Patent Description:
There are plenty of methods and devices for cell separation in the state of the art. Such methods and devices are based on the characteristic properties of motile cells as spermatozoa, such as rheotaxis (<NUM>) among others. Devices and methods for separation in the state of the art use filtering membranes, density gradients, perforated membranes which block the passage of cells, among others.

Patents <CIT>) and <CIT> describe a method and a device to separate the best spermatozoa using a membrane, where the spermatozoa which penetrate the membrane may be kept in a suitable medium. According to what is mentioned in the description, this device separates motile spermatozoa when they pass through a porous membrane of <NUM>- <NUM>-pore. This device does not separate spermatozoa by rheotaxis but filters non-motile spermatozoa.

Patent application <CIT>) describes a method and apparatus for regulating optimum flow of semen and separating motile sperms. The device consists of a cylindrical shape container, a membrane comprised of pores (holes of between <NUM> and <NUM> diameter), and a second cylindrical container which is assembled together with the first The separation method is based on the capacity of spermatozoa to swim upstream (swim-up). The method uses a system of currents which induces rheotaxis, but its realization becomes complex since it requires a degree of specialization and devices that the device of the present invention does not need. The quantity of medium required by this method is higher and it cannot proceed without using an oven. The application <CIT> describes a spermatic-cell separation process by a <NUM>-<NUM> micron pore filtering membrane. Said membrane may be of various materials such as PES, PVDF, MCE, PTFE, Nylon, polycarbonate, etc. This type of device does not allow the passage of motile cells but withholds them in the membrane pores.

Patent <CIT> describes a sperm filter trap having compressed glass wool filter material to separate low-motility spermatozoa from those of a high motility. It is neither based on rheotaxis; it filters non-motile spermatozoa.

Patent Application <CIT> describes a device for separating sperm which uses a radial array of microchannels disposed so as to direct sperm from a reservoir to another. Said microchannels are between <NUM> and <NUM> microns in width, from <NUM> to <NUM> microns in height, and between <NUM> and <NUM> in length. The microchannels are cylindrical in shape. This device is complex in design and manufacture. Spermatozoa do not swim upstream and therefore, the device directs them under the definition of "wall swimming", i.e., the shape of the channels redirects spermatozoa. In addition, this device needs a stove. On the contrary, the present invention achieves self-selection taking advantage of the spermatozoa's ability to penetrate and swim upstream, since an initial stream is generated within the device towards the site where the sperm is to be placed. Also, the channel shape of the present invention achieves extraordinary yields without requiring additional devices.

Application <CIT> discloses a method and a device to separate spermatozoa applying the swim-up technique.

<CIT> discloses a sperm separation device comprising a tubular conduit defining an upstream zone for receiving semen and a downstream zone from which sperm are arranged to be harvested. The upstream and downstream zones are separated by constriction means within the conduit, the constriction means being arranged to establish capillary flow of a fluid medium from the upstream zone to the downstream zone, whereby motile sperm in semen inserted into the conduit upstream from the constriction means and subjected to the flow of the fluid medium are enabled, having passed through the constriction means, to maintain a position in the downstream zone for harvesting.

<CIT> discloses a microfluidic chip for acquiring sperms with high motility in a sperm sample, which comprises: (a) an inlet region at one end of the microfluidic chip; (b) a first flow channel that is in fluidic communication with the inlet region; (c) a divergent channel, which is arranged at the downstream of the flow channel and in fluidic communication with the flow channel; and (d) one or more outlet region(s) located at one or both sides of the divergent channel. According to <CIT>, the sperm sample flows from the flow channel to the divergent channel, and that the divergent channel comprises one or more outlet region(s) located at one or both sides of the divergent channel to grade sperms with various motility scales.

<NPL>, describes a device for sorting sperm cells based on the progressive motility in <NUM> parallel microchannels. During separation no flow is present.

<NPL>, discloses a membrane filter with controlled (conical) pore shape and its application to cell separation and single cell trapping. Dong-Hoon Choi et al. describes that the membrane with conical shape pores shows strong potential as an analytical tool for a single cell analysis. The fabricated conical pores remarkably enhance the trapping ability, and thus the whole process, including filtration, single cell isolation, antibody staining, immunofluorescence imaging, and in situ hybridization, can be easily performed on the membrane after the filtration process thanks to the strong trapping ability.

<NPL>, discloses a microfluidic device for high efficiency and high purity capture of CTCs (circulating tumor cells) and the integration of a microfilter with conical-shaped holes and a micro-injector with crossflow components for size dependent capture of tumor cells without significant retention of non-tumor cells.

The devices and methods present in the state of the art have technical disadvantages compared with the present invention since the medium used for the separation can become clogged, they require manipulation by someone relatively skilled in laboratory instrument handling and therefore it is also necessary handling in controlled environments, making it impossible their use on the field or in consulting rooms. On the other hand, most of them require extra filtering and/or centrifugation operations, favouring the likelihood of sample contamination, material loss, DNA alteration, etc., whether due to the increased sample manipulation time or pressure caused by revolutions during centrifugation.

The present invention provides a method for the separation of motile cells which does not require the operations of filtration and/or centrifugation; its manipulation is simple and achieves excellent results of separation of those motile cells of a cell population, including the separation of motile cells with damaged DNA. A remarkable feature of the present invention is that it does not require an oven to perform motile cell separation. This allows taking the necessary actions for artificial insemination in humans as well as in animals under basic conditions, without the need of the usual equipment regarding this type of techniques. The method of the present invention even allows on-field artificial insemination.

The method of the present invention for the separation of motile cells from a cell population, preferably spermatozoa, uses a device which comprises a first reservoir and a second reservoir linked by at least a membrane, characterized in that said membrane comprises a multiplicity of channels with at least one end showing a reduction in its diameter towards the interior of said channels. And where said membrane is made preferably of a material selected from the group comprising of glass, PET, metal, polymer, carbon fiber and the mixtures or combinations thereof. Where said channels comprise preferably in at least one of their ends, a shape selected from the group comprised of conical and hyperboloid shapes, and said membrane comprises a multiplicity of channels. In a preferred embodiment, said membrane is a perforated plate.

In addition, said channel comprises, in another preferred embodiment, both ends of shapes selected from the group comprised of conical, paraboloid and hyperboloid shapes.

Said membrane comprises a multiplicity of channels which allow the separation of motile spermatozoa on one side of said membrane and of the elements or non-motile cells which remain on the other side of the membrane.

Preferably, said motile cells separation device used in the method of the invention has channels comprising a first outer diameter of between <NUM> and <NUM>; an inner diameter of between <NUM> and <NUM>, and length of between <NUM> and <NUM>; more preferably they comprise a first outer diameter of between <NUM> and <NUM> and a second outer diameter of between <NUM> and <NUM>; an inner diameter of between <NUM> and <NUM> and a length of between <NUM> and <NUM>.

Also, the motile cells separation device used in the method of the present invention presents preferably said membrane with between <NUM>,<NUM> and <NUM>,<NUM> channels /cm<NUM>; and preferably the volume of said first reservoir is between <NUM> and <NUM>, and the volume of said second reservoir is between <NUM> and <NUM>; and where said second reservoir comprises a higher level than said first reservoir. Also, said second reservoir is filled with a culture medium generating a current within said culture medium towards said first reservoir.

Where said membrane is made of a material selected from the group comprising of glass, PET, metal, ceramics, polymer, carbon fiber and the mixtures or combinations thereof.

In some embodiments, the motile cells separation method of the present invention allows achieving a decrease of sperm with damaged DNA in said second reservoir regarding a sample of fresh semen. The present invention succeeds in reducing up to nine times the value of the TUNEL Test (DNA damage) compared with the direct method. In particular, a decrease of at least <NUM> times is observed for the invention realized in PET (from <NUM>% to <NUM>%, for sample D, table <NUM>); and up to <NUM> times for the invention in glass (from <NUM>% to <NUM>% for the same sample D). In addition, the methodof the present invention increases the quantity of spermatozoa with normal morphology in at least <NUM>% (morphology values for Sample D).

Object of the present invention is a method to separate motile cells from a cell population according to claim <NUM>.

Where said separation method for motile cells from a cell population in step "b" comprises a counter-current in a culture medium flowing in the opposite sense of the passage of said motile cells, where said current is generated by pressure difference, or capillarity, and said pressure difference is caused by the difference in level between the reservoirs located at both sides of said membrane. In addition, said step "c" comprises incubating for a period of between <NUM> and <NUM> minutes at a temperature ranging from <NUM> to 37ºC. Preferably between <NUM> and <NUM> minutes. And, the method of the present invention uses the device of the present description.

According to the present invention, a cell population comprises a fluid which may be a culture medium, a biological fluid with a great variety of cells among which there are cells with locomotory capacity, hereinafter called motile cells. The example of the present invention comprises the separation of motile cells of the spermatic type; however, the device and method also apply to other types of motile cells such as microorganisms or parasites.

According to the present invention, membrane means any material characterized by containing at least one channel that passes from one face to the other of the membrane and connects both reservoirs of the device used in the method of the present invention.

The present invention describes a device and a method for the separation of motile cells, preferably spermatic cells, which is not based on the premises of swim-up or what is used by most other systems, but it is based on two aspects <NUM>) the search by a gamete or cell of a place to enter, at random, facilitated by the channel shape and <NUM>) the microcurrents generated by the spermatozoa themselves and that end up directing the following ones by rheotaxis (firstly induced by pressure difference and later self-induced by the passage through the narrow channel).

The device used in the method of the present invention does not require any other laboratory element to perform according to its intended use; i.e. no additional tubes, nor centrifuge, no stove required. It does not require electricity, either, which makes it a basic and innovative tool when it comes to extending assisted fertilization world widely, enabling health care professionals and veterinarians to access by themselves to a tool to be used in for example in rural areas far from laboratories. The incubation required by the method of the present invention may be carried out keeping the device within the closed fist of an adult or in contact with the human body.

The device used in the method of the present invention comprises a first reservoir (<NUM>), where the semen sample from which spermatozoa are to be separated are placed, a second reservoir (<NUM>) to where spermatozoa or motile cells migrate and are collected, being both reservoirs linked by a membrane (<NUM>). Said membrane comprises a multiplicity of perforations which constitute channels through which motile cells migrate. Said perforations, called channels, can be of the same or different diameter according to the section measured, i.e., taking into account the section that is linked to the first reservoir, the section that is linked to the second reservoir and the section located between the two sections already mentioned (both called outer sections), called hereinafter middle section.

In a preferred aspect of the present invention, the channels of the membrane are of the same diameter in all the sections of said channels.

In another aspect of the present invention, the diameter of the channels is different in the sections linked to the reservoirs regarding the middle section. In a preferred embodiment of the present invention, said outer sections of said channels are of a greater diameter than the diameter of the middle section of said channel.

In another aspect of the present invention, said channel outer sections are geometric in shape, such as conical, hyperbolic or parabolic.

The device used in the method of the present invention operates in the absence of filtering or the application of electromagnetism, or static currents or immunological labelling methods. The device used in the method of the present invention makes the spermatozoa swim in the same way they would do in vivo, migrating in groups from a reservoir passing through a membrane towards the other reservoir, leaving behind detritus, mostly spermatozoa with damaged DNA and non-motile cells. Rheotaxis -the property of having the design and natural behaviour of gametes to penetrate interfaces-is taken advantage of. Said behaviour is observed to a greater extent in spermatozoa with DNA that is not damaged. The device uses the property of rheotaxis of spermatozoa generated by a difference in pressure between the two reservoirs, achieving so due to the higher initial level of the second reservoir (where motile spermatozoa are retrieved), which persists until both pressures are balanced, though they are also involved as the driving forces of said capillary current and possibly difference in concentration in both reservoirs. Beyond the phenomenon caused by the current, it is a fact that there is a fluid current from the second reservoir towards the first one, which generates an impulse for the healthy spermatozoa to swim upstream. Up to that instant, the currents induced through each channel through the culture medium towards said first reservoir for whole semen collection, favours rheotaxis of the spermatozoa which retain such property intact, being the system from that moment (balanced pressure or zero rate flow) self-sustained by the microcurrents induced now by the group of spermatozoa which continues passing through the membrane.

This condition of the most apt spermatozoa to pass through the membrane of the present invention agrees with the results in the significant increase in the percentage of normal morphology and the highly significant decrease in the percentage of damaged DNA. The characteristics of the present invention allow the self-selection among gametes of rapid linear motility, which agree, as it is expected, with a percentage in an increased normal morphology and a higher percentage of DNA without alterations.

In the device used in the method of the present invention, the spermatozoa are following a microcurrent caused by the flow between the compartment of the retrieval medium and the semen compartment, and later self-sustained by those which follow. In other words, after one finds the entrance, the others see their way favoured by the microcurrents caused by the first flagellum, to which the microcurrent of the successive flagella add to, thus enhancing the current and therefore the rheotaxis effect. This way spermatozoa leave the seminal plasma by themselves and access the insemination culture medium, leaving behind in the seminal plasma all types of elements which are not spermatozoa or else non-motile spermatozoa (cells, detritus, immobile spermatozoa and also spermatozoa without rheotaxis capacity), since they do not show the capacity of spermatozoa of penetrating this type of orifice.

The device used in the method of the present invention comprises at least one membrane with a multiplicity of channels of shapes selected from the following ones: conical, hyperbolic, paraboloid of revolution, among many others with allow widening in at least one of the ends of the channels as it can be seen in <FIG>. The quantity, quality and type of channels vary according to the material used to manufacture the plate. The size of the reservoirs for the semen sample may also vary according to the volume to be processed, among other factors. The manufacturing methods are known in the state of the art and may be performed by laser, among other perforation mechanisms.

In a preferred embodiment of the present invention, the membrane comprises a channel density of between <NUM>,<NUM> and <NUM>,<NUM> per cm<NUM> of membrane.

In another aspect of the present invention, the perforations or channels of the membrane of the present invention comprise a length of between <NUM> and <NUM>.

As it was previously mentioned, the channels of the present invention comprise three sections of different diameters; a first outer diameter linked to said first reservoir (where semen is placed), an inner diameter and a second outer diameter which links the membrane with said second reservoir (where the motile cells are collected in a culture medium supplied to that aim). Thus, said first outer diameter comprises between <NUM> and <NUM>, said inner diameter comprises between <NUM> and <NUM>, and said second outer diameter comprises between <NUM> and <NUM>.

In another aspect of the present invention, the perforations of the membrane comprise different geometric shapes such as cylindrical, conical, parabolic, hyperbolic, hyperboloid of revolution. Preferably, the geometric inlet and outlet perforation shapes is a curve of revolution which may be straight (which generates a cone) or else a curve the type of a parabolic or hyperbolic shape, resulting in a figure of revolution with an outer diameter greater than the inner diameter (<NUM>).

Surprisingly, it has been found that the shapes described of said perforations which originate the channels of said membrane, when widened at the inlet favour a configuration which increases the efficiency of the device used in the method of the invention significantly to obtain a higher concentration of viable motile cells.

The reservoirs of the device used in the method of the present invention comprise a volumetric capacity which depends on various factors: the volume of the sample to be processed, the dimensions of the membrane, among others. In a preferred embodiment, the reservoirs used for a membrane of 1x3 cm vary between <NUM> and <NUM>. It is vital to highlight that the device used in the method of the present invention generates a current in the fluid or culture medium in such a way that the motile cells, preferably spermatozoa, use their ability to swim upstream for their separation. Said current is caused by a pressure difference in the reservoirs. In order to generate pressure difference, the second reservoir contains a higher liquid level, thus generating a current towards the first reservoir through the membrane. In a preferred embodiment, in the device used in the method of the present invention, the sample to be separated is placed in said first reservoir and fluid or culture medium is loaded into said second reservoir to retrieve motile cells separated from the rest of the population on their passage through said membrane. To generate difference in pressure and therefore cause a current flowing to the first reservoir, first the second reservoir has to be filled to same level or a higher level than said first reservoir; subsequently, the first reservoir is filled with the sample of the population of cells to be separated.

The device used in the method of the present invention separates motile spermatozoa within a period of between <NUM> and <NUM> minutes. Preferably between <NUM> and <NUM> minutes.

In another aspect of the present invention, the same can be performed in the absence of an stove, since it also works even when it is heated by the warmth released by operators themselves.

In another aspect of the embodiment of the present invention, the device is adapted to the intracytoplasmic sperm injection (ICSI) technique. For inseminating with semen microinjection into an ovocyte, separating high performing spermatozoa on the same injection membrane may be of use. It is based on an alternative embodiment of the present invention, but with dimensions suitable for the ICSI process. A preferred alternative embodiment not according to the present invention with only one channel that is shown in <FIG> comprises said two reservoirs, also called micro cuvettes (<NUM> and <NUM>) linked by a channel with a funnel-shaped inlet connecting both reservoirs (<NUM>). Both reservoirs are covered by a coverglass (<NUM>) thus enabling a <NUM>µ channel. This system allows retrieval of high quality spermatozoa in situ. In this alternative embodiment, the membrane has only one channel.

In another alternative embodiment of the present invention, the invention comprises a cylindrical shape as shown in <FIG>, comprising a cover (<NUM>), a hole close to the opening of said tube (<NUM>), a first reservoir (<NUM>) and a second reservoir (<NUM>) where it can be seen that the level of the second reservoir, outlet and collection reservoir for the selected spermatozoa is higher than the level of the first reservoir where the semen sample with the spermatozoa to be separated is loaded. <FIG> shows an enlargement of the section exhibiting the membrane (<NUM>) with channels (<NUM>) along which spermatozoa flow.

On a glass membrane The motile cells separation device of the present invention (MXM) comprises two reservoirs plate separated by a <NUM> ±<NUM> thick glass membrane plate with conical channels having the length of the glass width, of about <NUM>, a <NUM>-first diameter (inlet) and a <NUM>-second diameter (outlet). In the first place retrieval medium Ham F10 1X with HEPES was placed, supplemented with SSS and Penicillin / streptomycin was placed in the retrieval reservoir (second reservoir); then semen samples D, E y F were placed after having been processed according to WHO2010 Protocol, in the remaining reservoir (first reservoir).

In this example the membrane is <NUM> x <NUM>, with a density of <NUM> perforations/cm2. The first reservoir, where the sample to be processed is placed, has a maximum volumetric capacity of <NUM>, and the second reservoir comprises a maximum capacity of <NUM>.

The membrane made of PET was made with <NUM> channels, each <NUM> in length. Conical channels comprise a first (inlet) diameter of <NUM> and a second (outlet) diameter of <NUM>. The PET membrane is <NUM> x <NUM>. The reservoirs are the same ones used for the glass membrane in example <NUM>. In the first place retrieval medium Ham F10 1X with HEPES was placed, supplemented with SSS and Penicillin / streptomycin was placed in the retrieval reservoir (second reservoir); then semen samples A, B, C and D were placed after having been processed according to WHO2010 Protocol, in the remaining reservoir (first reservoir).

For both examples the procedure to use the device according to the method of the present invention is the following:.

For semen evaluation before and after retrieval, the WHO <NUM> guidelines were observed. To evaluate DNA the (Terminal Transferase dUTP Nick End Labeling) TUNEL Test was applied using a flow cytometer or a fluorescence microscope.

The results of the instrument show that applying the method of the present invention (MXM) for glass as the material of choice double the percentage of normal shapes (strict morphology) regarding the untreated semen, from <NUM>% in the direct method (average of samples D,E,F) of normal shapes to <NUM>% (n:<NUM>) with the device of the presentdescription. Comparing MXM of the present invention (glass), with swim-up, the present invention achieves an improvement of <NUM> % (samples D, E, F) in the morphology of the selected spermatozoa. (Table <NUM>).

In the case of the present invention using PET as the chosen material, the percentage of normally shaped spermatozoa tripled compared with the direct method Increasing from <NUM>% to <NUM>% (for samples A, B, C, D), average values of the results obtained in each test. While in the swim-up case, almost a two-fold value for normal shape spermatozoa is reached, from <NUM>% to <NUM>% of normal shape applying the method of the present invention. It is worth mentioning that swim-up is the normal basic technique for spermatozoa retrieval in view of high or low complexity artificial insemination, with the disadvantage that the exposure time and manipulation together with centrifugation may lead to DNA alterations.

On the other hand, morphology is evaluated applying Kruger's criteria, WHO. A sample whose Tunnel Test (<NUM>) for DNA integrity, yielded a value of <NUM> % (pathological, since the values of reference must be below <NUM>%) was taken to perform that same study on swim-up and on MXM with PET and glass, from the same sample (sample D). The values obtained, as it is seen in the following table, show a significant improvement when using MXM on PET: the original value decreases in over a third (from <NUM> to <NUM>), a remarkably significant value. On the other hand, the values of the Tunnel Test for the methods of swim-up as well as MXM on glass of the present invention for sample D, show an increase of said value with a reduction of almost nine times.

Even though it is observed that with the swim-up sample D achieves a decrease in DNA-damaged spermatozoa which is also equal to that found for MXM, the latter method of the present invention yields a sample with spermatozoa of a three-times better morphology for the said sample.

Claim 1:
A method to separate motile cells from a cell population using a device which comprises a first reservoir and a second reservoir linked by at least a membrane, characterized in that said membrane comprises a multiplicity of channels with at least one end that comprises a reduction in diameter towards the interior of said channels, comprising the following steps:
a) placing the culture medium to flood said second reservoir and said channels;
b) contacting the cell population in said first reservoir with a face of said membrane comprising a multiplicity of channels with at least one end having a reduction in their diameter towards the interior of said channels, wherein said first reservoir is filled with a sample of said population of cells to be separated at a level that is lower than the level of said second reservoir, whereby a pressure difference in said reservoirs is generated, so that continuous passing medium to the first reservoir is allowed, and the initial counter current effect is produced, so that said motile cells swim in counter-current for their separation;
c) incubating;
d) retrieving motile cells of a higher percentage of normal morphology from the other side of said membrane.