WO2012126478A1

WO2012126478A1 - Device for analysis of cellular motility

Info

Publication number: WO2012126478A1
Application number: PCT/DK2012/050085
Authority: WO
Inventors: Jacob Møllenbach LARSEN; Steen Broch Laursen
Original assignee: Motilitycount Aps
Priority date: 2011-03-21
Filing date: 2012-03-21
Publication date: 2012-09-27

Abstract

The present invention relates to a mesoscale fluidic system comprising a substrate having a first analysis chamber in fluid communication with a further analysis chamber via a passageway; the passageway having an entrance port in fluid communication with the first analysis chamber and an exit port in fluid communication with the further analysis chamber, wherein the cross-sectional area of the entrance port is larger than the cross-sectional area of the exit port. The system of the invention may be used in a method of estimating the quantity and motility of cells in a sample or a method of separating motile cells in a sample based on their motility.

Description

Device for analysis of cellular motility

Field of the invention

This invention relates to devices and methods for counting motile cells, such as sperm cells. In particular, the invention relates to a mesoscale fluidic system capable of separating motile cells based on their motility and allowing detection and quantification of the cells. The invention also relates to methods of estimating the quantity and motility of cells in a sample and a method of separating motile cells in a sample based on their motility using the system of the invention.

Prior art

Several devices for separating and manipulating motile cells, such as sperm cells, are described in the prior art. However, the prior art does not disclose any systems or devices which may provide both a count of cells in a sample and also an estimate of their motility. Devices disclosed in the prior art generally do not allow the cell number and the motility of the cells to be estimated without the need for auxiliary equipment such as microscopes, electrodes for impedance measurements or the like. In particular, the need to check motility and sperm counts in the comfort of the home environment is barely addressed by known devices.

US 5,296,375 describes devices and methods for the clinical analysis of sperm samples. The devices comprise a solid substrate, which is microfab- ricated to define a sample inlet port and a mesoscale flow channel extending from the inlet port. A sperm sample may be applied to the inlet port, and the competitive migration of the sperm sample through the mesoscale flow channel is detected to serve as an indicator of sperm motility. The flow channel may comprise a fractal region, comprising bifurcations leading to plural secondary channels, to enhance the detection or competitive migration of the sperm sample. The fractal region may comprise equal numbers of bifurca- tions and junctions disposed serially along the direction of sperm migration. In one embodiment, the branching channels in the fractal region progressively decrease in cross-sectional area at each bifurcation and increase at each junction. The function of this fractal pattern, which may be constructed with sequentially narrower channels towards the centre of the fractal is to enhance sensitivity to sperm migration.

W01996/14933 relates to devices and methods to facilitate analysis of a sample having cells characterised by their motility, e.g. sperm cells. W01996/14933 describes a device comprising a solid substrate having a flow system which includes at least one elongate flow channel of mesoscale cross- sectional dimension, and a receiving well communicating with the channel and defining a starting point in the channel. The device further comprises a cover for the substrate, which closes the channel and has a port in registry with the receiving well, for introducing the sample into the receiving well. Motile cells in the sample travel from the receiving well to various progress points along the channel.

The receiving well of the device may further include a plurality of flow - regulating solids having a size and shape effective to permit passage of non- aggregated motile cells of interest from the receiving well into the channel and concomitantly to substantially restrain passage into the channel of other particulate matter in the sample, such as cellular aggregates, large particles, gelatinous material and the like. These flow-regulating solids may take the form of a cell director comprising flow-guiding ribs longitudinally aligned with the channel for directing motile cells in the sample from the receiving well into the channel. The "flow-guiding ribs" of W01996/14933 should neither impede nor enhance the movement of cells in the flow channel.

W01996/14933 further teaches that the flow-guiding ribs orient motile cells and guide their entry into the flow channel. It is stated that without such a device fashioned into the receiving well sperm and other motile cells tend to swim into the corners of the receiving well, rather than entering the flow channel.

In operation a sample comprising the motile cell of interest (e.g. a sperm sample) is applied at an inlet port, optionally by way of a delivery ap- paratus, such as a pipette or syringe. Motile cells in the sample migrate from the receiving well into the flow channel towards the target chamber. The extent of progress of motile cells along the flow channel may serve as an indicator of cellular motility. The migration of motile cells may be detected optically. The device of W01996/14933 may further have a selection region that is adapted for selective separation of at least one motile cell type from a mixed population of cell types and comprising an electric field which selectively influences motility of the at least one motile cell type.

EP0739240 claims a device for preparing a test sample containing particulate components for analysis. The device comprises a sample flow passage having a sample inlet and an outlet in fluid communication and a separator disposed between the inlet and the outlet. The separator has an upstream-facing portion defining a separation zone in the flow passage in which particulate components are collected, and a flow channel in fluid communication with the separation zone for affording discharge of collected particulate components from the separation zone. The channel has an inlet section for directing a carrier fluid into the separation zone, and a discharge section for directing the carrier fluid from over the upstream facing portion of the sepa- rator and out of the separation zone. The flow passage and the flow channel sections have at least one mesoscale dimension between 0.1 and 1000 μηη. The device of EP0739240 does not include valves, and the inlet section of the flow channel is arranged to direct the carrier fluid into the separation zone over the upstream-facing portion of the separator.

WO2003/007711 discloses a microscale cell handling apparatus comprising at least two flow paths, each of which extend between an inlet region and an outlet region of a void that can be filled with fluid. The apparatus includes a microscale flow path and a non-microscale flow path. The apparatus may have two obstacles disposed within the void, which obstacles serve to define a microscale flow path so that fluid flow from the inlet region to the outlet region through the microscale flow path passes between the obstacles.

The apparatus of WO2003/007711 can be used to assess motility of cells in a sample. For example, it is described how a sample is delivered to the inlet region of the void for motile cells in the sample to move from the inlet region, and enter a microscale flow path of the apparatus. By assessing the number of cells in the microscale flow path, the motility of cells in the sample can be assessed.

WO2004/108011 relates to microfluidic devices for microfluidic sperm isolation and oocyte insemination. The device is an integral device which both sorts sperm based on motility and then uses the sorted sperm to inseminate an oocyte. The device has two gravity driven pumps, one for semen and one for a media fluid, and a common sort channel wherein more motile sperm swim across the interface between co-laminar flows of semen and media fluid. The media fluid being enriched with more motile sperm is then used to fertilise oocytes. It is noted that WO2004/108011 shows a device with a barrier structure, so that an oocyte chamber must, in general, contain a barrier which contains holes or passages which permit the flow of fluids and sperm, but which are sized so as not to allow the oocytes to travel through the barrier.

On this basis it is an aim of the present invention to provide a home diagnostic device for assessing the number and motility of cells in a sperm sample in order to estimate the male fertility potential. Such a device should be simple to use, and it should be available at a low cost in order to be of a disposable nature. Such devices may, however, also be relevant in the as- sessment of motile cells in other types of samples, such as prokaryotic cells, eukaryotic cells, or other motile mammalian cell types. Furthermore, it is an aim to provide a device for separating motile cells based on their motility allowing the separated cells to be used in subsequent procedures. Disclosure of the invention

The present invention relates to a mesoscale fluidic system comprising a substrate having

a first analysis chamber in fluid communication with a further analysis chamber via a passageway;

the passageway having an entrance port in fluid communication with the first analysis chamber and an exit port in fluid communication with the further analysis chamber, wherein the cross-sectional area of the entrance port is larger than the cross-sectional area of the exit port. The mesoscale fluidic system may be used for analysing the quantity and motility of motile cells in a sample or for separating motile cells based on their motility. Motile cells may be prokaryotic cells, e.g. bacterial cells, or eukaryotic cells, such as yeast cells, amoebae, micro- and macroparasites and the like or motile mammalian cell types. A preferred cell type is sperm cells, in particular human sperm cells or other mammalian sperm cells. In another embodiment each analysis chamber further comprises a solid-liquid separation unit defining an upstream surface facing the analysis chamber and a downstream surface facing an effluent channel, so as to allow fluid communication between the analysis chamber and the effluent channel via the solid-liquid separation unit.

The mesoscale fluidic system of the present invention will have a first analysis chamber in fluid communication with a further analysis chamber; in other embodiments the mesoscale fluidic system may have more than two analysis chambers, for example the mesoscale fluidic system may have 3, 4, 5, 6, 7, 8, 9 or 10 or even more analysis chambers. When more than two analysis chambers are present these will, in one embodiment, be in serial fluid communication, so that the first analysis chamber will be in fluid communication with a second analysis chamber, which in turn is in fluid communication with a third analysis chamber and so forth. These serially connected analysis chambers allow motile cells to move from the first analysis chamber to the second and on to the third, etc. Thus, a series of analysis chambers can provide an estimate of the motility of cells in a sample since the more motile the cells the further they may travel in the mesoscale fluidic system. The last analysis chamber in the series will typically not be in fluid communi- cation with another chamber than the previous analysis chamber before it in the series.

In a further embodiment several analysis chambers are present, which may be in parallel fluid communication, e.g. the first analysis chamber is in parallel fluid communication via passageways as defined above with two or more further analysis chambers. The mesoscale fluidic system may comprise several parallel series of serially connected analysis chambers. For example, in one embodiment a first analysis chamber is connected to from e.g. two to ten further analysis chambers where each further analysis chamber in turn may be in fluid communication with yet a further analysis chamber. Thus, in this embodiment the mesoscale fluidic system comprises from two to ten parallel series of one or more further analysis chambers where the different series share the first analysis chamber. This allows that a better estimate of cellular motility for a single sample is obtained due to the parallel analysis. Each series of analysis chambers need not contain the same number of analysis chambers. For example, one series may have the first and a second analysis chamber, where another series may have two or more further analysis chambers. When the mesoscale fluidic system comprises several parallel series of analysis chambers the passageways between the serially connected analysis chambers of different series may be of the same length although they need not be of the same length. When the passageways are of different lengths it is possible to obtain more detailed information of the motility of cells in a single sample as it is possible to get an estimate of cellular motility in the passageways without considering motility through an analysis chamber. Thus, in an embodiment the mesoscale fluidic system comprises a cen- tral analysis chamber into which a sample with motile cells is added. The central analysis chamber is in fluid communication with e.g. from two to ten further analysis chambers each connected to the central chamber via separate passageways of different lengths, with the passageways thus being in parallel fluid communication with the central analysis chamber. After addition of the sample with motile cells the cells will swim from the centre towards the analysis chambers via the passageways. The cells may then be detected in each analysis chamber where the presence of cells in the central chamber may provide a value for non-motile cells, and the motility of the population of cells may be estimated from the number of cells in each further analysis chamber since each of these chambers is located at a different distance from the centre.

In the embodiment where the mesoscale fluidic system comprises several parallel series of analysis chambers a sample with motile cells may be added to the first, e.g. the central, analysis chamber. However, in another embodiment with several parallel series of analysis chambers the central chamber is a receiving well not intended for subsequent detection of cells in the receiving well. The receiving well that may contain both semen sample and conditioning medium can be used as a positive control, meaning this receiving well will contain reagent.

The passageway between the analysis chambers provides a trapping function between two analysis chambers in fluid communication, so that the probability that a motile cell will travel from the first to the second of the two analysis chambers is larger than the probability that a cell having reached the second analysis chamber will travel back to the first analysis chamber. This trapping function is created by the relative sizes of the entrance port and the exit port of the passageway, with the entrance port being of a larger cross- sectional area than the exit port. Thus, a motile cell present in an analysis chamber in fluid communication with a previous and a further analysis chamber will be more likely to travel to the further analysis chamber than to the previous analysis chamber due to the respective cross-sectional areas of the entrance port and the exit port of the passageways leading to the further and the previous analysis chamber, respectively.

In a certain embodiment the mesoscale fluidic system comprises cell guidance structures to induce the cells to move from one analysis chamber to the next following the shortest route between the analysis chambers. In this embodiment a passageway, an analysis chamber, and/or the receiving well, when present, comprise one or more cell guidance structures having a first end and a second end, which guidance structure(s) are aligned so that the first end faces an entry point for cells in the passageway, the analysis cham- ber, or the receiving well, respectively, and the second end faces an exit point for cells in the passageway, the analysis chamber, or the receiving well, respectively. Motile cells, sperm cells in particular, tend to move along a wall or structure once it is encountered by the cell. Thus, by including cell guidance structures, e.g. in the passageway, the motile cells may be guided to travel from one analysis chamber to the next, which in turn may increase the signal and provide a better result in the quantification of the motility of or separation of the cells in a sample. In its simplest form a guidance structure may take the form of a rib, e.g. longitudinally aligned with the passageway, to provide a wall for the cell to swim along when moving between two analy- sis chambers. However, it is preferred that the cell guidance structure when present in a passageway, e.g. in the form of a rib, has a first end facing the entrance port of the passageway and a second end facing the exit port of the passageway, wherein the first end has a smaller cross-sectional area than the second end. This will further increase the difference in cross-sectional area available for a motile cell, so that the probability that the cell enters an exit port is even smaller when these guidance structures are present, than when no guidance structures are present. In another embodiment the analysis chambers and the receiving well, if present, also comprise one or more guidance structures. The same considerations apply when guidance structures are present in an analysis chamber or the receiving well as those applying the guidance structures are present in a passageway. However, in particular when two or more guidance structures are present in an analysis chamber or the receiving well these need not be parallel but may also have a larger distance between the first ends of the guidance structures than the distance be- tween the second ends of the guidance structures. Thus, the guidance structure will guide motile cells swimming from the first end of a guidance structure towards the second end of the guidance structure and further towards the entrance port of a further analysis chamber. Guidance structures present in a passageway may also have a larger distance between the first ends of two or more guidance structures located in the same passageway than the distance between the second ends of the guidance structures.

The analysis chambers of the mesoscale fluidic system may each have a solid-liquid separation unit. This solid-liquid separation unit is to be understood in the broadest terms as a unit capable of separating, e.g. retaining, solids, such as cells, from liquid when a liquid containing suspended solids passes through the unit. The solid-liquid separation unit has an upstream surface facing the analysis chamber and a downstream surface facing an effluent channel. Thus, when a sample liquid in the analysis chamber is withdrawn from the analysis chamber via the effluent channel solids, e.g. cells, are re- tained on the upstream side of the solid-liquid separation unit so that the solids are concentrated in liquid in the analysis chamber or on the upstream surface of the solid-liquid separation unit. This concentrating effect will allow that the solids in the sample are observed visually, e.g. on the upstream surface of the solid-liquid separation unit. The result may also be visible on the downstream surface of the solid-liquid separation unit. Alternatively, the solids may be suspended in a reduced volume of liquid remaining in the analysis chamber so that a higher concentration of the solids is provided in the analysis chamber compared to the concentration present in the liquid prior to withdrawal of liquid.

When the analysis chambers do not comprise a solid-liquid separation unit motile cells may be detected in the analysis chambers by direct observation of the motile cells. For example, the motile cells may have been dyed with an appropriate detection agent allowing detection and quantification of the population of cells present in an analysis chamber from the intensity of the dye. The dye may also be a fluorescent dye so that cells may be quantified from measurement of a fluorescence signal as appropriate.

In a specific embodiment the analysis chamber has a frustoconically shaped section having a narrow base and a broader top with the narrow base facing the bottom of the analysis chamber or the upstream surface of the solid-liquid separation unit. This may increase the visibility of solids suspended in the liquid remaining in the analysis chamber as may be derived from Lambert- Beer's law. A frustoconically shaped analysis chamber will also provide a further concentrating effect when the solids are retained on the surface of the solid-liquid separation unit, thus increasing the detectability of the solids in the sample.

A section of the analysis chambers, in particular when these have a frustoconically shaped section, may be located below the passageways between the analysis chambers. This will allow that enough liquid is withdrawn via the effluent channel to remove liquid from the passageway while retaining an amount of liquid in the optionally frustoconically shaped sections of the analysis chambers. Thereby further travel of cells between the analysis chambers may be prevented, and the mesoscale fluidic system may thus advantageously be used to separate cells according to their motility. Thereby the mesoscale fluidic system may separate more motile cells from less motile, e.g. for use in Assisted Reproductive Technologies (ART) when sperm cells are separated. However, motile cells may also be separated in the other embodiments of the device.

When the passageways are located above sections of the analysis chambers as described above, it is prevented that cells are dragged from one analysis chamber to the next via a flow of liquid between two analysis chambers, and therefore only motile cells will travel from one analysis chamber to a further analysis chamber. In a further embodiment, the interface between an analysis chamber, or the receiving well if present, and the entrance port of a passageway will contain an elevated ridge or hindrance forcing the cells to actively swim to enter the passageway. Such a ridge or hindrance will also prevent cells from being dragged into the passageway with a flow of fluid.

The bottom of the analysis chambers and the bottom of the passageways may also be in the same horizontal plane, so that the passageways are in plane with the analysis chambers, and the mesoscale fluidic system may then be described as "flat". This embodiment is particularly useful for analysis of cellular motility.

The mesoscale fluidic system may advantageously also comprise a receiving well in fluid communication with the first analysis chamber. A receiv- ing well will provide a location for adding a sample to be analysed for the quantity and motility of cells in the sample or to be separated based on their motility. Once the sample is added to the receiving well, the motile cells may travel towards the first analysis chamber and from there they may travel further into subsequent analysis chambers in serial fluid communication with the first. Having a receiving well separate from the analysis chamber may prevent false positive detection of non-motile cells, e.g. dead cells, as motile cells, since only live motile cells may travel from the receiving well to the analysis chambers.

The receiving well may also serve as a reservoir for a liquid, e.g. a sample conditioning medium containing components necessary for analysis of the cells. For example, the receiving well may contain pH buffers, salts, nutrients, detection agents capable of binding to motile cells of interest and allowing detection of the motile cells, or other components. A conditioning medium may also be present in the analysis chambers and passageways, both in case a receiving well is present or not. Thus, the mesoscale fluidic system may be prefilled with conditioning medium. The mesoscale fluidic system may also comprise separate reservoirs for sample conditioning media. In particular, the mesoscale fluidic system may comprise several separate reservoirs when different reactants are required at different stages of analysis of motile cells. In another embodiment, the sample conditioning medium is supplied in a separate capsule or the like. In this embodiment the mesoscale fluidic system may comprise a channel with an external fluid application port, which channel is in fluid communication with the first analysis chamber or the receiving well, if present. In particular, the capsule and the external fluid appli- cation port may be fitted with complementary connection devices. The capsule may be made from a flexible material allowing adjustment, e.g. reduction, of the volume to inject the contents of the capsule into the fluid application port. When the receiving well contains a sample conditioning medium, or when such liquids are contained in separate reservoirs, the receiving well or the reservoirs may be in fluid communication with the first analysis chamber via a channel or the like. This channel may comprise a valve or a membrane or similar structures preventing a flow in the channel until the valve or membrane is activated to allow a flow. This will ensure that premature entry of the conditioning medium into the analysis chambers is prevented. For example, an actuator may allow opening of the channel for flow of liquid from the reservoir to the analysis chambers. Thus, in one embodiment a sample conditioning medium is initially contained in a reservoir, e.g. the receiving well, whereas the analysis chambers do not contain liquid. Upon opening of the channel to flow, the conditioning medium will flow from the reservoir into the analysis chambers. Once the receiving well and the sample analysis chambers contain liquid, motile cells may travel from the receiving well to the analysis chambers. In an alternative embodiment the analysis chambers may be provided with a liquid, which may be the same liquid present in a reservoir or a different liquid. In yet another embodiment, no liquids are provided in the mesoscale fluidic system but added to the system before analysis of a sample. In a preferred embodiment, a detection reagent is not present in the analysis chambers or the passageways, but the detection reagent is present in a reservoir, e.g. a receiving well.

For certain embodiments it is preferred that mixing of sample and con- ditioning medium is avoided, e.g. substantially avoided, when applying the sample. Due to the mesoscale dimensions of the fluidic system liquids will generally move under laminar conditions and mixing of a sample with the conditioning medium will be avoided due to the laminar conditions allowing mixing by diffusion. For example, mixing of the sample and the conditioning medium may be substantially avoided by using microfluidics in order to put one fluid on top of the other in the laminar flow regime. Thus, addition, e.g. separate addition, of sample and conditioning medium without mixing causes that the cells may enter the conditioning medium by actively swimming from the sample and into the conditioning medium. By avoiding this mixing it is ensured that only motile cells will enter the analysis chambers, and thus preventing any non-motile cells and debris to have a negative influence on the test result in the form of false positive contributions. However, in other embodiments the mesoscale fluidic system may also comprise a mixer, e.g. a herring bone structure or the like, to ensure mixing of the sample with a con- ditioning medium when this is desired. In yet another embodiment the first analysis chamber, or the receiving well if present, further comprises a cell permeable filter which cell permeable filter defines a sample application side opposite a conditioning medium side. The cell permeable filter will have a pore size allowing motile cells to swim through the filter, e.g. a pore size from about 1 μηι to about 20 μηι or more. In general, the conditioning medium side will be facing the passageway or passageways connected to the further analysis chamber(s). The cell permeable filter may further provide a physical barrier between the sample application side and the conditioning medium side so that it will minimise, or even prevent, mixing of the liquid of a sample applied on the sample application side with a conditioning medium present at or added on the conditioning medium side. Thus, the cell permeable filter will ensure that cells are not transported from the sample application side to the conditioning medium side via convection, so that cells present at the conditioning medium side will be mo- tile cells that have traversed the cell permeable filter by swimming through it.

The mesoscale fluidic system may be upwards open allowing easy access to the analysis chambers and the passageways. However, it is preferred that the analysis chambers and the passageways are covered, e.g. with a lid or the like. When the analysis chambers and the passageways are upwards open this will allow that liquid in the analysis chambers is replaced with air upon withdrawal of the liquid via the effluent channel, when present. This same effect may be achieved when the analysis chambers and the passageways are covered by having an upwards open receiving well. Alternatively, the mesoscale fluidic system may be provided with an air inlet to allow with- drawal of liquid from the analysis chambers via the effluent channel. It is preferred that the receiving well is upwards open or otherwise allows an inflow of ambient air. The analysis chambers and the passageways may be permanently covered, or the mesoscale fluidic system may comprise a replaceable lid; a replaceable lid or the like is preferred when the mesoscale fluidic sys- tern is used to separate cells based on motility for use in other operations, such as ART. When the analysis chambers and the passageways are covered it is preferred that the cover, at least above the analysis chambers, is transparent for visual inspection of the analysis chambers and the solid-liquid separation unit. In one embodiment the area above the analysis chambers, or in another area allowing visual inspection of the analysis chamber and op- tionally also the solid-liquid separation unit, is provided with a magnifying lens for easier observation of the analysis chamber and the solid-liquid separation unit.

In one embodiment the system also comprises a means to provide a liquid driving force to move a liquid from one of the chambers, or a receiving well or reservoir if present, to another of the chambers via the passageway or the passageways, or the one or more of the channels if present. Thus, liquid may be driven from a chamber serving as a reservoir, e.g. a receiving well, to an analysis chamber. A liquid driving force may be provided by applying a positive relative pressure to the receiving well to disperse the liquid into an analysis chamber. Alternatively, a negative relative pressure applied to the effluent channel from the analysis chamber will create the same effect: move liquid from the receiving well to the analysis chamber and further withdraw liquid from the analysis chamber via the solid-liquid separation unit to the effluent channel.

Essentially all liquid may be withdrawn from the analysis chambers to concentrate the motile cells present in the analysis chamber on the surface of solid-liquid separation unit allowing detection of the cells. Alternatively, particularly when a section of the analysis chambers is located below the pas- sageways between the analysis chambers, e.g. when the analysis chambers comprise a frustoconically shaped section, withdrawal of liquid through a solid-liquid separation unit may provide a concentrating effect yielding a higher concentration of cells in liquid remaining on the upstream surface of the solid-liquid separation unit. This concentrating effect may be used in de- tecting and quantifying cells in the analysis chamber, and it may be used with particular advantage when separating cells based on their motility.

The means to provide a liquid driving force may be integrated into the mesoscale fluidic system, for example a syringe or the like may be integrated, e.g. in the substrate, to be in fluid communication with the effluent channel allowing a negative relative pressure to be applied to the effluent channel. An integrated syringe allows easy operation of the device by the end-user without requiring auxiliary pumps or the like. A syringe is preferably designed to be operable manually. The syringe may have a piston with predefined settings, to aid the user in operating the device. For example, the sy- ringe may have two settings with a first setting defining a "start position" and a second setting defining an "end position", so that a sample is applied in the receiving well with the piston of the syringe being in the start position; moving the piston to the end position will create a driving force to move the liquid from a reservoir, e.g. a receiving well, if present via the analysis chamber through the solid-liquid separation unit and into the effluent channel. The piston may also have more than two predefined settings with intermediate settings between the start and the end positions corresponding to various stages of the operation of the device. For example, an intermediate setting may correspond to a certain level of liquid in the analysis chambers. An in- termediate setting to indicate a certain level of liquid in the analysis chambers is particularly advantageous when a section of the analysis chambers is located below the passageways between the analysis chambers. Thus, the intermediate setting may correspond to a liquid level below the passageways.

Other means of providing a liquid flow in the device are also possible. For example, in the form of a peristaltic function acting on effluent channel. Alternatively, the system may be connected to an external auxiliary pump.

In another embodiment the mesoscale fluidic system also comprises means to regulate the temperature, e.g. to heat or cool, the analysis chambers. The mesoscale fluidic system may also be used at ambient tempera- ture. In one embodiment, the temperature regulating means will allow the analysis chambers to be retained at a desired temperature, e.g. 37°C or as high as 40°C or even above. This will allow the system to be used for cultur- ing cells, or the temperature regulation may be employed to modify the motility of the cells. For example, an increased temperature over a typical ambi- ent temperature will make motile cells, e.g. sperm cells, swim faster than at the ambient temperature. To be optimal for the purpose of separation and quantification of motile cells the temperature may also be chosen to correspond to the body temperature of a given mammal. Appropriate body temperatures for different species of mammals are well known to the skilled per- son, e.g. according to "Animal Heat." Encyclopedia Britannica. Chicago: Encyclopedia Britannica, 1965: A 965, the core temperatures of the mammals cattle, sheep, dog, cat, rabbit and pig are in the range 37.8-39.4°C depending on species of mammal.

The mesoscale fluidic system of the present invention is used in an- other aspect of the invention for estimating the motility of motile cells in a sample; the mesoscale fluidic system may also be used to quantify the amount or concentration of the motile cells. In another aspect the mesoscale fluidic system is used to separate cells based on motility, e.g. for providing live cells after the separation. In use the mesoscale fluidic system is initially provided with a conditioning medium, if not already present. A sample to be analysed is then added to the first analysis chamber or the receiving well if this is present. All analysis chambers, and the passageways between them, should contain liquid allowing motile cells to travel between the analysis chambers. After addition of the sample to the mesoscale fluidic system the cells will be allowed to travel from the point of addition to the further analysis chambers. The travelling time will be predetermined and depend on the type of cells and the specific application.

For detection and quantification the conditioning medium will typically comprise a detection agent. This may be a dye or a labelled antibody capable of binding to the cells of interest. When several cell types are present in the sample, the detection agent can advantageously bind specifically and possibly also selectively to the cells of interest. It is preferred that the detection agent will change colour upon binding to specific cell types, e.g. live motile sperm cells. Thus, a preferred detection agent will not have a visible colour when not being bound to a cell, but upon binding to the cell it will change to have a detectable, e.g. visible, colour. It is preferred that the detection reagent does not negatively affect the cells. In a preferred embodiment a conditioning medium containing a detection reagent is contained in a reservoir, e.g. a receiving well, whereas the liquid in the analysis chambers and the passageways do not contain the detection reagent. This will allow that the motile cells in the analysis chambers or on the surface of the solid-liquid separation unit do not need to be washed for detection since the only detection reagent in the analysis chambers has been carried there by the motile cells. Alternatively, a detection reagent is added to the receiving well, containing a detection re- agent free conditioning medium, upon addition of sample. This will achieve the same effect. In yet another embodiment, in particular when the mesoscale fluidic system is not filled with a conditioning medium, a detection reagent may be present in a dried form in a channel or chamber preferably downstreams of the first analysis chamber or in the cell permeable filter or the detection reagent may be present in the solid-liquid separation units, e.g. in the form of filters, or the mesoscale fluidic system may contain a detection reagent absorbed in a pad or the like. For example, the detection agent may be coated onto a chamber or channel wall. Having the detection agent in a dried form will allow easy operation of the system by the end user, since the detection agent will be present in a correct dosage and will be easily resolubi- lised upon application of sample conditioning medium. Unbound detection agent will typically be washed through the solid-liquid separation unit whilst cell bound dye will be retained. In further embodiments, agents with specific functions may be present, e.g. in a dried form, in an analysis chamber or passageway. For example, the last analysis chamber in the series may contain a chemokine to attract cells, or it may contain a toxic chemical to kill the cells and prevent that they swim backwards in the system.

When the mesoscale fluidic system is used to separate cells based on motility for use of the cells in subsequent applications, e.g. ART procedures, the conditioning medium typically will not contain a detection reagent.

Following the step to allow the cells to travel in the mesoscale fluidic system the liquid may be withdrawn from the analysis chambers via the effluent channel thereby forcing the liquid through the solid-liquid separation unit causing the cells to be retained on the upstream side of the solid-liquid separation unit.

When the mesoscale fluidic system is employed to quantify cells and their motility all liquid may be withdrawn from the analysis chambers to fix the cells on the solid-liquid separation unit for detection. However, in certain detection embodiments it may be advantageous not to withdraw liquid from the analysis chambers. The cells in each analysis chamber are then quantified, e.g. by visually inspecting the intensity of dye on or bound to the solid- liquid separation unit or by visually inspecting the intensity of dye in the analysis chambers, in particular when a solid-liquid separation unit is not present. For example, for a mesoscale fluidic system with five analysis cham- bers, visible dye in all five analysis chambers or on all five solid-liquid separation units, when present, will indicate a sample with an excellent result of cell concentration and motility, whereas only one or two died analysis chambers or solid-liquid separation units may indicate a poor result.

When the mesoscale fluidic system is employed to separate cells based on their motility the liquid is typically withdrawn to a level below the pas- sageways between the analysis chambers. Thereby motile cells are prevented from travelling between the analysis chambers. Cells may now be withdrawn, e.g. using a pipette, from an analysis chamber containing cells of interest. For example, for ART purposes it may of interest to withdraw cells from an analysis chamber containing cells having a high motility.

In a further aspect the invention relates to a system for analysis of motile cells in a sample, the system comprising a mesoscale fluidic system according to the first aspect of the invention and an external detector device comprising :

an optical detector;

a computer readable storage medium containing computer program code configured to quantify a detection reagent in the analysis chambers or on the solid-liquid separation units of the mesoscale fluidic system, if present;

a data processor for executing the computer program code. The external detector device may further comprise a display for presenting quantification results to an operator. For example, the results may be presented as a table with the estimated quantity of cells in each analysis chamber presented as an absolute quantity or as a relative quantity, e.g. of the relative distribu- tion of cells in the analysis chambers, or the result may be presented as an over all result to describe the sample, e.g. the sample may be described as "good", "normal", "average", "low", etc. In particular, quantification of the detection reagent in each of the analysis chambers allows that the motility of the cells in the sample is also analysed, since the more motile the cells the more cells will be present, and therefore the greater the intensity of the detection reagent, in the analysis chambers furthest away from the receiving well. The optical detector may be any detector capable of reading the results from the mesoscale fluidic system, e.g. quantifying the detection reagent on the solid-liquid separation units. Particularly useful optical detectors are those used for scanning barcodes or the like, and in a certain embodiment the optical detector is selected from the group consisting of a camera, a laser scanner, a CCD reader, a photodiode scanner. In a preferred embodiment the external detector device is a mobile user terminal. With "mobile user terminal" is meant any portable computing device, such as a mobile telephone, smart phone, personal digital assistant, portable computer, tablet computer or the like. The mobile user terminal is preferably a computing device employing Apple iOS, Android, Symbian, Windows Phone or similar operating systems,

A further aspect of the invention relates to a mobile user terminal, e.g. a smart phone, containing computer program code configured to quantify a detection reagent on the solid-liquid separation units of a mesoscale fluidic system according to the first aspect of the invention.

Brief description of the figures

In the following the invention will be explained in greater detail with the aid of examples of embodiments and with reference to the schematic drawings, in which

Fig. 1 shows top views of two different embodiments of the mesoscale fluidic system of the invention.

Fig. 2 shows a side view of an embodiment of the mesoscale fluidic system of the invention.

Fig. 3 shows a perspective of an embodiment of the mesoscale fluidic system of the invention.

Fig. 4 shows top views of two different embodiments of the mesoscale fluidic system of the invention.

Fig. 5 shows a top view of an embodiment of the mesoscale fluidic system of the invention.

Fig. 6 shows top views of two different embodiments of the mesoscale fluidic system of the invention.

Fig. 7 shows a perspective of an embodiment of the mesoscale fluidic system of the invention where the system has an integrated syringe.

Fig. 8 shows top views of further embodiments of the invention.

Fig. 9 shows the results of analyses using the mesoscale fluidic system of the invention. Detailed description of the invention

The present invention relates to a mesoscale fluidic system capable of separating motile cells based on their motility. This system may be used to analyse the content and also the motility of cells in a sample known or expected to contain motile cells. The system may also be used to separate cells based on their motility with the intention to provide a subset of cells from a sample containing such cells. This is particularly useful for separating sperm cells of high motility from sperm cells of lower motility for use in Assisted Reproductive Technologies (ART).

In the context of this invention the term "motile" and "motility" refers to cells that are capable of moving in a liquid independently of any flow of the liquid. In particular, motile cells are capable of moving in non-flowing liquids. The motile cells may also be said to be "travelling" or "swimming" etc. Motility may be considered to be random, or cells may respond to a stimulus by swimming, e.g. by swimming towards or away from a given condition. Common stimuli may be for motile cells to move in response to a chemical grad ient ("chemotaxis"), a temperature gradient ("thermotaxis"), a light gradient ("phototaxis"), a magnetic field line ("magnetotaxis"), or an electric field ("galvanotaxis"). Relevant stimuli will be known to the skilled person. In cer- tain embodiments cellular motility may be induced by providing a stimulus relevant to a motile cell of interest in order to make the cell swim from its point of addition towards subsequent analysis chambers in the system. For example, a chemokine or other chemical may be placed in the last analysis chamber in a series of analysis chambers to attract motile cells added in the first analysis chamber 21 or to a receiving well.

In the context of this invention the term "mesoscale" is intended to cover a range of sizes where the smallest dimension of channels is in the range from about 10 μηι to about 3 mm, e.g. about 100 μηι to about 1 mm, although the channels may also contain constrictions. Likewise an analysis chamber may be of a depth of about 100 μηι to about 20 mm or more, such as about 500 μηι to about 2 mm, e.g. about 500 μηι or about 1 mm, and the largest horizontal dimension may be from about 1 mm to about 50 mm, e.g. from about 1 mm to about 30 mm or from about 1 mm to about 20 mm, or from about 1 mm to about 10 mm, e.g. from about 2 mm to about 6 mm. The size of the receiving well, if present, should be sufficient to hold sample liquid to fill the analysis chambers of the system with fluid for analysis. It can generally be said that fluids in mesoscale fluidic systems will be flowing under laminar conditions, and fluidic systems with channels or chambers different from those defined above may well be described as "mesoscale" as long as fluids contained in the systems flow under laminar conditions. Referring now to the figures, Fig. 1 depicts a top view of two embodiments of the invention. The figures shows the mesoscale fluidic system 1 with a first analysis chamber 21 in fluid communication with a further analysis chamber 22 via a passageway 3. The passageway 3 has an entrance port 31 in fluid communication with the first analysis chamber 21 and an exit port 32 in fluid communication with the further analysis chamber 22. The two embodiments have differently designed passageways 3 to provide that the cross-sectional area of the entrance port 31 is larger than the cross-sectional area of the exit port 32. Fig. 1 further indicates the optional solid-liquid sepa- ration unit 4 in each analysis chamber 21,22. The mesoscale fluidic system with a solid-liquid separation unit 4 further comprises an effluent channel. However, this is not shown in Fig. 1. The mesoscale fluidic system 1 may further comprise an additional control chamber 211 that is not connected via channels or the like to either of the analysis chambers 21,22 or the receiving well if present. The control chamber 211 may be filled with detection reagent and can thus provide a reference for comparison with the analysis chambers 21,22 to quantify motile cells present in the analysis chambers 21,22 so that the control chamber 211 provides a negative control. The control chamber 211 may also comprise a solid-liquid separation unit connected to the effluent channel 5, if present (not shown in Fig. 1). It is also possible to add a known concentration of cells to the control chamber 211 so that the control chamber 211 may provide a reference for quantifying cells. A positive control may be provided by detecting cells in the receiving well or the first analysis chamber 21.

Fig. 2 shows a side view of a mesoscale fluidic system 1 of the invention, depicting the first analysis chamber 21 in fluid communication with the further analysis chamber 22 via a passageway 3. Although not depicted in Fig. 2, it is preferred that the mesoscale fluidic system 1 comprises multiple analysis chambers, e.g. five, serially connected via passageways 3. The side view in Fig. 2 further shows the upstream surface 41 and the downstream surface 42 of the optional solid-liquid separation unit 4. The downstream surfaces 42 of the optional solid-liquid separation units 4 are in fluid communication with an effluent channel 5. The substrate 10 of the mesoscale fluidic system 1 may comprise a cover 13, which is preferably transparent and may comprise, e.g. above the analysis chambers 21,22, magnifying lenses 14. Fig. 3 shows a perspective of the mesoscale fluidic system 1 of the invention, where the system comprises a first analysis chamber 21 in fluid communication with the further analysis chamber 22 via three intermediate analysis chambers 23. Fig. 3 also shows a receiving well 11. In the embodi- ment in Fig. 3, the channel leading from the receiving well 11 to the first analysis chamber 21 is provided with cell guidance structures 33.

In a certain embodiment the largest horizontal dimension of the analysis chambers 21,22 is in the range from about 2 to about 6 mm. In particular, when the mesoscale fluidic system is for analysis of motile cells, the larg- est horizontal dimension of the analysis chambers 21,22 may be about 2 mm, about 3 mm, about 4 mm, about 5 mm, or about 6 mm. Within the range of flow-rates typically employed in the mesoscale bioreactors of the invention the liquids will be moving in an essentially laminar flow.

In some embodiments of the invention the mesoscale fluidic system is particularly suited for ART-purposes. In this case the horizontal dimensions may be as defined above, although in some embodiments the horizontal dimensions of the analysis chambers 21,22 may be in the range from about 5 mm to about 30 mm, e.g. about 5 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm.

The passageway 3 of the mesoscale fluidic system of the present invention will generally be of a size comparable to the analysis chambers 21,22. The passageway 3 will define a distance between two analysis chambers, and it will comprise an entrance port 31 and an exit port 32, where the cross-sectional area of the entrance port 31 will be larger than the cross- sectional area of the exit port 32. The width of the entrance port 31 may be the same as the diameter of the first analysis chamber 21, or it may have a width which is smaller than the diameter of the first analysis chamber 21. The width of the exit port 32 will typically be smaller than the width of the entrance port 31. The height of the passageway 3 is typically the same at the entrance port 31 and the exit port 32, although the decrease in cross- sectional area between the two ports 31,32 may also be obtained by varying the height of the passageway 3. The height of the passageway 3 may be from about 10 μηι to about 3 mm or more, e.g. up to the height of the analysis wells, such as about 100 μηι, about 500 μηι or about 1 mm. When the mesoscale fluidic system is for separation of motile cells for subsequent re- covery of cells, e.g. for ART purposes, the height of the passageway may also be larger, such as from about 1 mm to about 10 mm. In some embodiments the passageway 3 may take the form of a channel where the superficial width and height are the same from the entrance port 31 to the exit port 32 where the difference in cross-sectional area is provided width cell guidance structures 33 as outlined below. The length of the passageway 3, i.e. the distance to be traversed by the motile cells between each analysis chamber may be up to about 1 cm or more, e.g. 15 mm, more typically up to about 5 mm or up to about 3 mm, in general the minimum length of the passageway will be about 1 mm, although the minimum length may also be shorter, such as about 500 pm or about 100 μηι.

Cell guidance structures 33 are depicted schematically in Fig. 4 showing two different embodiments of the structures. When cell guidance structures 33 are employed these will be of a size to fit at least one cell guidance structure 33 into the passageway 3. The passageway 3 may also contain a plurality of cell guidance structures 33. For example, the passageway 3 may contain 2, 3, 4, 5, 6, 7, 8, 9 or 10 or even more cell guidance structures 33. A cell guidance structure 33 may protrude from a wall, floor or ceiling of a passageway 3, and it may be of a size to reach and join the surface of the opposite wall, floor or ceiling as appropriate. It is preferred that a cell guidance structure 33 has a small dimension at the end 331 facing the entrance port 31 of the passageway 3 and a larger dimension at the end 332 facing the exit port 32 of the passageway 3 in order to decrease the possibility that a motile cell will enter an exit port 32 and move in the "wrong" direction to- wards a previous analysis chamber. Whatever the shape of the cell guidance structure 33 it will provide a guiding surface to guide a motile cell to the shortest route between the first and the further analysis chamber 22.

In one embodiment the analysis chambers 21,22 comprise a section located below the passageway 3 between the analysis chambers 21,22. The depth of the section below the passageway 3 may be from about 100 μηι to about 3 mm, such as about 500 μηι to about 2 mm, such as about 1 mm. This section is preferably frustoconical in shape with a narrow base of the frustum facing the bottom of the chamber or the upstream surface 41 of the solid-liquid separation unit 4, when this is present. The narrow base of the frustum will typically be from about 500 μηι to about 2 mm in diameter, and the top of the frustum from about 2 mm to about 6 mm or larger, such as about 8 mm or about 10 mm. In certain embodiments for ART-purposes the largest horizontal dimension and the depth of the analysis chamber 21,22 may be as defined above.

The analysis chambers 21,22 of the mesoscale fluidic system 1 of the present invention may each comprise a solid-liquid separation unit 4. The solid-liquid separation unit 4 defines an upstream surface 41 facing the analysis chamber 21,22 and a downstream surface facing an effluent channel 5. Any type of solid-liquid separation unit capable of separating a solid, e.g. cells, suspended in a liquid when the liquid passes through the solid-liquid separation unit 4 is appropriate for the present invention. Thus, the solid- liquid separation unit 4 may be a filter, e.g. a filter paper, a filter membrane etc., a sieve, a packed bed of particles capable of retaining particles in a liquid. Appropriate materials for the solid-liquid separation unit 4 may have a size cut-off of e.g. about 0.1 μηι to about 20 μηι, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 μηι although larger cut-off sizes may also be relevant, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μηι. Preferred cut-off sizes are 0.2 μηι, 0.45 μηι, 1 μηι and 3 μηι. The solid-liquid separation unit can be placed in the bottom of the analysis chamber. However, in a further embodi- ment it can also be placed in a ceiling of the analysis chamber, and in still further embodiments the solid-liquid separation unit can be placed in a vertical wall of the analysis chamber. The solid-liquid separation unit may contain a detection reagent.

The downstream surface 42 of a solid-liquid separation unit 4 will be in fluid communication with an effluent channel 5. The mesoscale fluidic system 1 preferably comprises only a single effluent channel 5 allowing that a single means to provide a liquid driving force, e.g. a pump or a syringe, may be employed to withdraw liquid from the analysis chambers 21,22. The effluent channel 5 will be of mesoscale dimensions so that liquid flow in the effluent channel 5 will be laminar as defined above. Typically, the diameter of the effluent channel 5 will be from about 500 μηι to about 3 mm, e.g. about 1 mm. In general, it is desirable that the diameter of the effluent channel 5 does not introduce an excessive pressure drop in the channel so that liquid from the analysis chambers 21,22 may be easily withdraw using simple means, e.g. a syringe. In one embodiment the mesoscale fluidic system 1 of the invention comprises a receiving well. The receiving well may be of a larger volume than the analysis chambers 21,22. In particular, the receiving well may also serve as a reservoir for liquid, e.g. a conditioning medium; in this embodiment the receiving well should be of a volume to hold sufficient liquid to fill the analysis chambers 21,22 and the passageway 3 with liquid. The receiving well may be upwards open to allow easy addition of a sample, and optionally also a conditioning medium, in the receiving well. In one embodiment the mesoscale fluidic system 1 is pre-filled with a conditioning medium, and in this case the receiving well may be provided with a removable cover, e.g. a plastic membrane, capable of preventing access of air and contaminants from the ambient surroundings until removal of the cover. In another embodiment the receiving well is pre-filled with a conditioning medium containing a detection agent and the analysis chambers 21,22 and the passageway 3 are pre-filled with a conditioning medium not containing a detection agent. In this embodiment the channel between the receiving well and the first analysis chamber 21 is preferably provided with means to provide a hindrance to flow between the receiving well and the first analysis chamber 21, e.g. with a membrane or plug or the like, until removal of the hindrance, e.g. piercing a membrane or removal of a plug. This embodiment ensures that only motile cells are detected, as non-motile cells remain in the receiving well, and furthermore it is advantageous since no detection reagent will reach the analysis chamber without the action of the motile cells. In this embodiment the system is advantageously a closed system, to make it more stable when used and more storage friendly.

In a specific embodiment, the mesoscale fluidic system 1 of the invention the first analysis chamber is in parallel fluid communication via passageways as defined in claim 1 with two or more further analysis chambers. This embodiment is illustrated in Fig. 5. The term "parallel fluid communication" indicates that the further analysis chambers 22 are in fluid communication with the first analysis chamber 21 via separate and distinct passageways 3, but that no channels or the like otherwise connect the further analysis chambers 22. Thus, once a motile cell has entered a passageway 3, it cannot readily swim to another further analysis chamber 22. Each further analysis cham- ber may however be in fluid communication with additional serially connected further analysis chambers (not shown). Fig. 5 further shows the presence of a control chamber 211.

The mesoscale fluidic system 1 of the invention may also comprise a cell permeable filter 7 defining a sample application side 71 opposite a condi- tioning medium side 72. Two different embodiments of the cell permeable filter 7 are depicted in Fig. 6. The cell permeable filter 7 serves to minimise or prevent mixing of sample and conditioning medium while allowing motile cells to swim through the cell permeable filter 7 and enter the conditioning medium present on the other side. The cell permeable filter 7 may be of any ap- propriate material, such as cellulose acetate/nitrate filter. The cell permeable filter 7 may have any pore size, e.g. from 1 μηι to 20 μηι, such as 1, 3, 5, 8, 10, 12, 15 μηι etc., allowing motile cells to swim through it while at the same time minimising flow of liquid through it. The cell permeable filter 7 may further contain compounds, e.g. hyaluronic acid, attracting cells to the cell per- meable filter 7 and to swim through it. The cell permeable filter 7 may also comprise a detection agent so that cells will be brought into contact with the detection agent upon passing the filter 7. The cell permeable filter 7 may have any shape allowing the definition of a sample application side 71 and a conditioning medium 72 side. For example, the cell permeable filter 7 may be span the entrance port 31 of the first passageway 3, or the filter 7 may divide a first analysis chamber 21 or a receiving well in two separate sections, e.g. in one embodiment the cell permeable filter 7 has a cylindrical shape and is located in a receiving well or the first analysis chamber 21 so that the cylindrical shape define a sample application point. This embodiment is illustrated in Fig. 6. The same effect can also be obtained using other filter shapes, e.g. rectangular, oval etc.

The mesoscale fluidic system 1 of the invention will comprise a substrate 10, which may be made from any convenient material, such as a polymer, a glass, a metal, a ceramic material or a combination of these. The substrate 10 will define a bottom surface and a sidewall of the analysis chamber and the passageway 3 between the chambers; when viewed from above the sidewall may form a perimeter for the chamber, which is round, square, polygonal, or oblong, etc. ; the perimeter is preferably round. Likewise, the passageway 3 may have any convenient shape when viewed from above, as long as the cross-sectional area of the entrance port 31 is larger than the cross-sectional area of the exit port 32.

In some embodiments the analysis chamber is constructed in a way to allow physical access to the chamber. When a receiving well is present it is preferably physically accessible. In this context the term "physical access" means that a tool may be inserted into the liquid in the analysis chamber or receiving well to manipulate the contents of the chamber or well. This manipulation may be to insert or remove one or more cells from the analysis chamber or from a receiving well, or it may involve manipulations of cells already present in the chamber or well.

It is preferred that the analysis chambers 21,22 and the passageways 3 are covered. A cover 13 may be permanent, e.g. be a ceiling over the analysis chambers 21,22 and the passageways 3, or the cover 13 may take the form of a closable member, such as a sliding or hinged lid or a removable lid, or the cover 13 may be provided by an elastic membrane allowing physical access to the analysis chambers 21,22 by piercing with an appropriate instrument; an elastic membrane may have a self-sealing capability.

It is preferred that the cover 13, at least above the analysis chambers 21,22, is transparent. Transparency means that the contents of an analysis chamber may be observed, e.g. with the naked eye or using a microscope or the like. However, in addition to being transparent to visible light the cover 13 may also be transparent to other wavelengths, such as ultraviolet light or infrared light. Transparency to ultraviolet light allows that certain fluorescent molecules, e.g. dyes or labels, can be excited with an appropriate light source. The remaining substrate 10 may likewise be transparent. The substrate 10 and the cover 13 may have the same or different characteristics regarding transparency. However, in certain embodiments the substrate 10 or the cover 13 may comprise filters to ranges of wavelengths to aid in excitation and observation of fluorescent dyes. For example, one part of the sub- strate 10 may be transparent to an excitation wavelength but not the emission wavelength and the cover 13 may in turn be transparent to the emission wavelength but not the excitation wavelength.

In certain embodiments the cover 13 above the analysis chambers 21,22 comprises a magnifying lens 14. This will allow easier observation of the contents of an analysis chamber and preferably also the solid-liquid separation unit 4. Magnifying lenses are well-known to the skilled person.

The cover 13 may also be marked with a colour-gradient indicating the correlation between the intensity of a dye employed as a detection agent, so that the user can easily estimate the amount of cells in a given analysis chamber or on the solid-liquid separation unit 4 after withdrawal of the liquid.

In a preferred embodiment, the quantification of motile sperm cells is performed by using a smart phone as an external detector device. In this embodiment the receiving well 11, the analysis cambers 21,22 (and option- ally 23), and the passageways 3 of the mesoscale fluidic system 1 are covered with a cover 13, where at least the part of the cover covering the analysis chambers is transparent and constitute a smart phone readable display. By using a smart phone provided with a digital camera, and computer program code, e.g. an app, enabling the smart phone to prosecute data obtained by the camera, the display can be read and data for the purpose of quantification and qualification (motility) of motile sperm cells can be obtained, and thereby give the user of the smart phone an indicative value of result, e.g. in terms of the concentration and motility of the sperm cells. The data can be stored in the smart phone and used to compare with data from prior tests. In a particular embodiment the computer program code is configured to upload the results to a database to allow comparison with results in the database, e.g. from other users or from prior tests. Data uploaded to the database may be stored in the database for future comparison.

The mesoscale fluidic system 1 of the present invention preferably comprises an integrated, e.g. integrated in the substrate 10, means to provide a liquid driving force to withdraw liquid from the analysis chambers 21,22. In a preferred embodiment the system 1 has an integrated syringe 6 in fluid communication with the effluent channel 5. The syringe 6 preferably has predefined settings, e.g. corresponding to a "start" position where a sample cells containing motile cells is added to the first analysis chamber 21 or to the receiving well 11, if present, and an "end" setting corresponding to a position where liquid is withdrawn from the analysis chambers 21,22 through the solid-liquid separation unit 4 and into the effluent channel 5. Thus, moving the piston from start to end will withdraw the liquid. In another embodiment the piston also has predefined intermediate settings. For exam- pie, the start position may correspond to a setting with no liquid in the analysis chambers 21,22, an intermediate setting may correspond to a setting where liquid is drawn into the analysis chambers 21,22 and the passageway 3; an end position may then provide a setting where the liquid is withdrawn from the analysis chambers 21,22 as described above. A further intermediate setting may provide a liquid level in the analysis chambers 21,22 where no liquid is contained in the passageway 3 so as to prevent motile cells from moving between the analysis chambers 21,22. Such an intermediate setting is particularly useful when the system 1 is used to separate cells in order to provide cells, e.g. live cells, of a desired motility for other procedure, e.g. sperm cells for use in ART procedures. Fig. 7 shows a perspective view of the system 1 of the present invention with an integrated syringe 6 with three settings. The embodiment depicted in Fig. 7 further comprises a capsule 15 and an external fluid application port 16; the capsule 15 may comprise a sample conditioning medium that may be applied to the receiving well 11 via the external fluid application port 16.

The syringe 6 may also comprise a resilient element, such as a spring, allowing the syringe 6 to be operated by pushing a button or by activating another actuator. For example, the system may comprise a syringe 6 with three different settings and a spring to move the piston so that a first activation of the piston will move from the first position to the intermediate position, and a second activation will move the piston from the intermediate position to the end-position with minimal effort to the user. When the syringe 6 comprises intermediate settings the piston may comprise a protrusion or pro- jection which may engage with a recess, a groove or a slot in the cylinder of the syringe 6. Thus, when the protrusion or projection engages the recess, groove or slot the intermediate setting has been reached. Being an intermediate setting the engagement should not completely prevent further movement of the piston in the cylinder, however it should be obvious to the user that the intermediate setting has been reached. The engagement is preferably accompanied with an audible click or the like to further inform the user that the intermediate setting has been reached.

In a certain embodiment the mesoscale fluidic system 1 comprises a means to regulate the temperature, in particular to increase the temperature over typical ambient temperature. Appropriate means to regulate tempera- ture may thus comprise a heating element, such as a coil of an electrically conductive wire, a Peltier element, tubes for a heating and/or cooling liquid, or similar. It is noted that a Peltier element may also be used to cool the system if desired.

The mesoscale fluidic system 1 of the present invention is preferably constructed from essentially transparent materials with hydrophilic surfaces, although well-defined regions of hydrophobic surfaces may also be used. In certain embodiments, the mesoscale fluidic system 1 may comprise sections of superhydrophilic surfaces to allow easy wetting of the analysis chambers and passageways with aqueous liquids. The mesoscale fluidic system 1 may also be constructed from non-transparent, e.g. white, materials. The construction material is preferably one or more thermoplastic polymers, although other materials, such as glass, silicon, metal, elastomeric polymers, may also be used.

Channels, chambers, passageways and other structures of the mesoscale fluidic system 1 of the present invention may be formed by joining a first substrate 10 comprising structures corresponding to the channels and chambers with a second substrate. In the following, such features are generally referred to as "channels and chambers" although this should not be con- sidered limiting. Thus, the channels and chambers may be formed between two substrates upon joining the substrates in layers. The mesoscale fluidic systems are not limited to two substrate layers. In certain embodiments multiple substrate layers may be used where each of the substrate layers may comprise structures for channels and chambers as appropriate. In particular, the solid-liquid separation unit 4 may be contained in a single layer, and likewise the effluent channel may be formed between two substrate layers. These multiple substrate layers are then joined so as to be assembled as a mesoscale fluidic system 1.

The structures corresponding to the channels and chambers in the substrate 10 may be created using any appropriate method. In a preferred embodiment the substrate materials are thermoplastic polymers, and the appropriate methods comprise milling, micromilling, drilling, cutting, laser ablation, hot embossing, injection moulding and microinjection moulding. These and other techniques are well known within the art. The channels may also be created in other substrate materials using appropriate methods, such as casting, moulding, soft lithography etc. It is also possible to employ different types of materials, e.g. thermoplastic materials, glasses, metals etc. to make a single mesoscale fluidic system.

The substrate materials may be joined using any appropriate method. In a preferred embodiment the substrate materials are thermoplastic polymers, and appropriate joining methods comprise gluing, solvent bonding, clamping, ultrasonic welding, and laser welding. Other relevant methods are fixing with screws or other fastening means.

The mesoscale fluidic system of the invention is employed with a con- ditioning medium. The term "conditioning medium" is not intended to be limiting, but "conditioning" refers to it that the medium may contain components necessary for analysis of the motile cells and also for keeping the cells viable. Thus, the conditioning medium may contain pH buffers, salts, nutrients as appropriate to a cell type of interest. The conditioning medium may also con- tain a detection agent, or a detection agent may be added separately to a conditioning medium in the system or present in a dried form in a channel or chamber. A detection agent will be capable of binding to motile cells of interest and allowing detection of the motile cells, and the detection agent may therefore be or comprise a dye or a binding partner for a cell labelled with a dye, e.g. an antibody against a surface marker on the cell, which antibody is labelled with a dye or radioactive isotope or the like. Appropriate dyes may be fluorescent dyes or other dyes; a dye may be capable of binding specifically or non-specifically to a cell type of interest. Exemplary dyes for use as detection labels for quantifying sperm cells comprise Coomassie blue, Trypan blue, Crystalviolet, Nile blue, Nile red, Hematoxylin, Acid fuchsine, Eosin, Sa- franin, Nigrosin, Acridine orange, Giemsa, Erythrosin, Papanicolaou, Methylene blue, Neutral red, Phenol red, Hoechst stain, Resazurin, Bismarck Brown, Dimethyl tetrazolium (MTT), Orange G, Periodic acid-Schiff, RoWright's stain, Jenner's stain, Leishman stain, Giemsa stain, Romanowsky stain, Sudan stain. When the mesoscale fluidic system is employed for separating motile cells in a sample, e.g. to be used for ART, it is preferred that no dye or label is present.

In use a sample containing motile cells is introduced into first analysis chamber 21, or optionally to the receiving well 11. The cells are then allowed to travel to the further analysis chambers 22,23. The time allowed after addi- tion of a sample will depend on the type of motile cells, but will typically be from about 10 minutes to about 1 hour, although shorter or longer times may also be used. When the system is used with sperm cells, the time allowed may constitute about 20 minutes, about 30 minutes, or about 40 minutes. When the time has been spent the liquid may be withdrawn from the analysis chambers 21,22,23, at least to a liquid level below the passageway 3, or to a level to bind the motile cells in the analysis chambers 21,22,23 to the upstream surfaces 41 of the solid-liquid separation units 4, when present. The cells in each analysis chamber 21,22,23 may then be detected and/or quanti- fied, or the cells may be withdrawn from the analysis chambers 21,22,23 using e.g. a pipette. Detection of motile cells on the solid-liquid separation units or in the analysis chambers may be performed simply by comparing the colour intensity observed, e.g. on the upstream surfaces of the 41 of the solid- liquid separation units 4, and comparing this to an indication of the correla- tion between cell number and dye intensity, e.g. provided on the substrate 10 or from a control chamber 211.

Further exemplary lay-outs of the mesoscale fluidic system of the invention are depicted in Fig. 8. In general these are shown without the presence of the solid-liquid separation unit. However, the embodiments may all comprise solid-liquid separation units and the effluent channel if desired. Likewise, the design features between the depicted embodiments may be combined freely, and in general the features of all embodiments described above may be combined with consideration of the limitations implied in certain embodiments.

Example

Six experiments were conducted (table 1). Each experiment represents a semen sample from different males. Two of the semen samples were of good quality according to the WHO criteria as determined by microscopy analysis, two of the semen samples were of normal quality according to the WHO criteria, and two of the semen samples were of low quality according to the WHO criteria (see table 1 below).

Each of the six experiments was conducted as follows: The semen samples were placed at ambient temperature in 30 minutes in order to liq- uefy. Then 0.3 ml liquefied semen was pipetted into the receiving well (4) of a mesoscale fluidic system (1) with 5 analysis chambers (21, 22, 23, see Fig. 3). With a pipette, the 5 analysis chambers were gentle filled with conditioning medium (water, NaCI, KCI, CaCI₂, NaH₂P0₄, NaHC0₃, MgS0₄, pyruvate, glucose, lactate, Hepes, Human Serum Albumin, pH 7.3-7.6) containing me- thylthiazolyldiphenyl-tetrazolium bromide (MTT) as dye for staining of living sperm cells, so the medium covered the liquefied semen.

The sperm cells were allowed to swim into the analysis chambers for 1 hour at ambient temperature. The content of the chambers was passed through the solid-liquid separation unit (4) (membrane filter) by vacuum using a disposable syringe. The medium was disposed, the fluidic system was disassembled and the filter was taken out, dried and stored.

The result is presented as 5 dots on a membrane filter (solid-liquid separation unit 4), where each dot represents the fraction of semen in an analysis chamber, and where the motility of the semen is increasing from the left to the right side of the filter. The concentration of living sperm cells in each of the 5 analysis chambers was reflected by the intensity of the staining on the membrane filter. The results are depicted in Fig. 9.

The experiments demonstrate that only the sperm cells of the high quality semen had a motility allowing them to swim to the last of the 5 chambers (right side the filter membrane in table 1). The intensity of the staining was also highest in this group compared to the two other groups, where both the staining intensity and the migration rate were lower decreasing with declining quality.

In conclusion the experiments demonstrate that both concentration and motility can be measured in one assay.

Table 1. Result of experiments determining sperm cell motility and concentration using the mesoscale fluidic system.

Semen WHO criteria (concen¬

Concentration of motile quality tration of motile sperm

sperm cells pr. ml, motility (WHO cells pr. ml semen, moquality in sample

criteria) tility quality)

126 x 10⁶ cells/ml semen,

Good >50 x 10⁶ cells/ml semen,

grade 4 + 3 + 2

Good 198 x 10⁶ cells/ml semen, Semen WHO criteria (concen¬

Concentration of motile quality tration of motile sperm

sperm cells pr. ml, motility (WHO cells pr. ml semen, moquality in sample

criteria) tility quality)

grade 4 + 3 + 2

20 x 10⁶ cells/ml semen, grade 4 + 3

Normal

>10 x 10⁶ cells/ml semen, grade + 2

4 + 3 + 2 16 x 10⁶ cells/ml semen, grade 4 + 3

Normal

+ 2

4.0 x 10⁶ cells/ml semen, grade 4 +

Low

< 10 x 10⁶ cells/ml semen, grade 3 + 2

4 + 3 + 2 3.3 x 10⁶ cells/ml semen, grade 4 +

Low

3 + 2

Semen quality WHO criteria:

Grade 4: Sperm with progressive motility. These are the strongest and swim fast in a straight line. Motility > 25 m/s.

Grade 3: Sperm cells with non-linear motility: These also move forward but tend to travel in a curved or crooked motion. Motility = 5-24

Grade 2: Sperm cells with non-progressive motility because they do not move forward despite the fact that they move their tails. Motility < 5

Grade 1 : These sperm cells are immotile and fail to move at all. Motility = 0 m/s.

Claims

P A T E N T C L A I M S

1. A mesoscale fluidic system comprising a substrate having a first analysis chamber in fluid communication with a further analysis chamber via a passageway;

the passageway having an entrance port in fluid communication with the first analysis chamber and an exit port in fluid communication with the further analysis chamber, wherein the cross-sectional area of the entrance port is larger than the cross-sectional area of the exit port.

2. The mesoscale fluidic system according to claim 1, wherein each analysis chamber further comprises a solid-liquid separation unit defining an upstream surface facing the analysis chamber and a downstream surface facing an effluent channel, so as to allow fluid communication between the analysis chamber and the effluent channel via the solid-liquid separation unit.

3. The mesoscale fluidic system according to any one of claims 1 or 2, wherein the substrate further comprises a receiving well in fluid communication with the first analysis chamber.

4. The mesoscale fluidic system according to any one of claims 1 to

3, wherein the smallest dimension of a passageway or channel is in the range from about 10 μηι to about 3 mm.

5. The mesoscale fluidic system according to any one of claims 1 to

4, wherein the analysis chamber has a depth of about 100 μηι to about 20 mm.

6. The mesoscale fluidic system according to any one of claims 1 to

5, wherein the analysis chamber has a largest horizontal dimension of from about 1 mm to about 50 mm.

7. The mesoscale fluidic system according to any one of claims 1 to

6, wherein the height of the passageway is from about 10 μηι to about 3 mm.

8. The mesoscale fluidic system according to any one of claims 1 to

7, wherein the length of the passageway is from about 100 μηι to about 1 cm.

9. The mesoscale fluidic system according to any one of claims 2 to

8, wherein the solid-liquid separation unit has a size cut-off from about 0.1 μηι to about 20 μηι.

10. The mesoscale fluidic system according to any one of claims 2 to 9, wherein the diameter of the effluent channel is from about 500 μηι to about 3 mm.

11. The mesoscale fluidic system according to any one of claims 1 to

10, wherein the substrate comprises multiple analysis chambers in serial fluid communication via passageways as defined in claim 1.

12. The mesoscale fluidic system according to any one of claims 1 to

11, wherein a passageway, an analysis chamber, and/or the receiving well, when present, comprise one or more cell guidance structures having a first end and a second end, which guidance structure(s) are aligned so that the first end faces an entry point for cells in the passageway, the analysis cham- ber, or the receiving well, respectively, and the second end faces an exit point for cells in the passageway, the analysis chamber, or the receiving well, respectively.

13. The mesoscale fluidic system according to claim 12, wherein the cell guidance structures take the form of ribs longitudinally aligned with the passageway with each rib having a first end facing the entrance port of the passageway and a second end facing the exit port of the passageway, wherein the first end has a smaller cross-sectional area than the second end.

14. The mesoscale fluidic system according to any one of claims 12 to 13, wherein two or more cell guidance structures located in an analysis chamber and/or the receiving well, when present, have a larger distance between first ends of the guidance structures than the distance between second ends of the guidance structures.

15. The mesoscale fluidic system according to any one of claims 1 to 14, wherein the first analysis chamber is in parallel fluid communication via passageways as defined in claim 1 with two or more further analysis chambers.

16. The mesoscale fluidic system according to claim 15, wherein the two or more passageways are of different length.

17. The mesoscale fluidic system according to any one of claims 1 to 16, wherein the first analysis chamber and/or the receiving well comprises a cell permeable filter which cell permeable filter defines a sample application side opposite a conditioning medium side.

18. The mesoscale fluidic system according to claim 17, wherein the cell permeable filter has a pore size from about 1 μηι to about 20 μηι.

19. The mesoscale fluidic system according to any one of claims 17 to 18, wherein the cell permeable filter comprises hyaluronic acid.

20. The mesoscale fluidic system according to any one of claims 1 to

19, wherein the analysis chambers and the passageways are covered with a transparent cover provided with a magnifying lens.

21. The mesoscale fluidic system according to any one of claims 1 to

20, wherein the analysis chamber, the passageway, and/or the receiving well are upwards open.

22. The mesoscale fluidic system according to any one of claims 1 to

21, wherein the analysis chambers comprise a frustoconically shaped section having a narrow base and a broader top with the narrow base facing the bottom of the analysis chamber or the upstream surface of the solid-liquid separation unit, if present.

23. The mesoscale fluidic system according to any one of claims 1 to

22, wherein mesoscale fluidic system further comprises a reservoir for a con- ditioning medium, which reservoir is in fluid communication with the first analysis chamber or the receiving well via a channel.

24. The mesoscale fluidic system according to claim 23, wherein the channel comprises a valve or membrane to prevent flow in the channel and an actuator to allow opening of the channel for flow of liquid.

25. The mesoscale fluidic system according to any one of claims 1 to

24, wherein mesoscale fluidic system further comprises a conditioning medium.

26. The mesoscale fluidic system according to any one of claims 1 to

25, wherein the conditioning medium further comprises a detection agent.

27. The mesoscale fluidic system according to claim 26, wherein the detection agent is in a dried form.

28. The mesoscale fluidic system according to any one of claims 1 to

27, wherein the mesoscale fluidic system further comprises means to provide a liquid driving force to move a liquid in the mesoscale fluidic system.

29. The mesoscale fluidic system according to any one of claims 1 to

28, wherein the mesoscale fluidic system further comprises means to regulate temperature.

30. The mesoscale fluidic system according to any one of claims 2 to

29, wherein the system further comprises a syringe in fluid communication with the effluent channel.

31. The mesoscale fluidic system according to any one of claims 1 to 30, wherein a section of the analysis chambers is located below the passageways between the analysis chambers.

32. A method of estimating the quantity and motility of cells in a sample comprising the steps of:

providing a mesoscale fluidic system according to any one of claims

1 to 31;

optionally adding a conditioning medium to the analysis chambers and the passageways between them;

mixing a sample containing motile cells with a detection reagent; adding the sample to the first analysis chamber, or optionally to the receiving well;

allowing the motile cells of the sample to travel to the further analysis chamber;

optionally withdrawing the liquid from the analysis chambers via the effluent channel, if present;

quantifying the detection reagent in the analysis chambers or on the solid-liquid separation units, if present.

33. A method of separating motile cells in a sample based on their motility comprising the steps of:

providing a mesoscale fluidic system according to claim 31;

adding the sample to the first analysis chamber, or optionally to the receiving well;

withdrawing the liquid from the analysis chambers via the effluent channel to a level below the passageway between the analysis chambers;

withdrawing cells from an analysis chamber.

34. The method according to any one of claims 32 or 33, wherein the cells are prokaryotic cells.

35. The method according to any one of claims 32 or 33, wherein the cells are eukaryotic cells.

36. The method according to claim 35, wherein the eukaryotic cells are selected from the group consisting of mammalian cells, yeast cells, amoebae, and micro- and macroparasites.

37. The method according to claim 35, wherein the eukaryotic cells are sperm cells.

38. The method according to claim 37, wherein the sperm cells are mammalian sperm cells, such as human sperm cells.

39. A system for analysis of motile cells in a sample, the system comprising a mesoscale fluidic system according to any one of claims 1 to 31, and an external detector device comprising :

an optical detector;

a data processor for executing the computer program code.

40. A system for analysis of motile cells in a sample according to claim 39, wherein the external detector device further comprises a display for presenting quantification results to an operator.

41. A system for analysis of motile cells in a sample according to claim 39 or 40, wherein the optical detector is selected from the group consisting of a camera, a laser scanner, a CCD reader, a photodiode scanner.

42. A system for analysis of motile cells in a sample according to claim 39 or 41, wherein the external detector device is a mobile user terminal.

43. A mobile user terminal containing computer program code configured to quantify a detection reagent on the solid-liquid separation units of a mesoscale fluidic system according to any one of claims 1 to 31.