Method and apparatus for investigating mammalian cells

Abstract

The invention relates to a microfluidic apparatus (10) having a diffusion chamber (30) into which multiple feeds (41-43) open, wherein at least one first feed (41) opens into the diffusion chamber (30) below at least one second feed (42), and at least one third feed (43) opens into the diffusion chamber (30) above the second feed (42). A method for investigating mammalian cells in the microfluidic apparatus (10) comprises introducing mammalian cells into the diffusion chamber through the first inlet (41) and lysing the mammalian cells.

Description

[0001] Description

[0002] title

[0003] Method and device for examining mammalian cells

[0004] The present invention relates to a microfluidic device. Furthermore, the present invention relates to a method for examining mammalian cells in the microfluidic device.

[0005] State of the art

[0006] Cancer models such as circulating tumor cells, organoid cultures, and patient-derived transplants can be used for personalized cancer therapy and drug development.

[0007] US Pat. No. 11,098,369 B2 describes a method for determining the viability of tumor cell spheroids in a three-dimensional microfluidic device. Viability determination can be performed using total fluorescence measurement.

[0008] K. Yin et al., Dynamic aqueous multiphase reaction system for one-pot CRISPR-Cas12a based ultrasensitive and quantitative molecular diagnosis, Analytical Chemistry 2020, pages 8561 -8568, describes how a quantitative genetic analysis of genomic DNA can be carried out using time-resolved fluorescence detection using separate reaction sequences for cell lysis, DNA amplification and CRISPR-Cas12a-based DNA detection in a sucrose gradient.

[0009] Disclosure of the Invention The microfluidic device has a diffusion chamber into which several inlets open. At least one first inlet opens into the diffusion chamber below at least one second inlet. At least one third inlet opens into the diffusion chamber above the second inlet.

[0010] The three inlets are arranged in particular in such a way that, when the device is used as intended, the second inlet opens into the diffusion chamber higher than the first inlet and the third inlet higher than the second inlet and thus the three inlets are preferably arranged vertically one above the other.

[0011] This microfluidic device can be used in particular to subject cells, preferably mammalian cells, which may, for example, originate from a 3D culture of tumor mammalian cells, to different analysis steps.

[0012] The diffusion chamber is preferably formed of at least one material selected from the group consisting of polycarbonates (PC), polymethyl methacrylates (PMMA), cycloolefin copolymers (COC), cycloolefin polymers (COP), polystyrenes (PS), and glass. These materials have the advantage of being transparent, thus enabling optical examination of the contents of the diffusion chamber, particularly by means of fluorescence spectroscopy.

[0013] The first inlet is preferably fluidically connected to a cell culture element, and in particular to a processing element for isolating and splitting mammalian cells from 3D cell cultures. This makes it possible to introduce mammalian cells from a cell culture, in particular from a 3D cell culture, as a cell suspension into the lower region of the diffusion chamber. The second inlet and the third inlet are each preferably fluidically connected to a reagent reservoir. This makes it possible to layer the mammalian cells in the diffusion chamber using reagents introduced through the second inlet and / or the third inlet.

[0014] In the context of the present invention, the term “splitting” refers to the dissolution of connections between cells of a cell agglomerate, such as an organoid or spheroid, and the resulting dissociation of the cell agglomerate into multicellular cell agglomerate fragments or individual cells.

[0015] The processing element can, for example, be a chamber or a structure in which separation or splitting takes place. The diffusion chamber preferably has at least one vent opening on its upper side. In this way, when a cell suspension or reagents are introduced, air can be displaced from the diffusion chamber through the vent opening. Particularly preferably, a filter is arranged in the vent opening to prevent contaminants from entering the diffusion chamber through the vent opening.

[0016] The first inlet opens in particular into a first segment of the diffusion chamber, the second inlet opens in particular into a second segment of the diffusion chamber, and the third inlet opens in particular into a third segment of the diffusion chamber. Each segment is intended to be filled with a different fluid. In principle, the segments can be sections of the diffusion chamber that are not separated from one another. However, it is preferred that a web is arranged between the first segment and the second segment and between the second segment and the third segment. The webs run in particular horizontally and are arranged in particular directly below the second inlet and directly below the third inlet. They enable improved capillary filling of the diffusion chamber and delimit the segments from one another.

[0017] In one embodiment of the microfluidic device, the third segment is divided into several sub-segments by at least one partition wall. This partition wall runs vertically, in particular. A third inlet opens into each sub-segment. This makes it possible to fill each sub-segment with a reagent.

[0018] It is further preferred that the first inlet has an interruptible fluidic connection line to the second inlet. This makes it possible to use the first inlet and the second inlet to circulate a fluid stored in the diffusion chamber.

[0019] The method for examining cells, in particular mammalian cells, in the microfluidic device comprises introducing cells, in particular mammalian cells, through the first inlet, in particular in the form of a cell suspension. The cells, in particular mammalian cells, are lysed in the diffusion chamber.

[0020] In principle, it is possible to carry out mixing processes within the diffusion chamber, in particular for mixing the mammalian cells with lysis reagents, by vibrating or abruptly rotating the diffusion chamber with the inlets closed. However, a first embodiment of the method provides for the use of a microfluidic device whose first inlet is connected to the second inlet by means of an interruptible fluidic connecting line. Lysis occurs by introducing at least one lysis reagent into the diffusion chamber, in particular through the first inlet. In order to thoroughly mix the mammalian cells and the lysis reagent and thus achieve rapid lysis, the contents of the diffusion chamber are led out of the diffusion chamber after the lysis reagent has been introduced through the first inlet and returned to the diffusion chamber via the connecting line and the second inlet.

[0021] In this embodiment of the method, it can be provided, in particular, that a viability determination of the mammalian cells is to be carried out. For this purpose, after the mammalian cells have been introduced and before lysis by the first inlet, reagents for viability determination are introduced into the diffusion chamber, and the viability determination is then carried out. These reagents preferably comprise a fluorophore dye that selectively stains dead cells and / or a fluorophore dye that selectively stains living cells. The fluorophore dye can, in particular, be conjugated to an antibody, be linked to a nanoparticle, or bind to free DNA. If two fluorophore dyes are used for living and dead cells, fluorophores that emit at different wavelengths are used. The total fluorescence of both fluorophore dyes can be measured to determine viability.If samples of a cell culture are examined several times in succession in this way, changes in the ratio of the fluorescence intensity at the two wavelengths can indicate cultivation progress.

[0022] In addition to determining the viability of mammalian cells, the culture medium can be analyzed for metabolites to obtain information about the metabolic activity of the mammalian cells. These metabolites can include, in particular, glucose, lactate, and / or lactate dehydrogenase (LDH).

[0023] In a further embodiment of the method, this comprises introducing sucrose into the diffusion chamber through the first inlet and mixing the sucrose with the lysed mammalian cells to obtain a lower phase containing sucrose and nucleic acids, in particular DNA and / or RNA. The introduction of the sucrose can occur before or after the introduction of the mammalian cells. In particular, the lysis of the mammalian cells can occur using the method according to the first embodiment before the lysate is subsequently admixed with sucrose.

[0024] CRISPR-Cas reagents are introduced into the diffusion chamber through the third inlet. This creates a particularly sucrose-free upper phase, which is positioned above the lower phase in the diffusion chamber. This is followed by diffusion of nucleic acid molecules from the lower phase to the upper phase, followed by analysis of the nucleic acid molecules using CRISPR-Cas-based diagnostics. This method exploits the fact that small nucleic acid fragments diffuse faster from the sucrose-containing lower phase to the upper phase than larger molecules. This enables, for example, quantitative genetic analysis using time-resolved fluorescence detection in a CRISPR-Cas12a or CRISPR-Cas13 system.This method enables the assessment of whether the quality of a cell culture changes over time in terms of viability and, depending on the genetic marker being investigated, for example, in terms of cell composition, tumor marker expression, or mutations, resulting in deviations from the native tumor immune microenvironment. This can be achieved by analyzing cellular nucleic acids for genetic tumor markers or single nucleotide polymorphisms (SNPs). This information enables timely adaptation of a tumor model. In addition to quality control, the method can also be used for endpoint analysis of medications, efficacy tests, or drug combination tests to analyze phenotypic and genetic characteristics of the same sample material.Another advantage is that data from model generation are comparable with the endpoint analyses because they are based on the same and automated procedure.

[0025] If the concentration of lysed mammalian cells is high or a target sequence is highly abundant in the lower phase, it may be sufficient for the upper phase to rest directly on the lower phase. While the upper phase is sucrose-free, the lower phase in this case preferably contains 10 wt% to 40 wt% sucrose.

[0026] If, however, it is necessary to increase the nucleic acid concentration for reliable analysis of the nucleic acid molecules, a sucrose solution containing amplification reagents is preferably introduced through the second inlet into the diffusion chamber before the CRISPR-Cas reagents are introduced. This creates a middle phase located between the lower phase and the upper phase. The sucrose concentration of the middle phase is lower than the sucrose concentration of the lower phase. In particular, the sucrose concentration in the lower phase is in the range of 30 wt% to 40 wt% and in the middle phase in the range of 10 wt% to 20 wt%, while the upper phase is sucrose-free even with this procedure. The amplification reagents can, in particular, be reagents for RPA (recombinase polymerase amplification). In particular, they also contain a buffer system.The nucleic acid molecules then diffuse from the lower phase through the middle phase into the upper phase. For isothermal amplification of the nucleic acid molecules in the middle phase, the diffusion chamber is preferably heated to a temperature in the range of 20 °C to 42 °C.

[0027] To implement the process in the microfluidic device, the volume of each phase, independently of each other, is preferably in the range of 10 pl to 30 pl. In particular, the volumes are identical.

[0028] Short description of the drawings

[0029] Embodiments of the invention are illustrated in the drawings and are explained in more detail in the following description.

[0030] Figure 1 schematically shows a microfluidic device according to an embodiment of the invention.

[0031] Figure 2 shows a transparent isometric view of a diffusion chamber of a microfluidic device according to an embodiment of the invention.

[0032] Figure 3 shows a flowchart of an embodiment of the method according to the invention.

[0033] Figure 4 shows a schematic representation of the diffusion chamber of a microfluidic device according to an embodiment of the invention in a step of an embodiment of the method according to the invention.

[0034] Figure 5 shows a schematic representation of a diffusion chamber of a device according to an embodiment of the invention in another step of an embodiment of the method according to the invention.

[0035] Figure 6 shows a flow diagram of another embodiment of the method according to the invention. Figure 7 shows a schematic representation of a diffusion chamber of an embodiment of the microfluidic device in a step of an embodiment of the method according to the invention.

[0036] Figure 8 shows a schematic representation of a diffusion chamber of a microfluidic device according to an embodiment of the invention in another step of an embodiment of the method according to the invention.

[0037] Figure 9 shows a schematic representation of a diffusion chamber of a microfluidic device according to an embodiment of the invention in yet another step of a method according to an embodiment of the invention.

[0038] Figure 10 shows a schematic representation of a diffusion chamber of an embodiment of the microfluidic device in yet another step of a method according to an embodiment of the invention.

[0039] Figure 11 shows a schematic representation of a diffusion chamber of an embodiment of the microfluidic device in a step of another embodiment of the method according to the invention.

[0040] Figure 12 shows a transparent isometric view of a diffusion chamber of a microfluidic device according to another embodiment of the invention.

[0041] Figure 13 shows a schematic representation of a diffusion chamber according to another embodiment of the invention in a step of the method according to the invention.

[0042] Embodiments of the invention

[0043] A microfluidic device 10 according to an embodiment of the invention is shown in Figure 1. It comprises a heater 11 and a microfluidic chip 12. A cell culture element 13, designed as a 3D cell culture chamber, is arranged on the microfluidic chip 12. This cell culture element 13 is fluidically connected to a processing element 14, which is configured to carry out cell isolation and splitting processes. An atmosphere control element 15 is connected to the cell culture element 13. It enables the microenvironment within the cell culture element 13 to be monitored by determining the oxygen concentration, the carbon dioxide concentration, and the pH value. Furthermore, the atmosphere control element 15 enables the viability of the cell culture to be monitored via its oxygen consumption. An inlet 16 supplies the cell culture element 13 with reagents, media, and cells.A microscopic control element 17 monitors the morphology of cells in the cell culture element 13 and the processing element 14. Cell samples from the cell culture element 13 or the processing element 14 can be fed to an analysis element 20. Furthermore, reagents can be fed to the analysis element 20 from a reagent reservoir 21. Waste products from the analysis element 20 and the processing element 14 can be collected in a waste reservoir 22.

[0044] Figure 2 shows the structure of the analysis element 20. This has a diffusion chamber 30 which is divided into three segments 31 to 33 arranged one above the other. A first horizontal web 34 is arranged between the lower first segment 31 and the middle second segment 32. A second horizontal web 35 is arranged between the second segment 32 and the upper third segment 33. A first inlet 41 opens into the first segment 31. It is fluidically connected to the cell culture element 13 and to the reagent reservoir 21. A second inlet 42 opens into the second segment 32 immediately above the first web 34. A third inlet 43 opens into the third segment 33 immediately above the second web 35. The second inlet 42 and the third inlet 43 are fluidically connected to different reagent tanks of the reagent reservoir 21. A connecting line 44 connects the first inlet 41 with the second inlet 42.An outlet 45 at the bottom of the first segment 31 is fluidly connected to the waste reservoir 22. Valves (not shown) are configured to interrupt the connection of the three inlets 41 to 43 and the outlet 45 to the diffusion chamber 30. Further valves (not shown) are configured to interrupt the connection of the connecting line 44 at both ends. A vent opening 46, which has a filter, is arranged at the top of the diffusion chamber 30.

[0045] The sequence of a first embodiment of the method according to the invention is shown in Figure 3. After the start 50 of the method, a suspension of cells from the cell culture element 13 or processing element 14 is first introduced 51 through the first inlet 41 into the diffusion chamber 30. As shown in Figure 4, the first segment 31 of the diffusion chamber 30 is filled with a liquid phase 60. With the exception of the valve that opens the fluidic connection of the inlet 41 to the diffusion chamber 30, all other valves are closed. Reagents for viability determination are then introduced 52 into the diffusion chamber 30 through one of the inlets 41-43. Subsequently, a viability determination 53 is carried out by means of fluorescence detection. This is possible because the diffusion chamber 30 is formed in a transparent substrate, which in the present embodiment is, for example, polycarbonate.A lysis reagent is now introduced 54 into the diffusion chamber 30 through the second inlet 42 or the third inlet 43. To mix the liquid phase 60 with the freshly introduced lysis reagent, the connections of the first inlet 41 and the second inlet 42 to the diffusion chamber are opened, and the connection of the first inlet 41 to the cell culture element 13 and the connection of the second inlet 42 to the reagent reservoir 21 are closed. Furthermore, the connections of the first inlet 41 and the second inlet 42 to the connecting line 44 are opened. The liquid phase 60 is now circulated 55 by being pumped out of the first segment 31 of the diffusion chamber 30 through the first inlet 41, passed through the connecting line 44 into the second inlet 42, and reintroduced into the second segment 32 of the diffusion chamber 30, from where it flows back into the first segment 31 following gravity. This is illustrated in Figure 5.This circulation thoroughly mixes all components of the liquid phase 60, allowing subsequent lysis 56 of the cells contained in the liquid phase 60 to release the nucleic acids contained therein. A sample of the nucleic acid solution can be taken via outlet 45 for further analysis if necessary. The process is then terminated 57.

[0046] The sequence of a second embodiment of the method according to the invention is shown in Figure 6. After the start 70 of the method, cells are introduced 71 from the cell culture element 13 into the first segment 31 of the diffusion chamber 30. These cells are then lysed. The lysing can be carried out using steps 54 to 56 of the first embodiment of the method according to the invention, or another lysing process can also be provided. This is followed by an introduction 72 of sucrose through the first inlet 41 into the diffusion chamber 30, so that a lower phase 61 is obtained which contains, for example, 40 wt.% sucrose. This is shown in Figure 7. The connection between the first inlet 41 and the diffusion chamber 30 is now closed, and the connection between the second inlet 42 and the diffusion chamber 30 is opened.Through this, sucrose and amplification reagents are introduced from the reagent reservoir 21 into the second segment 32 of the diffusion chamber 30 to obtain a middle phase 62 containing 10 wt.% sucrose 73. This is shown in Figure 8. After closing the connection of the second inlet 42 to the diffusion chamber 30 and opening the connection of the third inlet 43 to the diffusion chamber 30, CRISPR-Cas reagents are introduced 74 from the reagent reservoir 21 into the third segment 33 of the diffusion chamber 30 through the third inlet 43. This results in an upper phase 63. This is shown in Figure 9. After closing the connection of the third inlet 43 to the diffusion chamber 30, an analysis 75 of nucleic acid molecules from the lysed cells is carried out using CRISPR-Cas-based diagnostics.This involves diffusion D of the nucleic acid molecules from the lower phase 61 into the middle phase 62, where they are isothermally amplified, and then further into the upper phase 63, where they can be subjected to fluorescence analysis. The nucleic acid molecules follow the sucrose concentration gradient in the three phases 61 to 63.

[0047] In a third embodiment of the method according to the invention, the second embodiment is modified by omitting step 73. As shown in Figure 11, the lower phase 61 is immediately overlaid with the upper phase 63 without first creating a middle phase 62. Nucleic acid molecules diffuse from the lower phase 61 into the upper phase 63 without being amplified. However, with a sufficiently high concentration of nucleic acid molecules in the lower phase, a sufficiently accurate fluorescence analysis of the nucleic acid molecules in the CRISPR-Cas system can also be performed in the third embodiment of the method.

[0048] Figure 12 shows the structure of the analysis element 20 in a second embodiment of the microfluidic device 10. The diffusion chamber 30 differs from the first embodiment in that the third segment 33 is divided into two sub-segments 37, 38 by a horizontal partition wall 36. The diffusion chamber 30 has two vent openings 46, 47 on its upper side, with each of the sub-segments 37, 38 having one of the vent openings 46, 47. While the third inlet 43 opens into the first sub-segment 37, a further third inlet 48 opens into the second sub-segment 38. The second embodiment of the microfluidic device enables a modified implementation of the second embodiment of the method. As shown in Figure 13, two different upper phases 63a, 63b are generated by the two third inlets 43, 48 by introducing different CRISPR-Cas systems into the subsegments 37, 38.This makes it possible to examine the lysed cells for different targets simultaneously.

[0049] In all embodiments of the microfluidic device 10 and in all embodiments of the method according to the invention, after completion of an analysis, the entire contents of the diffusion chamber can be drained through the outlet 45 into the waste reservoir 22. The inlets 41 to 43 and optionally 48, as well as the interior of the diffusion chamber 30, can be flushed with a rinsing liquid from the reagent reservoir 21 to prepare the analysis element 20 for its next use. In this way, samples can be regularly taken from the cell culture element 13 and analyzed promptly in the analysis element 20.

Claims

Claims 1 . Microfluidic device (10) comprising a diffusion chamber (30) into which a plurality of inlets (41-43, 48) open, wherein at least one first inlet (41) opens into the diffusion chamber (30) below at least one second inlet (42) and at least one third inlet (43, 48) opens into the diffusion chamber (30) above the second inlet (42).

2. Microfluidic device (10) according to claim 1, characterized in that the first inlet (41) is fluidically connected to a cell culture element (13) and in particular to a processing element (14) for isolating and splitting mammalian cells and the second inlet (42) and the third inlet (43, 48) are each fluidically connected to a reagent reservoir (21).

3. Microfluidic device (10) according to claim 1 or 2, characterized in that the diffusion chamber (30) has at least one vent opening (46, 47) on its upper side.

4. Microfluidic device (10) according to one of claims 1 to 3, characterized in that the first inlet (41) opens into a first segment (31) of the diffusion chamber (30), the second inlet (42) opens into a second segment (32) of the diffusion chamber (30) and the third inlet (43, 48) opens into a third segment (43) of the diffusion chamber (30).

5. Microfluidic device (10) according to claim 4, characterized in that a web (34, 35) is arranged between the first segment (31) and the second segment (32) and between the second segment (32) and the third segment (33).

6. Microfluidic device (10) according to claim 4 or 5, characterized in that the third segment (33) is connected by means of at least one Partition wall (36) is divided into several sub-segments (37, 38), wherein a third inlet (43, 48) opens into each sub-segment (37, 38).

7. Microfluidic device (10) according to one of claims 1 to 6, characterized in that the first inlet (41) has an interruptible fluidic connection line (44) to the second inlet (42).

8. A method for examining mammalian cells in a microfluidic device (10) according to any one of claims 1 to 7, comprising introducing (51, 71) mammalian cells into the diffusion chamber through the first inlet (41) and lysing (56) the mammalian cells.

9. Method according to claim 8 using a microfluidic device (10) according to claim 7, characterized in that the lysing takes place by introducing at least one lysis reagent into the diffusion chamber (30) (54), and the contents of the diffusion chamber (30) are led out of the diffusion chamber (30) through the first inlet (41) and are led back into the diffusion chamber (30) through the connecting line (44) and the second inlet (42) (55).

10. Method according to claim 8 or 9, characterized in that after the introduction (51) of the mammalian cells and before lysing through the first inlet (41), reagents for viability determination are introduced (52) into the diffusion chamber (30) and a viability determination is then carried out (53).

11. Method according to one of claims 8 to 10, further comprising the following steps: Introducing (72) sucrose into the diffusion chamber (30) through the first inlet (41), Mixing the sucrose with the lysed mammalian cells to obtain a lower phase (61) containing sucrose and nucleic acids, Introducing (74) CRISPR-Cas reagents into the diffusion chamber (30) through the third inlet (43) to obtain an upper phase (63), Diffusion (D) of nucleic acid molecules from the lower phase (61) into the upper phase (63), and Analysis (75) of nucleic acid molecules using CRISPR-Cas-based diagnostics.

12. The method according to claim 11, characterized in that before introducing (73) the CRISPR-Cas reagents, a sucrose solution containing amplification reagents is introduced (74) through the second inlet (42) into the diffusion chamber (30) in order to obtain a middle phase (62), wherein a sucrose concentration of the middle phase is lower than a sucrose concentration in the lower phase.

13. The method according to claim 11 or 12, characterized in that a volume of each phase is in the range of 10 pl to 30 pl.

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