device 16.1, and in this case to at least one second lower-level microfluidic device 16.2.

[0056] Figure 5 shows the structure of the microfluidic device according to Figure 4. Containing a superior microfluidic device 15, which consists of several laminated plastic films 18.x, and several subordinate microfluidic devices 16.1, 16.2. In this embodiment, the microfluidic device contains several multi-electrode arrays 4.x.

[0057] Figure 6 Figure 1 shows an embodiment of the fluidic throttle, consisting of at least three (here four) laminated plastic films 18.x, a microfluidic channel 2 and an elastic membrane 12. In this embodiment, the elastic membrane can be deflected via an actuation channel 23. Figure 6 This shows the activated (displaced) and the non-activated (undisplaced) state of the fluidic throttle.

Claims

1. A microfluidic device, comprising a) at least one first culture chamber containing cardiomyocytes; b) at least one microfluidic channel; c) at least one pump for pumping a liquid through the at least one microfluidic channel and the at least one culture chamber; and d) at least one detector, which is configured to detect an activity of the cardiomyocytes contained in the culture chamber; wherein the microfluidic device further comprises at least one control device which is configured to control the at least one pump based on the activity of the cardiomyocytes detected by the at least one detector, wherein the microfluidic device comprises at least one device for influencing activity of the cardiomyocytes contained in the culture chamber, characterized in that the device for influencing activity of the cardiomyocytes contained in the culture chamber comprises or consists of an electrode.

2. The microfluidic device as claimed in claim 1, characterized in that the at least one control device is configured to i) increase the pumping capacity of the at least one pump when there is strong activity of the cardiomyocytes, preferably by increasing the pumping frequency and / or pump lift; and / or ii) decrease the pumping capacity of the at least one pump when there is weak activity of the cardiomyocytes, preferably by decreasing the pumping frequency and / or pump lift; and / or iii) reduce or increase perfusion of the at least one first culture chamber containing cardiomyocytes, preferably by opening or closing a by-pass channel connected fluidically parallel to the at least one first culture chamber; and / or iv) reduce or increase perfusion of at least one second culture chamber in the microfluidic device, preferably by opening or closing a by-pass channel connected fluidically parallel to the at least one second culture chamber.

3. The microfluidic device as claimed in one of the preceding claims, characterized in that the at least one first culture chamber contains cardiomyocytes, which form at least one heart muscle fiber, wherein the cardiomyocytes of the at least one heart muscle fiber preferably i) are oriented anisotropically; and / or ii) comprise biological cells that differ from cardiomyocytes, preferably cells selected from the group consisting of fibroblasts, endothelial cells and combinations thereof; and / or iii) comprise a hydrogel.

4. The microfluidic device as claimed in one of the preceding claims, characterized in that the microfluidic device has at least one reservoir, which contains a nutrient solution for the nutrition of cardiomyocytes, wherein i) the reservoir is connected preferably fluidically to the at least one microfluidic channel and the at least one culture chamber and especially preferably the at least one pump is configured to deliver the nutrient solution to the at least one culture chamber, in particular via the at least one microfluidic channel; and / or ii) the microfluidic device is optionally configured to increase the outflow of nutrient solution from the reservoir when there is strong activity of the cardiomyocytes, preferably by increasing the pumping frequency, the pump lift and / or by pressurizing the reservoir; and / or reduce the outflow of nutrient solution from the reservoir when there is weak activity of the cardiomyocytes, preferably by decreasing the pumping frequency, the pump lift and / or by reducing the pressure on the reservoir.

5. The microfluidic device as claimed in one of the preceding claims, characterized in that the microfluidic channel comprises at least one valve and / or at least one throttle, wherein the at least one valve and / or the at least one throttle preferably i) comprises an elastic membrane, which is preferably arranged in at least one wall of the at least one microfluidic channel and especially preferably comprises or consists of plastic, quite especially preferably a plastic selected from the group consisting of thermoplastic, elastomer and combinations thereof, especially a plastic selected from the group consisting of PC, PET, COC, PDMS, TPE and combinations thereof; and / or ii) is suitable for being controlled by a pneumatic, thermopneumatic, electromagnetic, electrostatic, magnetic, chemical and / or piezoelectric force, wherein the valve and / or the throttle is preferably connected to at least one actuating channel and / or at least one voltage source; wherein the at least one control device is optionally further configured to control the at least one valve and / or the at least one throttle, based on activity of the cardiomyocytes detected by the at least one detector, wherein the at least one control device is preferably configured to i) at least partially open the at least one valve and / or the at least one throttle when there is strong activity of the cardiomyocytes; and / or ii) at least partially close the at least one valve and / or the at least one throttle when there is weak activity of the cardiomyocytes.

6. The microfluidic device as claimed in one of the preceding claims, characterized in that the microfluidic device comprises at least i) an oxygenator for oxygenation or deoxygenation of the liquid in the microfluidic device, preferably a gas-permeable membrane and / or a hollow fiber; and / or ii) an O2 sensor for measuring the O2 content of the liquid in the microfluidic device, preferably an O2 sensor selected from the group consisting of optical O2 sensor, electrochemical O2 sensor and combinations thereof; wherein the at least one control device is optionally further configured to control the oxygenator as a function of the O2 content measured by the O2 sensor and / or as a function of the activity of the cardiomyocytes detected by the at least one detector.

7. The microfluidic device as claimed in one of the preceding claims, characterized in that the detector comprises or consists of i) an optical detector, preferably an optical detector configured to measure the calcium concentration; and / or ii) an electrical detector, preferably a multielectrode array; and / or iii) a mechanical detector, preferably a detector selected from the group consisting of strain gauges, tracking force microscope, spring beam and combinations thereof.

8. The microfluidic device as claimed in one of the preceding claims, characterized in that the detector is configured to send signals about activity of the cardiomyocytes contained in the at least one first culture chamber to a data acquisition unit, wherein the data acquisition unit is preferably configured to record and evaluating the signals as a function of time.

9. The microfluidic device as claimed in one of the preceding claims, characterized in that the device for influencing activity of the cardiomyocytes contained in the culture chamber comprises i) an actuator, especially preferably an actuator selected from the group consisting of piezoelectric actuators, electromechanical actuators, pneumatic actuators, hydraulic actuators, surface tension actuators and combinations thereof; and / or ii) a device for supply or removal of gas, preferably a gas exchange membrane, wherein the device for supply or removal of gas preferably has a source of a gas selected from the group consisting of (pure) oxygen, air (e.g. compressed air), nitrogen, carbon dioxide and combinations thereof.

10. The microfluidic device as claimed in one of the preceding claims, characterized in that the microfluidic device i) comprises at least one access for supplying a chemical, biochemical or biological substance, wherein the access optionally opens directly into the microfluidic channel, directly into the at least one first culture chamber and / or directly into the at least one reservoir; and / or ii) comprises at least one second microfluidic channel, which preferably branches from the at least one first microfluidic channel upstream of the at least one first culture chamber, and opens into the at least one first microfluidic channel downstream of the at least one first culture chamber; and / or iii) has at least one second culture chamber that contains biological cells, which are different from cardiomyocytes, preferably cells selected from the group consisting of liver cells, renal cells, nerve cells, fat tissue and combinations thereof, wherein the at least one second culture chamber especially preferably is connected fluidically to the at least one culture chamber, optionally via at least one second microfluidic channel; and / or iv) comprises at least one second detector, which is configured to detect activity of the biological cells contained in the second culture chamber, wherein the at least one second detector preferably comprises or consists of an optical detector and especially preferably is configured to send signals about activity of the biological cells contained in the at least one second culture chamber to a data acquisition unit, wherein the data acquisition unit is preferably configured to record and evaluate the signals as a function of time; and / or v) preferably the at least one microfluidic channel, the at least one first culture chamber and / or at least one reservoir, contains blood or blood constituents, preferably blood cells, especially preferably cancer cells and / or immunocytes, especially metastasizing cancer cells and / or monocytes.

11. The microfluidic device as claimed in one of the preceding claims, characterized in that the at least one control device comprises a flow velocity measuring device, preferably a particle image velocimetry apparatus, wherein the microfluidic device especially preferably is configured, on the basis of the flow velocity measured by the flow velocity measuring device, to i) control the at least one pump, preferably all pumps, of the microfluidic device; and / or ii) control at least one valve and / or at least one throttle, preferably all valves and / or all throttles, of the microfluidic device.

12. The microfluidic device as claimed in one of the preceding claims, characterized in that the microfluidic device comprises or consists of plastic, wherein the plastic preferably i) comprises or consists of at least one structured plastic film, which is laminated with at least one further, optionally unstructured, plastic film; and / or ii) is selected from the group consisting of thermosetting plastics, thermoplastics, elastomers and combinations thereof, especially preferably is selected from the group consisting of PC, PET, COC, PDMS, TPE and combinations thereof; and / or iii) is structured at least partially by a method that is selected from the group consisting of laser structuring, cutting-plotting, hot embossing, milling, thermoforming, injection molding, soft lithography, 3D printing and combinations thereof.

13. The microfluidic device as claimed in one of the preceding claims, characterized in that the control device is configured to adapt the distribution of liquid, solid and gaseous substances in the microfluidic device to a concrete requirement of cells or cell groups in the microfluidic device, preferably by selective control of the pump, of at least one valve, of at least one throttle and of an oxygenator, wherein the selective control especially preferably takes place via a mathematical model, which in particular is stored in the microfluidic device.

14. The microfluidic device as claimed in one of the preceding claims, characterized in that the microfluidic device has at least one further microfluidic device, with parallel or serial fluidic communication with the microfluidic device, preferably via at least one fluidic inlet and at least one fluidic outlet, wherein the microfluidic device or a part thereof especially preferably is a primary microfluidic device and the at least one further fluidic device or a part thereof is a subordinate microfluidic device, wherein quite especially preferably a subordinate part of the microfluidic device has the at least one first culture chamber and a primary part of the device has the at least one pump and / or the at least one detector.

15. Use of the microfluidic device as claimed in one of claims 1 to 14 for verifying whether i) a particular substance or metabolites thereof have an influence on the activity of cardiomyocytes, preferably for verifying whether the particular substance triggers tachycardia or bradycardia of the cardiomyocytes or gives rise to arrhythmia of the cardiomyocytes; and / or ii) the mechanical, electrical and / or gas-induced influence of activity of the cardiomyocytes has an effect on the activity of biological cells, which are different from cardiomyocytes, preferably for verifying whether and to what extent this influence has a negative or positive effect on the activity of the biological cells.