Apparatus and method for splitting three-dimensional agglomerates

Abstract

There is described a microfluidic method (500) for mechanically splitting three-dimensional agglomerates (7) into individual structures (9) and / or agglomerate fragments (8) by means of a microfluidic device (10), in particular for assisting the enzymatic splitting of three-dimensional agglomerates (7) into individual structures (9) and / or agglomerate fragments (8), comprising the following steps: a) feeding a first medium containing three-dimensional agglomerates (7) into a chamber (5) via a first fluid connection (1), wherein a vortex (6b) is generated when the first medium enters the chamber (5) from the first fluid connection (1), b) the first medium containing the three-dimensional agglomerates (7) is moved back and forth in a pulsating manner, in particular by means of a pump unit, via the first fluid connection (1) and the second fluid connection (2) and / or via the third fluid connection (3) and the fourth fluid connection (4), such that the vortex (6b) is generated, as a result, the three-dimensional agglomerates (7) are mechanically split.

Description

[0001] The present invention relates to a microfluidic device, a method for operating the microfluidic device and a control unit and a cartridge comprising the microfluidic device according to the preambles of the independent claims. Existing technology

[0002] In recent years, there has been a significant increase in interest in the use of three-dimensional aggregates such as organoids or spheroids for the study, diagnosis and treatment of diseases such as tumor diseases, since such three-dimensional aggregates can, for example, well reflect organ-specific properties.

[0003] They are typically handled manually using tools such as pipettes, reaction vessels, and laboratory instruments. Compared to traditional laboratory tests, microfluidics offers advantages such as smaller sample volumes and reagents required, shortened analysis times, and parallel processes.

[0004] However, implementing the necessary process steps in a microfluidic system presents numerous challenges that need to be overcome due to its complexity. For example, one of these challenges is culturing and growing such three-dimensional cell aggregates in a microfluidic system.

[0005] So-called lab-on-a-chip systems, or LoC systems for short, are microfluidic systems that house the functions of a macro-laboratory on a plastic substrate for automated processing. Such systems enable biochemical processes to be largely or completely automated.

[0006] Lab-on-a-chip systems typically consist of two main components. The first is a test carrier, such as a cartridge, which includes structures and mechanisms for manipulating the collected sample, typically passive components such as channels, reaction chambers, or stored reagents, or active components such as valves, pumps, or mixers. The second main component is a control unit for controlling the microfluidic processes within the cartridge.

[0007] US 2019 / 0329247 A1 describes a microfluidic chip. Cells or cell structures are guided from a chamber to a predetermined area to prevent accumulation of cells or cell structures on the sides of the chamber. By utilizing vortex flow in a liquid, the cells or cell structures are swirled into the center of the chamber and thus into the predetermined area.

[0008] US Patent No. 8277110 B2 discloses a "micromixer biochip" that utilizes active vortex mixing. To this end, the chip comprises a mixing chamber and a fluid channel connected to the mixing chamber at one end. Because the axis of the fluid channel does not pass through the center of the mixing chamber, the moving liquid forms a vortex, resulting in a mixing effect.

[0009] Invention Disclosure

[0010] For example, so-called tumor organoids and spheroids are used to study tumor diseases. These recreate various pathological tissue conditions very well, making them suitable for drug testing to assess drug effectiveness and dosage. These insights can, for example, enable the selection of personalized cancer drug treatments for individual patients, taking into account their individual characteristics and thus optimizing the effectiveness of the treatment.

[0011] One possible way to produce tumor organoids is to remove single cells or tissue fragments from a cancer patient's primary tumor and then culture them. This leads to the differentiation and proliferation of these cells or tissue fragments, which ultimately self-organize into three-dimensional structures. The resulting tumor organoids are therefore three-dimensional cell aggregates whose composition and structure are similar to the patient's primary tumor tissue. For example, they have a diameter of 100-750 μm.

[0012] Spheroids are three-dimensional cell aggregates that can be created by aggregating and organizing thousands of cells and have diameters of, for example, 100-750 μm. Compared to organoids, spheroids are less complex and typically contain only a single cell type.

[0013] To be able to work with organoids or spheroids over extended periods of weeks or months, established 3D cell culture methods are used.

[0014] The key process steps here are the cultivation of organoids or spheroids and their subsequent expansion. During expansion, organoids or spheroids are enzymatically and / or mechanically fragmented into organoid or spheroid fragments consisting of a few dozen cells and into single organoid or spheroid cells. These fragments and cells are then reseeded. In this way, the organoids or spheroids proliferate.

[0015] In the sense of the present invention, the term "splitting" is understood to mean the removal of the connections between the individual structures of a three-dimensional aggregate and the resulting dissociation of the three-dimensional aggregate into aggregate fragments and / or individual structures. In the sense of the present invention, the term three-dimensional aggregate is understood to mean, for example, cell aggregates, such as organoids or spheroids.

[0016] During division, for example, the connections between cells of a cell aggregate are broken, thereby causing the cell aggregate to dissociate into multicellular cell aggregate fragments or individual cells.

[0017] Three-dimensional aggregates, such as organoids and spheroids, are synthesized by adding enzyme solutions (e.g., trypsin or TrypLE TMExpress enzyme (Thermofischer)) for enzymatic splitting, and / or mechanical splitting by manually pipetting the suspension up and down. The splitting must be monitored under a microscope by an experienced professional to determine the degree of dissociation and then stop the splitting at the right time. Choosing the right stopping time and degree of dissociation is crucial because for some types of organoids, splitting into separate cells will impair their ability to re-form organoids. The dissociation of organoids will affect the formation of subsequent organoids and the properties of the final organoids. In order to stop the splitting, a buffer solution is first added to significantly dilute the enzyme solution. It is then centrifuged 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 accidentally losing any fragments or cells. The fragments or cells produced after the dissociation are resuspended in, for example, an extracellular matrix, and the 3D culture step is repeated to achieve organoid expansion that can be repeated many times.

[0018] The multi-stage dissociation process involves, for example, multiple additions and removals 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 containers, centrifugation steps, and pipetting processes with visual monitoring. These cultivation and expansion steps are very labor-intensive and time-consuming and can only be performed by well-trained and experienced personnel in suitably equipped laboratories.

[0020] The present invention relates to microfluidic implementation of a process for disrupting three-dimensional aggregates, such as tumor organoids or tumor spheroids.

[0021] According to the present invention, a microfluidic device for mechanically splitting three-dimensional aggregates into single structures and / or aggregate fragments, in particular for assisting the enzymatic splitting of three-dimensional aggregates into single structures and / or aggregate fragments, is provided, which has a chamber and a first fluid connection and a second fluid connection, as well as a method for operating the device, which have the features of the independent patent claims.

[0022] Here, the first fluid connection is arranged on a first side of the chamber, and the second fluid connection is arranged on a third side of the chamber, in particular opposite the first fluid connection. This is due in particular to the fact that the cross-sectional area of the chamber is 2 to 20 times the cross-sectional area of the first fluid connection, so that eddies can be generated.

[0023] The advantage lies in the vortex generated in the chamber, which, on the one hand, serves to disperse or mix the introduced three-dimensional aggregates and liquid, and, on the other hand, generates adjustable fluid-mechanical shear forces, which act on the three-dimensional aggregates, aggregate fragments, and individual structures, breaking them apart. These mechanical shear forces—in contrast to conventional manual pipetting—are precisely defined, can be adjusted in magnitude and duration, and are therefore reproducible.

[0024] The key reason for the formation of vortices is the significant and sudden change in cross-sectional area when connecting from the first fluid to the significantly enlarged chamber. The shape and intensity of the vortices that can be generated can be advantageously designed and adjusted by the geometric design of the fluid connections and the chamber, as well as the flow rate of the liquid conveyed by the pump unit.

[0025] Another advantage is that the microfluidic device can automate the steps involved in disrupting three-dimensional cell aggregates. This results in significant time savings due to the elimination of manual steps, shortened analysis times, and parallelization of processes. Furthermore, these steps can now be performed not only by highly trained and experienced professionals, thus reducing their burden.

[0026] Automation of process steps, which in turn can be standardized, offers enormous advantages, particularly for clinical and industrial applications, such as in the fields of drug testing and personalized medicine.

[0027] Another advantage is that three-dimensional aggregates and aggregate fragments as well as individual structures remain viable and can be used with virtually no loss in subsequent culture or analysis steps.

[0028] Furthermore, because these process steps are performed within the microfluidic device, the risk of unwanted or problematic contamination of the sample is reduced.

[0029] Further advantageous embodiments of the microfluidic device emerge from the dependent claims.

[0030] The cross section of the fluid connection can be, for example, rectangular or square, with rounded corners. Alternatively, the cross section of the fluid connection can be, for example, circular or oval.

[0031] Advantageously, the first fluid connection has a size of 100-750 μm so that three-dimensional agglomerates can pass through it, and the second fluid connection has a size of 25-150 μm so that only broken-off individual structures and / or agglomerate fragments can pass through it, but not three-dimensional agglomerates in their unbroken form.

[0032] In this way, it is ensured that only broken-off individual structures and / or agglomerate fragments are discharged via the second fluid connection.

[0033] The dimensions of the first and second fluid connections can be adjusted according to the respective requirements or dimensions of the three-dimensional agglomerates used.

[0034] In a particularly advantageous embodiment, the chamber further comprises a third fluid connection and a fourth fluid connection, in particular a fourth fluid connection opposite thereto. Here, the cross-sectional area of the chamber is 2 to 20 times the cross-sectional area of the third and fourth fluid connections, thereby being able to generate eddies.

[0035] The advantage of this is that liquids and media can also be introduced into the chamber via the third and fourth fluid connections, making the use of the microfluidic device highly flexible. Another advantage is that additional vortices are generated in the chamber, through which the liquid and components are dispersed or mixed in the chamber on the one hand. On the other hand, the vortices generate specifically adjustable fluid mechanical shear forces that act on the three-dimensional agglomerates, agglomerate fragments, and individual structures in the chamber, further driving and assisting the dissociation of agglomerates and agglomerate fragments. These mechanical shear forces are precisely defined and can be appropriately adjusted in terms of size and duration, making them reproducible.

[0036] Advantageously, the third and fourth fluid connections have integrated retaining elements and / or are sized between 5 and 75 μm. This has the advantage that three-dimensional agglomerates, agglomerate fragments, or individual structures cannot pass through these elements. This ensures that when media and liquids are introduced, discharged, or moved back and forth, three-dimensional agglomerates, agglomerate fragments, or individual structures cannot escape the chamber through the third and fourth fluid connections. This enables simple and loss-free supply and discharge, as well as back and forth movement of media and liquids.

[0037] The integrated retaining element is designed, for example, as a microsieve, microfilter and / or microstructured grid with pores. If the third and fourth fluid connections are equipped with such elements, these connections can have larger dimensions, thereby advantageously achieving higher flow rates.

[0038] In another advantageous embodiment, the third fluid connection is arranged on a second side (5b) transverse to the first and third sides of the chamber and the fourth fluid connection is arranged on a fourth side of the chamber opposite the second side (5b).

[0039] This has the advantage of creating favorable flow conditions. For example, during the splitting process, the medium or enzyme solution containing the three-dimensional aggregates can be moved back and forth alternately between the first and second fluid connections and the third and fourth fluid connections. This creates eddies throughout the chamber and prevents or minimizes unfavorable no-flow zones in the chamber where liquid, three-dimensional aggregates, enzyme solution, or individual structures can accumulate. This improves the efficiency of the splitting process.

[0040] In a further advantageous embodiment, the chamber has a rectangular shape, in particular with a length and width of 2-15 mm and a height of 0.75-2 mm.

[0041] These dimensions are advantageous since a cross-sectional area ratio of the chamber and the fluid connection is thereby achieved which is favorable for generating turbulence.

[0042] Alternatively, the chamber and fluid connections may also employ other geometric designs, in particular to allow for stronger or more precisely adjustable vortexes within the chamber. In an alternative embodiment, the microfluidic device comprises two or more microfluidic chambers, which can be used simultaneously, for example. This advantageously increases the throughput of the disruption process, thereby enabling shorter overall processing times.

[0043] Another advantage is that microfluidic chambers and fluid connections have significantly smaller volumes than conventional laboratory vessels. The volume of liquid required and consumed to break up three-dimensional aggregates is significantly smaller. Furthermore, the liquid exchange process takes less time, thus shortening processing time.

[0044] It is also advantageous if the fluid connection is arranged orthogonally to the chamber sidewall. It is advantageous if the fluid connection and the chamber transition are provided with a sudden change in cross-sectional area as large as possible, thereby promoting the generation of eddies in the chamber.

[0045] Alternatively, the number and position of the fluid connections can also be designed differently, for example with connections that are offset from one another or with more connections, in order to improve the formation of vortices.

[0046] For example, the first fluid connection and the second fluid connection are arranged opposite to each other, in particular, on opposite chamber walls of the microfluidic device. Alternatively, the first and second fluid connections can also be arranged staggered on opposite chamber walls of the microfluidic device.

[0047] In an advantageous embodiment, at least one side of the chamber is designed to be at least partially transparent and may comprise, in particular, a transparent polymer such as cycloolefin copolymer (COC), polycarbonate (PC), polymethyl methacrylate (PMMA), polystyrene (PS) or glass.

[0048] This has the advantage that the extent of dissociation of three-dimensional aggregates and aggregate fragments can be determined and monitored by an optically accessible chamber before, during and after disruption.

[0049] In another preferred 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 of the chamber. The control unit can monitor three-dimensional aggregates, aggregate fragments, and individual structures. This eliminates the need for experienced professionals to perform microscopic observation and control to determine the varying degree of dissociation and to stop the dissociation process at the appropriate time, thereby alleviating their burden.

[0050] Furthermore, in one embodiment, the microfluidic device advantageously comprises at least one reservoir for fluids. Advantageously, fluids such as media, flushing fluids, enzyme solutions, or staining solutions can be stored in the reservoir. This ensures a rapid and simple supply of these fluids.

[0051] In a particularly preferred embodiment, the microfluidic device comprises a reservoir containing an enzyme solution stored for the splitting. The enzyme solution particularly comprises trypsin or TrypLE TM Express enzyme, wherein the reservoir is in fluid communication with the third fluid connection (3) and / or the fourth fluid connection (4).

[0052] In an advantageous embodiment, at least one fluid connection of the microfluidic device comprises a pump unit or is connected to a pump unit. The pump unit is for example a peristaltic pump, a diaphragm pump or a syringe pump, in particular those that are electrically controllable.

[0053] In another advantageous embodiment, the microfluidic device includes at least one valve, which is particularly electrically controllable, thereby enabling the microfluidic device to be electrically operated. Advantageously, at least one fluid connection and / or at least one fluid reservoir can be individually closed and opened by one or more valves, thereby enabling the flow rate through the microfluidic device to be individually determined. In this way, various options for supplying and draining media and for controlling the flow through the chamber can be easily implemented.

[0054] Another advantageous embodiment provides that the microfluidic device has a heating device for regulating the temperature of the chamber. In the present invention, a heating device is understood to be a device that is capable of heating and / or cooling the chamber. Advantageously, an optimal temperature for the disruption of the three-dimensional aggregates can be provided in this way. The optimal temperature for the disruption depends, for example, on the culture of the three-dimensional aggregates. When culturing organoids in Matrigel® (Corning), for example, it is advantageous to cool the chamber of the microfluidic device because Matrigel liquefies at temperatures of approximately 4°C.

[0055] In embodiments where mechanical disruption is used to assist enzymatic disruption of three-dimensional aggregates, enzymatic disruption can be further improved by adjusting the optimal temperature for the enzyme. For example, for enzymatic disruption of organoids and / or spheroids, trypsin or TrypLE TMExpress enzyme (Thermofischer), which has an optimal operating temperature of 37°C.

[0056] Alternatively or additionally, the microfluidic device comprises a unit for generating and introducing ultrasound waves into the chamber of the microfluidic device and / or a unit for generating and introducing vibrations into the chamber of the microfluidic device. The advantage of introducing ultrasound waves and / or vibrations is that the ultrasound waves and / or vibrations act on the three-dimensional aggregates in the chamber and assist and improve the disruption.

[0057] The present invention also relates to a microfluidic method for mechanically disrupting three-dimensional aggregates into individual structures and / or aggregate fragments by means of a microfluidic device, in particular for assisting the enzymatic disruption of three-dimensional aggregates into individual structures and / or aggregate fragments, comprising the following steps:

[0058] a) A first medium containing three-dimensional aggregates is connected to a chamber of a microfluidic device via a first fluid connection, wherein a vortex is generated when the first medium is connected to enter the chamber from the first fluid connection. The three-dimensional aggregates are dispersed in the chamber by the vortex. The chamber may have been filled with a medium, in particular a medium identical to the medium containing the three-dimensional aggregates. The medium may be discharged from the chamber via a second fluid connection. The decisive reason for the generation of the vortex is that a significant and sudden change occurs in the cross-sectional area of the chamber that is significantly expanded from the first fluid connection.

[0059] b) The first medium containing the three-dimensional agglomerates is subjected to a pulsating back-and-forth movement, in particular by means of a pump unit, via the first and second fluid connections and / or via the third and fourth fluid connections, thereby generating a vortex.

[0060] On the one hand, the eddy current generates a movement of the three-dimensional agglomerates or agglomerate fragments in the chamber, and on the other hand, it generates a specific and adjustable shear force, which acts on the three-dimensional agglomerates or agglomerate fragments, causing them to mechanically dissociate, or in other words, to split them up. The first medium has a higher speed than the three-dimensional agglomerates, which also causes the individual structures or agglomerate fragments on the surface of the three-dimensional agglomerates to be washed away or fall off. In addition, the three-dimensional agglomerates themselves will also collide with each other, thereby generating further friction. Due to these friction processes, the connections between the individual structures that constitute the cohesion of the three-dimensional agglomerates are gradually released. More and more individual structures and agglomerate fragments fall off from the three-dimensional agglomerates. In this way, the three-dimensional agglomerates are mechanically dissociated. The pulsating back and forth movement of the first medium means that the transport direction of the first medium through the microfluidic device is temporarily alternated by the successive intermittent forward and backward transport of the first medium.

[0061] The first medium containing the three-dimensional agglomerates is moved back and forth between the two fluid connections in the chamber, for example, at a flow rate of 0.05 to 0.2 m / s. The flow rate can be, for example, constant or variable, in particular pulsating. In the case of a pulsating back-and-forth movement involving the first, second, third, and fourth fluid connections, the back-and-forth movement occurs, for example, in an alternating order between the first and second fluid connections and between the third and fourth fluid connections.

[0062] The advantage of the method of the present invention is that vortices are generated and utilized within the chamber to, on the one hand, disperse or mix the introduced three-dimensional aggregates and liquid, and, on the other hand, to generate adjustable fluid mechanical shear forces that act on the three-dimensional aggregates, aggregate fragments, and individual structures, leading to the dissociation of the aggregates and aggregate fragments. Compared to traditional manual pipetting processes, these mechanical shear forces are precisely defined and can be appropriately adjusted in magnitude and duration, resulting in reproducible results.

[0063] A further advantage is that the shape and intensity of the vortex that can be generated can be advantageously designed and adjusted by the geometrical design of the fluid connections and the chamber as well as the flow rate of the liquid conveyed by the pump unit.

[0064] Another advantage is that the method for dissociating 3D cell aggregates can be automated. This saves significant time by eliminating manual steps (such as the repeated addition and removal of various liquids, centrifugation and washing processes, and dissociation of organoids by pipetting up and down), shortening analysis time, and parallelizing processes. Consequently, these steps can be performed by more than just highly trained and experienced professionals, reducing their burden.

[0065] Automation of method steps, which in turn allows for their standardization, offers enormous advantages, particularly for clinical and industrial applications, for example in the areas of drug testing and personalized medicine.

[0066] Another advantage is that the three-dimensional aggregates and aggregate fragments as well as the individual structures remain viable and can be used with little loss for subsequent cultivation or analysis steps.

[0067] Since the method steps are carried out within the microfluidic device, the risk of unnecessary or problematic contamination of the sample is also reduced.

[0068] In a particularly advantageous embodiment, in step a'), the enzyme solution is added via a fluid connection, in particular via the third and / or fourth fluid connection, such that a vortex is generated when the enzyme solution enters the chamber from the fluid connection, and wherein in step b), the enzyme solution moves back and forth in a pulsating manner.

[0069] To this end, for example, a disrupting enzyme solution is first added to the three-dimensional aggregates, which are then incubated with the enzyme solution for a period of, for example, ten minutes. During or after the incubation period, the enzyme solution containing the three-dimensional aggregates is pulsed back and forth in the chamber to mechanically assist the disruption process. Advantageously, the disruption process is enzymatically assisted and improved, and in particular accelerated, by exerting, in particular simultaneous, enzymatic and mechanical action on the three-dimensional aggregates and aggregate fragments.

[0070] In another embodiment, during the disruption process in method step c), the degree of dissociation of the three-dimensional aggregates into individual structures and / or aggregate fragments is determined by a control unit, in particular by image evaluation methods. This method has the advantage that the degree of dissociation of the three-dimensional aggregates and aggregate fragments can be observed and monitored, for example, by image analysis methods, and the time at which the disruption is complete can be determined reliably and simply. Traditionally, this step is usually performed by experienced professionals, thereby alleviating this burden.

[0071] In another advantageous embodiment, when determining by a control unit that the three-dimensional agglomerate has reached the desired degree of dissociation to dissociate into individual structures and / or agglomerate fragments, the splitting in method step d) stops, particularly automatically stops. If enzymatically assisted mechanical splitting, then for example, by introducing a buffer solution or a medium (especially culture medium) into the chamber, splitting is stopped. Here, the buffer solution or medium can replace the enzyme solution in the chamber. The buffer solution or medium are introduced by one of fluid connections, wherein when it enters the chamber, a vortex is generated, which ensures rapid and sufficient mixing or displacement.

[0072] In step e), the individual structures and / or aggregate fragments are discharged by a fluid connection, in particular by a second fluid connection. For example, the first, third and fourth fluid connections can be used simultaneously or in a suitable alternating order as an inlet for the medium to flow into the chamber. Thus, favorable flow conditions and eddies can be generated in the chamber to remove all or as many organoid fragments and organoid cells as possible from the chamber. Alternatively, the individual structures and / or aggregate fragments can be further processed in the chamber. Further cultivation or analysis steps can be performed on the individual structures and / or aggregate fragments outside or inside the microfluidic device.

[0073] Advantageously in one embodiment, by fluid connection, particularly by second and / or third and / or fourth fluid connection, other medium (such as flushing fluid) is introduced into the chamber and is discharged from the chamber, particularly for carrying out cleaning process, wherein when other medium enters the chamber, eddy current is generated.By flushing fluid, possible residues of the first medium can be removed, and three-dimensional agglomerates and microfluidic devices are cleaned.In the embodiment that three-dimensional agglomerates are carried out combined mechanical and enzymatic splitting, it is advantageous to use flushing fluid to carry out cleaning step, because thus remove the protein of the first medium that adheres to the three-dimensional agglomerates, otherwise these proteins can inhibit enzymatic reaction.

[0074] In a particularly advantageous embodiment, the three-dimensional aggregates are cell aggregates, in particular organoids or spheroids, and the individual structures that are broken apart are cells, in particular organoid cells or spheroid cells, and the aggregate fragments are cell aggregate fragments, in particular organoid fragments or spheroid fragments. The advantage of using cell aggregates (such as organoids or spheroids) is that their viability can be well maintained, so further culture and expansion steps can be performed.

[0075] The subject of the invention is also a control unit configured to perform and / or control the steps of the method according to the invention in a respective unit, in particular in a microfluidic cartridge, in particular by electrical actuation of at least one valve and / or at least one pump.

[0076] Another subject of the present invention is a cassette, in particular a microfluidic cassette, as described, for example, in DE 10 2016 222 072 A1 or DE 10 2016 222 075 A1, comprising a microfluidic device according to the present invention. For example, the microfluidic cassette forms a system for the cultivation, expansion, drug treatment, and analysis of tumor organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Embodiments of the invention are illustrated in the accompanying drawings and are explained in more detail in the following description of the drawings. The drawings show:

[0079] Figure 1 : A schematic cross-sectional view of a microfluidic device according to the present invention in a first embodiment, illustrating the generation of vortexes.

[0080] Figure 2a : Schematic cross-section of a microfluidic device according to the invention in a second embodiment, for illustrating method step a)

[0081] Figure 2b :according to Figure 2a Schematic diagram for illustrating method step b)

[0082] Figure 2c :according to Figure 2aSchematic diagram for illustrating method step e),

[0083] Figure 3 : A schematic diagram of a box according to the present invention, comprising Figure 2a microfluidic devices, and

[0084] Figure 4 : A schematic flow chart of an embodiment of the method according to the present invention.

[0085] Embodiments of the present invention

[0086] Figure 1 A first embodiment of a microfluidic device 10 according to the present invention is shown. The microfluidic device 10 comprises a first fluid connection 1 and a second fluid connection 2, which lead to a chamber 5. The first fluid connection 1 is arranged on a first side 5a of the chamber 5, and the second fluid connection 2 is arranged on a side 5c of the chamber 5 opposite thereto. The fluid connections 1, 2 are arranged orthogonally to the sides 5a, 5c of the chamber 5. The cross-sectional area 55 of the chamber 5 is 2 to 20 times the cross-sectional area 11 of the first fluid connection 1. The chamber 5 has, for example, a rectangular shape with a length and width of, for example, 2 to 15 mm and a height of 0.75 to 2 mm. The dimensions of the first fluid connection 1 are, for example, 100 to 750 μm, and the dimensions of the second fluid connection 2 are, for example, 25 to 150 μm. Advantageously, although Figure 1 It is not shown in the drawing, but at least one side surface 5a, 5b, 5c, 5d of the chamber 5 is designed to be at least partially transparent.

[0087] Figure 1 To illustrate the generation of vortex 6b. The figure shows the inflow 6a of liquid into chamber 5 through fluid connection 1. When the liquid transitions from fluid connection 1 to chamber 5, vortex 6b is generated due to the significant and abrupt change in cross-sectional area from the first fluid connection 1 to the significantly enlarged chamber 5. The shape and intensity of the vortex 6b that can be generated can be adjusted by the geometric design of fluid connections 1, 2 and chamber 5, as well as by the flow rate of the liquid. Liquid flows out of chamber 5 through second fluid connection 2, 6c.

[0088] Figure 2a Shown Figure 1 In the second embodiment of the microfluidic device 10 according to the present invention, it has two additional fluid connections, namely a third fluid connection 3 and a fourth fluid connection 4. The third fluid connection 3 is arranged on the second side 5b of the chamber 5, and the fourth fluid connection 4 is arranged on the fourth side 5d opposite thereto of the chamber 5. The dimensions of the third fluid connection 3 and the fourth fluid connection 4 are, for example, 5-75 μm.

[0089] An example of a method according to the invention using the second embodiment of the microfluidic device 10 shown in Figure 2 is described below. Here, the mechanical disruption of organoids 7 into organoid cells 9 and / or organoid fragments 8 is described, for example, for the mechanical disruption of three-dimensional aggregates 7 into individual structures 9 and / or aggregate fragments 8.

[0090] In a first step a) of the microfluidic method, the first fluid connection 1 and the second fluid connection 2 are open, and the third fluid connection 3 and the fourth fluid connection 4 are closed or their pump units are inactive. The dimensions of the first fluid connection 1 are suitably selected so that organoids 7 of the desired size can pass through. In contrast, the dimensions of the second fluid connection 2 are chosen to be suitably smaller so that only dissociated or split organoid fragments 8 and organoid cells 9 or very small organoids 7 pass through it and out of the chamber 5. Using a pump unit (not shown), a first medium containing the organoids 7 is introduced into the chamber 5 of the microfluidic device 10 via the first fluid connection 1. The first medium is, for example, a culture medium or a buffer solution, in which the organoids 7 are suspended. The chamber 5 is, for example, already filled, in particular filled with the first medium without organoids. When the first medium containing the organoids 7 flows from the first fluid connection 1 into the chamber 5, a flow is generated. Figure 1 The vortex 6b is shown. The organoids 7 are dispersed in the chamber 5 by the vortex 6b. The first medium can be discharged from the chamber 5 via the second fluid connection 2.

[0091] Optionally, in step a'), an enzyme solution is added to the chamber 5. For example, the addition is carried out via the third fluid connection 3 as inlet and the fourth fluid connection 4 as outlet. This is done until the desired enzyme concentration is present in the chamber 5. The vortex 6b thus generated contributes to the formation of a uniformly dispersed enzyme concentration in the chamber 5. The dimensions of the third fluid connection 3 and the fourth fluid connection 4 are suitably selected so that, during the addition of the enzyme solution, organoids 7, organoid fragments 8 or organoid cells 9 are not transported out of the chamber 5 and lost. Alternatively, retention elements such as microfilters or microsieves are integrated into the third microfluidic connection 3 and the fourth microfluidic connection 4 so that they can also have larger dimensions.

[0092] Figure 2b The second step b of the method according to the invention is shown. For example, the enzyme solution containing the organoids 7 is subjected to a suitably executed pulsating back-and-forth movement in an alternating sequence via the third fluid connection 3 and the fourth fluid connection 4. The first fluid connection 1 and the second fluid connection 2 are closed, or the pump units connected thereto are inactivated. This back-and-forth movement is carried out, in particular, by at least one pump unit. The vortex 6b thus generated, on the one hand, generates a movement of the organoids 7 and optionally the organoid fragments 8 in the chamber 5, and on the other hand, generates specific and adjustable shear forces that act on the organoids 7 and organoid fragments 8 and mechanically assist the enzymatic cleavage, thereby leading to the dissociation of the organoids 7 and organoid fragments 8.

[0093] In order to optically observe and monitor the dissociation process of the organoid 7 , at least one side surface 5 a , 5 b , 5 c , 5 d of the chamber 5 is designed to be at least partially transparent.

[0094] In step c), which is performed simultaneously with step b), the interior of chamber 5 containing organoids 7, organoid fragments 8, and organoid cells 9 is monitored, for example, by a control unit (not shown). The control unit may comprise, for example, a microscope or a microscope unit connected to chamber 5. Optical monitoring can, in particular, determine when the desired degree of dissociation of organoids 7 and organoid fragments 8 has been achieved. This can be accomplished automatically using suitable image analysis methods, for example, by detecting the size and number of organoids 7, organoid fragments 8, and organoid cells 9 in chamber 5.

[0095] When it is determined by the control unit that the desired degree of dissociation of the organoid 7 into organoid cells 9 and / or organoid fragments 8 has been reached, Figure 2b The splitting process in step d) (not shown) is stopped, in particular automatically. To this end, a buffer solution or a medium, in particular a culture medium, is introduced into the chamber 5 via one of the fluid connections 1, 2, 3, 4, in particular via the third fluid connection 3 or the fourth fluid connection 4, wherein a vortex 6b is generated when the buffer solution or medium enters the chamber 5. Thus, the enzyme solution is first diluted and ultimately removed from the chamber 5. For liquid exchange and possible further cleaning processes, the fluid connections 3 and 4 can also be used alternately as inlet or outlet in a suitable manner. The vortex 6b generated in the chamber 5 helps to achieve liquid exchange efficiency in terms of improving thorough mixing and shortening the required time.

[0096] Figure 2c Another method step e) is shown, in which organoid fragments 8 and organoid cells 9 are removed from the microfluidic chamber 5. They are output via the outflow 6c of the second fluid connection 2. The first fluid connection 1, the third fluid connection 3 and the fourth fluid connection 4 are used simultaneously or in a suitable alternating order as an inlet for a buffer solution or a medium (in particular a culture medium) to flow into 6a the chamber 5. Favorable flow conditions and vortices 6b can thus be generated in the chamber 5 so that all or as many organoid fragments 8 and organoid cells 9 as possible are removed from the chamber 5. The interior of the optically accessible chamber 5 can be observed, for example, by microscopy, so as to monitor and optimize the output of organoid fragments 8 and / or organoid cells 9 from the chamber 5. The generated and output organoid fragments 8 and / or organoid cells 9 are then used for subsequent culture or analysis steps. In an alternative embodiment, the organoid fragments 8 and / or organoid cells 9 are not removed from the microfluidic chamber 5, but are subjected to further culture or analysis steps within the chamber 5.

[0097] Figure 3A cartridge 100 according to the invention is shown, which comprises, for example, for all embodiments of the microfluidic device 10 according to the invention, a cartridge according to Figure 2a The second embodiment of the microfluidic device 10 is shown. The microfluidic device 10 is mounted on a plastic substrate or a chip, for example.

[0098] Figure 4 A flow chart showing an embodiment of a method 500 according to the present invention for mechanically splitting three-dimensional agglomerates 7 into individual structures 9 and / or agglomerate fragments 8, in particular assisting enzymatic splitting of three-dimensional agglomerates 7 into individual structures 9 and / or agglomerate fragments 8, for example by Figures 2a-2c The microfluidic device shown in and the embodiments and method steps described therein.

[0099] Figure 4 Step a) is shown, in which three-dimensional aggregates 7 are introduced 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 assist the mechanical disruption that occurs in step b). Here, the medium or enzyme solution containing the three-dimensional aggregates 7 is pulsating back and forth so that vortices 6b are formed. In particular, at the same time, in step c), the degree of dissociation of the three-dimensional aggregates 7 into individual structures 9 and / or aggregate fragments 8 is determined by the control unit. In step d), the disruption process stops when it is determined by the control unit that the desired degree of dissociation has been reached. In step e), the individual structures 9 and aggregate fragments 8 are discharged from the chamber 5 again.

Claims

1. A microfluidic method (500) for mechanically splitting three-dimensional agglomerates (7) into individual structures (9) and / or agglomerate fragments (8) by means of a microfluidic device (10) according to any one of claims 6 to 15, in particular for assisting the enzymatic splitting of three-dimensional agglomerates (7) into individual structures (9) and / or agglomerate fragments (8), comprising the following steps: a) feeding a first medium containing three-dimensional agglomerates (7) into a chamber (5) through a first fluid connection (1), wherein a vortex (6b) is generated when the first medium enters the chamber (5) from the first fluid connection (1), b) causing the first medium containing the three-dimensional agglomerates (7) to move back and forth in a pulsating manner, in particular by means of a pump unit, through the first fluid connection (1) and the second fluid connection (2) and / or through the third fluid connection (3) and the fourth fluid connection (4), thereby generating vortices (6b), thereby mechanically breaking up the three-dimensional agglomerates (7).

2. A method (500) according to claim 1, wherein in step a') after step a), the enzyme solution is added via the fluid connections (1, 2, 3, 4), in particular via the third fluid connection (3) and / or the fourth fluid connection (4), so that a vortex (6b) is generated when the enzyme solution enters the chamber (5) from the fluid connections (1, 2, 3, 4), and wherein in step b), the enzyme solution moves back and forth in a pulsating manner.

3. The method (500) according to claim 1 , wherein during the fragmentation process, the degree of dissociation of the three-dimensional agglomerates (7) into individual structures (9) and / or agglomerate fragments (8) is determined by a control unit, in particular by an image evaluation method, And wherein the splitting process is stopped, in particular automatically, when the control unit detects that the desired degree of dissociation has been reached.

4. A method (500) according to any of the preceding claims, wherein the splitting is stopped by introducing a buffer solution or medium into the chamber (5) via one of the fluid connections (1, 2, 3, 4), wherein a vortex (6b) is generated when the buffer solution or medium enters the chamber (5).

5. The method (500) according to any one of the preceding claims, characterized in that The three-dimensional aggregates (7) are cell aggregates, in particular organoids (7) or spheroids, And wherein the disrupted individual structures (9) are cells, in particular organoid cells (9) or spheroid cells, and wherein the aggregate fragments (8) are cell aggregate fragments, in particular organoid fragments (8) or spheroid fragments.

6. A microfluidic device (10) for mechanically splitting three-dimensional aggregates (7) into single structures (8) and / or aggregate fragments (9), in particular for assisting the enzymatic splitting of three-dimensional aggregates (7) into single structures (8) and / or aggregate fragments (9), comprising a chamber (5) having a first fluid connection (1) and a second fluid connection (2), wherein the first fluid connection is arranged on a first side (5a) of the chamber (5), and the second fluid connection is arranged on a third side (5c) of the chamber (5), in particular opposite to the first fluid connection (1), wherein the cross-sectional area of the chamber (5) is 2 to 20 times the cross-sectional area of the first fluid connection (1), so that a vortex (6b) for splitting can be generated.

7. The microfluidic device (10) according to claim 6, wherein the size of the first fluid connection (1) is 100-700 μm, and wherein the size of the second fluid connection (1) is 25-150 μm.

8. A microfluidic device (10) according to any one of claims 6 or 7, wherein the chamber (5) further includes a third fluid connection (3) and a fourth fluid connection (4), in particular a fourth fluid connection (4) opposite thereto, wherein the cross-sectional area of the chamber (5) is 2 to 20 times the cross-sectional area of the third fluid connection (3) and the fourth fluid connection (4), so that a vortex (6b) can be generated.

9. The microfluidic device (10) according to claim 8, wherein the third fluid connection (3) and the fourth fluid connection (4) have integrated retaining elements and / or have a size of 5-75 μm.

10. A microfluidic device (10) according to any one of claims 8 or 9, wherein the third fluid connection (3) is arranged on a second side (5b) transverse to the first side (5a) and the third side (5c) of the chamber (5), and wherein the fourth fluid connection (4) is arranged on a fourth side (5d) of the chamber (5) opposite to the second side (5b).

11. The microfluidic device (10) according to any one of claims 6 to 10, wherein the chamber (5) has a rectangular shape, in particular with a length and width of 2 to 15 mm and a height of 0.75 to 2 mm.

12. The microfluidic device (10) according to any one of claims 6 to 11, wherein the fluid connections (1, 2, 3, 4) are arranged orthogonally to the sides (5a, 5b, 5c, 5d) of the chamber (5).

13. A microfluidic device (10) according to any one of claims 6 to 12, wherein at least one side (5a, 5b, 5c, 5d) of the chamber (5) is designed to be at least partially transparent, in particular comprising a transparent polymer, such as cyclic olefin copolymer (COC), polycarbonate (PC), polymethyl methacrylate (PMMA), polystyrene (PS) or glass.

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

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

16. Cassette (100), in particular a microfluidic cassette, comprising a microfluidic device (10) according to any one of claims 6 to 15.

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