Sperm cell separation device and high-quality sperm cell selection method

Abstract

The present invention relates to a sperm cell selection device having a support plate comprising an inlet for receiving a sample of sperm cells, an outlet for collecting at least some of the sperm cells contained in the sample, and a microfluidic system located between the inlet and the outlet, wherein the microfluidic system comprises a tank for receiving a culture medium, and the tank has an inlet region and an outlet region in the vicinity of the inlet and the outlet, respectively, and wherein the inlet and the outlet are arranged such that when a culture medium is added into the tank through the outlet, the culture medium is introduced into the tank through the outlet. Providing a hydrostatic pressure difference between the inlet region and the outlet region to cause the medium to flow from the outlet region to the inlet region to initiate sperm cells in a sperm cell sample to migrate from the inlet region to the outlet region, and wherein the inlet region and the outlet region are in fluid communication with each other via a main channel arranged in the slot, the channel having a region of reduced width arranged between the inlet region and the outlet region to form a taxis region, whereby the sperm cell sample has undergone selection by passing through the taxis region during the sperm cell migration so that a premium sperm cell sample can be collected from the exit region at the exit.

Description

[0001] The invention relates to a sperm cell selection device and a method for separating high-quality sperm cells by using the device.

[0002] Semen analysis and the selection and separation of high-quality sperm are essential for the practice of artificial reproductive technology (ART). ART, including intracytoplasmic sperm injection (ICSI), intrauterine insemination (IUI), and in vitro fertilization (IVF), are being implemented to address health issues related to male infertility and treat oligospermia.

[0003] The ART market has been growing since the last decade. Rising infertility rates, technological advances, and government financial support serve as market drivers. On the contrary, higher costs and failure rates serve as potential constraints. The European Society of Human Reproduction and Embryology (ESHRE) reports that the average probability of pregnancy and birth per embryo transfer is 37% and 21%, respectively (DeGeyter et al., ART in Europe, 2014: “ESHRE generated results from European registries”, Human Reproduction, 2018). Substandard IVF conditions, female / male gamete quality, and embryo damage are potential factors leading to ART failure. Screening of semen samples and selection of subpopulations of high-quality sperm (or spermatids) is an important step and is closely related to the efficacy of ART (Oseguera-Lopez et al., "New technologies for sperm selection to improve IVF and ICSI outcomes", Frontiers in Cell and Developmental Biology, 2019; Perez-Cerezales et al., "The fallopian tube: epigenetic landscape from sperm selection to embryos", Reproductive Biology, 2018; Sakkas et al., "What can we learn from nature to improve outcomes of assisted reproduction?", Human Reproduction Update, 2015).

[0004] There are three main known mechanisms for guiding sperm cells to the oocyte: rheotaxis, thermotaxis, and chemotaxis (Giojalas and Guidobaldi, "Reaching and leaving the oocyte: Interactions between several sperm transport mechanisms and complex fallopian tube physiology," Molecular and Cellular Endocrinology, 2020; Suarez, "Interactions of mammalian sperm with the female reproductive tract," Cell Tissue Research, 2016, 363; Suarez and Pacey, "Sperm transport in the female reproductive tract," Human Reproduction Update, 2006).

[0005] Fluorotaxis involves the swimming and repositioning of sperm cells against the direction of fluid flow. Thermotaxis involves the migration of sperm cells caused by temperature gradients. It is believed that thermotaxis is primarily responsible for guiding the swimming of sperm cells in the follicular tubes. Chemotaxis involves redirecting sperm cells toward the oocyte and triggering the accumulation of sperm cells. Fluorotaxis, thermotaxis, and chemotaxis are known to occur through biological synergies between sperm cell swimming and the microenvironment of the female reproductive tract. In addition, the anatomical structure of the female reproductive tract promotes the migration of high-quality sperm cells toward the oocyte. In summary, the female reproductive tract provides a microenvironment that is conducive to quality selection for conception in vivo.

[0006] Despite the known mechanisms of sperm cell transport through the female reproductive tract, reproductive health clinicians still follow the "World Health Organization (WHO) screening and quality subpopulation collection protocol" (World Health Organization, WHO Laboratory Manual for the Examination and Processing of Human Semen, Sixth Edition, 2021). This protocol has been revised from time to time because the evaluation of the minimum survival threshold ignores potentially relevant parameters such as ethnicity, environmental toxins, and the navigation ability of sperm cells under optimized conditions (Douglas et al., "A new method to improve the reliability of artificial semen analysis: a paradigm shift in the examination of infertile men", World Journal of Men's Health, 2019; Levine et al., "Temporal trends in sperm count: a systematic review and meta-regression analysis", Human Reproduction Update, 2017; Wang and Swerdloff, "Limitations of semen analysis as a test of male fertility and the anticipated need for new tests", Fertility and Sterility, 2014). In addition, as the applicants have shown, the standardized protocol is manual and time-consuming (Shukla et al., "Automated analysis of rat sperm motility in microchannels", Biomedical Physics and Engineering Letters, 2018; 4). However, the WHO manual refers to “computer-assisted semen analysis (CASA)”, which provides rapid and automated screening of semen samples. CASA provides the kinematics of motile sperm cells, however these parameters do not take into account the microenvironment and physiological conditions of the female reproductive tract, and therefore, the biological significance of these parameters remains unknown. In addition, reproductive clinicians have questioned and criticized the accuracy and reproducibility of CASA analysis (Talarczyk-Desole et al., “Manual versus computer-assisted sperm analysis: Can CASA replace manual evaluation of human semen in clinical practice?”, Ginekologia Polska, 2017;88).

[0007] For sperm separation and isolation, the WHO manual refers to standard sperm washing, density gradient centrifugation (DGC), and sperm swim-up procedures, which result in DNA fragmentation in sperm (Alvarez et al., "Centrifugation induces sublethal damage to human spermatozoa; isolation of human spermatozoa from seminal plasma by the dextran swim-up procedure without centrifugation prolongs their motility," Human Reproduction, 1993). The prior art describes the side effects of using damaged male gametes in a mouse model, where the incompetent cells are able to proficiently fertilize the egg; however, this results in altered gene expression and promotes defective fetal / placental development (Fernandez-Gonzalez et al., "Long-term effects of intracytoplasmic sperm injection of DNA-fragmented spermatozoa on the health and behavior of adult offspring in mice," Biology of Reproduction, 2008).

[0008] Despite these technical drawbacks, CASA, DGC, and swim-up are still the most commonly used protocols for sperm screening and selection, respectively. Therefore, there is a huge potential for technological upgrading to improve the saturation success rate of ART.

[0009] The in vivo mechanism of sperm motility is a complex phenomenon; therefore, implementing this proposal is not straightforward. Nevertheless, Lab-on-a-Chip researchers have taken advantage of microfluidics and established a proof of concept (PoC) related to sperm motility, semen analysis, and sperm sorting.

[0010] US 8,535,622, US 2014 / 0248656 and WO 2020 / 041303 disclose sperm cell selection systems.

[0011] Microfluidics involves the manipulation of small volumes, from mL to nL. Small volume sizes and submillimeter channel dimensions include a unique feature: fluid motion in parallel flow (laminar flow), in which the ratio of inertial and viscous forces is small. This dimensionless ratio is called the Reynolds number (Re), which calculates the propensity of fluid motion to produce turbulence. Laminar flow through microchannels facilitates a high degree of control, which offers many advantages over traditional laboratory practices. Microfluidic systems use smaller amounts of sample and reagents, thereby reducing operating costs and increasing the sensitivity and rapidity of related biological protocols. Microfluidics provides parallel processing and therefore high throughput; in addition, the technology can be combined with external perturbation techniques, including acoustic (Clark et al., “Acoustic capture of sperm cells in simulated sexual assault samples,” Int J Forensic Genetics, 2019; 41), optical (Schiffer et al., “Rotational motion and rheology of human sperm do not require functional CatSper channels and transmembrane Ca 2+Signaling, European Molecular Biology Society, 2020), magnetic (Xu et al., “Magnetic micromotors for multimotile sperm cell capture, transport and enzyme release”, Angewandte Chemie International Edition, 2020;59), electrical (Chen, Chen et al., “Direct characterization of sperm motility-related parameters in a microfluidic device: proof of principle”, Clinical Chemistry, 2013; De Wagenaar et al., “Microfluidic sperm purification: impedance-based sperm cell analysis and sorting”, Lab on a Chip, 2016;16; De Wagenaar et al., “Spermometer: electrical characterization of single boar sperm motility”, Fertility & Sterility, 2016;106), for single cell manipulation or separation. Microfluidics is developing into the most commonly used technology to replicate and control the microenvironment of human organs and physiological conditions in vivo.

[0012] However, the devices of the prior art are not satisfactory, especially in the recovery of sperm cells. Generally, the devices usually have low recovery performance.

[0013] In addition, the implementation of ART methods such as IUI and IVF requires a certain concentration of high-quality sperm. Although microfluidics-based methods can generally improve the quality of sperm cells, the throughput is still unsatisfactory. In other words, high throughput is still an unmet need. Nevertheless, multiplexing can be used to improve the recovery of high-quality cells, but this will increase the workload and complexity of the protocol. In addition, there is no technology in the prior art that combines active and passive sperm navigation mechanisms for sperm quality separation.

[0014] The present invention improves this situation.

[0015] To this end, the present invention relates to a sperm cell selection device, which has a support plate, the support plate including an inlet for receiving a sperm cell sample, an outlet for collecting at least some sperm cells contained in the sample, and a microfluidic system located between the inlet and the outlet, wherein the microfluidic system includes a tank for receiving a culture medium, the tank having an inlet area and an outlet area near the inlet and the outlet, respectively, and wherein the inlet and the outlet are configured to provide a hydrostatic pressure difference between the inlet area and the outlet area when the culture medium is added to the tank through the outlet, thereby prompting the culture medium to flow from the outlet area to the inlet area to initiate the migration of sperm cells in the sperm cell sample from the inlet area to the outlet area, and wherein the inlet area and the outlet area are fluidically connected to each other via a main channel arranged in the tank, the channel having a region of reduced width arranged between the inlet area and the outlet area to form a hydrotaxis area, whereby the sperm cell sample undergoes selection by passing through the hydrotaxis area during the migration of the sperm cells, thereby enabling the collection of a high-quality sperm cell sample from the outlet area at the outlet.

[0016] Thus, the present invention replicates the natural selection process of sperm cells by at least partially mimicking the female reproductive tract. Specifically, the device of the present invention includes rhizotaxis properties that enable the separation of high-quality sperm from a sperm cell sample.

[0017] The present invention further proposes different embodiments of the device: - The inlet may comprise a tube having a first diameter (preferably 5 mm to 12 mm) and a first height (preferably 1.5 mm to 2.5 mm), and the outlet comprises a tube having a second diameter (preferably 4 mm to 5 mm) and a second height (preferably 3.5 mm to 5 mm (preferably 3.5 mm to 4.5 mm)), wherein the first diameter is greater than or equal to the second diameter and the first height is less than the second height, so as to generate the hydrostatic pressure difference between the inlet area and the outlet area when the fluid culture medium is added. This arrangement improves the characteristics regarding the hydrostatic pressure difference, ultimately resulting in a liquid flow close to the flow of the natural female reproductive tract. This embodiment improves the selection of sperm cells.

[0018] - The support plate consists of a top layer and a bottom layer, the top layer comprising the inlet and the outlet, and the bottom layer comprising the slot. This improves and / or facilitates the production of the device, such as a molding process or high-resolution SLA / DLP resin 3D printing, etc.

[0019] - the main channel comprises a microchannel extending from the hydrotaxis region to the outlet region; and / or the main channel comprises a microchannel extending from the inlet region to the hydrotaxis region. Sperm cells tend to swim near the walls of the microchannel. In addition, the restriction of the microchannel (from 35 μm to 45 μm, more typically from 35 μm to 120 μm) improves the progressiveness and directionality of the sperm cells. The hydrotaxis region has converging sub-microchannels that can control the progressiveness of the sperm cells.

[0020] In a particularly preferred embodiment of the present invention, the device also includes a support having at least one container configured to accommodate the support plate, the support including a first heating device and a second heating device, wherein, when the support plate is placed in the container, the first heating device is located near the inlet and the second heating device is located near the outlet, the first heating device and the second heating device are respectively configured to heat at a first temperature and a second temperature, the first temperature being lower than the second temperature, so as to set a temperature gradient in the microfluidic system of the support plate to simulate thermotropism.

[0021] In the preferred embodiment, the device of the present invention comprises both hydrotaxis and thermotaxis means.The quality of sperm cell selection is greatly improved.

[0022] Another object of the present invention is a method for sperm cell selection comprising the following steps: i. providing a device as described above, the device comprising a support plate, the support plate having an inlet, an outlet and a microfluidic system between the inlet and the outlet, the microfluidic system comprising a groove, the groove having an inlet area and an outlet area near the inlet and the outlet, respectively, the inlet and the outlet being arranged to provide a hydrostatic pressure difference between the inlet area and the outlet area when a fluid culture medium is added to the groove via the outlet, thereby causing the fluid culture medium to flow from the outlet area toward the inlet area, the inlet area and the outlet area being in fluid communication with each other via a main channel arranged in the groove, wherein the channel has a region of reduced width arranged between the inlet area and the outlet area so as to form a rhizotaxis area; ii. pre-filling the groove with a fluid culture medium (preferably sperm separation medium); iii. providing a sperm cell sample to the inlet to distribute the sample to the inlet area of ​​the groove; iv. providing a fluid culture medium to the outlet to cause the culture medium to flow from the outlet region to the inlet region, thereby causing sperm cells to migrate from the inlet region to the outlet region; v. selecting sperm cells from the sperm cell sample when the sperm cells pass through a rhizotaxis region during the sperm cell migration process; and vi. Collecting a sample of high-quality sperm cells from the outlet area at the outlet.

[0023] The method also includes the step of setting a temperature gradient on the support plate to simulate thermotaxis and improve the migration of the sperm cells. Preferably, the groove includes sub-microchannels. The integration of sub-microchannels is crucial because the present invention not only promotes sub-channels along the XY plane, but also includes sub-channels in the YZ plane. The present invention allows for the accommodation of multiple sub-microchannels without compromising the quality of the microfluidic-based method.

[0024] Other features and advantages of the invention will stand out and / or become clear from a reading of the following description, which includes specific examples given by way of illustration and non-limiting manner, and the accompanying drawings, in which: Figure 1 A perspective view showing an embodiment of the device according to the invention; Figure 2 shows a perspective view of a device according to the invention; Figure 3 A side view showing the top layer of the device according to the invention; Figure 4 A top view of the trough of the present invention is shown; Figure 5 A top view of an embodiment of a groove of the present invention is shown, and further details of a specific part / section of the groove are shown; Figure 6 A top view of another embodiment of a groove of the present invention is shown, and further details of a specific part / section of the groove are shown; Figure 7 A perspective view of a heating rack according to the present invention is shown; Figure 8 A graph showing the temperature variation over time in a heating rack according to the present invention; Fig. 9 A graph showing temperature versus position in a heating rack according to the present invention is shown; Fig.10 Micrographs showing sperm cells and their tracks; Fig.11 Graphs showing the sperm cell concentration from a mother solution and from sperm cell samples treated by three different embodiments of the device according to the invention; Fig.12 Graphs showing sperm cell movement from a mother solution and sperm cell movement from sperm cell samples treated by three different embodiments of the device according to the invention; Fig.13 Graphs showing the instantaneous velocity of sperm cells from a mother solution and from sperm cell samples treated by three different embodiments of the device according to the invention; Fig.14 Graphs showing the advancing speed of sperm cells from a mother solution and from sperm cell samples treated by three different embodiments of the device according to the invention; Fig.15 The results of the DNA fragment test are shown; Fig.16 A perspective view showing an embodiment of the device according to the invention; Fig.17 shows a perspective view of a device according to the invention; Fig.18 A side view showing the top layer of the device according to the invention; Fig.19 A top view of the trough of the present invention is shown; Fig. 20 A top view of an embodiment of a groove of the present invention is shown, and further details of a particular portion / section of the groove are shown; Fig.21 A top view of another embodiment of a groove of the present invention is shown, and further details of a specific part / section of the groove are shown; Fig. 22 A perspective view showing a heating rack according to the present invention; and Fig.23 A perspective view of a heating rack with a single heating device according to the present invention is shown.

[0025] The drawings and descriptions herein mostly contain elements of a definite nature. Therefore, the description and drawings are used not only to better understand the invention but also to help define the invention when appropriate.

[0026] The term "mother solution" as used herein may refer to a sperm cell sample. More specifically, once the mother solution is applied to the device of the present invention, it becomes a sperm cell sample. The sperm cell sample is then processed by the device of the present invention to select and / or separate sperm cells of high quality, for example, in terms of motility or velocity.

[0027] The term "spermatid" and the term "spermatozoon" are often used to refer to the same type of cell.

[0028] The present invention replicates the microenvironment of the female reproductive tract. The design of the device according to the present invention utilizes the basic principles of sperm motility: rhizotaxis and thermotaxis. The device according to the present invention is capable of collecting sperm cell subpopulations with high DNA integrity from a sperm cell sample.

[0029] Sperm motility is initiated by the coordinated action of the cytoskeleton and motor proteins and other complementary molecules. Any defects in the basic structure and function of these proteins will weaken the motility of the cell. In order to solve health problems such as female / male infertility and treat oligospermia, "artificial reproductive technology (ART)" has been introduced, including "intracytoplasmic sperm injection (ICSI)", "intrauterine insemination (IUI)" and "in vitro fertilization (IVF)". As mentioned above, reproductive health clinicians follow the "World Health Organization (WHO)" or "European Association of Urology (EAU)" standard semen sample screening and high-quality subpopulation isolation protocols. However, the WHO-proposed protocol for separating high-quality sperm cells by sperm swim-up and density gradient centrifugation is not satisfactory. For example, these methods include centrifugation, which leads to the generation of reactive oxygen species (ROS) and DNA fragmentation of sperm cells. The present invention simulates natural selection to select high-quality sperm cells.

[0030] Accordingly, the present invention is arranged to achieve both hydrotropism and thermotropism simultaneously. As a result, cell migration of spermatozoa is accelerated, resulting in high throughput (~5×106 / ml) subpopulation collection with ~100% motility and ~100% DNA integration. More typically, high throughput can reach about 3% to 7% of the cell concentration contained in the sample. The qualitative threshold of subpopulation collection produced by it cannot be matched by any device disclosed in the prior art or any conventional or other existing microfluidic-based system.

[0031] The device of the present invention includes at least one main channel (height: about 80μm to 100μm), and preferably a neutrino channel (height: about 40μm to 50μm). More specifically, the main channel is subjected to a flow driven by hydrostatic pressure and includes a so-called hydrotaxis region, which provides a gate-like filtration system for sperm cells. The hydrotaxis region is equivalent to a region with a Venturi effect. As described above, the device may include a neutrino channel. The neutrino channel is arranged in the main channel. More specifically, the neutrino channel can be attached to the bottom of the main channel. The neutrino channel facilitates the swimming of sperm. The applicant surprisingly found that the structure of the subchannel accelerates the swimming of sperm. This will result in a higher subpopulation collection, that is, the selection of sperm cell concentration can be improved, and more importantly, without losing quality characteristics.

[0032] The device of the present invention is usually prepared by computer 3D design and then by high-resolution 3D printing or molding technology. The "computer-aided design (CAD)" of the microfluidic circuit can be drawn in the open source FreeCAD software and further exported to the ".STL" format for injection molding or 3D printing-based microfabrication. Specifically, the support plate can be prepared using this technology.

[0033] According to a preferred embodiment, the device comprises a support having a pocket or container to accommodate a support plate. The support may comprise a gradient plate made of an aluminum alloy (6800, 7075). Two pieces of polyimide tape may be glued in a thickness pattern to two corners or opposite sides of the support and then heated at preferably 37°C and 39°C, respectively. The temperature may be controlled by a Meerstetter® 1091 thermoelectric cooler (TEC) driver (available from Meerstetter Engineering GmbH) connected to a PT-100 sensor (010010TD Element14). More generally, the invention is improved when the temperature is actively kept as constant as possible (i.e., constant gradient) by means of a thermal element and independently of temperature fluctuations. To meet these requirements, the support of the invention may use heating and / or cooling elements. The TEC controller controls the temperature by delivering current and voltage to the thermal element, regulated by feedback from the temperature sensor. In the present invention, a gradient of approximately 2°C is achieved on the gradient plate. The gradient is diffused to the support plate (also referred to as chip) by conduction.

[0034] The temperature gradient activates thermoreceptors in sperm cells, which further stimulates cell migration through the main channel and / or the neutrino channels.

[0035] Reference now Figures 1 to 7 The device is described.

[0036] Figure 1 1 shows a perspective view of an embodiment of a device according to the present invention, which comprises a support 7. A support plate 2 has an elongated shape, preferably a rectangular shape, and comprises an inlet 3 and an outlet 4. The inlet 3 and the outlet 4 each have a wall of a circular cross section and are used to apply or extract a liquid sample, such as a sperm cell sample and a fluid culture medium, respectively. The inlet 3 and the outlet 4 lead to a groove ( Figure 1 The inlet and outlet areas (not shown) within Figure 1 The inlet area and the outlet area are in fluid communication with each other. The shape and / or configuration of the inlet 3 and the outlet 4 can be selected to provide a hydrostatic pressure difference when the liquid (e.g., sperm cell sample) is dispensed onto the support plate through the inlet 3, or the outlet 4, or both. Preferably, as Figure 1As shown, the inlet 3 and the outlet 4 comprise tubes. More specifically, the inlet 3 comprises a tube having a first diameter (preferably 5 mm to 12 mm) and a first height (preferably 1.5 mm to 2.5 mm). The outlet 4 comprises a tube having a second diameter (preferably 4 mm to 5 mm) and a second height (preferably 3.5 mm to 4.5 mm, or more typically 3.5 mm to 5 mm). The first diameter is equal to or greater than the second diameter, and the first height is less than the second height, so as to generate a hydrostatic pressure difference between the inlet area and the outlet area when the fluid culture medium is added.

[0037] The support 7 is elongated, preferably rectangular. The support 7 includes at least one pocket or container 6 configured to accommodate the support plate 2. Figure 1 In the illustrated embodiment, the support 7 has five containers 6. This provides a ladder-like design for the support 7. The support 7 also includes a first heating device 8 and a second heating device 10. The first heating device 8 is located on one side of the support and the second heating device 10 is located on the opposite side of the support. More generally, the heating devices are arranged so that when the support plate 2 is placed in the container 6, the first heating device 8 is located near the inlet and the second heating device 10 is located near the outlet. The first heating device and the second heating device are configured to heat at a first temperature and a second temperature, respectively, the first temperature being lower than the second temperature, so as to set a temperature gradient in the microfluidic system of the support plate to simulate heat drive.

[0038] Figure 1 The sperm selection scheme of an embodiment of the device of the present invention is generally shown. After pre-filling the disposable support plate 2 with the fluid culture medium, the inlet 3 and the outlet 4 are emptied using a pipette 1. Subsequently, the fluid culture medium (~0.065ml or ~0.08ml) and the sperm cell sample (~0.05ml or ~0.035ml) are injected at the outlet 4 and the inlet 3 respectively using a pipette 1. The height difference between the outlet 4 and the inlet 3 causes a flow 5 (represented by the reference numerals 5a, 5b). The sperm cells migrate from the inlet area to the outlet area against the flow direction, and eventually arrive near the outlet 4, where the sperm cells can be collected. The temperature gradient T also triggers sperm migration. Here, the temperature gradient T is established by the TEC card provided by Meersttetter®. The disposable support plate 2 is held in a stage pocket / container 6, and the heating is controlled along the disposable support plate via a polyamide belt placed on the opposite side of the bracket 7. The TEC card reads and acquires the temperature through the PT-100 sensors, namely the first heating device 8 and the second heating device 10, and for verification, a third heating device 9 is installed between the first and second heating devices. The third heating device is a negative temperature coefficient (NTC 10K) sensor.

[0039] Reference now Figure 2 and Figure 3 , Figure 2 shows a perspective view of the device according to the invention, Figure 3 A side view of the top layer of a device according to the invention is shown. Figure 2 The support plate 2 is shown. The support plate 2 includes an upper plate 11 (or top layer 11) and a lower plate 12 (or bottom layer 12). The top layer 11 includes an inlet 3 and an outlet 4. The height difference 14 between the inlet 3 and the outlet 4 is as shown in FIG. Figure 3 As shown. The bottom layer 12 includes a groove 13. The groove 13 can be filled or partially filled with fluid via the inlet 3 or the outlet 4. More generally, the inlet and the outlet are both configured to receive a fluid culture medium, such as a sperm sample or a sperm culture medium. However, the flow of the fluid according to the present invention (i.e., the direction of flow from the outlet area to the inlet area) is triggered only when the outlet is filled with the culture medium. In most cases, the top layer 11 ensures the fluid flow function via the hydrostatic pressure difference between the fluid terminals, i.e., the height difference 14. The bottom layer has a groove 13 that ensures the fluid design. The coupling of the two layers completes the fluid circuit and the fluid communication between the inlet 3 and the outlet 4.

[0040] Figure 4A top view of the groove 13 of the present invention is shown. The groove 13 is an elongated shape. Here, it can be described as an 8-shaped shape or having the overall appearance of a spoon. The groove 13 includes a main channel 13a used as a microfluidic system. The groove is generally arranged to include a volume of about 20μl. More generally, depending on its length, the groove can accommodate a volume of about 15μl to 20μl. The groove 13 also includes an inlet area 103 and an outlet area 104. The inlet area 103 is located near the inlet 3. When the fluid is dispensed to the inlet 3, it is filled with fluid. The outlet area 104 is located near the outlet 4. When the fluid is dispensed to the outlet 4, it is filled with fluid. The inlet area 103 and the outlet area 104 are fluidically connected to each other via the main channel 13a. Therefore, when the fluid is dispensed to either the inlet 3 or the outlet 4, after a given time, the inlet area 103 and the outlet area 104 both contain fluid. In the microfluidic system, the groove 13 is preferably arranged in a width 16 of 5 mm and a length 15 of 5 cm (or 3 cm in another embodiment). In this arrangement, the groove includes a restricted width 17 of about 1.5 to 1.8 mm. More generally, the ratio of the length to the width of the groove 13 can be between 10 and 6 (or a minimum of 5), preferably about 6. The ratio between the restricted width 17 and the average groove width 16 is between 0.3 and 0.36, preferably about 0.3, and in addition, the ratio between the length of the rhizotaxis region 17a and the average groove width 16 is between 0.6 and 0.8, preferably 0.6 (not shown in the figure). The configuration of the groove 13 is such that the main channel 13a is suppressed. The width of the restriction 17 is less than the width of the groove 13. More generally, the inlet area 103 and the outlet area 104 are in fluid communication with each other through a main channel 13a arranged in the groove 13, and the channel 13a has a region of reduced width arranged between the inlet area 103 and the outlet area 104 to form a hydrotaxis area 117. The purpose of the hydrotaxis area 117 is to select a sample of sperm cells by passing through the hydrotaxis area 117. Therefore, the hydrotaxis area 117 acts as a gate for sperm selection. The structure of the present invention only allows sperm cells that swim upward and have a motility sufficient to exceed a given speed at the hydrotaxis area 117 to enter the other side (the outlet area).

[0041] Figure 5 A top view of an embodiment of a slot of the present invention is shown, and further details of a particular portion / section of the slot are shown. The slot 13 comprises a fluid circuit, wherein the main channel 13a comprises a neutrino channel 13b (also referred to as a sub-microchannel). The neutrino channel 13b extends from the rhizotaxis region 117 to the outlet region 104. Figure 5 The enlarged view [A] shows the width 18a of the neutrino channel 13b near the rhizotactic region 117. Figure 5 The enlarged view [C] shows the width 18b of the neutrino channel 13b near the exit region 104. Figure 5In the embodiment of the present invention, the width 18b of the neutrino channel near the outlet region is greater than the width 18a of the neutrino channel near the hydrotropism region. In other words, the width of the neutrino channel 13b gradually widens from the hydrotropism region 117 to the outlet region 104. The neutrino channel 13b can be formed by placing a first set of rods 19a along the main channel 13. Figure 5 The enlarged view [A] shows the details of the rod 19a placed in the main channel 13. The height of the main channel is usually between 80 μm and 100 μm, preferably about 80 μm. Correspondingly, the height of the rod is usually between 40 μm and 50 μm, preferably about 40 μm (enlarged view [B]). The microchannel assists the sperm cells to navigate to the exit area 104 and ultimately to the exit 4.

[0042] As described above, the integration of sub-channels according to the present invention is beneficial not only to sub-channels along the XY plane, but also to sub-channels in the YZ plane. Figure 5 (Enlarged view [B]) shows the so-called XY subchannel 201 and YZ subchannel 202. The XY subchannel 201 is arranged between the rods. The YZ subchannel 202 is arranged above the rods. Thus, Figure 5 As shown (enlarged view [B]), the rods are arranged in a toothed manner, which enables the combination of the XY subchannel 201 and the YZ subchannel 202. Thus, the present invention allows accommodating multiple subchannels without compromising the quality of the microfluidics-based method.

[0043] Figure 6 A top view of another embodiment of the slot of the present invention is shown, and further details of a specific part / cross section of the slot are shown. In this preferred embodiment, the main channel 13a of the slot 13 includes not only a neutrino channel 13b located on the side of the outlet region 104 (after the rhizotaxis region 117), but also a neutrino channel 13b located on the side of the inlet region 103 (before the rhizotaxis region 117). The neutrino channel 13b on the side of the inlet region 103 is formed by placing a second set of rods 19b in the main channel 13. The rods 19b extend from the inlet region 103 to the rhizotaxis region 117. Figure 6 It is also shown that no rods are placed within the hydrotactic region 117 itself. The length 17a of the hydrotactic region 117 is typically approximately between 1 mm and 4 mm, preferably about 3 mm, or between 1 mm and 2.75 mm, preferably about 2.75 mm. Figure 6 The enlarged view [D] shows the arrangement of the first group of rods 19a and the second group of rods 19b near the rhizotactic region 117. Figure 6 The enlarged view [E] of FIG. 1 shows the arrangement of the second set of rods 19b near the inlet region 103. Typically, the second set of rods 19b converge from the inlet region 103 to the rhizotaxis region 117. However, at least some of the rods in the second set of rods 19b may be arranged in parallel, such as Figure 6More generally, Figure 6 Subchannels located on both sides of the hydrotaxis region are described. The subchannels located on the inlet side facilitate the propagation of sperm cells toward the hydrotaxis region. This arrangement facilitates filtering out immotile sperm cells and unwanted fuzziness from the semen sample.

[0044] Figure 7 FIG. 2 shows a three-dimensional view of a heating rack according to the present invention. Figure 1 The bracket shown is similar. It is made of an aluminum plate of about 1 cm thickness and has a rectangular shape. The bracket 7 includes a container 6 cut from the aluminum plate so as to accommodate at least one support plate 2 of the present invention. The bracket 7 also includes a first polyamide heating belt 25 and a second polyamide heating belt 26 arranged on opposite length sides of the bracket 7. A Meestteter® TEC board 36 is applied to control and drive the polyamide heating belts 25, 26. The heating module is connected to the first polyamide belt and the second polyamide belt. The belts 25, 26 are powered by the TEC board via the first heating connection 31 and the second heating connection 30, respectively. The first PT-100 sensor 8 and the second PT-100 sensor 10 and the NTC-10K sensor 9 are connected to three "general purpose input / output (GPIO)" 27, 28, 29, respectively. The coupling of the three sensors 8, 9, 10 to the TEC card 36 enables real-time reading of the temperature. The "proportional-integral-differential (PID)" function of the card enables the temperature to be controlled by the current flowing to the resistance belts 25, 26. The functions of the card are processed by a computing framework 35. Under the working conditions of the preferred embodiment, the support is arranged to heat the first polyamide tape 25 and the second polyamide tape 26 at 37° C. and 39° C. respectively. Thus, the support made of aluminum together with the container 6 is heated to 37° C. on one length side and to 39° C. on the other length side.

[0045] Figure 8 and Fig. 9 The graphs of the temperature change over time and the graphs of the temperature change over position in the heating rack according to the present invention are shown respectively. Both graphs show the temperature data results. Figure 8 The temperature measurement stability of the first PT-100 sensor 8 and the second PT-100 sensor 10 and the NTC-10K sensor 9 is shown. Fig. 9 It is shown that the temperatures at the corners (the length sides of the bracket 7) are maintained at 37°C and 39°C respectively, and the NTC-10K sensor data shows that the temperature of the midpoint (middle) of the bracket 7, that is, the aluminum plate, is 38°C. Figure 8 , Fig. 9 A regular temperature gradient is shown installed in the support 7. When the support plate 2 is placed in a container 6 of the support 7, the regular temperature gradient spreads regularly in the support plate 2.

[0046] Fig.10 This is a microscopic image of a sperm cell. Fig.10 The trajectory of the sperm cells is also shown. The initial concentration of sperm cells was assessed using a Makler chamber (Sefi Medical Instruments Ltd.). The motility and swimming dynamics of sperm cells were assessed using a computational framework, which helped to understand the trajectory of sperm cells. The purpose of this microscopic image can be to facilitate information related to data analysis (extraction of velocity and motion parameters).

[0047] Example The collected sperm cell sample (or semen sample) and sperm culture medium are placed in an incubator at 37°C for liquefaction and preheating. The sperm culture medium will be cultured in 5% CO2. At the same time, a temperature gradient is started in the device of the present invention. The temperatures of the first heating device and the second heating device of the support 7 are set to 37°C and 39°C, respectively. After about 12 minutes, the temperature gradient will be regularly distributed on the support plate. The motility and concentration of the mother liquid are evaluated using a Makler chamber. Videos of moving sperm are recorded by a microscope camera (SwiftCamSC500), and image processing methods involving background subtraction, image denoising, and intensity and area segmentation are performed for motility and sperm kinematic analysis. The device (or more precisely the tank) is pre-filled with (80μl) pre-heated (37°C) sperm separation solution (Sperm Rinse™ 510312 Vitrolife ). The support plate is kept on the gradient stage. After 20 minutes, 0.05 ml (or 0.35 ml in another embodiment) of liquefied semen sample is loaded at the inlet of the device and 80 μl (or 65 μl in another embodiment) of sperm culture medium is immediately added at the outlet. Sperm migration based on rhizotaxis and thermotaxis occurs and after about 45 minutes (or even after 30 minutes), about 80 μl (or 65 μl in another example) of sperm sample is collected from the outlet. Motility of subpopulation collection is also done by Makler chamber and microscope (Amscope T720Q). An object tracking algorithm module based on Kalman filter and Hungarian algorithm is implemented to extract XY trajectories from the images.

[0048] Fig.11 , Fig.12 , Fig.13 and Fig.14 Measurements and results are shown for the above examples implemented in three different embodiments of the invention.

[0049] Fig.11 , Fig.12 , Fig.13 and Fig.14 Shown in Figure 4 , Figure 5 and Figure 6The measurements and results of the above examples implemented in the device of the present invention are shown in the embodiment of the present invention. RS represents the data from the original sperm sample (mother fluid). MF represents the data from the original sperm sample (mother fluid). Figure 4 The data of sperm cells selected and collected at the outlet of the device of the present invention (i.e., with main channel but without neutrino channel) are shown. MFS stands for Figure 5 The data of sperm cells selected and collected at the outlet of the device of the present invention (i.e., having a main channel and a neutrino channel on the outlet side) are shown. MFBS stands for Figure 6 Data showing sperm cells selected and collected at the outlet of a device of the present invention (i.e., having a main channel and neutrino channels on the outlet and inlet sides).

[0050] Fig.11 Sperm cell concentration results are shown. Fig.12 Motility of sperm cells is shown. Almost ~100% of motile cells were separated and isolated from the initial sperm sample. Fig.13 Improved transient velocity of sperm cells in the collected high-quality sperm subpopulation separated from the initial sperm sample is shown. Fig.14 It shows that the forward speed of sperm cells is increased.

[0051] Fig.15 Figures [A] and [B] are representative microscopic images (1280×940px) of the mother solution (RS) and microfluidics embodiment (MF). 2 , 2.48px / μm). The Haloserm® G2 DNA fragmentation kit and protocol (provided by Halotech®) were followed to assess DNA fragmentation. A large halo head represents no breakage or degradation, on the other hand, a small halo head or no halo head represents breakage and degradation. Figure [C] demonstrates that sperm selection using Example 13 favors a sperm subpopulation with ~100% DNA integrity. Other settings have been implemented. According to an embodiment, the temperature set by the heating device of the support 7 is between 36°C and 40°C. Specifically, the first temperature is set between 36°C and 38°C, preferably 37°C, and the second temperature is set between 38°C and 40°C, preferably 39°C.

[0052] In a more general sense, the present invention is directed to a device for sperm cell selection having means for simulating rheotaxis and means for simulating thermotaxis.

[0053] Another object of the present invention is a kit for sperm cell selection, comprising: A. A device having a support plate, the support plate comprising an inlet for receiving a sperm cell sample, an outlet for collecting at least some sperm cells contained in the sample, and a microfluidic system located between the inlet and the outlet, wherein the microfluidic system comprises a tank for receiving a culture medium and having an inlet region and an outlet region near an inlet and an outlet, respectively, and wherein the inlet and the outlet are configured to provide a hydrostatic pressure difference between the inlet region and the outlet region when culture medium is added to the tank through the outlet, thereby causing the culture medium to flow from the outlet region to the inlet region to initiate migration of sperm cells in the sperm cell sample from the inlet region to the outlet region, and wherein the inlet region and the outlet region are in fluid communication with each other via a main channel arranged in the slot, the channel having a region of reduced width arranged between the inlet region and the outlet region to form a rhizotactic region, whereby the sperm cell sample is selected during the migration of the sperm cells through the rhizotaxis region, thereby enabling the collection of a high-quality sperm cell sample from the outlet region at the outlet; and B. A support having at least one container configured to accommodate a support plate, the support comprising a first heating device and a second heating device, wherein when the support plate is placed in the container, the first heating device is located near an inlet and the second heating device is located near an outlet, the first heating device and the second heating device are respectively configured to heat at a first temperature and a second temperature, the first temperature being lower than the second temperature, so as to set a temperature gradient in the microfluidic system of the support plate to simulate thermotropism.

[0054] The applicant has further developed an embodiment which is close to the above described embodiment, but shows particularly good results.

[0055] Fig.16 A perspective view of an embodiment of a device according to the present invention is shown, which includes a support 7. The support plate 2 has an elongated rectangular shape, which includes an inlet 3 and an outlet 4. The inlet 3 and the outlet 4 both have walls with a circular cross-section and are used to apply or extract liquid samples, such as sperm cell samples and liquid culture media, respectively. The inlet 3 and the outlet 4 lead to an inlet area and an outlet area (not shown) in a groove (not shown) inside the support plate, respectively. The inlet area and the outlet area are fluidically connected to each other. The shape and / or configuration of the inlet 3 and the outlet 4 can be selected to provide a hydrostatic pressure difference when a liquid (such as a sperm cell sample) is distributed onto the support plate through the inlet 3, or the outlet 4, or both the inlet 3 and the outlet 4. Preferably, as Fig.16As shown, the inlet 3 and the outlet 4 comprise tubes. More specifically, the inlet 3 comprises a tube having a first diameter (preferably 5 mm to 12 mm) and a first height (preferably 1.5 mm to 2.5 mm). The outlet 4 comprises a tube having a second diameter (preferably 4 mm to 5 mm) and a second height (preferably 3.5 mm to 4.5 mm, or more typically 3.5 mm to 5 mm). The first diameter is equal to or greater than the second diameter, and the first height is less than the second height, so as to generate a hydrostatic pressure difference between the inlet area and the outlet area when liquid culture medium is added.

[0056] The support 7 is elongated, preferably rectangular. The support 7 includes at least one pocket or container 6 configured to receive the support plate 2. Fig.16 In the illustrated embodiment, the support 7 has a continuous container 6 to accommodate a plurality of support plates 2. This provides a ladder-like design for the support 7. The support 7 also includes a first heating device 8 and a second heating device 10. The first heating device 8 is located on one side of the support and the second heating device 10 is located on the opposite side of the support. More generally, the heating devices are arranged so that when the support plate 2 is placed in the container 6, the first heating device 8 is located near the inlet and the second heating device 10 is located near the outlet. The first heating device and the second heating device are configured to heat at a first temperature and a second temperature, respectively, the first temperature being lower than the second temperature, so as to set a temperature gradient within the microfluidic system of the support plate to simulate heat drive.

[0057] Fig.16 The sperm selection scheme of an embodiment of the device of the present invention is illustrated. After pre-filling the disposable support plate 2 with liquid culture medium, the inlet 3 and the outlet 4 are emptied using a pipette 1. Subsequently, the liquid culture medium (~0.065ml or ~0.08ml) and the sperm cell sample (~0.05ml or ~0.035ml) are injected into the outlet 4 and the inlet 3 respectively using a pipette 1. The height difference between the outlet 4 and the inlet 3 causes a flow 5 (indicated by the reference numerals 5a, 5b). The sperm cells migrate from the inlet area to the outlet area against the flow direction and eventually arrive near the outlet 4, where the sperm cells can be collected. The temperature gradient T also triggers sperm migration. Here, the temperature gradient T is established by a TEC card provided by Meersttetter®. The disposable support plate 2 is held in a stage pocket / container 6, and the heating is controlled along the disposable support plate via a polyamide belt placed on the opposite side of the bracket 7. The TEC card reads and acquires the temperature through PT-100 sensors, namely the first heating device 8 and the second heating device 10, and for verification, a third heating device 9 is installed between the first and second heating devices. The third heating device is a negative temperature coefficient (NTC 10K) sensor.

[0058] Fig.17shows a perspective view of a device according to the invention, Fig.18 A side view of the top layer of a device according to the invention is shown. Fig.17 The support plate 2 is shown. The support plate 2 includes an upper plate 11 (or top layer 11) and a lower plate 12 (or bottom layer 12). The top layer 11 includes an inlet 3 and an outlet 4. The height difference 14 between the inlet 3 and the outlet 4 is as shown in FIG. Fig.18 As shown in . The bottom layer 12 includes a groove 13. The groove 13 can be filled or partially filled with fluid via the inlet 3 or the outlet 4. More generally, the inlet and the outlet are both configured to receive a fluid culture medium, such as a sperm sample or a sperm culture medium. However, the flow of the fluid according to the present invention (i.e., the direction of flow from the outlet area to the inlet area) is triggered only when the outlet is filled with the culture medium. In most cases, the top layer 11 ensures the fluid flow function via the hydrostatic pressure difference between the fluid terminals, i.e., the height difference 14. The bottom layer has a groove 13 that ensures the fluid design. The coupling of the two layers completes the fluid circuit and the fluid communication between the inlet 3 and the outlet 4.

[0059] Fig.19A top view of the groove 13 of the present invention is shown. The groove 13 is an elongated shape. Here, the groove 13 can be described as being in an 8-shaped shape or having the overall appearance of a spoon. The groove 13 includes a main channel 13a used as a microfluidic system. The groove is generally arranged to include a volume of about 20μl. More generally, depending on its length, the groove can accommodate a volume of about 15μl to 20μl. The groove 13 also includes an inlet area 103 and an outlet area 104. The inlet area 103 is located near the inlet 3. When the fluid is dispensed to the inlet 3, it is filled with fluid. The outlet area 104 is located near the outlet 4. When the fluid is dispensed to the outlet 4, it is filled with fluid. The inlet area 103 and the outlet area 104 are fluidically connected to each other via the main channel 13a. Therefore, when the fluid is dispensed to either the inlet 3 or the outlet 4, after a given time, the inlet area 103 and the outlet area 104 both contain fluid. In the microfluidic system, the groove 13 is preferably arranged in a width 16 of 5 mm and a length 15 of 5 cm (or 3 cm in another embodiment). In this arrangement, the groove includes a restricted width 17 of about 1.5 to 1.8 mm. More generally, the ratio between the length and width of the groove 13 can be between 10 and 6 (or a minimum of 5), preferably about 6. The ratio between the restricted width 17 and the average groove width 16 is between 0.3 and 0.36, preferably about 0.3, and in addition, the ratio between the length of the rhizotaxis region 17a and the average groove width 16 is between 0.6 and 0.8, preferably 0.6 (not shown in the figure). The configuration of the groove 13 is such that the main channel 13a is confined. The width of the restriction 17 is less than the width of the groove 13. More generally, the inlet area 103 and the outlet area 104 are in fluid communication with each other through a main channel 13a arranged in the groove 13, the channel 13a having a region of reduced width arranged between the inlet area 103 and the outlet area 104 to form a hydrotaxis area 117. The purpose of the hydrotaxis area 117 is to select a sample of sperm cells by passing through the hydrotaxis area 117. Therefore, the hydrotaxis area 117 acts as a gate for sperm selection. The structure of the present invention allows only sperm cells that flow upstream and have a motility sufficient to exceed a given speed at the hydrotaxis area 117 to enter the other side (the outlet area).

[0060] Fig. 20 A top view of an embodiment of a slot of the present invention is shown, and further details of a particular portion / section of the slot are shown. The slot 13 comprises a fluid circuit, wherein the main channel 13a comprises a neutrino channel 13b (also referred to as a sub-microchannel). The neutrino channel 13b extends from the rhizotaxis region 117 to the outlet region 104. Fig. 20 The enlarged view [A] shows the width 18a of the neutrino channel 13b near the rhizotactic region 117. Fig. 20 The enlarged view [C] shows the width 18b of the neutrino channel 13b near the exit region 104. Fig. 20In the embodiment of the present invention, the width 18b of the neutrino channel near the outlet region is greater than the width 18a of the neutrino channel near the hydrotropism region. In other words, the width of the neutrino channel 13b gradually widens from the hydrotropism region 117 to the outlet region 104. The neutrino channel 13b can be formed by placing a first set of rods 19a along the main channel 13. Fig. 20 The enlarged view [A] shows a detail of the rod 19a placed in the main channel 13. The height of the main channel is typically between 80 μm and 100 μm, preferably about 80 μm. Therefore, the height of the rod is typically between 40 μm and 50 μm, preferably about 40 μm (enlarged view [B]). The microchannel helps the sperm cells navigate to the exit area 104 and ultimately to the exit 4.

[0061] As described above, the integration of neutrino channels according to the present invention not only facilitates channels along the XY plane, but also facilitates channels in the YZ plane. Fig. 20 (Enlarged view [B]) shows the so-called XY neutrino channel 201 and YZ neutrino channel 202. The XY neutrino channel 201 is arranged between the rods. The YZ neutrino channel 202 is arranged above the rods. Fig. 20 As shown (enlarged view [B]), the rods are arranged in a toothed manner, which enables the combination of the XY subchannel 201 and the YZ subchannel 202. Thus, the present invention allows accommodating multiple subchannels without compromising the quality of the microfluidics-based method.

[0062] Fig.21 A top view of another embodiment of the slot of the present invention is shown, and further details of a specific part / portion of the slot are shown. In this preferred embodiment, the main channel 13a of the slot 13 includes not only a neutrino channel 13b located on the side of the outlet region 104 (after the rhizotaxis region 117), but also a neutrino channel 13b located on the side of the inlet region 103 (before the rhizotaxis region 117). The neutrino channel 13b on the side of the inlet region 103 is formed by placing a second set of rods 19b in the main channel 13. The rods 19b extend from the inlet region 103 to the rhizotaxis region 117. Fig.21 It is also shown that no rods are placed within the hydrotactic region 117 itself. The length 17a of the hydrotactic region 17 is typically approximately between 1 mm and 4 mm, preferably about 3 mm, or between 1 mm and 2.75 mm, preferably about 2.75 mm. Fig.21 The enlarged view [D] shows the arrangement of the first group of rods 19a and the second group of rods 19b near the rhizotactic region 117. Fig.21 The enlarged view [E] of FIG. 1 shows the arrangement of the second set of rods 19b near the inlet region 103. Typically, the second set of rods 19b converge from the inlet region 103 to the rhizotaxis region 117. However, at least some of the rods in the second set of rods 19b may be arranged in parallel, such as Fig.21More generally, Fig.21 Subchannels located on both sides of the hydrotaxis region are described. The subchannels located on the inlet side facilitate the propagation of sperm cells toward the hydrotaxis region. This arrangement facilitates filtering out immotile sperm cells and unwanted fuzziness from the semen sample.

[0063] Fig. 22 FIG. 2 shows a three-dimensional view of a heating rack according to the present invention. Fig.16 The bracket 7 is similar to the one shown. The bracket 7 is made of an aluminum sheet of about 1 cm thickness and has a rectangular shape. The bracket 7 includes a container 6 cut from the aluminum sheet so as to accommodate at least one support plate 2 of the present invention. The bracket 7 also includes a first polyamide heating belt 25 and a second polyamide heating belt 26 arranged on opposite length sides of the bracket 7. A Meestteter® TEC board 36 is applied to control and drive the polyamide heating belts 25, 26. The heating module is connected to the first polyamide belt and the second polyamide belt. The belts 25, 26 are powered by the TEC board via a first heating connection 31 and a second heating connection 30, respectively. The first PT-100 sensor 8 and the second PT-100 sensor 10 and the NTC-10K sensor 9 are connected to three "General Purpose Input / Output (GPIO)" 27, 28, 29, respectively. The coupling of the three sensors 8, 9, 10 to the TEC card 36 enables real-time reading of the temperature. The "Proportional-Integral-Derivative (PID)" function of the card enables the temperature to be controlled by the current flowing to the resistance belts 25, 26. The functions of the card are processed by a computing framework 35. Under the working conditions of the preferred embodiment, the support is arranged to heat the first polyamide tape 25 and the second polyamide tape 26 at 37° C. and 39° C. respectively. Thus, together with the container 6, the support made of aluminum is heated to 37° C. on one length side and to 39° C. on the other length side.

[0064] In a specific embodiment, thermotropism can be achieved by integrating only one heating source unit, where the heating stage will be placed in an incubator at a lower temperature, while the heating stage is at a relatively higher temperature.

[0065] Therefore, the present invention further provides an embodiment, wherein the device comprises a support (7), the support having at least one container (6) configured to accommodate a support plate (2), the support comprising a single heating device, wherein when the support plate (2) is placed in the container (7), the single heating device is located near the inlet (3) or near the outlet (4). The single heating device is configured to heat at a selected temperature above room temperature. The preferred temperature is 39°C. Surprisingly, this specific embodiment inhibits the formation of bubbles in the culture medium. Therefore, the results of this embodiment are very satisfactory. The selected temperature establishes a specific gradient within the microfluidic system of the support plate, thereby effectively simulating thermotropism.

[0066] Fig.23 Another configuration for producing thermotropism is shown. This configuration includes an aluminum metal rod 7a which includes a container 6 (or pocket) and is connected to a polyamide heating tape 26. The metal rod 7a is connected to an adjacent non-conductive plate 38 which acts as a container 6 (or holding pocket) for a support plate (sometimes called a fluid cell). A TEC Meerstetter® 36 printed circuit board is used to control and drive the polyamide heating tape 26. The aluminum metal rod 7a contains a PT 100 sensor 10 and data acquisition is performed via (GPIO) 29. Coupling the sensor 10 to the TEC board 36 enables real-time temperature reading and control. The operation of the card has been described in conjunction with the previous Figure 7 Here, the second temperature is activated by the incubator 37.

[0067] The device of the present invention is a pioneer in the field of bionics.

Claims

1. A device for sperm cell selection, the device comprising a support plate (2), the support plate comprising an inlet (3) for receiving a sperm cell sample, an outlet (4) for collecting at least some of the sperm cells contained in the sample, and a microfluidic system located between the inlet and the outlet, in, The microfluidic system comprises a tank (13) arranged to receive a culture medium and having an inlet region (103) and an outlet region (104) near the inlet and the outlet, respectively. And wherein the inlet (3) and the outlet (4) are arranged to provide a hydrostatic pressure difference between the inlet area (103) and the outlet area (104) when culture medium is added to the tank (13) via the outlet (4), thereby inducing the flow of the culture medium from the outlet area (104) toward the inlet area (103) to initiate the migration of sperm cells in the sperm cell sample from the inlet area to the outlet area (104), and wherein the inlet region (103) and the outlet region (104) are in fluid communication with each other via a main channel (13a) arranged in the groove (13), the channel having a region of reduced width arranged between the inlet region (103) and the outlet region (104) to form a rhizotaxis region (117), And wherein the main channel (13a) includes a neutrino channel (13b) extending from the rhizotaxis region (117) to the outlet region (104), and further includes a neutrino channel (13b) extending from the inlet region to the rhizotaxis region (117), Thus, the sperm cell sample undergoes selection by passing through the hydrotaxis region (117) during the migration of the sperm cells, so that a high-quality sperm cell sample can be collected from the outlet region (104) at the outlet (4).

2. The device according to claim 1, wherein: The inlet (3) comprises a tube having a first diameter and a first height, the first diameter is preferably 5 mm to 12 mm, and the first height is preferably 1.5 mm to 2.5 mm; and the outlet (4) comprises a tube having a second diameter and a second height, the second diameter is preferably 4 mm to 5 mm, and the second height is preferably 3.5 mm to 4.5 mm, wherein the first diameter is greater than or equal to the second diameter, and the first height is less than the second height, so as to generate the hydrostatic pressure difference between the inlet area (103) and the outlet area (104) when the culture medium is added.

3. A device according to any one of the preceding claims, wherein: The support plate (2) is composed of a top layer (11) and a bottom layer (12), the top layer comprising the inlet (3) and the outlet (4), and the bottom layer comprising the groove (13).

4. A device according to any one of the preceding claims, wherein: Neutrino channels converge toward the rhizotactic region.

5. A device according to any one of the preceding claims, wherein: The main channel comprises rods (19) arranged along the groove, the rods extending from the inlet region towards the rheotactic region and / or from the rheotactic region (117) towards the outlet region (104), and wherein the neutrino channel (13b) is arranged between (201) and above (202) the rods.

6. A device according to any one of the preceding claims, wherein: The neutrino channel (13b) is arranged in the XY plane and the YZ plane of the slot (13).

7. A device according to any one of the preceding claims, wherein: The device also includes a support (7), the support (7) having at least one container (6) configured to accommodate the support plate (2), the support including a first heating device and a second heating device, wherein when the support plate (2) is placed in the container (7), the first heating device is located near the inlet (3) and the second heating device is located near the outlet (4), the first heating device and the second heating device are respectively configured to heat at a first temperature and a second temperature, the first temperature being lower than the second temperature, so as to set a temperature gradient in the microfluidic system of the support plate to simulate thermotropism.

8. A method for sperm cell selection, the method comprising the following steps: i. A device according to any one of claims 1 to 7, wherein the device comprises a support plate (2), the support plate (2) comprising an inlet (3), an outlet (4) and a microfluidic system between the inlet (3) and the outlet (4), the microfluidic system comprising a groove (13), the groove having an inlet area (103) and an outlet area (104) near the inlet (3) and the outlet (4), respectively, the inlet (3) and the outlet (4) being arranged such that when a fluid culture medium is added to the groove (13) via the outlet (4), When the flow of the fluid culture medium from the outlet region (104) to the inlet region (103) is increased, a hydrostatic pressure difference is provided between the inlet region (103) and the outlet region (104), thereby causing the fluid culture medium to flow from the outlet region (104) toward the inlet region (103), the inlet region (103) and the outlet region (104) being in fluid communication with each other via a main channel (13a) arranged in the groove (13), wherein the channel has a region with reduced width arranged between the inlet region (103) and the outlet region (104) so ​​as to form a rhizotaxis region (117); ii. pre-filling the tank (13) with a fluid culture medium; iii. providing a sperm cell sample to the inlet to distribute the sample to the inlet area (103) of the groove (13); iv. providing a fluid culture medium to the outlet (4) to cause the culture medium to flow from the outlet region (104) to the inlet region (103), thereby causing sperm cells to migrate from the inlet region (103) to the outlet region (104); v. selecting sperm cells from the sperm cell sample when the sperm cells pass through the rhizotaxis region (117) during the sperm cell migration process; and vi. Collecting a sample of high-quality sperm cells from the outlet area (104) at the outlet (4).

9. The method according to claim 8, further comprising the step of setting a temperature gradient on the support plate (2) to simulate thermotaxis and improve the migration of the sperm cells.

10. The method according to claim 8 or 9, wherein: The fluid culture medium in step ii is a sperm separation medium.

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