Device and method for culturing cells

Abstract

There is described a microfluidic device (10) for culturing cells (9), comprising a channel (1) with at least one cavity (5a, 5b, 5c) for accommodating at least one microparticle (3), which protrudes outwardly from an outer surface of the channel (1), in which the at least one cavity (5a, 5b, 5c) has a fluid interface (55a, 55b, 55c) connected to a pump unit, the fluid interface is led out of the channel (1).

Description

Field of the Invention

[0001] The present invention relates to a microfluidic device for culturing cells, a method for operating a microfluidic device, and a cartridge comprising a microfluidic device, as described in the preamble of the independent claims. Background Art

[0002] WO2019 / 010587A1 proposes a microfluidic channel with a cavity in which a hydrogel precursor is pre-stored. Cells enter the cavity and then enter the hydrogel precursor, which is finally solidified. Subsequently, cell culture is carried out in the formed hydrogel beads.

[0003] Cancer patients with solid tumors vary greatly in their response to drug anti-cancer treatments. Currently, there is a very high demand for personalized cancer treatment, which particularly requires considering factors such as the cell characteristics of individual patients in order to be able to develop individualized optimized treatment plans.

[0004] In recent years, there has been a significant increase in the interest in using three-dimensional cell aggregates (such as organoids or spheroids) to study, diagnose, and treat diseases (such as tumor diseases), because such three-dimensional cell aggregates can well reproduce organ-specific characteristics, etc. Generally, the manipulation of such three-dimensional cell aggregates needs to be manually completed with the help of auxiliary tools such as pipettes, reaction vessels, and laboratory equipment. Compared with traditional laboratory tests, microfluidic technology shows many advantages here, such as less required sample volume and reagent amount, shorter analysis time, and the ability to implement parallel processes, etc. However, when integrating the required process steps into a microfluidic system, due to its complexity, there are many challenges that need to be overcome. One of the challenges is, for example, culturing and expanding such three-dimensional cell aggregates in a microfluidic system.

[0005] The so-called lab-on-a-chip system (abbreviated as LoC system) is a microfluidic system that integrates the functions of a macroscopic laboratory onto a plastic substrate to achieve automated processing. Such a system can enable biochemical processes to be processed to a large extent or completely automatically. A lab-on-a-chip system usually includes two main components. The first is a test carrier, for example in the form of a cartridge, which contains structures and mechanisms for manipulating the collected samples, especially passive components such as channels, reaction chambers, or pre-stored reagents, or can also be active components such as valves, pumps, or mixers. The second main component is a control unit for controlling the microfluidic flow in the cartridge. Summary of the Invention

[0006] For the study of tumor diseases, so-called tumor organoids are used, for example. One method of manufacturing tumor organoids is to extract single cells or tissue fragments from the primary tumor of a cancer patient and then culture them. In this case, these cells or tissue fragments will differentiate and proliferate, and eventually self-organize into three-dimensional structures. The resulting tumor organoids are three-dimensional cell aggregates, whose composition and structure are similar to those of the patient's primary tumor tissue. With the help of tumor organoids, the in vivo situation can be well reproduced. Currently, established three-dimensional cell culture methods are used to manufacture organoids or tumor organoids. In this case, the main process steps are to culture the cells or tissue fragments extracted from the original material and then amplify the resulting tumor organoids.

[0007] During the culture process, the cells or cell aggregates are suspended in a so-called extracellular matrix (ECM, English: extracellular matrix). The ECM is, for example, a hydrogel containing appropriate contents. Droplets of this suspension are pipetted into a cell culture container (such as a multi-well plate), and gel polymerization occurs by incubation at 37 °C. In this case, gel structures (English: geldomes) are formed. After the gel has solidified, an appropriate culture medium is pipetted into the container so that the gel structure is completely covered by the culture medium. Next, the culture phase in the incubator is carried out. During this process, the cells proliferate within the gel structure, forming organoids of different sizes and shapes. The growth of the organoids is monitored, for example, by microscopy, and the passage of the organoids is started at an appropriate time point. For this purpose, the gel structure is transferred to another container, and the organoids are washed, broken up, and resuspended. Subsequently, the organoids are amplified and dissociated into multicellular fragments or single cells. These process steps of culture and amplification are very labor-intensive and time-consuming and can only be carried out by trained and experienced professionals in a laboratory equipped with appropriate equipment.

[0008] The present invention relates to cell culture processes, such as the culture process of three-dimensional cell aggregates (especially organoids or spheroids), which are considered extremely crucial for the implementation of microfluidic technology. The present invention also describes a technical solution for realizing the culture-related working steps in a microfluidic manner.

[0009] According to the present invention, there is provided a microfluidic device for culturing cells having the features of the independent claims, which includes a channel with at least one cavity for accommodating at least one microparticle, the at least one cavity protruding outward from the outer surface of the channel, and a method for operating the microfluidic device.

[0010] This is particularly based on the following: At least one cavity of the microfluidic device has a fluid interface connected to the pump unit, and this fluid interface leads outwards from the channel. Preferably, the microfluidic device has a plurality of cavities, and each cavity has a fluid interface connected to the pump unit. Cavities protruding outwards from the outer surface of the channel can be particularly understood as lateral protrusions or depressions in the channel, which are arranged especially as recesses in the inner wall of the channel for accommodating at least one microparticle. The diameter of the cavity is, for example, 200–1500 μm, especially 400–750 μm. The shape and size of the cavity are preferably selected such that each cavity accommodates one microparticle respectively, and the subsequently flowing microparticles are transported to the next available cavity. Alternatively, the size of the cavity can also be designed to accommodate multiple microparticles. Each cavity has a fluid interface, which is connected to the pump unit directly or through a suitably connected valve. Thereby, liquid can be output from the cavity or input into the cavity. Suitable pump units for this purpose are, for example, syringe pumps, diaphragm pumps, or peristaltic pumps.

[0011] Advantageously, the fluid interface enables flow-induced processes to be achieved within the cavity or within the microfluidic device. This is achieved by inputting liquid into the cavity or outputting liquid from the cavity via the respective fluid interfaces. For example, this enables the flow-induced movement and positioning of one or more microparticles within the cavity to be achieved by outputting liquid via the fluid interface. Similarly, the microparticles or their dissolved components (especially cells or three-dimensional cell aggregates) can be flushed from the cavity into the channel by supplying liquid via the fluid interface. In addition, the culture conditions and the culture process of the microparticles within the cavity or the cells and / or three-dimensional cell aggregates within the microparticles can be influenced. This is achieved by precisely and quantitatively supplying a suitably selected liquid. Thereby, in particular, different culture conditions can be achieved for each cavity, thus enabling the effects on the behavior or properties of the cells or three-dimensional cell aggregates to be studied. In addition, by supplying a suitable liquid via the fluid interface of at least one cavity, it is also ensured that fresh nutrients and gases required for culturing are always present within the cavity containing the microparticles. They are introduced to where they are needed and do not have to diffuse into the cavity through the channel or other structures. Therefore, the fluid interface improves the supply of cells to be cultured. In addition, advantageously, a defined volume of liquid can be taken out of the cavity via the fluid interface of each cavity for subsequent analysis, so that the culture process can be monitored and adjusted if necessary.

[0012] The device according to the invention also has the following advantages: The cultivation of cells and / or three-dimensional cell aggregates is automated inside the microfluidic system, making the cultivation process reproducible, automatable, standardizable, and user-friendly. Therefore, there is no need for experienced professionals and laboratories equipped with appropriate equipment, thus reducing the burden on professionals and saving time and costs. Since the process steps do not need to be manually performed, for example, there will be no operation-induced effects or errors, and the entry of possible contaminants can be prevented. In particular, when three-dimensional cell aggregates (such as organoids) are used for reliable drug testing, standardization is essential.

[0013] Other advantageous embodiments of the microfluidic device result from the dependent claims.

[0014] In an advantageous embodiment, the fluid interface of at least one cavity has an integrated barrier element. Alternatively or additionally, the fluid interface of at least one cavity has a size of 5–75 μm. It is advantageous here that the size of the microparticles is larger than the fluid interface or cannot pass through the fluid interface due to the barrier element, so they cannot leave the cavity via the fluid interface. In this way, liquids can be introduced into and withdrawn from the cavity without loss of microparticles.

[0015] Furthermore, it is advantageous that the channels of the microfluidic device have a size of 100–1000 μm. In the context of the present invention, the term "size" refers to the height and / or width of the microfluidic channels.

[0016] In another advantageous embodiment, the channel has at least one bend, and at least one cavity is arranged on the outer arc surface of the channel bend. It is advantageous here that due to the effect of the bend, the components (such as microparticles) transported through the channel are subjected to a centrifugal force that transports the components towards at least one cavity and causes them to enter the cavity.

[0017] In another advantageous embodiment, the device has means for temperature control of the channel and / or at least one cavity. Thereby, a suitable temperature for cultivating cells and / or three-dimensional cell aggregates can be set. This is achieved, for example, by temperature control (especially below the microfluidic device), for example, by using one or more Peltier elements (especially arranged below the microfluidic device).

[0018] Furthermore, in another embodiment, it is advantageous that the channel and / or at least one cavity is designed to be at least partially optically transparent and includes a control unit (especially a camera and / or a microscope unit). It is advantageous here that the cultivation process of cells and / or three-dimensional cell aggregates can be optically observed. For example, the size of the cultivated three-dimensional cell aggregates can be known, and the cultivation process can be stopped when they reach a defined size.

[0019] Furthermore, in another advantageous embodiment, the microfluidic device comprises at least one reservoir for a fluid, which is fluidly connected to the fluid interface of at least one cavity. The reservoir is pre-filled with a culture medium, growth factors, nutrients and / or pharmacologically active substances. This ensures a rapid and easy supply of the culture medium, growth factors, nutrients and / or pharmacologically active substances.

[0020] Another subject of the invention is a method for culturing cells using the microfluidic device according to the invention, comprising the following steps:

[0021] a) Conveying a medium containing microparticles through a channel, wherein one microparticle encapsulates at least one cell and introducing at least one microparticle into at least one cavity. The microparticles are transported, for example, in a culture medium.

[0022] b) Culturing at least one cell in the microparticles in the cavity, in particular into three-dimensional cell aggregates. The culture medium, growth factors, nutrients and / or pharmacologically active substances are metered into the cavity via the fluid interface of the cavity. During the culturing process, the surrounding culture medium diffuses into the cavity and the microparticles, thereby supplying the cells with dissolved nutrients and gases. The culture medium is, for example, continuously or at defined times continued to be conveyed through the microfluidic channel. Via the fluid interface of the cavity, fresh culture medium of the same or different composition is supplied to the microparticles (and thus to the cells or three-dimensional cell aggregates) during the culturing process, such that the consumed culture medium is replaced by fresh culture medium, for example, at defined times. Alternatively or additionally, growth factors, nutrients and / or pharmacologically active substances are metered precisely. Different growth factors can be added, for example, at defined concentrations.

[0023] It is advantageous here that in this way the culturing of the cells or three-dimensional cell aggregates can be influenced in a targeted manner. The culture medium, growth factors, nutrients and / or pharmacologically active substances enter the cavity directly via the fluid interface and thus reach their defined positions to act on the cells or three-dimensional cell aggregates in a targeted manner during the culturing process. This ensures that the cells or three-dimensional cell aggregates come into contact with the corresponding culture medium, growth factors, nutrients and / or pharmacologically active substances in sufficient concentration. By adding a drug solution of defined concentration via the fluid interface to the cavity, in particular the influence on the culturing process and the properties of the cells or three-dimensional cell aggregates can be studied. For example, chemotherapeutic drugs of single or multiple selectable concentrations can be introduced into the cavity via the fluid interface. Furthermore, it is advantageous that different culturing conditions can be realized, in particular, for each cavity, thereby studying the influence on the behavior or properties of the cells or three-dimensional cell aggregates. For culturing, the cavity is, for example, temperature-controlled to a suitable temperature, for example, to 37 °C. In addition, the fluid interfaces of the channel and the cavity can also be heated.

[0024] c) Terminate the cultivation and introduce at least one microparticle or at least one cultivated cell, in particular a three-dimensional cell aggregate, from the cavity into the channel. The termination of the cell or three-dimensional cell aggregate cultivation process is carried out at an optional time point, for example when the three-dimensional cell aggregate reaches a desired size or morphology. The termination can be carried out for a single or multiple cavities. Finally, the microparticles in the microfluidic channel can be further transported and processed.

[0025] In addition to the advantages already described in the individual method steps, this method also has the following advantages: The cells or three-dimensional cell aggregates can be used for subsequent cultivation or analysis steps, in particular for amplification or drug testing. Furthermore, the method according to the invention also has the following advantages: The cultivation of the cells and / or three-dimensional cell aggregates is carried out automatically, which makes the cultivation process reproducible, automatable, standardizable and user-friendly. From this, the advantages already described for the microfluidic device result.

[0026] Other advantageous embodiments of the method according to the invention result from the dependent claims.

[0027] In an advantageous embodiment, in step a), at least one microparticle is introduced into at least one cavity based on the centrifugal force acting on the microparticle due to the channel bend when flowing through the channel. Here, the cavity is arranged on the outer arc surface of the channel. Here, it is advantageous that the centrifugal force acting on the microparticle transports the microparticle directly towards the at least one cavity and causes it to enter the cavity.

[0028] In an alternative or additional advantageous embodiment, in step a), at least one microparticle is introduced into at least one cavity in a flow-induced manner by outputting the culture medium via the fluid interface of the cavity. Here, it is advantageous that the microparticle thus moves towards the cavity in a flow-induced manner and is positioned therein.

[0029] Alternatively or additionally, the movement and positioning of the microparticles in the cavity can also be achieved or assisted by other techniques, for example by means of dielectrophoresis and an applied non-uniform electric field to move and capture the microparticles, and / or by means of an applied magnetic field and magnetic labeling of the microparticles, and / or by means of standing wave ultrasound to generate pressure nodes that attract and fix the microparticles.

[0030] In another advantageous embodiment, in step b), the oxygen and / or carbon dioxide content in the culture medium supplied via the fluid interface of the cavity is changed. Thereby, hypoxic culture conditions can be advantageously achieved in the cavity, which characterize the in vivo situation of solid tumors.

[0031] In another advantageous embodiment, during step b), a liquid volume is output from the cavity via the fluid interface of at least one cavity during cultivation, and the determined parameters are detected. These parameters are, for example, the pH value in the culture medium, the concentration of glucose and / or lactate, the oxygen and / or carbon dioxide content in the culture medium, and / or the presence or concentration of proteins (especially cytokines) in the culture medium. Advantageously, analysis and monitoring can be carried out during the cultivation process without stopping or disturbing the cultivation. In addition, the detection of characteristic properties can provide information about the cultivation quality in advance, so that the cultivation conditions can be adjusted directly during the cultivation process.

[0032] Furthermore, in another embodiment, it is advantageous that in step c), the cultivation is terminated by generating a liquid flow in the direction of the channel via the fluid interface of the cavity. Thereby, the culture medium around the microparticles is washed away, thus stopping the cultivation process. The microparticles themselves or the cells or three-dimensional cell aggregates after dissolution of the microparticles can also be transported out of the cavity and into the channel by means of the liquid flow. The supplied liquid can be, for example, a culture medium or a buffer solution. Advantageously, these steps can be completed in a flow-induced manner via the fluid interface of the cavity, and compared with the flow caused only via the channel, almost complete extraction of the microparticles or cells and three-dimensional cell aggregates from the cavity can be ensured.

[0033] In another advantageous embodiment, the microparticle is a spherical hydrogel structure containing at least one cell and / or three-dimensional cell aggregate. Furthermore, the three-dimensional cell aggregate is, for example, an organoid or a spheroid. As the hydrogel of the hydrogel structure, Matrigel TM (Corning Incorporated) is preferably used. Alternatively, other hydrogels such as agarose, gelatin or polyethylene glycol can also be used. The size of the hydrogel structure is selected such that one or more three-dimensional cell aggregates, especially organoids, can be cultivated therein. The diameter of tumor organoids, which are particularly suitable for drug testing, is, for example, between 100 - 750 μm.

[0034] Furthermore, in one embodiment, it is advantageous that in step c), the hydrogel structure is depolymerized and the cells and / or three-dimensional cell aggregates, especially organoids or spheroids, are released in the cavity. To depolymerize, a reagent is supplied via the fluid interface of the cavity. The reagent has, for example, a temperature that initiates or promotes depolymerization.

[0035] For Matrigel hydrogel, such a depolymerizing reagent is, for example, Corning TM Dispase solution (Corning Incorporated) or Corning TM Cell Recovery Solution (Corning Incorporated). Alternative reagents are, for example, enzyme solutions containing trypsin or TrypLE TM Express enzyme (Thermo Fisher Scientific). Additionally or alternatively, MatrigelTM (Corning Incorporated) The depolymerization can be achieved by reducing the temperature to about 4°C. For this purpose, a culture medium at a temperature of about 4°C is supplied, for example, through the fluid interface of the cavity and / or the channels of the microfluidic device. Alternatively, the microfluidic device (especially the cavity) is temperature-controlled to a temperature of about 4°C.

[0036] The subject matter of the present invention also includes a cartridge, especially a microfluidic cartridge, for example as described in DE102016222072A1 or DE102016222075A1, which includes a microfluidic device according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Embodiments of the present invention are shown in the drawings and are elaborated in detail in the following description of the drawings, wherein:

[0038] Figure 1 : A cross-sectional schematic view of a microfluidic device according to the present invention, which includes channels and cavities, and the cavities respectively have fluid interfaces;

[0039] Figure 2 : A schematic view of a cartridge according to the present invention, which includes a Figure 1 microfluidic device;

[0040] Figure 3 : A schematic view of a flowchart of an embodiment of a method according to the present invention. DETAILED DESCRIPTION

[0041] Figure 1 shows a microfluidic device 10 for culturing cells 9. The microfluidic device 10 includes a channel 1 with three cavities 5a, 5b, 5c, wherein the cavities 5a, 5b, 5c protrude outward from the outer surface of the channel 1. Figure 1 The number of cavities 5a, 5b, 5c in is only an example. The channel 1 preferably has a plurality of cavities 5a, 5b, 5c. Each cavity 5a, 5b, 5c has a fluid interface 55a, 55b, 55c connected to a pump unit, and the fluid interface extends outward from the channel 1. The fluid interfaces 55a, 55b, 55c have, for example, an integrated blocking element (not shown) and / or a size of 5–75 μm, such that the size of the hydrogel structure 3 is larger than the fluid interfaces 55a, 55b, 55c, and the hydrogel structure cannot pass through and leave the fluid interfaces. The pump unit is not shown in Figure 1 The channel 1 has a bend 11, such that the channel 1 has an inner arc surface 12, a neutral axis 13, and an outer arc surface 14 in the region of the bend 11. The cavities 5a, 5b, 5c are arranged on the outer arc surface 14 of the bend of the channel 1. Preferably, the device includes means for temperature-controlling the channel 1 and / or at least one cavity 5a, 5b, 5c, Figure 1Not shown. In addition, channels 1 and / or cavities 5a, 5b, 5c can be designed to be at least partially optically transparent and have a control unit (not shown), in particular a camera and / or microscope unit.

[0042] The following describes an embodiment of a method for culturing cells 9 with the aid of a microfluidic device 10 according to the invention. Here, the microparticles 3 surrounding at least one cell 9 are exemplarily described as spherical hydrogel structures 3, and the three-dimensional cell aggregates 7 are described as organoids 7. Matrigel TM (Corning Incorporated) is preferably used as the hydrogel. The size of the hydrogel structure 3 is selected such that one or more organoids 7 can be cultured therein. The diameter of the organoids 7 is, for example, 100–750 μm. Figure 1 In [the figure], cavities 5a, 5b, 5c represent different steps of the method: the first cavity 5a shows the first step a) of the method, the second cavity 5b shows the second method step b), and the third cavity 5c shows the third step c) of the method.

[0043] In the first step a), a medium, in particular a culture medium, containing the spherical hydrogel structures 3 is conveyed through the channel 1. The direction of flow is as indicated by the arrow 15. Each hydrogel structure 3 encloses a cell 9 and is transported along with the liquid flow in the channel 1. The channel 1 has a bend 11. When the hydrogel structure 3 is transported through the bend 11, a centrifugal force acts on the hydrogel structure 3, which transports the hydrogel structure towards the outer arc surface 14 of the channel 1 (where the cavities 5a, 5b, 5c are located) and pushes it into the cavity. In addition, the medium is output through the fluid interfaces 55a, 55b, 55c of the cavities 5a, 5b, 5c, as indicated by the outflow arrows 17, thereby creating a suction force in the direction of the cavities 5a, 5b, 5c, which sucks the hydrogel structure 3 therein. Preferably, the cavities 5a, 5b, 5c are dimensioned to exactly accommodate one hydrogel structure 3, and subsequent hydrogel structures 3 are continuously transported to the next free cavity 5a, 5b, 5c. Alternatively, the cavities 5a, 5b, 5c can also be dimensioned such that the cavities 5a, 5b, 5c accommodate multiple hydrogel structures 3, Figure 1 Not shown.

[0044] In the second step b), the cells 9 in the hydrogel structures 3 in the cavities 5a, 5b, 5c are cultured and grown into organoids 7. For example, the culture medium is provided for the culture process through the channel 1. The culture medium diffuses into the cavities 5a, 5b, 5c and within the hydrogel structure 3. The temperature suitable for culturing the organoids 7 is set by regulating the temperature of the channel 1 and / or the cavities 5a, 5b, 5c, preferably at about 37 °C. During the culture process, liquids are precisely metered and supplied to the hydrogel structure 3 or the cells 9 via the fluid interfaces 55a, 55b, 55c of the cavities 5a, 5b, 5c, as shown by the inflow arrow 19, thereby specifically influencing the culture of the cells 9 into organoids 7. These liquids are, for example, culture media with altered compositions, and / or contain growth factors, nutrients, and / or pharmacologically active substances. The supply or inflow 19 of the liquids can be carried out continuously or at defined time points. When adding the culture medium, the "consumed" culture medium is replaced by "fresh" culture medium, thereby supplying the cells 9 with dissolved nutrients and gases. In this way, the culture medium replacement is achieved microfluidically. Additionally, culture media with altered oxygen and / or carbon dioxide contents can be supplied. In this way, possible hypoxic culture conditions are set. During the culture, for example, the liquid volume is output from the cavities through the fluid interfaces 55a, 55b, 55c of the cavities 5a, 5b, 5c and parameters are detected, in particular the pH value, the concentrations of glucose and / or lactate, the oxygen and / or carbon dioxide contents, and the presence and concentration of proteins, especially cytokines.

[0045] In the third step c), at a defined time point, for example when the organoids 7 reach the desired size or morphology, the culture process is terminated. For this purpose, the individual or all hydrogel structures 3 in the cavities 5a, 5b, 5c are depolymerized and dissolved to release the generated organoids 7. To depolymerize the hydrogel structure 3, a suitable liquid reagent is supplied via the fluid interfaces 55a, 55b, 55c of the cavities 5a, 5b, 5c, as shown by the inflow arrow 19. Additionally or alternatively, the hydrogel depolymerization can be achieved by lowering the temperature to about 4 °C. For this purpose, for example, a culture medium at a temperature of about 4 °C is supplied via the fluid interfaces 55a, 55b, 55c of the cavities 5a, 5b, 5c and / or the channel 1. Additionally or alternatively, for this purpose, the microfluidic device 10 can also be regulated to 4 °C. The organoids 7 released from the hydrogel structure 3 after depolymerization are then transported from the cavities 5a, 5b, 5c to the microfluidic channel 1. For this purpose, for example, the culture medium is input via the fluid interfaces 55a, 55b, 55c of the cavities 5a, 5b, 5c and a liquid flow in the direction of the channel 1 is generated via the inflow arrow 19. Finally, the organoids 7 located in the microfluidic channel 1 will be further transported and can be used for subsequent culture or analysis steps, especially for amplification or drug testing. Alternatively, in Figure 1 not shown, the hydrogel structures 3 in the cavities 5a, 5b, 5c are not depolymerized first, but flow into the channel 1 in the form of the intact hydrogel structures 3.

[0046] Figure 2 shows the cartridge 100 according to the present invention, which comprises according to Figure 1 the microfluidic device 10 according to the present invention as shown. The microfluidic device 10 is, for example, arranged on a plastic substrate or a chip.

[0047] Figure 3 shows a flowchart of a method 500 for culturing cells 9 according to the present invention, which is accomplished by means of Figure 1 the microfluidic device as shown and the examples and method steps described therein.

Claims

1. A microfluidic device (10) for culturing cells (9), comprising a channel (1) with at least one cavity (5a, 5b, 5c), said at least one cavity being adapted to receive at least one microparticle (3), said at least one cavity protruding outwardly from an outer surface of said channel (1), characterized in that, The at least one cavity (5a, 5b, 5c) has a fluid interface (55a, 55b, 55c) connected to the pump unit, and the fluid interface leads outwards from the channel (1).

2. The microfluidic device (10) according to claim 1, characterized in that The fluid interfaces (55a, 55b, 55c) of the at least one cavity (5a, 5b, 5c) have integrated barrier elements and / or are sized 5–75 μm.

3. The microfluidic device (10) according to any one of the preceding claims, characterized in that The channel (1) has at least one bend (11), and the at least one cavity (5a, 5b, 5c) is arranged on the outer arc surface (14) of the bend (11) of the channel (1).

4. The microfluidic device (10) according to any one of the preceding claims, characterized in that, The device (10) has a device for temperature control of the channel (1) and / or the at least one cavity (5a, 5b, 5c).

5. The microfluidic device (10) according to any one of the preceding claims, characterized in that The channel (1) and / or the at least one cavity (5a, 5b, 5c) is configured to be at least partially optically transparent and includes a control unit, in particular a camera and / or a microscope unit.

6. The microfluidic device (10) according to any one of the preceding claims, characterized in that, The microfluidic device further includes a reservoir, which is fluidly connected to the fluid interfaces (55a, 55b, 55c) of the at least one cavity (5a, 5b, 5c), and a culture medium, growth factors, nutrients, and / or pharmaceutically active substances are pre-stored in the reservoir.

7. A microfluidic method (500) for culturing cells (9), the method being completed by means of the device (10) according to any one of claims 1-6, the method comprising the following steps: a) Conveying a medium containing micro-particles (3) through the channel (1), wherein one micro-particle (3) encapsulates at least one cell (9), and introducing at least one micro-particle (3) into at least one cavity (5a, 5b, 5c); b) Culturing at least one cell (9) in the micro-particles (3) in the cavity (5a, 5b, 5c), in particular culturing into three-dimensional cell aggregates (7), wherein a culture medium, growth factors, nutrients, and / or pharmaceutically active substances are quantitatively supplied via the fluid interfaces (55a, 55b, 55c) of the cavity (5a, 5b, 5c); c) Terminating the culture and leading out the at least one micro-particle (3) or at least one cultured cell (9), in particular the three-dimensional cell aggregate (7), from the cavity (5a, 5b, 5c).

8. The microfluidic method (500) according to claim 7, characterized in that In step a), the at least one micro-particle (3) is introduced into the at least one cavity (5a, 5b, 5c) based on the centrifugal force acting on the micro-particle (3), and the centrifugal force is generated due to the bend (11) of the channel (1) when flowing through the channel.

9. The microfluidic method (500) according to any one of claims 7 or 8, characterized in that, In step a), the at least one micro-particle (3) is introduced into the at least one cavity (5a, 5b, 5c) in a flow-induced manner by outputting the medium via the fluid interfaces (55a, 55b, 55c) of the cavity (5a, 5b, 5c).

10. The microfluidic method (500) according to any one of claims 7-9, characterized in that, In step b), a culture medium with a varying oxygen and / or carbon dioxide content is supplied, in particular low oxygen culture conditions are set.

11. The microfluidic method (500) according to any one of claims 7-10, characterized in that, In step b), during the cultivation, a liquid volume is output from the cavity (5a, 5b, 5c) via the fluid interfaces (55a, 55b, 55c) of the cavity, and parameters are detected, in particular the pH value, the concentration of glucose and / or lactic acid, the oxygen and / or carbon dioxide content, and / or the presence and concentration of proteins, in particular the presence and concentration of cytokines.

12. The microfluidic method (500) according to any one of claims 7-11, characterized in that, In step c), the cultivation is terminated and / or the at least one microparticle (3) or at least one cultivated cell (9), in particular a three-dimensional cell aggregate (7), is withdrawn from the cavity (5a, 5b, 5c) by a liquid flow in the direction of the channel (1) via the fluid interfaces (55a, 55b, 55c) of the cavity (5a, 5b, 5c).

13. The microfluidic method (500) according to any one of claims 7 - 12, characterized in that, The microparticle (3) is a hydrogel structure (3), in particular spherical, comprising at least one cell (9) and / or a three-dimensional cell aggregate (7), and / or the three-dimensional cell aggregate (7) is an organoid (7) or a spheroid.

14. The microfluidic method (500) according to claim 13, wherein, In step c), the hydrogel structure (3) is depolymerized, and the cell (9) and / or the three-dimensional cell aggregate (7), in particular the organoid (7) or the spheroid, is released in the cavity (5a, 5b, 5c), wherein a reagent is supplied via the fluid interfaces (55a, 55b, 55c) of the cavity (5a, 5b, 5c) to effect the depolymerization, in particular by adjusting the temperature to a temperature that initiates or promotes the depolymerization.

15. The microfluidic method (500) according to any one of claims 7-14, characterized in that, The movement of the microparticle (3) in the microfluidic device (10) and the positioning of the microparticle (3) in the cavity (5a, 5b, 5c) are achieved by dielectrophoresis and an applied non-uniform electric field and / or by an applied magnetic field and the magnetic labeling of the microparticle (3) and / or by a standing wave ultrasound generating pressure nodes that attract and fix the microparticle (3).

16. A cartridge (100), in particular a microfluidic cartridge, comprising the microfluidic device (10) according to any one of claims 1-6.

Images