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WO2021124111A1 - Dispositif microfluidique pour la sélection de spermes hautement motiles - Google Patents

Dispositif microfluidique pour la sélection de spermes hautement motiles Download PDF

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Publication number
WO2021124111A1
WO2021124111A1 PCT/IB2020/061977 IB2020061977W WO2021124111A1 WO 2021124111 A1 WO2021124111 A1 WO 2021124111A1 IB 2020061977 W IB2020061977 W IB 2020061977W WO 2021124111 A1 WO2021124111 A1 WO 2021124111A1
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Prior art keywords
chamber
sperm
microchannels
sperm cells
micro
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Inventor
Almas RAKHYMZHANOV
Ulrich BUTTNER
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King Abdullah University of Science and Technology KAUST
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King Abdullah University of Science and Technology KAUST
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0612—Germ cells sorting of gametes, e.g. according to sex or motility
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0874—Three dimensional network
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0457—Moving fluids with specific forces or mechanical means specific forces passive flow or gravitation

Definitions

  • Embodiments of the subject matter disclosed herein generally relate to a system and method for selecting highly motile sperm, and more particularly, to a microfluidic system that uses long microchannels and no external forces for separating progressive sperm from the raw sperm.
  • microfluidic devices have been gaining traction, especially in the biomedical fields, as these new devices can control the fluid transport, which is useful for cell analysis systems, drug delivery systems, and assisted reproductive technologies.
  • Microfluidic technology is currently used in many biological applications, specifically with regard to miniaturization and simplification of laboratory techniques.
  • reproductive medicine One field that is taking advantage of the microfluidic devices is reproductive medicine. Reproductive medicine is concerned with infertility in males and females. One of the main reasons for male infertility is the deformity and/or the deficient amount of sperm cells.
  • ART assisted reproductive technology
  • IVF in vitro fertilization
  • ICSI intracytoplasmic sperm injection
  • the methods discussed above are prone to damaging the DNA of the sperm cells, which is highly undesirable.
  • a sperm-sorting microchannels device that includes a collecting chamber configured to receive a sorting medium, a loading chamber configured to receive raw sperm cells, and plural microchannels extending between the collecting chamber and the loading chamber. A bottom of the collecting chamber and a bottom of the loading chamber are in a same plane.
  • a sperm-sorting microchannels device that includes a lower chamber configured to receive raw sperm cells, an upper chamber located over the lower chamber, and configured to receive motile sperm cells, and a a micro-tunnel part sandwiched between the lower chamber and the upper chamber, wherein the micro-tunnel part includes plural micro-tunnels that extend between the lower chamber and the upper chamber.
  • a thickness T of the micro-tunnel part is at least twice a length I of an average sperm cell.
  • a method for sperm sorting with a microfluidic device includes placing raw sperm cells into a loading chamber, placing a sorting medium into a collecting chamber, generating a gravitational fluid flow from the loading chamber toward the collecting chamber, along plural microchannels that extend between the collecting chamber and the loading chamber, where a bottom of the collecting chamber and a bottom of the loading chamber are in a same plane, and collecting motile sperm cells from the collecting chamber, after the sperm cells swim against the gravitational fluid flow.
  • the gravitational fluid flow is generated exclusively due to the gravity.
  • Figure 1 is a schematic diagram of a passive sperm-sorting microfluidic device
  • Figure 2 shows a microchannel used by the passive sperm-sorting microfluidic device of Figure 1;
  • Figure 3 shows various components of the passive sperm-sorting microfluidic device as they are being assembled
  • Figure 4 is an image of the passive sperm-sorting microfluidic device after being assembled
  • Figure 5 illustrates the disposition and density of the microchannels formed between various chambers of the passive sperm-sorting microfluidic device
  • Figure 6 illustrates the disposition and density of holes made in a track- etched membrane in a traditional device used for sperm-selection
  • Figures 7 A to 7C illustrate a dynamic sperm-sorting microfluidic device in which a fluid flow is generated exclusively by gravitational means for selecting the motile sperm cells;
  • Figure 8 illustrates the various elements of the dynamic sperm-sorting microfluidic device during assembly
  • Figure 9 is a table that indicates the motility of the sperm cells when processed with traditional methods and with the devices discussed herein;
  • Figure 10 is a table that compares the sperm morphology assessment of the sperm cells selected with the traditional methods and the devices discussed herein;
  • Figure 11 is a flow chart of a method for selecting highly motile sperm cells with one of the devices discussed herein.
  • a passive microfluidic device for selecting highly motile sperm has input and output ports located at the same level, a lower compartment that receives sperm having different mobility grades, an upper compartment that receives only highest grade mobility sperm cells, and micro tunnels that separate the lower compartment from the upper compartment.
  • the micro-tunnels are designed to select the highest grade mobility sperm cells.
  • the motility of the sperm is divided into four different grades, A, B, C, and D. Grade A sperm cells have good motility and they swim fast in a straight line while Grade D are damaged sperm cells that cannot move.
  • a passive sperm-sorting microfluidic device 100 has a lower chamber 110 and an upper chamber 120, which are separated from each other by a micro-tunnel part 130.
  • the upper chamber 120 is formed on top of the lower chamber 110.
  • the lower chamber 110 has two or more input ports 112 that are formed in a thin top layer 146.
  • Lower microfluidic channels 114 fluidly connect the input ports 112 to the lower chamber 110.
  • Each of the lower microfluidic channels 114 may have a length in the range of 100 to 500 pm, with a preferred length of about 250 pm.
  • the lower chamber 110 is formed on top of a thin bottom layer 140, and extends into a lower layer 142.
  • the lower layer 142 is directly located over the thin bottom layer 140.
  • the lower microfluidic channels extend horizontally into the lower layer 142, and then vertically through the lower layer 142, an upper layer 144, which is located directly over the lower layer 142, and also through the thin upper layer 146.
  • the upper chamber 120 has two or more output ports 122, also formed in the thin top layer 146 so that the input ports 112 and the output ports 122 are located in the same upper surface of the thin top layer 146.
  • Upper microfluidic channels 124 fluidly connect the two or more output ports 122 to the upper chamber 120.
  • the upper chamber 120 is formed directly on top of the lower chamber 110 and extends into the upper layer 144.
  • the upper microfluidic channels extend horizontally into the upper layer 144, and then vertically through the thin upper layer 146.
  • each chamber and associated microfluidic channels and the associated ports are disposed symmetrically relative to a vertical axis Y, as shown in Figure 1.
  • a length of the lower microfluidic channels is longer than a length of the upper microfluidic channels.
  • micro is used herein to indicate that a size of the ports and/or the channels that this term characterizes is in the micrometer range.
  • the micro-tunnel part 130 is shown in more detail in Figure 2 and includes a layer of material 132 in which plural micro-tunnels 134 are formed.
  • the micro-tunnel part 130 is arranged in the device 100 so that the plural micro-tunnels 134 are perpendicular to an interface between the lower chamber 110 and the upper chamber 120, i.e. , the micro-tunnels 134 are parallel to the gravity.
  • a thickness T of the layer of material 132 is selected based on a length of the sperm cell.
  • an average length I of the sperm cell 150 is 50 pm and the thickness T of the layer of material 132 is selected to be 100 pm or more, i.e., at least twice the length L of the sperm cell 150.
  • a diameter D of the micro-tunnel 134 is selected to be about three times the diameter d of the sperm cell 150.
  • the diameter d is about 7 pm and the diameter D is selected to be about 20 pm.
  • Other sizes for the diameter D, up to 70 p , for the micro-tunnels 134 may be selected as long as the diameter D is larger than the diameter d.
  • the sperm-sorting microfluidic device 100 receives at the inlet ports 112 unprocessed, raw semen 152, which moves due to the gravity to the lower chamber 110.
  • the raw semen 152 includes the sperm cells 150, and one or more of other substances, which are not important for the purpose of this embodiment.
  • the desired motile sperm cells 156 capable of fertilization, swim upwards, from the lower chamber 110, through the micro-tunnels 134 of the micro tunnel part 130, into the upper chamber 120.
  • the sperm cells 154 cannot move through the micro-tunnels 134, as they are damaged or not motile enough, and thus, they accumulate in the lower chamber 110, while the highly motile sperm cells 156 accumulate in the upper chamber 120. Thus, the high quality sperm cells 156 are later collected from the output ports 122 of the upper chamber 120.
  • the upper chamber 120 may also hold a buffer fluid 158, in which the highly motile sperm cells 156 swim and thrive.
  • the buffer fluid 158 may include various chemicals that are typically used for maintaining the sperm cells alive.
  • the sperm cells selection microfluidic device 100 relies only on self-propelled sperm cells swimming up against gravity through the micro-tunnels 134. In other words, there is no external force or no sperm flow exerted on the sperm cells selection microfluidic device 100 to make the sperm cells to move through the device.
  • the micro-tunnels 134 are selected in terms of size and orientation to imitate the anatomy of the female reproductive tract and resemble the natural process in vivo.
  • the sperm-sorting microfluidic device 100 can be manufactured as now discussed with regard to Figure 3.
  • the four layers 140 to 146 may be cut with a laser device (e.g., CO2 laser) from polymethyl methacrylate (PMMA) sheets.
  • the thin bottom layer 140 and the thin top layer 146 may have a thickness of about 2 mm while the lower layer 142 and the upper layer 144 may have a thickness of about 4 mm.
  • the same laser or a different one may be used to form the inlet ports 112 and the outlet ports 122 into the thin upper layer 146.
  • the lower microfluidic channels 114 are formed in the lower layer 142 and partially into the upper layer 144, as shown in Figure 3, while the upper microfluidic channels 124 are formed into the upper layer 144. All the channels are formed with known methods.
  • the lower chamber 110 is formed into the lower layer 142 and the upper chamber 120 is formed into the upper layer 144.
  • the micro-tunnel part 130 which is a foil with plural micro-tunnels 134, is placed between the lower layer 142 and the upper layer 144, to fully separate the lower chamber 110 from the upper chamber 120.
  • the micro-tunnel part 130 may be manufactured to have the desired thickness T and the plural micro tunnels 134 to have the desired diameter D discussed above. Then, all the parts are placed directly on top of each other, in the order shown in Figure 3, and the parts are hermetically sealed together by thermal bonding, which results in the device 100 shown in Figure 4.
  • the micro-tunnels 134 are formed in the micro-tunnel part 130 as now discussed.
  • a high-quality UV-curable photosensitive dry film is used to fabricate a freestanding, thick foil, with highly precise tunnel size, shape, and spatial order.
  • a dry film is used as a negative resist type and its UV-light exposed area hardens, while the unexposed area dissolves during a development process.
  • a borosilicate glass photomask may be used to imprint a pore pattern, which corresponds to the micro-tunnels 134, on the photosensitive dry film.
  • the photomask which contains patterned micron-size opaque holes on its surface, blocks the collimated UV-light in these parts and allows it to shine through the transparent areas.
  • the dry film After exposure to UV light through the photomask, the dry film is developed in sodium carbonate solution (Na2CC>3) to remove the unpolymerized parts of the photoresist.
  • Na2CC sodium carbonate solution
  • the resulting foil 130 has a high resolution, low-cost processing, and does not require expensive equipment.
  • the micro-tunnel part 130 has the tunnels 134 formed along a grid or net of lines, some formed parallel to the X-axis and some formed parallel to the Y-axes.
  • the plural micro-tunnels are made in the micro-tunnel part at regular locations along the grid (e.g., at the nodes of the grid).
  • a distance x1 along the X-axis, between two adjacent tunnels 134, and a distance y1 along the Y- axis, between the other two adjacent tunnels 124, can be controlled as desired with the UV mask discussed above.
  • This is in stark contrast to the porous membrane 600 shown in Figure 6, which has randomly formed holes 602, which cover about 14% of the surface area of the membrane.
  • the porous membrane 600 is used in U.S. Patent no. 10,422,737 for separating the motile sperm cells.
  • the process selected to fabricate the micro-tunnel part 130 it is possible to form the channels 134 in half or more of the surface area 130A, which dramatically increases the efficiency of the device 100.
  • the inventors Based on the details of the U.S. Patent no. 10,422,737, the inventors have estimated that the density of the tunnels 134 per surface area 130A in Figure 5 is three times or higher than the density of the holes 602 per surface area 600A of the membrane 600.
  • the inventors Based on the characteristics of the present sperm-sorting microfluidic device 100 and the device disclosed in the U.S. Patent no. 10,422,737, the inventors note the following advantages of the sperm-sorting microfluidic device 100.
  • the thickness of the membrane 600 of the 737 patent is short, e.g., less than 10 pm, which makes it easy for a 50 pm long sperm cell to cross the filter once its head passes such a micropore. This is undesired because even a low mobility sperm cell can pass through such a thin barrier.
  • a 100 pm or longer tunnel 134 as is the case in the present device 100, which is twice the length of a sperm cell 150, allows passing only highly motile sperm cells moving in a straight line.
  • the length of the tunnel 134 in the sperm-sorting microfluidic device 100 can be further increased by using a thicker dry film or by using multi-laminated parts 130.
  • Increasing the length of the tunnel 134 is an effective way to improve the quality of the sperm selection.
  • the long tunnels in the device 100 minimize the diffusive exchange of liquids between the upper and lower chambers 110 and 120. This reduces contamination of the collecting chamber with dead sperm, debris, somatic cells, and other undesired macromolecules from the lower chamber 110. Note that in the device of the 737 patent, such debris may move from one compartment to another one through the thin membrane 600 simply because of the diffusive exchange of liquids, which is undesired.
  • the micro-tunnel part 130 used in the device 100 has tunnels 134 formed in half of the surface area, as illustrated in Figure 5, which is more than three times higher than the pore density of the track-etched pores of the membrane 600 used in 737 patent.
  • a large pore area allows a significantly higher number of sperm to swim through at a given time, which allows the reduction in sperm sorting time.
  • Another difference between device 100 and the device in the 737 patent is the top layer 146, which is present over the upper chamber 120, and which protects the medium in the upper chamber from evaporation and the gas exchange between the buffer solution 156 and the atmosphere. Such control of the gas exchanges helps to maintain the stability of pC>2, pCC>2, and pH in the solution 156.
  • the device in the 737 patent has no such top layer.
  • the device 100 differs from the device of the 737 patent.
  • no toxic or hazardous chemicals such as glue or adhesive, are used at any stage of fabrication.
  • all the layers may be made of PMMA, the micro-tunnel part 130 can be made of a UV- curable photosensitive film, and all these components are directly attached to each other by thermal bonding. This process eliminates any possibility of chemical contamination during the manufacturing of the sperm-sorting microfluidic device.
  • Another embodiment of a sperm-sorting microfluidic device 700 is now discussed with regard to the figures.
  • This sperm-sorting microfluidic device dynamically selects sperm cells based on sperm motility with significantly higher vitality, better morphology, and higher DNA integrity.
  • the motility of the sperm cells is divided into three different grades: progressive, non-progressive motility, and immotile. Progressive sperm cells, the healthiest cells, have good motility and swim fast in a straight line. As discussed above, selecting progressive sperm cells from the rest of the heterogeneous semen samples to fertilize the egg cell is essential in assisted reproductive technology (ART).
  • the dynamic sperm-sorting microfluidic device 700 is superior in terms of sperm vitality, morphology, and DNA integrity than those sorted using current clinical sperm selection methods.
  • the device 700 has a body 702 which is divided, by a dividing wall 704, into a collecting chamber 710 and a loading chamber 720.
  • the collecting chamber 710 has a bottom 710A, which is at the same level (or in the same plane 711) as a bottom 720A of the loading chamber 720.
  • the collecting chamber 710 and the loading chamber 720 are positioned in the body 702 of the device 700 to have their bottoms at the same level, along the gravity axis Z.
  • the loading chamber 720 is also divided, with another dividing wall 721 , into a raw chamber 722, and a waste chamber 724.
  • the raw chamber 722 is configured to receive raw sperm while the waste chamber 724 is configured to receive waste from the raw chamber 722, as discussed later.
  • the collecting chamber 710 is configured to receive a sorting medium 712, which includes one or more chemicals that promote sperm cell’s survival.
  • the sorting medium may include glycerol, sucrose, disaccharides, trisaccharides, egg yolk, soy lecithin, etc. Note that the sorting medium does not initially include any sperm cells.
  • the collecting chamber 710 fluidly communicates with the raw chamber 722 through one or more microchannels 730.
  • the microchannels 730 extend horizontally, through the dividing wall 704, from the collecting chamber 710 to the raw chamber 722.
  • Each microchannel 730 may be a straight line that has a length L between 10 and 30 mm, with a preferred length of about 15 mm, and a diameter D between 100 and 500 pm. Note that the diameter D is larger than a diameter of a healthy and highly motile sperm cell 750 to allow the sperm cell to swim through the microchannels 730. Also, the diameter D is constant along the length L.
  • the microchannels may be distributed at different levels relative to the bottom of the body 702. For the embodiment illustrated in Figures 7 A and 7B, the microchannels are located at the bottom of the collecting chamber 710 and the loading chamber 720. Additional microchannels may be provided above these bottom microchannels, as shown in Figure 7A.
  • the sorting medium 712 is placed into the collecting chamber 710 and the raw sperm cells 752 are placed into the raw chamber 722.
  • the bottom of the collecting chamber 710 and the bottom of the raw chamber 722 are in the same plane.
  • the amount of the sorting medium 712 that is added to the collecting chamber 710 is such that the top surface 712A of the sorting medium 712 is higher than a top surface 752A of the raw sperm cells 752, i.e., a height H of the surface 712A is higher than a height h of the surface 752A, when the heights are measured from a common bottom surface of the body 702, and along the gravity axis, Z. in one embodiment, the height H and h start in the plane 711.
  • This height difference in the two surfaces generates a hydrostatic pressure in the collecting chamber 710, at the output port 730B of the microchannels 730, to be larger than a hydrostatic pressure in the raw chamber 722, at the input port 730A of the microchannels 730.
  • This difference in the hydrostatic pressure at the opposite ends 730A and 730B of the microchannels 730 generate a flow of the sorting medium 712, along the microchannels, from the collecting chamber 710 to the raw chamber 722, as indicated by arrow 732.
  • sperm cells 750 when swimming from the raw chamber 722, to the collecting chamber 710, through the microchannels 730, encounter a fluid flow 732 of the sorting medium 712, which makes less healthy and/or less motile sperm cells not be able to arrive in the collecting chamber 710.
  • the sorting medium 712 is flowing through the microchannels 730 into the raw chamber 722, due to the different hydrostatic pressures at the ends of the microchannels, the level of fluid in the raw chamber 722 is increasing and eventually overflows the dividing wall 721 into the waste chamber 724.
  • the highly motile sperm cells 750 swim against the flow 732 into the collecting chamber 710, and the sperm cells 754 that fail to pass through the microchannels 730, are taken by the flow 732 into the waste chamber 724.
  • the flow 732 of the sorting medium 712 allows only the best sperm cells 750 to move into the collecting chamber 710 and removes the other sperm cells 754 from the raw chamber, to make room for new raw sperm cells.
  • Figure 7C shows the flow 732, the diameter D and the length L of a microchannel 730, and a sperm cell 750 having a length I, which swims against the flow 732.
  • the microfluidic device 700 relies on self-propelled sperm motility swimming up against the natural flow 732, through the microchannels 730. Note that there is no active force or pressure exerted from outside by the operator of the device to generate the flow 732. Thus, there is no need for pumps or other electrical or mechanical devices for sperm cell selection.
  • These microchannels imitate the anatomy of the female reproductive tract and resemble the natural process in vivo.
  • the body 702 of the device 700 is made from three separate parts 800, 810, and 820.
  • a base 800 can be cut by a CO2 laser from a 2 mm thick PMMA sheet. Other materials may be used.
  • a first layer of microchannels 730-1 may also be created with the same CO2 laser with different power and speed settings, in the top surface of the bottom part 800.
  • the middle part 810 which also can be cut by a CO2 laser from a 2 mm thick PMMA sheet, is shaped to form the collecting chamber 710, the raw chamber 722, and the waste chamber 724. A second layer of microchannels 730-2 may be formed in the top surface of this part.
  • the middle part 810 is placed on top of the bottom part 800.
  • the top part 820 is cut by the CO2 laser from a 4 mm thick PMMA sheet.
  • the laser is also used to cut the top part of the collecting chamber 710, and the top part of the loading chamber 720, which is common to both the raw and waste chambers.
  • the top part is placed over the middle part and then all the parts are hermetically sealed together by thermal bonding.
  • the device 700 shown in Figure 8 can be made from more parts, for example, more middle parts 810 for adding supplementary layers of microchannels.
  • the number of microchannels formed in the device 700 depends on the desired efficiency of the device, and the overall size of the device. The more microchannels, the more sperm cells can be processed.
  • Various other fabrication technologies such as a high-resolution 3D printer with biocompatible and autoclavable resin can be used to make the sperm sorting microfluidic device 700. While the embodiments discussed herein appear to show that the microchannels have a rectangular cross-section, one skilled in the art would understand that a cross-section of the microchannels can be square, circular, elliptical, triangular, rhombic, etc.
  • PR Progressive motility
  • Non-progressive motility all other patterns of motility with an absence of progression, e.g., swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar beat can be observed.
  • the inventors analyzed the sperm 750 collected from the collecting chamber 710 of the sperm-sorting microfluidic device 700.
  • the results show that the sorted sperm cells have a progressive motility of 90%, which is significantly higher than the stock sperm motility of 27%, as illustrated in Table 1 in Figure 9.
  • the higher motility in the sorted sperm cells obtained with the device 700 is due to the presence of the microchannels 730 between the two chambers 710 and 722, which selectively allow the most motile sperm to swim through the microchannels against the liquid flow.
  • the sperm cells vitality has also been assessed.
  • Sperm vitality is the percentage of living sperm in a semen sample. This parameter is especially important to be measured to differentiate between live non-motile sperm and dead sperm.
  • Sperm vitality is not the same characteristic as motility. Motility measures how many sperm are swimming and their swimming prowess, while vitality is a measurement of all living sperm, irrespective of whether they are moving or not.
  • the two-compartment microfluidic sperm-sorting device 700 was found to promote high-vitality sperm cells separation.
  • the sperm cells sample injected into the loading chamber 720 and the sorted motile sperm collected from the collecting chamber 710 were analyzed and the results show that the sorted sperm cells have 100% vitality. This is because only the superior sperm cells 750 that traveled forward in a straight line against the fluid flow were capable of reaching the collecting chamber 710, while the weak sperm cells with curved (non-linear) motion and dead sperm cells stay in the loading chamber 720.
  • Another parameter analyzed for the device 700 was the sperm morphology.
  • the assessment of sperm morphology is a standard procedure in semen analysis.
  • the sperm morphology assessment used by the inventors is a simple normal/abnormal classification, with optional tallying of the location of the abnormalities in the abnormal spermatozoa.
  • the Spermac StainTM (FertiPro N.V., Belgium) spermatozoa staining method was used to be able to differentiate the morphologically normal sperm cells from the abnormal sperm cells.
  • 10 qL of semen was spread onto a glass slide and allowed to air-dry at room temperature.
  • the smears were then stained with Spermac Stain and the sperm morphology was assessed according to the WHO criteria.
  • a morphologically normal spermatozoon has an oval head and an acrosome covering 40%-70% of the head area.
  • a normal spermatozoon has no neck, midpiece, tail abnormalities nor cytoplasmic droplets larger than 50% of the sperm head.
  • the results of the sperm morphology assessment were compared for the sperm cells obtained with the novel microfluidic device 700, with the traditional swim-up method, and also with the traditional density gradient-based centrifugation method.
  • the swim-up method and density gradient-based centrifugation method are two conventional sperm sorting methods which are the most commonly used in IV fertility clinics around the world.
  • the inventors observed that a higher percentage of the sperm cells sorted using the microfluidic sperm-sorting device 700 is morphologically normal compared to the stock and sperm sorted using the other methods. A comparison of these groups in terms of the outcomes is shown in Table 2 in Figure 10.
  • the method includes a step 1100 of placing raw sperm cells 752 into a loading chamber 720, a step 1102 of placing a sorting medium 712 into a collecting chamber 710, a step 1104 of generating a gravitational fluid flow 732 from the loading chamber 720 to the collecting chamber 710, along plural microchannels 730 that extend between the collecting chamber 710 and the loading chamber 720, where a bottom 710A of collecting chamber 710 and a bottom 720A of the loading chamber 720 are in a same plane 711, and a step 1106 of collecting motile sperm cells 750 from the collecting chamber 710, after swimming against the gravitational fluid flow 732, where the gravitational fluid flow 732 is generated exclusively to the gravity.
  • a length L of a microchannel of the plural microchannels is between 10 and 30 mm and a diameter D of the microchannel is between 100 and 500 pm.
  • the disclosed embodiments provide sperm-sorting microfluidic systems that use one or more microchannels, with no external forces or pressures applied by an operator, for selecting the sperm cells. It should be understood that this description is not intended to limit the invention. On the contrary, the embodiments are intended to cover alternatives, modifications, and equivalents, which are included in the spirit and scope of the invention as defined by the appended claims. Further, in the detailed description of the embodiments, numerous specific details are set forth in order to provide a comprehensive understanding of the claimed invention. However, one skilled in the art would understand that various embodiments may be practiced without such specific details.

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Abstract

Un dispositif à microcanaux de tri de spermes (700) comprend une chambre de collecte (710) conçue pour recevoir un milieu de tri (712) ; une chambre de chargement (720) conçue pour recevoir des cellules spermatiques brutes (752) ; et plusieurs microcanaux (730) s'étendant entre la chambre de collecte (710) et la chambre de chargement (720). Un fond (710A) de la chambre de collecte (710) et un fond (720A) de la chambre de chargement (720) sont dans un même plan (711).
PCT/IB2020/061977 2019-12-16 2020-12-15 Dispositif microfluidique pour la sélection de spermes hautement motiles Ceased WO2021124111A1 (fr)

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JP2023180256A (ja) * 2022-06-08 2023-12-20 アイプレグ インコーポレーション 進行性精子選別のための垂直温度勾配装置および方法
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EP4429590A4 (fr) * 2021-11-09 2025-08-13 Motilitycount Aps Dispositif de séparation de cellules motiles
WO2023114266A1 (fr) * 2021-12-14 2023-06-22 Awadalla Michael S Nouveau procédé de nage pour le lavage du sperme
JP2023180256A (ja) * 2022-06-08 2023-12-20 アイプレグ インコーポレーション 進行性精子選別のための垂直温度勾配装置および方法
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WO2024007019A3 (fr) * 2022-07-01 2024-02-08 DxNow, Inc. Systèmes multi-puits et procédés de tri de sperme

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