Device and method for culturing cells

Abstract

The invention relates to a microfluidic device (10) for culturing cells (9), comprising a channel (1) having at least one cavity (5a, 5b, 5c) for receiving at least one microparticle (3), which cavity bulges out from an outer surface of the channel (1), wherein the at least one cavity (5a, 5b, 5c) comprises a fluidic connection (55a, 55b, 55c), which is connected to a pump unit and leads away from the channel (1).

Description

[0001] Description

[0002] title

[0003] Device and method for culturing cells

[0004] The present invention relates to a microfluidic device for cultivating cells, a method for operating the same and a cartridge comprising the microfluidic device according to the preamble of the independent claims.

[0005] State of the art

[0006] WO 2019 / 010587 A1 proposes a microfluidic conduit with cavities containing a hydrogel precursor. Cells enter the cavities and thus the hydrogel precursor, which is then cured. The cells are then cultured in the resulting hydrogel spheres.

[0007] Cancer patients with solid tumors respond to drug therapy with varying degrees of success. There is a strong need for increased personalization of cancer treatment, which, among other things, takes into account the cellular characteristics of the individual patient in order to derive an individually optimized therapy.

[0008] Interest in the use of three-dimensional cell agglomerates, such as organoids or spheroids, for the research, diagnosis and treatment of diseases such as tumors has increased significantly in recent years, as such three-dimensional cell agglomerates can, for example, well represent organ-specific properties.

[0009] Typically, these are handled manually using tools such as pipettes, reaction vessels, and laboratory equipment. Compared to conventional laboratory tests, microfluidics offers advantages such as smaller sample volumes and reagents required, shorter analysis times, and parallel processes.

[0010] However, due to their complexity, implementing the required process steps in a microfluidic system presents numerous challenges that must be overcome. One of these challenges, for example, is the cultivation and propagation of such three-dimensional cell agglomerates in a microfluidic system.

[0011] So-called lab-on-a-chip systems, or LoC systems for short, are microfluidic systems that integrate the functionalities of a macroscopic laboratory onto a plastic substrate for automated processing. Such systems enable biochemical processes to be largely or completely automated.

[0012] Lab-on-a-chip systems typically comprise two main components. The first is a test carrier, for example, in the form of a cartridge, which contains structures and mechanisms for manipulating a sample, particularly passive components such as channels, reaction chambers, or upstream reagents, or active components such as valves, pumps, or mixers. The second main component is a control unit for controlling the microfluidic processes in the cartridge.

[0013] Disclosure of the invention

[0014] For example, tumor organoids are used to research tumor diseases. One method for producing tumor organoids is to remove individual cells or tissue fragments from a cancer patient's primary tumor and then cultivate them. This involves differentiation and proliferation of these cells or tissue fragments, which ultimately self-organize into three-dimensional structures. The resulting tumor organoids are thus 3D cell agglomerates that exhibit a similar composition and architecture to the patient's primary tumor tissue. Tumor organoids can be used to accurately represent in vivo conditions. Currently established methods of 3D cell cultivation are used to produce organoids or tumor organoids.

[0015] The main process steps are the cultivation of the removed cells or tissue fragments from the primary material and the subsequent expansion of the resulting tumor organoids.

[0016] During cultivation, cells or cell agglomerates are suspended in a so-called extracellular matrix (ECM). The ECM is, for example, a hydrogel with suitable ingredients. Drops of this suspension are pipetted into a cell culture vessel, such as a multiwell plate, and incubation at 37°C causes the gel to polymerize. This process forms gel structures (gel domes).

[0017] After the gel has solidified, a suitable culture medium is pipetted into the vessel so that the gel structures are completely covered. This is followed by a cultivation phase in an incubator. During this phase, the cells proliferate within the gel structures, and organoids of varying sizes and shapes form. The growth of the organoids is monitored microscopically, for example, and passaging of the organoids begins at an appropriate time. For this purpose, the gel structures are transferred to another vessel, washed, broken open, and the organoids are resuspended.

[0018] The organoids are then expanded and dissociated into multicellular fragments or individual cells.

[0019] These cultivation and expansion process steps are very labor- and time-intensive and can only be carried out by trained and experienced specialists in appropriately equipped laboratories.

[0020] The present invention addresses processes for cultivating cells, for example into three-dimensional cell agglomerates, in particular organoids or spheroids, which are considered to be very critical for microfluidic implementation, and describes a technical solution for the microfluidic implementation of the work steps relevant for cultivation.

[0021] According to the invention, a microfluidic device for cultivating cells, comprising a channel with at least one cavity for receiving at least one microparticle, which protrudes from an outer surface of the channel, and a method for operating the same are provided with the characterizing features of the independent patent claims.

[0022] This is based in particular on the fact that the at least one cavity of the microfluidic device has a fluidic connection connected to a pump unit, which leads away from the channel. Preferably, the microfluidic device has a plurality of cavities, each with a fluidic connection connected to a pump unit.

[0023] A cavity protruding from an outer surface of the channel can be understood in particular as a lateral bulge or bay in the channel, which is arranged in particular as a recess in an inner wall of the channel for receiving the at least one microparticle.

[0024] The cavities have a diameter of, for example, 200–1500 μm, and in particular 400–750 μm. The shape and dimensions of the cavities are preferably selected so that each microparticle can accommodate one, and subsequently flown microparticles are transported to the next available cavity. Alternatively, the cavities can also be dimensioned so that multiple microparticles can be accommodated.

[0025] Each of the cavities has a fluid connection connected to a pump unit either directly or via appropriately switched valves. This makes it possible to pump fluids into or out of a cavity. Suitable pump units for this purpose include syringe, diaphragm, or peristaltic pumps.

[0026] The advantage of the fluidic connections is that they enable the realization of flow-induced processes within the cavities or the microfluidic device. These processes occur by pumping fluids into or out of the cavities via the individual fluidic connections.

[0027] This enables, for example, flow-induced movement and positioning of one or more microparticles in a cavity by pumping out a liquid via the fluidic connection. Likewise, the microparticles or, after their dissolution, components of the microparticles, in particular cells or three-dimensional cell agglomerates, can be flushed back out of the cavity into the channel by supplying a liquid via the fluidic connection.

[0028] Furthermore, the cultivation conditions, and thus the cultivation process, can be influenced by a microparticle located in the cavity or by a cell and / or three-dimensional cell agglomerate located within the microparticle. This is achieved by precisely metering appropriately selected liquids.

[0029] This allows for the realization of different cultivation conditions for each cavity, thus allowing the investigation of influences on the behavior or properties of the cells or three-dimensional cell agglomerates. The supply of suitable liquids via the fluidic connection of at least one cavity also ensures that the cavities containing the microparticles always contain fresh nutrients and gases required for cultivation. They are thus introduced directly where they are needed and do not have to first diffuse into the cavities via a channel or other structures. The fluidic connections thus improve the supply to the cells being cultivated.

[0030] Furthermore, a defined volume of liquid can be removed from each cavity for subsequent analysis via the fluidic connection, so that the cultivation process can be monitored and adjusted if necessary.

[0031] The device according to the invention also offers the advantage that the cultivation of cells and / or three-dimensional cell agglomerates is automated within a microfluidic system, making the cultivation reproducible, automatable, standardizable, and user-friendly. Thus, no experienced experts or appropriately equipped laboratories are required, which relieves the burden on specialist personnel and saves time and money. By eliminating manual process steps, for example, no interference or errors from operation occur, and the introduction of potential contamination is prevented. Standardization is particularly necessary for the use of three-dimensional cell agglomerates, such as organoids, for reliable drug testing.

[0032] Further advantageous embodiments of the microfluidic device emerge from the subclaims. In an advantageous embodiment, the fluidic connection of the at least one cavity has an integrated retaining element. Alternatively or additionally, the fluidic connection of the at least one cavity has dimensions of 5-75 μm.

[0033] The advantage here is that the microparticle(s) are larger than the fluidic connection or cannot pass through it due to the retaining element, thus preventing them from leaving the cavity via the fluidic connection. This makes it possible to introduce and remove liquids from the cavity without causing any loss of microparticles.

[0034] It is also advantageous if the channel of the microfluidic device has dimensions of 100 - 1000 μm. For the purposes of the present invention, the term "dimension" refers to the height and / or width of the microfluidic channel.

[0035] In a further advantageous embodiment, the channel has at least one bend, wherein the at least one cavity is arranged in the bend of the channel on the outer curved surface.

[0036] The advantage here is that components such as microparticles that are conveyed through the channel are subjected to a centrifugal force due to the bending, which transports the components in the direction of at least one cavity and into it.

[0037] In a further advantageous embodiment, it is provided that the device has a device for tempering the channel and / or the at least one cavity.

[0038] This allows a suitable temperature to be set for cultivating the cells and / or three-dimensional cell agglomerates. This is achieved, for example, by temperature control, particularly below the microfluidic device, for example by using one or more Peltier elements, particularly positioned below the microfluidic device.

[0039] Furthermore, in a further embodiment, it is advantageous if the channel and / or the at least one cavity is at least partially optically transparent and comprises a control unit, in particular a camera and / or a microscope unit. This is advantageous because optical observation of the cultivation process of the cells and / or three-dimensional cell agglomerates is possible. For example, the dimensions of the cultivated three-dimensional cell agglomerates can be determined, and the cultivation process can be stopped when they have reached a defined size.

[0040] Furthermore, in one embodiment, it is advantageous if the microfluidic device comprises at least one reservoir for a fluid, which is in fluidic communication with the fluidic connection of the at least one cavity. A culture medium, growth factors, nutrients, and / or pharmacologically active ingredients are stored upstream in the reservoir.

[0041] This ensures quick and easy supply of the culture medium, growth factors, nutrients and / or pharmacological agents.

[0042] The invention further relates to a method for cultivating cells using the microfluidic device according to the invention, comprising the following steps: a) conveying a medium containing microparticles via the channel, wherein a microparticle encloses 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. b) cultivating the at least one cell in the microparticle located in the cavity, in particular to form a three-dimensional cell agglomerate. This involves a metered addition of a culture medium, growth factors, nutrients, and / or pharmacologically active ingredients into the cavity via the fluidic connection of the cavity.

[0043] During cultivation, the surrounding culture medium diffuses into the cavities and into the microparticles, supplying the cells with dissolved nutrients and gases. The culture medium is conveyed continuously or at specific times, for example, via the microfluidic channel.

[0044] During the cultivation process, fresh culture medium of the same or a different composition is supplied to the microparticles and thus to the cells or three-dimensional cell agglomerates via the fluidic connections of the cavities. This allows for the replacement of used culture medium with fresh culture medium at specific times. Alternatively or additionally, growth factors, nutrients, and / or pharmacological agents are added in precisely measured doses. For example, different growth factors can be added in defined concentrations.

[0045] The advantage here is that the cultivation of the cells or three-dimensional cell agglomerates can be specifically influenced in this way. The culture medium, growth factors, nutrients and / or pharmacological agents reach the cavity via the fluidic connection directly and thus at their destination in order to specifically influence the cells or three-dimensional cell agglomerates during cultivation. This ensures that the cells or three-dimensional cell agglomerates come into contact with the respective culture medium, growth factors, nutrients and / or pharmacological agents in sufficient concentrations. By adding drug solutions with defined concentrations to a cavity via the fluidic connection, the influence on the cultivation process and the properties of the cells or three-dimensional cell agglomerates can be investigated.For example, single or multiple chemotherapeutic agents with selectable concentrations can be introduced into the cavities via the fluidic connections.

[0046] Furthermore, it is particularly advantageous to realize different cultivation conditions for each cavity and thus to investigate influences on the behavior or properties of the cells or three-dimensional cell agglomerates.

[0047] For cultivation, the cavities are heated to a suitable temperature, for example, 37°C. Additionally, the channel and the fluidic connections of the cavity can also be heated. c) Terminating the cultivation and discharging the at least one microparticle or the at least one cultured cell, in particular the three-dimensional cell agglomerate, from the cavity into the channel. The cultivation process of the cells or three-dimensional cell agglomerates is terminated at a selectable time, for example, when a desired size or morphology of the three-dimensional cell agglomerates is reached. Termination can occur for a single or multiple cavities.

[0048] Finally, the microparticles located in the microfluidic channel can be further transported and processed.

[0049] In addition to the advantages explained for the individual process steps, such a process offers the further advantage that the cells or three-dimensional cell agglomerates are available for subsequent cultivation or analysis steps, in particular for expansion or for drug testing.

[0050] The method according to the invention also offers the advantage that the cultivation of cells and / or three-dimensional cell agglomerates is automated, making the cultivation process reproducible, automatable, standardizable, and user-friendly. This results in the advantages already described for the microfluidic device.

[0051] Further advantageous embodiments of the method according to the invention emerge from the subclaims.

[0052] In an advantageous embodiment, in step a), the introduction of the at least one microparticle into the at least one cavity occurs due to a centrifugal force acting on the microparticles, which occurs due to a bend in the channel as the fluid flows through it. In this case, the cavity is arranged on the outer curved surface of the channel.

[0053] The advantage here is that the centrifugal force acting on the microparticles transports the microparticles directly towards and into at least one cavity.

[0054] In an alternative or additional advantageous embodiment, in step a), the introduction of the at least one microparticle into the at least one cavity is carried out in a flow-induced manner by conveying medium out through the fluidic connection of the cavity. Advantageously, the microparticles are thereby moved toward the cavities in a flow-induced manner and positioned within them.

[0055] Alternatively or additionally, the movement and positioning of the microparticles into the cavities can also be carried out or supported by other technologies, for example by dielectrophoresis and an applied inhomogeneous electric field to move and capture the microparticles and / or by applying magnetic fields and magnetic labeling of the microparticles and / or by using standing ultrasonic waves to generate pressure nodes that attract and hold the microparticles.

[0056] In a further advantageous embodiment, in step b) the oxygen and / or carbon dioxide content in the culture medium supplied via the fluidic connection of the cavity is varied.

[0057] This allows advantageous hypoxic cultivation conditions to be realized within the cavity, which are characteristic of the in vivo situation in solid tumors.

[0058] In a further advantageous embodiment, in step b), during cultivation, a volume of liquid is pumped out of the at least one cavity via the fluidic connection and analyzed for specific parameters. These parameters include, for example, the pH value, the concentration of glucose and / or lactate in the culture medium, the oxygen and / or carbon dioxide content in the culture medium, and / or the presence or concentration of proteins in the culture medium, in particular cytokines.

[0059] The advantage here is that analysis and monitoring can be carried out during the cultivation process without stopping or disrupting it. Furthermore, examining characteristic properties can provide early insight into the quality of the cultivation, allowing adjustments to the cultivation conditions to be made directly during cultivation.

[0060] Furthermore, in a further embodiment, it is advantageous if, in step c), the cultivation is terminated by generating a liquid flow via the fluidic connection of the cavity in the direction of the channel. This flushes away the culture medium around the microparticles, thus stopping the cultivation process. The microparticles themselves or the cells or three-dimensional cell agglomerates of dissolved microparticles can also be transported from the cavity into the channel by means of the liquid flow. The supplied liquid can be, for example, a culture medium or a buffer. It is advantageous that these steps can be flow-induced via the fluidic connections of the cavities, and the discharge of the microparticles or cells orthree-dimensional cell agglomerates from the cavities is almost completely ensured compared to flows induced only via the channel.

[0061] In a further advantageous embodiment, the microparticles are hydrogel structures comprising at least one cell and / or a three-dimensional cell agglomerate, in particular spherical ones. Furthermore, the three-dimensional cell agglomerates are, for example, organoids or spheroids. Matrigel™ (Corning) is preferably used as the hydrogel of the hydrogel structure. Alternatively, other hydrogels such as agarose, gelatin, or polyethylene glycol can also be used.

[0062] The hydrogel structures are chosen to be large enough to accommodate one or more three-dimensional cell agglomerates, particularly organoids. The diameters of tumor organoids, for example, which are particularly suitable for drug testing, range between 100 and 750 pm.

[0063] Furthermore, in one embodiment, it is advantageous if, in step c), the hydrogel structures are depolymerized, and the cells and / or three-dimensional cell agglomerates, in particular organoids or spheroids, are released into the cavity. For depolymerization, a reagent is supplied via the fluidic connection to the cavity. The reagent, for example, has a temperature that induces or supports depolymerization.

[0064] In the case of the Matrigel hydrogel, this depolymerizing reagent is, for example, Corning™ Dispase (Corning) or Corning™ cell recovery solution (Corning). Alternative reagents are enzyme solutions containing, for example, trypsin or TrypLE™ Express Enzyme (Thermofisher). Additionally or alternatively, the depolymerization of the Matrigel™ (Corning) can be achieved by lowering the temperature to approximately 4°C. For this purpose, culture medium at a temperature of approximately 4°C is supplied via the fluidic connections of the cavities and / or the channel of the microfluidic device. Alternatively, the microfluidic device, in particular the cavity, is heated to a temperature of approximately 4°C.

[0065] The invention further relates to a cartridge, in particular a microfluidic cartridge, as described for example in DE102016222072A1 or DE102016222075A1, comprising the microfluidic device according to the invention.

[0066] Short description of the drawing

[0067] Embodiments of the present invention are illustrated in the drawing and explained in more detail in the following description of the figures. It shows:

[0068] Fig. 1 : the schematic representation of a cross section through a microfluidic device according to the invention with a channel and cavities, each having a fluidic connection,

[0069] Fig. 2: the schematic representation of an inventive

[0070] Cartridge comprising the microfluidic device according to Figure 1 , and

[0071] Fig. 3: the schematic representation of a flow diagram of an embodiment of the method according to the invention.

[0072] Embodiments of the invention

[0073] Figure 1 shows a microfluidic device 10 for cultivating cells 9. The microfluidic device 10 comprises a channel 1 with three cavities 5a, 5b, 5c, wherein the cavities 5a, 5b, 5c extend outward from an outer surface of the channel 1. In Figure 1, the number of cavities 5a, 5b, 5c is shown merely as an example; preferably, the channel 1 has a plurality of cavities 5a, 5b, 5c. Each cavity 5a, 5b, 5c has a fluidic connection 55a, 55b, 55c connected to a pump unit, which leads away from the channel 1. The fluidic connections 55a, 55b, 55c have, for example, an integrated retaining element (not shown) and / or dimensions of 5 - 75 pm, so that the hydrogel structures 3 have larger dimensions than the fluidic connections 55a, 55b, 55c and cannot pass through or exit them. The pump unit is not shown in Figure 1.The channel 1 has a bend 11, whereby the channel 1 has an inner curved surface 12, a neutral axis 13, and an outer curved surface 14 in the region of the bend 11. The cavities 5a, 5b, 5c are arranged in the bend of the channel 1 on the outer curved surface 14.

[0074] Preferably, and not shown in Figure 1, the device comprises a device for tempering the channel 1 and / or the at least one cavity 5a, 5b, 5c.

[0075] Furthermore, the channel 1 and / or the cavities 5a, 5b, 5c can be designed to be at least partially optically transparent and can have a control unit (not shown), in particular a camera and / or a microscope unit.

[0076] The following describes an embodiment of the method according to the invention for cultivating cells 9 using the microfluidic device 10. Spherical hydrogel structures 3 are described as examples for the microparticles 3 surrounding the at least one cell 9, and organoids 7 are described as examples for the three-dimensional cell agglomerates 7. Matrigel™ (Corning) is preferably used as the hydrogel.

[0077] The hydrogel structures 3 are chosen to be large enough to accommodate one or more organoids 7. The diameters of the organoids 7 are, for example, 100–750 pm.

[0078] In Figure 1, the cavities 5a, 5b, and 5c depict different steps of the process. The first cavity 5a depicts the first step a) of the process, the second cavity 5b shows the second step b), and the third cavity 5c depicts the third step c) of the process.

[0079] In the first step a), a medium, in particular a culture medium, with spherical hydrogel structures 3 is conveyed through the channel 1. The flow direction is indicated by the arrows 15. The hydrogel structures 3 each enclose a cell 9 and are transported with the liquid flow in the channel 1. The channel 1 has a bend 11. During the transport of the hydrogel structures 3 through the bend 11, a centrifugal force acts on the hydrogel structures 3, which transports them towards the outer curved surface 14 of the channel 1, on which the cavities 5a, 5b, 5c are located, and into this. In addition, medium is conveyed out via the fluidic connections 55a, 55b, 55c of the cavities 5a, 5b, 5c, shown as outflow 17, so that a suction is created in the direction of the cavities 5a, 5b, 5c, which draws the hydrogel structures 3 into them.

[0080] Preferably, the cavities 5a, 5b, 5c are dimensioned such that exactly one hydrogel structure 3 fits therein, and subsequent hydrogel structures 3 are transported to the next free cavity 5a, 5b, 5c. Alternatively, and not shown in Figure 1, the cavities 5a, 5b, 5c can also be dimensioned such that multiple hydrogel structures 3 are accommodated in one cavity 5a, 5b, 5c.

[0081] In a second step b), the cell 9 is cultured in the hydrogel structure 3 located in the cavity 5a, 5b, 5c and grows into an organoid 7. For example, culture medium is provided for the cultivation process via channel 1. The culture medium diffuses into the cavities 5a, 5b, 5c and into the hydrogel structures 3. The temperature suitable for culturing the organoids 7, preferably approximately 37°C, is set by controlling the temperature of channel 1 and / or the cavities 5a, 5b, 5c. Via the fluidic connections 55a, 55b, 55c of the cavities 5a, 5b, 5c, liquids are supplied to the hydrogel structures 3 or cells 9 in a precisely dosed manner during the cultivation process, represented as inflow 19, and thus the cultivation of the cells 9 into organoids 7 is specifically influenced.This liquid is a culture medium, for example, with a modified composition and / or containing growth factors, nutrients, and / or pharmacologically active ingredients. The supply or inflow 19 of the liquid can occur continuously or at specific times. When culture medium is added, "used" culture medium is replaced with "fresh" culture medium, so that the cells 9 are supplied with dissolved nutrients and gases. In this way, a media change is implemented microfluidically. Furthermore, for example, a culture medium with varied oxygen and / or carbon dioxide content can be supplied. In this way, it is possible to establish hypoxic cultivation conditions.During cultivation, for example, a volume of liquid is pumped out of the cavity 5a, 5b, 5c via the fluidic connection 55a, 55b, 55c and examined for parameters, in particular the pH value, the concentration of glucose and / or lactate, the oxygen and / or carbon dioxide content and / or the presence and concentration of proteins, in particular cytokines.

[0082] In a third step c), the cultivation process is terminated at a specific time, for example, when the desired size or morphology of the organoids 7 is reached. For this purpose, individual or all hydrogel structures 3 within the cavities 5a, 5b, 5c are depolymerized and dissolved in order to release the resulting organoids 7. For the depolymerization of the hydrogel structures 3, suitable liquid reagents, represented as inflow 19, are supplied via the fluidic connections 55a, 55b, 55c of the cavities 5a, 5b, 5c. Additionally or alternatively, the depolymerization of the hydrogel can be achieved by lowering the temperature to approximately 4°C. For this purpose, for example, culture medium with a temperature of approximately 4°C is supplied via the fluidic connections 55a, 55b, 55c of the cavities 5a, 5b, 5c and / or channel 1. Alternatively or additionally, the microfluidic device 10 can also be tempered to 4°C.The organoids 7 released from the hydrogel structures 3 after depolymerization are then transported from the cavities 5a, 5b, 5c into the microfluidic channel 1. For this purpose, for example, a culture medium is introduced via the fluidic connections 55a, 55b, 55c of the cavities 5a, 5b, 5c, and a liquid flow is generated towards the channel 1 via this inflow 19. Finally, the organoids 7 located in the microfluidic channel 1 are transported further and are available for subsequent cultivation or analysis steps, in particular for expansion or drug testing. Alternatively, and not shown in Figure 1, the hydrogel structures 3 in the cavities 5a, 5b, 5c are not initially depolymerized, but are flowed into the channel 1 as entire hydrogel structures 3.

[0083] Figure 2 shows a cartridge 100 according to the invention, which comprises the microfluidic device 10 according to the invention shown in Figure 1. The microfluidic device 10 is accommodated, for example, on a plastic substrate or chip.

[0084] Figure 3 shows a flow diagram of the method 500 according to the invention for cultivating cells 9 by means of the microfluidic device shown in Fig. 1 and the embodiments and method steps described therein.

Claims

Claims 1. Microfluidic device (10) for cultivating cells (9), comprising a channel (1) with at least one cavity (5a, 5b, 5c) for receiving at least one microparticle (3), which protrudes from an outer surface of the channel (1), characterized in that the at least one cavity (5a, 5b, 5c) has a fluidic connection (55a, 55b, 55c) connected to a pump unit, which leads away from the channel (1).

2. Microfluidic device (10) according to claim 1, characterized in that the fluidic connection (55a, 55b, 55c) of the at least one cavity (5a, 5b, 5c) has an integrated retaining element and / or dimensions of 5 - 75 pm.

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

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

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

6. Microfluidic device (10) according to one of the preceding claims, characterized in that it further comprises a reservoir which is in fluidic connection with the fluidic connection (55a, 55b, 55c) of the at least one cavity (5a, 5b, 5c) and in which a culture medium, growth factors, nutrients and / or pharmacological active substances are stored.

7. Microfluidic method (500) for cultivating cells (9) by means of a device (10) according to one of claims 1-6, comprising the following steps: a) conveying a medium with microparticles (3) via the channel (1), wherein a microparticle (3) encloses at least one cell (9), and introducing at least one microparticle (3) into at least one cavity (5a, 5b, 5c); b) cultivating the at least one cell (9) in the microparticle (3) located in the cavity (5a, 5b, 5c), in particular to form a three-dimensional cell agglomerate (7), wherein a dosed supply of a culture medium, growth factors, nutrients and / or pharmacologically active ingredients takes place via the fluidic connection (55a, 55b, 55c) of the cavity (5a, 5b, 5c). c) terminating the cultivation and removing the at least one microparticle (3) or the at least one cultured cell (9), in particular the three-dimensional cell agglomerate (7), from the cavity (5a, 5b, 5c).

8. Microfluidic method (500) according to claim 7, characterized in that in step a) the introduction of the at least one microparticle (3) into the at least one Cavity (5a, 5b, 5c) occurs due to a centrifugal force acting on the microparticles (3), which occurs due to a bend (11) of the channel (1) as it flows through.

9. Microfluidic method (500) according to one of claims 7 or 8, characterized in that in step a) the introduction of the at least one microparticle (3) into the at least one cavity (5a, 5b, 5c) is carried out in a flow-induced manner by conveying out the medium via the fluidic connection (55a, 55b, 55c) of the cavity (5a, 5b, 5c).

10. Microfluidic method (500) according to one of claims 7 - 9, characterized in that in step b) a culture medium with varied oxygen and / or carbon dioxide content is supplied, in particular wherein hypoxic cultivation conditions are set.

11. Microfluidic method (500) according to one of claims 7 - 10, characterized in that in step b) during the cultivation via the fluidic connection (55a, 55b, 55c) of the cavity (5a, 5b, 5c) a volume of liquid is conveyed out of the cavity (5a, 5b, 5c) and is examined for parameters, in particular the pH value, the concentration of glucose and / or lactate, the oxygen and / or carbon dioxide content and / or the presence and concentration of proteins, in particular of cytokines.

12. Microfluidic method (500) according to one of claims 7-11, characterized in that in step c) the termination and / or the removal of the at least one microparticle (3) or the at least one cultured cell (9), in particular the three-dimensional cell agglomerate (7), from the cavity (5a, 5b, 5c) is carried out by a liquid flow via the fluidic connection (55a, 55b, 55c) of the cavity (5a, 5b, 5c) in the direction of the channel (1).

13. Microfluidic method (500) according to one of claims 7-12, characterized in that the microparticles (3) are hydrogel structures (3) comprising at least one cell (9) and / or a three-dimensional cell agglomerate (7), in particular spherical, and / or the three-dimensional cell agglomerates (7) are organoids (7) or spheroids.

14. Microfluidic method (500) according to claim 13, characterized in that in step c) the hydrogel structures (3) are depolymerized and the cells (9) and / or three-dimensional cell agglomerates (7), in particular organoids (7) or spheroids, are released in the cavity (5a, 5b, 5c), wherein for depolymerization a reagent is supplied via the fluidic connection (55a, 55b, 55c) of the cavity (5a, 5b, 5c), in particular tempered to a temperature that induces or supports the depolymerization.

15. Microfluidic method (500) according to one of claims 7-14, characterized in that the movement of the microparticles (3) in the microfluidic device (10) and the positioning of the microparticles (3) in the cavities (5a, 5b, 5c) takes place by means of dielectrophoresis and an applied inhomogeneous electric field and / or by means of applying magnetic fields and magnetic labeling of the microparticles (3) and / or by means of standing ultrasonic waves to generate pressure nodes which attract and hold the microparticles (3).

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

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