Method and device for extracting motile cells and for storing motile cells in a straw

Abstract

The invention comprises a housing with a separation chamber. The separation chamber may comprise a first chamber, a second chamber and a membrane between the first chamber and the section chamber. The membrane prevents non-motile cells from a semen sample in the first chamber, and allows motile cells from the semen sample, to enter the second chamber. The housing has an inlet for the semen sample to enter the separation chamber and at least one outlet opening for extracting the motile cells from the separation chamber. A hollow interior of a straw is connected with the at least one outlet opening of the separation chamber of the housing. A fluid pressure difference extracts motile cells from the separation chamber to the hollow interior of the straw.

Description

[0001]METHOD AND DEVICE FOR EXTRACTING MOTILE CELLS AND FOR STORING MOTILE CELLS IN A STRAW FIELD OF THE INVENTION The invention relates to a method of extracting and collecting motile cells from a raw semen sample, the method comprising the step of providing a housing with a separation chamber, the separation chamber preferably comprising a first chamber, a second chamber and a membrane between the first chamber and the section chamber, themembrane intended for preventing non-motile cells from the semen sample, andallowing motile cells from the semen sample, to enter the separation chamber, the housing having an inlet for the semen sample to enter the separation chamber and at least one outlet opening for extracting the motile cells from the separation chamber. The invention also relates to a device extracting and collecting motile cells from the separation chamber and to a use of such device. BACKGROUND OF THE INVENTIONWO 2023 / 084325 A1 discloses a mesoscale fluidic system for extracting motile cells fromnon-motile cells. The mesoscale fluidic system comprises a sample chamber. The sample chamber comprises a cell permeable filter defining a first chamber a second chamber. The first chamber has a sample inlet opening. The second chamber is in fluid communication with an entry port to an analysis chamber via a channel. The first chamber is below the cell permeable filter and the second chamber is above the cell permeable filter. Motile cells are extracted via an outlet of the second chamber into a syringe. Subsequently, the semen sample in the syringe must be extracted from the syringe and handled, to be used for artificial insemination after a period of incubation. EP 0527676 A1 discloses a straw filled by suction through a suction nozzle, fitted with a needle penetrating the straw behind a composite plug and fitted with a seal. A biological product such as sperm rises from a semen sample through a flexible tube made of elastomer towards an injection nozzle in response to the reduced pressure in the straw. The flexible tube is held centered over at least five times its external diameter, in a hoop held in a support. The flexible tube dips into a flask containing the biological product. The parts which have been in contact with the sperm may be disposed of after oneejaculation has been exhausted, instead of requiring expensive cleaning operations. Thesemen sample in flask is a raw sample. After extraction into the straw, subsequently further handling is needed for the semen sample to be used for artificial insemination.WO 2022 / 154755 A1 discloses methods and devices for separation of motile sperm. Amethod of separating motile sperm comprises: introducing a fluid sample comprising motile sperm to an inlet portion of a microfluidic device; and causing the fluid sample to flow through a separation portion of the microfluidic device at a flow velocity within a rheotaxis range such that motile sperm in the sample undergo rheotaxis and remain in the separation whereas a part of the fluid sample flows out of the separation zone through an outlet portion of the microfluidic device. Because separation is provided by rheotaxis, and a pressure difference is applied manually, there is both a risk of motilecells, together with non-motile cells, being extracted during the first extraction of non-motile cells and also a risk of non-motile cells, possibly still present in the separationportion, being extracted during the second extraction of motile cells.US 2019 / 0329256 A1 discloses a method for separating and enriching sperm from atissue sample comprises: obtaining a microfluidic separating system having an inlet end and an outlet end, and a membrane filter (e.g., hollow fiber membrane filter) fluidly connected to the outlet end; separating the tissue sample via the microfluidic separating system into a debris fluid volume and a sperm fluid volume; and enriching the spermfluid volume by removing excess media via the membrane filter. The filter hasmicrochannels or pores sized to permit the passage of some materials based on size and sized to restrict passage of other / larger materials, such as sperm cells. The pores can have a pore size from 0.2 μm to 1 μm, in some cases from 2 to 5 μm, and in one example approximately 0.2 μm. The filter is not capable of separating motile cells from non-motile cells, due to the very small pore size, which is too small for mammaliansperm cell. The enriched sperm sample volume, void of debris, can be collected in astorage container and / or stored in cryotips for cryogenic storage. Collection to either astorage container or cryotips is not disclosed, possibly by a syringe or pipette.SUMMARY OF THE INVENTION One object of the present invention may be to reduce the risk of a semen sample to be used for in intrauterine insemination (IUI), in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) is subject to contamination. Another object of the present invention may be to extract and collect only an amount of sample sufficient for obtaining optimal results in intrauterine insemination (IUI), in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Still another object of the present invention may to be able to limit as much as possible handling of a semen sample for in intrauterine insemination (IUI), in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The objects of the present invention may be obtained by a method comprising the steps of- providing a housing with a separation chamber, and the housing having an inlet for theraw semen sample to enter the separation chamber and at least one opening for extracting the motile cells from the separation chamber,- providing at least a longitudinal straw for storing a sample with motile cells, the strawbeing an elongated tubular member with a first end and an opposite second end andwith a hollow interior extending between the first end and the second end of the straw, -connecting the first end of the hollow interior of the straw with the at least one openingfor extracting motile cells from the separation chamber of the housing,- creating a fluid pressure difference between the at least one outlet opening of theseparation chamber and the second end of the hollow interior of the straw, a fluidpressure at the at least one outlet opening being higher than a fluid pressure at thesecond end of the hollow interior of the straw,- extracting, because of the fluid pressure difference created, an amount of motile cellsfrom the separation chamber, through the at least one opening of the separation chamber of the housing, into the first end of the hollow interior of the straw and into the hollow interior of the straw. Providing both a housing with key features as mentioned and a straw for extracting motile cells directly from the separation chamber of the housing into the straw, and forcollecting the motile cells in the straw, significantly reduces handling of the motile cellsand reduces a risk of contamination of the motile cells during handling and a risk ofdifferent samples with motile cells being interchanged. ‘Connection’ and ‘connected with’ is to be understood as a fluid connection between two separate parts of the device, e.g., the housing and the straw, but the two parts not necessarily being physically attached to each other. Other items may be part of aconnection, such as a connector, transition elements and possible tubes between the onepart, e.g., the housing, and the other part, e.g., the straw, connected with each other.One way of connecting and proving a connection between the housing and the strawmay be attaching an inlet of a connector to an outlet opining of the housing and attaching an end of the straw to an outlet of the connector. Another way of connectingthe housing and the straw may be by attaching an end of the straw directly, and notindirectly via a connector, to an outlet opening of the housing.According to one aspect of the method, the method comprises the steps of- providing a connector for connecting the at least one opening for extracting motile cellsfrom the separation chamber of the housing with the first end of the straw and forpassing a sample with motile cells along a passage in the connector, between an inletand an outlet of the connector, and- attaching one end of the connector, the one end having an inlet orifice, to the at leastone opening for extracting motile cells from the separation chamber of the housing andattaching another end of the connector, the other end having an outlet orifice, to the firstend of the hollow interior of the longitudinal straw.Providing a connector between the outlet of the housing and the first end of the strawhas the advantage, that the one end having the inlet orifice of the connector may beconfigured to the size and shape of the outlet of the housing and the other end with theoutlet orifice may be configured to the size and shape of the first end of the straw. Motilecells are then extracted from the outlet of the housing into a passage in the connector, between the one end with the inlet orifice and the other end with the outlet orifice, and into the hollow interior of the straw through the first end of the straw. According to another aspect of the method, the method comprises the steps of- attaching the first end of the hollow interior of the straw to the at least one opening forextracting motile cells from the separation chamber of the housing.Possibly, the size and shape of the outlet of the housing is congruent with the first and ofthe straw, so that the first end of the straw may be attached to the outlet of the housing without use of a connector as transition element between the outlet of the housing andthe first end of the straw, thus extracting motile cells directly from the outlet of thehousing into the hollow interior of the straw through the first end of the straw. Preferably, the separation chamber comprises a first chamber, a second chamber and a cell permeable membrane between the first chamber and the second chamber, the cell permeable membrane dividing the separation chamber into the first chamber and the second chamber, the membrane intended for preventing non-motile cells from the semen sample, and allowing motile cells from the semen sample, to enter the second chamber.According to one aspect of the invention, the step of creating a fluid pressure differencebetween a fluid pressure at the at least one opening of the separation chamber and afluid pressure at the second end of the straw is performed by the following steps:- connecting the first end of the hollow interior of the straw to at least one opening ofthe separation chamber of the housing, and connecting the second end of the hollowinterior of the straw to an orifice of a fluid pressure adjusting device,- creating a decreased fluid pressure in the hollow interior at the second end of thestraw, compared to a fluid pressure at the at least one opening of the separationchamber of the housing, by means of the fluid pressure adjusting device,- extracting, because of the decreased pressure created, an amount of motile cells fromthe separation chamber, through the at least one opening of the separation chamber of the housing, into the first end of the hollow interior of the straw and into the hollow interior of the straw.Preferably, the fluid pressure difference is created by decreasing the fluid pressure at theinlet of the hollow interior of the straw. Thereby, a risk is limited of the end of the straw detaching from the connection with the at least one opening of the separation chamber.According to another aspect of the invention, the step of creating a fluid pressuredifference between a fluid pressure at the at least one opening of the separationchamber and a fluid pressure at the second end of the straw is performed by thefollowing steps:- connecting the first end of the hollow interior of the straw to at least one opening ofthe separation chamber of the housing and connecting another opening of the separationchamber of the housing to an orifice of a fluid pressure adjusting device,- creating an increased fluid pressure in the separation chamber of the housing,compared to a fluid pressure at in the hollow interior at the second end of the straw, bymeans of the fluid pressure adjusting device,- extracting, because of the increased pressure created, an amount of motile cells fromthe separation chamber, through the at least one opening of the separation chamber of the housing, into the first end of the hollow interior of the straw and into the hollow interior of the straw.Possibly, the fluid pressure difference is created by increasing the fluid pressure in theseparation chamber of the housing. Thereby, a probability is increased of a sufficient fluid pressure difference, ensuring that the motile cells are forced towards the straw. In a preferred aspect of the invention, the at least one opening of the separation chamber of the housing is one of the following openings: an outlet opening of the separation chamber of the housing, or a medium opening of the separation chamber of the housing, and where the other opening is one of the following openings: the medium opening or an inlet opening, with the proviso of the at least one opening being the outlet opening, or the outlet opening, with the proviso of the medium opening being the at least one opening. Using already provided openings of the separation chamber of the housing makes it easy to adopt the method of the invention to various separation devices having at least two openings in the separation chamber, independent of whether the separation chamber is divided into two or more chambers or is only constituted by one chamber. According to a possible aspect of the invention, the method comprises the step of- providing a fluid pressure adjusting device being a syringe comprising a cylinder withan orifice, and a plunger with a piston fitting gastight inside the cylinder, the piston being displaceable along the inside of the cylinder, and- creating a decreased pressure by pulling the plunger along the cylinder of the Syringe,in a direction away from the orifice, so that a decreased pressure is created at the orifice and at the second end of the hollow interior of the straw, or- creating an increased pressure by pushing the plunger along the cylinder of thesyringe, in a direction towards the orifice, so that an increased pressure is created at one of the at least one opening of the separation chamber of the housing. According to another possible aspect of the invention, the step of connecting the first end of the straw to the at least one opening of the separation chamber of the housing comprises the following steps:- connecting a first end of the straw with the at least one opening of the separationchamber of the housing by providing a connector, the connector having one end, another opposite end, and a passage for a fluid between the one end and the other opposite end,- applying, preferably attaching, the one end of the connector to the at least one openingof the separation chamber of the housing, and applying, preferably attaching, the first end of the straw to the other opposite end of the connector,- thereby establishing a fluid connection between the at least one opening of theseparation chamber of the housing and the first end of the hollow interior of the straw.A connector gives a possibility of providing a safe and fluid tight connection between theat least one opening of the separation chamber and the one end of the straw. The connector may be designed for a specific outlet opening of the separation chamber. According to an additional aspect of the invention, the method comprises the further step of- subsequent to extracting an amount of motile cells from the separation chamber intothe hollow interior of the straw along at least a major part of an extension of the straw,- dividing the straw into a plurality of elongated sections being limited parts of theextension of the straw, each section being closed at ends of the elongation, and- each section of the straw being a limited part of the hollow interior of the straw andcontaining a limited amount of the motile cells extracted from the separation chamber.Dividing the straw into two or more sections, subsequent to having extracted an amount of motile cells, gives a possibility of using the amount of motile cells for more than one insemination or other artificial reproduction technique. The objects of the invention may also be obtained by a device for extracting and collecting motile cells from a raw semen sample, the device comprising a housing with a separation chamber, and- a longitudinal straw for storing a portion of motile cells, the straw being an elongatedtubular member with a first end and an opposite second end and with a hollow interior extending between the first end and the second end of the straw,- the first end of the hollow interior of the straw (11) connected with the at least oneoutlet opening (5,6) of the separation chamber of the housing, , and- an orifice of a fluid pressure adjusting device connected with one of the followingopenings: a second end of the hollow interior of the straw or one of the at least one opening of the separation chamber of the housing.According to one embodiment of the device, the device comprises a connector forpassing a sample with motile cells from the at least one opening for extracting motilecells from the separation chamber of the housing to the first end of the straw by passingthe sample with motile cells along a passage in the connector, between one end havingan inlet orifice of the connector and another end having an outlet orifice of the connector, and with the one end of the connector attached to the at least one outlet opening of the housing, and with the first end of the hollow interior of the straw being attached to the other end of the connector. Motile cells are then extracted from the outlet of the housing into a passage in the connector, between the one end with the inlet orifice and the other end with the outlet orifice, and into the hollow interior of the straw through the first end of the straw. According to another embodiment of the device, a connector is omitted, and the first end of the hollow interior of the straw attached to the at least one outlet opening of theseparation chamber of the housing. Motile cells are extracted directly from the outlet ofthe housing into the hollow interior of the straw through the first end of the straw. Preferably, the separation chamber comprises a first chamber, a second chamber and a cell permeable membrane between the first chamber and the second chamber, cell permeable membrane dividing the separation chamber into the first chamber and the second chamber, the membrane intended for preventing non-motile cells from the semen sample and allowing motile cells from the semen sample to enter the second chamber. Such separation provides separation of very high quality.According to a preferred embodiment of the device, the fluid adjusting device is asyringe having a cylinder with an orifice and a plunger provided gastight inside the cylinder, the plunger being displaceable along the cylinder, and where displacement of the plunger along the cylinder in a direction away from the orifice provides a decreased pressure inside the cylinder, and where displacement of the plunger along the cylinder, in a direction towards the orifice provides an increased pressure inside the cylinder. Asyringe is an easy, safe and ready-at-hand means for providing a fluid pressuredifference. According to one possible embodiment of the invention, the device comprises a connector having one end with a fluid inlet, another opposite end with a fluid outlet, and a fluid passage extending between the inlet and the outlet of the connector, and the one end of the connector being connected to the at least one opening of the separation chamber of the housing, and a first end of the straw connected with the other end of the connector. A connector is a means of providing a connection, which can be designed to the size and shape of the at least one opening of the housing for extracting motile cells from the separation chamber, and to the size and shape of the first end of the straw.Preferably, an orifice of the one end of the connector faces one direction and an orifice ofthe other end of the connector faces another direction, and where that the connector isprovided with a bend between the orifice of the one end of the connector and the orificeof the other end of the connector, with the one direction being at an angle between 5degrees and 175 degrees to the other direction, preferably the one direction being at anangle between of 90 degrees to the other direction. A connector with a bend provides the possibility of the straw being positioned in a suited direction, possibly a lateral direction, preferably a horizontal direction, so that extraction of the motile cells need not overcome gravitational forces from the connector to the hollow interior of the straw. According to another possible embodiment of the invention, the device comprises aconnector specifically configured for a HarvesterTM housing, and having one end with afluid inlet, another opposite end with a fluid outlet, and a fluid passage extending between the inlet and the outlet of the connector, and the one end of the connector being applied to the housing at the at least one opening of the separation chamber of the housing, and a first end of the straw connected with the other end of the configured connector.A configured connector provides a possibility of not having to make a fixed attachment,which is the case with a more common connector according to the one embodiment,between the at least one opening of the separation chamber and an inlet opening of theconnector. The configured connector is manually positioned over the at least one openingof the separation chamber so that an inlet opening of the configured connector is inconnection with the at least one opening, and then the configured connector is manuallymaintained in such position during extraction of the motile cells.Because the configured connector is manually maintained in position over the at leastopening of the at least one opening, the configured connector, if configured according toa shape of the housing, may also be used for manually maintaining the housing towards the support surface. Preferably, when the housing is placed on a lateral surface such as on a table or other lateral support surface for the housing, the at least one opening of the separation chamber of the housing is facing upwards, preferably vertically upwards, and the fluid inlet at the one end of the connector is connected with the at least one opening of the separation chamber of the housing and is facing downwards, preferably vertically downwards, and the fluid outlet at the other end of the connector is facing sideways, preferably horizontally, and the hollow interior of the straw being connected with the fluid outlet at the other end of the connector extends sideways, preferably horizontally.Objects of the invention may be obtained by a connector intended for being applied to ahousing for extracting and collecting motile cells from a raw semen sample, and theconnector intended for providing a connection between an at least one opening of theseparation chamber and a longitudinal straw for storing a portion of motile cells, theconnector having a fluid inlet and a fluid outlet and a fluid passage extending betweenthe fluid inlet and the fluid outlet, the fluid inlet of the connector intended for beingapplied to the at least one opening of the separation chamber of the housing, and thefluid outlet of the connector intended for being connected with a first end of the straw.BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A-1B are drawings of an embodiment of a cell separation housing, known per se, Fig. 2 is a photo showing the housing with a connector, a straw, and a syringe, all connected, ready for extracting of motile cells from the housing, Fig. 3 is a photo showing the housing with the connector, and the straw with the syringe disconnected, after having extracted motile cells from the housing, Fig. 4 is a photo showing the housing with a connector connected to the housing, Fig. 5 is a photo showing the housing with the connector, an interface and the straw, Fig. 6 is a photo showing a first embodiment and a second embodiment of a connector, Fig. 7 is photo showing the second embodiment of a connecter connected to the housing, Fig. 8-9 are photos showing the housing with a specifically configured connector providing a connection between one opening of a second chamber of the housing and the straw, Fig. 10-11 are photos showing the housing with the specifically configured connector providing a connection between another opening of a second chamber of the housing and the straw, andFig. 12 is a photo showing a connector configured for operating with a selected outletopening of a selected housing, providing a connection between one opening of a secondchamber of the housing and the straw. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION Fig. 1A and 1B shows a housing, known per se, for separating cells from a raw semen sample with both non-motile cells and motile cells, and for extracting motile cells from a separation chamber inside the housing, from an opening of the separation chamber. In the embodiment shown, the housing is a housing named HarvesterTM, from the company MotilityCount ApS, Denmark, Europe. The separation chamber of HarvesterTMis divided into a first chamber and a second chamber with a membrane in-between the first and second chamber, the membrane constituting an obstruction for the cells (see description below). In other embodiments of separation devices, only one chamber is provided, and separation is performed by other obstructive means than a membrane, as example, by stops and / or channels provided in the separation chamber, which other obstructive means also separate non-motile cells from motile cells. In still other embodiments of a separation device, the separation chamber is divided into more than two individual chambers, possibly with a membrane or other obstructive means between each of the chambers, or at least between some of the chambers. In still yet other embodiments of separation devices, the obstructive means is a combination of two or more obstructive means, as example, a combination of an initial obstruction being a channel and a subsequent obstruction being stops. Fig. 1A shows in detail, in an exploded view, various key features of the HarvesterTMhousing. However, a housing is non-dependent of being a HarvesterTMhousing from the company MotilityCount Aps and may be another housing from another company but having the following key features: a separation chamber comprising a first chamber 1, asecond chamber 2 and a membrane 3 between the first chamber 1 and the sectionchamber 2. During use of the housing, the first chamber 1 is situated on one side of thevell permeable membrane 3, preferably, but not necessarily, below the second chamber2, and the second chamber 2 is situated on an opposite site side of the cell permeablemembrane 3, preferably, but not necessarily, above the first chamber 1.The housing has one opening constituting an inlet 4 to the first chamber 1 for allowing the semen sample to enter the first chamber 1, and another opening constituting anoutlet 5 from the second chamber 2 for extracting the motile cells from the secondchamber 2. In the embodiment shown, the housing has an additional opening constituting a medium opening 6 of the second chamber 2. The membrane 3 is intended for preventing non-motile cells from the semen sample to enter the second chamber 2,but still allowing motile cells from the semen sample to enter the second chamber 2.In the embodiment shown in Fig. 1, which is the housing named HarvesterTM, the housing also has a medium opening 6. During use, if and when motile cells are extracted from the outlet 5 of the second chamber 2, the medium opening 6 of the HarvesterTMis intended for being in fluid communication with the ambient, providing a pressure release function so that when motile cells are extracted from the second chamber 2 through the at least one opening of the second chamber 2, a pressure in the second chamber 2 isrelieved by the medium opening 6 by air entering the second chamber 2 through themedium opening 6, thereby preventing active suction of cells from the first chamber 1 through the cell permeable membrane 3 and into the second chamber 2. The outlet 5 of the second chamber 2 and the medium opening 6 of the second chamber 2 each constitute one of at least one opening of the second chamber 2. In other types of separation devices, a medium opening 6 may not be present. In the embodiment shown,each of the openings, that is, the inlet 4 to the first chamber 1, the outlet 5 of thesecond chamber 2, and the medium opening 6 of the second chamber 2 are provided with flanges 7,8,9, respectively, all flanges intended for insertion of a spout of a syringe 12. In other types of separation devices, flanges at the openings may not be present. Fig. 2 shows the HarvesterTMhousing with a connector 10 with a fluid passage is used for connecting an opening of the second chamber of the housing (see Fig. 1) with one end of a hollow interior of the straw 11. Another end of the hollow interior of the straw 11 isconnected to a syringe 12. A first transition element 13, known per se, with a fluidpassage is used for connecting the passage of the connector 10 with the one end of the hollow interior of the straw 11. At another end of the straw 11, an orifice of a spout of the syringe 12 is connected with the hollow interior of the straw 11. A second transitionelement 14, known per se, with a fluid passage is used for connecting the orifice of thespout of the syringe 12 with the other end of the hollow interior of the straw 11. In the embodiment shown, one end of the connector 10, with a fluid inlet, is attached tothe medium opening 6 and another opposite end of the connector 10, with a fluid outlet,is attached to the first transition element 13 providing a transition between theconnector 10 and the hollow interior of the straw 11. Thus, motile cells may be extracted from the second chamber 2 through the medium opening 6, and not from the outlet 5 of the second chamber 2. When motile cells are extracted through the medium opening 6, pressure in the second chamber 2 is relieved by air entering the second chamber 2 through the at least one opening of the second chamber 2, which pressure relief in the embodiment shown will be air entering through the outlet 5. That is opposite to extracting motile cells from the second chamber 2 through the outlet 5 of the second chamber 2 and relieving pressure in the second chamber 2 by air entering the second chamber 2 through the medium opening 6. In an alternative use of the device, motile cells are extracted from the second chamber 2 through the outlet 5 of the second chamber 2, and not from the medium opening 6 of the second chamber 2. When motile cells are extracted through the outlet 5 of the second chamber 2, pressure in the second chamber 2 is relieved by air entering the second chamber 2 through the medium opening 6. As previously mentioned, other embodiments of separation devices may have only one chamber for separating motile cells from non-motile cells and having other obstructions than a membrane, such as stops or channels, for the cells. Such other embodiments may have an inlet opening of the separation chamber leading to the one and same chamber as the chamber which an outlet opening, and a possible medium opening, leads to or from. In such embodiment of separation devices, also the inlet opening may function as pressure relief opening for air entering the separation chamber, if an outlet opening or a medium opening of the one and same chamber is used for extracting motile cells. Use of the device is performed by the following method: Initially, a semen sample containing both motile cells and non-motile cells are injected into the lower first chamber 1 through the inlet 4 to the first chamber 1 (see also fig. 2). A sample medium is provided in the first chamber 1, and which the semen sample ismixed with for assisting the cells in moving in the first chamber 1 by the motile cellsactively transporting themselves in any given direction in the liquid of the sample, the motile cells actively transporting themselves overcoming the gravitational force exerted on them, and the motile cells transporting themselves through the cell permeable membrane 3 by swimming by the cells themselves. After a certain period of time, all or most motile cells have transported themselves from the lower first chamber 1, through the cell permeable membrane 3 to the upper second chamber 2. A conditioning medium is provided in the second chamber 2, and which the semen sample is mixed with for assisting the cells in moving in the second chamber 2 by the motile cells actively transporting themselves in any given direction in the liquid of the second chamber 2 and possibly towards the outlet 5 or the medium opening 6. Thereafter, the motile cells are to be extracted from the second chamber 2.The longitudinal straw 11 for storing a portion of motile cells is per se known. The straw11 is an elongated tubular member with a first end and an opposite second end and with a hollow interior extending between the first end and the second end of the straw 11. The first end of the straw 11 is connected with the other end of the connector 10 and thesecond end of the straw 11 is connected with an orifice in a spot of the syringe 12. Thesyringe 12 comprises a cylinder with the spout having an orifice, and a plunger with a piston fitting gastight inside the cylinder end being displaceable along the inside of the cylinder. The plunger of the syringe 12 is displaced along the cylinder, in a direction away fromthe spout of the syringe 12. Thereby, a decreased pressure, compared to a fluid pressureinside the hollow interior and at the first end of the straw 11, is created at the orifice of the spout of the syringe 12. A decreased pressure is then created in the hollow interior in the straw 11, in the hollow interior of the connector 10, at the medium opening 6 and in the second chamber 2. Thereby, motile cells are extracted from the second chamber 2, through the medium opening 6, through the hollow interior of the connector 10, into the hollow interior of the straw 11. When a desired or needed number of motile cells are extracted into the hollow interior of the straw 11, displacement of the lunger of the cylinder is stopped. A plurality of motile cells is now contained in the hollow interior of the straw 11. One part of the straw 11, close to the first end attached to the connector 10, and another part of the straw 11, close to the second end attached to the spout of the syringe 12, are then squeezed together, mechanically and by providing a thermal bonding of sidewalls of the straw 11. Thereby, an enclosure is provided for the motile cells. Then, the first end of the straw 11 may be detached from the connector 10 and second end may be detached from the spout of the syringe 12. Subsequently, the straw 11 may be handled without the motile cells being spilled from the hollow interior of the straw 11, initially often being handled for freezing. In addition to closing the straw 11 by thermally bonding sidewalls of the straw 11 at one part close to the first end and another part close to the second end, the straw 11 may be divided into more than one enclosure of motile cells by bonding sidewalls together at one or more positions between the one part close to the first end and the other part close to the second end. Thereby, more than one enclosure of motile cells is provided, obtaining more samples with motile cells. However, each divisional sample of motile cells thereby obtained will be smaller than only one sample obtained between the one part close to the first end and the other part close to the second end.Fig. 3 is a close-up view of the housing, the connector 10, the first transition element 13and the first end of the straw 11. The figure shows the inlet 4 for a semen sample into the lower first chamber 1 (not visible). The cell permeable membrane 3 is visible through a top surface of the housing. Above the cell permeable membrane 3, the second chamber 2 is provided. At least one opening of the second chamber 2 from the second chamber 2 (not visible, see Fig. 2) is situated at an opposite end of the housing than the inlet 4 to the first chamber 1. Fig. 4 shows the housing with a connector 10 with one end attached to the mediumopening 6 and the other opposite end being free, ready for a first transition element 13to be attached. Attachment of the one end of the connector 10 to the flange 15 of the medium opening 6 is obtained by a snug fit, that is, dimensioning an inner circumference of the orifice in the one end of the connector 10, See Fig. 6) so that the connector 10 fits, and is kept, by frictional forces, to an outer circumference of the flange 15 of the medium opening 6. Fig. 5 shows the housing with a connector 10 with one end attached to the mediumopening 6 and the other opposite end having the first transition element 13 attached.Attachment of the first transition element 13 to the other end of the connector 10 isobtained by a snug fit, that is, dimensioning an inner circumference of an orifice in oneend of the first transition element 13 so that an inner circumference of the first transitionelement 13 fits and is kept, by frictional forces, to an outer circumference of the otherend of the connector 10. Fig. 6 shows two various embodiments of a connector 10. The one connector 10 to the right in the figure is the embodiment of a connector 10 shown in the previous figures. The connector 10 has one end intended for being attached to the flange of the medium opening 6, and another end intended for being attached to afirst transition element 13, see as example Fig. 5. The one end and the other end haveorifices facing obliquely at an angle in relation to each other, as example, 90 degrees as shown in the figure. During use, an orifice of the one end is facing downwards, and an orifice of the other end is facing horizontally sideways. Between the one end and the other end a passage is provided in which the motile cells can pass through the connector 10. The other connector 10 to the left in the figure is an alternative embodiment of a connector 10. The alternative connector 10 also has one end intended for being attached to the flange of the medium opening 6, and another end intended for being attached to afirst transition element 13. The one end and the other end have orifices facing oppositelyto each other, that is, at an angle of 180 degrees in relation to each other as shown in the figure. During use, an orifice of the one end is facing downwards, and an orifice of the other end is facing upwards. Between the one end and the other end a passage is provided in which the motile cells can pass through the connector 10. Fig. 8-9 show one alternative to extracting motile cells from the second chamber 2 through the medium opening 6. The figures show an alternative embodiment of aconnector 10 specifically configured for being attached to the outlet of the HarvesterTMhousing, with one end with a fluid inlet to be connected with the medium opening 6, andanother end with a fluid outlet connected with an end of the straw 11. Between the fluidinlet in the one end of the configured connector 10 and the fluid outlet in the other endof the configured connector 10, a passage for a fluid, including a sample with motilecells, is provided.The configured connector 10 is specifically configured for a housing named HarvesterTMfrom the company MotilityCount ApS. Thus, the one end of the configured connector 10extends sideways along an upper surface of the housing at least at a distance so that thefluid inlet of the configured connector 10 at the one end is aligned with the mediumopening 6 of the housing. Furthermore, the fluid outlet at the other end of the configuredconnector 10 extends downwards along a side surface of the housing a distance so that alowest surface of the other end of the configured connector 10 rests on, and is supportedby, the same support surface as the housing rests on and is supported by. Thereby, auser holding on the configured connector 10 assists in holding the housing towards thesupport surface. In the embodiments shown of the device, either an angled embodiment of a connector 10 or a linear embodiment of a connector 10 (see Fig. 6) or a specifically configuredembodiment of a connector 10 (See Fig. 8-9 and Fig. 10-11 and Fig. 12) is used forconnecting the medium opening 6 with the hollow interior at one end of the straw 11, the connector 10having a passage for the motile cells to pass through the connector 10.In the embodiments of the configured embodiment of a connector 10 shown, one end ofthe straw 11 is connected directly to the fluid outlet at the other end of the connector10, the fluid outlet of the connector 10 having a shape and a size corresponding to theone end of the straw 11. In an alternative use of the configured connector 10, a firsttransition element 13 may be used for providing a transition between the configuredconnector 10 and the one end of the straw 11.Fig. 10-11 show another alternative to extracting motile cells from the second chamber 2, however, in this alternative embodiment through outlet 5. The figures show anembodiment of connector 10 specifically configured for a HarvesterTM housing with oneend with an orifice to be connected with the outlet 5, and another end with an orifice connected directly to the straw 11, allowing motile cells top pass directly from theconfigured connector 10 to the hollow interior of the straw 11.Fig. 12 shows one embodiment of a configured connector 10 as viewed from anunderside, when in use as shown in fig. 10 and Fig. 11, of the connector 10. Theunderside extends generally in a plane corresponding to a plane top side of theHarvesterTM housing around the inlet 4 or around the outlet 5. An outer flange 16 and aninner flange 17 of the configured connector 10 delimit an annular groove 18.A size and circumference of the annular groove 18 correspond to a size andcircumference of the part of the outlet 5 protruding from the top side of the HarvesterTMhousing. Therefore, when the configured connector 10 is attached to the part of theoutlet 5 protruding from the top side of the housing, as shown in Fig. 10 and Fig. 11, asnug fit is obtained when the flanges 16,17 of the configured connector 10 extendaround the part of the outlet 5 protruding from the top side of the housing, the part ofthe outlet 5 protruding extending into the annular groove 18 between the flanges 16,17.Another embodiment of a configured connector may be for the medium opening 6. A sizeand circumference of the annular groove 18 then correspond to a size and circumferenceof the part of the medium opening 6 protruding from the top side of the HarvesterTMhousing. When the configured connector 10 is attached to the part of the mediumopening 6 protruding from the top side of the housing, as shown in Fig. 8 and Fig. 9, asnug fit is obtained when the flanges 16,17 of the configured connector 10 extendaround the part of the medium opening 6 protruding from the top side of the housing, the part of the medium opening 6 protruding extending into the annular groove 18 between the flanges 16,17.In an alternative embodiment of the HarvesterTM housing a size and circumference of thepart of the outlet 5 protruding from the top side of the HarvesterTM housing and a sizeand circumference of the part of the medium opening 6 protruding from the top side ofthe HarvesterTM housing are the same, so that the one and same configured connectormay be used either together with the outlet 5 or together with the medium opening 6.In general, during use of all embodiments, extraction of motile cells from the second chamber 2 of the housing is obtained by creating a decreased pressure, compared to a fluid pressure inside the hollow interior and at the first end of the straw 11, in the second chamber 2. The decreased pressure is created by the plunger of the syringe 12 being displaced along the cylinder, in a direction away from the spout of the syringe 12, thereby creating a decreased pressure at the orifice of the spout of the syringe 12, and thereby creating a decreased pressure is then created in the hollow interior in the straw 11, in the hollow interior of the connector 10, and at the medium opening 6. The decreased pressure at the medium opening 6 draws the motile cells from the second chamber 2 to the medium opening 6, through the medium opening 6 and to the hollow interior of the straw 11. An alternative use of the device may obtain extraction of motile cells from the secondchamber 2 of the housing by creating an increased pressure, compared to a fluidpressure inside the hollow interior and at the first end of the straw 11, in the second chamber 2. The increased pressure may be created by placing a spout of syringe 12 within the flange 9 of the medium opening 6 or the flange 8 of the outlet 5 of the second chamber 2 of the housing, and the plunger of the syringe 12 being displaced along the cylinder, in a direction towards the spout of the syringe 12, thereby creating an increased pressure at the medium opening 6 or the outlet 5 of the second chamber 2 of the housing, and thereby creating an increased pressure in the second chamber 2. The increased pressure in the second chamber 2 pushes the motile cells from the second chamber 2 to the medium opening 6, through the medium opening 6 and to the hollow interior of the straw 11. In the embodiments shown in the figures, and the method of operating the embodiments of the device, a connector is used as an interface between the opening of the housing and the first end of the hollow interior of the straw. In an alternative method, a connector is not used as an interface between the opening of the housing and the first end of the hollow interior of the straw. In the alternative method, the first end of the straw is inserted into the flange of one of the openings of the second chamber. Thereby, the sample with motile cells are extracted directly, and not via a connector, into the hollow interior of the straw. In such alternative or additional use, the flange 9 of the medium opening 6 of the housing or the flange 8 of the outlet 5 of the second chamber 2 may have a shape and a size corresponding to the one end of the straw 11. Thereby, the one end of the straw 11 may be connected with the medium opening 6, alternatively to the outlet 5 of the second chamber 2, by connecting the one end of the straw 11 directly to the flange 9 of the medium opening 6, alternatively directly to the flange 8 of the outlet 5 of the secondchamber 2. Thus, use of a connector 10 according to the one embodiment or aconfigured connector 10 according to the embodiment may be omitted.

Claims

CLAIMS 1. Method of extracting and collecting motile cells from a raw semen sample, the method comprising the steps of- providing a housing with a separation chamber, and the housing having an inlet (4) forthe raw semen sample to enter the separation chamber and at least one opening (5,6) for extracting the motile cells from the separation chamber,- providing at least a longitudinal straw (11) for storing a sample with motile cells, thestraw (11) being an elongated tubular member with a first end and an opposite second end and with a hollow interior extending between the first end and the second end of the straw (11),- connecting the first end of the hollow interior of the straw (11) with the at least oneopening for extracting motile cells from the separation chamber of the housing,- creating a fluid pressure difference between the at least one outlet opening (5,6) of theseparation chamber and the second end of the hollow interior of the straw (11), a fluidpressure at the at least one outlet opening (5,6) being higher than a fluid pressure atthe second end of the hollow interior of the straw (11),- extracting, because of the fluid pressure difference created, an amount of motile cellsfrom the separation chamber, through the at least one opening (5,6) of the separation chamber of the housing, into the first end of the hollow interior of the straw (11) and into the hollow interior of the straw (11).

2. A method according to claim 1, the method comprising the steps of- providing a connector (10) for connecting the at least one opening (5,6) for extractingmotile cells from the separation chamber of the housing with the first end of the straw(11) and for passing a sample with motile cells along an internal passage in theconnector (10), between an inlet orifice and an outlet orifice of the connector (10), and- attaching one end of the connector (10), the one end having the inlet orifice, to the atleast one opening (5,6) for extracting motile cells from the separation chamber of thehousing, and attaching another end of the connector (10), the other end having theoutlet orifice, to the first end of the hollow interior of the longitudinal straw (11)- thereby establishing a fluid connection between the at least one outlet opening (5,6) ofthe separation chamber of the housing and the first end of the hollow interior of thestraw (11) along the internal passage in the connector (10).

3. A method according to claim 1, the method comprising the step of- attaching the first end of the hollow interior of the straw (11) to the at least oneopening (5,6) for extracting motile cells from the separation chamber of the housing.

4. Method according to any of the preceding claims, where the separation chamber comprises a first chamber (1), a second chamber (2) and a cell permeable membrane (3) between the first chamber (1) and the second chamber (2), the cell permeablemembrane (3) intended for preventing non-motile cells from the semen sample, andallowing motile cells from the semen sample, to enter the second chamber (2) and to be extracted from the second chamber (2) through the at least one outlet opening (5,6) of the second chamber.

5. Method according to any of the preceding claims, where the step of creating a fluidpressure difference between a fluid pressure at the at least one outlet opening (5,6) ofthe separation chamber and a fluid pressure at the second end of the straw (11) isperformed by the following steps:- connecting the first end of the hollow interior of the straw (11) to at least one outletopening (5,6) of the separation chamber of the housing and connecting the second endof the hollow interior of the straw (11) to an orifice of a fluid pressure adjusting device,- creating a decreased fluid pressure in the hollow interior at the second end of the straw(11), compared to a fluid pressure at the at least one outlet opening (5,6) of theseparation chamber of the housing, by means of the fluid pressure adjusting device,- extracting, because of the decreased pressure created, an amount of motile cells fromthe separation chamber, through the at least one outlet opening (5,6) of the separation chamber of the housing, into the first end of the hollow interior of the straw (11) and into the hollow interior of the straw (11).

6. Method according to any of claims 1-4, where the step of creating a fluid pressuredifference between a fluid pressure at the at least one outlet opening (5,6) of theseparation chamber and a fluid pressure at the second end of the straw (11) isperformed by the following steps:- connecting the first end of the hollow interior of the straw (11) to the at least oneoutlet opening (5,6) of the separation chamber of the housing and connecting anotheropening (5,6) of the separation chamber of the housing to an orifice of a fluid pressureadjusting device,- creating an increased fluid pressure in the separation chamber of the housing,compared to a fluid pressure at the second end of the straw (11), by means of the fluidpressure adjusting device,- extracting, because of the increased pressure created, an amount of motile cells fromthe separation chamber, through the at least one outlet opening (5,6) of the separation chamber of the housing, into the first end of the hollow interior of the straw (11) and into the hollow interior of the straw (11).

7. Method according to any of the preceding claims, where the at least one outlet opening of the separation chamber of the housing is one of the following openings:- an outlet (5) opening of the separation chamber of the housing, or a medium opening(6) of the separation chamber of the housing, and where the other opening is one of the following openings:- the medium opening (6) or an inlet opening (4), with the proviso of the at least oneopening being the outlet opening (5), or- the outlet opening (5), with the proviso of the medium opening (6) being the at leastone outlet opening.

8. Method according to any of the preceding claims, comprising the step of- providing a fluid pressure adjusting device being a syringe (12) comprising a cylinderwith an orifice, and a plunger with a piston fitting gastight inside the cylinder, the piston being displaceable along the inside of the cylinder, and- creating a decreased pressure by pulling the plunger along the cylinder of the Syringe(12), in a direction away from the orifice, so that a decreased pressure is created at the orifice and at the second end of the hollow interior of the straw (11), or- creating an increased pressure by pushing the plunger along the cylinder of the Syringe(12), in a direction towards the orifice, so that an increased pressure is created at one of the at least one outlet openings (5,6) of the separation chamber of the housing.

9. A method according to any of the preceding claims, the method comprising the further step of- subsequent to extracting an amount of motile cells from the separation chamber intothe hollow interior of the straw (11) along at least a major part of an extension of the straw (11),- dividing the straw (11) into a plurality of elongated sections being limited parts of theextension of the straw (11), each section being closed at ends of the elongation, and- each section of the straw (11) being a limited part of the hollow interior of the straw(11) and containing a limited amount of the motile cells extracted from the separationchamber.

10. A device for extracting and collecting motile cells from a raw semen sample, the device comprising- a housing with a separation chamber, and the housing having an inlet (4) of theseparation chamber for the semen sample with both non-motile cells and motile cells toenter the separation chamber, and at least one outlet opening (5,6) of the separation chamber for extracting motile cells from the separation chamber,- a longitudinal straw (11) for storing a portion of motile cells, the straw (11) being anelongated tubular member with a first end and an opposite second end and with a hollow interior extending between the first end and the second end of the straw (11),- the first end of the hollow interior of the straw (11) connected with the at least oneoutlet opening (5,6) of the separation chamber of the housing, , and- an orifice of a fluid pressure adjusting device connected with one of the followingopenings: a second end of the hollow interior of the straw (11) or one of the at least one outlet opening (5,6) of the separation chamber of the housing.

11. A device according to claim 10, the device comprising a connector (10) having oneend with a fluid inlet, another opposite end with a fluid outlet, and a fluid passage extending between the inlet and the outlet of the connector (10), and the one end of theconnector (10) attached to the housing at the at least one outlet opening (5,6) of theseparation chamber of the housing, and the other end of the connector (10) attached tothe first end of the straw (11).

12. A device according to claim 10, the device void of a connector between the at least one outlet of the separation chamber of the housing and the first end of the hollow interior of the straw, the first end of the hollow interior of the straw (11) attached directly to the at least one outlet opening (5,6) of the separation chamber of the housing.

13. A device according to any of claims 10-12, where the separation chamber comprisesa first chamber (1), a second chamber (2) and a membrane (3) between the first chamber (1) and the second chamber (2), the membrane (3) intended for preventing non-motile cells from the semen sample, and allowing motile cells from the semen sample, to enter the second chamber (2), and the at least one outlet opening (5,6) for extracting the motile cells from the second chamber (2) is provided in the second chamber (2)14. A device according to any of claims 10-13, where the fluid adjusting device is asyringe (12) having a cylinder with an orifice and a plunger provided gastight inside the cylinder, the plunger being displaceable along the cylinder, and where displacement of the plunger along the cylinder in a direction away from the orifice provides a decreased pressure inside the cylinder, and where displacement of the plunger along the cylinder, in a direction towards the orifice provides an increased pressure inside the cylinder.

15. A device according to any of claims 10 and 12-14, where an orifice of the one end ofthe connector (10) faces one direction and an orifice of the other end of the connector(10) faces another direction, and where that the connector (10) is provided with a bendbetween the orifice of the one end of the connector (10) and the orifice of the other endof the connector (10), with the one direction being at an angle between 5 degrees and175 degrees to the other direction, preferably the one direction being at an anglebetween of 90 degrees to the other direction.

16. A device according to any of claims 10-15, where, when the housing, during use ofthe device, being placed on a lateral surface such as on a table or other lateral supportsurface, the at least one outlet opening (5,6) of the separation chamber of the housing,during use of the device, facing upwards, preferably vertically upwards, and the fluidinlet at the one end of the connector (10), during use facing downwards, preferablyfacing vertically downwards, and being attached to the at least one outlet opening (5,6)of the separation chamber of the housing, and the fluid outlet at the other end of theconnector (10), during use of the device, facing sideways, preferably facing horizontally,and the hollow interior of the straw (11) being attached with the fluid outlet at the otherend of the connector (10) and, during use of the device, extending sideways, preferablyhorizontally.

17. A connector (10) intended for being attached to a housing for extracting motile cellsfrom a raw semen sample, the housing comprising a separation chamber and at leastone outlet opening (5,6) of the separation chamber, and the connector (10) intended forproviding a connection between the at least one outlet opening (5,6) of the separationchamber and for being attached to a longitudinal straw (11) for storing a portion ofmotile cells, the connector (10) having one end with a fluid inlet and another end with afluid outlet and a fluid passage extending between the one end and the other end of theconnector (10), the one end of the connector (10) intended for being attached to the atleast one outlet opening (5,6) of the separation chamber of the housing, and the otherend of the connector (10) intended for being attached to a first end of the straw (11).

18. A connector (10) according to claim 17,- the connector (10) having an outer flange (16) and an inner flange (17) and an annulargroove (18) delimited by an outer flange 16 and an inner flange 17, a size and shape ofthe annular groove (18) corresponding to a size and shape of part of an outlet protrudingfrom a housing for extracting the motile cells from the separation chamber, and- when the connector (10 is attached to the housing, the part of the outlet protrudingfrom the housing extending into the annular groove (18), providing a snug fit betweenthe flanges (16,17) of the connector (10) and the part of the outlet protruding from thehousing.

19. Use of a device according to any of claims 10-16 for obtaining an amount of motilecells to be stored in at least a part of a hollow interior of a straw (11), said motile cells, after having been stored in at least part of the hollow interior of the straw (11), to beused for at least one of the following artificial insemination methods: in intrauterineinsemination (IUI), in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

19. Use of a connector according to claim 17 or 18 for obtaining an amount of motilecells to be stored in at least a part of a hollow interior of a straw (11), said motile cells, after having been stored in at least part of the hollow interior of the straw (11), to beused for at least one of the following artificial insemination methods: in intrauterineinsemination (IUI), in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

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