Device and process for splitting three-dimensional agglomerates

Abstract

The invention relates to a microfluidic process (500) for mechanically splitting, in particular for assisting with an enzymatic splitting of, three-dimensional agglomerates (7) to form individual structures (9) and / or agglomerate fragments (8) by means of a microfluidic device (10), the process having the following steps: a) supplying a first medium having three-dimensional agglomerates (7) into a chamber (5) via a first fluidic connection (1), wherein eddy flows (6b) are generated when the first medium flows out of the first fluidic connection (1) into the chamber (5); b) pulsatingly moving the first medium having three-dimensional agglomerates (7) back and forth, in particular by means of a pump unit, via the first (1) and a second fluidic connection (2) and / or via a third (3) and fourth fluidic connection (4) so that eddy flows (6b) are generated, on account of which the three-dimensional agglomerates (7) are mechanically split.

Description

[0001] Description

[0002] title

[0003] Device and method for splitting three-dimensional agglomerates

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

[0005] State of the art: Interest in the use of three-dimensional 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 agglomerates can, for example, accurately represent organ-specific properties. Typically, they are handled manually using tools such as pipettes, reaction vessels, and laboratory equipment. Here, microfluidics offers advantages over conventional laboratory tests, such as smaller sample volumes and reagents required, shorter analysis times, and parallel processes.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] US 2019 / 0329247 A1 describes a microfluidic chip. Cells or cell structures are to be guided from a chamber to a predetermined region to prevent the cells or cell structures from accumulating at the sides of the chamber. Using a vortex of fluid, the cells or cell structures are swirled into the center of the chamber and thus into the predetermined region.

[0010] US Patent No. 8,277,110 B2 discloses a "micromixer biochip" that mixes substances using an active vortex flow. For this purpose, it comprises a mixing chamber and a fluid channel connected to the mixing chamber at one end. Since the axis of the fluid channel does not pass through the center of the mixing chamber, the moving fluid forms a vortex flow, which creates the mixing effect.

[0011] Disclosure of the invention

[0012] For example, so-called tumor organoids and spheroids are used to research tumor diseases. These allow for the excellent reproduction of various pathological tissue conditions, making them ideal for drug tests to evaluate the efficacy and dosage of medications. Using these observations, a personalized drug-based cancer therapy can then be selected for each patient, taking individual characteristics into account and thus optimizing the effectiveness of the therapy. One method for producing tumor organoids is the removal of individual cells or tissue fragments from a cancer patient's primary tumor and their subsequent cultivation. This process involves the differentiation and proliferation of these cells or tissue fragments, which ultimately self-organize into three-dimensional structures.The resulting tumor organoids are thus three-dimensional cell agglomerates that exhibit a similar composition and architecture to the patient's primary tumor tissue. They have a diameter of 100–750 pm, for example.

[0013] Spheroids are three-dimensional cell agglomerates that can be generated by the aggregation and organization of several thousand cells, for example, with a diameter of 100–750 pm. Compared to organoids, spheroids are less complex and typically contain only one type of cell.

[0014] In order to be able to work with organoids or spheroids over a longer period of several weeks or months, currently established methods of 3D cell cultivation are used.

[0015] The main process steps here are the cultivation of organoids or spheroids and their subsequent expansion. During expansion, the organoids or spheroids are enzymatically and / or mechanically split into organoid or spheroid fragments consisting of a few tens of cells and into individual organoid or spheroid cells. The split organoid or spheroid fragments and cells are then reseeded. This process results in the proliferation of the organoids or spheroids.

[0016] For the purposes of the present invention, the term "splitting" refers to the dissolution of connections between individual structures of a three-dimensional agglomerate and the resulting dissociation of the three-dimensional agglomerate into agglomerate fragments and / or individual structures. For the purposes of the present invention, the term "three-dimensional agglomerates" refers, for example, to cell agglomerates such as organoids or spheroids.

[0017] During splitting, for example, connections between cells in a cell agglomerate are broken down, resulting in the dissociation of the cell agglomerate into multicellular cell agglomerate fragments or individual cells. The splitting of three-dimensional agglomerates, such as organoids and spheroids, is carried out enzymatically by adding an enzyme solution, for example, trypsin or TrypLE™ Express Enzyme (Thermofisher), and / or mechanically by manually pipetting the suspension up and down. Splitting must be monitored microscopically by experienced specialists to determine the degree of dissociation and then terminate splitting at the appropriate time. Choosing the appropriate time and degree of dissociation for termination is crucial, since for certain organoid types, splitting down to individual cells impairs the ability to form new organoids.The dissociation of the organoids influences the subsequent organoid formation and the ultimately resulting organoid properties. To stop the splitting, a buffer solution is first added to greatly dilute the enzyme solution. Centrifugation then occurs to form a pellet of fragments or cells, and the supernatant is aspirated and discarded. The supernatant must be removed carefully to avoid damaging the pellet and inadvertently losing any fragments or cells. The fragments or cells resulting from dissociation are resuspended, for example, in an extracellular matrix, and 3D cultivation steps are repeated for repeatable organoid expansion.

[0018] The multi-step splitting process includes, for example, repeated addition and removal of different liquids, centrifugation and washing processes, and dissociation of the organoids by pipetting up and down.

[0019] On a laboratory scale, this involves changing vessels, centrifugation steps, and visually monitored pipetting processes. These cultivation and expansion steps are highly labor- and time-intensive and can only be performed by trained and experienced specialists in appropriately equipped laboratories.

[0020] The present invention addresses a microfluidic implementation of the processes for splitting three-dimensional agglomerates, such as tumor organoids or tumor spheroids.

[0021] According to the invention, a microfluidic device for mechanical splitting, in particular for supporting enzymatic splitting, of three-dimensional agglomerates into individual structures and / or agglomerate fragments with a chamber and a first and second fluidic connection, as well as a method for operating the same with the features of the independent patent claims are provided.

[0022] Here, the first fluidic connection is arranged on a first side surface of the chamber, and the second fluidic connection is arranged on a third side surface of the chamber, in particular opposite the first fluidic connection. This is based in particular on the fact that the cross-section of the chamber is 2 to 20 times larger than the cross-section of the first fluidic connection, so that vortex flows can be generated.

[0023] The advantage here is that the vortex flows in the chamber are generated and utilized to distribute and mix the introduced three-dimensional agglomerates and liquids, and to generate adjustable fluid-mechanical shear forces that act on the three-dimensional agglomerates, agglomerate fragments, and individual structures, causing them to dissociate. These mechanical shear forces are precisely defined and can be adjusted in terms of magnitude and duration, thus making them reproducible.

[0024] The key reason for the generation of vortex flows lies in the significant and abrupt cross-sectional change from the first fluidic connection to the significantly larger chamber. The shape and strength of the generated vortex flows can be advantageously designed and adjusted by the geometric configuration of the fluidic connections and the chamber, as well as by the flow rate of the liquid pumped by a pump unit. Another advantage is that the microfluidic device allows the work steps for splitting three-dimensional cell agglomerates to be carried out automatically. This saves considerable time due to the elimination of manual work steps, shortened analysis times, and parallel processes. Furthermore, these work steps can be performed by more than just trained and experienced specialists, thus reducing the workload for them.

[0025] The automation of process steps, in turn, enables their standardization, which offers significant advantages, particularly for clinical and industrial applications, for example, in drug testing and personalized medicine. Another advantage is that the three-dimensional agglomerates and agglomerate fragments, as well as the individual structures, remain viable and are available for subsequent cultivation or analysis steps with virtually no loss.

[0026] Because the process steps take place within the microfluidic device, the risk of unwanted or problematic contamination of the sample is also reduced.

[0027] Further advantageous embodiments of the microfluidic device emerge from the subclaims.

[0028] The cross-section of the fluidic connections is, for example, rectangular or square, with rounded corners. Alternatively, the cross-section of the fluidic connections is, for example, round or oval.

[0029] It is advantageous if the first fluidic connection has dimensions of 100 - 750 pm so that three-dimensional agglomerates can pass through it and if the second fluidic connection has dimensions of 25 - 150 pm so that only split individual structures and / or agglomerate fragments can pass through it, but not three-dimensional agglomerates in unsplit form.

[0030] In this way, it is ensured that only split individual structures and / or agglomerate fragments are removed via the second fluidic connection.

[0031] The dimensions of the first and second fluidic connections can be adapted to the respective requirements or sizes of the three-dimensional agglomerates used.

[0032] In a particularly advantageous embodiment, the chamber further comprises a third and a fourth fluidic connection, particularly one located opposite the third and fourth fluidic connections. The cross-section of the chamber is 2 to 20 times larger than the cross-section of the third and fourth fluidic connections, allowing vortex flows to be generated.

[0033] The advantage here is that liquids and media can also be introduced into the chamber via the third and fourth fluidic connections, allowing the microfluidic device to be used with great flexibility. Another advantage is that additional vortex flows are generated in the chamber, which, on the one hand, distribute and mix the liquids and components in the chamber. On the other hand, the vortex flows generate defined, adjustable fluid-mechanical shear forces that act on the three-dimensional agglomerates, agglomerate fragments, and individual structures in the chamber, further driving and supporting the dissociation of the agglomerates and agglomerate fragments. These mechanical shear forces are precisely defined and, in terms of magnitude and duration, can be suitably adjusted, thus being reproducible.

[0034] It is advantageous if the third and fourth fluidic connections have an integrated retaining element and / or dimensions of 5 - 75 pm. This is advantageous because three-dimensional agglomerates, agglomerate fragments, or individual structures cannot pass through them. This ensures that the three-dimensional agglomerates, agglomerate fragments, or individual structures cannot leave the chamber via the third and fourth fluidic connections during the introduction, removal, and reciprocating movement of media and liquids. This enables simple and loss-free introduction, removal, and reciprocating movement of media and liquids.

[0035] The integrated retention element is designed, for example, as a microsieve, microfilter, and / or microstructured mesh with pores. If the third and fourth fluidic ports are equipped with such a mesh, these ports can have larger dimensions, advantageously enabling higher flow rates.

[0036] In a further advantageous embodiment, the third fluidic connection is arranged on a second side surface (5b) lying transversely to the first and third side surface of the chamber, and the fourth fluidic connection is arranged on a fourth side surface of the chamber opposite the second side surface (5b).

[0037] The advantage here is that favorable flow conditions are created. During the splitting process, for example, the medium containing the three-dimensional agglomerates or the enzyme solution can be alternately moved back and forth between the first and second fluidic connections and the third and fourth fluidic connections. This generates vortex flows throughout the chamber and prevents or minimizes disadvantageous flow-free areas in the chamber where liquids, three-dimensional agglomerates, agglomerate fragments, or individual structures accumulate. This improves the efficiency of the splitting process.

[0038] In a further advantageous embodiment, the chamber has a rectangular shape, in particular with a length and a width of 2 - 15 mm and a height of 0.75 - 2 mm.

[0039] These dimensions are advantageous because they realize a cross-sectional ratio of the chamber and fluidic connections that is advantageous for the generation of vortex flows.

[0040] Alternatively, other geometric configurations of the chamber and the fluidic connections are also possible, particularly to allow the generation of stronger or more precisely adjustable vortex flows inside the chamber. In an alternative embodiment, the microfluidic device comprises two or more microfluidic chambers, which can be used simultaneously, for example. The advantage here is that the throughput of the splitting process is increased, thus enabling shorter overall processing times.

[0041] Another advantage is that the microfluidic chamber and fluidic connections have a significantly smaller volume compared to conventional laboratory vessels. Significantly smaller fluid volumes are required and consumed for the process of splitting the three-dimensional agglomerates. Furthermore, fluid exchange processes require less time, thus reducing processing times.

[0042] It is also advantageous if the fluidic connections are aligned orthogonally to the side walls of the chamber. This allows for the greatest possible abrupt change in cross-section at the transition from a fluidic connection to the chamber, promoting the generation of vortex flow in the chamber.

[0043] The number and position of the fluidic connections can alternatively be designed differently, such as offset connections or multiple connections to improve the formation of vortex flows. The first fluidic connection is arranged opposite the second fluidic connection, for example, in particular on opposite chamber walls of the microfluidic device. Alternatively, the first and second fluidic connections can be arranged offset from one another on opposite chamber walls of the microfluidic device.

[0044] In an advantageous embodiment, at least one side surface of the chamber is at least partially transparent. This can, in particular, comprise a transparent polymer such as a cycloolefin copolymer (COC), a polycarbonate (PC), a polymethyl methacrylate (PMMA), a polystyrene (PS), or glass.

[0045] The advantage here is that the optically accessible chamber allows the degree of dissociation of the three-dimensional agglomerates and agglomerate fragments to be determined and monitored before, during and after splitting.

[0046] In a further advantageous embodiment, the microfluidic device comprises a control unit. The control unit is, in particular, a microscopic unit arranged on or near the at least partially transparent side surface of the chamber. The three-dimensional agglomerates, agglomerate fragments, and individual structures are monitored by the control unit. This eliminates the need for microscopic observation and control by experienced personnel to determine the changing degree of dissociation and to stop the splitting process at an appropriate time, thus reducing the workload on the personnel.

[0047] Furthermore, in one embodiment, it is advantageous if the microfluidic device comprises at least one reservoir for a fluid. This is advantageous because fluids such as media, rinsing fluids, enzyme solutions, or staining solutions can be pre-stored in the reservoir. This ensures quick and easy supply of these fluids.

[0048] In a particularly advantageous embodiment, the microfluidic device comprises a reservoir containing an upstream enzyme solution for splitting. The enzyme solution comprises, in particular, trypsin or TrypLE™ Express enzyme, wherein the reservoir is fluidically connected to the third (3) and / or fourth fluidic port (4).

[0049] In an advantageous embodiment, at least one of the fluidic connections of the microfluidic device comprises or is connected to a pump unit. The pump unit is, for example, a peristaltic, diaphragm, or syringe pump, in particular an electrically controllable one.

[0050] In a further advantageous embodiment, the microfluidic device comprises at least one valve, which is in particular electrically controllable, so that the microfluidic device can be operated electrically. Advantageously, at least one fluidic connection and / or at least one reservoir for fluids can be individually closed and opened by means of one or more valves, so that the flow through the microfluidic device can be individually determined. In this way, various options for supplying and removing media, as well as for flow through the chamber, can be easily implemented.

[0051] A further advantageous embodiment provides that the microfluidic device has a heating device for controlling the temperature of the chamber. For the purposes of the present invention, a heating device is understood to be a device that can heat and / or cool the chamber.

[0052] The advantage here is that an optimal temperature for splitting the three-dimensional agglomerates can be provided in this way.

[0053] The optimal temperature for splitting depends, among other things, on the cultivation of the three-dimensional agglomerates. When cultivating organoids in Matrigel® (Corning), for example, it is advantageous to cool the chamber of the microfluidic device, as Matrigel liquefies at temperatures of approximately 4°C.

[0054] In an embodiment in which enzymatic splitting of the three-dimensional agglomerates is assisted by mechanical splitting, the enzymatic splitting can be further improved by setting an optimal active temperature of the enzyme.

[0055] For the enzymatic splitting of organoids and / or spheroids, the enzyme trypsin or TrypLE™ Express Enzyme (Thermofisher), for example, is used, whose optimal operating temperature is 37° Celsius. An alternative or additional embodiment provides that the microfluidic device comprises a device for generating and introducing ultrasound and / or a device for generating and introducing vibration into the chamber of the microfluidic device.

[0056] The advantage of introducing ultrasound and / or vibration is that the ultrasonic waves and / or vibrations act on the three-dimensional agglomerates in the chamber and support and improve the splitting.

[0057] The invention further relates to a microfluidic method for mechanically splitting, in particular to support enzymatic splitting, three-dimensional agglomerates into individual structures and / or agglomerate fragments by means of a microfluidic device, comprising the following steps: a) feeding a first medium containing three-dimensional agglomerates into the chamber of the microfluidic device via the first fluidic connection, wherein vortex flows are generated when the first medium passes from the first fluidic connection into the chamber. The three-dimensional agglomerates are distributed in the chamber by means of the vortex flow. The chamber can already be filled with a medium, in particular with the same medium as that in which the three-dimensional agglomerates are contained. Medium can be discharged from the chamber via the second fluidic connection.The decisive reason for the formation of vortex flows lies in the significant and abrupt cross-sectional change from the first fluidic connection to the significantly larger chamber. b) Pulsatile back-and-forth movement of the first medium with three-dimensional agglomerates, in particular by means of a pump unit, across the first and second fluidic connections and / or across the third and fourth fluidic connections, so that.

[0058] Vortex flows are generated. The vortex flows generate, on the one hand, a movement of the three-dimensional agglomerates or agglomerate fragments in the chamber and, on the other hand, defined and adjustable shear forces that act on the three-dimensional agglomerates or agglomerate fragments and lead to their mechanical dissociation, or in other words, their splitting. The first medium here absorbs a higher speed than the three-dimensional agglomerates, which also leads to the washing away or detachment of individual structures or agglomerate fragments on the surface of the three-dimensional agglomerates. In addition, the three-dimensional agglomerates themselves collide with one another, generating further friction. Due to these friction processes, the connections between the individual structures, which are responsible for the cohesion of the three-dimensional agglomerate, are increasingly dissolved.More and more individual structures and agglomerate fragments split off from the three-dimensional agglomerates. In this way, the three-dimensional agglomerates are mechanically dissociated.

[0059] Pulsatile back and forth movement of the first medium means that the conveying direction of the first medium through the microfluidic device is temporarily changed by successive pulsed forward and backward conveying of the first medium.

[0060] The first medium containing the three-dimensional agglomerates is moved back and forth between two fluid connections in the chamber at a flow velocity of 0.05–0.2 m / s, for example. The flow rate can be constant or varying, particularly pulsating.

[0061] In the case of a pulsatile reciprocating movement involving the first, second, third and fourth fluidic connection, the reciprocating movement occurs, for example, in an alternating sequence between the first and second fluidic connection and the third and fourth fluidic connection.

[0062] An advantage of the method according to the invention is that the vortex flows are generated and used in the chamber to, on the one hand, distribute or mix the introduced three-dimensional agglomerates and liquids and, on the other hand, to generate adjustable fluid-mechanical shear forces that act on the three-dimensional agglomerates, agglomerate fragments, and individual structures, causing the agglomerates and agglomerate fragments to dissociate. These mechanical shear forces are precisely defined and, in comparison to conventional manual pipetting processes, can be suitably adjusted in terms of magnitude and duration, thus making them reproducible. A further advantage is that the shape and strength of the generated vortex flows can be advantageously designed and adjusted through the geometric configuration of the fluidic connections and the chamber, as well as through the flow rate of the liquid pumped by a pump unit.Another advantage is that the process for splitting three-dimensional cell agglomerates can be automated. This saves considerable time by eliminating manual steps (such as repeated addition and removal of various liquids, performing centrifugations and washing processes, and dissociating the organoids by pipetting up and down), shortening analysis times, and eliminating parallel processes. These steps can therefore be performed by more than just trained and experienced specialists, thus reducing the workload for them.

[0063] The automation of process steps makes their standardization possible, which offers great advantages, particularly for clinical and industrial applications, for example in the field of drug testing and personalized medicine.

[0064] Another advantage is that the three-dimensional agglomerates and the agglomerate fragments, as well as the individual structures, remain viable and are available for subsequent cultivation or analysis steps with almost no losses.

[0065] The fact that the process steps take place within the microfluidic device also reduces the risk of unwanted or problematic contamination of the sample.

[0066] In a particularly advantageous embodiment, in a step a') an enzyme solution is added via a fluidic connection, in particular via the third and / or fourth fluidic connection, so that when the enzyme solution passes from the fluidic connection into the chamber, vortex flows are generated and wherein in step b) the enzyme solution is moved back and forth in a pulsating manner.

[0067] For this purpose, for example, the enzyme solution with a splitting effect is first added to the three-dimensional agglomerates, which are then incubated with the enzyme-containing solution, for example, for a period of ten minutes. During or after the incubation, the enzyme-containing solution is pulsated back and forth with the three-dimensional agglomerates in the chamber to mechanically support the splitting process. The advantage is that the splitting process is enzymatically supported, improved, and especially accelerated by the simultaneous enzymatic and mechanical action on the three-dimensional agglomerates and agglomerate fragments.

[0068] In a further embodiment, it is provided that the degree of dissociation of the three-dimensional agglomerates into individual structures and / or agglomerate fragments is determined during the splitting in a method step c) by means of a control unit, in particular by means of an image evaluation method.

[0069] The advantage of this approach is that the degree of dissociation of the three-dimensional agglomerates and agglomerate fragments can be observed and monitored, for example, using image analysis, and the time to complete the splitting process can be reliably and easily determined. Traditionally, this step was performed by experienced specialists, which can reduce the workload.

[0070] In a further advantageous embodiment, the splitting in a process step d) is stopped, in particular automatically, when the control unit determines that the desired degree of dissociation of the three-dimensional agglomerates into individual structures and / or agglomerate fragments has been reached.

[0071] If mechanical splitting was supported by enzymes, the splitting process can be stopped, for example, by introducing a buffer solution or a medium, especially a culture medium, into the chamber. The buffer solution or medium displaces the enzyme solution from the chamber. The buffer solution or medium is introduced via one of the fluidic connections; as it enters the chamber, vortex flows are generated, ensuring rapid mixing or displacement.

[0072] In a step e), the individual structures and / or agglomerate fragments are removed via a fluidic connection, in particular via the second fluidic connection.

[0073] The first, third, and fourth fluidic ports are used, for example, simultaneously or in a suitable alternating sequence as inlets for the flow of a medium into the chamber. This can generate favorable flow conditions and vortex flows in the chamber to remove all or as many of the organoid fragments and organoid cells as possible from the chamber.

[0074] Alternatively, the individual structures and / or agglomerate fragments can be further processed within the chamber.

[0075] Further cultivation or analysis steps with the individual structures and / or agglomerate fragments can be carried out outside or inside the microfluidic device.

[0076] In one embodiment, it is advantageous if further media, for example a rinsing liquid, are introduced into and discharged from the chamber via the fluidic connections, in particular via the second and / or third and / or fourth fluidic connection, in particular for carrying out washing processes, wherein vortex flows are generated when the further media pass into the chamber.

[0077] A rinsing liquid can be used to remove any residues of the first medium and to clean the three-dimensional agglomerates as well as the microfluidic device.

[0078] In an embodiment in which a combined mechanical and enzymatic splitting of the three-dimensional agglomerates takes place, a washing step using a rinsing liquid is advantageous, since this removes proteins of the first medium adhering to the three-dimensional agglomerates, which would otherwise, for example, inhibit the enzyme reaction.

[0079] In a particularly advantageous embodiment, the three-dimensional agglomerates are cell agglomerates, in particular organoids or spheroids and the split

[0080] Individual structures are cells, in particular organoid cells or spheroid cells, and the agglomerate fragments are cell agglomerate fragments, in particular organoid fragments or spheroid fragments.

[0081] The advantage of using cell agglomerates such as organoids or spheroids is that their viability is well maintained and thus further cultivation and expansion steps are possible.

[0082] The invention further relates to a control unit configured to carry out and / or control the steps of the method according to the invention in corresponding units, in particular in a microfluidic cartridge, in particular by electrical actuation of the at least one valve and / or the at least one pump.

[0083] A further subject matter of the invention is a cartridge, in particular a microfluidic cartridge, as described for example in DE102016222072A1 or DE102016222075A1, comprising the microfluidic device according to the invention.

[0084] The microfluidic cartridge, for example, represents a system for culturing, expanding, drug treatment, and analyzing tumor organoids.

[0085] Short description of the drawing

[0086] Embodiments of the present invention are illustrated in the drawing and explained in more detail in the following description of the figures. It shows:

[0087] Fig. 1 : the schematic representation of a cross section through a microfluidic device according to the invention in a first embodiment to illustrate the generation of vortex flows,

[0088] Fig. 2a: the schematic representation of a cross section through a microfluidic device according to the invention in a second embodiment to illustrate the method step a)

[0089] Fig. 2b: the schematic representation according to Figure 2a to illustrate the process step b)

[0090] Fig. 2c: the schematic representation according to Figure 2a to illustrate the process step e),

[0091] Fig. 3: the schematic representation of a cartridge according to the invention comprising the microfluidic device according to Figure 2a, and

[0092] Fig. 4: The schematic representation of a flow diagram of an embodiment of the method according to the invention. Embodiments of the invention Figure 1 shows the microfluidic device 10 according to the invention in a first embodiment. The microfluidic device 10 comprises a first fluidic connection 1 and a second fluidic connection 2, which open into a chamber 5. The first fluidic connection 1 is arranged on the first side surface 5a of the chamber 5, and the second fluidic connection 2 is arranged on the opposite side surface 5c of the chamber 5. The fluidic connections 1, 2 are aligned orthogonally to the side surfaces 5a, 5c of the chamber 5. The cross-section 55 of the chamber 5 is 2 to 20 times larger than the cross-section 11 of the first fluidic connection 1. The chamber 5 has a rectangular shape, for example with a length and a width of 2 - 15 mm and a height of 0.75 - 2 mm.The first fluidic connection 1 has, for example, dimensions of 100 - 750 pm, and the second fluidic connection 2 has, for example, dimensions of 25 - 150 pm. Advantageously, but not shown in Figure 1, at least one side surface 5a, 5b, 5c, 5d of the chamber 5 is at least partially transparent.

[0093] Figure 1 illustrates the generation of vortex flows 6b. An inflow 6a of a liquid into chamber 5 is shown via fluidic connection 1. As the liquid transitions from fluidic connection 1 into chamber 5, vortex flows 6b are generated due to the significant and abrupt change in cross-section from the first fluidic connection 1 to the significantly larger chamber 5. The shape and strength of the generated vortex flows 6b can be adjusted by the geometric design of the fluidic connections 1, 2 and chamber 5, as well as by the flow rate of the liquid. The outflow 6c of the liquid from chamber 5 occurs via the second fluidic connection 2.

[0094] Figure 2a shows the microfluidic device 10 according to the invention as shown in Figure 1 in a second embodiment with two further fluidic connections, namely a third fluidic connection 3 and a fourth fluidic connection 4. The third fluidic connection 3 is arranged on the second side surface 5b of the chamber 5, and the fourth fluidic connection 4 is arranged on the fourth side surface 5d of the chamber 5 opposite this. The third fluidic connection 3 and the fourth fluidic connection 4 have dimensions of 5-75 pm, for example. An exemplary embodiment of the method according to the invention using the microfluidic device 10 in the second embodiment shown in Figure 2 is described below.Here, the mechanical splitting of organoids 7 into organoid cells 9 and / or organoid fragments 8 is described as an example for the mechanical splitting of three-dimensional agglomerates 7 into individual structures 9 and / or agglomerate fragments 8.

[0095] In a first step a) of the microfluidic process, the first fluidic port 1 and the second fluidic port 2 are open, and the third 3 and fourth fluidic ports 4 are closed, or their pump units are inactive. The dimensions of the first fluidic port 1 are suitably selected so that organoids 7 of the desired size can pass through. The dimensions of the second fluidic port 2 are suitably smaller in comparison, so that only dissociated or split organoid fragments 8 and organoid cells 9, or very small organoids 7, can pass through it out of the chamber 5. A first medium containing organoids 7 is introduced into the chamber 5 of the microfluidic device 10 via the first fluidic port 1 using a pump unit (not shown). The first medium is, for example, a culture medium or a buffer solution in which the organoids 7 are suspended.Chamber 5, for example, is already filled, in particular with the first medium without organoids. When the first medium containing organoids 7 passes from the first fluidic connection 1 into chamber 5, the vortex flows 6b described in Figure 1 are generated. The organoids 7 are distributed within chamber 5 by means of the vortex flow 6b. The first medium can be discharged from chamber 5 via the second fluidic connection 2.

[0096] Optionally, in a step a'), an enzyme solution is added to chamber 5. The addition occurs, for example, via the third fluidic connection 3 as the inlet and the fourth fluidic connection 4 as the outlet. This continues until the desired enzyme concentration is reached within chamber 5. The vortex flows 6b generated thereby support the formation of a homogeneously distributed enzyme concentration within chamber 5. The dimensions of the third 3 and fourth fluidic connections 4 are suitably selected so that no organoids 7, organoid fragments 8, or organoid cells 9 are transported out of chamber 5 and lost during the addition of the enzyme solution. Alternatively, retention elements such as microfilters or sieves are integrated into the third 3 and fourth microfluidic connections 4, so that these can also have larger dimensions.

[0097] Figure 2b shows a second step b) of the method according to the invention. For example, a suitably executed pulsatile back-and-forth movement of the enzyme solution with organoids 7 takes place in alternating sequence via the third 3 and fourth fluidic connection 4. The first 1 and the second fluidic connection 2 are closed, or the pump unit connected to them is inactive. The back-and-forth movement takes place in particular via at least one pump unit. The resulting vortex flows 6b generate, on the one hand, a movement of the organoids 7 and, if applicable, organoid fragments 8 in the chamber 5 and, on the other hand, defined and adjustable shear forces that act on the organoids 7 and organoid fragments 8 and mechanically support the enzymatic splitting, resulting in a dissociation of the organoids 7 and organoid fragments 8.

[0098] For the optical observation and control of the dissociation process of the organoids 7, at least one side surface 5a, 5b, 5c, 5d of the chamber 5 is at least partially transparent.

[0099] In a step c), which takes place simultaneously with step b), the interior of the chamber 5 containing the organoids 7, organoid fragments 8, and organoid cells 9 is monitored, for example, by means of a control unit not shown in the figures. The control unit comprises, for example, a microscope or a microscope unit connected to the chamber 5. The optical monitoring allows, in particular, the decision as to when the desired degree of dissociation of the organoids 7 and organoid fragments 8 has been reached. This can be automated using suitable image analysis methods, for example, by detecting the dimensions and number of organoids 7, organoid fragments 8, and organoid cells 9 in the chamber 5.

[0100] When the control unit determines that the desired degree of dissociation of the organoids 7 into organoid cells 9 and / or organoid fragments 8 has been achieved, the splitting process is stopped, in particular automatically, in a step d) not shown in Figure 2b. For this purpose, a buffer solution or a medium, in particular a cultivation medium, is introduced into the chamber 5 via one of the fluidic connections 1, 2, 3, 4, in particular via the third 3 or the fourth fluidic connection 4, whereby vortex flows 6b are generated when the buffer solution or the medium passes into the chamber 5. As a result, the enzyme solution is first diluted and finally removed from the chamber 5. To carry out the fluid exchange and possibly further washing processes, the fluidic connections 3 and 4 can also be used alternately as inlet and outlet in a suitable manner.The resulting vortex flows 6b in the chamber 5 support the efficiency of the fluid exchange in terms of improved mixing and reduction of the required duration.

[0101] Figure 2c shows a further method step e), in which the organoid fragments 8 and organoid cells 9 are removed from the microfluidic chamber 5. These are removed via an outflow 6c via the second fluidic connection 2. The first 1, the third 3, and the fourth fluidic connection 4 are used simultaneously or in a suitable alternating sequence as inlets for the inflow 6a of a buffer solution or a medium, in particular cultivation medium, into the chamber 5. This allows advantageous flow conditions and vortex flows 6b to be generated in the chamber 5 in order to remove all or as many as possible of the organoid fragments 8 and organoid cells 9 from the chamber 5.In this case, the interior of the optically accessible chamber 5 can be observed, for example, microscopically, whereby monitoring and optimization of the removal of the organoid fragments 8 and / or organoid cells 9 from the chamber 5 takes place.

[0102] The generated and extracted organoid fragments 8 and / or organoid cells 9 are then available for subsequent cultivation or analysis steps.

[0103] In an alternative embodiment, the organoid fragments 8 and / or organoid cells 9 are not removed from the microfluidic chamber 5, but further cultivation or analysis steps are carried out within the chamber 5.

[0104] Figure 3 shows a cartridge 100 according to the invention, which, as an example for all embodiments of the microfluidic device 10 according to the invention, comprises a microfluidic device 10 in the second embodiment shown in Figure 2a. The microfluidic device 10 is housed, for example, on a plastic substrate or chip.

[0105] Figure 4 shows a flow diagram with an embodiment of the method 500 according to the invention for mechanical splitting, in particular for supporting enzymatic splitting, of three-dimensional agglomerates 7 into individual structures 9 and / or agglomerate fragments 8, for example by means of the microfluidic device shown in Figs. 2a - 2c and the embodiments and method steps described therein.

[0106] Figure 4 shows step a) in which the three-dimensional agglomerates 7 are fed into the chamber 5 of the microfluidic device 10. In an optional step a'), an enzyme solution is added to the chamber 5 to enzymatically support the mechanical splitting process that occurs in step b). Here, the medium or enzyme solution containing the three-dimensional agglomerates 7 is moved back and forth in a pulsating manner, creating vortex flows 6b. In particular, in step c), the degree of dissociation of the three-dimensional agglomerates 7 into individual structures 9 and / or agglomerate fragments 8 is determined simultaneously by means of a control unit. In step d), the splitting process is stopped when the control unit determines that the desired degree of dissociation has been achieved. In step e), the individual structures 9 and agglomerate fragments 8 are removed from the chamber 5.

Claims

Claims 1. A microfluidic method (500) for mechanically splitting, in particular to assist enzymatic splitting, three-dimensional agglomerates (7) into individual structures (9) and / or agglomerate fragments (8) by means of a microfluidic device (10) according to one of claims 6 to 15, comprising the following steps: a) feeding a first medium with three-dimensional agglomerates (7) into a chamber (5) via a first fluidic connection (1), wherein vortex flows (6b) are generated when the first medium passes from the first fluidic connection (1) into the chamber (5), b) pulsatile back and forth movement of the first medium with three-dimensional agglomerates (7), in particular by means of a pump unit, via the first (1) and a second fluidic connection (2) and / or via a third (3) and fourth fluidic connection (4), so that vortex flows (6b) are generated, due to which the three-dimensional agglomerates (7) be split mechanically.

2. Method (500) according to claim 1, wherein in a step a') following step a) an enzyme solution is supplied via a fluidic connection (1, 2, 3, 4), in particular via the third (3) and / or fourth fluidic connection (4), so that when the enzyme solution passes from the fluidic connection (1, 2, 3, 4) into the chamber (5), vortex flows (6b) are generated and wherein in step b) the enzyme solution is moved back and forth in a pulsating manner.

3. Method (500) according to one of the preceding claims, wherein the degree of dissociation of the three-dimensional agglomerates (7) into individual structures (9) and / or agglomerate fragments (8) is determined during the splitting by means of a control unit, in particular by an image evaluation method, and wherein the splitting process is stopped, in particular automatically, when the control unit determines that the desired degree of dissociation has been reached 4. Method (500) according to one of the preceding claims, wherein the splitting is stopped by introducing a buffer solution or a medium into the chamber (5) via one of the fluidic connections (1, 2, 3, 4), wherein vortex flows (6b) are generated when the buffer solution or the medium passes into the chamber (5).

5. Method (500) according to one of the preceding claims, characterized in that the three-dimensional agglomerates (7) are cell agglomerates, in particular organoids (7) or spheroids, and wherein the split individual structures (9) are cells, in particular organoid cells (9) or spheroid cells, and wherein the agglomerate fragments (8) are cell agglomerate fragments, in particular organoid fragments (8) or spheroid fragments.

6. Microfluidic device (10) for mechanical splitting, in particular for supporting enzymatic splitting, of three-dimensional agglomerates (7) into individual structures (8) and / or agglomerate fragments (9), comprising a chamber (5) with a first fluidic connection (1), which is arranged on a first side surface (5a) of the chamber (5), and a second fluidic connection (2), which is arranged, in particular opposite the first fluidic connection (1), on a third side surface (5c) of the chamber (5), wherein the cross section of the chamber (5) is 2 to 20 times larger than the cross section of the first fluidic connection (1), so that vortex flows (6b) for splitting can be generated.

7. Microfluidic device (10) according to claim 6, wherein the first fluidic port (1) has dimensions of 100 - 700 pm, and wherein the second fluidic port (1) has dimensions of 25 - 150 pm.

8. Microfluidic device (10) according to one of claims 6 or 7, wherein the chamber (5) further comprises a third (3) and a fourth fluidic connection (4), in particular opposite thereto, wherein the cross-section of the chamber (5) is 2 to 20 times larger than the cross-section of the third (3) and the fourth fluidic connection (4), so that vortex flows (6b) can be generated.

9. Microfluidic device (10) according to claim 8, wherein the third (3) and the fourth fluidic port (4) have an integrated retaining element and / or dimensions of 5 - 75 pm.

10. Microfluidic device (10) according to one of claims 8 or 9, wherein the third fluidic connection (3) is arranged on a second side surface (5b) lying transversely to the first (5a) and third side surface (5c) of the chamber (5), and wherein the fourth fluidic connection (4) is arranged on a fourth side surface (5d) of the chamber (5) opposite the second side surface (5b).

11. Microfluidic device (10) according to one of claims 6-10, wherein the chamber (5) has a rectangular shape, in particular with a length and a width of 2 - 15 mm and a height of 0.75 - 2 mm 12. Microfluidic device (10) according to one of claims 6-11, wherein the fluidic connections (1, 2, 3, 4) are aligned orthogonally to the side surfaces (5a, 5b, 5c, 5d) of the chamber (5).

13. Microfluidic device (10) according to one of claims 6-12, wherein at least one side surface (5a, 5b, 5c, 5d) of the chamber (5) is at least partially transparent, and in particular comprises a transparent polymer, for example a cycloolefin copolymer (COC), a polycarbonate (PC), a polymethyl methacrylate (PMMA), a polystyrene (PS) or a glass.

14. Microfluidic device (10) according to one of claims 6-13, wherein the microfluidic device (10) comprises a control unit, in particular a microscopic control unit.

15. Microfluidic device (10) according to one of claims 6-14, further comprising a reservoir with an enzyme solution stored upstream for splitting, in particular comprising trypsin or TrypLE™ Express enzyme, wherein the reservoir is in fluidic connection with the third (3) and / or fourth fluidic connection (4).

16. Cartridge (100), in particular microfluidic cartridge, comprising a microfluidic device (10) according to one of claims 6-15.

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