Device for isolating motile cells

Abstract

The present invention relates to a mesoscale fluidic device for separating motile from non-motile cells, the mesoscale fluidic device comprising a substrate having a sample application compartment below a media compartment and a cell permeable filter with a pore size in the range of 1 μm to 20 μm, the cell permeable filter having an upper surface facing the media compartment and a lower surface facing the sample application compartment, the sample application compartment and the media compartment being in fluid communication via the cell permeable filter, the sample application compartment having a floor and at least one flow structure extending from the floor towards the lower surface of the cell permeable filter facing the sample application compartment. The device is particularly useful for obtaining a medium enriched in motile sperm cells from a mammalian sperm sample.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a mesoscale fluidic device for separating motile cells from non-motile cells. The mesoscale fluidic device is useful for obtaining a sperm sample enriched in motile cells compared to non-motile cells. The present invention also relates to a method of extracting motile cells from non-motile cells, particularly using the mesoscale fluidic device. [Background technology]

[0002] background Over the past decades, there has been an increasing awareness of the decline in male fertility, which has created a demand for sperm sample analysis to improve the quality of sperm samples, especially since the concentration of motile sperm cells in a sample is known to be the best predictor of male fertility (Tomlinson et al., 2013, Human Fertility, 1-19).

[0003] Although home diagnostic devices for estimating male fertility do exist, analyses for assessing the number and motility of cells in a sperm sample can be performed by professionals in a laboratory environment. For example, WO 2014 / 177157 discloses a system capable of estimating the amount of motile cells in a sample. The device of WO 2014 / 177157 has a sample application compartment below a cell-permeable filter and a conditioning medium compartment above the cell-permeable filter, and it has been found that applying the sperm sample below the filter provides a more efficient separation of motile cells from non-motile cells.

[0004] However, although the system of WO 2014 / 177157 is efficient for quantifying the number of motile cells in a sample, it suffers from limitations in the volume that it can handle, and the system of WO 2014 / 177157 is not suitable for isolating motile sperm cells from sample volumes larger than 0.5 mL. Furthermore, the physical integrity of the cell-permeable filter in the device may be compromised. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide an improved system for separating motile from non-motile cells while offering the possibility of extracting motile cells from a larger sample volume than in the prior art. Furthermore, it is an object of the present invention to provide a device in which the cell-permeable filter does not substantially bulge or stretch upon introduction of sample and / or medium into the respective compartments, in particular to prevent the cell-permeable filter from touching the floor or ceiling of the device, which could disrupt the proper functioning of the device. [Means for solving the problem]

[0006] overview A first aspect of the present invention relates to a mesoscale fluidic device for separating motile cells from non-motile cells, the mesoscale fluidic device comprising a substrate having a sample application compartment below a media compartment and a cell-permeable filter having a pore size in the range of 1 μm to 20 μm, the cell-permeable filter having an upper surface facing the media compartment and a lower surface facing the sample application compartment, the sample application compartment and the media compartment being in fluid communication via the cell-permeable filter; The sample application compartment has a floor and at least one flow structure extending from the floor towards a lower surface of the cell permeable filter that faces the sample application compartment.

[0007] The sample application compartment of the mesoscale fluidic device can include an inlet port and the media compartment can include a pressure relief vent that defines a sample flow direction between the inlet port and the pressure relief vent, the pressure relief vent being in gas phase communication with the surroundings.

[0008] The sample application compartment having a floor may have at least one flow structure, and the interface in-plane distance between the individual flow structures, between the individual flow structures and the perimeter of the sample application compartment, or between the perimeter of the sample application compartment is up to 15 mm, preferably 1 mm to 12 mm. In larger animals such as horses, it may be advantageous to increase the perimeter to more than 15 mm to obtain sufficient sample volume. In one implementation of the invention, the media compartment includes a sealing, an access port, and a pressure relief vent, the pressure relief vent being in gas phase communication with the surroundings.

[0009] In a possible realisation of the first aspect, the sample application compartment comprises an inlet port and the media compartment comprises a pressure relief vent.

[0010] In another implementation of the first aspect, the flow structure (25) is a wall or pillar along or across the sample flow direction (S), which wall or pillar extends from the floor of the sample application compartment towards the cell permeable filter.

[0011] In another implementation of the first aspect, the flow structure (25) is a wall or pillar along or across the sample flow direction (S), which wall or pillar extends from the floor of the sample application compartment so as to contact the cell-permeable filter when installed between the sample application compartment and the media compartment.

[0012] In a further possible realisation of the first aspect, the inlet port is located at an inlet end of the sample application compartment and the pressure relief vent is located at an opposite or inlet end of the sample application compartment.

[0013] In a further possible realisation of the first aspect, the access port and the pressure relief vent define a media flow direction between the access port and the pressure relief vent, and the media compartment includes at least one flow structure.

[0014] In a further realization of the first aspect, at least one wall is positioned along or across the media flow direction (M) or at least one pillar, which wall or pillar extends between the sealing of the media compartment and the upper surface of the cell permeable filter.

[0015] In a possible realization of the first aspect, at least one wall is positioned along or across the media flow direction (M) or at least one pillar, which wall or pillar extends from the ceiling of the media compartment towards the upper surface of the cell permeable filter.

[0016] In a further realisation of the first aspect, the wall has a shape that provides a tortuous flow path for the medium from the access port to the pressure relief vent.

[0017] In a further realisation of the first aspect, the walls or pillars are spaced apart from each other by a distance (F) in the range of 50 μm to 15 mm.

[0018] In a possible implementation of the first aspect, the sample application compartment has a depth between the bottom of the sample application compartment and the lower surface of the cell permeable filter, the depth being in the range of 100 μm to 5 mm. A larger depth may be useful to accommodate larger sample volumes. In a preferred implementation of the first aspect, the depth is in the range of 3.5 mm to 4.5 mm, for example 4.2 mm.

[0019] In a possible implementation of the first aspect, the height between the sealing of the media compartment and the upper surface of the cell-permeable filter is in the range of 50 μm to 2 mm, for example in the range of 50 μm to 1000 μm or in the range of 1 mm to 2 mm. At a height between the sealing of the media compartment and the upper surface of the cell-permeable filter in the range of 50 μm to 1000 μm, small amounts of conditioning medium may be more easily removed from the media compartment. At a height in the range of 1 mm to 2 mm, larger volumes of conditioning medium may be more easily removed from the media compartment. In a possible implementation of the first aspect, the pressure relief vent has a minimum lateral dimension of at least 1 mm, and the pressure relief vent opens to the surroundings.

[0020] In another implementation of the first aspect, the access port is at an access end of the media compartment and the pressure relief vent is at the other end.

[0021] In a possible implementation of the first aspect, the mesoscale fluidic device includes a receiving well in fluid communication with the sample application compartment.

[0022] In a second aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: - providing a mesoscale fluidic device according to the present invention as disclosed above; - applying a conditioning medium to the medium compartment via the access port, the conditioning medium comprising one or more of nutrients, salts, buffers, and / or viscosity modifiers; - applying a sample comprising motile and non-motile cells to a sample application compartment via an inlet port; - Swim motile cells through the cell permeable filter to the conditioned medium in the medium compartment within a transition time; - extracting the motile cell enriched conditioned medium from the medium compartment via the access port after the transition time; The present invention relates to a method for preparing a conditioned medium enriched in motile cells from a sample containing motile and non-motile cells, comprising:

[0023] The device is particularly useful for obtaining motile sperm cells from a sperm sample from a mammal. Thus, the device of the present invention is particularly useful for obtaining highly motile cells from a sample of sperm cells having mixed motility for use in assisted reproductive technology (ART).

[0024] Since sperm cells have a natural preference to swim towards a less dense medium environment, swimming sperm cells upward through a filter after being added to a sample chamber below the filter is advantageous over swimming sperm cells through variously arranged filters as described in WO 2014 / 177157. A mesoscale fluidic device for separating motile cells from non-motile cells by swimming cells through a filter should preferably have a large surface area relative to the volume of the chamber adjacent to the two surfaces of the filter to increase the amount of cells that swim through the filter. However, when a sample containing motile cells is added below the filter in a system with a large surface area relative to the volume, for example when the height of the sample chamber is at most 1 / 10 of either the width, e.g. diameter, or length of the sample chamber, it is generally necessary to press the conditioning medium into the media chamber, i.e. to apply high pressure to the medium when filling the media chamber, in order to fill the media chamber and avoid bubbles in the sample chamber. For example, in prior art systems, it has been necessary to apply the sample to the sample chamber using a syringe or the like. Increasing the pressure when applying the sperm sample would create shear forces that could damage the cells, and the cell-permeable filter may be stretched or bulged, so it is undesirable to increase the pressure substantially.

[0025] The sample application compartment includes one or more flow structures. The flow structures have a primary purpose to inhibit or prevent the filter from stretching or bulging, i.e. to prevent or hinder physical contact of the filter to the floor or ceiling of the device. Another purpose of the one or more flow structures may be to form a capillary flow structure that draws liquid into the device by capillary forces. The capillary flow structure is generally defined by the distance in the interface plane between the flow structures or between the flow structures and the perimeter of the sample application compartment. Thus, the perimeter of the flow structures and the sample application compartment generally determines the space available to liquid in the sample application compartment. The term "in the interface plane" with respect to the flow structures and the distance between the individual flow structures should be understood to mean that the location of the flow structures, e.g., on the floor of the sample application compartment, extends towards the interface plane. The flow structures do not need to contact the cell-permeable filter in order to draw liquid into the device by capillary forces. When the distance between the flow structures or between the flow structures and the perimeter is short, for example, in the range of 50 μm to 1000 μm, the liquid will be drawn into the space between the flow structures or between the flow structures and the perimeter by capillary force. Therefore, when the distance between the flow structures or between the flow structures and the perimeter is short, for example, in the range of 50 μm to 1000 μm, the liquid will be drawn into the space between the flow structures or between the flow structures and the perimeter, and the flow structures can also be called "capillary structures". However, when a sperm sample is added to the sample application compartment of the device of the present invention, i.e., added through the inlet port, the inventors have surprisingly found that the sperm sample is drawn into the space between the flow structures or between the flow structures and the perimeter even at distances up to and including 15 mm.In such cases, depending on the intended sample size and / or sample viscosity, it may be advantageous to limit the depth between the bottom of the sample application compartment and the lower surface of the cell permeable filter to a range of 50 μm to 4.5 mm, such as 50 μm to 1000 μm, or such as 1 mm to 4.5 mm. Larger sample sizes may require a greater depth between the bottom of the sample application compartment and the lower surface of the cell permeable filter, such as a depth in the range of 1 mm to 4.5 mm. Similarly, the use of higher viscosity fluids may require a greater depth, such as a depth in the range of 1 mm to 4.5 mm. On the other hand, smaller sample sizes and less viscous samples may benefit from a smaller depth, such as a depth in the range of 50 μm to 1000 μm.

[0026] In a possible realization of the first aspect, the distance between the flow structures or between the flow structures and the perimeter is in the range of 50 μm to 2000 μm, such as 0.5 mm to 2 mm, such as 1.2 mm, i.e. the flow structures are referred to as "capillary structures". In another possible realization of the first aspect, the distance between the flow structures or between the flow structures and the perimeter is in the range of 1 mm to 15 mm, such as 1 mm to 12 mm. Mesoscale fluidic devices with flow structures separated by distances falling within these ranges may be particularly well equipped to enable even distribution of sample throughout the sample application compartment while reducing the appearance of air bubbles in said compartment upon sample loading.

[0027] Therefore, by suitable design and positioning of the flow structure, the sperm sample will be drawn in the sample flow direction. The inventors have now found that when adding a sample to the sample application compartment, even when the mesoscale fluidic device has a large surface area relative to the volume of the sample application compartment, for example when the height of the sample chamber is at most 1 / 10 of either the width, e.g. diameter, or the length of the sample application compartment, particularly both, the sperm sample added at the inlet port is uniformly distributed in the sample application compartment without the formation of air bubbles, with little or no pressure applied to the sample, particularly through the inlet port. Surprisingly, it has been noticed that when the sample is introduced at the inlet, the air present in the sample application compartment can permeate the pores of the cell-permeable filter. This is particularly applicable when the sample application compartment has a depth between the bottom of the sample application compartment and the lower surface of the cell-permeable filter in the range of 100 μm to 2 mm, for example up to 1.5 mm or up to 1 mm. Thereby, a device is provided for providing a sperm sample enriched with motile cells without the need for sample application with substantially increased pressure. Thus, the present invention provides a device for enriching a sperm sample with motile cells with limited shear-induced damage to sperm cells. Thus, in a preferred implementation of the second aspect, the sperm sample is applied to the sample application compartment without increasing pressure or with only a non-substantially increased pressure due to air escaping through the pores of the cell-permeable filter. For example, the mesoscale fluidic device may include a receiving well in fluid communication with the sample application compartment, and the sperm sample is applied to the receiving well, and the sperm sample is drawn into the sample application compartment by capillary forces. The receiving well may be in fluid communication with the sample application compartment via a channel having a maximum cross-sectional dimension of, for example, up to 1 mm.Such a restriction to the cross-sectional dimension advantageously ensures reduced contamination of the clean sample above the filter, for example under transport that may cause fluid to move more easily within the channel. The receiving well is preferably located on the upper surface of the substrate. The receiving well may have a diameter or cross-sectional diameter in the range of 1 mm to 5 mm.

[0028] In a specific implementation of the first aspect, the media compartment comprises a plurality of walls or pillars along the media flow direction, the walls or pillars extending between the sealing of the media compartment and the upper surface of the cell permeable filter, and the walls or pillars spaced apart from each other at a distance in the range of 50 μm to 1000 μm. The walls or pillars thereby provide a capillary effect within the media compartment. Thus, when the conditioning medium is applied to the access port, the conditioning medium will be drawn into the media compartment via capillary forces. However, in some embodiments, the media compartment does not comprise a capillary structure, and specifically, the walls or pillars spaced apart from each other at a distance in the range of 50 μm to 1000 μm. When capillary structures are present in the media compartment, the capillary structures may generate capillary forces even after motile cells of a sperm sample added to the sample application compartment have crossed the cell-permeable filter, which may be harmful to the cells, for example, by exposing the motile sperm cells to shear forces, since removing the motile sperm cells from the media compartment would require a greater negative relative pressure to be applied to the liquid containing the motile sperm cells. Thus, when the media compartment does not include capillary structures, a mesoscale fluidic device for separating motile from non-motile cells is provided, which suggests that the risk of exposing the motile sperm cells to shear forces is reduced.

[0029] The media compartment has a pressure relief vent and an access port. The pressure relief vent of the media compartment is in gas phase communication with the surroundings. In particular, the sample application compartment is in fluid communication with the media compartment through a filter, so that the pressure relief vent will provide fluid communication from the sample application compartment to the surroundings. The pressure relief vent provides pressure relief, so that when liquid is applied to the inlet port or the access port, air can exit the respective compartment through the pressure relief vent. In a specific implementation of the first aspect, the pressure relief vent opens to the surroundings and has a minimum lateral dimension of at least 1 mm. Thus, a pressure relief vent having a minimum lateral dimension of at least 1 mm will minimize the risk of motile cells escaping from the media compartment. For example, the pressure relief vent has a minimum lateral dimension in the range of 1 mm to 3 mm. It is also contemplated that the pressure relief vent may have a minimum lateral dimension of less than 1 mm. For example, the pressure relief vent may have a minimum lateral dimension in the range of 0.5 mm to 1 mm.

[0030] In use, a suitable medium, for example a conditioning medium containing one or more of nutrients, salts, buffers, and / or viscosity modifiers, is added through the access port. The conditioning medium preferably has a density lower than that of the sperm sample to induce motile sperm cells to swim toward the conditioning medium. The medium flows into the medium compartment, whereupon the pressure relief vent will replace the air in the medium compartment with the medium. A sample containing motile and non-motile cells, particularly a sperm sample from a mammal, is added to the sample application compartment through the inlet port. The mesoscale fluidic device preferably includes a receiving well in fluid communication with the sample application compartment, and the sperm sample is added to the receiving well, and the receiving well is further in fluid communication with the sample application compartment through a capillary channel, so that the sperm sample is at least partially drawn into the sample application compartment with the aid of capillary forces.

[0031] After application of the sperm sample, the motile cells are allowed to swim through the cell permeability filter to the media compartment. The time required for the sperm sample to swim through the cell permeability filter is referred to as the transition time in the context of the present invention. The transition time can be freely selected, but typically ranges from 1 minute to 60 minutes.

[0032] When the transition time has elapsed, the liquid, i.e. the conditioned medium now containing the motile cells, is extracted from the medium compartment via the access port. The access port may be dimensioned to allow the insertion of a suitable tool, e.g. any kind of pipette or syringe, into the liquid in the medium compartment. In a specific realization of the first aspect, the access port is fluidly connected to the access well, e.g. via a channel. In particular, the access well may be located on the upper surface of the substrate. The access well or optionally the access port may have a minimum cross-sectional dimension, preferably within the range of 1 mm to 5 mm. In particular, the access well will generally be larger than the tip of the pipette or syringe. When the access well is larger than the tip of the pipette or syringe, the manipulation of the mesoscale fluidic device is easier than when the tip of the pipette or syringe has the same approximate size as the access well, e.g. the diameter of the access well, ensuring that the pipette or syringe cannot be stuck in the access well, in particular preventing shear damage to the sperm cells. When the mesoscale fluidic device has an access well, the access well can be in fluid communication with the access port via a channel, which can have any diameter or cross-sectional dimension.

[0033] In one implementation of the first aspect, the inlet port of the sample application compartment is located at the inlet end of the sample application compartment, and the pressure relief vent is located at the other end of the device. The access port can be at the access end of the opposing media compartment, and the pressure relief vent can be at the other end of the media compartment. In a preferred implementation of the first aspect, the inlet port of the sample application compartment and the access port of the media compartment are located adjacent to each other. A further advantage of the device of the present invention is that the flow structure in the sample application compartment allows the sperm sample added to the sample application compartment through the inlet port to form a uniform distribution, in particular no air bubbles in the sample application compartment. This effect is particularly applicable when the distance between the flow structures or between the flow structure and the perimeter is in the range of 1 mm to 12 mm. In this case, the sperm sample is drawn into the sample application compartment and distributed uniformly, and the total length of the movement of the sperm sample into the sample application compartment can be shorter than when the distance is less than 1 mm, so that a better enrichment effect is obtained in the mesoscale fluidic device. This is even more applicable when the distance between the flow structures or between the flow structures and the perimeter is in the range of 2 mm to 12 mm, for example 3 mm to 12 mm. Therefore, the sperm sample fills the sample application compartment in an amount corresponding to the volume of the sperm sample. The media compartment can be filled with the conditioning medium, but due to the laminar flow conditions prevailing in the media compartment, only the liquid from the media compartment above the section of the sample application compartment where the sperm sample was drawn can be extracted. Therefore, when the inlet end and the access port are located adjacent to each other, only the volume of liquid where the sperm cells have migrated can be extracted from the media compartment. Thereby, the volume of liquid enriched with motile sperm cells is kept to a minimum. Thus, a flexible system is provided for enriching motile sperm cells in a sperm sample.

[0034] In the context of the present invention, the terms "motile" and "motility" refer to cells capable of moving in a liquid independent of any flow of the liquid. In particular, motile cells are capable of moving in a non-flowing liquid. Motile cells may also be said to "migrate", "swim", etc. Motility may be considered random, or cells may respond to a stimulus by swimming, e.g., by swimming towards or away from a given condition. In mesoscale fluidic devices, the conditioning medium is preferably of a density lower than that of a typical sperm sample. When a conditioning medium of a density lower than that of the sperm sample is added to the media compartment, motile sperm cells in a sperm sample added to the sample application compartment will preferentially swim towards the media compartment. Other common stimuli may be those in which motile cells move in response to a chemical gradient ("chemotaxis"), a temperature gradient ("thermotaxis"), a light gradient ("phototaxis"), a magnetic field ("magnetotaxis"), or an electric field ("electrotaxis"). Suitable stimuli will be known to those skilled in the art. Cell motility may be induced by providing a suitable stimulus to motile sperm cells to cause the cells to swim from the sample application compartment to the media compartment. For example, a chemokine or other chemical agent may be placed in the media compartment to attract motile sperm cells that are added to the sample application compartment. In one implementation of the first aspect, the media compartment contains an attractant that attracts sperm cells based on whether they contain an X or Y chromosome. Such attractants are well known to those skilled in the art (see, for example, T. Umehara, N. Tsujita, M. Shimada, PLOS Biology, 13 August 2019, https: / / doi.Org / 10.1371 / journal.pbio.3000398) and are also known as spawning attractants.

[0035] In the context of the present invention, the term "mesoscale" is intended to cover a size range in which the smallest dimension of the channel is within the range of about 10 μm to about 10 mm, e.g., about 100 μm to about 5 mm, typically about 2 mm, although the channel may also contain restrictions. Similarly, the compartments may be about 100 μm to about 20 mm or more deep, e.g., about 500 μm to about 2 mm, e.g., about 500 μm or about 1 mm, and the largest horizontal dimension may be about 1 mm to about 50 mm, e.g., about 1 mm to about 30 mm or about 1 mm to about 20 mm or about 1 mm to about 10 mm, e.g., about 2 mm to about 6 mm. In some implementations, e.g., equine implementations, mesoscale devices may have compartments that are greater than 20 mm deep and / or the largest horizontal dimension may be greater than 50 mm. Fluids in mesoscale fluidic systems may generally be said to flow under laminar conditions, and fluidic systems with channels or chambers other than those defined above may be described as "mesoscale" so long as the fluids contained in the systems flow under laminar conditions.

[0036] A "filter" according to the present invention should be understood in the broadest terms as a unit capable of separating solids, e.g. cells and liquids. Thus, a filter can be, e.g., a filter paper, a filter membrane, a sieve, a packed bed of particles. The system includes a cell-permeable filter that allows cells to cross the filter. The cell-permeable filter has a pore size of 1 μm to 20 μm, e.g., 1 μm to 3 μm, e.g., 1, 3, 5, 8, 10, 12, 15 μm, etc., thereby allowing motile sperm cells to swim through it while providing a pressure drop across the filter. A preferred pore size is in the range of 8 μm to 10 μm, e.g., about 10 μm. In one realization of the first aspect, the cell-permeable filter is a nucleopore filter. The thickness of the cell-permeable filter can be freely selected. However, in a preferred implementation of the first aspect, the sample application compartment has 4 to 12 pillars as flow structures to provide support for the cell permeable filter, and the cell permeable filter has a thickness in the range of 10 μm to 25 μm.

[0037] The mesoscale fluidic system of the present invention is utilized with a conditioning medium. The term "conditioning medium" is not intended to be limiting, and "conditioning" means that the medium may contain components necessary for the analysis of motile cells and also to keep the cells viable. Thus, the conditioning medium may contain pH buffers, salts, nutrients appropriate for the cell type of interest. The conditioning medium may also contain a detection agent, or the detection agent may be added separately to the conditioning medium of the system or may be present in a dry form within the channel or chamber.

[0038] When a sample containing motile cells is added to the sample application compartment, the motile sperm will encounter the cell permeability filter and swim through the encountered surface by swimming along the cell permeability filter. After migrating through the cell permeability filter to the media compartment, the motile sperm cells can be extracted with the liquid via the access port, for example with a pipette, syringe, etc., thereby providing a liquid enriched in motile cells compared to the sample added to the sample application compartment.

[0039] The cell permeable filter has a surface facing the sample application compartment, i.e., the "lower surface of the cell permeable filter," and a surface facing the media compartment, i.e., the "upper surface of the cell permeable filter." In general, the upper surface of the cell permeable filter and the lower surface of the cell permeable filter will be as large as possible compared to the size of the respective compartments. Also, specifically, the upper surface of the cell permeable filter and the lower surface of the cell permeable filter may have substantially the same size.

[0040] Typically, the access port and the pressure relief vent are at opposite ends of the media compartment. For example, the access port can be at the access end of the media compartment and the pressure relief vent can be at the opposite end of the media compartment. Similarly, the inlet port can be located at the inlet end of the sample application compartment and the pressure relief vent can be located at the opposite end of the device such that the sample flow direction is defined from the inlet port to the pressure relief vent. In particular, the inlet end and the pressure relief vent can be at opposite positions of the device, so that the media can fill the sample application compartment and the sample can fill the media compartment, while the air in the sample application compartment can escape through the pressure relief vent after passing through the pores of the filter.

[0041] In another implementation of the first aspect, the sample inlet port and the pressure relief vent are adjacent to each other.

[0042] The flow structures may have any desired shape and there may be any number of flow structures. For example, the flow structures may be bumps extending from a surface of the sample application compartment. In one implementation of the first aspect, the flow structures extend along the sample flow direction. The flow structures may be located at any section over the length of the sample flow direction. For example, the flow structures may be present over 10% to 100% of the length of the sample flow direction. It is preferred that the flow structures are found up to 50% of the length of the sample flow direction from the outlet.

[0043] When the flow structure extends from the floor of the sample application compartment, the extension will typically be within 40% to 100% of the distance between the floor of the sample application compartment and the lower surface of the cell permeable filter. The distance between the top surface of the flow structure extending from the floor of the sample application compartment, i.e., the surface facing the lower surface of the cell permeable filter, can be up to 1.2 mm, for example, within the range of 0 mm to 1 mm or within the range of 0.2 mm to 0.4 mm, and when the distance is greater than 0 mm, it creates the same flow effect in the interface plane as observed between the flow structures or between the perimeter of the flow structure and the sample application compartment. That is, the sperm sample is drawn into the space between the top surface and the lower surface of the cell permeable filter.

[0044] In a specific implementation of the first aspect, the flow structures are walls or pillars extending from the floor of the sample application compartment, for example between the floor of the sample application compartment and the lower surface of the cell permeable filter along the sample flow direction. The flow structures can also be a combination of walls and pillars. When the flow structures are walls, the walls can extend along the sample flow direction. The flow structures, whether walls, pillars, or a combination of walls and pillars, can be spaced apart at regular or irregular intervals.

[0045] In one implementation of the first aspect, the flow structure is a pillar extending from the floor of the sample application compartment, for example the pillar may have a cylindrical or frusto-conical shape with a diameter in the range of 0.1 mm to 2.0 mm, for example in the range of 0.2 mm to 0.6 mm.

[0046] In a possible realization of the first aspect, the pillars are clearly conical, i.e. the diameter difference between the base and the tip is large, creating a cone with a significant inclination. A frustoconical shape with such and similar proportions allows an optimal compromise between a large surface area to ensure good capillary forces and a relatively small contact area between the conical tip and the porous filter. Thereby, the area of ​​the filter that remains unobstructed is maximized while maintaining good capillary performance by remaining presented with a large surface area. In a specific realization of the first aspect, the pillars have a diameter of 1.68 mm at their base and 0.40 mm at their tip, and a height of 0.45 mm. Pillars with a frustoconical shape may advantageously allow an increase in the surface area available in the sample application compartment (compared to the same number of pillars with a cylindrical shape), thus allowing an increase in the capillary forces to draw fluids uniformly into said compartment and an increase in the fluid flow from the sample application compartment to the filter.

[0047] In general, pillars with larger diameters present a larger surface area and increased capillary forces, while thinner pillars, i.e. pillars with smaller diameters, allow for a larger working volume to be available in the sample application compartment, thus allowing for the introduction of a larger volume of sample.

[0048] In a possible realization of the first embodiment, the pillar has an essentially cylindrical shape, i.e. the diameter of the base of the pillar is similar to the diameter of the tip of the pillar, resulting in a perfect cylinder or a cone with a very shallow inclination. Such shapes and shapes with similar proportionality may allow an increase in the end surface of the tip, thereby maximizing the contact area between the pillar and the supporting filter. This design may be more adapted to large filters, and may be advantageous to include a glue or sealant or similar adhesive to fix the filter in place and to avoid deformation of the filter due to hydration and / or drying.

[0049] In a specific implementation of the first embodiment, the pillar has a diameter of 0.44 mm at its base and 0.40 mm at its tip, and is 0.45 mm high.

[0050] In a possible realization of the first aspect, the number of pillars or flow structures is maximized. In such a case, the average pillar diameter can be reduced to maintain the available working volume.

[0051] In another possible implementation of the first aspect, the number of pillars or flow structures is relatively small in favor of the working volume, in which case the surface area and capillary forces are compensated for by a larger average pillar diameter.

[0052] In a specific realization of the first embodiment, the sample application compartment contains 4 to 12 pillars as flow structures.

[0053] The pillars are preferably uniformly distributed within the sample application compartment. In the context of the present invention, the term "uniformly distributed", when used to describe the distance between flow structures, especially between pillars, means that groups of flow structures can be defined in which the minimum distance between any pair of flow structures within a group varies from the minimum distance between any other pair of flow structures within a group by ±25% from a mean value.

[0054] When the sample application compartment has at least four pillars as flow structures, especially when the pillars are uniformly distributed and when the extension from the floor of the sample application compartment is at least 80% of the distance between the floor of the sample application compartment and the lower surface facing the sample application compartment, the pillars provide suitable structural support to the cell-permeable filter. This is particularly applicable when the sample application compartment has at least six pillars or at least eight pillars as flow structures, or when the flow structures are any number of walls. In the context of the present invention, "support to the cell-permeable filter" means that the sample application compartment cannot be collapsed by bending the cell-permeable filter. This simplifies the manufacture of the mesoscale fluidic device of the present invention, since the mesoscale fluidic device does not need to be rigid. Moreover, this further limits the variation due to bending of the cell-permeable filter, so that the volume of the sample application compartment is constant. In particular, the support provided by pillars, e.g. 4-12 uniformly distributed pillars, or when the flow structure is any number of walls, allows the sample application compartment to be wide. For example, the width, i.e. the typical distance between the perimeters of the sample application compartment, can be in the range of 25 mm to 100 mm without risking bending of the cell permeable filter and collapsing of the sample application compartment. This allows a large surface area to be available for the volume of the chamber adjacent to the two surfaces of the filter. When the width, i.e. the typical distance between the perimeters of the sample application compartment, is in the range of 25 mm to 100 mm, the sample application compartment has at least 4 uniformly distributed pillars, and a higher concentration power is provided in the mesoscale fluidic device for separating motile from non-motile cells.

[0055] The support further allows the use of thinner cell-permeable filters in mesoscale fluidic devices. Therefore, manufacturing costs are reduced when the sample application compartment contains 4-12 uniformly distributed pillars as flow structures, or if the flow structures are any number of walls. Therefore, in a preferred implementation of the first aspect, the flow structures are pillars, the number of pillars is in the range of 4-8, and the pillars are uniformly distributed, for example with a distance between pillars or between pillars and perimeter in the range of 6 mm to 12 mm. The pillars may extend from the floor of the sample application compartment at a distance in the range of, for example, 0.4 mm to 1.6 mm, for example 0.8 mm to 1.2 mm, in particular the extension from the floor of the sample application compartment is preferably at least 80% of the distance between the floor of the sample application compartment and the lower surface of the filter facing the sample application compartment.

[0056] In a preferred implantation configuration of the first embodiment, the pillars extend from the floor of the sample application compartment up to 100% of the distance between the floor of the sample application compartment and the lower surface of the filter facing the sample application compartment, thereby providing support to and contacting the filter.

[0057] The upper surface of the cell permeable filter can be said to define the bottom of the media compartment, and the media compartment will have a ceiling such that the media compartment has a height between the ceiling and the upper surface of the cell permeable filter. Similarly, the lower surface of the cell permeable filter can be said to define the ceiling of the sample application compartment, and the sample application compartment has a floor such that the sample application compartment has a depth between the bottom and the lower surface of the cell permeable filter. When the lower surface of the cell permeable filter and the upper surface of the cell permeable filter have substantially the same size, a concentration effect is provided when the height of the media compartment is smaller than the depth of the sample application compartment. Motile cells will move from a volume in the sample application compartment to a volume in the media compartment, which will be proportionally smaller depending on the difference between the height of the media compartment and the depth of the sample application compartment. For example, in one implementation of the first aspect, the ratio between the depth of the sample application compartment and the height of the media compartment is in the range of 1.5:1 to 20:1, for example, 2:1 to 10:1, or 3:1 to 5:1. This is particularly advantageous when the inlet end of the sample application compartment and the access end of the media compartment are located adjacent to each other, and the exit end and the outlet end are also adjacent to each other and opposite the position of the adjacent inlet end and access end, because there is only a limited volume, i.e., the volume above the sample application compartment into which the sperm sample is drawn depending on its volume. In another implementation of the first aspect, the ratio between the depth of the sample application compartment and the height of the media compartment is approximately 1 mm.

[0058] In general, the shorter the distance between the bottom or floor of the sample application compartment and the lower surface of the cell permeability filter, the more efficient the separation of motile cells from non-motile cells. Thus, in one implementation of the first aspect, the sample application compartment has a depth between the bottom of the sample application compartment and the lower surface of the cell permeability filter, which depth is in the range of 100 μm to 2 mm.

[0059] The substrate can be made of any convenient material, such as polymer, glass, metal, ceramic material, or combinations thereof, and the channels and chambers in the substrate can be made using any suitable method. Suitable methods include milling, micromilling, drilling, cutting, laser ablation, hot embossing, injection molding and micro-injection molding, and 3D printing. For example, the substrate can be made of thermoplastic polymers, such as poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), polycarbonate, polystyrene, polyethylene, polyethylene terephthalate (PET), and the like. It is preferred that the substrate material has a hydrophilic surface, and thus COC and PMMA are preferred materials. However, one skilled in the art will know how to increase the hydrophilicity of a polymeric material relative to a less hydrophilic material, for example, a hydrophobic material can be plasma treated or coated.

[0060] The cell-permeable filter can be any filter having a suitable pore size, such as 1, 3, 5, 8, 10, 12, 15 μm, etc. For example, the cell-permeable filter can be a polycarbonate membrane with pores created by irradiation. Such filters are known under the trademarks Nucleopore Filter and Isopore Filter. The thickness of the filter will generally be in the range of 1 μm to 50 μm, such as 5 μm to 20 μm, for example about 10 μm or about 8 μm. It is more preferable that the cell-permeable filter has a hydrophilic surface, and for example, a polycarbonate membrane can be used.

[0061] In one implementation of the first aspect, the substrate includes two polymer layers of a thermoplastic polymer having channels and chambers formed therein, for example by injection molding, and the two polymer layers are bonded together with a filter between them.

[0062] Any implementation may be used with any aspect of the invention, and the advantages of any of the specific embodiments apply equally when an embodiment of a specific aspect is used.

[0063] BRIEF DESCRIPTION OF THE DRAWINGS In the following the invention is explained in more detail with the help of examples and with reference to schematic drawings. [Brief description of the drawings]

[0064] [Figure 1] 1 shows a schematic cross-sectional view of a mesoscale fluidic device according to the present disclosure. [Diagram 2] 1 shows a top view of a sample application compartment of one implementation of a mesoscale fluidic device according to the present disclosure. [Diagram 3] 1 shows a top view of a sample application compartment of one implementation of a mesoscale fluidic device according to the present disclosure. [Figure 4] FIG. 1 shows a top view of a mesoscale fluidic device and sample application compartment according to the present disclosure. [Diagram 5] 1 shows an exploded view of one implementation of the device and an oblique view showing the underside of the upper substrate layer and the underside of the lower substrate layer, note that the filter has been removed for clarity. [Figure 6] 1 shows an exploded view of one implementation of the device and an oblique view showing the underside of the upper substrate layer and the underside of the lower substrate layer, note that the filter has been removed for clarity. [Figure 7] 1 illustrates a conical pillar according to the present disclosure. [Figure 8] 1 shows a sample application compartment with cylindrical pillars according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Detailed Description The present invention relates to a mesoscale fluidic device 1 for separating motile cells from non-motile cells and a method of separating motile cells from non-motile cells. Figure 1 illustrates a schematic cross-sectional side view of an embodiment of the mesoscale fluidic device 1, and Figures 2 and 3 illustrate top views of two embodiments of a sample application compartment 2 of the mesoscale fluidic device 1. Figure 4 shows an upper substrate layer 11 (upper panel) and a lower substrate layer 12 (lower panel) of an embodiment of the mesoscale fluidic device 1. The figures are not drawn to scale. The mesoscale fluidic device 1 includes a substrate 10 having two substrate layers, the upper substrate layer 11 and the lower substrate layer 12, of a thermoplastic polymer, such as poly(methyl methacrylate) (PMMA), that are injection molded to contain a sample application compartment 2 and a media compartment 3, as well as channels and other structures. 1, mesoscale fluidic device 1 is shown with a dashed line separating upper and lower substrate layers 11 and 12, which is for illustrative purposes only and is not part of mesoscale fluidic device 1. Substrate layers 11, 12 have dimensions of 25 mm by 50 mm and a thickness of 2 mm.

[0066] The substrate layers 11, 12 are ultrasonically welded or glued to a nucleopore filter with a thickness of 10 μm and a pore size of 10 μm or 8 μm, which provides a cell-permeable filter 4, thereby forming a sample application compartment 2 and a media compartment 3. The cell-permeable filter 4 has an upper surface 31 facing the media compartment 3 and a lower surface 22 facing the sample application compartment 2. The distance from the lower surface 22 to the floor 21 of the sample application compartment 2 defines the height H of the sample application compartment 2, and the distance from the upper surface 31 to the ceiling 32 of the media compartment 3 defines the depth D of the media compartment 3. Thus, the sample application compartment 2 and the media compartment 3 are formed on the surface of the substrate layers 11, 12 with a depth D and height H of 1 mm or 1.5 mm from the surface, respectively. The perimeter 26 of the sample application compartment 2 defines the sample application compartment 2 with a width of 20 mm and a length of 40 mm. The media compartment 3 also has a width of 20 mm and a length of 40 mm.

[0067] The cell-permeable filter 4 defines an interface plane A, illustrated by a box drawn as a dashed line in Figures 2 and 3. Thus, the interface plane A is defined between the sample application compartment 2 and the media compartment 3. In the embodiment of Figures 2 and 3, the flow structure 25 extends from the floor 21 of the sample application compartment 2 towards the cell-permeable filter 4, so that Figures 2 and 3 illustrate the projection of the flow structure 25 onto the interface plane A. In the embodiment of Figures 2 and 3, the sample application compartment 2 and the media compartment 3 have the same size, so that the entire surface area of ​​each compartment is available for sperm cells to traverse the cell-permeable filter 4.

[0068] The media compartment 3 has an access point 33 at the access end 331 and a pressure relief vent 24 at the other end 341 opposite the access end 331, and the sample application compartment 2 has an inlet port 23 at the inlet end 231. The inlet end 231 and the access end 331 are in the same location in the mesoscale fluidic device 1.

[0069] The inlet port 23 comprises a 500 μm diameter channel in the lower substrate layer 11 and is in fluid communication with a receiving well 232 in the top surface of the upper substrate layer 12. The access port 33 comprises a 500 μm diameter channel in the upper substrate layer 12 and is in fluid communication with an access well 332 in the top surface of the upper substrate layer 12. The receiving well 232 is adjacent to the access well 332. The inlet port 23 and the pressure relief vent 24 define a sample flow direction S between the inlet port 23 and the pressure relief vent 24, and the access port 33 and the pressure relief vent 24 define a medium flow direction M between the access port 33 and the outlet 34. The filter has pores sized to allow air to escape from the sample compartment 2 when it is loaded with semen. Air penetrating the filter pores can pass through the pressure relief vent 24 to balance the ambient pressure and the pressure within the device.

[0070] The pressure relief vent 24 comprises a 500 μm diameter channel in the upper substrate layer 12. The pressure relief vent 24 is shown in the end wall of the upper substrate layer 12, however it may also be present in the sealing of the media compartment 3, so the pressure relief vent 24 may be in the top surface of the upper substrate layer 12. The access port 33 may be used as an inlet as well as an outlet, so that cell media may be supplied via the access port 33 and after the sperm cells have had sufficient time to permeate the filter 4, it may then be extracted from the access port.

[0071] In the embodiment of Figure 2, the sample application compartment 2 contains as flow structures 25 six frustoconical pillars each having a height of 90% of the depth of the sample application compartment 2, e.g. 0.9 mm or 1.35 mm, and a width of 0.4 mm at the base width. The pillars 25 are uniformly distributed in the sample application compartment 2 with a distance F between the flow structures 25. Figure 2 shows a pressure relief vent 24. The pressure relief vent 24 should be understood to be above the floor 21 of the sample application compartment 2. The distance F between the flow structures is 8 mm.

[0072] In the embodiment of Figure 3, the sample application compartment 2 contains three walls as flow structures 25, each having a height of 90% of the depth of the sample application compartment 2, e.g. 0.9 mm or 1.35 mm. The walls 25 have a length of 80% of the distance between the inlet port 23 and the pressure relief vent 24. Figure 3 shows the pressure relief vent 24. It should be understood that the pressure relief vent 24 is above the floor 21 of the sample application compartment 2.

[0073] FIG. 4 discloses the upper substrate layer 11 and the lower substrate layer 12 of a specific embodiment of the mesoscale fluidic device. The filter has been removed for clarity. The upper surface of the upper substrate layer comprises a receiving well 232 for the inlet of semen and an access well 332 for the supply of a conditioning medium and for the extraction of the conditioning medium enriched in sperm cells after a certain incubation time. Also visible on the drawing is a pressure relief vent 24 for the escape of air from inside the device when the conditioning medium and / or semen is introduced into the device 1.

[0074] In the lower panel of Figure 4, the upper part of the lower substrate layer 11 is shown. The drawing shows the position of pillars 25 on the floor 21. The pillars extend upwardly from the floor and are adapted to support a cell-permeable filter. An inlet port 23 is provided on the left hand side of the drawing and is in fluid communication with the receiving well 232. In use, a semen sample is introduced into the receiving well 232 and the medium is introduced into the access well 332. During a certain incubation time, cells are allowed to traverse the filter from the sample application compartment 2 to the medium compartment. When the incubation time is over, the cell-enriched medium can be extracted via the access port 332.

[0075] In Figures 5 and 6, the same embodiment of the mesoscale fluidic device 1 is disclosed, with the right panel being slightly tilted with respect to the mesoscale fluidic device 1 in the left panel so that the lower part of the upper substrate layer 11 is exposed. This embodiment includes four parts: the upper substrate layer 11, the cell-permeable filter 4, the lower substrate layer 12, and the lid 35. The upper substrate layer includes a receiving well 232 for the introduction of the semen sample, an access port 33 for the delivery of the medium and for the subsequent collection of the medium enriched with semen cells, and a pressure relief vent 24 for the escape of air from the inside of the mesoscale fluidic device 1 when the semen sample and / or the medium is introduced. The lower side of the upper substrate layer is provided with flow structures in the form of pillars. The pillars extend from the ceiling 32 to the inside of the mesoscale fluidic device 1. The cell-permeable filter is positioned between the upper and lower substrate layers. However, the cell-permeable filter 4 is not included in the drawings to increase clarity.

[0076] The lower substrate layer 12 includes an inlet port 23 in fluid communication with the receiving well 232. The inlet allows the sample to flow into the sample application compartment 2, i.e., into the space between the floor 21, the filter 4, and the perimeter of the compartment. The floor 21 of the lower substrate layer 12 includes flow structures 25 in the form of pillars. The pillars 25 of the lower substrate layer are positioned opposite the pillars of the upper substrate layer 11 to provide support for the filter from both sides. The support from each side results in a filter that does not substantially bulge or stretch when sample and / or medium are introduced into the respective compartments. Upon incubation, cells are migrated through the filter, enriching the medium with cells. The medium enriched with cells is extracted via the access port 33.

[0077] The lid 35 participates in forming the inlet port 23 when positioned in a corresponding recess in the lower portion of the lower substrate layer.

[0078] In Figures 7 and 8, the same embodiment of the mesoscale fluidic device 1 is disclosed, with the right panel being slightly tilted with respect to the mesoscale fluidic device 1 in the left panel so that the lower part of the upper substrate layer 11 is exposed. The main difference with the previous embodiment illustrated in Figures 5 and 6 is the presence of a serpentine flow channel 36 in the lower part of the upper substrate layer 11. The serpentine flow channel 36 extends from the access port 33 for the medium and the pressure relief vent 24. The serpentine flow path is formed by a set of flow structures in the form of walls that extend from alternating perimeters to near opposing perimeters. The set of flow structures is alternating in the sense that a first flow structure 25 extends from a first perimeter and a nearby second flow structure extends from an opposing perimeter. By repeating the set of flow structures, the serpentine flow path is formed as a space between the walls. The walls can be rounded at their base to create a flow path without sharp corners.

[0079] The lower substrate layer 12 includes pillars 25 positioned against the walls of the lower portion of the upper substrate layer to support the cell-permeable filter from both sides. EXAMPLES

[0080] Example 1 The above-described mesoscale fluidic device 1 having a depth D and height H of 1.5 mm and 1 mm, and a cell-permeable filter 4 with a pore size of 10 μm and 8 μm, was tested with a sperm sample. A sperm sample was provided and initially analyzed for the number of motile cells. Conditioning medium was added to each access well 332 of the mesoscale fluidic device 1 to fill the media compartment 3 with the conditioning medium. The sperm sample was then added to the receiving well 232 of the mesoscale fluidic device 1, and the sperm cells were allowed to migrate from the sample application compartment 2 to the media compartment 3. After incubation, the liquid 3 in the media compartment was extracted through the access well 332 to provide a sample enriched for motile sperm cells, and the enriched sample was analyzed for motile sperm cells and non-motile sperm cells.

[0081] The percentage of motile cells for the spiked and treated samples and the recovery rate for the treated samples are shown in Table 1 for Mesoscale Fluidic Device 1 with H and D of 1.5 mm and in Table 2 for Mesoscale Fluidic Device 1 with H and D of 1.0 mm. In Table 1, Sample 1 had a 35 minute incubation time, Sample 2 had a 1 hour incubation time, and Samples 3-5 had a 30 minute incubation time. In Table 2, the incubation time was 30 minutes.

[0082] [Table 1]

[0083] [Table 2]

[0084] Therefore, the mesoscale fluidic device 1 of the present invention consistently provided enrichment of motile sperm cells to greater than 90%. [Explanation of symbols]

[0085] Reference symbol list Mesoscale Fluidic Device 1 Base material 10 Upper base layer 11 Lower base material layer 12 Sample application compartment 2 Media Compartment 3 Cell-permeable filter 4 Upper surface of cell permeable filter 31 Lower surface of cell permeable filter 22 Inlet port 23 Pressure Relief Vent 24 Floor 21 Flow Structure 25 Perimeter 26 Ceiling 32 Access Port 33 exit 34 Outlet end 341 Entrance end 231 Exit end 241 Receiving well 232 Access end 331 Access Well 332 Interface Plane A Distance between flow structures F Sample flow direction S Media flow direction M Depth D Height H

Claims

1. A mesoscale fluidic device (1) for separating motile cells from non-motile cells, the mesoscale fluidic device (1) comprising: a substrate (10) having a sample application compartment (2) below a media compartment (3); and a cell-permeable filter (4) having a pore size in the range of 1 μm to 20 μm, the cell-permeable filter (4) having an upper surface (31) facing the media compartment (3) and a lower surface (22) facing the sample application compartment (2), the sample application compartment (2) and the media compartment (3) being in fluid communication via the cell-permeable filter (4); The mesoscale fluidic device (1) includes a sample application compartment (2) having a floor (21) and at least one flow structure (25) extending from the floor toward a lower surface (22) of the cell permeable filter facing the sample application compartment (2).

2. 2. The mesoscale fluidic device of claim 1, wherein a distance (F) in an interface plane (A) defined by the cell-permeable filter (4) between an individual flow structure (25) and a perimeter (26) of the sample application compartment (2) is in the range of 50 μm to 15 mm.

3. 2. The mesoscale fluidic device of claim 1, wherein the media compartment (3) includes a sealing (32), an access port (33), and a pressure relief vent (24), the pressure relief vent (24) being in gas-phase communication with the ambient.

4. 2. The mesoscale fluidic device of claim 1, wherein the sample application compartment (2) includes an inlet port (23) and the media compartment (3) includes a pressure relief vent (24).

5. 2. The mesoscale fluidic device (1) of claim 1, wherein the flow structure (25) is a wall or pillar arranged along or across the sample flow direction (S), the wall or pillar extending from the floor (21) of the sample application compartment (2) toward the cell permeable filter.

6. 5. The mesoscale fluidic device (1) of claim 4, wherein the inlet port (23) is located at the inlet end (231) of the sample application compartment (2), and the pressure relief vent (24) is located at the opposite end (241) of the sample application compartment (2) to the inlet end (231).

7. 4. The mesoscale fluidic device (1) of claim 3, wherein the access port (33) and the pressure relief vent (24) define a medium flow direction (M) between the access port (33) and the pressure relief vent (24), and the medium compartment (3) includes at least one flow structure (25).

8. 8. The mesoscale fluidic device (1) of claim 7, wherein at least one wall or at least one pillar extends between the ceiling (32) of the media compartment (3) and the upper surface (31) of the cell-permeable filter (4).

9. The mesoscale fluidic device of claim 1 , wherein the wall is arranged to provide a tortuous flow path for a medium from the access port (33) to the pressure relief vent (24).

10. The mesoscale fluidic device of claim 9 , wherein the walls are arranged transverse to the medium flow direction (S).

11. 9. The mesoscale fluidic device of claim 8, wherein the at least one wall or at least one pillar is arranged to extend from a ceiling (32) of the media compartment (3) toward a corresponding wall or pillar (25) extending from a floor of the application compartment (2) on the other side of the cell-permeable filter (4).

12. 2. The mesoscale fluidic device of claim 1, wherein the flow structure (25) is a cone-shaped pillar having a base diameter perpendicular to a height dimension of the pillar (25) greater than a tip diameter of the pillar (25).

13. The mesoscale fluidic device of claim 1 , wherein the flow structure (25) contacts the cell-permeable filter (4) when installed.

14. The mesoscale fluidic device of claim 13, wherein a sealant or adhesive is layered between the tip of the flow structure (25) and the cell-permeable filter (4).

15. - providing a mesoscale fluidic device (1) according to any one of claims 1 to 9; - applying a conditioning medium to the medium compartment via the access port (33), the conditioning medium comprising one or more of nutrients, salts, buffers, and / or viscosity modifiers; - applying a sample containing motile cells to the sample application compartment (2) via the inlet port (23); - allowing the motile cells to swim through the cell-permeable filter (4) into the conditioned medium in the medium compartment (3) within a transition time; - after said transition time, extracting the conditioned medium containing the motile cells from said medium compartment (3) through said access port (33); 1. A method for preparing a conditioned medium enriched in motile cells from a sample containing motile and non-motile cells, comprising:

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