Microfluidic Devices

Abstract

The present invention relates to a microfluidic device (10) suitable for containing an aqueous liquid, the device having an essentially planar shape and comprising at least one conduit (11) fluidly connecting at least two structures selected from compartments, reservoirs (14) and / or ports (15, 62). The conduit comprises at least one bend (12) that presents an angle between 85° and 275° around an axis perpendicular to the planar shape. The planar shape is arranged at an angle between +45° and -45° with respect to the direction of gravity (G) or centrifugal force (Fc) applied to the microfluidic device.

Description

[Technical field]

[0001] The present invention relates to the field of microfluidic devices for use in cell and tissue culture or testing thereof. [Background technology]

[0002] 3D tissue cultures are increasingly used in testing or screening compounds for their toxicity, immunogenicity or therapeutic effect.In a particular embodiment, arrays of different or similar 3D tissue cultures connected to each other can be exposed to the same test compound, (i) to investigate the different effects of the compound on different tissues, and (ii) to consider the potential communication or crosstalk between different tissues in response to such exposure.

[0003] These devices are sometimes called "organ-on-a-chip" or even "organism-on-a-chip" or "body-on-a-chip". More recently, they have been lumped together as "microphysiological systems" (MPS). Microphysiological systems combine microfluidic technology with cell / tissue culture and are able to mimic human (or any other animal species) biology in vitro, at the smallest biologically acceptable scale defined by the objectives.

[0004] For example, many devices and methods have been published that allow microtissue spheroids to be formed inside a chip and cultured under flow for a period of time. See Ruppen et al. (2015), Occhetta et al. (2015), Kwapiszewska et al. (2014), Jin et al. (2011), Hsiao et al. (2009), Torisawa et al. (2007) or Wu et al. (2008).

[0005] However, only a few exist that use dedicated platforms to form micro-tissue spheroids externally and then transfer and load them into microfluidic culture chips.

[0006] Therefore, hereinafter, microtissue spheroid, organoid, precision-cut tissue slice, 3D tissue spheroid, embryoid body and pancreatic islet are referred to as "microtissue".All disclosures and conclusions made herein regarding microtissue spheroid are applicable to organoid, precision-cut tissue slice, 3D tissue spheroid, embryoid body and pancreatic islet as specimen.

[0007] Microfluidic devices are systems that are typically characterized by small liquid volumes and small diameter liquid conduits. They are often characterized by laminar flow of liquid within the conduits.

[0008] In general, the flow of liquids in long thin tubes can be turbulent or laminar. The Reynolds number (Re) helps predict such flow patterns in different fluid flow regimes. At low Reynolds numbers, the flow is dominated by shear forces and is laminar, whereas at high Reynolds numbers, the flow is dominated by inertial forces and is turbulent. The Reynolds number is defined as:

number

[0009] Due to the small diameter of the conduits, microfluidic devices often have a Reynolds number <2300, which marks the boundary where laminar flow dominates. In contrast to systems where turbulence predominates, in microfluidic devices non-soluble components of the liquid tend to settle or sediment due to gravity (or centrifugal forces), and this is especially true for cells contained in the liquid flowing through the microfluidic device.

[0010] Microfluidic devices are often used in tissue or cell biology, where microtissues are exposed to media containing agents such as drugs or toxins. As long as these agents are soluble in the liquid, their sedimentation is not a major issue, even in laminar flow systems.

[0011] However, some applications require the study of synergies between suspended cells or non-soluble particles, e.g. nanomaterials, and such micro-tissues.

[0012] In immuno-oncology, the efficacy of cells of the immune system against cancerous (solid) tissues is often investigated in combination with drug candidates, such as immune checkpoint inhibitors, bispecific T cell-engaging antibodies, or antibodies with ADCC (antibody-dependent cellular cytotoxicity) activity. Another example is circulating tumor cells (CTCs), which may or may not interact with microtissues, for example in their role as organ models.

[0013] Also in other applications, for example when the efficacy of drugs against overshooting immune responses is investigated, cells of the immune system are brought into contact with target micro-tissues.

[0014] In other applications, cells are investigated under flow conditions with or without interaction with drug candidates.

[0015] In all these applications, sedimentation of soluble cells can indeed be problematic, since there is a risk that the respective cell will not reach the microtissue under investigation and / or that sedimentation and detachment of the flow will have adverse effects on the cells (e.g. cell death, cell accumulation).

[0016] This also applies to non-soluble particles contained in a fluid, for example to study the interactions between microstructures and such particles.

[0017] Examples of such particles include the following: • Liposomes containing, for example, mRNA or other genetic material; Viral vectors, ● Colloids (silver, silicate), ● Nanoplastic particles and microplastic particles; ● ZnO, SiO2, CeO2, BaSO4, TiO2, and / or • Beads coated with an active agent, for example an antibody.

[0018] Therefore, the overall discussion provided herein for cells in culture also applies to non-soluble particles that are prone to gravitational settling. Summary of the Invention

[0019] One object of the present invention is to provide a microfluidic device suitable for use with cells and / or particles circulated through the conduits of the system.

[0020] One object of the present invention is to provide a microfluidic device that allows to study interactions between microtissues, cells and / or particles, optionally in the presence of agents that affect or are affected by such interactions.

[0021] Another object of the present invention is to provide a microfluidic device that reduces settling of cells and / or particles circulating through the conduits of the system.

[0022] These and further objects are achieved by the subject matter of the independent claims, and the dependent claims and the description disclose further preferred embodiments. [Brief description of the drawings]

[0023] [Figure 1] 1 shows a microfluidic device for containing aqueous liquids. [Diagram 2] (A) State-of-the-art microfluidic device, (B) the phenomenon of sedimenting cells and / or particles (dots) suspended in a liquid medium flowing through a microfluidic conduit in a microfluidic device according to the invention, (C) and (D) the rotation concept that generates a liquid flow. [Diagram 3] 1 shows a cross section of a microfluidic device. [Figure 4] 1 shows a cross section of a microfluidic device. [Diagram 5] 1 shows different possible shapes of conduits, with different numbers of bends and different angles. [Figure 6] (A) shows a microfluidic device with a conduit, (B) shows another cross-section of the microfluidic device along line C-C' in FIG. 6A. [Figure 7] 1 illustrates various means for generating liquid flow within a conduit; [Figure 8] Different arrangements of conduits, micro-tissue compartments, reservoirs, reservoir ports and compartment ports are shown. [Figure 9] 1 shows an example cross section of a conduit. [Figure 10] (A) and (B) show plan views of a microfluidic device according to the present invention, and (C) and (D) show perspective views of a microfluidic device according to the present invention. [Figure 11] 1 shows a cross-section of a microfluidic device according to the present invention. [Figure 12]1A shows a top view of a microfluidic device according to the invention, (B) a close-up of the area around the compartment for containing the microtissue, and (C) a cross-section of said area. [Figure 13] 13 shows another embodiment of a conduit and a bend. [Figure 14] (A) An array of microfluidic devices, (B) a platform that can rotate or tilt the microfluidic devices, (C) a variation of the microfluidic device, and (D) another platform that can rotate or tilt the microfluidic devices. [Figure 15] 1 shows different examples of a microfluidic device according to the present invention. [Figure 16] 1 shows a perspective view (A) and a front view (B) of a cross section of a microfluidic device according to the invention with a reservoir port, a compartment and a compartment port, as well as a close-up of the micro-tissue compartment (C) and the reservoir (D) of the microfluidic device. A close-up of the micro-tissue compartment (E) and the reservoir (F) when placed in an operational mode. A close-up of a dome structure (G). A comparative configuration with a lower section having a conical or funnel shape (H). [Figure 17] (A) Bottom view of the microfluidic device, (B) profiles of ports, compartments and conduits. [Figure 18] 1 shows the microfluidic device in two different operational mode positions. [Figure 19] Tumor growth in microtissue compartments is shown. [Figure 20] PBMC distribution in microtissue compartments is shown. [Figure 21] PBMC cell numbers are shown as determined by precision counting beads by flow cytometry. [Figure 22] The time viability of PBMC under different conditions is shown. [Diagram 23] Cytotoxic T cell activation markers are shown. [Figure 24] 1 shows a cross-section of a microfluidic device according to the invention in a level position and in an operational mode position. [Diagram 25]1 shows a cross-section of a microfluidic device according to the invention in a level position and in an operational mode position. [Figure 26] The results of an experiment using primary human PBMCs are shown. [Figure 27] The results of an experiment using primary human PBMCs are shown. [Figure 28] The results of an experiment using primary human PBMCs are shown. [Figure 29] The experimental results using a 3D liver model are shown. [Diagram 30] 1 shows the results of an experiment using HCT116 colon cancer cell microtissues. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Before the invention is described in detail, it should be understood that the invention is not limited to specific component parts or structural features of the described devices or compositions, or process steps of the described methods, and that such devices and methods may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include singular and / or plural referents unless the context clearly dictates otherwise. Moreover, in the claims, the term "comprising" does not exclude other elements or steps.

[0025] Moreover, when parametric ranges bounded by numerical values ​​are given, it will be understood that the ranges are intended to be inclusive of those limits.

[0026] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate embodiments unrelated to each other. Features discussed in one embodiment are intended to be disclosed in relation to other embodiments shown herein. In cases where a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, those skilled in the art will understand that the feature is not necessarily intended to be disclosed for the other embodiment. Those skilled in the art will understand that it is the gist of the present application to disclose the features for the other embodiments, but this has not been done merely for the sake of clarity and to keep the specification to a manageable length.

[0027] Furthermore, the contents of the prior art documents referred to in this specification are incorporated herein by reference, particularly those prior art documents that disclose standard or routine methods, in which case the incorporation by reference is primarily intended to provide a full and enabling disclosure and to avoid lengthy repetition.

[0028] According to one aspect of the present invention, there is provided a microfluidic device suitable for containing an aqueous liquid, the device having an essentially planar shape, the device comprising at least one conduit fluidly connecting at least two structures selected from compartments, reservoirs and / or ports.

[0029] The conduit includes at least one bend ("serpentine") that subtends an angle between 85 degrees and 275 degrees about an axis perpendicular to the planar shape, the planar shape being oriented at an angle between +45 degrees and -45 degrees relative to the following directions: a) Gravity (G), or b) Centrifugal force (Fc) applied to the microfluidic device.

[0030] In another embodiment, the bends are 85 degrees, 86 degrees, 87 degrees, 88 degrees, 89 degrees, 90 degrees, 91 degrees, 92 degrees, 93 degrees, 94 degrees, 95 degrees, 96 degrees, 97 degrees, 98 degrees, 99 degrees, 100 degrees, 101 degrees, 102 degrees, 103 degrees, 104 degrees, 105 degrees, 106 degrees, 107 degrees, 108 degrees, 109 degrees, 110 degrees, 111 degrees, 112 degrees, 113 degrees, 114 degrees, 115 degrees, 116 degrees, 117 degrees, 118 degrees, 119 degrees, 120 degrees, 121 degrees, 122 degrees, 123 degrees, 124 degrees, 125 degrees, 126 degrees, 127 degrees, 128 degrees, 129 degrees, 130 degrees, 131 degrees, 132 degrees, 133 degrees, 134 degrees, 135 degrees, 136 degrees, 137 degrees, 138 degrees, 139 degrees, 140 degrees, 141 degrees, 142 degrees, 143 degrees, 144 degrees, 145 degrees, 146 degrees, 147 degrees, 148 degrees, 149 degrees, 150 degrees, 151 degrees, 152 degrees, 153 degrees, 154 degrees, 155 degrees, 156 degrees, 157 degrees, 158 degrees, 159 degrees, 160 degrees, 161 degrees, 162 degrees, 163 degrees, 164 degrees, 165 degrees, 166 degrees, 167 degrees, 168 degrees, 169 degrees, 170 degrees, 3 degrees, 134 degrees, 135 degrees, 136 degrees, 137 degrees, 138 degrees, 139 degrees, 140 degrees, 141 degrees, 142 degrees, 143 degrees, 144 degrees, 14 5 degrees, 146 degrees, 147 degrees, 148 degrees, 149 degrees, 150 degrees, 151 degrees, 152 degrees, 153 degrees, 154 degrees, 155 degrees, 156 degrees, 157 degrees, 158 degrees, 159 degrees, 160 degrees, 161 degrees, 162 degrees, 163 degrees, 164 degrees, 165 degrees, 166 degrees, 167 degrees, 168 degrees, 169 degrees, 170 degrees, 171 degrees, 172 degrees, 173 degrees, 174 degrees, 175 degrees, 176 degrees, 177 degrees, 178 degrees, 179 degrees, 180 degrees, 181 degrees , 182 degrees, 183 degrees, 184 degrees, 185 degrees, 186 degrees, 187 degrees, 188 degrees, 189 degrees, 190 degrees, 191 degrees, 192 degrees, 193 degrees , 194 degrees, 195 degrees, 196 degrees, 197 degrees, 198 degrees, 199 degrees, 200 degrees, 201 degrees, 202 degrees, 203 degrees, 204 degrees, 205 degrees , 206 degrees, 207 degrees, 208 degrees, 209 degrees, 210 degrees, 211 degrees, 212 degrees, 213 degrees, 214 degrees, 215 degrees, 216 degrees, 217 degrees , 218 degrees, 219 degrees, 220 degrees, 221 degrees, 222 degrees, 223 degrees, 224 degrees, 225 degrees, 226 degrees, 227 degrees, 228 degrees, 229 degrees, 230°, 231°, 232°, 233°, 234°, 235°, 236°, 237°, 238°, 239°, 240°, 241°, 242°, 243°, 244°, 245°, 246°, 247°, 248°, 249°, 250°, 251°, 252°, 253°, 254°, 255°, 256°, 257°, ​​258°, 259°, 260°, 261°, 262°, 263°, 264°, 265°, 266°, 267°, 268°, 269°, 270°, 271°, 272°, 273°, 274° and / or 275° angles.

[0031] Preferred sub-ranges are the ranges 85 degrees to 185 degrees, 130 degrees to 185 degrees and / or 85 degrees to 140 degrees.

[0032] As used herein, the term at least one bend exhibiting an angle between 85 degrees and 275 degrees about an axis "orthogonal to the planar geometry" should be understood broadly and should include embodiments in which the conduit is not parallel to the surface of the device but is angled thereto, for example by forming a helical conduit structure, see FIG. 26 for a better illustration of this embodiment.

[0033] Such a microfluidic device is a three-dimensional body defined by three axes: X, Y and Z. The planar geometry is defined by X and Z. Z is the axis oriented at an angle of +45 degrees to -45 degrees relative to gravity or the centrifugal force applied to the microfluidic device. The angle defining the bend in the conduit is orthogonal to X and Z and therefore about the Y axis.

[0034] In this regard, the term "planar" geometry means that the microfluidic device may also be referred to as a chip, a plate or a slide.

[0035] In other embodiments, the planar shape is oriented at an angle of +40 degrees to -40 degrees, +35 degrees to -35 degrees, +30 degrees to -30 degrees, +25 degrees to -25 degrees, +20 degrees to -20 degrees, +15 degrees to -15 degrees, +10 degrees to -10 degrees or +5 degrees to -5 degrees.

[0036] According to one embodiment, the device further comprises at least one compartment disposed within the conduit or between the two sections, said compartment being capable of housing at least one micro-tissue (the "micro-tissue compartment").

[0037] As used herein, the term "disposed within a conduit" refers to an embodiment in which the diameter of the conduit expands to accommodate one or more compartments, such as those shown in Figure 8C or Figures 10C and 10D.

[0038] As used herein, the term "disposed between two sections of a conduit" refers to an embodiment in which at least one compartment is disposed between two sections of a conduit and does not expand in width, such as shown, for example, in Figures 6, 8A and 8B.

[0039] According to one embodiment, the conduit branches into two or more conduits arranged in parallel, each of the two or more conduits including at least one compartment arranged between its two sections.

[0040] According to one embodiment, at least one compartment has a circular cross section and has a compartment port having a circular cross section arranged concentrically with the compartment, the diameter of the compartment being greater than the diameter of the compartment port.

[0041] In such an embodiment, the compartment may have a lower section adjacent the conduit and an upper section adjacent the compartment port, as shown, for example, in Figure 12C or Figure 16C. In such an embodiment, the compartment may have a dome structure in its lower section to reduce the risk that the microtissue contained in the compartment will be washed away during operation of the microfluidic device. Tests have shown that the dome structure significantly reduces such risk, relative to a lower section having a conical or funnel shape tapering towards the compartment port.

[0042] Such dome structures may have a spheroidal or concave shape as compared to a simple conical or funnel-shaped taper.

[0043] It is important to note that the above embodiment, having a compartment with a circular cross section, a lower section and an upper section for containing a microtissue, a compartment port with a circular cross section arranged concentrically in the compartment, and a dome structure, is also considered to be disclosed in a separate form, i.e. independent of other elements of the microfluidic device, since such a structure can also be advantageously used in other devices for storing or culturing microtissues, reducing the risk that the microtissue contained in the compartment will be washed away during operation of the device.

[0044] In some embodiments, the compartment has the shape of a spheroid, an ellipsoid, a prism, a cube, or a cylinder.

[0045] In one embodiment, the compartment has a lower section adjacent to the two sections of the conduit and an upper section adjacent to the compartment port, for example as shown in FIG. 16C.

[0046] In one embodiment, for example as shown in Figures 16C and 16E, the compartment has a dome structure in its lower section to reduce the risk that microtissues contained within the compartment will be washed away during operation of the microfluidic device.

[0047] The diameter of the compartment may be selected for the type of tissue or microtissue being used.

[0048] In another embodiment, the compartments comprise an ultra-low attachment surface coating suitable for long-term maintenance of micro-tissues, for example as used in ULA plates (Akura™ 96 Spheroid Microplates, Insferro Inc.).

[0049] In a further embodiment, the compartment comprises an optical detection region suitable for high content imaging. In one embodiment, such an optical detection region is formed by a compartment for containing at least one microtissue and a transparent bottom layer, which may be formed, for example, by a transparent liner as described elsewhere herein. As shown in FIG. 16B, the microfluidic device may be placed, for example, on the stage of an inverted microscope.

[0050] In other embodiments, the microfluidic device comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 32, 48, 64, 96, 128, 192, 256 or 384 micro-tissue compartments.

[0051] The micro-tissue compartments can be arranged sequentially along the straight and / or curved portions of the conduit (e.g., Figure 6 or Figure 8A) or can be arranged in parallel (e.g., Figures 8B, 8C or Figure 10).

[0052] The distance between the sections may range from 0.5 to 20 mm, 1 to 10 mm, 2 to 8 mm, 3 to 5 mm, and in one preferred embodiment may be 9.0 mm, and in another preferred embodiment may be 4.5 mm.

[0053] In one embodiment, the conduit includes a series of bends ("serpentines") having angles selected from the list above, hi some embodiments, the conduit includes 2 bends, 3 bends, 4 bends, 5 bends or 6 bends.

[0054] In one embodiment, the liquid is a cell culture medium. In one embodiment, the cell culture medium comprises cells and / or particles. In one embodiment, the cells are immune cells or circulating tumor cells (CTCs) or a mixture thereof.

[0055] In some embodiments, the microfluidic device comprises a polymeric material such as, for example, a cycloolefin polymer, a cyclic olefin copolymer, polycarbonate, polydimethylsiloxane, polyethylene, polyethylenefluoroethylene, polymethylmethacrylate, polypropylene, polysiloxane, polystyrene, polyurethane, polyvinyl chloride, or a combination of two or more thereof.

[0056] According to one embodiment, at least one conduit has a polygonal, circular, elliptical, semicircular or semi-elliptical cross-section with at least one cross-sectional dimension less than 250 μm.

[0057] As used herein, the term "cross-sectional dimension" refers to one of the following: height (h) (in conduits having a polygonal cross section), diameter (d) (in conduits having a circular or semicircular cross section), or axis (b) (in conduits having an elliptical or semi-elliptical cross section). In this regard, see FIG. 9 and discussion.

[0058] The cross-sectional dimensions are selected such that typical sized micro-tissues cannot pass through the conduit and remain within the compartment (see FIG. 6B, FIG. 12C, FIG. 13 or FIG. 16C). In one embodiment the cross-section is less than 150 μm, which is particularly useful for example for pancreatic islet micro-tissues. In one embodiment the cross-section is less than 220 μm, which is particularly useful for example for micro-tissues containing hepatocytes. The compartment is bounded by a stationary droplet (of cell culture medium).

[0059] According to one embodiment, the device further comprises or is connected or attachable to a means for generating a flow of aqueous liquid in the conduit.

[0060] In one embodiment, said means for generating a liquid flow is a tilting or tiltable device. Preferably, such a tilting device is capable of performing a tilting movement, preferably around the X-axis. In one embodiment, said means for generating a liquid flow is a rotational device. In one embodiment, said direction of rotation is preferably around the Y-axis. In one embodiment, said means for generating a liquid flow is a pump. In one embodiment, such a pump comprises a syringe pump. Two or three of these approaches can be combined. In one embodiment, a back and forth flow ("oscillating flow") is generated.

[0061] According to one embodiment, the reservoir is suitable for containing a liquid, optionally containing cells and / or particles. Such an aqueous liquid is preferably a culture medium, preferably a cell culture medium or a microtissue culture medium.

[0062] According to one embodiment, at least one port is suitable for or connected to an external tube or hose, which tube or hose is optionally connected to a pump.

[0063] As described herein, a port may be a conduit port (e.g., for connecting a pump), a compartment port (e.g., for loading microtissues), or a reservoir port (e.g., for removing cells and / or medium, or for adding drugs, etc.).

[0064] According to one embodiment, the conduit has two ends, each of which fluidly connects to a reservoir.

[0065] According to one embodiment, at least one reservoir includes a reservoir port. In one embodiment, the reservoir has a circular or elliptical cross section. In one embodiment, the reservoir port is disposed coaxially with the reservoir.

[0066] According to one embodiment, the device includes a base plate having a surface including at least one elongated groove, with a liner disposed on at least a portion of each surface of the base plate to seal the elongated groove to form a conduit.

[0067] In some embodiments, the base plate including the at least one elongated groove can be manufactured by 3D printing, grinding or molding.

[0068] In some embodiments, the elongate groove has a polygonal, semicircular or semi-elliptical cross-section. When sealed, at least one conduit has a polygonal, circular, elliptical, semicircular or semi-elliptical cross-section with at least one cross-sectional dimension smaller than a cross-sectional dimension of the microtissue so as to prevent the microtissue from being carried out of the compartment by the aqueous liquid.

[0069] In one embodiment, the compartment includes a port (compartment port) through which the microtissue can be loaded into the compartment.

[0070] In some embodiments, the base plate comprises a material selected from the group consisting of polystyrene, PMMA, COC, COP, PTFE and aluminum, or a material selected from the group consisting of polydimethylsiloxane (PDMS) rubber, vinylmethylsiloxane, phenylvinylmethylsiloxane, fluorosilicone rubber and / or nitrile rubber and natural rubber.

[0071] In some embodiments, the liner comprises a material selected from the group consisting of polystyrene, PMMA, COC, COP, PTFE and aluminum, or a material selected from the group consisting of polydimethylsiloxane (PDMS) rubber, vinylmethylsiloxane, phenylvinylmethylsiloxane, fluorosilicone rubber and / or nitrile rubber and natural rubber.

[0072] In another embodiment, the microfluidic device further comprises a lid that completely or partially covers at least one port selected from the group of the compartment port and the reservoir port.

[0073] According to one embodiment, the lid comprises a foil or mat. In some embodiments, the foil or mat comprises a material selected from the group consisting of polystyrene, PMMA, COC, COP, PTFE and aluminum, or a material selected from the group consisting of polydimethylsiloxane (PDMS) rubber, vinylmethylsiloxane, phenylvinylmethylsiloxane, fluorosilicone rubber and / or nitrile rubber and natural rubber.

[0074] According to another aspect of the invention there is provided an assembly or array comprising a plurality of microfluidic devices according to the above description. Such an array may be provided as a frame capable of housing several microfluidic devices.

[0075] The array or frame may each include or house, for example, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or more microfluidic devices according to the above description.

[0076] According to one embodiment, the array includes or is attachable or connectable to at least one tilt unit, tiltable unit, rotation unit and / or pump.

[0077] According to another aspect of the present invention there is provided a method for culturing at least one microtissue and / or at least one cell in a microfluidic device according to the above description, the method comprising the steps of: - loading at least one micro-tissue and / or at least one cell into a microfluidic device and simultaneously or subsequently introducing an aqueous liquid into the microfluidic device; - Establishing a flow of liquid through the microfluidic device.

[0078] In this method, settling of non-soluble components in the aqueous liquid is avoided by a bend in at least one of the conduits.

[0079] According to one embodiment, the flow of liquid is driven or supported by at least one of the following: ● Pumps, and / or ● Tilt, swivel, pivot or rotate the device.

[0080] Tilting, turning, rotating or swirling induces flow through gravity, especially when there are two or more reservoirs. Turning, turning or swirling causes one to constantly change their height relative to the other, causing a change in hydrostatic pressure between the reservoirs, which causes the liquid to flow.

[0081] In one embodiment, the direction of the liquid flow is repeatedly changed, thus creating a back and forth flow ("oscillating flow").

[0082] In one embodiment, after the microfluidic device has been tilted or rotated, a pause or break is introduced before the tilt or rotation is resumed in the opposite direction. Such a pause or break can have a length between 0.5 seconds and 3600 seconds. In a preferred embodiment, the pause or break has a length between 10 seconds and 240 seconds.

[0083] According to one embodiment, the aqueous liquid comprises cells and / or particles.

[0084] Such aqueous liquid is preferably a culture medium, preferably a cell culture medium or a microtissue culture medium. Cells are insoluble and in laminar flow systems, such as microfluidic channels, tend to settle on the inner surface of the channel facing the direction of gravity or centrifugal force. As mentioned above, the same applies to particles.

[0085] In one embodiment, the cell is an immune cell. In one embodiment, the cell is a cytotoxic T cell (CD8+). In one embodiment, the cell is a T helper cell (CD4+). In one embodiment, the cell is a NK cell (e.g., CD16+). In one embodiment, the cell is a peripheral blood mononuclear cell (PBMC). In one embodiment, the cell is a granulocyte (basophil, eosinophil, neutrophil, and mast cell). In one embodiment, the cell is an agranulocyte (lymphocyte, macrophage, monocyte, and natural killer (NK) cell). In one embodiment, the cell is a stem cell-derived immune cell or immune cell line.

[0086] In one embodiment, the cells express an immune checkpoint, e.g., PD-1, CTLA-4, Lag-3, and in one embodiment, the cells express CD3, or a subdomain thereof, e.g., CD3α, CD3β, CD3ε, CD3γ, or CD3δ. In such an embodiment, the agent being investigated is a bispecific antibody comprising a CD3 binding domain, e.g., a bispecific T cell engager (biTE).

[0087] In one embodiment, the cells express one or more Fcγ receptors, such as, for example, CD16 or FcγRIII, In such an embodiment, the agent being investigated is, for example, an ADCC-inducing antibody that contains an Fc domain.

[0088] In another embodiment, the cell is a circulating tumor cell (CTC).

[0089] definition As used herein, the term at least one bend exhibiting an angle between 85 degrees and 275 degrees about an axis "orthogonal to the planar geometry" should be understood broadly and should include embodiments in which the conduit is not parallel to the surface of the device but is angled thereto, for example by forming a helical conduit structure, see FIG. 26 for a better illustration of this embodiment.

[0090] The term "planar" geometry means that the microfluidic device may also be referred to as a chip, a plate or a slide.

[0091] As used herein, the term "aqueous liquid" refers to a liquid that is composed mostly of water. Such liquids may further include organic or inorganic components.

[0092] The term "particles" as used herein is meant to encompass non-soluble, non-degradable or degradable particles of matter such as "microparticles" (1-1000 μm), "fine particles" (100-2500 nm) and "coarse particles" (2500-10000 nm). Such particles may be, for example, selected from: • Liposomes containing, for example, mRNA or other genetic material; Viral vectors, ● Colloids (silver, silicate), ● Nanoplastic particles and microplastic particles; ● ZnO, SiO2, CeO2, BaSO4, TiO2, and / or • Beads coated with an active agent, for example an antibody.

[0093] The term "microtissue" as used herein is meant to encompass 3D cell culture models including organoids, 3D tissue spheroids, embryoid bodies, pancreatic islets, precision-cut tissue slices, etc. Microtissues can be naturally derived or engineered from isolated cells, or can be passively or actively reconstructed.

[0094] The microtissues may comprise primary cells obtained from organs and reconstituted in an appropriate manner to form such microtissues, for example as described in Messner et al. (2013). The microtissues may comprise or consist of stem cells, immortalized cells or cell lines or mixtures thereof. The microtissues may also comprise cancer cells obtained from tumors in a similar manner, may comprise or consist of tumor microbiopsies, or may be derived from tumor biopsies. In one embodiment, the microtissues may comprise cancer cells and healthy cells.

[0095] In other embodiments, the microtissue cells are mammalian cells, preferably human cells, cynomolgus monkey cells, porcine cells, canine cells, rat cells or mouse cells. In other embodiments, the microtissue comprises hepatic cells, Kupffer cells and / or stellate cells. In other embodiments, the microtissue comprises pancreatic islet microtissues.

[0096] Experiments and figures While the present invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description should be considered as illustrative or exemplary and not restrictive, and the present invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure and the appended claims. In the claims, the term "comprises" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be interpreted as limiting the scope.

[0097] figure FIG. 1 shows a microfluidic device 10 for containing aqueous liquids to demonstrate some of the principles of the present invention.

[0098] In Fig. 1A, a microfluidic device 10 has a planar shape extending essentially in the X and Z directions. The device comprises a microfluidic device including a conduit 11 fluidly connecting at least two structures selected from compartments, reservoirs and / or ports. The conduit includes at least one bend 12 exhibiting an angle α between 85 degrees and 275 degrees about a Y axis arranged orthogonal to the planar shape. The device further includes a compartment 13 for accommodating a microtissue.

[0099] In this embodiment, the open end of the conduit 11 serves as a conduit port 16, through which culture medium can, for example, be loaded into the microfluidic device 10. The port can adopt different technical configurations not shown in this figure, for example to allow either the insertion of a pipette or the connection of a tube.

[0100] The planar shape, preferably its Z axis, is oriented at an angle of +45 degrees to -45 degrees with respect to the direction of gravity (G) or the direction of centrifugal force (Fc) applied to the microfluidic device.

[0101] 1B shows another embodiment of the invention without a compartment for accommodating micro-tissues. It is important to understand that the principles of the invention also encompass embodiments without such a compartment for accommodating micro-tissues, suitable for example for studying suspension cell interactions.

[0102] Figure 2 shows the phenomenon of sedimenting cells and / or particles (dots) suspended in a liquid medium flowing through a microfluidic conduit (rectangular cross section) in a cross section of a microfluidic device according to the invention (A is "State of the art"). Due to the relatively small diameter and flow rate, a laminar flow exists, causing the cells and / or particles to sediment by gravity or centrifugal forces.

[0103] The microfluidic device (B) according to the invention comprises at least one bend that presents an angle α between 85° and 275° around the Y axis perpendicular to the planar shape, making it possible to avoid sedimentation, since the lower inner surface of the conduit, on which the cells and particles tend to settle, becomes, after each bend, the upper inner surface from which they leave.

[0104] Figure 2 further illustrates a rotation concept (C, D) that further contributes to avoiding sedimentation and further generates a flow of liquid within the conduit. Figures 2C and 2D can be seen as a sequence, where position 2C is maintained (pause, rest) until all liquid has flowed from top to bottom, then the device rotates 180 degrees to position 2D, inducing cells and liquid to flow back. As shown in Figure 2, the preferred rotation direction is preferably around the X-axis. It can be seen that cells and / or particles prone to settling (Figure 2C, top right box) are resuspended by the reversal of relative position from top to bottom, generating a hydrostatically driven reversal of flow through the meander (Figure 2D, bottom right box).

[0105] 3 shows a cross section of a microfluidic device. The planar shape of the microfluidic device, preferably its Z axis, is arranged at an angle of +45 degrees to -45 degrees with respect to the direction of gravity (G) or the direction of centrifugal force (Fc) applied to the microfluidic device.

[0106] Figure 4 shows a cross-section of a microfluidic device according to the invention. The device comprises a base plate 41 having a surface 42 including at least one elongated groove 43. A liner 44 is disposed on at least a portion of each surface 42 of the base plate 41 so as to seal the elongated groove. The conduits (and compartments and reservoirs, if present) may be formed partly or entirely in the groove sealed by the liner. The base plate including at least one elongated groove may be manufactured, for example, by grinding or molding.

[0107] The liner 44 may comprise a foil. The foil may comprise a material selected from the group consisting of polystyrene, PMMA, COC, COP, PTFE and aluminum, or may comprise a mat, which may comprise a material selected from, for example, polydimethylsiloxane (PDMS) rubber, silicone rubber such as vinylmethylsiloxane, phenylvinylmethylsiloxane, fluorosilicone rubber and / or nitrile rubber, and natural rubber. It may also comprise a foil or mat irreversibly bonded to the base plate, for example by a hot melt or acrylic adhesive.

[0108] At least one conduit has a polygonal, circular, elliptical, semicircular or semi-elliptical cross-section having at least one cross-sectional dimension smaller than a cross-sectional dimension of the microtissue, thereby preventing the microtissue from being carried out of the compartment by the aqueous liquid.

[0109] Figure 5 shows different possible shapes of conduits, with different numbers of bends and different angles. Compartments, reservoirs and ports are not shown for simplicity.

[0110] FIG. 6A shows a diagram of a microfluidic device with a conduit 11 fluidly connecting at least two structures selected from compartments, reservoirs and / or ports. The conduit comprises at least one bend 12 exhibiting an angle α between 85 degrees and 275 degrees around a Y axis arranged orthogonally to the planar geometry. The device further comprises a series of compartments 13 with compartment ports 62 for accommodating several microtissues. The device further comprises two reservoirs 14 with reservoir ports 15. An aqueous liquid, optionally containing cells, can be loaded into at least one reservoir through the respective port. A flow of culture medium is then generated through the conduit and the compartments by the mechanisms described herein.

[0111] FIG. 6B shows another cross section of the microfluidic device along line C-C' of FIG. 6A, where the conduit 11 fluidly connects at least two structures selected from the compartments, reservoirs and / or ports. The conduit includes at least one bend, not shown in this figure. FIG. 6B shows the compartment 13 with a compartment port 62 for receiving the microtissue 61, through which the microtissue 61 is loaded into the compartment. FIG. 6B further shows the reservoir 14 including the reservoir port 15. An aqueous liquid, optionally containing cells 63 (e.g., immune cells), can be loaded into the reservoir through the respective reservoir port 15. The liner 44 is placed on the underside of the device to seal the device from the bottom, thereby transforming the elongated channel into the conduit 11. It should also be noted that the sizes of the reservoir 14 and the compartment 13 are not drawn to scale, and in some embodiments, the reservoir 14 is much larger than the compartment 13 (see, e.g., FIG. 10).

[0112] The conduit may have a polygonal, circular, elliptical, semicircular or semi-elliptical cross-section with at least one cross-sectional dimension smaller than a cross-sectional dimension of the microtissue 61. The cross-sectional dimension is therefore selected such that a typical sized microtissue cannot pass through the conduit but remains within the compartment.

[0113] Figure 7 shows various means for generating a liquid flow in a conduit. (A) The device can be tilted (preferably by 90-180 degrees, as indicated by the mark "X"), rocked back and forth, or rotated about the Y axis. (B) The device can be tilted (preferably by 90-180 degrees), rocked back and forth, or rotated about the X axis. (C) The device can be connected to at least one pump to generate a liquid flow. Two or three of these approaches can be combined.

[0114] For options (A) and (B), hydrostatic pressure is generated which drives the flow of liquid. Once all the liquid has passed through the tip, the tilt must be repeated.

[0115] It can be seen that the device shown in Figure 7 does not include a compartment for accommodating micro-tissues, it is important to understand that although the gradient principle shown in Figure 7 applies to all embodiments of the invention, in particular with or without a compartment for accommodating micro-tissues, the principles of the invention also cover embodiments without such a compartment, e.g. for accommodating micro-tissues suitable for studying suspension cell interactions.

[0116] 8 shows different arrangements of conduits 11, micro-tissue compartments 13, reservoirs 14, reservoir ports 15 and compartment ports 62. In one embodiment, some compartments and conduits are arranged parallel to one another. In another embodiment, the conduits expand in width to accommodate the compartments. In these and other embodiments, it can be seen that the reservoirs generally have a larger diameter than the micro-tissue compartments.

[0117] In Figures 8A and 8B, compartment 13 is disposed between two sections of conduit 11. In Figure 8B, the conduit branches into two or more conduits arranged in parallel, with each branched conduit including at least one compartment disposed between its two sections.

[0118] In FIG. 8C, compartment 13 is disposed within conduit 11 , ie conduit 11 expands in width to accommodate compartment 13 .

[0119] Example cross sections of conduits are shown in Figure 9. The conduits can have polygonal, circular or elliptical cross sections. The conduits can also consist of elongated grooves with open-ended semicircular, semi-elliptical or rectangular cross sections. In such cases, they are sealed with liners, shown in dotted lines, to produce the conduits.

[0120] FIG. 10 shows one embodiment of a microfluidic device according to the present invention.

[0121] Figures 10A and B show plan views of a microfluidic device according to the present invention, and Figures 10C and D show perspective views of a microfluidic device according to the present invention.

[0122] 10B and D show the underside 101 of the microfluidic device with the conduits 11, bends 12, compartments 13 and reservoirs 14. The underside 101 can be sealed by a liner 44 (not shown), which is preferably transparent, allowing, among other things, microscopic examination of the compartments and the microtissues contained therein.

[0123] 10A and C show the top surface 102 of the microfluidic device. The reservoir 14 includes a rim that functions as a reservoir port 15, through which medium, optionally including cells, can be loaded. The compartment 13 includes a rim that functions as a compartment port 62, through which a microtissue 61 (not shown) can be loaded.

[0124] The regions in which the compartments 13 are located (in this embodiment seven compartments are provided per region) are provided with pillars 17 (in this embodiment four pillars are provided per region) providing a constant distance between the liner and the upper surface of the region to ensure that the space provided by the compartments 13 for the microtissues 61 is constant.

[0125] Thus, in Figure 10, compartment 13 is placed within conduit 11, i.e. conduit 11 expands in width to accommodate compartment 13. A device such as that shown in Figure 10 provides permanent access to cells and media and is suitable for use directly on a microscope, in particular an inverted microscope and / or a confocal microscope.

[0126] FIG. 11A shows a cross-section of a microfluidic device according to the invention with a conduit 11 fluidly connecting at least two structures selected from compartments, reservoirs and / or ports. The conduit includes at least one bend that is not shown in this figure. FIG. 11 shows a compartment 13 with a compartment port 62 for containing a microtissue 61, through which the microtissue 61 is loaded into the compartment. A liner 44 is placed on the underside of the device to seal, among other things, the reservoirs, the conduits and the compartments. FIG. 11 further shows a reservoir 14 including a reservoir port 15. An aqueous liquid, optionally including cells 63 (e.g. immune cells), can be loaded into the reservoir via the respective reservoir port 15, for example to interact with the microtissue 61.

[0127] The reservoir port 15 and the compartment port 62 include a relief structure 111 (not drawn to scale), which is exemplarily shown in the form of an anti-overflow edge, allowing the formation of a droplet 112 of culture medium at the top of the port by establishing a maximum contact angle, thus preventing the release of the droplet by capillary pinning. This phenomenon increases the size of the liquid-air interface and thus improves the gas exchange between the culture medium and the surrounding gas medium. The droplet is drawn in an exaggerated form. It should also be noted that in particular the size of the reservoir 14 and the compartment 13 is not drawn to scale, and in some embodiments the reservoir 14 is much larger than the compartment 13 (see, for example, FIG. 10).

[0128] It should be noted that, as mentioned above, the relief structure 111 can adopt different shapes having different angles, heights and roundness, so long as the shape fulfills its purpose. The relief structure can alternatively include non-wettable areas (i.e., including hydrophobic areas, or areas with a lotus effect) to achieve the same effect.

[0129] Furthermore, the relief structure avoids spillage of liquid when the device is tilted or when pressure is applied within the microfluidic device, for example by pumping an active from a reservoir with liquid flow through the channel. In both situations, the droplet formed on the top of the well may increase in volume or swell, but will not spill or overflow. In this aspect, the surface tension of the liquid has a contributing effect, creating a pressure barrier.

[0130] Instead of leak-proof edges, such relief structures may also include non-wettable areas (ie including hydrophobic areas, or areas with a lotus effect).

[0131] The relative sizes of the reservoir 14 and the micro-tissue compartment 13 are not to scale. In many embodiments, the reservoir 14 has a larger volume than the micro-tissue compartment 13.

[0132] Figure 11B shows a cross-section of a microfluidic device according to the invention with a conduit 11 fluidly connecting at least two structures selected from compartments, reservoirs and / or ports. The conduit includes at least one bend that is not shown in this figure. Figure 11 shows a compartment 13 with a compartment port 62 for receiving a microtissue 61, through which the microtissue 61 is loaded into the compartment by a pipette 113. A liner 44 is placed on the underside of the device to seal inter alia the reservoirs, conduits and compartments.

[0133] A device such as that shown in FIG. 11 provides permanent access to cells and media and is suitable for direct use on a microscope, in particular an inverted microscope and / or a confocal microscope.

[0134] Figure 12A shows a top view of a microfluidic device 10 according to the invention. Figure 12B shows an enlarged view of the area around a compartment 13 for containing a microtissue 61. Figure 12C shows a cross-section of that area. The compartment has a circular cross-section and has a compartment port 62 with a circular cross-section arranged concentrically in the compartment. The absolute dimensions given are merely illustrative and not binding. The relief structure 111 is optional.

[0135] In one embodiment, the compartment has a dome structure 162 in its lower section 13A to reduce the risk that the microtissues 61 contained in the compartment are washed away during operation of the microfluidic device. Tests have shown that the dome structure 162 significantly reduces such risk, relative to a lower section having a cone or funnel shape tapering towards the compartment port 62. Note that the dome structure is not drawn to scale in this image. It preferably terminates at the intersection of the lower section 13A and the upper section 13B. See FIG. 16C, which better reflects the actual dimensions.

[0136] The diameter of the compartment 13 for receiving the micro-tissue is d1, the diameter of the compartment port is d2, and the diameter of the micro-tissue is d3. In one embodiment, (d1-d2) / 2>d3. In this way, it is ensured that the micro-tissue does not escape through the compartment port.

[0137] A device such as that shown in FIG. 12 provides permanent access to cells and media and is suitable for direct use on a microscope, in particular an inverted microscope and / or a confocal microscope.

[0138] Figure 13A shows a diagram of a simplified device according to the invention, having a conduit 11 and a bend 12. Figure 13B shows a cross section thereof, demonstrating that in this embodiment the conduit is not oriented parallel to the surface of the device, but traverses the device in a plane that is inclined relative to the surface of the device.

[0139] 14A shows an array of several microfluidic devices 10 according to the invention arranged on a frame 140 suitable for being rotated. The microfluidic devices may be secured to the frame by snap-in connectors, although other suitable securing means are also contemplated, including Velcro or magnetic plates.

[0140] FIG. 14B shows a platform 141 capable of rotating or tilting the microfluidic device 10 disposed on a frame 140 .

[0141] Figure 14C shows another variation of the device shown in Figure 14A, where the microfluidic device 10 includes an injection molded polystyrene body and a pressure sensitive adhesive film 163 (e.g., 100 mm thick) to seal the channel structure of the microfluidic device 10. The frame 140 accommodates an assembly / array of four such microfluidic devices and adopts the standard SLAS / ANSI format.

[0142] The injection molding process and standardized format allows for pre-series mass production of reproducible devices in large quantities with minimal production effort, and allows the use of inert marshals with low compound adsorption and reliance on effective surface coating protocols and chemistries to achieve cell repellent properties.

[0143] FIG. 14D shows a platform 141 that can rotate or tilt the microfluidic device 10 placed on the frame 140. The microfluidic devices 10 are stacked in multiple frames 140 placed on a frame holder 164. In this way, multiple experiments can be performed in parallel by stacking multiple frames. Bidirectional perfusion of the microfluidic device 10 is achieved by periodic vertical tilts that recirculate single cells in suspension.

[0144] FIG. 15 shows a different example of a microfluidic device according to the invention.

[0145] 16A and B show a cross-section of a microfluidic device 10 according to the invention, having a reservoir port 15, a compartment 13 for containing a microtissue, and a compartment port 62. A liner 44 is barely visible, but seals the elongated groove, thus forming a conduit 11. The device can be placed, for example, on an inverted microscope 136 to allow tissue examination.

[0146] 16C and D show, in cross section, an enlarged view of a micro-tissue compartment (FIG. 16C) and a reservoir (FIGS. 16D and D') of a microfluidic device according to the invention.

[0147] The microtissue compartment comprises a lower section 13A and an upper section 13B. The upper section 13B comprises a compartment port 62 with a relief structure 111 that avoids leakage of the culture medium 112. A lid 160 is provided, which takes the form of a foil that seals the die on its upper section so as to avoid leakage or loss of the culture medium due to evaporation as well as contamination from above. The lower section comprises a dome structure 162, which reduces the risk that the microtissue 61 contained in the compartment is washed away during operation of the microfluidic device. Tests have shown that the dome structure 162 significantly reduces such risk, with respect to a lower section having a conical or funnel shape that tapers towards the compartment port 62. As mentioned above, preferably the upper end of the dome structure defines the beginning of the upper section.

[0148] The reservoir 14 includes a reservoir port 15 that is also sealed with a lid 160, which includes an opening 161 that allows for the addition of medium 112. Preferably, the lid has a circular shape and is concentrically disposed around the reservoir and the reservoir port, which also have a circular shape.

[0149] Figures 16E and F show enlarged cross-sectional views of the micro-tissue compartments (Figure 16E) and reservoirs (Figure 16F) of a microfluidic device according to the present invention when the device is positioned in operating mode, i.e., when its Z-axis is parallel to the direction of gravity or centrifugal force.

[0150] In the micro-tissue compartments, leakage of the culture medium 112 is avoided by a relief structure provided in the form of an overpinning edge, i.e. by surface tension or capillary pinning of the liquid and by coherent forces between the liquid and the surface of the relief structure. It is noted that instead of a leak-proof edge, such a relief structure can also include non-wettable areas (i.e. including hydrophobic areas, or areas with a lotus effect).

[0151] Although no such relief structure is provided in the reservoir, the lid opening 161 is arranged concentrically with the reservoir and the reservoir port, thereby preventing leakage of the culture medium.

[0152] 16G again shows an embodiment with a dome structure 162 and reducing the risk that the microtissue 61 contained in the compartment is washed away during operation. Tests have shown that the dome structure 162 can significantly reduce such risk, relative to a lower section having a conical or funnel shape tapering towards the compartment port 62.

[0153] Fig. 16H shows, for comparison purposes only, an embodiment not according to the invention with a lower section having a conical or funnel shape tapering towards the compartment port 62. In such an embodiment, it can be seen that the microtissue 61 contained in the compartment is easily washed away during operation.

[0154] 17A shows a bottom view of a microfluidic device 10 according to the present invention, with the liner 44 removed for clarity, comprising a reservoir 14 including a reservoir port 15, a conduit 11 including a bend 12, and a compartment 13 for containing a microtissue, including a compartment port 62. The liner for sealing the elongated channel and thus forming the conduit is not shown.

[0155] FIG. 17B shows a cross section of the same microfluidic device to show the profiles of the ports, compartments and conduits.

[0156] 18 shows a diagram of a microfluidic device according to the invention in two different operating mode positions, i.e. with its Z-axis parallel and perpendicular to the direction of gravity or centrifugal force. Since in reservoir 14 lid opening 161 is arranged concentrically with reservoir 14 and reservoir port 15 leakage of medium 112 is avoided in either of the two positions representing any of the operating positions between 0 degrees and ±180 degrees.

[0157] Figure 19 shows tumor growth in the microtissue compartment. HCT116 tumor microtissue 61 was grown in compartment 13 of the microfluidic device 10 for 3 days and showed similar growth as in static conditions.

[0158] Figure 20 shows PBMC distribution in compartment 13. The distribution of immune cells 63 (peripheral blood mononuclear cells, "PBMC") within compartment 13 of microfluidic device 10 was assessed by labeling the cells with a fluorescent dye and monitoring them under perfusion conditions using a handheld fluorescent microscope. The cells migrated freely through conduit 11 and compartment 13.

[0159] FIG. 21 shows the cell count of PBMCs measured with precision counting beads by flow cytometry. Human primary PBMCs in naïve or stimulated conditions were loaded onto a microfluidic device according to the invention (herein referred to as "chip"), or onto a 96-well ULA plate (Akura™ 96 Spheroid Microplate, Insfero, Inc., herein referred to as "static"). The 0 h 6WPctrl measurement shows the number of cells transferred onto the microfluidic device or plate. Cells were harvested from the microfluidic device after 24 and 72 h of culture. Cell counts show representative cell counts taken from the microfluidic device system and static well plate control and processed for flow cytometry. After 72 h under perfused and static conditions, proliferation of stimulated cells was observed.

[0160] The results obtained with the microfluidic device according to the invention were comparable to the control condition cultured in 96-well ULA plates. The results show that when cells were cultured in the microfluidic device and could be easily removed, no sedimentation or aggregation occurred over the time course of the experiment. The results of four independent experiments show the robustness of cell recovery from the microfluidic device for downstream analysis.

[0161] FIG. 22 shows the time period viability of PBMC under different conditions. The viability of PBMC was measured by flow cytometry using a fixable viability stain. Human primary PBMC in naive or stimulated conditions were loaded onto the microfluidic device according to the invention or onto a 96-well ULA plate. The measurement "0h 6WP ctrl" shows the viability at the time of transfer. Cells were harvested from the microfluidic device after 24 and 72 hours of culture. Viability assessment showed high viability for at least 72 hours of culture. The results obtained with the microfluidic device according to the invention were comparable to the control condition cultured in a 96-well ULA plate. The results of four independent experiments show the robustness of the culture conditions and the suitability of the microfluidic device for PBMC culture.

[0162] Figure 23 shows activation markers of cytotoxic T cells. Cytotoxicity (CD3 + / CD8 + ) Phenotypic analysis of T cell activation status. Naïve and stimulated (S and IL-2) PBMCs were cultured under perfusion and static culture conditions for up to 144 h. Cells were harvested after 24 h, 72 h and 144 h and analyzed by flow cytometry. The early activation marker (CD69) showed the highest expression on T cells stimulated after 24 h. The late activation marker (CD25) was highly expressed after 72 h and 144 h. Naïve cells did not show expression of T cell activation markers.

[0163] FIG. 24 shows a cross section of the microfluidic device according to the invention in level position and in operating mode (i.e. with Z-axis parallel to the direction of gravity or centrifugal force). In the micro-tissue compartment 13, leakage of the culture medium 112 is avoided by a relief structure provided in the form of an overpinning edge, i.e. by surface tension or capillary pinning of the liquid and by coherent forces between the liquid and the surface of the relief structure. It should be noted that instead of a leak-proof edge, such a relief structure can also include non-wettable areas (i.e. including hydrophobic areas, or areas with a lotus effect). In the reservoir 14, no such relief structure is provided, but the lid opening 161 is arranged concentrically with the reservoir and the reservoir port, so that leakage of the culture medium is avoided.

[0164] Figures 26-28 show the results of experiments with primary human PBMCs. Cells were thawed and cultured in ultra-low attachment 6-well plates. After 24 h of rest, they were harvested, counted and transferred to the microfluidic device (perfusion) at different cell densities and a total volume of 300 mL per channel (conduit). Static controls were loaded into Akura™ 96 plates at the same concentration and a total volume of 100 mL per well. After 3 and 6 days of culture, cells were harvested from the device or plate, respectively, and processed for flow cytometry analysis.

[0165] Figure 26 shows the results of PBMC culture on the chip, as well as cell number and viability. Cells were mixed with counting beads to determine the number of harvested cells and stained with a fixable viability stain (FVS575V, BD Biosciences) for viability measurements. Data was analyzed on a BD Fortessa (BD Biosciences) equipped with a high-throughput sampler. Data analysis was performed in FlowJo (BD Biosciences) by gating on singlets and then separating cell and bead events in forward and side scatter. Viable cells were not stained for FVS575V. As a result, the viability of PBMCs was generally high under both static and perfusion conditions.

[0166] Figure 27 shows the results of a T cell activation experiment. Cells were mixed with counting beads to determine the number of harvested cells, and they were stained with a fixable viability stain (FVS575V) for viability measurements and a panel of surface marker antibodies to determine the T cells and their activation status. Cells were activated with CD3 / CD28. Data were analyzed on a BD Fortessa equipped with a high throughput sampler. Viable cytotoxic T cells (Tc cells; FVS575V-, CD3+, CD8+, CD4-) were analyzed, and the fractions of naive Tc cells (CD45RA+, CD62L+) and activated Tc cells (CD25+; CD69+) were determined. As a result, similar levels of naive Tc cells were obtained in all conditions, but a somewhat more pronounced density dependence of activated Tc cells was observed under static culture conditions.

[0167] Figure 28 shows the cell composition. Cells were mixed with counting beads to determine the number of cells recovered, and they were stained with a fixable viability stain (FVS575V) for viability measurements and a panel of surface marker antibodies to determine and quantitate the different cell subpopulations of PBMCs. Data was analyzed on a BD Fortessa equipped with a high throughput sampler. Cells were determined as T cells (CD3+, CD56-), cytotoxic T cells (Tc cells; CD3+, CD56-, CD8+, CD4-), helper T cells (Th cells; CD3+, CD56-, CD4+, CD8-), B cells (CD3-, CD19+), monocytes (CD3-, CD14+), natural killer cells (NK cells; CD3-, CD56+), natural killer T cells (NKT cells; CD3+, CD56+). As a result, similar cell composition was obtained under all culture conditions, and under stimulated perfusion conditions, the Tc cell fraction was larger and the Th cell fraction was smaller.

[0168] Figure 29 shows the results of an experiment with a 3D liver model. Viability and response to known drug effects were tested in the microfluidic device and compared to static conditions in well plates. Primary liver microtissues were loaded into a dedicated compartment of the microfluidic device and cultured under perfusion (flow) for 7 days. Medium exchange was performed on the 2nd and 6th days after loading. Brightfield images were acquired on the 1st and 7th days. On the 7th day, the microtissues were retrieved from the microfluidic device and the ATP content, which correlates with tissue viability, was determined using the CellTiter® GLO kit from Promega. In parallel, the microtissues were cultured in well plates under static conditions using the same protocol. Microtissues cultured under flow as well as under static conditions showed high viability and healthy morphology after 7 days. Microtissues exposed to 150 mM tolcapone resulted in a complete loss of ATP and could therefore be considered no longer viable, as expected. These results are supported by the damaged morphology of the tissues.

[0169] FIG. 30 shows the results of an experiment with HCT116 colon cancer cell microtissues. HCT116 colon cancer cell microtissues (MT) were transferred to a microtissue device containing seven compartments per channel (perfused MT). After 24 hours of rest, PBMCs were added to the media reservoir of the channel and distributed throughout the channel (perfused MT+PBMC). Brightfield images of MTs were taken daily for size measurements and MT viability was determined by intracellular ATP measurements after 3 and 6 days of co-culture. Under static control conditions, they were cultured in Akura™ 96 plates with the same PBMC concentration and 1 MT per well (static MT, static MT+PBMC). As a result, MTs grew faster under perfused conditions and were characterized by a higher ATP concentration than under static conditions. The presence of naive PBMCs had no observable effect on the growth behavior of MTs. Under perfusion conditions, PBMCs were visible in the MT compartment, but there were no clusters and no PBMC aggregation around the MTs.

[0170] References ● Ruppen J. et al., A microfluidic platform for chemotherapy testing of multicellular pleural cancer spheroids, Lab Chip, 14, 1198-1205 (2014) ● Occhetta P., Centola M., Tonnarelli B., Redaelli A., Martin I., Rasponi M., A high-throughput microfluidic platform for 3D culture of mesenchymal stem cells, towards the engineering of developmental processes, Scientific Reports, May 2015, 18;5, 10288 ● Kwapiszewska K., Michalczuk A., Rybka M., Kwapiszewski R., Brzozka Z., A microfluidic-based platform for tumor spheroid culture, monitoring and drug screening, Lab Chip, June 2014, 21;14(12):2096-104 ● Jin HJ, Cho YH, Gu JM, Kim J., Oh YS, 2011, Multicellular spheroids for conjugation and extraction using a detachable cell-trapping barrier, Lab Chip 11, 115-119, http: / / dx.doi.org / 10.1039 / c0lc00134a ● Hsiao AY et al., Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids, Biomaterials 30, 3020-3027 (2009) ● Torisawa Y. et al., Multicellular spheroid array capable of spheroid formation, culture and viability assay on a chip, Biomaterials 28, 559-566 (2007) ● Wu LY, Di Carlo D. and Lee LP, Microfluidic self-assembly of tumor spheroids for anticancer drug discovery, Biomed.Microdevices, 10, 197-202 (2008) ● Messner S., Agarkova I., Moritz W., Kelm JM, Multicellular human liver microtissues for hepatotoxicity testing, Arch Toxicol, 2013 Jan;87(1):209-13, doi:10.1007 / s00204-012-968-2, Epub 2012 Nov 11, PMID:23143619, PMCID:PMC3535351 [Explanation of symbols]

[0171] 10 Microfluidic Devices 11 Conduit 12 Bend 13 Compartments for housing microtissues (Also called "microtissue compartments" or "compartments") 13A Lower section adjacent to conduit 13 13B Upper section of compartment 13 adjacent to compartment port 62 14 Reservoir 15 Reservoir port 16 Conduit Port 17 Pillar 41 Base Plate 42 Surface 43 A long, narrow groove forming part of a conduit 44 Liner 61 Microtissue 62 partition ports 63 Cells in culture medium 101 Underside of microfluidic device 102 Top surface of microfluidic device 111 Relief Structure 112 Culture solution 113 Pipette 136 Inverted Microscope 140 A frame for housing several microfluidic devices 141 Platform for rotating or tilting 180 Microstructures that Generate Turbulence 160 Lid 161 Lid opening 162 Dome Structure 163 Adhesive film for sealing channel structure (100mm) 166 A frame holder for accommodating a plurality of frames 140 G gravity direction Fc centrifugal force

Claims

1. A microfluidic device (10) suitable for containing aqueous liquids, comprising: The device has an essentially planar shape and includes at least one conduit (11) fluidly connecting at least two structures selected from reservoirs (14) and / or ports (15, 62); the conduit includes at least one bend (12) that subtends an angle between 85 degrees and 275 degrees about an axis perpendicular to the planar shape; A microfluidic device, wherein the planar shape is positioned at an angle of +45 degrees to -45 degrees with respect to the direction of a) gravity (G) or b) centrifugal force (Fc) applied to the microfluidic device.

2. 2. The microfluidic device of claim 1, further comprising at least one compartment (13) arranged within the conduit or between two sections thereof, said compartment being capable of accommodating at least one microtissue.

3. 3. The microfluidic device of claim 2, wherein at least one compartment (13) has a circular cross section and a compartment port (62) having a circular cross section arranged concentrically with said compartment (13), and wherein the diameter of said compartment is larger than the diameter of said compartment port.

4. 10. The microfluidic device of claim 1, wherein at least one conduit has a polygonal, circular, elliptical, semicircular, or semi-elliptical cross-section with at least one cross-sectional dimension less than 250 μm.

5. 2. The microfluidic device of claim 1, wherein the device further comprises or is connected to or attachable to a means for generating a flow of aqueous liquid in the conduit, preferably wherein said means for generating a flow of liquid is selected from the group consisting of a tilting device, a tiltable device, a rotating device and a pump.

6. The microfluidic device of claim 1 , wherein the reservoirs and / or conduits are suitable for containing a liquid, optionally containing cells and / or particles.

7. 10. The microfluidic device of claim 1, wherein at least one port is suitable for connection to an external tube or hose, said tube or hose optionally being connected to a pump.

8. The microfluidic device of claim 1 , wherein the conduit has two ends that fluidly connect to reservoirs, and optionally at least one reservoir includes a port.

9. 2. The microfluidic device of claim 1, comprising a base plate (41) having a surface (42) including at least one elongated groove (43), and a liner (44) disposed on at least a portion of each surface (42) of the base plate (41) so as to seal the elongated groove and form a conduit (11).

10. The microfluidic device of claim 9 , wherein the liner (44) comprises a foil or a mat.

11. An assembly or array comprising a plurality of microfluidic devices according to any one of claims 1 to 10, and optionally comprising at least one of a tilt unit, a tiltable unit, a rotation unit and / or a pump.

12. A method for culturing at least one microtissue and / or at least one cell in a microfluidic device according to any one of claims 1 to 10, comprising: loading at least one microtissue and / or at least one cell into a microfluidic device and simultaneously or subsequently introducing an aqueous liquid into the microfluidic device; and establishing a flow of liquid through the microfluidic device; wherein the bends in at least one conduit (11) prevent the settling of insoluble components in the aqueous liquid; Optionally, the aqueous liquid comprises cells and / or particles.

13. 13. The method of claim 12, wherein the flow of the liquid is driven or supported by a pump and at least one of tilting, turning, pivoting and rotating the device.

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