Cell culturing device for multi-assay assessment

Abstract

The invention provides a microfluidic device (10) comprising a device top side (11) and a device bottom side (19), wherein the microfluidic device (10) comprises at least one test arrangement (100); wherein for each test arrangement applies that: the test arrangement (100) comprises two reservoirs (110a, 110b), a test chamber (150), and a fluid channel (190); wherein (A) each reservoir (110a, 110b) comprises (i) a bottom end (119), (ii) a bottom partition (118) comprising the bottom end (119), (iii) a top end (111) configured at the device top side (11), and a (iv) top partition (112) comprising the top end (111), wherein the top end (111) comprises an opening (113), wherein the top partition (112) has a top equivalent circular diameter (Dtop) and the bottom partition (118) has a bottom equivalent circular diameter (Dbot), wherein for at least one of the reservoirs (110a, 110b) a reservoir ratio DtOp / Dbotis larger than 1; (B) the fluid channel (190) spans between the two reservoirs (110), wherein the test chamber (150) extends from the fluid channel (190) in a direction of the device bottom side (19), and wherein the fluid channel (190) fluidically connects the two reservoirs (110a, 110b) and the test chamber (150).

Description

[0001] Cell culturing device for multi-assay assessment

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method for analyzing biological material and to a microfluidic device that may be used in the method.

[0004] BACKGROUND OF THE INVENTION

[0005] Microfluidic tissue culture devices are known in the art. EP 3907277A1, for instance, describes systems and methods for providing culturing of a number of various tissue types in an air-liquid configuration in a high-throughput format and allowing co-culture of cells as well as application of physiologically relevant flow. A microfluidic cell culturing device is described that includes a first channel having a first inlet port and a second inlet port, the first channel defined in a first layer. The microfluidic cell culturing device includes a membrane layer having a first surface coupled to the first layer defining the first channel, the membrane layer comprising semipermeable membrane that forms at least a portion of a surface of the first channel. The microfluidic cell culturing device includes a chamber defined in a second layer that exposes a portion of the membrane layer to an external environment, wherein the chamber overlaps a portion of the first channel across the membrane layer.

[0006] US20240010962A1 describes a microfluidic cell culture device including two or more media reservoirs, a first microfluidic chamber having a non-planar surface, a second microfluidic chamber being a pressure chamber and a flexible non-porous membrane that separates the first microfluidic chamber and the second microfluidic chamber. The flexible non- porous membrane is opposite to the non-planar surface of the first microfluidic chamber. One or more microfluidic channels connect the first microfluidic chamber to the two or more media reservoirs.

[0007] WO2024085844A2 describes a microfluidic platform comprising at least one microfluidic chip with at least one-end closed filling channel and at least one sample reservoir, along with at least one centrifuge tube designed for placing the microfluidic chip into the centrifuge device, to biological analyses conducted on this platform by filling the liquid in a closed-end channel using the centrifugal principle.

[0008] WO2023073178A1 describes a microfluidic cell culturing device comprising a cell culture cavity, a first perfusion channel having an inlet and an outlet, a first capillary pressure barrier essentially vertically connecting the first perfusion channel with the cell culture cavity, a second perfusion channel having an inlet and an outlet, and a second capillary pressure barrier essentially vertically connecting the second perfusion channel with the cell culture cavity.

[0009] SUMMARY OF THE INVENTION

[0010] Microfluidic tissue culture devices may be used to culture biological tissue and, for instance, to study the effects of external factors on the development of the tissue, such as in safety, toxicity, or potency assessment studies of drugs or other substances. Current standard potency bioassays typically make use of a monolayer of cells. The standard assays may use 2D culture plates where cells are seeded, and a drug may then be added to the system. This may not be representative of what is normally happening in vivo. In particular, for the mimicry of cell therapies affecting 3D solid tumor this may not be representative. Also more complex systems are known in the art which may allow creation of aggregates in the system. This may already increase the complexity of the system. However, these systems may not allow adding therapy and / or studying cellular dynamics without perturbing the test environment. These systems may, for instance, be specifically configured for culturing and optionally for aggregate forming.

[0011] It appears that there is a need for alternative platforms or systems to study, among others, immune-cellular behavior in real-time (and especially in a parallelized fashion) that facilitates rapid assays as well as more complex environments to better mimic actual (human) physiology, and that are not negatively affected by operating conditions such as a flow of, or addition of, a growing media or drugs (i.e., configurations / operations that in prior art systems may provide perturbation of the system).

[0012] Hence, it is an aspect of the invention to provide an alternative microfluidic device, which preferably further at least partly obviates one or more of the above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0013] According to a first aspect, the invention provides a microfluidic device. In embodiments, the microfluidic device (or: “device”) may comprise a device top side and a device bottom side. The device bottom side is especially configured opposite to the device top side. The device may in embodiments comprise a virtual top plane abutting the device top side. Likewise, in embodiments, the device may comprise a virtual bottom plane abutting the device bottom side. The microfluidic device may in embodiments comprise at least one test arrangement. The test arrangement is especially configured between the device top side and the device bottom side. In further embodiments, for each test arrangement applies that the test arrangement may comprise two reservoirs, a test chamber, and a fluid channel. Further, especially each reservoir may comprise (i) a bottom end, (ii) a bottom partition comprising the bottom end, (iii) a top end, and a (iv) top partition comprising the top end. The top end is especially configured at the device top side. The top end may further comprise an opening (to external of the microfluidic device). The opening is further especially configured in the device top side. In further embodiments, the (each, especially individually) top partition has a top equivalent circular diameter (Dtop) and the (each, especially individually) bottom partition has a bottom equivalent circular diameter (Dbot). The reservoirs may (each individually) have a reservoir ratio Dtop / Dbot (of the top equivalent circular diameter (Dtop) to the bottom equivalent circular diameter (Dbot)). In specific embodiments for at least one of the reservoirs the reservoir ratio Dtop / Dbotmay be larger than 1. Further, especially, each bottom end is separated from the device bottom side by a bottom end distance (dbe), especially an individual bottom end distance (for each of the bottom ends). The bottom end distance (dbe) may especially be defined between the bottom end and the virtual bottom plane. Further, the bottom end distance (dbe) may especially be defined perpendicular to the virtual bottom plane. Further, (for each test arrangement) the fluid channel may in embodiments span between the two reservoirs . The test chamber may especially extend from the fluid channel in a direction of the device bottom side. In further embodiments, the fluid channel may fluidically connect the two reservoirs and the test chamber. In further embodiments, (for each test arrangement) the fluid channel comprises a top channel wall and a bottom channel wall. The top channel wall is especially a closed wall (closed in a direction towards the device top). Further, in embodiments, the bottom channel wall is separated from the device bottom side by a channel wall distance (dWaii). The channel wall distance (dwaii) may especially be defined between the bottom channel wall and the virtual bottom plane. The channel wall distance (dwaii) may especially be defined perpendicular to the virtual bottom plane. In further specific embodiments for each reservoir applies that 0.75<dbe / dWaii<1.25, and in embodiments 0.9<dbe / dWaii<l .1.

[0014] In embodiments, the invention provides a microfluidic device comprising a device top side and a device bottom side, wherein the microfluidic device comprises at least one test arrangement; wherein for each test arrangement applies that the test arrangement comprises two reservoirs, a test chamber, and a fluid channel; wherein (A) each reservoir (110a, 110b) comprises (i) a bottom end, (ii) a bottom partition comprising the bottom end, (iii) a top end configured at the device top side, and a (iv) top partition comprising the top end, wherein the top end comprises an opening, wherein the top partition has a top equivalent circular diameter (Dtop) and the bottom partition has a bottom equivalent circular diameter (Dbot), wherein the reservoirs have a reservoir ratio Dtop / Dbot of the top equivalent circular diameter (Dtop) to the bottom equivalent circular diameter (Dbot), wherein for at least one of the reservoirs the reservoir ratio Dtop / Dbotis larger than 1; (B) the fluid channel spans between the two reservoirs, wherein the test chamber extends from the fluid channel in a direction of the device bottom side, and wherein the fluid channel fluidically connects the two reservoirs and the test chamber; and (C) the fluid channel comprises a top channel wall and a bottom channel wall, wherein the top channel wall is a closed wall.

[0015] The bottom ends of the reservoirs and the bottom channel wall may have similar distances to the device bottom side, such as to a bottom virtual plane abutting the device bottom side.

[0016] Hence, in embodiments, the invention provides a microfluidic device comprising a device top side and a device bottom side, wherein the microfluidic device comprises at least one test arrangement; wherein for each test arrangement applies that the test arrangement comprises two reservoirs, a test chamber, and a fluid channel; wherein (A) each reservoir (110a, 110b) comprises (i) a bottom end, (ii) a bottom partition comprising the bottom end, (iii) a top end configured at the device top side, and a (iv) top partition comprising the top end, wherein the top end comprises an opening, wherein the top partition has a top equivalent circular diameter (Dtop) and the bottom partition has a bottom equivalent circular diameter (Dbot), wherein the reservoirs have a reservoir ratio Dtop / Dbot of the top equivalent circular diameter (Dtop) to the bottom equivalent circular diameter (Dbot), wherein for at least one of the reservoirs the reservoir ratio Dtop / Dbotis larger than 1 ; and wherein each bottom end is separated from the device bottom side by a bottom end distance (dbe), wherein the bottom end distance (dbe) is defined between the bottom end and a virtual bottom plane abutting the device bottom side; (B) the fluid channel spans between the two reservoirs, wherein the test chamber extends from the fluid channel in a direction of the device bottom side, and wherein the fluid channel fluidically connects the two reservoirs and the test chamber; and (C) the fluid channel comprises a top channel wall and a bottom channel wall, wherein the top channel wall is a closed wall, wherein the bottom channel wall is separated from the device bottom side by a channel wall distance (dwaii) defined between the bottom channel wall and the virtual bottom plane; and wherein for each reservoir (110a, 110b) applies 0.9<dbe / dWaii<l. l.

[0017] Such device may provide various advantages over prior art devices. The microfluidic device may facilitate culturing cell aggregates, adding therapies, and studying cellular dynamics without perturbing the system. The test chamber being configured extending from the fluid channel in a direction of the device bottom side may minimize perturbation of cell material in the test chamber when a fluid is flown through the fluid channel. For instance, nutrients and / or drugs (in a fluid) may be provided to seeded biological cells in the test chamber, substantially without exerting unwanted external forces (such as shear or turbulence) on the cells which could result in removing of cells (cell material) from the test chamber and / or which may affect the cells in an uncontrolled way. Moreover, the configuration may facilitate removing remaining material (fluid and / or cell material) from the fluid channel, subsequent to loading the test chamber (especially using centrifugation) with cell material and / or fluid. Furthermore, the configuration of the least one of the reservoirs may provide a plateau at a bottom end of the top partition. The configuration of the plateau may facilitate a provision of a fluid and / or especially a discharge of the fluid to / from the fluid channel via the top partition, especially via the plateau, without substantially perturbation of fluid in the fluid channel and in the test chamber.

[0018] Moreover, the microfluidic device may facilitate the studying of more complex systems. For instance, hydrogel or another material may be provided in the channel to mimic a vasculature channel, to better mimic human physiology compared to prior art systems. The device may host simple assays considering one cell type, e.g., as a monolayer, as well as more complex configurations. The device may, e.g., be used to introduce 3D spheroids (single - biological- cells or plurality of cells), combinations of 3D spheroids and a hydrogel (essentially any flowable hydrogel), optionally in combination with cells (single cells or cell aggregates). This may allow for an increase of complexity of the system while maintaining the same structure. This may allow scientists with a degree of versatility to apply the device. A scientist may for instance start with a single cell type and progressively increase the type of cells as well as other variables (e.g., hydrogel) while maintaining the same structural configuration and process. This would help not only in user-friendliness, as the scientist would use the same format, but also in establishing a reproducible and validated model. Embodiments of the device may further also be put on a plate rocker in an incubator and fluid flow could be performed by simply tilting the plate as per plate-rocker protocol. This may help in mimicry of shear stress if the assay requires it.

[0019] Studies may be performed in embodiments of the device without perturbing the environment being studied. The device may be applied to do safety, toxicity, dosing, and potency assessment of drugs, including, but not limiting, to biologies, small molecules, mRNA, and cell therapies. The device may be applied for studies on CAR-T cell (chimeric antigen receptor - T cell) potency assay and, e.g., insulin assessment using beta-cell islets. Moreover, the device may be applied to study disease progression as well as understanding metabolic pathways. The device may further be used for studying personalized medicine; small biopsies of a tumor may in embodiments be added and different pharmaceutical cocktails can be used to assess the most potent ones. The studies may be performed in embodiments of the device especially without perturbing the environment being studied.

[0020] Further, the test arrangement may facilitate modifying / replacing fluid, e.g. medium, in the channel with limited to no perturbation to cells in the test chamber, while also facilitating removing cells, e.g. a cell aggregate, from the test chamber when desired, such as for further analysis outside of the device. In particular, by arranging a pipette tip on a plateau in a reservoir, liquid can be removed or added to the system with limited to no perturbation to the cells. In contrast, by arranging a pipette tip directly into the channel, a cellular aggregate may be recovered from the test chamber.

[0021] Yet further, in embodiments, the test arrangement may facilitate providing a particularly constant (or “smooth”) flow of liquid through the fluid channel. In particular, a continuous fluid flow may be applied from one reservoir to the corresponding reservoir, in part due to the absence of other channels through which the fluid may flow. The ability to provide of a smooth continuous flow may contribute to the ability to analyze and / or cultivate specific tissue types. For instance, the application of a smooth continuous flow may be relevant for vascularization.

[0022] Further, specific embodiments comprising a plurality of test arrangements, and comprising a channel between the test arrangements, may allow for having tissue-tissue communication. This may further allow multi-tissue assessment to compounds. In embodiments, studies may be carried out and analyzed real-time in a parallelized fashion. The device may, in further embodiments, allow to do high-content imaging (e.g., using a high resolution and a (e.g., 60-times) magnification). Moreover, the device may in embodiments allow full automation of the studies. Automation may result in a reduction of potential humanerror increasing reproducibility.

[0023] As indicated above, the invention relates to a microfluidic device (herein also indicated with the term “device”) comprising at least one test arrangement. The test arrangement may especially be used to culture biological cells and to study the effects of imposed treatments, actions, therapies, etc. on the biological material (such as the cells or material originating from the cells or cell aggregates).

[0024] Microfluidic devices are known in the art. The term “microfluidic device”, herein, may especially refer to a device for the manipulation of small amounts of fluids (including materials comprised by the fluids) using channels and chambers with sizes of ten to hundreds or thousands of micrometers. The terms “channel” and “chamber” are known in the art and may refer herein to a space or volume in the microfluidic device. The term “channel” may especially refer to an elongated space, such as a tube, duct, pipe, or conduit, suitable for the transportation of a fluid. A channel may especially be elongated along one dimension with respect to the other dimensions. A channel may comprise a channel axis of elongation (also: “channel axis”). The term “chamber” may especially refer to a space having sizes in three dimensions, wherein the respective sizes in embodiments may be in the same order of magnitude. A chamber may especially be suitable to host a fluid. Yet, in embodiments a fluid may flow through the chamber. Such chamber may also be referred to as “flow-through chamber”. Herein, especially the test chamber as well as the reservoirs may have a chamber configuration. Moreover, in embodiments, the bottom partitions and the top partitions may also have a chamber configuration. The reservoirs (and their partitions) may be suitable to flow through, as well as to host fluid. The test chamber is especially configured to host a fluid (and, among others, biological material).

[0025] Herein also the (simple) term “channel” may be used to refer to the “fluid channel” unless it is clear from the context that the term refers to another channel (e.g., to a connection channel configured to fluidically connect a first test arrangement with a further test arrangement, see further below).

[0026] The terms “top” and “bottom”, such as in “device top” and “bottom end”, and comparable terms, are used especially referring to a relative position during use of the device. During use, the bottom side, for instance, may be arranged closer to the ground than the top side of the device. Likewise, the bottom partition of the reservoirs may be arranged closer to the ground than the top partitions, i.e., when providing a fluid in (the top partition of) the reservoir, it may flow to the bottom partition based on gravity. It may further flow via the fluid channel to the test chamber (based on gravity). Furthermore, herein also the terms “lower”, “under”, “higher”, and “above” may be used when referring to a configuration of a first element relative to a second element. Also, these terms may especially refer to the device during use. Moreover, the terms “lower”, “under”, “bottom”, and comparable terms (in relations to elements of the device) may refer to a configuration (of the element) closer to the bottom side of the device, and “higher”, “above”, top”, and the like may refer to a configuration closer to the top side of the device. For instance, the phrase “the test chamber extends from the fluid channel in a direction of the device bottom side” and the phrase “the test chamber is arranged below the fluid channel (while being fluidically connected to the fluid channel)” may relate to the same relative configuration of the test chamber to the fluid channel. Further, for instance, a “top end” or “top extreme” of the top partition is especially configured closer to the top side of the device than a “bottom end” or “bottom extreme” of the top partition.

[0027] The fluid channel especially spans between the two reservoirs, especially between the bottom partitions of the two reservoirs. The fluid channel may comprise a fluid channel axis (of elongation). The fluid channel axis may especially comprise a straight axis. Yet in further embodiments, the fluid channel axis may comprise a curved axis. The fluid channel axis may in specific embodiments be configured curved in a plane parallel to the virtual bottom plane. Moreover, a virtual line comprising the fluid channel axis may in embodiments intersect each bottom partition of the test arrangement.

[0028] As indicated above, the fluid channel may have a top channel wall and a bottom channel wall. The fluid channel may further comprise side channel walls connecting the top channel wall to the bottom channel wall. In embodiments, the top channel wall, the bottom channel wall, and the side channel walls define the fluid channel. Further, especially, a (shortest) distance between the side channel walls may define a channel width (WChan). The distance may especially be defined perpendicular to the (fluid) channel axis. In further embodiments, a (shortest) distance between the top channel wall and the bottom channel wall defines a channel height (HChan). The channel height is especially configured perpendicular to the channel width. The channel height may in embodiments be at least 10 pm, such as at least 50 pm, especially at least 100pm. The channel height may further be 5 mm at maximum, especially 3 mm at maximum, and in embodiments 1 mm at maximum. The channel height may especially be no more than 900 pm. In specific embodiments, the channel height (HChan) is selected from the range of 0. 1 - 3 mm, especially from the range of 0. 1-0.9 mm. The channel width may comprise a value as described in relation to the channel height. In embodiments, the channel width may (also) be selected from the range of 0. 1 - 3 mm, especially from the range of 0. 1-0.9 mm. In embodiments, WChan / HChan is approximately 1. In further embodiments, Wchan / Hchan may be selected from the range of 0.1-10, such as from the range of 0.2-5, especially from the range of 0.3-3, even more especially from the range of 0.5-2. In specific embodiments, a channel ratio WChan / HChan of the channel width (WChan) to the channel height (HChan) is selected from the range of 0.5 to 2.

[0029] The fluid channel may further comprise a channel length, defined along the channel axis. The channel length (LChan) may especially be in the range of at least three times the channel width. In embodiments LChan / WChan >3, such as LChan / WChan >4, especially LChan / WChan >5. In further embodiments LChan / WChan < 20, such as LChan / WChan < 10. For instance, in embodiments WChan may be about 1 mm, wherein LChan is about 5 mm. In embodiments, the channel width (WChan) and the channel height (HChan) may be (essentially) constant over the channel length (LChan). In particular, in embodiments, WChan and Hchan may be constant along at least 80% of LChan, such as along at least 90% of LChan, especially along at least 95% of LChan, including 100% of LChan. In further embodiments, along the channel length (LChan), WChan may vary less than 5% (relative to a maximum value of WChan), such as less than 3%, especially less than 1%. Similarly, in embodiments, along the channel length (Lchan), HChan may vary less than 5% (relative to a maximum value of HChan), such as less than 3%, especially less than 1%. A uniform height and width in the channel may contribute to providing a stable flow through the channel.

[0030] The fluid channel may in embodiments comprise a round(ed) tubular shape. The fluid channel may in further embodiments have an elliptical cross-section (perpendicular to the fluid channel axis). In yet further embodiments, the fluid channel may comprise a cross-section having a (regular) polygonal shape, such as a pentagonal shape or an octagonal shape or a triangular shape, or tetragonal (including rectangular) shape. In further specific embodiments, the fluid channel may especially comprise a (rectangular) cuboid shape. The cross-sectional area of the fluid channel perpendicular to the fluid channel axis may in embodiments comprise a rectangle (including a square).

[0031] The fluid channel may in embodiments at least fluidically connect the bottom partitions of the respective reservoirs. Therefore, the ratio of the bottom end distance dbe to the channel wall distance dwaii may in embodiments especially be at least 0.75, such as at least 0.8, and in embodiments at least 0.9. In specific embodiments, dbe / dWaii > 0.95, such as dbe / dWaii >0.99. Further especially dbe / dWaii may be equal to or smaller than 1.25, such as equal to or smaller than 1.2, and especially equal to or smaller than 1.15. In specific embodiments dbe / dWaii < 1.1, such as dbe / dwaii < 1.05, especially dbe / dWaii <1.01. Hence, in embodiments the ratio dbe / dwaii may be approximately 1. Especially based on these ratios (especially for the at least one of the reservoirs) a quiescent fluid flow may be provided in the fluid channel when providing a fluid in the reservoir. In specific embodiments, the bottom end (especially at least of the at least one of the reservoirs) may be aligned with the bottom channel wall.

[0032] Moreover, in further embodiments a height of the bottom partition may be approximately equal to the channel height. A bottom partition height (HbP) (of a respective reservoir) may be defined by a (minimal) distance between the top partition and the bottom end (of the respective reservoir, and especially defined perpendicular to the virtual bottom plane. A ratio Hbp / Hchan (especially at least for the at least one of the reservoirs) may especially be at least 0.8, such as at least 0.9, and in embodiments at least 0.95. In further embodiments, Hbp / Hchan (especially at least for the at least one of the reservoirs) may be 1.25 at maximum, such as 1.15 at maximum, and in embodiments Hbp / HChan (especially at least for the at least one of the reservoirs) may be 1.1 at maximum, such as 1.05 at maximum.

[0033] Hence, in embodiments at least for the at least one of the reservoirs, and especially for each of the reservoirs applies that a distance between the top partition and the bottom end, and especially defined perpendicular to the virtual bottom plane, defines a bottom partition height (HbP), wherein a shortest distance between the top channel wall and the bottom channel wall defines a channel height (Hchan), and wherein Hbp / Hchan is selected from the range of 0.8 - 1.25, such as from the range of 0.9-1.1, especially from the range of 0.95-1.05. In embodiments HbP / HChan is approximately equal to 1. Based on such ratio, the fluid may quiescently be flown in the fluid channel by providing the fluid to the top partition. Moreover, fluid may also be removed from the top partition without disturbing fluid in the fluid channel. In specific embodiments, HbP / HChan is approximately equal to 1 and dbe / dWaii may be approximately 1 (at least for the at least one of the reservoirs).

[0034] The fluid channel further especially fluidically connects the test chamber to the reservoirs. When providing a fluid in one of the reservoirs, it may flow to the test chamber. The fluid may further optionally also flow to the other reservoir. When removing fluid from the reservoir, in embodiments, the fluid channel may keep the fluid as described above. Yet, in embodiments fluid may also be removed from the channel when removing fluid from the reservoir. The test chamber may especially be configured such that when removing fluid from the channel, fluid hosted in the test chamber may be maintained in the test chamber.

[0035] The device may thus comprise a device top side and a device bottom side. The device may further comprise a virtual top plane (or “top plane”) abutting the device top side. Moreover, the device may also comprise a virtual bottom plane (or: “bottom plane”) abutting the bottom side. The top plane may in embodiments be configured parallel to the bottom plane. The device top side may in further embodiments comprise a planar surface, e.g., in embodiments the (top) surface of a well plate (with access to the wells). Furthermore, the device bottom side may (also) comprise a planar surface, e.g., in embodiments the (closed and / or open) bottom surface of the well plate. The device top side may further comprise a plurality of openings, for accessing the reservoirs. The openings may be defined by (the opening in) the top ends of the reservoirs. The device may in specific embodiments comprise a plurality of test arrangements (see also further below). The invention may in embodiments provide a well plate (configuration) comprising a plurality of test arrangements. The reservoirs may define the wells of the well plate. Especially, the wells may define sets of (each) two wells, wherein the two wells are connected via a fluid channel having a test chamber extending from the fluid channel. Moreover, a test arrangement described herein may be defined by a set of wells, a fluid channel, and a test chamber extending from the fluid channel.

[0036] Herein, when it is described that a specific element is configured parallel to, at an angle from, or, e.g., perpendicular, to the device top side (or to the device bottom side), this may especially indicate that the specific element is configured parallel to, at an angle from, or e.g. perpendicular to the virtual top plane (or to virtual bottom plane, respectively).

[0037] As indicated above, the device especially comprises one or more test arrangements. Each test arrangement may comprise two reservoirs fluidically connected to each other via the fluid channel. Furthermore, the reservoirs may also be fluidically connected to the test chamber that especially extends from the channel downwards (towards the device bottom side). The reservoirs are in embodiments configured at (opposite) extremes of the fluid channel. Furthermore, the test chamber is especially configured (extending from the channel) at a location between the two reservoirs. Based on such configuration a fluid provided to one (or to both) of the reservoirs may flow to the test chamber (based on gravity). The test chamber may in embodiments be configured closer to one of the reservoirs, for instance a distance between one of the reservoirs (of the two reservoirs) and the test chamber relative to a second distance between the other one of the (two) reservoirs may be selected from the range of 0.1 to 10. Yet, in embodiments said ratio may be approximately one. In specific embodiments, the test chamber in the at least one test arrangement is configured centered between the two reservoirs, especially centered along the axis of elongation of the fluid channel.

[0038] The fluid may in embodiments comprise a biological material (e.g., a biological cell to be seeded or cultured in the test chamber, a cell aggregate, cell tissue, etc.). The fluid may in specific embodiments comprise a liquid, especially an aqueous liquid. The fluid may in further embodiments comprise further elements. The further elements may, e.g., be dissolved in the fluid. Yet, additionally, or alternatively, the further elements may not dissolve in the fluid. For instance, the fluid may in embodiments comprise a (first) liquid comprising a further liquid dispersed in the (first) liquid. Additionally, or alternatively, the further elements may comprise solid material. In yet further embodiments, the further elements may comprise a gaseous phase. The further elements may comprise gas bubbles. The further elements may in embodiments comprise nutrients or cells (see also further below) that may be provided to test chamber by providing the fluid to one or more of the reservoirs. Essentially, the fluid may comprise a fluid, especially a liquid, optionally comprising a substance, or mixture of substances, as desired. The substance(s) may have a solid, and / or liquid and / or gaseous state. The fluid may in embodiments comprise a liquid comprising (gas) bubbles, or e.g., a liquid comprising droplets of a second liquid. Herein, the term “fluid” may especially refer to a liquid. The liquid may in embodiments comprise one or more further elements.

[0039] The test arrangement described herein is especially configured to prevent (uncontrolled) disturbance or perturbation of matter in the test chamber during use. Moreover, it is a relevant aspect of the invention to minimize perturbation of activity or of a process (being studied) taking place in the test chamber, especially during a change of fluid in the channel. Preventing perturbation may especially be the result of the configuration of the reservoir that is used to provide a fluid to the fluid channel and / or to remove a fluid from the channel. Preventing perturbation may further be the result of the configuration of the fluid channel (relative to the reservoirs) as is described above. In embodiments both reservoirs may be used to provide fluid in and / or remove fluid from the channel. In other embodiments only one of the two reservoirs may be used to provide fluid in or remove fluid from the channel. It is noted that the device may in embodiments allow providing a controlled shear stress onto matter in the test chamber, for instance by tilting the device (in a controlled way).

[0040] At least the reservoir configured for providing and / or removing fluid in or from the channel (herein also indicated as “the at least one of the reservoirs”) may in embodiments comprise a reservoir ratio DtOp / Dbot larger than one. In embodiments DtOp / Dbot (of the at least one of the reservoirs) is at least 1.2, such as at least 1.5, and in embodiments at least 2. In further embodiments, DtOp / Dbot (of the at least one of the reservoirs) may be equal to or smaller than 50, especially equal to or smaller than 20, such as equal to or smaller than 10, and in embodiments DtOp / Dbot (of the at least one of the reservoirs) may be equal to or smaller than 5. It is noted that in embodiments, DtOp / Dbot of the other reservoir may have a value as described above (for the at least one of the reservoirs). Yet, in further embodiments DtOp / Dbot of the other reservoir may be 1, or smaller than 1. Hence, in embodiments (at least) for the at least one of the reservoirs applies that the reservoir ratio DtOp / Dbot is selected from the range of 1.2-20, such as from the range of 1.5-20, especially from the range of 1.5-10. In an embodiment, e.g., Dtopmay be 4 or 2 mm, and Dbot may be 2 or 1 mm, respectively. Yet in other embodiments Dtopmay be 5 to 10 mm, whereas Dbot may be 0.5 to 4 mm.

[0041] The reservoir ratio is especially a ratio between the equivalent circular diameter of the top partition and the equivalent circular diameter of the bottom partition. The equivalent circular diameter (or ECD) (or “circular equivalent diameter”) of an (irregularly shaped) two- dimensional shape is the diameter of a circle of equivalent area. For instance, the equivalent circular diameter of a square with side a is 2*a*SQRT(l / 7t). For a circle, the diameter is the same as the equivalent circular diameter. Would a circle in an xy-plane with a diameter D be distorted to any other shape (in the xy-plane), without changing the area size, then the equivalent circular diameter of that shape would be D.

[0042] For a reservoir ratio larger than one, a plateau (or “terrace” or “ledge”) may be defined at a bottom end of the top partition (or at a top end of the bottom partition). The top partition may be defined by a (circumferential) top partition wall, especially by an inner surface of the circumferent top partition inner wall, in combination (in embodiments) with the bottom end of the top partition. The bottom end of the top partition may provide an open (fluid) connection to the bottom partition. The bottom end of the top partition (or “top partition bottom end”) may in embodiments define the top end of the bottom partition (or “bottom partition top end”). In embodiments, the bottom end of the top partition may be open in a central part of the bottom end of the top partition, whereas it may define a closed surface at a peripheral part of the bottom end of the top partition. For instance if the top partition wall defines a cylinder (and the bottom partition is also cylindrically shaped), the bottom end of the top partition may in embodiments comprise an annular shape, especially if the top partition and the bottom partition are configured coaxially. Hence, in embodiments, the top partition and the bottom partition may be configured coaxially, especially wherein the top partition and the bottom partition both approximate, especially have, cylindrical shapes, and wherein the bottom end of the top partition defines an annular shape. In particular, in such embodiments, the plateau may approximate, especially have, an annular shape.

[0043] The term “approximate” and its conjugations herein, such as in “to approximate a shape”, refers to being nearly identical to, especially identical to, the following term, for example nearly identical to a cylindrical shape. For example, a top partition may define a cylindrical shape but for a defect. In particular, an object approximating a first shape may herein refer to: a first shape realization encompassing the object, wherein the first shape realization is defined as the smallest encompassing shape of the (2D or 3D, respectively) object wherein the first shape realization has the shape of the first shape, wherein a ratio of the area (volume) of the first shape realization to the area (volume) of the object is < 1.2, especially < 1.1, such as <1.05, especially <1.02. For instance, a top partition may approximate a cylindrical shape, wherein the first shape realization may be defined as the smallest encompassing cylindrical shape of the top partition, wherein a ratio of the volume of the first shape realization to the volume of the top partition is t 1.2, especially, especially < 1.1, such as <1.05, especially <1.02, including 1. Further, if the dimensions of the first shape are defined, the term approximate may refer to the object and the first shape being superimposable (in 2D or 3D, respectively) such that an intersection between the object and the first shape covers at least n% of the object and at least n% of the shape, wherein n is at least 90%, such as at least 95%, especially at least 98%, such as at least 99%, including 100%.

[0044] The annular shape may especially extend (towards a central axis of the top partition) from the top partition wall. The annulus shape of the top partition bottom end may define the plateau. In further embodiments, the plateau may extend over at least part of the top partition wall in a direction towards a central axis of the top partition. Hence in embodiments, the plateau, terrace or ledge may extend radially along 360° of the top partition wall. Yet, in other embodiments, the plateau may extend radially along less than 360° of the top partition wall, such as for instance along no more than 270° of the top partition wall, especially along no more than 180° of the top partition wall. Moreover, the plateau may extend a first distance from the top partition wall at a first location. The plateau may further extend (over) a further distance from the top partition wall at a further location. In embodiments, the first distance and the further distance may be equal in size. Yet in further embodiments, the first distance may be smaller or larger than the further distance. The first distance may in embodiments be zero. The first distance may further especially be no more than 0.5 * Dtop, such as 0.3*Dtopat maximum, especially 0.2*Dtopat maximum. Likewise, the further distance may in embodiments be zero and further especially be no more than 0.5 * Dtop, such as 0.3*Dtopat maximum, especially 0.2*Dtopat maximum. Hence, in embodiments, 0< the first distance < 0.3*Dtop. In further embodiments, 0< the (especially any) further distance < 0.3*Dtop. Especially at least one of the first distance and the further distance (or any further distance) is non-zero.

[0045] In embodiments, the top partition may approximate a cylindrical shape. In specific embodiments, the top partition may approximate a plurality of stacked cylindrically shaped sections (see further below). In further embodiments, the bottom partition may (also) approximate a cylindrical shape.

[0046] The (circumferential) top partition wall may in embodiments extend from this peripheral part of the bottom end of the top partition (especially from an edge of the peripheral part of the top partition bottom end). The peripheral part of the top partition bottom end may therefore define the plateau. The plateau may in embodiments define a fluid supply and / or fluid remove location configured for receiving or supporting a fluid supply device or a fluid extraction device. Herein, the term “fluid transport device” may be used for a fluid supply device and / or for a fluid extraction device. The fluid transport device may for instance comprise a tube, or (a tip of) a pipette or a needle, an automated fluid dispenser, or a customized fluid outlet (or inlet). For example, the plateau may be configured to support a tip of a (manual or automated) pipette during a supply of fluid to the reservoir (and / or during removal of fluid from the reservoir). This way the fluid may enter (or exit) the channel gradually via the top partition and a smooth flow of the fluid may be transported through the channel along the test chamber, especially without perturbing matter in the test chamber. Moreover, this way fluid may be removed from the top partition while maintaining fluid in the bottom partition and in the channel.

[0047] As indicated above, the (at least one of the) reservoirs may in embodiments be defined by a reservoir wall, especially by an inner surface of the reservoir wall, especially in combination with the bottom end (of the reservoir). In specific embodiments, the reservoir wall may comprise a discrete step (“ledge” or ridge”). The discrete step may especially be configured at the bottom partition top end (or at the top partition bottom end). The discrete step may define the plateau. In further specific embodiments, the reservoir wall may comprise more than one discrete step and / or a plurality of ledges. The top partition may in embodiments, e.g., have a first top equivalent circular diameter defined at the top end of the reservoir and at least one further top equivalent circular diameter defined at (at least) a location between the top end of the reservoir and the top partition bottom end. The term “top equivalent circular diameter” may in embodiments refer to a plurality of (different) top equivalent circular diameters. In specific embodiments, the top equivalent circular diameter may increase (especially step by step) in a direction from the top partition bottom end to the top end of the reservoir. A top partition comprising a plurality of different equivalent circular diameters may facilitate estimating a total amount of fluid being present in the test arrangement. The reservoir, especially the top partition, may in embodiments comprise a plurality of plateaus. Especially, one of the plateaus is configured at the top partition bottom end. For calculating the reservoir ratio (Dtop / Dbot), the top equivalent circular diameter at the top partition bottom end (and especially the bottom equivalent circular diameter at the bottom partition top end) may especially be used. The term “plateau” may herein also refer to a plurality of plateaus. In particular, in embodiments, the top partition may comprise a plurality of (spatially separated) plateaus.

[0048] The plateau may in embodiments be configured parallel to the virtual bottom plane. In further embodiments, the plateau may be configured at an angle with the virtual bottom plane. The angle between the plateau and the virtual bottom plane (or: “plateau angle”) may in embodiments be selected from the range of 0-30°, such as selected from the range of 0- 15°, especially from the range of 0-5°, including (essentially) 0°. The plateau, especially the plateau angle, may in embodiments be configured to taper in a direction towards the top end of the reservoir. In further embodiments, the plateau may comprise a rim extending from the plateau in the direction of the top end of the reservoir. The rim may in embodiments be configured over a segment of the plateau. The rim may be configured to allow a fluid to flow along the rim (to or from the bottom partition) while minimizing turbulence in the flowing fluid and in the fluid channel. The rim may facilitate removing the fluid while minimizing perturbation of the system The plateau is especially a continuous plateau, especially configured radially over / along at least 90° (especially at least 180°) of the reservoir wall, and in embodiments over / along 360° of the reservoir wall.

[0049] The top partition and the bottom partition are not necessarily cylindrically shaped. Both partitions may essentially comprise any arbitrary shape. A cross-sectional area of the top partition and / or the bottom partition may in embodiments for instance define a triangle or a rectangle (including a square). In specific embodiments, the cross-sectional area of the top partition and / or the bottom partition may approximate a circle or an ellipse. The cross-sectional area of the top partition and of the bottom partition may in further specific embodiments be similar. The cross-sectional areas of the top partition and the bottom partition may especially be circular. The top partition and the bottom partition may have a cylindrical shape. The (circular) cross-sectional areas of the top partition and the bottom partition may vary (e.g., gradually or step-wise) along the top partition wall and / or along the bottom partition wall. In further embodiments, the top partition and the bottom partition may have a (slight) conical shape. The top partition may comprise a (central) top partition axis perpendicular to the virtual bottom plane (or to the virtual top plane) and especially intersecting a center of the cross- sectional area of the top partition. Likewise, the bottom partition may comprise a (central) bottom partition axis perpendicular to virtual bottom plane and especially intersecting a center of the cross-sectional area of the bottom partition. In specific embodiments, the top partition axis and the bottom partition axis are configured aligned. Hence in embodiments, (at least part of) the top partition may define a first (conical) cylinder, and the bottom partition may define a second (conical) cylinder, and the plateau may bridge a space between the first (conical) cylinder and the second (conical) cylinder. In further specific embodiments, the top partition axis and the bottom partition axis may be arranged parallel to each other, and especially at a distance from each other.

[0050] In further embodiments, a height of the top partition may be larger than the bottom partition height (at least) for the at least one of the reservoirs. A ratio of the top partition height Htp to the bottom partition height HbPmay especially be larger than 1, such as equal to or larger than 1.1 or equal to or larger than 1.25, especially equal to or larger than 2, and in embodiments equal or larger than 5. The ratio Htp / Hbp may in further embodiments be 25 at maximum, such as 10 at maximum, and in specific embodiments 5 at maximum. A high ratio Htp / Hbp may in embodiments allow to host a large fluid volume in the top partition relative to the bottom partition. Such large volumes may in embodiments prevent a loss of fluid from the channel (and the chamber) due to evaporation. A large ratio Htp / Hbp may in embodiments facilitate buffering of (an additional volume of) the fluid in the top partition, allowing for, e.g., long term culturing experiments in the device. In specific embodiments, for each of the reservoirs applies that a shortest distance between the top end and the bottom partition defines a top partition height (Htp), and (at least) for the at least one of the reservoirs applies that a reservoir height ratio Htp / Hbp of the top partition height (Htp) to the bottom partition height (HbP) is larger than 1, especially larger than 1.25, such as selected from the range of 1.25 to 10.

[0051] Herein, the term “test arrangement” may refer to a plurality of test arrangements. The test arrangements may in embodiments all be the same. Yet, in further embodiments at least one of the test arrangements may have a configuration that differs from other test arrangements of the device. The test arrangement(s) is (are) especially configured in the device, especially between the topside and the bottom side of the device.

[0052] The term “reservoir” may refer in embodiments to a single reservoir, especially to the “at least one of the reservoirs” of a test arrangement. The term may in further embodiments refer to both reservoirs of a test arrangement. In yet further embodiment, the term may refer to a plurality of (optionally different) reservoirs of a plurality of test arrangements. The term “reservoir” may thus in embodiments refer to a plurality of (different) reservoirs. In embodiments, for instance at least one of the reservoirs may differ from other reservoirs of a plurality of test arrangements. Moreover, also elements comprised by a first reservoir may have another configuration than a comparable element comprised by another reservoir. For instance, the bottom partition or the top partition of the first reservoir and of the other reservoir, respectively, may be configured differently. Moreover, in specific embodiments, a first reservoir (the at least one of the reservoirs) may comprise the plateau, whereas the equivalent circular diameters of the top partition (especially at the top partition bottom end) and of the bottom partition (especially at the bottom partition top end) of another reservoir may have the same value (wherein the other reservoir lacks a plateau). One of the reservoirs (especially not the at least one of reservoirs) may in embodiments lack the plateau. Moreover, in such embodiments, the top partition equivalent circular diameter and the bottom partition equivalent diameter may have (approximately) the same value. The top partition may especially be distinguished from the bottom partition. In embodiments, the bottom end of the top partition may be configured aligned with the top channel wall, and especially the top end of the bottom partition may (also) be configured aligned with the top channel wall. In further embodiments, the plateau may be part of the top partition, and especially the plateau may also define or (contact) the top end of the bottom partition. In further embodiments, the plateau and the top channel wall are aligned.

[0053] As indicated above, the test chamber may be configured for culturing cells. The test chamber may in embodiments also be indicated as “culturing chamber”. Herein, also the term “chamber” may be used to refer to the test chamber. The test chamber may be defined by a (circumferential) chamber wall, especially by an inner chamber surface of the (circumferential) chamber wall, in combination with a chamber bottom end, especially an inner chamber surface of the chamber bottom end. The test chamber may further comprise a chamber top end. The chamber top end may especially provide the open fluidic connection to the fluid channel. The chamber top end may be aligned with the bottom channel wall. The chamber top end may especially comprise an open chamber top end. Hence, the bottom channel wall may especially comprise an opening at a location of the chamber. The test chamber may further especially have a chamber height. In embodiments, a shortest distance between the chamber top end and the chamber bottom end defines the chamber height. In further embodiments, the chamber height is configured in a first direction. The first direction may in embodiments be configured perpendicular to the virtual bottom plane. The first direction may in further specific embodiments be configured perpendicular to the fluid channel axis.

[0054] The chamber may in further embodiments especially have a chamber height in the range of 0.1 to 2 times the channel height. The chamber height may in specific embodiments be equal to or smaller than the channel height, such as selected to be equal to or smaller than 0.95 * Hchan. In embodiments, the chamber height is at least 0.5 times the channel height. For instance, in embodiments, the channel height may be 0.8 mm, and the chamber height may be in the range of 0.4 to 0.7 mm. Having the channel height smaller than the chamber height may facilitate maintaining the biological material in the chamber. Depending on a size or volume of the biological material (e.g., tissue) relative to a channel cross sectional area, the biological material may not be able to move from the chamber to the fluid channel. Yet, in embodiments, it may also be advantageous to facilitate removing the biological material from the chamber (to the channel). Hence, in embodiments, the channel height may be larger than the chamber height. In further embodiments, the chamber height may be larger than the channel height. In embodiments, HCham / HChan is selected from the range of 0.1-10, especially from the range of 0.2- 5, such as from the range of 0.5-2. A cross-sectional area (perpendicular to the first direction or parallel to the virtual bottom plane) of the chamber may, in embodiments, be circular. In further embodiments, the cross-sectional chamber area of the chamber may be rectangular or e.g., elliptical. Essentially, the cross-sectional area of the chamber may have any arbitrary shape. The cross-sectional area of the chamber may in specific embodiments be circular. The chamber may in embodiments have a (conical) cylinder shape. Especially, in embodiments, the chamber may have a flat bottom surface. In particular, in embodiments, the chamber may have a planar bottom surface. The cross-section of the test chamber may especially have a (maximum) chamber equivalent circular diameter (DCham). The (maximum) chamber equivalent circular diameter (DCham) may especially be configured to agree with the channel width WChan. In embodiments, DCham may be approximately equal to WChan. In further embodiments, Wchan>DCham. Moreover, in embodiments a (maximal) width, WCham, of the chamber defined perpendicular to the channel axis may especially be equal to or smaller than the channel width. This may especially allow good contact between fluid in the channel and fluid in the chamber. Yet, in alternative embodiments, WChan<DCham. This may provide a relatively large chamber (relative to the channel). The chamber is especially configured centered with respect to the channel. In embodiments wherein WCham=WChan, the chamber wall may contact both channel side walls. In (circular) embodiments DCham=WCham. Especially a virtual line through centers of cross-sections of the chamber may intersect the fluid channel axis (through centers of crosssections of the fluid channel).

[0055] Hence, in specific embodiments, the fluid channel comprises side channel walls connecting the top channel wall to the bottom channel wall, wherein a shortest distance between the side channel walls defines a channel width (WChan), wherein a cross-section of the test chamber in a plane parallel to virtual bottom plane comprises a (maximum) chamber equivalent circular diameter (DCham), wherein the chamber equivalent circular diameter (DCham) is equal to or smaller than the channel width (WChan).

[0056] In further specific embodiments, the test chamber comprises a chamber top end and a chamber bottom end, wherein a shortest distance between the chamber top end and the chamber bottom end defines a chamber height (HCham); wherein a chamber ratio HCham / HChan of the chamber height (HCham) relative to the channel height (HChan) is equal to or smaller than 1, such as selected from the range of 0.1-1, especially from the range of 0.5-0.95.

[0057] The microfluidic device may especially be used to study multiple processes in a parallel session. As indicated above, the device may comprise a plurality of test arrangements. The device may e.g., comprise at least two test arrangements or at least four test arrangements. In further embodiments, the device may comprise at least 16 test arrangements, or at least 32 test arrangements, especially at least 64 test arrangements. In embodiments, all test arrangements may be identical. This way, for instance different operating conditions may be varied between the various test arrangements, while keeping the structure of the test arrangement constant. Alternatively, at least one of the test arrangements may comprise a configuration that differs from at least one other test arrangement of the plurality of test arrangements.

[0058] In embodiments, all test arrangements may be used individually. The test arrangements may (functionally) be separated from each other, in embodiments. Alternatively, at least two, especially neighboring, test arrangements may be configured fluidically connected to each other. In embodiments, the device may comprise a connection channel fluidically connecting a first test arrangement of the plurality of test arrangements to a second test arrangement of the plurality of test arrangements. The connection channel may in embodiments especially be configured for connecting the fluid channel of the first test arrangement with the fluid channel of the second test arrangement. The fluid channels may especially be connected at locations of the respective fluid channels between their two reservoirs . In specific embodiments the connection channel may be connected to the fluid channels at a location of (especially above) the respective test chambers. In particular, a cross-section perpendicular to the axis of elongation of one of the (respective) fluid channels (see above) may intersect the connection channel and the (respective) test chambers. This may advantageously be used to provide tissue-tissue communication between tissues cultured in the chambers of the respective test arrangements. This may further allow multi-tissue assessment to compounds provided to the tissues.

[0059] Hence, in embodiments, the device comprises a plurality of test arrangements, wherein the device further comprises a connection channel fluidically connecting a first test arrangement of the plurality of test arrangements to a second test arrangement of the plurality of test arrangements, wherein the connection channel is configured for connecting the fluid channel of the first test arrangement at a location between the two reservoirs (especially at a location of (or “above”) the test chamber) of the first test arrangement with the fluid channel of the second test arrangement at a location between the two reservoirs (especially at a location of (or above) the test chamber) of the second test arrangement.

[0060] The connection channel may further comprise a bottom wall (a wall configured closest to the device bottom side), a top wall (closest to the device top side) and a connection channel height Hccdefined as a (shortest) distance between the bottom wall of the connection channel and the top wall of the connection channel. In specific embodiments, the bottom wall of the connection channel is configured at a location between the bottom walls and the top walls of the respective fluid channels. Furthermore, the top wall of the connection channel may especially be aligned with the top walls of the (respective) fluid channels. Further, the height (Hcc) of the connection channel may especially be smaller than any one of the respective channel heights (HChan). For instance, in embodiments the channel heights of both fluid channel may be around 1 mm or around 0.7 mm, wherein the connection channel height is about 0.6, or 0.3 mm, respectively.

[0061] The term “connection channel” may especially refer to a plurality of connection channels. Moreover, in embodiments a first subset of the plurality of test arrangements may comprise a number of first test arrangements and a second subset of the plurality of test arrangements may comprise a number of second test arrangements, and especially any first test arrangement is connected to an individual one of the second test arrangements via an individual connection channel. The first subset especially does not overlap with the second subset.

[0062] As indicated above, the device may be used to study biological material. In a further aspect, the invention provides a method. The method may in embodiments be configured for analyzing biological material (including analyzing biological cells or for instance tissue cultured from biological cells). The device may be applied in the method of the invention. The method may further especially be configured for analyzing biological material (including cell material originating from a biological cell, such as cell aggregates, tissue, etc.) using a test arrangement of the device described herein. The term “a test arrangement” may in embodiments relate to a plurality of test arrangements. In embodiments, all test arrangements of the device may be used, In further embodiments a subset of the test arrangements of the device may be used. Hence, in embodiments a subset of the test arrangements of the device are used. The term “test arrangement” may in embodiments refer to a “subset of the test arrangements” (of the device). The subset may comprise any arbitrary number of test arrangements configured in the device (such as one, two, 50% of the test arrangements of the device, or e.g., all of the test arrangements of the device, or all but one or more of the test arrangements of the device).

[0063] In embodiments, the method may comprise a preparation stage. In specific embodiments, the preparation stage may comprise providing a first fluid to the channel of the test arrangement. The first fluid may especially comprise the biological material or a precursor for the biological material. In further embodiments, the method may (further) comprise a chamber filling stage. The chamber filling stage may especially comprise providing the first fluid (comprising the (precursor for the) biological material) (from the fluid channel) into the test chamber (of the test arrangement). The chamber filling stage may comprise replacing a gas in the test chamber with the first fluid. The chamber filling stage may in specific embodiments comprise centrifuging the device to provide the first fluid into the test chamber of the test arrangement. Centrifuging the device may especially comprise configuring the device in a centrifuge, wherein the device top side is configured closer to a center of the centrifuge than the device bottom side (at least during centrifuging). Centrifuging the device may comprise moving the fluid in the test chamber based on centripetal force. In further embodiments, the first fluid may be removed from the channel (after providing the first fluid into the test chamber, especially after centrifuging the device). In specific embodiments, the chamber filling stage may comprise removing the first fluid from the channel of the (first) test arrangement (especially after centrifuging the device). In embodiments, the method may (also) comprise an analysis stage. The analysis stage may especially comprise analyzing the biological cell material in the test chamber of the (first) test arrangement.

[0064] Accordingly, the invention provides in embodiments a method for analyzing a biological material, especially originating from a biological cell, using a test arrangement (especially one or more test arrangements) of the (microfluidic) device of the invention, wherein the method comprises a preparation stage; a chamber filling stage; and an analysis stage; wherein: (A) the preparation stage comprises providing a first fluid to the channel of the test arrangement(s) wherein the first fluid comprises the biological material or a precursor for the biological material; (B) the chamber filling stage comprises: (i) centrifuging the device to provide the first fluid into the test chamber(s) of the test arrangement(s), and (successively) (ii) removing the first fluid from the fluid channel of the test arrangement s); (C) the analysis stage comprises analyzing the biological material in the test chamber(s) of the test arrangement(s).

[0065] The first fluid may in embodiments be provided to the channel using a fluid transport device, such as described herein. The first fluid is especially (directly) supplied in the bottom partition of the test arrangement. Likewise, the first fluid may especially be removed from the fluid channel by removing the first fluid from the bottom partition of one of the reservoirs. The fluid transport device may e.g., comprise a pipette tip, and especially the pipette tip may be configured in the bottom partition (of one of the reservoirs) during providing the first fluid to the channel and / or during removing the first fluid from the fluid channel. This way in embodiments substantially only the fluid channel (and the bottom partitions) may be filled during providing the first fluid to the channel. Moreover this way in embodiments substantially the complete fluid channel (and the bottom partitions) may be emptied during removing the first fluid from the fluid channel.

[0066] In further embodiments, the method may comprise a pre-preparation stage, especially configured prior to the preparation stage. The pre-preparation stage may comprise pre-treating the device. In embodiments, e.g., a coating may be provided to (walls of) the channel and / or (walls of) the chamber of the device. Additionally, or alternatively, a coating may be provided to the wall of the reservoir(s). Hence, in embodiments, the pre-preparation stage may comprise providing a pre-treatment fluid in one of the reservoirs (especially in the at least one of the reservoirs) of the test arrangement, and especially flushing the test arrangement with the pre-treatment fluid. The pretreatment fluid may especially comprise a coating material. In further embodiments, the pre-treatment fluid may comprise a disinfectant. The term pre-treatment fluid may refer to a plurality of different pre-treatment fluids. For instance, in embodiments first a coating comprising fluid may be provided to the test arrangement, and subsequently a further fluid may be provided to the reservoir to displace the coating comprising fluid. In embodiments, the pre-preparation stage may further comprise centrifuging the device to provide the pre-treatment fluid in the chamber.

[0067] In further specific embodiments, the preparation stage may comprise providing the first fluid in one of the reservoirs (especially in the at least one of the reservoirs) of the test arrangement, especially wherein the first fluid is provided in the bottom partition of the reservoir and in the channel.

[0068] The term “biological material” especially refers to material relating to any form of life. The term may e.g., refer to a biological cell, a cluster or an aggregate of biological cells, cell tissue, (cell) material originating from cell proliferation, tumor cells, etc. The term especially refers to material comprising biological cells. The term “precursor of the biological material” may especially indicate that biological material initially provided in the chamber may alter (such as based on culturing, based on proliferation, based on an impact of a drug, stress, and / or any kind of therapy described herein) in time (during carrying out the method) and especially the changes (to the biological material) may be analyzed / monitored. The biological material analyzed may originate from biological material initially provided in the chamber. The term “cell” herein especially refers to a biological cell. A cell is the basic structural and functional unit of any forms of life. The cell may comprise a eukaryotic cell. The cell may comprise a prokaryotic cell. The term “cell” may in embodiments refer to a single cell, The term “cell” may in further embodiments refer to a plurality of (different) (types of) biological cells. The biological material may in embodiments comprise a plurality of (different) (types of) biological cells or material originating from a plurality of (different) (types of) biological cells. Moreover, in embodiments (a precursor of) a first type of biological material may be provided into a first test chamber, and (a precursor of) another type of biological material may be provided to a further test chamber. The term “biological material” may further refer to cultured cells, aggregates of cells, or, e.g., tissue. The cell (or precursor of the biological material) may in embodiments comprise a stem cell, e.g., a primary stem cell, an immortalized stem cell, an induced pluripotent stem cell (iPSC). The (stem) cell may especially relate to an animal cell, especially a mammalian cell, including a human cell. Moreover, the device may be used to investigate one or more of a bacterial infection, a fungal infection and a viral infection. The term cell may therefore also refer to a plurality of (different) cells, such as a mammalian cell, e.g., a human cell, and one or more of a bacterial cell or a fungal cell, such as a yeast cell. The cell may in embodiments comprise a primary cell, for instance comprising a chondrocyte, a fibroblast, a cardiomyocyte, or a dorsal root ganglia cell. The term “cell” may especially refer to any arbitrary kind or type of biological cell, especially that may be grown and / or cultured. Hence, the term biological material (such as in analyzing the biological material) may further refer to (analyzing) biological material originating from biological material initially provided to the chamber. Hence, analyzing the biological material (in the chamber) may in embodiments refer to analyzing, especially monitoring, changes in the biological material (in the chamber).

[0069] In embodiments, the biological cell may comprise a microbial cell. The biological cell may, for instance, comprise one or more of a prokaryotic cell and an archaeal cell. Hence, the biological cell may comprise a bacterial cell. In further embodiments, the biological cell may comprise a fungal cell or an algal cell, especially a fungal cell, or especially an algal cell.

[0070] In further embodiments, the biological cell may comprise a plant cell.

[0071] In embodiments, the biological material may comprise a virus particle, especially a virion.

[0072] After providing the first fluid to the channel, the fluid may in embodiments not enter the chamber as a result of the dimensions of the chamber and the physical properties (e.g., surface tension and / or viscosity) of the fluid. As indicated above, the chamber filling stage may therefore comprise forcing the first fluid into the chamber. The first fluid may especially be forced into the chamber by centrifuging the device. In embodiments, after filling the chamber, the reservoirs and / or the fluid channel may be flushed, especially to remove any remaining cells from the reservoirs and / or from the fluid channel. Flushing may especially be configured such that cells (present in the test chamber) are maintained in the test chamber. Hence, in embodiments, the chamber filling stage may further comprise flushing the reservoirs and the fluid channel of the test arrangement after providing the first fluid into the test chamber, especially after centrifuging the device. The chamber filling stage may in further embodiments comprise removing any of the cells (present) from the fluid channel and from the reservoirs (of the test arrangement(s)).

[0073] Flushing the reservoirs and / or the fluid channel may comprise providing a flushing fluid to the channel of the test arrangement and successively removing the flushing fluid from the channel. During flushing, the fluid transport device may in embodiments (also) be configured in the bottom partition of the reservoir(s) in a way as is described in relation to providing the first fluid to the channel and removing the first fluid from the channel. Flushing may in embodiments comprise providing the flushing fluid to the channel via a first one of the reservoirs and removing the flushing fluid from the channel via the other one of the reservoirs (of the test arrangement(s)).

[0074] Using the first fluid, especially one or more biological cells may be seeded in the test chamber. In further embodiments, after seeding the biological cell (and optionally flushing the reservoir and / or channel), a further fluid may be provided. The further fluid may in embodiments for instance comprise a culturing medium for the cell. The further fluid may in further embodiments comprise a drug (or “active compound”), especially to test a reaction of biological material originating from the cell to the drug. Many drugs may be tested this way. The term “drug” may especially refer to a small molecule. The term may refer to a biologic. In further embodiments, the term “drug” may refer to ribonucleic acid (RNA), such as a messenger RNA (mRNA), or small interfering RNA (SiRNA). The term “drug” may further relate to providing cell therapy. The drug may refer to a T cell. In embodiments, the drug may comprise cytokines or a chemical agent, e.g., to trigger an inflammatory state. The drug may comprise a chemical drug, e.g., a chemical solved in the further fluid. The drug may comprise a biological drug, such as comprising (inflammatory) cytokines, a chemical product, or a biological product. The drug may be a candidate drug. The further fluid may in further embodiments comprise a hydrogel.

[0075] The further fluid may in yet further embodiments comprise a solid material (e.g., one or more metal particles, or comprising a nano plastic). This way, in embodiments, for instance a (possible inflammatory) reaction of the solid material on the cultured cell, e.g., tissue (such as lung tissue) may be studied. In general, any prosthesis inserted in the body may release microparticles. In the knee joint, they may release titanium which may impact cellular survival as well as that it may trigger a potential immune response. Yet another example to be studied may relate to the breast implant. A breast implant may release a lot of microparticles that may then generate fibrotic tissues. The microfluidic device may allow for studying such reactions. The further fluid may essentially comprise any compound for studying the effect of the compound on the biological cell. In embodiments, for instance, the cell may comprise a cancer cell (cancer cell material or tumor), and the further fluid may comprise a chemical agent to study the effect of the chemical agent on the tumor. For instance, the cell may comprise a liver cancer cell (e.g., an“HepG2” cell or an “HBG” cell) or a breast cancer cell (e.g. an “MDA- MDB 231” cell or a “BT-20” cell) and effects of a cell therapy may be studied. Moreover, in embodiments the cell may comprise a combination of different (types) of cells.

[0076] Hence, in embodiments, the analysis stage may comprise providing a further fluid to the channel of the test arrangement(s) via one of the reservoirs (especially via the at least one of the reservoirs), wherein the further fluid comprises one or more of a culturing medium for the cells and a drug for the cells. In further embodiments, the further fluid may (at least partly) be extracted from the test arrangement via one of the reservoirs (especially via the at least one of the reservoirs).

[0077] The further fluid may especially be provided via the top partition of the reservoir. Likewise, the further fluid may in further embodiments be extracted via the top partition of the reservoir. This may in embodiments allow to completely fill the fluid channel with the further fluid (especially while maintaining the cell in the test chamber). This may in further embodiments (also) allow to extract only part of the further fluid from the test arrangement, while maintaining remaining further fluid in the fluid channel. This may further prevent disturbance of the (process going on in) the test chamber.

[0078] Hence, in embodiments, the further fluid may be provided via one of the reservoirs having a reservoir ratio DtOp / Dbot being larger than 1, especially wherein the further fluid is provided in the top partition of said reservoir, especially (directly) at the plateau (defined at a bottom end of the top partition). In yet further embodiments, the further fluid is extracted from the test arrangement via one of the reservoirs (100a, 110b) having a reservoir ratio Dtop / Dbot being larger than 1, especially wherein the further fluid is extracted in the top partition of said reservoir, especially (directly) at the plateau (defined at a bottom end of the top partition).

[0079] The further fluid may in embodiments be provided to the channel using a fluid transport device, such as described herein. The fluid transport device may e.g., comprise a pipette tip, and especially the pipette tip may be configured at the bottom end of the top partition. The (tip of the) fluid transport device may especially be arranged at the plateau defined at a bottom end of the top partition (see above), (of one of the reservoirs) during providing the further fluid to the channel and / or during removing the further fluid from the test arrangement. This may minimize perturbation of the process and / or the biological material in the test chamber. In further embodiments, (a tip of) the fluid transport device may be configured in the bottom partition, especially during removing the further fluid from the test arrangement. This may allow to also extract the further fluid from the channel.

[0080] The term further fluid may refer to a plurality of different (or the same) further fluids. Hence in embodiments initially a first further fluid may be provided to the fluid channel (and optionally (at least partly) be extracted from the test arrangement), and successively a second (or third, etc.) further fluid may be provided to the channel (and optionally extracted from the test arrangement), etc. The (further) fluid extracted from the test arrangement may in embodiments be optionally analyzed.

[0081] Extracting a fluid (such as the first fluid, the flushing fluid, and the further fluid) from the channel may especially comprise maintaining the cells in the test chamber.

[0082] In embodiments, the analysis stage may (thus) comprise repeatedly (i) providing the further fluid to the channel of the (first) test arrangement, and (ii) extracting (at least part of) the further fluid from the test arrangement and optionally analyzing the further fluid extracted from the test arrangement. In embodiments, various further fluids may be used. For instance initially the further fluid may comprise a culturing medium for the cell, whereas after a period of time, the further fluid is changed into a further fluid comprising a drug for the cell.

[0083] To study effects of (elements in) the fluid, it may be advantageous to allow visual observation of processes taking place in the device, or for example to count distinct cells provided in the test chamber. At least the test chamber may be configured to study the biological cell, especially an aggregate, using a microscope. Therefore, in embodiments, the device is at least partly transparent. Especially, the test chamber may be configured transparent. The device may especially comprise a transparent device material. In embodiments, the device material may comprise a polymeric material. The device material may comprise a plastic. The device material may in embodiments comprise (especially be) any plastic transparent material. The device material may comprise any material, especially transparent material, that may be used in biological applications, known to the skilled person. The device material may be biologically compatible, especially biologically inert. The device material may especially be selected for not being toxic (to the cell material). The device material may in embodiments comprise glass. The device material may further for instance comprise a polymeric material selected from the group consisting of polyethylene (PE), polypropylene (PP), polydimethylsiloxane (PDMS), and an epoxy-based negative photoresist such as SU8 polymer.

[0084] In further embodiments, the device may be configured for optical inspection of the test chamber from the device top side, e.g., by a microscope. In embodiments, the device may be devoid of moving (device) parts above the test chamber. In particular, the test chamber may define an optical axis, especially centrally arranged in the test chamber, wherein the optical axis passes (only) through the device bottom side, the fluid channel, and the device top side. Accordingly, in embodiments, the device may comprise a transparent device material configured to provide optical access to (all of) the test chamber, especially from the top device side.

[0085] In further embodiments, the device may be configured to be compatible with confocal imaging. In particular, the device may be configured to facilitate confocal imaging of the test chamber.

[0086] It may further be advantageous to control interactions between the device and the biological cell (material). For instance, it may be desired to prevent sticking of cell material at a surface of the reservoirs, and / or a surface of the fluid channel. Additionally or alternatively, a surface of the chamber may repel cells. Hence, in further embodiments, a surface of one or more of (i) the reservoirs, (ii) the test chamber, and (iii) the channel comprises a cell-repellant surface.

[0087] BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig.lA and Fig. IB depict aspects of the microfluidic device; Fig. 2 depicts an embodiment of the microfluidic device; Fig. 3 A and Fig. 3B depict some further aspects of the device; and Fig. 4A to Fig 4F depict an embodiment of the method of the invention. The schematic drawings are not necessarily to scale.

[0089] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0090] Figs 1A to IB schematically depict a section of the microfluidic device 10 and an embodiment of one test arrangement 100. Fig. IB depicts a top view, and Fig. 1A a side view along cross-section A-A indicated in Fig. IB. The device 10 comprises a device top side 11 and a device bottom side 19, schematically indicated in Fig. 1A. The device 10 further comprises a virtual top plane 12 abutting the device top side 11 and a virtual bottom plane 18 abutting the device bottom side 19.

[0091] The device 10 may comprise in embodiments a single test arrangement 100 and in further embodiments a plurality of test arrangements 100. Especially, test arrangement 100 comprises two reservoirs 110a, 110b, a test chamber 150, and a fluid channel 190. Further, reservoir 100a and reservoir 100b may have different configurations, but especially each reservoir 110a, 110b comprises a bottom partition 118 comprising a bottom end 119 and a top partition 112 comprising a top end 111. The top end 11 lof the top partition 112, configured at the device top side 11, is open to location external of the device 10 via the opening 113. The opening 113 is especially configured in the device top side 11. Further, the top partition 112 depicted in the figure has a circular cross-section with a top (equivalent) diameter Dtop. Also the bottom partition 118 of the depicted embodiment is cylindrical and comprises a bottom (equivalent) diameter (Dbot). The diameters of the respective partition may define a reservoir ratio Dtop / Dbot of the reservoir 110. In the depicted embodiment, the reservoir ratio DtOp / Dbotis larger than 1 for both reservoirs. For other embodiments only one of the reservoirs 110a, 110b may have a reservoir ratio DtOp / Dbot larger than 1.

[0092] In Fig. 1A, further the bottom distance dbe of the reservoirs 110a, 110b is indicated. The bottom distance dbe is the distance between the bottom end 119 and the virtual bottom plane 18 abutting the device bottom side 19. The bottom distance dbe of the two reservoirs 110a and 110b may have different values in embodiments.

[0093] The fluid channel 190 comprises a top channel wall 191, a bottom channel wall 199, and (two) side channel walls 195 connecting the top channel wall 191 to the bottom channel wall 199. The top channel wall 191 is a closed wall, see Fig. 1A, whereas the test chamber 150 extends from the fluid channel 190 (at the bottom channel wall 199) in a direction of the device bottom side 19. In embodiments, the test chamber 150 is configured centered between the two reservoirs 110a, 110b (like in the depicted embodiment), especially in at least one test arrangement 100. The fluid channel 190 is bridging between the two reservoirs 110a, 110b. Therefore, the fluid channel 190 fluidically connects the two reservoirs 110a, 110b and the test chamber 150. In Fig. 1A, further the channel wall distance dwaii is indicated. The channel wall distance dWaii is defined between the bottom channel wall 199 and the virtual bottom plane 18. The channel wall distance dwaii may especially be configured such that 0.9<dbe / dWaii<l .1. In the depicted embodiment, for each reservoir 110a, 110b dbe / dWaii is about 1.

[0094] In Fig. 1 A, further the bottom partition height HbPand the channel length LChan are indicated. The bottom partition height HbPis defined as the distance between the top partition 112 and the bottom end 119, and especially perpendicular to the virtual bottom plane 18. In embodiments, HbP / HChan may also be approximately 1, such as selected from the range of 0.8 - 1.25.

[0095] The plateau in the reservoir is indicated with reference numeral 114. The plateau 114 is aligned with the top channel wall 191 and has an annulus shape. The figure further demonstrates that the wall of the reservoir 110 comprises a discrete step at a top end 1181 of the bottom partition 118 (or the bottom end 1129 of the top partition 112). The discrete step is defined by the plateau 114. The plateau 114 of the reservoir 110a depicted at the left hand side further comprises a small rim 1141 extending (upwards) from the plateau 114. The rim 1114 may further facilitate a reduction in perturbation when a fluid is provided or removed from the fluid channel 190 with, e.g. a pipette, wherein the pipette tip is arranged behind the rim 1141, especially between the rim 1141 and (the nearest part of) the reservoir wall (as is, e.g., depicted in Fig. 4E in an embodiment not comprising the rim 1141). It is noted that the plateau is configured as an annulus in the figure because the top partition 112 and the bottom partition are configured around the same central axis 117 of the reservoir 110. It will be understood that also other configurations of the top partition 112 and the bottom partition 118 are possible still resulting in the presence of a plateau 114.

[0096] Further, in the depicted embodiment, especially for the reservoir 110 used to supply or extract a fluid in during operating the device 10 (herein also indicated as “the at least one of the reservoirs” (from the arrangement 100)), it applies that the top partition 112 is larger than the bottom partition 118. The top partition 112 may have a larger (equivalent) diameter DtOp than the bottom partition 118; e.g., the reservoir ratio Dtop / Dbot may be at least 1.5 in embodiments. Additionally, or alternatively, the height Htp of the top partition 112 may be larger than the height HbPof the bottom partition 11. In the depicted embodiment, Dtop / Dbot is about 1.75 and Htp / Hbt is also about 1.75, for both reservoirs 110a, 110b.

[0097] Fig. IB, further schematically depicts that for the depicted embodiment, the channel width WChan is equal to the chamber equivalent circular diameter DCham. The channel width Wchan is especially defined as the shortest distance between the side channel walls 195. Further, the (maximum) chamber equivalent circular diameter DCham may be determined based on the (maximum) cross-section of the test chamber 150 in a (virtual) plane parallel to the bottom channel wall 199 (or parallel to the virtual bottom plane 18). In the depicted embodiment, the chamber 150 has a cylindrical shape. In further embodiments, the chamber 150 may have a conical shape, especially tapering in a direction towards the device bottom side 19. The chamber equivalent circular diameter DCham may therefore especially be on the cross- section of the test chamber 150 at a top end 151 of the chamber 150, especially a location aligned with the bottom channel wall 199. Likewise, the top partition 112 and / or the bottom partition 118 may in further embodiments have a conical shape, especially tapering in a direction towards the device bottom side 19. Moreover, in embodiments, the top partition 112 may comprise a plurality of plateaus 114 defining the conical shape. Therefore, the equivalent circular diameters Dtopand Dbotfor the reservoir ratio DtOp / Dbot are especially determined at the top partition bottom end 1129 and the bottom partition top end 1181, respectively.

[0098] The chamber 150 of depicted embodiment of Figs 1 further illustrates that the chamber 150 comprises a chamber ratio HCham / HChan that is equal to or smaller than 1 (in the depicted embodiment, this ratio is between 0.9 and 0.95). The chamber height Hcham is especially defined as a shortest distance between the chamber top end 151 and the chamber bottom end 159. Furthermore, Figs 1 also depict an embodiment of the device 10, wherein a channel ratio WChan / HChan is selected from the range of 0.5 to 2 (in the figure, this ratio is approximately 1).

[0099] Further, Fig. 1 A schematically depicts the test chamber 150 to have a flat bottom surface. In particular, the chamber bottom end 159 may have (or “define”) a flat bottom surface.

[0100] In specific embodiments, the device 10 is at least partly transparent. This may facilitate optically monitoring processes in the device 10, such as the presence of a fluid in the elements of the device 10, and the presence biological material 90 such as cells or tissue in the device 10, especially in the chamber 150. Moreover, the device 10 may comprise cell-repellent surfaces contacting the fluids. For that the device material may be cell-repellant, or e.g., the device material may have been surface treated with a cell-repellant material, e.g., with a surfactant comprising amphiphilic properties, such as a poloxamer, e.g., Pluronic 127. The device material or the surfactant may in embodiments be highly hydrophobic or ultra hydrophobic which may help reducing binding of the biological material 90, such as the cells 90,91. Especially the surface of one or more of the reservoirs 110a, 110b, the test chamber 150, and the channel 190 may comprise a cell-repellant surface. The cell-repellant surface especially be hydrophobic. The cell-repellant surface may further comprise a smooth surface.

[0101] In Fig. 2 an embodiment of the device 10 is depicted comprising a plurality ( in this case 56) of test arrangements 100 configured in a well plate 15 configuration. Each one of the test arrangements 100 in the embodiment may be individually used. As such, 56 experiments may be carried out in a parallel way at the same time. In further specific embodiments, see e.g., Figs 3, neighboring test arrangements 100 may be connected to each other. Herein, the term neighboring in relation to elements, such as in “neighboring test arrangements” may especially indicate that no further same elements are configured between the neighboring elements.

[0102] Figs. 3A and 3B depict an aspect of an embodiment of the device 10 comprising a connection channel 200 fluidically connecting a first test arrangement 100a (of the plurality of test arrangements 100) to a second test arrangement 100b (of the plurality of test arrangements 100). The connection channel 200 connects the fluid channel 190 of the first test arrangement 100a with the fluid channel 190 of the second test arrangement 100b. The connection is configured between the two reservoirs 110a, 110b of the respective test arrangements 100a, 100b. In the depicted embodiment, the fluid channels 190 are connected via the connection channel 200 at a location of (or “above”) the test chambers 150 of the test arrangements 100a, 100b. It is further depicted that the bottom wall 209 of the connection channel 200 is configured at a location between the bottom walls 199 and the top walls 191 of the respective fluid channels 190. The top wall 201 of the connection channel 200 is in the depicted embodiment aligned with the top walls 191 of the fluid channels 190. In further embodiments, the top wall 201 may also be configured at a location between the bottom walls 199 and the top walls 191 of the respective fluid channels 190. The connection channel height Hccis smaller than any one of the respective channel heights Hchan. Using such configuration allows to culture cells 90,91 in the two test arrangements 100a, 100b and to study communication between tissues 90,92 growing in the respective chambers 150.

[0103] Fig. 4A to Fig. 4F schematically depict the use of an embodiment of the device 10 in an embodiment of the method of the invention. In the figures a side view of a test arrangement 100 is depicted. The method of the invention may be described herein as a method for analyzing a biological material 90 using a test arrangement 100 of the device 10, and comprises a preparation stage; a chamber filling stage; and an analysis stage. The preparation stage is schematically indicated in Fig. 4A and Fig. 4B, depicting that the first fluid 50 comprising biological material 90 or a precursor for the biological material 90 is provided to the channel 190 of the test arrangement 100. Here, a pipette tip containing the first fluid 50 comprising a cell-medium (comprising, e.g., cells 90,91 or, e.g., cell aggregates 90,93) is placed in bottom partition 118 of reservoir 110a, that is fluidically connected to the entrance of fluid channel 190. The cell-medium is pushed outside the pipette tip, filling the channel 190. Due to the configuration, the chamber 150 may in embodiments (still) be filled with a pocket of air 70 as indicated in Fig. 4B.

[0104] Fig. 4C and Fig. 4D depict embodiments of the chamber filling stage comprising centrifuging the device 10 to provide the first fluid 50 into the test chamber 150 of the test arrangement 100 (the result of centrifuging is depicted in Fig. 4C), and removing the first fluid

[0105] 50 from the channel 190 of the test arrangement 100 (Fig. 4D). The device 10 may be centrifuged to allow the pocket of air 70 to be released while filling the chamber 150 with the first fluid 50. The concentration of cells 90,91 in the test chamber 150 may depend on a seeding concentration earlier established. In the analysis stage, the biological material 90, such as cells 90,91, cell aggregates, 93, proliferated cells 90,91, and cell tissue 90,92 in the test chamber 150 may be analyzed. Fig. 4D, schematically depicts that a pipette tip configured in the bottom partition 118 of reservoir 110a is used to withdraw the remaining first fluid 50 that is not in the test chamber 150.

[0106] In further embodiments (not depicted in Figs 4), the chamber filling stage may further comprise flushing the reservoirs 110 and the channel 190 of the test arrangement 100 after providing the first fluid 50 into the chamber 150. This may remove any of the biological material 90 (such as cells 90,91) from the channel 190 and from the reservoirs 110 (while maintaining the biological material 90 in the test chamber 150).

[0107] As indicated in the figures, in embodiments only one of the reservoirs 110a, 110b may be used to fill or remove fluid from the test arrangement 100. Herein, that one reservoir 110 is also indicated as the at least one of the reservoirs 110a, 110b of the test arrangement 100. In the depicted figures, the at least one of the reservoirs 110 of the test arrangement 100 is indicated with reference numeral 110a.

[0108] In the depicted embodiment, the first fluid 50 is provided in the bottom partitions 118 of the reservoir 110a to provide it in the bottom partitions 118 of both reservoirs 110a, 110b and in the channel 190. Furthermore, also (part) of the first fluid 50 is removed from the test arrangement 100 via the bottom partition 118 of the reservoir 110a. In embodiments, the first fluid 50 may be removed from the channel 190 this way, while maintaining the first fluid 50 in the chamber 150.

[0109] The analysis stage may further comprise (see Fig. 4E) providing a further fluid

[0110] 51 to the channel 190 of the test arrangement 100 via one of the reservoirs 110a, 110b, here via the at least one of the reservoirs 110a. The further fluid 51 may for instance (initially) comprise a culturing medium comprising nutrients 80 for the cells 90,91, to culture the cells 90,91. The further fluid 51 may further also be partly removed from the test arrangement 100, as is indicated in Fig. 4F and / or replaced by a second further fluid 51, such as comprising a drug for the biological material 90 originating from the cells 90,91 to study the effect of the drug on the biological material 90 in the chamber 150. The term further fluid 51 may thus refer to a plurality of further fluids 51, that may (sequentially) be provided to the fluid channel 190 in the analysis stage. The further fluid 51 may comprise a load 80, to study the effect of the load on the biological material 90 in the chamber 150. Examples of such load are inflammatory cytokines, a chemical solved in the further fluid 51, a biological product, a hydrogels, and a solid material (e.g., metal particles, nano plastics, etc.). The further fluid 51 may in embodiments correspond to the first fluid 50 without the cells 90,91 (or aggregates 90,93, etc.).

[0111] Fig. 4F schematically depicts the extraction of at least part of the further fluid 51 from the test arrangement 100 via the at least one of the reservoirs 110a (while maintaining the biological material 90 in the test chamber 150). In embodiments of the analysis stage the further fluid 51 extracted from the channel 190 is analyzed.

[0112] It is noted that in the depicted embodiments (especially in Fig. 4E), different embodiments of biological materials 90 are schematically indicated in the chamber 150, i.e. cells 91, cell aggregates 93, and tissue 92 for explanatory reasons. In embodiments one or more of these biological materials 90 may be present in the chamber. In embodiments, e.g. initially cells 91 may be provided in the chamber 150, and after proliferation, the chamber may comprise tissue 92.

[0113] Hence, in embodiments, the analysis stage comprises repeatedly providing the further fluid 51 (especially including different further fluids 51) to the fluid channel 190 of the test arrangement 100, extracting at least part of the further fluid 51 from the test arrangement 100, and optionally analyzing the further fluid 51 extracted from the test arrangement 100.

[0114] Hence, in embodiments fresh medium 51, not containing cells 90,91, may be provided to the treatment arrangement 100 using the pipette tip facing the plateau 114 in one or in both of the reservoirs 110a, 110b. The medium 51 may then be provided in the channel 190 without affecting the (number of) cells 90,91. This may allow cells 90,91 to be counted as well as allowing them to aggregate in the chamber 150 (see e.g., Fig. 4E). At a later stage, a pipette tip may be used to withdraw the medium 51 configuring the tip at the plateau 114 again. This may allow long term culture and removal of medium 51 for potential analysis. In further embodiments, the test arrangement 100 may be provided with additional loads (e.g., for cell therapies, and / or comprising small molecules and / or biologies).

[0115] The microfluidic device 10 described herein may also be called the “pMASS”. In embodiments, the device 10 may comprise a transparent plastic device 10 hosting a plurality of test chambers 150 or “micro-testers” 150. Experiments have been performed using a device 10 comprising 64 test chambers 150. The number of chambers 150 may in other embodiments be different, and may e.g., be based on a size of the chamber 150. Each tests arrangement 100 may comprise two side reservoirsl 10 connected with each other by a fluid channel 190. The reservoirs 110 may be used to access to the channel 190. The micro-tester 150 may be configured in the middle of the channel 190. In embodiments, the micro-tester 150 may comprise a circular test chamber 150. The circular test chamber 150 may especially have a diameter DCham corresponding to the channel width WChan. It is noted that the reservoirs 110, channel 190, and micro-tester 150 may have other dimensions in further embodiments. It is anticipated that a maximum size of the test chamber 150 and of the fluid channel 190 may be in the mm scale. For instance, in embodiments the channel height HChan may be in the range of a couple of millimeters. In further embodiments the channel height HChan may be in the sub millimeter range. Embodiments may have a channel height HChan in the range of 0.1 - 3 mm, especially in the range of 0.1- 0.9 mm. Also the equivalent circular diameter DCham of the chamber may have a value in such a range.

[0116] In specific embodiments, the device 10 may be configured according to typical guidelines of a 96-well plate commercially available. The device may e.g., have standard dimensions e.g.,. 127.8 * 85.5 cm), which may facilitate handling of the device. The device 10 may be configured to allow for multi-channel pipetting and for robot pipetting procedures. Hence, the device 10 may also be used for large assay assessments. The micro-tester 150 may have a conical configuration. Nonetheless, the micro-tester 150 may vary presenting multiple different geometrical configurations. The device 10 may embodiments comprise a (detachable) lid (for clarity not depicted in the figures) to cover the device top side 11 and protect against potential contamination.

[0117] The device 10 may in embodiments be produced using soft-lithography. The device 10 may in further embodiments (also) be produced using 3D printing. In specific embodiments, also other manufacturing processes currently available for plastic materials or glass, especially for biological applications (e.g., injection molding) may be used. The device 10 may especially be configured for being bio-compatible and gas permeable allowing for cell culture.

[0118] In embodiments, aggregates 90,93 may be cultured in the chamber 150. Furthermore, the method may in embodiments comprise adding therapies and studying cellular dynamics without perturbing the system. In specific embodiments, the first fluid 50 or further fluid 51 may comprise a hydrogel or other material that may be provided to the channel 190 and a vasculature channel may be mimicked. This may facilitate more accurately mimicking human physiological processes.

[0119] The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably. The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

[0120] The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’.

[0121] The term “and / or” especially relates to one or more of the items mentioned before and after “and / or”. For instance, a phrase “item 1 and / or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of' but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species". Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0122] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0123] The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

[0124] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0125] The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0126] The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. 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.

[0127] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0128] The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

[0129] The various aspects discussed in this patent document can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims

CLAIMS:

1. A microfluidic device (10) comprising a device top side (11) and a device bottom side (19), wherein the microfluidic device (10) comprises at least one test arrangement (100); wherein for each test arrangement applies that: the test arrangement (100) comprises two reservoirs (110a, 110b), a test chamber (150), and a fluid channel (190); wherein each reservoir (110a, 110b) comprises (i) a bottom end (119), (ii) a bottom partition (118) comprising the bottom end (119), (iii) a top end (111) configured at the device top side (11), and a (iv) top partition (112) comprising the top end (111), wherein the top end (111) comprises an opening (113), wherein the top partition (112) has a top equivalent circular diameter (Dtop) and the bottom partition (118) has a bottom equivalent circular diameter (Dbot), wherein the reservoirs (110a, 110b) have a reservoir ratio Dtop / Dbot of the top equivalent circular diameter (Dtop) to the bottom equivalent circular diameter (Dbot), wherein for at least one of the reservoirs (110a, 110b) the reservoir ratio Dtop / Dbotis larger than 1; the fluid channel (190) spans between the two reservoirs (110), wherein the test chamber (150) extends from the fluid channel (190) in a direction of the device bottom side (19), and wherein the fluid channel (190) fluidically connects the two reservoirs (110a, 110b) and the test chamber (150); and the fluid channel (190) comprises a top channel wall (191) and a bottom channel wall (199), wherein the top channel wall (191) is a closed wall.

2. The device (10) according to claim 1, wherein at least one of the reservoirs (110a, 110b) is defined by a reservoir wall, wherein the reservoir wall comprises a discrete step defining a plateau 114.

3. The device (10) according to any one of the preceding claims, wherein for each of the reservoirs (110a, 110b) applies that a distance between the top partition (112) and the bottom end (119) defines a bottom partition height (HbP), wherein a shortest distance between the top channel wall (191) and the bottom channel wall (199) defines a channel height (HChan), and wherein HbP / Hchan is selected from the range of 0.8 - 1.25.

4. The device (10) according to claim 3, wherein for the at least one of the reservoirs (110a, 110b) applies that the reservoir ratio DtOp / Dbot is selected from the range of 1.5-10.

5. The device (10) according to any one of the claims 3-4, wherein for each of the reservoirs (110a, 110b) applies that a shortest distance between the top end (111) and the bottom partition (118) defines a top partition height (Htp), wherein for the at least one of the reservoirs (110a, 110b) applies that a reservoir height ratio Htp / Hbp of the top partition height (Htp) to the bottom partition height (HbP) is larger than 1.25.

6. The device (10) according to any one of the preceding claims, wherein the fluid channel (190) comprises side channel walls (195) connecting the top channel wall (191) to the bottom channel wall (199), wherein a shortest distance between the side channel walls (195) defines a channel width (WChan), wherein a cross-section of the test chamber (150) has a chamber equivalent circular diameter (DCham), wherein the chamber equivalent circular diameter (DCham) is equal to or smaller than the channel width (WChan).

7. The device (10) according to claim 6, wherein the test chamber (150) comprises a chamber top end (151) and a chamber bottom end (159), wherein a shortest distance between the chamber top end (151) and the chamber bottom end (159) defines a chamber height (HCham);wherein a chamber ratio HCham / HChan of the chamber height (HCham) relative to the channel height (HChan) is selected from the range of 0.5-.95.

8. The device (10) according to any one of the claims 6-7, wherein a channel ratio Wchan / Hchan of the channel width (WChan) to the channel height (HChan) is selected from the range of 0.5 to 2, and wherein the channel width (WChan) and the channel height (HChan) are substantially constant along a channel length (LChan) of the fluid channel (190).

9. The device (10) according to any one of claims 3-8, wherein the channel height (Hchan) is selected from the range of 0.1 - 3 mm.

10. The device (10) according to any one of the preceding claims, wherein the test chamber (150) in the at least one test arrangement (100) is configured centered between the two reservoirs (110a, 110b).

11. The device (10) according to any one of the preceding claims, wherein the device (10) is at least partly transparent, wherein the test chamber (150) has a flat bottom surface.

12. The device (10) according to any one of the preceding claims, wherein a surface of one or more of (i) the reservoirs (110a, 110b), (ii) the test chamber (150), and (iii) the channel (190) comprises a cell-repellant surface.

13. The device (10) according to any one of the preceding claims, wherein the device (10) comprises a plurality of test arrangements (100), wherein the device (10) further comprises a connection channel (200) fluidically connecting a first test arrangement (100a) of the plurality of test arrangements (100) to a second test arrangement (100b) of the plurality of test arrangements (100), wherein the connection channel (200) is configured for connecting the fluid channel (190) of the first test arrangement (100a) at a location between the two reservoirs (110a, 110b) of the first test arrangement (100a) with the fluid channel (190) of the second test arrangement (100b) at a location between the two reservoirs (110a, 110b) of the second test arrangement (100b), and wherein a height (Hcc) of the connection channel (200) is smaller than any one of the respective channel heights (HChan).

14. The device (10) according to any one of the preceding claims, wherein each bottom end (119) is separated from the device bottom side (19) by a bottom distance (dbe), wherein the bottom distance (dbe) is defined between the bottom end (119) and a virtual bottom plane (18) abutting the device bottom side (19), and wherein the bottom channel wall (199) is separated from the device bottom side (19) by a wall distance (dwaii) defined between the bottom channel wall (199) and the virtual bottom plane (18); and wherein for each reservoir (110a, 110b) applies 0.9<dbe / dWaii<l. l15. A method for analyzing biological material (90) using a test arrangement (100) of the device (10) of any one of the preceding claims, wherein the method comprises a preparation stage; a chamber filling stage; and an analysis stage; wherein: the preparation stage comprises providing a first fluid (50) to the fluid channel (190) of the test arrangement (100) wherein the first fluid (50) comprises the biological material (90) or a precursor for the biological material (90);the chamber filling stage comprises: centrifuging the device (10) to provide the first fluid (50) into the test chamber (150) of the test arrangement (100), and removing the first fluid (50) from the fluid channel (190) of the test arrangement (100); the analysis stage comprises analyzing the biological material (90) in the test chamber (150) of the test arrangement (100).

16. The method according to claim 15, wherein the analysis stage comprises providing a further fluid (51) to the fluid channel (190) of the test arrangement(100) via one of the reservoirs (110a, 110b), wherein the further fluid (51) comprises one or more of a culturing medium for the biological material (90) and a drug for the biological material (90); and optionally extracting at least part of the further fluid (51) from the test arrangement (100) via one of the reservoirs (110), and optionally analyzing the fluid (51) extracted from the test arrangement (100).

17. The method according to claim 16, wherein the further fluid (51) is provided via one of the reservoirs (110a, 110b) having a reservoir ratio DtOp / Dbot being larger than 1, wherein the further fluid (51) is provided in the top partition (112) of said reservoir (110a, 110b); and wherein optionally the further fluid (51) is extracted from the test arrangement via one of the reservoirs (110a, 110b) having a reservoir ratio DtOp / Dbotbeing larger than 1, wherein the further fluid (51) is extracted in the top partition (112) of said reservoir (110a, 110b).

18. The method according to any one of claims 16-17, wherein the analysis stage comprises repeatedly (i) providing the further fluid (51) to the fluid channel (190) of the test arrangement (100), and (ii) extracting at least part of the further fluid (51) the test arrangement (100), and optionally analyzing the further fluid (51) extracted from the test arrangement (100).

19. The method according to any one of the claims 15-18, wherein the chamber filling stage comprises flushing the reservoirs (110) and the fluid channel (190) of the test arrangement (100) after centrifuging the device (10).

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