WO2022078144A1 - 一种从精液样本中分离精子的装置和方法 - Google Patents

一种从精液样本中分离精子的装置和方法 Download PDF

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WO2022078144A1
WO2022078144A1 PCT/CN2021/118151 CN2021118151W WO2022078144A1 WO 2022078144 A1 WO2022078144 A1 WO 2022078144A1 CN 2021118151 W CN2021118151 W CN 2021118151W WO 2022078144 A1 WO2022078144 A1 WO 2022078144A1
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sperm
chamber
microchannel
reservoir
motile
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PCT/CN2021/118151
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English (en)
French (fr)
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瓦西列斯库S
易卜拉欣米•瓦尔基亚尼M
努斯拉蒂R
樊华
巴扎兹S•R
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微创世纪有限公司
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    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • 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

Definitions

  • the present invention relates to a device and method for isolating sperm from a semen sample, and more particularly to a device and method for selecting sperm having predetermined characteristics from the separated sperm.
  • the actual sperm isolation cannot be performed due to the time constraints of the sample (eg obtained by testicular surgery), but only sperm purification.
  • the sperm purification process is generally more time-consuming; screening sperm from testicular samples is a manual procedure that typically takes 2-3 hours and only removes seminal plasma from the sperm (WHO, 2010).
  • DGC typically selects up to about 36% of the sperm population from 0.5mL of raw semen within 30 minutes, while upstream assays typically select about 12% of the sperm population from 1mL of semen within 1 hour, allowing sperm motility 18-19% and 5%, respectively (WHO, 2010).
  • US Patent No. 2015/0140655 A1 describes a method and apparatus for isolating sperm from a semen sample.
  • the device includes an outer annular chamber for a semen sample, a plurality of spoke-like microchannels (ie, a radial array or network of such channels), and a central chamber to collect sperm that has passed through the array of microchannels.
  • This device functions by autonomously swimming sperm (motile sperm) swimming through effectively stagnant liquids (which can be relatively high viscosity liquids) in microchannels, and exploits the natural swimming behavior of sperm in confined spaces , such as sperm surface aggregation behavior and swimming behavior along boundaries.
  • a device for separating motile sperm from a semen sample comprising an inlet reservoir for receiving a semen sample; an outlet for collecting sperm separated from the sample a reservoir; and at least one but preferably a plurality of microchannels disposed between the inlet reservoir and the outlet reservoir to provide fluid communication between the two reservoirs, the microchannel including at least one wall, the the wall defines a boundary surface along which sperm is directed towards the outlet reservoir; and wherein the at least one wall has at least one surface structure selected from, for example, one or more recesses, protrusions, and combinations thereof, the surface The structure extends over at least a portion of the length of the microchannel to provide at least one additional boundary surface along which the motile sperm travel toward the outlet reservoir.
  • the device includes a housing, preferably made of a polymeric material such as cyclic olefin copolymer (COC), polycarbonate (PCA), polymethylmethacrylate (PMMA), poly Dimethylsiloxane (PDMS) or photopolymer resins used in additive manufacturing techniques that can be advantageously used to manufacture the devices of the present invention (but not to the exclusion of other manufacturing techniques such as casting, moulding manufacturing and machining),
  • the housing is designed to be integrally formed or completely encasing the inlet reservoir, the outlet reservoir and one or more microchannels extending between the inlet and outlet reservoirs, the housing At least one port is arranged to supply a semen sample into the inlet reservoir, and at least one extraction port is used to remove sperm separated from the semen from the outlet chamber.
  • a housing preferably made of a polymeric material such as cyclic olefin copolymer (COC), polycarbonate (PCA), polymethylmethacrylate (PMMA), poly Dimethylsilox
  • each microchannel can be constant or variable, eg, square, rectangular, circular, oval, and the like. It should be understood that when the cross-section of the microchannel is circular or oval, there will be one annular wall defining an adjoining boundary surface, and in the case of a rectangular cross-section, there are four main boundary surfaces, which The surfaces define sharp corners (or fillets) at the planes where the adjoining walls meet. Sperm exhibiting wall-like behavior will tend to move towards and along the boundary surface.
  • Additional surface structures such as one or more recesses, one or more protrusions, and one or more combinations of recesses and protrusions, are provided in the other planar walls of the microchannel, extending over at least a portion of the length of the microchannel , designed to increase the number of boundary surfaces of the microchannel paths along which sperm can travel.
  • sperm motility is affected by the surface with which it interacts, and sperm generally exhibit wall-like behavior, or when in constrained channel geometries such as microfluidic channels, sperm tend to move closer to the boundary surface direction move.
  • Profiles of microchannel walls capable of providing or forming additional edges and boundary surfaces facilitate motility-based sperm separation more than profiles where the microchannel walls are smooth or flat (as described in U.S. Patent '655) .
  • the device of US Patent Document '655 employs the concept of sequentially connecting the initially greater number of microchannels in the radially outer region of the radial microchannel network such that their number drains the radially inner region reduction, seeking to keep the width and height (ie, the cross-sectional geometry) of the substantially rectangular cross-sectional channel approximately constant in order to sort sperm based solely on the directionality of the wall process.
  • a device does not seek to differentiate sperm based on the direction of travel within the microchannel, but rather changes the geometry of the microchannel to include additional boundary surfaces that the sperm will be based on Its wall behavior swims indiscriminately along this boundary surface.
  • Sperm with the desired properties for fertilization generally travel a greater distance than sperm whose motility decreases with distance traveled, and the increase in the boundary surface allows more sperm with the desired properties to reach the outlet reservoir and then in The sperm can be collected or further processed at the outlet reservoir.
  • the configuration or shape of the surface structure is not limited to any particular geometry, as long as the structure can form an additional boundary surface for guiding sperm, many geometries can accomplish this, as described below. Additionally, the number of sperm swimming boundaries can be controlled to regulate sperm throughput.
  • the structures may include one or more grooves, ribs, and combinations thereof, such as rectangular, semicircular, or stepped cross-sections, extending the entire length of the microchannel from inlet to outlet, Or just extend along part of it.
  • the grooves/ribs define an additional surface path within the microchannel from the inlet reservoir to the outlet reservoir along which sperm can travel based on wall procession.
  • These structures may extend along part or the entire length of the microchannel along one or more axially straight, serrated, wavy, or combinations thereof.
  • the depth of the grooves/height of the ribs can be constant or variable.
  • the cross-sectional shape or profile of the grooves/ribs can be chosen to be suitable as long as there is at least one sharp or rounded edge with channel walls along its extension which extends beyond the edge defined by the smooth walled channel.
  • Surface structures that are discontinuous in depth or height can be provided by the stepped sidewalls of the microchannels. This is to add more boundary surfaces in the sidewalls of the microchannels that the sperm can follow as they pass through the microchannels.
  • the device is designed such that a plurality of microchannels within the housing are spoked from an inlet reservoir in the form of an annular radially outer chamber (which can be subdivided into segments with separate semen sample inlets) Extends to an outlet reservoir in the form of a circular radially inner outlet chamber.
  • a common separating sidewall may be shared between adjacent microchannels, and surface structures providing additional boundary surfaces may be present in one or both sidewalls of a microchannel having a rectangular cross-section.
  • surface structures may also be present in one or both of the bottom and top boundary walls of such a rectangular cross-section microchannel.
  • microchannel arrangements may also be employed, without excluding other microchannel arrangements, for example, multiple microchannels extending parallel to each other between inlet and outlet chambers at opposite ends of the microchannel (as compared to radial networks), or multiple microchannels.
  • the microchannels may be helically arranged from the radially outer annular inlet chamber towards the radially inner cylindrical outlet chamber.
  • microchannels do not need to extend in a straight line, but can be arranged in a serpentine or meandering arrangement between the inlet and outlet reservoirs.
  • the number of microchannels of the device is 400 to 700, more preferably 100 to 500, but the number of microchannels may range as a minimum of 5 , the maximum can be up to 5000.
  • the plurality of microchannels may be between about 10 ⁇ m and about 5000 ⁇ m in width, preferably between 50 ⁇ m and 250 ⁇ m, and about 10 ⁇ m in height between about 5000 ⁇ m, preferably between 100 ⁇ m and 1000 ⁇ m.
  • one or both of the width and height of all microchannels are the same.
  • the length of the microchannel is between about 0.1 mm and 15 mm, preferably between about 5 mm and 9 mm.
  • microchannels are arranged in a radially converging arrangement
  • two or more microchannels originating at the inlet reservoir end of the device may join along their paths to meet at the outlet reservoir form a single microchannel.
  • microchannels may be arranged within the device housing in a single-layer or multi-layer stack, with the microchannels originating in a common or separate inlet chamber and draining into a common outlet chamber.
  • a device for screening a semen sample for motile sperm of defined characteristics comprising: an inlet reservoir for receiving a semen sample; a binding chamber arranged to pass through a guide structure in downstream fluid communication with the inlet chamber, the guide structure being designed to direct motile sperm from the semen sample to the binding chamber, preferably based on wall behavior of the motile sperm; and an outlet reservoir in downstream fluid communication with the binding chamber,
  • the outlet reservoir is designed to receive motile sperm that have passed through a binding chamber; wherein the binding chamber is used to accommodate selection particles having a selectivity for binding to motile sperm that (i) have passed through the guide structure, and (ii) ) with predetermined, undesired labels, binding chambers and/or selection particles designed to substantially confine particle-bound sperm in the binding chamber and prevent further sperm travel to the outlet reservoir without hindering unbound sperm The passage of motile sperm to the selection pellet.
  • the guide structure will advantageously take the form described with reference to the first aspect of the invention, ie comprising a plurality of microchannels extending between the inlet reservoir and the junction chamber (rather than the outlet chamber).
  • the inlet side of the bonding chamber is bounded by the downstream ends of the plurality of microchannel walls (thus providing a grid-like inlet structure), and the outlet side is defined by a series of spaced columns or columns, the columns or The columns are arranged in a configuration that defines a grid-like outlet structure, so that the binding chamber is a "cage structure" into which motile spermatozoa can enter from a plurality of microchannels, and which are prevented by a plurality of blocking columns from exiting the particle-bound spermatozoa.
  • the cage structure flows out.
  • the end portions of the walls separating adjacent microchannels adjoining the binding chamber are advantageously configured such that (i) the microchannels narrow toward their respective discharge ends, and (ii) collectively define the compartment A grid boundary structure that inhibits the re-entry of motile spermatozoa that have exited the microchannel.
  • the ends of the microchannel narrow in cross-section and provide an inner surface along which motile sperm can travel from the microchannel into the binding chamber in a preferred converging path, due to the reduced cross-section. Narrow, making it substantially (but not absolutely) impossible for motile sperm to re-enter the microchannel.
  • the microchannel sidewalls are substantially rectangular in vertical cross-section and the ends widen in thickness, the wall ends are substantially triangular in plan view.
  • the plurality of blocking posts are shaped and/or arranged with respect to each other in a manner functionally similar to the shaping of the ends of the microchannel-defining sidewalls to restrict the return of motile sperm that has exited the binding chamber to the binding chamber.
  • the gap between the blocking columns needs to be large enough to allow unbound motile sperm to flow through the binding chamber to the outlet reservoir.
  • the gap between the blocking columns needs to be small enough to accommodate the selection particles within the binding chamber.
  • the gap between adjacent blocking posts is between 10 and 100 ⁇ m.
  • the cross-section of the microchannel at its ends needs to be equally small to accommodate selection particles in the binding chamber.
  • the posts are chevron, semi-circular, or crescent-shaped in cross-sectional shape, and the posts are arranged so as not to obstruct passage between the posts toward the exit chamber, but still inhibit the return of motile sperm from the exit chamber into the binding chamber.
  • Selection particles of suitable shape, size and packing density are selected for incorporation into the binding chamber so that they do not substantially impede the passage of motile spermatozoa that do not exhibit undesired labels as they swim out of the binding chamber, but the selection particles
  • the shape, size and packing density should be large enough to properly accommodate motile sperm exhibiting undesired characteristics in the binding chamber.
  • the selection particles contained in the binding chamber comprise spheres, in particular microspheres or microbeads, conjugated to ligands comprising proteins capable of attaching to labels, the labels being selected Autolectin, anti-CRISP (cytosine-rich secreted protein), annexin A5, anti-chemokine receptor antibody and anti-CD63, anti-CD9, ALIX, or TSG101m, or the ligand may be a ligand including requinimod ( resiquimod), imiquimod, and gardiquimod.
  • the ligand is Annexin A5, which chemically binds to a marker expressed by motile sperm.
  • ligand refers to a substance that forms a complex with a biomolecule to function in a biochemical pathway.
  • a ligand might be a molecule that triggers a cellular response when bound to a specific cellular site.
  • the ligand may be an ion or protein or any other suitable molecule with a chemical structure capable of interacting with the binding site.
  • the ligand is a protein, preferably Annexin A5, by means of which the ligand can bind and capture spermatozoa expressing phosphatidylserine on the outer surface of the spermatozoa in the binding chamber.
  • these phosphatidylserine expressing spermatozoa are prevented from traveling further towards the outlet reservoir, whereas spermatozoa that do not express phosphatidylserine are able to travel substantially unhindered through the binding chamber towards the outlet reservoir and then in the binding chamber. It can be collected or further processed.
  • the device further comprises an oocyte retention chamber in fluid communication with or as part of the outlet reservoir.
  • a configuration can be selected for regulating the passage of screened (ie, unparticle-bound) motile sperm from the outlet reservoir into the oocyte storage chamber.
  • Oocytes can be introduced directly into the oocyte storage chamber, so that motile sperm isolated from the sample and "screened" during passage through the binding chamber can bind to the oocyte for fertilization.
  • chemoattractants can be applied to the outlet reservoir and/or the oocyte preservation chamber. Chemoattractants help guide the isolated and screened sperm to oocytes.
  • the device may have in its housing a first region comprising an inlet reservoir, a second region comprising a microchannel or guide structure, a third region comprising a binding chamber, a fourth region comprising an outlet reservoir, and Includes the fifth area of the oocyte storage chamber.
  • the third and fourth zones may preferably be juxtaposed, which means that the oocyte storage chamber will be located within the confines of the outlet reservoir.
  • the outlet reservoir is designed as a substantially cylindrical disc chamber, there may be one or more separate wall structures therein, each for containing oocytes, but at the same time motile sperm can pass through the wall structures to contact oocytes.
  • the first region eg, the inlet reservoir in the form of an annular chamber
  • the second region e.g., an annular outer inlet chamber with radially inward guide channels at the same height, the ends of which are at the same height the annular junction or outlet chamber on the
  • the second region may be located at the same height as the third region (ie, the binding chamber may also be in the form of an annular chamber).
  • the microchannel ie, the second region
  • the microchannel is located between these two heights, so the channel itself will extend obliquely with respect to the horizontal.
  • the microchannels are arranged in a radial array
  • the inlet reservoir is an annular chamber in the region above the annular binding chamber or the outlet chamber
  • the 3D channel array will be enclosed by a frustoconical (straight channel) or frustoparaboloid (or similar) envelopes are defined, and the channels have curvatures in planes perpendicular to the horizontal.
  • the third area (the binding chamber) may be located at the same height as the fourth area (the outlet reservoir), and the fourth area (the outlet reservoir) may be located at the same level as the fifth area (the oocyte storage chamber) or at Above the fifth area.
  • these regions are in fluid communication with each other, and buffer solutions fill various structures to enable (a) motile sperm to be separated from the semen sample within the microchannels or guide structures, ( b) motile spermatozoa that have entered the binding chamber and have predetermined characteristics can be separated from motile spermatozoa that bind to the selection particles and thus remain in the binding chamber, and (c) motile spermatozoa that have not bound to the particles can reach the outlet reservoir device.
  • the (non-particle-bound) spermatozoa thus screened can then be removed from the outlet reservoir and/or can "swim" into the oocyte storage chamber, where they can interact with the One or more oocytes combine to achieve fertilization.
  • ports into the various chambers may also be provided to enable removal of particle-bound sperm from the binding chamber and unbound sperm from the outlet reservoir and/or oocyte storage chamber, for example using Pipette or other known IVF output/removal instrument.
  • a method for screening and/or isolating motile sperm from a semen sample comprising the steps of: isolating motile sperm from a semen sample only, or isolating and screening motile sperm according to specific circumstances (ie, isolating motile sperm from a semen sample only) ), providing a device according to the foregoing first or second aspect of the invention; filling the microchannel or guide structure (as the case may be) and the inlet and outlet reservoirs of the device with a sperm buffer solution to produce a basic A non-flowing fluid in which motile sperm can travel; introducing a semen sample into the inlet reservoir in a manner that does not cause or minimize fluid flow in the device, such as removing a portion of the buffer solution at the same time or only to a limited extent Prefilling the associated chambers and microchannels; waiting for a predetermined period of time during which motile spermatozoa will pass through
  • a method for in vitro fertilization of oocytes using motile sperm isolated and screened from a semen sample comprising the steps of: providing a device according to the second aspect of the present invention; viewing Situation Fill the microchannel or guide structure with sperm buffer solution, and the inlet and outlet reservoirs of the device; fill the binding chamber with selection particles with selectivity for binding to motile sperm, which have the ability to bind to oocytes Predetermined undesired features associated with fertilization, such as the expression of apoptosis-related markers, the expression of sex-selective-related markers, or the presence of a permeable membrane that confines particle-bound motile sperm to the binding compartment without hindering the passage of motile sperm bound to the selection pellet; introduction of one or more oocytes into the oocyte storage chamber; introduction of a semen sample into the inlet reservoir; waiting time for screened motile sperm (i.e.
  • Figure 1 shows in a highly schematic simplified diagram a sperm screening device according to the present invention, and in an overview diagram the sperm screening performed during operation of the device when a sperm screening method is performed according to another aspect of the present invention step;
  • FIGS. 2A to 2F show schematically and partly in detail a first embodiment of a sperm separation and screening device according to the present invention.
  • Figure 2A is a top perspective view of the top housing portion of the device
  • Figure 2B is a bottom perspective view of the top housing portion showing a plurality of spoked microchannel sperm conduits from radially outer annular sperm receiving reservoirs or chambers to radially inner annular sperm screening (or binding) a chamber extending, which in turn adjoins a substantially cylindrical, radially inner, motile sperm outlet (or collection) reservoir or chamber;
  • Figure 2C is a top perspective view of the lower bottom shell portion combined with the top shell portion, according to the highlighted detail view, containing nine individual oocyte-preserving structures in the radially central region, when the two shell portions are as shown
  • the sperm collection chamber of the top housing portion accommodates and is in fluid communication with the oocyte storage chamber at the radially centermost position;
  • Figure 2D is an enlarged detail view of the top housing section showing (i) the radially inner end of the microchannel, which provides a circular grid boundary structure for the screening (or binding) chamber, (ii) the screening chamber and (iii) a radially inner circular grid boundary structure in the screening chamber formed by a plurality of closely spaced blocking posts, and (iv) a more radially inner circular sperm outlet reservoir;
  • Figure 2E is a cross-section along the diameter of the device with the upper and lower halves separated from each other;
  • Figure 2F is a schematic microscopic illustration (enlarged view) depicting the device of Figure 2B or Figure 2D having, from left to right, (i) spoke-like multiplexes extending from a radially outer annular sperm receiving reservoir or chamber two microchannel sperm conduits, (ii) a radially inner annular sperm screening (or binding) chamber, (iii) a radially inner circular grid boundary structure formed by a plurality of closely spaced blocking posts in the screening chamber, and ( iv) radially more inward circular sperm outlet reservoir;
  • Figures 3A to 3I show schematically and partly in detail another embodiment of a device for sperm separation and screening according to the present invention, the device having more than 100 microchannels;
  • Figure 3A is a bottom perspective view of the top housing portion similar to Figure 2B, but showing another embodiment of a device according to the present invention, showing the direction from the radially outer annular sperm receiving reservoir or chamber to the A plurality of microchannel sperm ducts extending in a radial pattern with a radially inner annular sperm screening (or binding) chamber, which in turn is connected to a radially further inner motile sperm outlet (or collection) that is also substantially annular ) reservoir or chamber adjoining;
  • Figures 3B and 3C are different perspective views of the detail highlighted in Figure 3A showing an embodiment of a microchannel comprising ribs and grooves in opposing sidewalls according to another aspect of the invention a structure in the form that extends over a portion of the length of the microchannel and increases the number of boundary surfaces;
  • Figure 3D shows three of a number of other configurations of the facing surfaces of the opposing sidewalls of the microchannel shown in Figures 3B and 3C, with additional motile sperm added by forming surface structures at each sidewall Can be based on the number of boundary surfaces along which the wall behavior travels;
  • Figure 3E is a view similar to Figure 3B but further enlarged to better illustrate how the sidewalls of the microchannels are shaped in the radially outer end region compared to the adjoining radially inward region to provide motile sperm Provides additional boundary surfaces for travel based on wall-like behavior;
  • Figures 3F-3I are schematic top views and detailed perspective views of four different embodiments of radially inner multi-column grid barrier structures forming one of the grid boundaries of the annular bonding (or screening) chamber;
  • Figures 3J to 3L show schematically and in partial detail another embodiment of a device for sperm isolation and screening according to the present invention, the device having 45 microchannels, wherein the features shown are further (but not to scale) Enlarged to better illustrate the wall structure and wall insert of this embodiment;
  • Figure 4 schematically illustrates an embodiment of a manufacturing process of a device according to various embodiments of an aspect of the present invention, the manufacturing process using additive manufacturing techniques, ie 3D printing;
  • Figure 5A schematically illustrates the steps for the preparation of selection (binding) particles which, when using the devices of Figures 1 , 2, 3 and 4, will be in the form of proteins according to one embodiment of this aspect of the invention
  • the coated microbead form is received and contained in the annular binding chamber of the device;
  • Figure 5B is a schematic view of a microscope (enlarged view) depicting the attachment of sperm cells to protein-conjugated microbeads fabricated by the method shown in Figure 5A through protein-protein interactions;
  • Figure 6 illustrates method steps for loading selection particles in the form of microbeads into the annular binding chamber of the device shown in Figures 1 to 4 in accordance with one embodiment of the present invention
  • Figure 7 shows, by way of an illustrative flow chart, a method according to one embodiment of the present invention that first isolates motile sperm from a semen sample and then selects motile sperm with the desired Motile spermatozoa for fertility indicators, allowing "desired" motile spermatozoa to pass through the binding chamber of the device according to Figures 1 to 4;
  • Figure 8 includes three graphs embodying performance testing of the device of the present invention in terms of sperm recovery, sperm motility and sperm vitality, wherein Figure 8A is a graph of sperm concentration versus time, inset 8B is a graph of sperm exit count over time, FIG. 8C is a graph of sperm motility and vitality over time, and FIG. 8D is a graph of DNA fragmentation index before and after sperm screening with the device; and
  • FIGS 9A to 9E show schematically and partly in detail a second embodiment of a device for sperm separation and screening according to the present invention, which device has a stacked array of microchannels compared to the device of Figures 2A to 2E,
  • the microchannel extends in a parabolic curve between an annular semen collection reservoir located in the upper region or level of the device and an annular binding chamber divided into four separate arcuate regions located lower in the middle of the device In area or height, there is a bottom cover similar to that shown in Figure 2C with an oocyte storage chamber (not shown), the bottom cover forming the bottom of the device in use to receive the device wherein,
  • Figure 9A is a schematic perspective view of the device comprising three stacked housing parts showing microchannels extending along the height of the substantially cylindrical intermediate housing part between the upper and base housing parts (shown in dashed outline), the upper and base housing portions are substantially mirror images of the top housing portion shown in Figure 2B, but without the microchannels and bottom housing portion of Figure 2C;
  • Figure 9B is an exploded perspective view showing three housing parts of the device of Figure 9A, each fabricated by 3D printing;
  • Figure 9C is a partially cutaway side view of the device of Figure 9A;
  • Figure 9D is a bottom view of the top housing portion showing a radially outer annular sperm receiving reservoir or chamber with two adjacent inlets separated by a continuous web with surrounding circular ports The radially inner annular wall is connected, and the circular port communicates with a tubular conduit extending through the mid-height position of the device housing, as shown in the translucent portion of Figures 9A and 9B and the plan view of Figure 9E;
  • FIGE is a top view of an intermediate housing portion with an enlarged schematic detail showing an array of microchannels in layered regions of concentric quarter arcs whereby the microchannels are bounded by the housing webs separate and extend along a substantially parabolic path between the upper ends of the housing parts starting from the adjacent semen sample receiving reservoirs of the upper housing parts towards the annular, quarter-arc semen a binding chamber integrally formed in and recessed in the lower end face of the middle portion, as schematically shown in Figure 9F;
  • 9F is a bottom view of the intermediate housing portion, wherein another enlarged schematic detail is a cross-sectional view taken along the line shown, showing the conduit extending between the top and bottom surfaces of the intermediate housing portion The lower end, which extends to an enlarged, motile semen outlet chamber, in which is located the oocyte retention chamber on the upper surface of the bottom closed plate schematically shown in Figure 9C.
  • Various embodiments of the devices, methods of isolating sperm, methods of screening sperm, and methods of fertilization described herein may be practiced in fertility clinics or research centers to study in vitro fertilization (IVF), intrauterine insemination (IUI), and egg Treatment of male infertility using intracytoplasmic sperm injection (ICSI).
  • IVF in vitro fertilization
  • IUI intrauterine insemination
  • ICSI intracytoplasmic sperm injection
  • the information disclosed herein can be used to isolate motile sperm and subsequently screen sperm for IVF, ICSI, IUI and/or other reproductive treatments.
  • the device and method, and variants and methods thereof may also be used in non-human applications, particularly agriculture, such as assisted reproductive technology (ART) treatments for cattle and other animal breeding.
  • ART assisted reproductive technology
  • Figure 1 schematically shows an apparatus 10 for isolating motile sperm from a semen sample and subsequently performing a screening procedure to produce oocytes capable of satisfying Specific criteria for in vitro fertilization of motile sperm and outlines the steps in the process to perform sperm isolation and screening using individual structures within the device.
  • the device 10 includes the following main functional structures:
  • a semen sample receiving chamber also referred to as an inlet reservoir 12;
  • a motile sperm guide and separation structure 14 in fluid communication with the inlet reservoir 12, and in the preferred embodiment shown in the other figures, the structure consists of a plurality of microchannels;
  • a motile sperm screening chamber 6 (also referred to as a binding chamber) in fluid communication with the separation structure 14, in which a plurality of selection particles 17 bind to and prevent motile sperm having specific characteristics that have passed through the guide structure 14 Exit chamber 16, whereby chamber 16 is a grid structure (ie a liquid permeable cage) designed to allow unbound motile sperm to swim/pass through the chamber and prevent selection of particles 17 and particles bound to these particles sperm out;
  • a sperm outlet reservoir or chamber 18 in fluid communication with the selection chamber 16 and arranged to receive unbound motile sperm that has passed through the binding chamber 16;
  • An optional oocyte retention chamber 20 in a preferred embodiment, the oocyte retention chamber 20 being in fluid communication with the outlet chamber 18, or disposed within the outlet chamber 18, or as part of the outlet chamber 18, which It is designed to receive and independently deposit oocytes for fertilization by union with sperm accumulated in the exit chamber 18 .
  • FIG. 1 also shows a flow chart of the sperm screening process performed within the device 10 .
  • the process includes (at step 1 ), motility-based sperm screening using radial array microchannels of a separation structure 14 located between the inlet reservoir 12 and the binding chamber 16 .
  • the process also includes (step 2) using a substrate contained (filled with) bioactive selection particles 17 in the grid binding chamber 16, the selection particles 17 being designed to assist in the negative selection of sperm by means of sperm capture ( Induced, for example, by protein-protein interactions between the sperm plasma membrane and the protein surface layer of the selection granule).
  • the process also includes (step 3) the use of a motile sperm accumulation chamber 18 (outlet reservoir) into which unbound motile sperm can enter from the grid screening chamber 16 .
  • the shape of the outlet reservoir allows for immediate collection of motile sperm cells.
  • Figure 1 also schematically depicts another process (step 4) in which the motile spermatozoa in the exit chamber 18 further swim towards the integrated oocyte storage chamber 20 of the device 10, in which oocytes can be deposited, thereby The separated sperm meet and combine with the oocyte to achieve fertilization, thereby completing at least one in vitro fertilization of the oocyte.
  • At least some of the embodiments described herein utilize a combination of sperm guiding (and separation) structures 14 (provided in the illustrated embodiment by a radial array of microchannels) and sperm screening structures 16 (by a grid Sperm binding chamber formation) devices to improve conception outcomes in an in vitro environment, particles 17 that exhibit selective affinity for certain types of sperm are included in sperm screening structures 16, as discussed in greater detail elsewhere, particles 17 are used to bind those Although sufficiently motile, such sperm exhibit "undesirable" factors when used for in vitro oocyte fertilization. In the description of these examples, the word “undesirable” does not simply mean lack of viability or impaired sperm, but rather represents a negative screening criterion that includes other factors such as sex selectivity.
  • the process of "negative" selection is achieved by binding undesired sperm, thereby effectively Particle-bound sperm are immobilized and restricted from exiting the binding chamber 16, while other motile sperm without specific risk factors do not bind to the particles and swim forward through the binding chamber 16 to the device's outlet reservoir 18 without restriction.
  • Motile sperm that do not have undesired characteristics accumulate in the outlet chamber 18 for further processing, such as removal from the device for subsequent freezing or in vivo fertilization procedures.
  • the oocyte storage chamber 20 is present within the device, either as a separate structure, or within the outlet chamber 18 so that the accumulated activity in the outlet reservoir 18 can be used sperm to fertilize the oocytes placed in the oocyte storage chamber 20 in vitro.
  • Figures 2 and 3 illustrate one embodiment of the device 10 in greater detail, wherein Figures 3B to 3E and Figures 3J to 3L illustrate various alternative embodiments of the specific configuration of the microchannels 38, radially extending microchannels 38 Forming the sperm separation structure 14 of the device, various embodiments of the binding chamber 16 are shown in Figures 3F to 3I. A general description has been made above with reference to the schematic diagram of FIG. 1 .
  • a housing 25 which consists of two generally flat cylindrical plates 22, 24, and is entirely or partly made of a suitable hard-cured, Thermoset or thermoplastic polymers are formed into concave or convex structures using additive manufacturing techniques (i.e. 3D printing).
  • Preferred materials for manufacture are plastics such as cyclic olefin copolymer (COC), polycarbonate (PCA) or polymethylmethacrylate (PMMA), but the device can also be made of materials such as polydimethylsiloxane (PDMS) or photopolymer resin materials.
  • COC cyclic olefin copolymer
  • PCA polycarbonate
  • PMMA polymethylmethacrylate
  • the device can also be made of materials such as polydimethylsiloxane (PDMS) or photopolymer resin materials.
  • PDMS polydimethylsiloxane
  • the plates 22 , 24 are bonded (or otherwise secured) to each other in face-to-face relationship into a flat cylindrical housing 25 .
  • Other fabrication techniques such as molding, machining, etc., are not excluded, although the internal construction of the various structures in the device 10 are less preferred for fabrication by these techniques.
  • the semen sample chamber 12 is embodied in the form of an annular chamber in which an annular channel is recessed into the bottom surface of the upper housing portion 26 (only for a small radially extending web separating the channel into two parts) case), this bottom surface is covered and closed by the top surface of the lower housing part 22 after bonding together.
  • Two cylindrical semen sample inlets 28 extend into the sample chamber 12 from the upper surface of the upper housing portion 26 on either side of the web. Thus, a semen sample can be deposited into the annular chamber 12 through the inlet 28 .
  • the semen sample chamber must be sized to accommodate the buffer and the semen introduced into the inlet reservoir 12 in a volume between 50 microliters and 4 mL, while the semen volume typically introduced is between Between 0.5mL and 1mL.
  • the sperm guiding (and separation) structure 14 is comprised of a plurality of spoke-like radially extending microchannels 38, 38', 38" Microchannels 38, 38' and 38" are formed to extend between the radially outer annular semen sample chamber 12 and the radially inner sperm binding chamber 16 (described in more detail below) for the migration of motile sperm from the semen storage chamber 12 Entry into the sperm combination chamber 16 provides multiple travel paths.
  • the radially inner "boundary" structure of the annular semen sample chamber 12 is defined along its inner circumference by the radially outer end faces 54 of a plurality of spaced apart radially extending webs 40, 50, Stands on the concave inner center face of the upper housing portion 26 .
  • a plurality of microchannels 38 , 38 ′, 38 ′′ of the guide and separation structure 14 are defined between circumferentially adjacent pairs of these radially extending webs 40 , 50 and are defined by the lower (bottom) housing portion 22
  • the upwardly facing portion of the upper housing portion 26 and the downwardly facing portion of the upper housing portion 26 define which portions extend vertically between the webs 40, 50 when the housing portions 22, 26 are assembled, thereby providing a plurality of closed transverse Cross-section microchannel.
  • the webs 40 are referred to herein as microchannel side walls 40
  • the webs 50 are referred to as microchannel intermediate walls 50 . While the sidewall 40 extends over the entire radial length/extension of the sperm guiding (and separation) structure 14 between the annular sample chamber 12 and the radially inwardly positioned sperm binding chamber 16, the wall thickness varies as described below , the intermediate wall 50 extends only partially and is present only in the radially outer region of the microchannel 38 as shown in FIG. 3E and described below, and has a constant wall thickness.
  • the radially outer end faces 54 of the radially extending side walls 40 and intermediate wall 50 form substantially circular grid strips that only partially connect the annular semen sample holding chamber 12 with the plurality of radially extending microchannels 38,
  • the guide structures 14 formed by 38', 38" are physically separated.
  • the plurality of microchannels 38 , 38 ′, 38 ′′ extend in a radially converging manner, starting from the annular semen sample receiving chamber 12 and extending radially inwardly further before the center of the upper housing portion 26 , thereby defining a cylindrical void and bounded by the radially inner end face 52 of the sidewall 40.
  • the number of entry points to the guide structure 14 at the annular semen storage chamber 12 is greater, the number of exit points into the radially inner end of the sperm binding chamber 16 is reduced by an amount equivalent to the number of channels along the The number of engagement points that the radial extension of the guide structure 14 has.
  • passages 38' and 38' are each bounded by side walls 40 and share between them a common intermediate wall 50 having a finite radial length and terminating in the junction zone 39, where the junction Channels 38 ′, 38 ′′ merge into radially inner channel 38 at zone 39 .
  • the side walls 40 have two rectangles on opposite sides facing the respective channels 38', 38" Ribs 44, these two rectangular ribs 44 extend/project vertically from the central web 43 of the side wall 40 and extend parallel to each other radially inward from the annular semen storage chamber 12, as the central web 43 thickens, they become is narrower (at 44'), as shown by the merged land 39 in Figure 3E.
  • the sidewall 40 tapers from the junction 39 where the channels 38', 38" merge into the radially inner channel 38 towards their radially inner ends. This is illustrated in Figures 3J to 3L. are best illustrated in the specific examples shown.
  • FIG. 3D shows the opposite side walls 44 (and intermediate wall 50, if Three of a variety of other configurations of opposing surfaces if desired).
  • Figure 3D shows a microchannel cross-sectional embodiment that omits the intermediate wall 50, and further, as the dashed vertical line As shown, only half of the cross-section of the wall 40 is shown.
  • motile sperm swimming in a closed channel with eg a rectangular cross-section will be limited by four bounding surfaces. If the cross-section is small enough to simulate the in vivo environment in which motile sperm swim in a substantially stagnant liquid, e.g. the microchannel forms a rectangular cross-section of e.g. 100 ⁇ m x 75 ⁇ m, the natural swimming properties of sperm will result in Mural behavior of the boundary surface. However, if the cross-section is larger, only the more motile sperm will move towards the boundary surface and then swim along the boundary surface.
  • At least one of the walls providing the boundary surface has at least one structure selected from the recesses 42 and/or the protrusions 44 extending slightly along at least a portion of the length of the channel 38 and providing at least one additional A boundary surface (compared to a channel without such surface structure) along which motile spermatozoa can swim based on wall marching behavior towards the binding chamber 16 at the radially inner end of the guide structure 14/microchannel 38 .
  • a boundary surface as used or defined herein is defined by the confluence of any two planes that intersect to form a corner/angle between them (corners need not be stepped discontinuities, but may include rounded edges) In a manner converging, the angle between the separate boundary surfaces is preferably between 30 degrees and 150 degrees.
  • a portion of the upper housing portion is identified as 26, and as previously described, the sidewall 40 is upstanding and formed with the base plate of the upper housing portion 26. one.
  • the lower housing part 22 closes the channel 38 at the bottom (the channel 38 would otherwise be open).
  • the upper housing 26 provides the top boundary of the channel 38
  • the lower housing portion 22 provides its bottom boundary
  • the opposing wall 40 provides its side boundary.
  • Three rectangular cross-section ribs 44 protrude/project from the side 48 of each wall 40 and are integrally formed thereon (in the embodiment of Figures 3B and 3C, there are only two such ribs).
  • the rib 44 effectively subdivides or divides the two boundary surfaces along the height of the channel, however , the upper and lower surfaces of ribs 44 define additional boundary surfaces 49 along which motile spermatozoa can swim once they have entered channel 38 .
  • the presence of six ribs 44 would add a total of twelve additional boundary surfaces along which motile spermatozoa can swim.
  • the ribs 44 are integrally formed with the side walls 44 and project into the channel 38 .
  • a C-shaped slot 42 is defined between the two ribs 44 and the side 48 of the side wall 40 . In other words, identifying the number of surface feature elements that increase the boundary surface depends on whether the spacing between the opposing end faces 45 of the ribs 44 or the sides 48 of the side walls 44 is considered to define the interior width of the channel 38 .
  • microchannel cross-sectional view in the middle of Figure 3D shows different embodiments of such surface structural elements present in a microchannel that increases the number of boundary surfaces along which motile sperm swim, with sidewalls 44 along
  • the side wall 40 is stepped 46 in height, wider at the lower shell end and narrower at the upper end of the side wall at the web portion.
  • the example of the cross-sectional configuration of the rightmost microchannel 30 illustrates that the ten additional boundary surfaces provided by the sidewall surface structural elements may also be provided by corresponding curved cross-sectional ribs 44 (or defined between adjacent ribs 44). Complementary shaped grooves 42) are provided.
  • the channel is rectangular in cross-section
  • the top or bottom of the microchannel 38 will also be the surfaces that form the normal boundary surfaces, and it is conceivable to provide structural elements on these surfaces to increase the number of boundary surfaces.
  • the "basic" channel cross-sectional configuration is not limited to a rectangular cross-section.
  • its geometric shape may be circular, triangular or hexagonal.
  • the microchannels 38 can be between 2 mm and 15 mm in length and 50 ⁇ m to 5000 ⁇ m in height, but are typically between 100 ⁇ m and 1000 ⁇ m.
  • the width of the microchannels can be between 50 and 5000 ⁇ m, usually between 50 and 250 ⁇ m, assuming a rectangular channel geometry (which is only one possibility).
  • the free space (or distance) between oppositely arranged (or facing) surface feature elements 42, 44 should also be chosen to be of a size that facilitates the travel of motile sperm to the additional boundary surfaces 49 arranged at these structures, and is minimized
  • the value can be set to 50 ⁇ m (may be higher).
  • the choice of maximum and minimum microchannel dimensions will also be influenced in part by the sperm phenotype to be isolated and screened from the semen sample and the semen source (eg, bovine, human, canine, etc.).
  • the width of the microchannels may also be non-uniform across the length of the device, resulting in uneven boundaries or converging and diverging boundaries for sperm to travel along.
  • microchannel pathways may vary. Although devices with 5 to 5000 microchannels have been used to isolate sperm from human and animal semen samples, acceptable performance has been observed, although specific performance will vary depending on the sample processed. In this case, one or more of these factors can be varied to improve the sperm separation process depending on the volume, concentration and viscosity of the sample.
  • the sperm binding chamber 16 will next be described with reference to Figures 2B, 2D and 3F to 3J.
  • the binding chamber 16 is substantially annular in shape, allowing liquid to permeate from the inlet to the outlet side, and is disposed downstream of the guide structure 14 (when viewed in terms of the various functional structures of the motile sperm swimming through the device 10) and upstream of the outlet reservoir 18 . It is primarily designed to receive and contain therein a large number of selection particles 17 that are selective for binding to motile spermatozoa having predetermined, undesired labels that have passed through the guide structure 14 .
  • Binding chamber 16 and/or selection particles 17 are also designed to substantially confine particle-bound sperm in chamber 16 and prevent such sperm from further travel toward outlet reservoir 18 without hindering unbound sperm from selection particles. Motile sperm pass through.
  • the binding chamber 16, filled with binding particles 17, can be considered a filter, serving a dual purpose, acting as an additional physical barrier, similar to the mass of cumulus cells through which sperm must pass during natural in vivo egg fertilization, and as a Capture DNA (or other) damaged sperm.
  • the radially inner ends 52 of the microchannels 38 of the guide structure 14 provide a circular grid boundary structure 34 radially outside the chamber 16, as shown in Figure 2D, which is the top housing portion shown in Figure 2B
  • the enlarged highlighted detail of 26 is also visible in Figures 3F to 3I.
  • the inlet side of the bonding chamber 16 is bounded or bounded by the downstream ends of the walls 44 of the plurality of microchannels 38 (thereby providing a grid inlet structure).
  • the outlet side at the radially inner end of the annular bonding chamber 16 is defined by a circular array of closely spaced cylinders or posts 56 arranged in a form defining a grid outlet structure 36 .
  • the binding chamber 16 may be properly described as a "cage structure" into which motile spermatozoa can enter from the plurality of microchannels 38 on the inlet side, and the spermatozoa bound to the particles are blocked on the outlet side by the plurality of blocking posts 56 Emerging from the cage, the blocking posts 56 have spacings 58 between circumferentially adjacent posts 56 that are sufficiently small to accommodate (ie prevent passage of) sperm-binding selection particles 17 contained in the chambers 16 .
  • the radially inner end portion 52 of the wall 40 separates the microchannels 38 adjoining the bonding chamber 16, and is configured such that (i) the microchannels 38 change towards their respective discharge ends are narrow, and (ii) collectively define a grid boundary structure that inhibits re-entry of motile spermatozoa that have exited microchannel 38.
  • the ends of the microchannels 38 are narrowed in cross-section and provide an inner surface along which motile spermatozoa swim out of the microchannels 38 into the binding chamber 16 in a preferred converging path, due to the narrowing in cross-section, such that It is substantially (but not absolutely) impossible for motile sperm to re-enter the microchannel 38 .
  • the microchannel sidewalls are substantially rectangular in vertical cross-section, the thickness of the ends widens, whereby the microchannels are substantially triangular in plan view at the ends of the walls.
  • the posts 56 may be integrally formed with the upper housing portion 26 by additive manufacturing techniques. 2D and 3F/3G, which function similarly to the shape of the ends 52 of the microchannel-defining sidewalls 40, in a preferred embodiment a plurality of blocking posts 56 are opposed are formed and/or arranged with respect to each other in such a way that motile sperm that have exited the binding chamber 16 are restricted from returning to the binding chamber.
  • the gap (spacing) 58 between the blocking posts 56 needs to be wide enough to allow unbound motile sperm to pass through the binding chamber 16 towards the radially more inward outlet reservoir 18 .
  • the grid structure 36 provided by the circular array of closely spaced posts 56 provides a boundary structure 36 between the bonding chamber 16 and the outlet chamber 18 that does not obstruct the passage of liquid.
  • the gap 58 between adjacent blocking posts 56 is between 10 and 100 ⁇ m.
  • the cross-section of the end of the microchannel 38 needs to be approximately small to accommodate the selection particles 17 in the binding chamber 16 .
  • the shape of the blocking post 56 is not limited to any particular geometry, however, a shape that directs sperm to the outlet reservoir 18 and prevents sperm from re-entering the binding chamber 16 is preferred. Suitable cross-sectional shapes include trapezoid, semicircle, triangle, chevron, semicircle and crescent. An example of a crescent cross-sectional shape is shown in Figure 3I.
  • selection (or binding) particles 17 may be filled into and removed from binding chamber 16 through inlet 32 in upper housing plate 26 connected to chamber 16 .
  • the upper hole of the inlet 32 of the bonding chamber outside the device may be between 0.5 and 5 mm, and the lower hole of the inlet may be between 0.5 and 5 mm.
  • the inlet may be funnel-shaped or cylindrical, but is preferably funnel-shaped and more preferably also tapered to allow better flow of selection particles 17 into binding chamber 16 and reduce the likelihood of clogging during filling.
  • the binding chamber 16 in the device 10 enables sperm that express predetermined, undesired biomarkers outside the sperm outer membrane to be bound in the binding chamber 16 .
  • This can be accomplished, for example, by providing selection particles 17 coated with ligands capable of binding and capturing sperm expressing these predetermined, undesired biomarkers.
  • These markers can be expressed at any point on the sperm anatomy, such as the head, midsection (the mitochondria that house the sperm), and tail.
  • the term "surface marker” refers to, for example, a protein that is transferred to the outer layer of the sperm plasma membrane and interacts with the sperm's local environment.
  • the device 10 which includes a binding chamber 16 in which selection particles 17 are housed.
  • the selection particles 17 may be spherical, preferably microspheres or microbeads.
  • the beads are formulated/manufactured to enable molecular screening of sperm cells based on the presence of conjugated ligands on the surface of the beads.
  • Conjugated ligands can be selected that comprise proteins capable of attaching to labels selected from lectins (eg peanut agglutinin and pea lectin), antibodies and annexin A5 (preferably annexin A5.
  • Ligands may also be are drugs, including imidazoquinolinamines (eg, resiquimod, imiquimod, and gadmodmod).
  • the selection particles 17 may be coated with annexin A5, which is a cellular protein in the annexin group. Annexin A5 is able to bind to outer membrane phosphatidylserine expressed on the surface of motile sperm passing through the device. In other embodiments, selection particles can be coated with antibodies to target specific antigens expressed on the surface of motile sperm.
  • the diameter of the selection particles 17 used may be between 50 ⁇ m and 300 ⁇ m, which will also determine the spacing 59 between the blocking posts 56 in the radially inner grid circular boundary structure 36 of the bonding chamber 16 , and the maximum height/width of the microchannels 38 that provide the radially outer grid circular boundary structures 34 of the combined chamber 16 .
  • the particles are selected to be spherical and between 150 ⁇ m and 200 ⁇ m in diameter.
  • the gap 59 between the pillars 56 is between 6 ⁇ m and 100 ⁇ m. The size and diameter of these columns are factors that determine the required distance between them and the size of the beads used.
  • the shape, size and packing density of the particles 17 in the binding chamber 16 are selected so as not to substantially impede the passage of motile spermatozoa that do not exhibit undesired labels when exiting the binding chamber 16, but need to be large enough to be in the binding chamber 16 Motile sperm exhibiting undesired characteristics are appropriately accommodated.
  • the ligands can be "attached" to the surface of the selection particle 17 (prior to injection into the device 10) using various methods known in the art, and in the present case, the preferred form is by polymer encapsulation. implemented by the method.
  • Ligand polymers typically consist of metal complexes with affinity for proteins and carboxylated bead surfaces.
  • the strong multivalent bonds formed with the electron donating carboxyl groups enable the polymer adhesion layer to remain stably on the surface of the selection particle 17 .
  • the polymer layer utilizes ligands to bind the Ab Fc domain.
  • the selection particle 17 is typically introduced into the binding chamber 16 and suspended in a solution (carrier liquid) suitable for anchoring to one or more molecular entities on the surface of the selection particle 17 .
  • a solution carrier liquid
  • the binding chamber 16 is designed to hold 10 to 1000 ⁇ l of solution, including the microbeads 17 .
  • the distance between the radially outer grid circular boundary structure 34 and the radially inner grid circular boundary structure 36 of the bonding chamber 16 may be between 0.1 and 2 mm.
  • the sperm outlet reservoir 20 is described below as being located radially inward of the radially inner grid circular boundary structure 36 of the bonding chamber 16 . Essentially, it is a cylindrical void in the center of the housing 25, surrounded by a cylindrical array of posts 56, with inlets 30 formed through its material, and the plate-like upper housing portion 26 is formed along the flat cylindrical housing 25. on the central axis. The upper surface of the lower housing portion 22 provides the bottom surface of the reservoir 20 .
  • a chemoattractant such as progesterone, RANTES, lilylaldehyde, p-tert-butylphenylpropanal, atrial natriuretic peptide, hyaluronic acid, or a combination thereof, is introduced into the outlet reservoir 20 through the inlet port 30, which Helps to attract motile sperm that are not bound to the selection particles 17 out of the binding chamber 16 .
  • each oocyte holding structure 24 is adapted to receive and hold an oocyte for fertilization by the combination of motile sperm accumulated in the exit chamber 18 with the oocyte.
  • the preservation structure 18 is advantageously 3D printed during manufacture of the lower housing portion 22 . It can be said that the storage structures 24 collectively define the oocyte storage chamber 20 .
  • Each retention structure 24 is formed by eight arcuate wall sections or posts arranged in a circular, cylindrical fashion with a small spacing between them, but the spacing is large enough to allow motile sperm to enter the egg The interior of the mother cell storage chamber. Although eight arcuate wall sections are shown in this embodiment, in another preferred embodiment, the oocyte storage chamber includes six columns. Likewise, the appropriate spacing between the wall sections is chosen to limit the oocytes housed therein from passing through the gap. Usually, a pitch of 10 ⁇ m to 200 ⁇ m is sufficient, but preferably 30 ⁇ m to 50 ⁇ m.
  • the oocyte retention structure is located below the inlet 30 through which the oocyte will be inserted into the structure, and that the height of the structure may be the same as or greater than the height of the microchannel. However, in a preferred embodiment, the height of the retention structure 24 and the height of the extended sperm outlet chamber itself is between 0.5 and 5 mm.
  • the number of oocyte storage chambers can vary, depending on the quality of the clinical sample and the time required to store oocytes throughout the in vitro fertilization (IVF) cycle.
  • the distance between the oocyte retention structures 24 is typically between 0.1 mm and 1 mm.
  • the sperm outlet chamber 18 and the oocyte retention structure 24/oocyte retention chamber 20 are in fluid communication, whereby the outlet chamber 18 is filled with a suitable, A buffered liquid of viscosity (as are the microchannels 38 of the guide structure 14 and the semen retention chamber 12) to create a virtually no flow of stagnant fluid for the sperm to pass from the semen retention chamber 12, through the guide structure 14 and the binding chamber 16, through the device Various separation and screening areas within 10 swim into sperm holding chamber 18 and oocyte holding structure 24 .
  • the sperm buffer is a hydroxyethylpiperazine ethanethiosulfonic acid (HEPES) based buffer or a sperm cryoprotectant medium.
  • HEPES hydroxyethylpiperazine ethanethiosulfonic acid
  • Figures 9A to 9E show schematically and partly in detail a second embodiment of a device 100 for sperm separation and screening according to the present invention.
  • device 100 In contrast to the device of Figures 2A to 3J, device 100 consists of three stacked housing parts 126, 123 and 122, also fabricated using 3D printing as previously described.
  • the device 100 has a plurality of concentrically stacked microchannel layers 114 (similar to the layers in an onion, see Figures 9E and 9F, rather than a planar layer with a single radially extending microchannel) to provide the sperm guiding structure 14 that Layer 114 extends in a parabolically curved pattern in cylindrical intermediate housing portion 123 .
  • Layer 114 extends between annular semen collection reservoir 112, which is located/defined on the upper region or height of device 100, and annular semen collection reservoir 116, which is disposed on the upper circle.
  • the annular bonding chamber 116 is divided into four separate arcuate regions formed at the lower region or height of the cylindrical intermediate housing portion 123 (see Figure 9F).
  • a cylindrical sperm outlet holding chamber 118 is also formed in the intermediate housing portion 123 below the binding chamber.
  • a bottom circular base plate (bottom shell portion) 122 similar to that shown in Figure 2C carries the oocyte retention structure (shown only schematically at 124 in Figure 9C), which is located in the sperm outlet chamber 118 (thus having the preceding provision). to achieve a dual function) and provide the bottom of the device 100 on which the oocyte-preserving structure is located when in use.
  • FIGD shows the top housing portion 126 in a bottom view to show the radially outer annular sperm receiving reservoir or chamber 112 having two adjacent inlets 128 separated by a continuous web 129 open, the web 129 is connected to a radially inner annular web 131 surrounding a circular port 130, which in turn communicates with a tubular conduit 132 extending through the height of the middle portion 123 of the housing 125 of the device 100, As shown in the translucent portion of Figures 9A and 9B and the plan view of Figure 9E. Conduit 132 communicates with the sperm outlet chamber and allows removal of motile sperm from the chamber if desired.
  • the six-layer structure 114 is shown in dashed outline extending along the height of the intermediate housing portion 123, each layer including a plurality of circumferentially adjacent microchannels 138, while Figure 9E and its enlarged, etc.
  • the scale detail and the enlarged detailed side view of FIG. 9F more clearly show the geometry and layout of the curved microchannel layer 114 .
  • the upper ends of the six microchannel layers 114 all fall within the contours (or confines) of the annular semen sample receiving chamber 112 so that sperm dispensed within the chamber 112 can enter each microchannel 138 toward the upper end of the device 100
  • the bonding chamber 116 at a low level travels.
  • microchannel 138 itself is constructed similarly to the microchannels 38, 38', 38" previously described with reference to Figures 2 and 3 .
  • Device 100 is characterized by different functional units/structures 112 , 114 , 116 and 118 / 120 located at different heights (also referred to as zones) within device housing 125 .
  • the device 100 has within its cylindrical housing 125 a first distinct region including the inlet reservoir 112, a second region including the microchannels of the guide structure 114, a third region including the binding chamber 116, including The fourth region of the outlet reservoir 118 and the fifth region 120 comprising the oocyte storage chamber, note that the third, fourth and fifth regions may preferably be arranged in a common region, which means that the oocytes
  • the holding chamber will be located within the confines of the outlet reservoir, and the outlet reservoir 116 (as is the case in the embodiment shown in Figures 2 and 3) may be at the same height, but surround the outlet chamber 118 radially outward in an annular fashion , and has a similar boundary structure to the previous one.
  • the outlet reservoir is designed as a substantially cylindrical disc chamber, one or more separate wall structures may exist within
  • FIGS. 9A-9F show layer 114 extending within a frusto-parabolic (or similar) envelope
  • a multi-layer 3D channel array may be formed with rectilinear microchannels (rather than in a plane perpendicular to the horizontal plane).
  • Vertically curved microchannels) are defined by frustoconical envelopes. More specifically, as in the first embodiment of the device 10, the regions (whether at the same height or at different heights) are in fluid communication with each other, and buffer solutions fill the various structures to enable (a) motile spermatozoa in the microchannels.
  • motile spermatozoa that have entered the binding chamber 116 and have predetermined characteristics are able to separate from the motile spermatozoa that bind to the selection particles 117 and thus remain in the binding chamber 116
  • unbound motile sperm reaches the outlet reservoir 118.
  • the (unbound) sperm thus screened can then be removed from the outlet reservoir 118 and/or allowed to "swim" into the oocyte storage chamber 120 where it is received with One or more oocytes combine to achieve fertilization.
  • the present invention also provides a method for separating and/or screening motile sperm from a semen sample, comprising the steps of: providing a device 10, 100 according to the first or second embodiment described above ; fill the microchannels 38, 138 of the guide structures 14, 114 and the semen sample inlet reservoirs 12, 112 and motile sperm outlet reservoirs 18, 118 of the device with sperm buffer solution to allow for various functional areas of the device Creating a substantially non-flowing liquid environment within the structure for motile sperm to swim in; introducing a semen sample into the inlet reservoirs 12, 112; waiting for a predetermined period of time; and removing the accumulation in the outlet reservoirs 118, 118 of sperm for further processing or use.
  • a solution containing selection particles 17 is injected into the binding chambers 16, 116 through dedicated inlets prior to introduction of sperm buffer or semen into the device.
  • the solution containing the selection particles 17 is introduced into the device by a continuous slow injection, and the reservoir is filled to the desired percentage by this injection.
  • the device itself can be evacuated and then pre-filled with a buffer solution (to maintain sperm motility and vitality) that can be preheated to a desired temperature, such as room temperature or near core body temperature, to simulate an in vivo environment.
  • a buffer solution to maintain sperm motility and vitality
  • a desired temperature such as room temperature or near core body temperature
  • the device can be placed under controlled atmospheric conditions at room temperature. For example, after injection of a semen sample, the levels of oxygen and carbon dioxide in the atmosphere and humidity can be preset to optimal levels for sperm to travel through the microchannel to the outlet of the collection chamber.
  • the method may also include covering the sperm outlet and selection particle (bead) inlet with closures prior to injecting the semen sample into the annular holding chamber, and exposing only the outlet reservoir prior to placing the oocyte.
  • the closure may be a lid, tape, special plug or snap-on lid.
  • a method for in vitro fertilization of oocytes using isolated and screened motile sperm obtained from a semen sample comprising the steps of: providing The device of the present invention; fills the microchannel or guide structure, as the case may be, with sperm buffer solution, and the inlet and outlet reservoirs of the device; fills the binding chamber with selection particles having a Selective, the motile sperm have predetermined undesired characteristics associated with oocyte fertilization, such as the expression of apoptosis-related markers, the expression of sex-selective-related markers, or the presence of a permeable membrane that binds motile sperm to particles Confinement in the binding chamber without impeding the passage of motile sperm that are not bound to the selection particle; introduce the oocyte into the oocyte preservation chamber; introduce the semen sample into the inlet reservoir; wait for a period of time for the selected motile Sperm accumulates in the outlet
  • naked oocytes are pipetted directly into the oocyte storage chamber/conservation structure 24, 124 of the device 10, 100.
  • the placement of the oocytes is preferably performed after the device is filled with a solution containing selection particles (if used), sperm buffer and semen sample.
  • the outlet reservoir can optionally be sealed with mineral oil and the oocyte storage chamber covered.
  • chemoattractants may assist in directing unbound sperm out of the selection particle holding/binding chambers 16, 116 and into the oocyte storage area of the device.
  • the chemoattractant can be added to the middle of the outlet as a concentrated droplet before or after oocyte placement. This allows the chemoattractant to diffuse outward in the gradient, providing the sperm with a chemical gradient to follow.
  • Figure 4 schematically illustrates a potential fabrication method using 3D printing techniques.
  • the process includes 3D printing, cleaning the resin material, curing the resin material, and attaching the associated housing parts 22, 26, 122 of the device 10, 100 with double-sided tape (if the device is desired to be removable) or with a permanent adhesive when pressure is applied. , 123, 126 for bonding.
  • FIG. 5 shows an example of the production process of protein-coated microbeads used as selection particles 17 .
  • microbeads polystyrene cores in this case
  • the conjugated beads are added to the second module assembly along with the antibody or protein solution in a second mixing module to produce fully coated beads.
  • Antibody-conjugated microbeads are now capable of binding externally expressed surface markers, such as those found on sperm.
  • sperm cells were firmly attached to the protein-conjugated microbeads through protein-protein interactions.
  • FIG. 6 schematically illustrates an exemplary method for loading microbeads (selection particles) into the binding chamber (reservoir) 16 of the device 10 .
  • the method includes steps such as resuspending the bead solution, pipetting the solution and injecting the solution into the binding chamber inlet (in a smooth manner).
  • the volume of the bead reservoir can be customized to accommodate different solution volumes. For example, 200 to 300 ⁇ l of the solution can be injected. Additionally, the volume of the reservoir can be adjusted to ensure that the beads adequately fill the area of the chamber.
  • the concentration of beads used in the bead solution depends on the size of the beads. Solution concentration and volume can be adjusted according to the desired sample and size.
  • An example of this process involves injecting a high concentration of microbeads into the chamber to completely fill the chamber or reservoir.
  • the end of the flow chart also shows a clear image of the bead reservoir 16 loaded with the protein-coated beads 17 and the boundary structures 34 and 36 described with reference to FIGS. 2 and 3 .
  • Figure 7 is a schematic flow chart showing the process of performing a method of isolating sperm and fertilizing oocytes using the device 10 described above. This includes filling the device 10 with sperm buffer in a vacuum chamber, loading the semen sample into the semen reservoir 12, dripping a chemoattractant into the central outlet 18 (see accompanying illustration for its role), and finally removing the oocyte Placed into a holding structure 24 located within the oocyte holding chamber 20 surrounded by the annular sperm outlet chamber 18 .
  • the addition of chemoattractants can enhance the guidance of sperm traveling toward the oocyte storage chamber 20 through a chemical gradient, prompting sperm redirection in response to changes in concentration.
  • Figures 8A, 8B and 8C show performance measurements (performance in sperm recovery, motility and vitality) using the device 10 fabricated according to the illustrations of Figures 2 and 3, according to an exemplary embodiment of the present invention.
  • the concentration of sperm recovered from the sperm collection chamber of the device is typically time, the length of the sperm travel path from the semen storage chamber through the guide structure, the binding chamber and finally into the sperm collection (or exit) chamber, and the starting concentration of the semen sample The function.
  • sperm are separated primarily based on motility, and in the binding chamber, negative selection is performed by binding and retaining "defective" or “undesired” sperm in the binding chamber.
  • the mean concentration (mL) of sperm recovered is directly related to the starting concentration of the original semen. Even among different samples from the same person, the concentration of human raw semen can vary widely. Sperm recovery increased with increasing sample incubation time within the device and resulted in sperm concentrations higher than those required for conventional in vitro fertilization.
  • sperm motility at the exit was higher than 95%, while viability was close to 100%, but sperm lost energy when placed for a long time. This shows a significant increase in the motility and vitality of the recovered sperm population compared to the original semen; in multiple tests, the motility was over 95% and consistently close to 100%. Due to the hybrid screening mechanism, only sperm cells that are highly motile and healthy are recovered from the device. The percentage of viable sperm cells throughout the device remained high over time, also indicating the biocompatibility of the 3D printing material used in this example.
  • Figure 8D shows the results of DNA fragmentation of untreated primordial spermatozoa compared to spermatozoa screened using the device according to the present invention. The results showed that sperm screened by the device exhibited a lower DFI compared to untreated sperm, suggesting that the device was able to screen higher quality sperm with a greater chance of conception.
  • channel entry may include more than one channel entry.
  • Ranges provided herein should be understood as shorthand for all values within that range.
  • the range 1 to 50 should be understood to include any number, combination of numbers, or subrange from the group of 1, 2, 3, 4, etc. to 48, 49, or 50.
  • Muratori M., Tarozzi, N., Cambi, M., Boni, L., Iorio, A.L., Passaro, C., Luppino, B., Nadalini, M., Marchiani, S., Tamburrino, L., Forti , G., Maggi, M., Baldi, E. & Borini, A. 2016, "Changes in DNA Fragmentation Levels During Density Gradient Sperm Screening for Assisted Reproductive Technology", Journal of Medicine (USA), Vol. 95, Issue 20, pp. 1-9.
  • Muratori M., Tarozzi, N., Cambi, M., Boni, L., Iorio, A.L., Passaro, C., Luppino, B., Nadalini, M., Marchiani, S., Tamburrino, L., Forti ,G.,Maggi,M.,Baldi,E.&Borini,A.2016,'Variation of DNA Fragmentation Levels during Density Gradient Sperm Selection for Assisted Reproduction Techniques',Medicine(United States),vol.95,no.20, pp.1–9.
  • Muratori M., Tarozzi, N., Carpentiero, F., Danti, S., Perrone, F.M., Cambi, M., Casini, A., Azzari, C., Boni, L., Maggi, M., Borini , A. & Baldi, E. 2019, 'Sperm selection with density gradient centrifugation and swim up: effect on DNA fragmentation in viable spermatozoa', Scientific Reports, vol.9, no.1, pp.1–12.

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Abstract

一种用于从精液样本中分离活动精子的装置和方法，该装置包括用于接收精液样本的入口储液器；用于收集从样本中分离的精子的出口储液器；以及至少一个但优选多个设置在入口储液器和出口储液器之间的微通道，以提供两个储液器之间的液体连通，该微通道包括至少一个壁，该壁限定了边界表面，精子沿着该边界表面导向出口储液器；并且其中该至少一个壁具有至少一个表面结构，该表面结构选自诸如一个或多个凹部、凸部及其组合，该表面结构在微通道的至少一部分长度上延伸，从而提供至少一个额外的边界表面，活动精子沿着该边界表面向出口储液器行进。

Description

一种从精液样本中分离精子的装置和方法 技术领域
本发明涉及一种用于从精液样本中分离精子的装置和方法，尤其涉及用于从分离的精子中选择具有预定特征的精子的装置和方法。
背景技术
精子筛选是辅助生殖技术(ART)的重要组成部分，对于生殖治疗的成功率和胚胎的健康都有很大影响(Davies，2012年和Mcdowell，2014年)。生殖治疗和生物学的大部分焦点都围绕着卵子获取和胚胎培养，然而自从1973年成功实现体外受精(IVF)以来，雄配子的健康及其分离策略的重要性就被严重低估了。
最近有研究(Oseguera-López，2019年和Sakkas，2015年)描述了精子在传递第二组染色体之外，其重要且复杂的作用。然而，目前的临床精子筛选技术(其中最常用的是密度梯度离心(DGC)和上游法(SU)自1978年以来几乎没有创新。这些方法中的一些受到许多限制，例如一些方法会引入活性氧，耗时且成本高。精子筛选过程也很大程度上依赖于胚胎学家的技术和非标准化的操作方案，这样通常就导致在不同操作者之间的体外受精成功率存在差异(Kashaninejad，2018年)。这种差异在精子处理阶段最为明显，该阶段通常包括三次或更多次梯度离心、细胞重悬和精细等分(世界卫生组织，2010年)。
现有的微流控精子筛选策略仍然只是复刻了DGC和SU技术，在通量或选择性方面没有明显优势。目前使用的这些技术绕过了自然条件下的精子筛选过程，主要根据精子的活动力或形态来选择精子，而忽略了其他可能更重要的因素，例如DNA完整性、精子凋亡程度和质膜成熟度。事实上，DGC和SU都无法降低精子DNA碎片化(sDF)，甚至在某些样本中会导致精子DNA碎片化(Robinson，2012年和Oleszczuk，2013年)。
DGC和SU技术中存在这种缺陷的原因在于：它们在离心过程中往往会产生活性氧，研究表明，这些活性氧可能通过氧化DNA加合物和使DNA碎片化而损害精子功能(Rappa，2016年和Aitken，2013年)。由于这些精子筛选技术会对精子造成损害(Esteves，2015年；Muratori等，2016年，2019年)，并且在很大程度上取决于技术人员在选择精子方面的经验，这使得该过程容易出错，直接影响到受精率。在2012年，澳大利亚的临床成功率仅在4％至30.9％之间(Wade，2015年)。此外，这使得选定的精子有可能出现形态正常、适合体外受精(IVF)但存在明显DNA损伤的风险。尽管这些技术得到了广泛的应用(特别是DGC)，但近年来人们已经从认可其适用性转变成意识到其不足之处。
在其他当前技术中，由于样本(例如通过睾丸手术获得的样本)有时间限制，无法进行实际的精子分离，而只是进行精子纯化。精子纯化过程通常更为耗时；从睾丸样本中筛选精子是一项人工操作，通常需要2-3个小时，并且只能清除精子中的精浆(世界卫生组织，2010年)。
这意味着这种精子制备方法和当前其他的精子制备方法可能无法从样本中挑选出最有生育能力的精子。DGC通常在30分钟内从0.5mL的原始精液中选出多达约36％的精子群，而上游试验通常在1小时内从1mL的精液中选出约12％的精子群，使得精子活动力分别提高18-19％和5％(世界卫生组织，2010年)。
美国专利2015/0140655 A1号专利文献描述了一种用于从精液样本中分离精子的方法和装置。该装置包括用于精液样本的外部环形室、多个辐条状微通道(即这种通道的径向阵列或网络)、以及收集已经穿过微通道阵列的精子的中心室。这种装置的功能通过自主游动的精子(活动精子)游过微通道中有效停滞的液体(可以是相对高粘度的液体)来实现，并且利用了精子在受限空间中的自然游动行为，例如精子的表面积聚行为和沿边界游动行为。这种贴壁游动的行为在下文中简称为“微流体结构中精子的壁游行为”。考虑到所涉及的几何形状，从环形精液样本 室开始的微通道以级联的方式连接在一起，使得排入中心室的微通道的数量减少。也就是说，在微通道路径的入口和出口之间至少有一个基于精子的壁游行为引导精子的连接点。
因此需要一种能够改进美国专利文献‘655中公开的，从精液样本中分离精子的装置和方法，并且具有可接受的产率。
还需要一种装置和方法，既能够从精液样本中分离精子，同时减少在处理阶段对精子可能造成的损伤。
还需要一种改进的装置和方法，能够通过基于运动的分离过程(如前述美国专利文献‘655中所述)从精液样本中选取精子，以产出具有某些(或可限定的)所需特征的活动精子。本发明解决了一个或多个这些未满足的需求。
在本说明书中，对任何先前出版物(或由其衍生的信息)或任何已知物质的引用均不是也不应视为本文认可或确认或以任何形式暗示先前出版物(或由其衍生的信息)或已知物质构成本说明书所涉及的技术领域中的公知常识的一部分。
发明内容
根据本发明的第一个方面，提供了一种用于从精液样本中分离活动精子的装置，该装置包括用于接收精液样本的入口储液器；用于收集从样本中分离的精子的出口储液器；以及至少一个但优选多个设置在入口储液器和出口储液器之间的微通道，以提供两个储液器之间的液体连通，该微通道包括至少一个壁，该壁限定了边界表面，精子沿着该边界表面导向出口储液器；并且其中该至少一个壁具有至少一个表面结构，该表面结构选自诸如一个或多个凹部、凸部及其组合，该表面结构在微通道的至少一部分长度上延伸，从而提供至少一个额外的边界表面，活动精子沿着该边界表面向出口储液器行进。
在一些实施例中，该装置包括壳体，该壳体优选由聚合物材料制成，例如环烯烃共聚物(COC)、聚碳酸酯(PCA)、聚甲基丙烯酸甲酯(PMMA)、聚二甲基硅氧烷(PDMS)或在增材制造技术中使用的光敏聚合物树脂，该增材制造技术可有利地用于制造本发明的装置(但不排除其他制造技术，例如铸造、模制和机械加工)，该壳体设计成整体成型或者完全包住入口储液器，该出口储液器和一个或多个微通道在入口储液器和出口储液器之间延伸，壳体中的至少一个端口布置成将精液样本供应到入口储液器中，并且至少一个取出端口用于从出口室移除从精液中分离的精子。
在下文中，将主要参考具有多个所述微通道的实施例，应当理解，具有单个或少量微通道的实施例也属于本发明的构思范围内。
各个微通道的横截面可以是恒定或可变的，例如正方形、矩形、圆形、椭圆形等。应当理解，当微通道的横截面为圆形或椭圆形时，将有一个环形壁限定一个邻接的边界表面，而在矩形横截面的情况下，有四个主边界表面，这四个主边界表面在邻接壁相交的平面处限定有尖角(或圆角)。表现出壁游行为的精子将趋向于向边界表面移动并沿着边界表面移动。
在微通道的其他平面壁中设置有额外的表面结构，例如一个或多个凹部、一个或多个凸部以及一个或多个凹部和凸部的组合，其在微通道的至少一部分长度上延伸，旨在增加精子可以沿其行进的微通道路径的边界表面的数量。如上所述，精子的运动受到与其相互作用的表面的影响，并且精子通常表现出壁游行为，或者当处于微流体通道等受限的通道几何形状中时，精子趋向于向更靠近边界表面的方向移动。与微通道壁为光滑或平坦的轮廓(如美国专利文献‘655中所述)相比，能够提供或形成额外边缘和边界表面的微通道壁的轮廓更能促进基于活动力为标准的精子分离。
应当理解，美国专利文献‘655的装置采用了这样的构思：按顺序将径向微通道网络的径向外部区域中最初数量较多的微通道连接起来，使得它们的数量沿径向内部排出区域减少，从而寻求保持基本为矩形横截面通道的宽度和高度(即横截面几何形状)大致恒定，以便完全基于壁游行为的方向性来分选精子。
相比之下，根据本发明第一个方面的装置并不寻求基于微通道范围内的行进方向来区分精子，而是改变微通道的几何形状以将额外的边界表面包括在内，精子将基于其壁游行为沿着该边界表面无区别地游动。与活动力随行进距离降低的精子相比，具有受精所需特性的精子通常会行进更远的距离，并且边界表面的增加使更多具有所需特性的精子能够到达出口储液器，然后在出口储液器处可以对精子进行收集或进一步处理。
此外，如上所述通过在微通道中提供额外的表面结构，与光滑壁通道相比，有可能增加各个微通道的最大横截面尺寸，而不减损寻求满足活动精子的壁游行为的通道的微流体性质，因为增加的表面结构在通道边界内提供了额外的边界表面，以便活动精子基于其壁游行为行进。
表面结构的构型或形状不限于任何特定的几何形状，只要该结构可以形成用于引导精子额外的边界表面，许多几何形状都可以实现该目的，如下文所述。另外，可以控制精子游动边界的数量以调节精子的通过量。
从制造的角度来看，该结构可以包括一个或多个诸如矩形、半圆形或阶梯形横截面的凹槽、肋及其组合，这些结构从入口到出口在微通道的整个长度上延伸，或者仅沿着其一部分延伸。
凹槽/肋在微通道内限定了从入口储液器到出口储液器的额外表面路径，精子可以基于壁游行为沿着该路径行进。这些结构可以沿着微通道的部分或整个长度沿着一个或多个轴向呈直、锯齿状、波浪状或其组合的路径延伸。凹槽的深度/肋的高度可以是恒定的或可变的。凹槽/肋的横截面形状或轮廓可以选择适合的，只要沿着其延伸部存在至少一个带有通道壁的尖锐或圆形边缘，该边缘超出了由光滑壁通道限定的边缘。
通过微通道的阶梯形侧壁可以提供深度或高度不连续的表面结构。这是为了在微通道侧壁中增加更多边界表面，以使精子在通过微通道时得以沿着行进。
在一个优选实施例中，该装置设计成使得多个微通道在壳体内呈辐条状从环形径向外部室(可以将其细分成具有单独的精液样本入口的段)形式的入口储液器延伸到圆形径向内部出口室形式的出口储液器。
相邻的微通道之间可以共享一个共同的分离侧壁，并且提供额外边界表面的表面结构可以存在于横截面为矩形的微通道的一个或两个侧壁中。当然，替代地或附加地，在这种矩形截面微通道的底部和顶部边界壁的一个或两个中也可以具有表面结构。
其他微通道布置模式也可以采用，不排除其他微通道布置模式，例如，多个微通道在微通道相对端的入口室和出口室之间彼此平行延伸(与径向网络相比)，或者多个微通道可以从径向外部环形入口室朝向径向内部圆柱形出口室呈螺旋状布置。
此外，微通道不需要沿直线延伸，而是可以在入口和出口储液器之间呈蛇形或曲折状布置。
为了在可接受的(较短的)收集时间范围内产出活动精子，该装置的微通道个数为400至700个，更优选为100至500个，但微通道的数量范围最少可以是5个，最大可以至5000个。
根据已有微通道的数量和待从精液样本中收集或分离的精液种类，多个微通道的宽度可以在约10μm至约5000μm之间，优选地在50μm至250μm之间，并且高度在约10μm至约5000μm之间，优选地在100μm至1000μm之间。优选地，所有微通道的宽度和高度中的一个或两个是相同的。
通常，微通道的长度在约0.1mm至15mm之间，优选地在约5mm至9mm之间。
此外，特别是在微通道呈径向汇聚布置的实施例中，起始于装置的入口储液器端部的两个或更多个微通道可以沿着它们的路径接合以在出口储液器处形成单个微通道。
还应当理解，微通道可以单层或多层堆叠布置在装置壳体内，微通道的起点在共同的或单独的入口室中，并排入到共同的出口室中。
根据本发明的第二个方面，提供了一种用于从精液样本中筛选具有确定特征的活动精子的装置，该装置包括： 入口储液器，用于接收精液样本；结合室，设置成通过引导结构与入口室在下游流体连通，该引导结构设计成将活动精子从精液样本导向结合室，优选基于活动精子的壁游行为进行导向；以及与结合室在下游流体连通的出口储液器，该出口储液器设计成接收已经通过结合室的活动精子；其中该结合室用于容纳选择颗粒，选择颗粒具有与以下活动精子结合的选择性：(i)已经穿过引导结构，并且(ii)具有预定的、非期望的标记，结合室和/或选择颗粒设计成基本上将与颗粒结合的精子限制在结合室中，并且防止精子进一步向出口储液器行进，而不会阻碍未结合到选择颗粒的活动精子的通过。
在一些实施例中，引导结构将有利地采用参照本发明的第一个方面所描述的形式，即包括多个在入口储液器和结合室(而不是出口室)之间延伸的微通道。
在一个实施例中，结合室的入口侧以多个微通道壁的下游端为边界(因此提供了格栅式入口结构)，出口侧由一系列间隔开的列或柱限定，所述列或柱以限定格栅式出口结构的构造布置，因此结合室是“笼形结构”，活动精子可以从多个微通道进入该笼形结构，并且通过多个阻塞柱阻止与颗粒结合的精子从该笼形结构流出。在一些实施例中，将与结合室邻接的相邻微通道分隔的壁的末端部分被有利地构造成使得(i)微通道朝着其各自的排出端变窄，并且(ii)共同限定格栅式边界结构，该格栅式边界结构抑制已经离开微通道的活动精子重新进入微通道。换句话说，微通道的末端横截面变窄，并提供活动精子可以沿其行进的内表面，活动精子沿着该内表面以优选的汇聚路径从微通道行进到结合室中，由于横截面变窄，使得活动精子基本上(但不是绝对)不可能重新进入微通道。例如，假设微通道侧壁在垂直截面上基本上为矩形，末端在厚度上变宽，那么在壁末端的平面图中基本上为三角形。
在一个优选实施例中，以功能上类似于微通道限定侧壁末端成形的方式，将多个阻塞柱彼此成形和/或布置以限制已经离开结合室的活动精子重新返回到结合室。阻塞柱之间的间隙需要足够大，使未结合的活动精子能够通过结合室流向出口储液器。同时，阻塞柱之间的间隙需要足够小，才能将选择颗粒容纳在结合室内。在优选实施例中，相邻阻塞柱之间的间隙在10至100μm之间。同样，微通道在其末端的横截面需要同样小，才能将选择颗粒容纳在结合室中。
在一些实施例中，这些柱的横截面形状是人字形、半环形或新月形，并且这些柱布置成不会阻碍柱之间朝向出口室的通道，但仍可以抑制活动精子从出口室返回到结合室中。
选择具有合适形状、大小和堆积密度的选择颗粒加入结合室中，使其基本上不会阻碍活动精子的通过，这些活动精子在游出结合室时不会表现出非期望的标记，但选择颗粒的形状、大小和堆积密度应足够大以在结合室中适当地容纳表现出非期望的特征的活动精子。
根据本发明的多个实施例，包含在结合室中的选择颗粒包括与配体缀合的小球，特别是微球或微珠，所述配体包括能够附着到标记上的蛋白质，标记选自凝集素、抗CRISP(富含胞嘧啶的分泌蛋白)、膜联蛋白A5、抗趋化因子受体抗体和抗CD63、抗CD9、ALIX或TSG101m，或者配体可以是包括瑞喹莫德(resiquimod)、咪喹莫特(imiquimod)和嘎德莫特(gardiquimod)在内的药物。优选地，配体是膜联蛋白A5，其以化学方式结合活动精子表达的标记。
当在本发明的实施例中使用时，术语“配体”是指与生物分子形成复合物以在生物化学通路中起作用的物质。例如，配体可能是一种当结合到特定的细胞位点时会触发细胞反应的分子。在其他示例中，配体可以是具有能与结合位点相互作用的化学结构的离子或蛋白质或任何其他合适分子。
在一个优选实施例中，配体是蛋白质，优选膜联蛋白A5，通过配体可以在结合室中结合和捕获在精子外表面表达磷脂酰丝氨酸的精子。从而阻止了这些表达磷脂酰丝氨酸的精子进一步朝着出口储液器行进，而不表达磷脂酰丝氨酸的精子则能够基本上不受阻碍地通过结合室朝着出口储液器行进，然后在结合室可以对其进行收集或进一步处理。
根据本发明的另一个方面，该装置还包括与出口储液器流体连通或作为出口储液器一部分的卵母细胞保存室。 可以视情况选择一种结构，用于调节筛选的(即未与颗粒结合的)活动精子从出口储液器进入卵母细胞保存室的通道。卵母细胞可以直接引入卵母细胞保存室，使得从样本中分离出来并在通过结合室的过程中“筛选”到的活动精子可以与卵母细胞结合实现受精。在一些实施例中，可以将化学引诱物施加到出口储液器和/或卵母细胞保存室。化学引诱物有助于将分离和筛选到的精子导向卵母细胞。
在本发明装置的一种形式中，在装置壳体内划分了不同的功能单元或结构。例如，该装置可以在其壳体中具有包括入口储液器的第一区域、包括微通道或引导结构的第二区域、包括结合室的第三区域、包括出口储液器的第四区域和包括卵母细胞保存室的第五区域。第三区域和第四区区域可优选为并置，这意味着卵母细胞保存室将位于出口储液器的范围内。例如，当出口储液器设计成基本上圆柱形的圆盘室时，其中可以存在一个或多个单独的壁结构，每个壁结构用于容纳卵母细胞，但同时活动精子能够通过壁结构以接触卵母细胞。
在其他实施例中，其中一些区域相对于基准平面或参考平面(例如，使用时放置该装置的支撑表面)处于不同的高度上，第一区域(例如呈环形室形式的入口储液器)可以位于与第二区域(微通道)相同的高度上或位于第二区域(微通道)上方或下方，例如，在相同高度上具有径向向内引导通道的环形外部入口室，其末端位于相同高度上的环形结合室或出口室。在另一个实施例中，第二区域(微通道)可以位于与第三区域(即也可以是呈环形室形式的结合室)相同的高度上。然而，在第一区域和第三区域位于不同高度的实施例中，微通道(即第二区域)则位于这两个高度之间，因此通道本身将相对于水平面倾斜延伸。
在一个实施例中，微通道呈径向阵列设置，入口储液器是在环形结合室或出口室上方的区域中的环形室，3D通道阵列将由截头圆锥(直通道)封套或截头抛物面(或类似的)封套限定，且通道在垂直于水平面的平面上具有弯曲。第三区域(结合室)可以位于与第四区域(出口储液器)相同的高度上，而第四区域(出口储液器)可以位于与第五区域(卵母细胞保存室)相同或位于第五区域上方。
更贴切地说，这些区域(无论处于相同还是不同的高度)彼此流体连通，缓冲溶液填充各种结构，以使得(a)活动精子能够在微通道或引导结构内从精液样本中分离出来，(b)已经进入结合室并具有预定特征的活动精子能够与结合到选择颗粒并因此保留在结合室中的活动精子中分离开来，以及(c)未与颗粒结合的活动精子能够到达出口储液器。然后，这样筛选到的(未与颗粒结合的)精子可以从出口储液器中取出和/或能够“游”到卵母细胞保存室中，在那里它们可以与卵母细胞保存室中容纳的一个或多个卵母细胞结合实现受精。
在一些实施例中，还可以提供进入各个室的端口，以便能够从结合室取出与颗粒结合的精子，从出口储液器和/或卵母细胞保存室取出未与颗粒结合的精子，例如使用移液管或其他已知的体外受精输出/取出仪器。
根据本发明的另一个方面，提供了一种从精液样本中筛选和/或分离活动精子的方法，包括以下步骤：根据具体情况(即仅从精液样本中分离活动精子，或者分离并筛选活动精子)，提供根据本发明前述第一个或第二个方面的装置；用精子缓冲溶液填充微通道或引导结构(视情况而定)以及装置的入口储液器和出口储液器，以产生基本上无流动的液体，使活动精子可以在该液体中行进；以不引起或最小化装置中液体流动的方式将精液样本引入入口储液器，例如同时移除一部分缓冲溶液或者仅在一定程度上预填充相关的室和微通道；等待一段预定的时间，在此期间活动精子将穿过装置的各个区域进入出口室；以及回收出口储液器中积累的精子。
根据本发明的另一个方面，提供了一种利用从精液样本中分离和筛选的活动精子使卵母细胞体外受精的方法，该方法包括以下步骤：提供根据本发明第二个方面的装置；视情况用精子缓冲溶液填充微通道或引导结构，以及装置的入口储液器和出口储液器；用选择颗粒填充结合室，选择颗粒具有与活动精子结合的选择性，活动精子具有与卵母细胞受精相关的预定的非期望的特征，例如凋亡相关标记的表达、性别选择性相关标记的表达或具有渗透性膜，从而将与颗粒结合的活动精子限制在结合室中，而不会阻碍未结合到选择颗粒的活动精子的通过；将一个或多个卵母细胞引入卵母细胞保存室；将精液样本引入入口储液器；等待一段时间，以使筛选到的活动精子(即未与选择颗粒结合的活动精子)积累在出口储液器中；允许活动精子从出口储液器进入卵母细胞保存室；以及允许所筛选的活 动精子有足够的时间与容纳在卵母细胞保存室中的卵母细胞结合受精。
与本发明中定义的微通道壁的“表面轮廓”相关的方面，以及包含能够与“非期望的”精子结合的选择颗粒的结合室，可以添加到Nosrati等人的专利文献US 2015/0140655 A1中说明的各种精子分离装置中，该专利文献公开了基于精子在受限空间中的壁游行为来提供用于引导精子的多个微通道路径的基本原理，以及这种微通道的典型尺寸、微通道网络内限定的防回流室和储液器的类型。相应地，美国专利文献‘655的内容通过简短的交叉引用将其整体并入本文，但不因此说明该文献中提出的所有理论实际上都能得到证据支持。
通过参考以下附图提供的本发明的各种非限制性实施例的描述，本发明的各个方面的其他方面和其他特征将变得更加清楚。
附图说明
图1以高度示意性的简化图示出了根据本发明的精子筛选装置，并以概括图示出了在根据本发明的另一个方面执行精子筛选方法时，在装置操作过程中执行的精子筛选步骤；
图2A至2F示意性且部分详细地示出了根据本发明的精子分离和筛选装置的第一实施例；其中，
图2A是该装置的顶部壳体部分的俯视透视图；
图2B是顶部壳体部分的仰视透视图，示出了多个呈辐条状的微通道精子导管，其从径向外部环形精子接收储液器或室向径向内部环形精子筛选(或结合)室延伸，该室又与基本上为圆柱形的径向更靠内的活动精子出口(或收集)储液器或室邻接；
图2C是与顶部壳体部分结合的下部底部壳体部分的俯视透视图，根据突出的详细视图，在径向中心区域包含九个单独的卵母细胞保存结构，当两个壳体部分如图2E示意性示出的那样放在一起时，顶部壳体部分的精子收集室在径向最中心位置容纳卵母细胞保存室并与其流体连通；
图2D是将顶部壳体部分放大的突出详细视图，示出了(i)微通道的径向内端，其为筛选(或结合)室提供了圆形格栅边界结构，(ii)筛选室和(iii)筛选室中由多个紧密间隔的阻塞柱形成的径向内部圆形格栅边界结构，以及(iv)径向更靠内的圆形精子出口储液器；
图2E是沿装置直径的横截面，其中上半部分和下半部分彼此分开；
图2F是描绘图2B或图2D的装置的示意性显微镜图示(放大图)，其从左到右具有(i)从径向外部环形精子接收储液器或室延伸的呈辐条状的多个微通道精子导管，(ii)径向内部环形精子筛选(或结合)室，(iii)筛择室中由多个紧密间隔的阻塞柱形成的径向内部圆形格栅边界结构，以及(iv)径向更靠内的圆形精子出口储液器；
图3A至3I示意性且部分详细地示出了根据本发明的用于精子分离和筛选的装置的另一实施例，该装置具有100多个微通道；
图3A是类似于图2B的顶部壳体部分的仰视透视图，但示出的是根据本发明的装置的另一个实施例，其示出了从径向外部环形精子接收储液器或室向径向内部环形精子筛选(或结合)室以径向模式延伸的多个微通道精子导管，精子筛选(或结合)室又与基本上也是环形的径向更靠内的活动精子出口(或收集)储液器或室邻接；
图3B和3C是图3A中突出显示的详细部分的不同透视图，示出了根据本发明另一方面的微通道的实施例，该微通道包括在相对的侧壁中呈肋和凹槽的形式的结构，该结构在微通道的一部分长度上延伸并且增加了边界表面的数量；
图3D示出了图3B和3C中所示的微通道的相对侧壁的正对表面的多种其他结构形式中的三种，通过在每个侧壁处形成表面结构从而额外增加了活动精子可以基于壁游行为沿其行进的边界表面的数量；
图3E是类似于图3B的视图，但是进一步放大以更好地说明，相比于邻接的径向向内区域，微通道侧壁在径向外端区域内是如何成形的，从而为活动精子基于壁游行为的行进提供额外的边界表面；
图3F至3I是径向内部多柱格栅障碍物结构的四个不同实施例的示意性俯视图和详细透视图，该障碍物结构形成了环形结合(或筛选)室的格栅边界之一；
图3J至3L示意性且部分详细地示出了根据本发明用于精子分离和筛选的装置的另一实施例，该装置具有45个微通道，其中所示特征被进一步(但不是按比例)放大，以更好地示出该实施例的壁结构和壁插入物；
图4示意性地示出了根据本发明一个方面的各种实施例的装置的制造过程的实施例，该制造过程使用了增材制造技术，即3D打印；
图5A示意性地示出了根据本发明该方面的一个实施例的选择(结合)颗粒的制备步骤，在使用图1、2、3和4的装置时，该选择(结合)颗粒将以蛋白质包被的微珠的形式被接收并包含在装置的环形结合室中；
图5B是显微镜示意图(放大图)，描绘了通过蛋白质与蛋白质之间的相互作用，使精子细胞附着到用图5A所示方法制造的蛋白质缀合微珠上；
图6示出了根据本发明一个实施例中用于将微珠形式的选择颗粒装入图1至4所示装置的环形结合室的方法步骤；
图7以说明性流程图的方式示出了根据本发明的一个实施例的方法，该方法首先从精液样本中分离活动精子，然后通过选择性结合“非期望的”活动精子来选择具有所需生育力指标的活动精子，允许“需要的”活动精子通过根据图1至4所示装置的结合室；
图8包括体现了本发明装置的性能测试的三个图表，从精子回收量、精子活动力和精子生命力的方面来测试本发明装置的性能，其中图8A是精子浓度随时间变化的图表，插图8B是精子出口计数随时间变化的图表，图8C是精子活动力和生命力随时间变化的图表，图8D是用装置进行精子筛选前后DNA碎片化指数的图表；以及
图9A至9E示意性且部分详细地示出了根据本发明的用于精子分离和筛选的装置的第二实施例，与图2A至2E的装置相比，该装置具有堆叠的微通道阵列，该微通道以抛物面状弯曲的方式在位于装置上部区域或水平的环形精液收集储液器和环形结合室之间延伸，该结合室分成四个独立的弧形区域，位于该装置的中间较低区域或高度上，具有类似于图2C所示的底盖，该底盖带有卵母细胞保存室(未示出)，该底盖形成在使用时该装置承放该装置的底部其中，
图9A是该装置的示意性透视图，包括三个堆叠的壳体部分，示出了在上部和基部壳体部分之间沿着基本上为圆柱形的中间壳体部分的高度延伸的微通道(以虚线轮廓示出)，该上部和基部壳体部分基本上与图2B所示的顶部壳体部分成镜像，但是没有微通道和图2C的底部壳体部分；
图9B的立体分解图示出了图9A装置的三个分别由3D打印制造的壳体部件；
图9C是图9A的装置的局部剖切侧视图；
图9D是顶部壳体部分的仰视图，示出了径向外部环形精子接收储液器或室，其具有两个相邻的入口，入口由连续的腹板分开，腹板与围绕圆形端口的径向内部环形壁连接，圆形端口又与管状导管连通，管状导管延伸穿过装置壳体的中部高度位置，如图9A和9B的半透明部分以及图9E的平面图所示；
图9E是中间壳体部分的俯视图，具有放大的示意性详图，该详图示出了同心的四分之一弧的分层区域中的微通道阵列，由此微通道被壳体腹板分开，并且沿着壳体部分的上端之间基本上为抛物线的路径延伸，该路 径从上壳体部分的相邻的精液样本接收储液器开始，朝向环形的、四分之一弧的精液结合室，该精液结合室整体形成于中间部分的下端面中并凹入该下端面，如图9F示意性所示；
图9F是中间壳体部分的仰视图，其中另一个放大的示意性详图是沿着所示线截取的截面图，示出了在中间壳体部分的顶面和底面之间延伸的导管的下端，该下端延伸到扩大的、活动精液的出口室，在该出口室中定位了卵母细胞保存室，该卵母细胞保存室位于图9C中示意性示出的底部封闭板的上表面。
具体实施方式
以下提供了本发明实施例的详细描述，以帮助本领域技术人员实施本发明的各个方面。
本文所述的装置、分离精子的方法、筛选精子的方法和受精的方法的各种实施例可以在生育诊所或研究中心实施，以研究在体外受精(IVF)、子宫内授精(IUI)和卵胞浆内单精子注射(ICSI)应用方面男性不育症的治疗。本文公开的信息可用于分离活动精子，并随后筛选精子用于IVF、ICSI、IUI和/或其他生殖治疗。该装置和方法及其变体和方法也可用于非人类应用，特别是农业，如牛和其他动物育种的辅助生殖技术(ART)治疗。
如本说明书的引言部分所述，目前精子筛选技术与在男性和女性生殖系统内的体内过程存在很大差异，甚至可能对精子造成损伤。尤其值得关注的是DNA完整性，因为在自然授精和人工授精中，当DNA碎片化指数(DFI)超过30％时都可能降低受孕机会。此外，与体细胞不同，精子缺乏精子形成后的DNA修复机制；这一弱点使得在ART治疗中保护精子DNA的完整性成为当务之急。
在更详细地描述本发明的优选实施例之前，参考图1，其示意性地示出了装置10，其用于从精液样本中分离活动精子并随后执行筛选程序，以产生能够满足卵母细胞体外受精的特定标准的活动精子，并概括地示出了在该过程中使用装置内各个单独的结构执行精子分离和筛选的步骤。
装置10包括以下主要功能结构：
(1)精液样本接收室(也称为入口储液器)12；
(2)与入口储液器12流体连通的活动精子引导和分离结构14，在其他附图所示的优选实施例中，该结构由多个微通道构成；
(3)与分离结构14流体连通的活动精子筛选室6(也称为结合室)，在该室中，多个选择颗粒17与已经通过引导结构14并具有特定特征的活动精子结合并阻止其离开室16，由此室16是格栅结构(即液体可以透过的笼)，其设计成允许未结合的活动精子游动/穿过该室并防止选择颗粒17和结合到这些颗粒上的精子出去；
(4)与选择室16流体连通的精子出口储液器或室18，其布置成接收已经穿过结合室16的未结合的活动精子；以及
(5)在一个优选实施例中任选的卵母细胞保存室20，该卵母细胞保存室20与出口室18流体连通，或者布置在出口室18内，或者作为出口室18的一部分，其被设计用于接收和独立地存放卵母细胞，以便通过与积累在出口室18中的精子进行结合实现受精。
图1还示出了在装置10内执行精子筛选过程的流程图。该过程包括(在步骤1)，使用分离结构14的径向阵列微通道基于活动力进行精子筛选，该分离结构14位于入口储液器12和结合室16之间。该过程还包括(步骤2)，使用在格栅式结合室16中包含的(填充有)生物活性选择颗粒17的基底，选择颗粒17被设计用于通过精子捕获的方式来帮助负筛选精子(例如通过精子质膜和选择颗粒蛋白质表层之间蛋白质与蛋白质的相互作用来诱导)。该过程还包括(步骤3)，使用活动精子积累室18(出口储液器)，未结合的活动精子可以从格栅式筛选室16进入该室。在一些实施例中，出口储液器的形状使得活动精子细胞可以立即收集。图1还示意性地描述了另一个过程(步骤4)，其中出口室18中的活动精子进一步游向装置10的集成卵母细胞保存室20，卵母细胞可以放置在该室中，由此分离的精子遇到卵母细胞并与卵母细胞结合实现受精，从而完成至少一次卵母细胞体外受精。
本文所述的至少一些实施例通过使用具有本文所称的精子引导(和分离)结构14(在图示的实施例中由微通道的径向阵列提供)和精子筛选结构16(由格栅式精子结合室形成)的装置来改善体外环境中的受孕结果，在精子筛选结构16中包含对某些类型的精子表现出选择性亲和力颗粒17，如别处更详细讨论的，颗粒17用于结合那些虽然具有足够的活动力，但使这种精子用于体外卵母细胞受精时表现出“非期望的”因素的精子。在这些实施例的描述中，“非期望”一词并不仅仅意味着缺乏生存能力或精子受损，而是代表一种负筛选标准，其包括性别选择性等其他因素。
如上所述，在填充有选择性结合颗粒17的结合室16中(可以参照图5和图6更详细的描述)，通过结合非期望的精子来实现“负”筛选的过程，由此有效地固定结合了颗粒的精子，限制其离开结合室16，而不具有特定风险因素的其他活动精子不与颗粒结合也不受限制地通过结合室16向前游动到装置的出口储液器18。
不具有非期望特征的活动精子积累在出口室18中，用于进一步处理，例如从装置中取出随后用于冷冻或体内受精程序。然而，如上所述，在具体优选的实施例中，卵母细胞保存室20存在于装置内，或者作为单独的结构，或者位于出口室18内，如此可以使用出口储液器18中积累的活动精子来使放置于卵母细胞保存室20中的卵母细胞实现体外受精。
图2和3更详细地示出了装置10的一个实施例，其中图3B至3E和图3J至3L示出了微通道38的具体构造的多个替代实施例，径向延伸的微通道38形成了该装置的精子分离结构14，图3F至3I示出了结合室16的各种实施方式。以上参照图1的示意图进行了一般性描述。
在参照附图描述装置部件和结构时，使用诸如“上”、“下”、“底部”、“顶部”、“径向内部”、“径向外部”等相关术语和类似表达以及参考平面，以便于理解部件或结构彼此的相对位置。应当理解，这些术语并不意味着对由此引用的特征有任何限制，除非在装置的实际使用中，上下文另有指示。以类似的方式，在附图中使用相同的附图标记来表示功能等同的结构，尽管这些结构在实施例之间可以不同。
在下文中，将首先描述装置10的主要功能结构12、14、16、18和20，然后详细描述该装置如何用于进行卵母细胞的体外受精。
所有这些功能结构12、14、16、18和20都位于壳体25内，该壳体25由两个通常为扁平圆柱形的板22、24组成，并且全部或部分地由合适的硬固化、热固性或热塑性聚合物使用增材制造技术(即3D打印)形成凹入或凸出结构。用于制造的优选材料是塑料，例如环烯烃共聚物(COC)、聚碳酸酯(PCA)或聚甲基丙烯酸甲酯(PMMA)，但是该装置也可以由诸如聚二甲基硅氧烷(PDMS)或光敏聚合物树脂的材料制造。如在图2A和2C之间延伸的箭头所示，板22、24以面对面的关系彼此粘合(或以其他方式固定)成一个扁平圆柱形壳体25。不排除其他制造技术，例如模制、机械加工等，尽管装置10中各种结构的内部构造并不太优选这些技术来制造。
首先参见图2B和3A，精液样本室12具体为环形室的形式，其中环形通道凹入上壳体部分26的底面(仅对于有一小的径向延伸的腹板将通道分隔为两个部分的情况)，在粘合到一起后，该底面被下壳体部分22的顶面覆盖和封闭。两个圆柱形精液样本入口28从腹板的任一侧的上壳体部分26的上表面延伸到样本室12中。因此，精液样本可以通过入口28沉积到环形室12中。根据要从中分离和筛选活动精子的精液类型，精液样本室的尺寸必须能够容纳缓冲液和引入入口储液器12的精液，精液体积在50微升至4mL之间，而通常引入的精液体积在0.5mL至1mL之间。
然后参见图3A以及图3B、3C和3E的放大详图，在图示的实施例中，精子引导(和分离)结构14由多个辐条状径向延伸的微通道38、38’、38”形成，微通道38、38’和38”在径向外部环形精液样本室12和径向内部精子结合室16(下面将更详细地描述)之间延伸，从而为活动精子从精液保存室12游入精子结合室16提供了多个行进路径。
可以看出，环形精液样本室12的径向内部“边界”结构沿着其内周由多个间隔开的径向延伸腹板40、50的径向外部末端面54限定，腹板40、50立在上壳体部分26的凹入的内部中心面上。引导和分离结构14的多个微通道38、38’、38”被限定在这些径向延伸的腹板40、50的周向相邻的腹板对之间，并且由下(底部)壳体部分22的朝上侧的部分和上壳体部分26的朝下侧的部分限定，在组装壳体部分22、26时，这些部分在腹板40、50之间垂直延伸，从而提供多个封闭的横截面微通道。
腹板40在这里被称为微通道侧壁40，而腹板50被称为微通道中间壁50。虽然侧壁40在环形样本室12和径向向内定位的精子结合室16之间的精子引导(和分离)结构14的整个径向长度/延伸范围内延伸，但壁厚如下文所述变化，中间壁50仅部分延伸，并且如图3E所示和如下所述仅存在于微通道38的径向外部区域，并且具有恒定的壁厚。
径向延伸的侧壁40和中间壁50的径向外部末端面54实质上形成圆形格栅带，其仅部分地将环形精液样本保存室12与由多个径向延伸的微通道38、38’、38”形成的引导结构14物理分离。
然后对于活动精子引导结构14的构造/组成。如上所述，多个微通道38、38’、38”以径向汇聚的方式延伸，从环形精液样本接收室12开始，并延伸到上部壳体部分26的中心之前较远处径向向内处，由此限定了圆柱形空隙，并且以侧壁40的径向内部末端面52为边界。由于希望(i)在引导结构14中容纳尽可能多的微通道38、38’、38”，(ii)使微通道38的网络径向汇聚，以及(iii)希望保持通道的宽度和高度大概恒定以模拟体内输卵管环境，从而触发或有利于精子的自然壁游行为，可以选择一种结构/布局，由此从精液保存室12开始的相邻微通道38’、38”沿着它们的径向路径相互接合，并在过渡或合并区域之后形成与通道38’、38”具有相似横截面的微通道38。也就是说，尽管环形精液保存室12处的引导结构14的入口点的数量较多，但是进入精子结合室16的径向内端处的排出点的数量减少了，减少的数量相当于通道沿着引导结构14的径向延伸部所具有的接合点的数量。
如图3E的放大视图所示，其中引导结构14的径向外部区域中的微通道38’、38”的数量是径向内部区域中微通道的两倍，这是因为相邻的通道38’、38”从环形精液保存室12开始，在接合区39处向下游合并成单个通道38。可以看出，通道38’和38’分别由侧壁40界定，并在它们之间共享一个共同的中间壁50，该中间壁50具有有限的径向长度，并终止于接合区39，在接合区39处通道38’、38”合并成径向内部通道38。
还应注意的是，虽然中间壁(或腹板)50具有朝向微通道38’、38”的光滑侧面，但是侧壁40在面对相应通道38’、38”的相对侧上具有两个矩形肋44，这两个矩形肋44从侧壁40的中心腹板43垂直延伸/凸出，并且从环形精液保存室12径向向内彼此平行延伸，随着中心腹板43变厚，它们变得更窄(在44’处)，如图3E的合并接合区39所示。在顶部平面图中，侧壁40从接合区39逐渐变窄，在接合区39处，通道38’、38”朝着其径向内端合并成径向内部通道38。这在图3J至3L所示的具体实施例中得到最好的说明。
肋44的功能将参照图3D进行说明，肋44在本文中一般也称为“表面结构元件”，并且在所示实施例中沿微通道38的部分长度延伸，但是在其他实施例中，肋44可以沿着微通道38的整个径向长度延伸，图3D示出了图3B、3C和3E所示的微通道38、38’、38”的相对的侧壁44(以及中间壁50，如果需要的话)的相对表面的多种其他结构形式中的三种。与图3B、3C和3E相比，图3D示出了省略中间壁50的微通道横截面实施例，此外，如虚线垂直线所示，仅示出了壁40横截面的一半。
首先，显而易见的是，在具有诸如矩形横截面的封闭通道中游动的活动精子将受到四个边界表面的限制。如果横截面足够小，以模拟活动精子在基本上停滞的液体中游动的体内环境，例如微通道形成例如100μm×75μm的矩形横截面，精子的自然游动特性将导致如上述沿着四个边界表面的壁游行为。然而，如果横截面更大，则只有活动力更强的精子才会向边界表面移动，然后沿着边界表面游动。也就是说，基于活动力的精子选择性不仅受精子从入口点游向排出点的通道尺寸的影响，还受通道横截面内和沿其长度存在的边界表面数量的影响。因此，根据本发明的一个方面，提供边界表面的至少一个壁具有至少一个选自凹部42和/或凸部44的结构，该结构微沿通道38的至 少一部分长度延伸，并提供至少一个额外的边界表面(与不具有这种表面结构的通道相比)，活动精子可以基于壁游行为沿着该边界表面向位于引导结构14/微通道38的径向内端的结合室16游动。
这里所使用或定义的边界表面由相交的任意两个平面汇合来限定，两个平面以在它们之间形成拐角/角度(角不必是阶梯式不连续的，而是可以包括圆形边缘)的方式汇合，独立的边界表面之间的夹角优选在30度和150度之间。
然后对于图3D所示的三个微通道横截面实施例中的右边一个，上壳体部分的一部分被标识为26，如前所述，侧壁40直立并且与上壳体部分26的基板成一体。下壳体部分22在底部封闭了通道38(否则通道38会打开)。因此，上壳体26提供通道38的顶部边界，下壳体部分22提供其底部边界，相对的壁40提供其侧边界。三个矩形截面肋44从每个壁40的侧面48伸出/凸出并整体形成在其上(在图3B和3C的实施例中，只有两个这样的肋)。假定侧面48通常提供沿着通道38的整个高度的边界表面，供活动精子基于壁游行为游动，应注意的是，肋44沿着通道高度有效地细分或分割了两个边界表面，然而，肋44的上表面和下表面限定了额外的边界表面49，一旦活动精子进入通道38就可以沿着该边界表面49游动。因此，存在的六个肋44将总共额外增加十二个活动精子可以沿着其游动的边界表面。应注意到，肋44与侧壁44是一体成形的，并凸出到通道38中。然而，很明显，在两个肋44和侧壁40的侧面48之间限定了一个C形槽42。换句话说，鉴定增加边界表面的表面结构元件的数量取决于考虑是肋44的相对端面45还是侧壁44的侧面48之间的间距来限定通道38的内部宽度。
图3D中间的微通道截面图示出了存在于微通道中的这种表面结构元件的不同实施例，该微通道增加了活动精子沿其游动的边界表面的数量，其中侧壁44沿着侧壁40的高度呈阶梯状46，在下壳体端的部分较宽，而在侧壁上端的腹板部分较窄。
最后，最右边的微通道30横截面构造的实施例说明了由侧壁表面结构元件提供的十个额外边界表面也可以由相应的弯曲横截面肋44(或限定在相邻肋44之间的互补形状的凹槽42)提供。
应当理解，可以存在这种额外边界表面的其他实施例。例如，如果通道的横截面是矩形的，则微通道38的顶部或底部也将是形成法向边界表面的表面，并且可以想到在这些表面上提供结构元件以增加边界表面的数量。还值得注意的是，“基本的”通道横截面构型不限于矩形横截面。例如，其几何形状可以是圆形、三角形或六边形。
就尺寸而言，微通道38的长度可以在2mm至15mm之间，高度为50μm至5000μm，但是通常在100μm至1000μm之间。如上所述，假设矩形通道呈几何形状(只是其中一种可能性)，微通道的宽度可以在50至5000μm之间，通常在50至250μm之间。相对布置的(或面对的)表面结构元件42、44之间的自由空间(或距离)也应该选择成有利于活动精子向布置在这些结构处的额外边界表面49行进的尺寸，并且其最小值可以设定为50μm(可以更高)。最终，微通道最大和最小尺寸的选择也将部分地受到从精液样本中要分离和筛选的精子表型以及精液来源(例如牛、人、犬等)的影响。如上所述，微通道的宽度在整个装置的长度上也可能是不统一的，从而产生不均匀的边界或汇聚和发散的边界，供精子沿着行进。
本领域技术人员将会理解，微通道路径的具体几何布置、尺寸或构型可以变化。尽管利用具有5至5000个微通道的装置从人和动物精液样本中分离精子，均能观察到其表现出可接受的性能，但具体性能会由于所处理的样本不同而有所不同。在这种情况下，可以根据样本的体积、浓度和粘度，改变这些因素中的一个或多个，以改善精子分离过程。
接下来将参照图2B、2D和3F至3J来描述精子结合室16。结合室16基本上是环形结构，液体可以从入口透过到出口侧，并且结合室16设置在引导结构14的下游(当以活动精子游过装置10的各种功能结构的角度来看时)和出口储液器18的上游。它主要被设计用于在其中接收和容纳大量选择颗粒17，这些选择颗粒17对已经穿过引导结构14的，具有预定的、非期望的标记的活动精子具有结合的选择性。结合室16和/或选择颗粒17还设计成基本 上将与颗粒结合的精子限制在室16中，并防止这种精子进一步向出口储液器18行进，而不会阻碍未结合到选择颗粒的活动精子通过。填充有结合颗粒17的结合室16可视为过滤器，具有双重作用，一是可以作为额外的物理屏障，类似于在自然体内卵子受精过程中精子必须通过的卵丘细胞团，二是用于捕获DNA(或其他)受损的精子。
引导结构14的微通道38的径向内部末端52在室16的径向外侧提供了圆形的格栅边界结构34，如图2D所示，该图是图2B中所示的顶部壳体部分26的放大的突出详图，在图3F至3I中也同样可见。换句话说，结合室16的入口侧由多个微通道38的壁44的下游端界定或限定(从而提供了格栅入口结构)。环形结合室16的径向内端处的出口侧由紧密间隔的圆柱或柱56的圆形阵列限定，圆柱或柱56以限定格栅出口结构36的形式布置。因此，结合室16可以恰当地描述为“笼形结构”，活动精子可以从入口侧的多个微通道38进入该笼形结构，并且通过多个阻塞柱56在出口侧阻止与颗粒结合的精子从该笼形结构中流出，该阻塞柱56在周向相邻的柱56之间具有间距58，该间距58足够小以容纳(即阻止通过)容纳在室16中的精子结合选择颗粒17。
在图2D中可以更清楚地看到，壁40的径向内部末端部分52将邻接结合室16的微通道38分隔开，其构造成使得(i)微通道38朝向它们各自的排出端变窄，并且(ii)共同限定了格栅边界结构，该格栅边界结构能够抑制已经离开微通道38的活动精子重新进入微通道38。换句话说，微通道38的末端横截面变窄，并提供内表面，活动精子沿着该内表面以优选的汇聚路径从微通道38游出到结合室16中，由于横截面变窄，使得活动精子基本上(但不是绝对)不可能重新进入微通道38。例如，假设微通道侧壁在垂直截面上基本上是矩形的，末端的厚度变宽，由此在壁末端的平面图中，微通道基本上呈三角形。
至于柱56，它们可以通过增材制造技术与上壳体部分26一体成形。虽然可以使用具有如图2D和3F/3G所示的圆柱形结构的柱56，其功能与微通道限定侧壁40的末端52的形状类似，但是在优选实施例中，多个阻塞柱56相对于彼此成形和/或布置的方式使其可以限制已经离开结合室16的活动精子重新回到结合室中。阻塞柱56之间的间隙(间距)58需要足够宽，以允许未结合的活动精子穿过结合室16朝向径向更靠内的出口储液器18。换句话说，由紧密间隔的柱56的圆形阵列提供的格栅结构36在结合室16和出口室18之间提供了不会阻碍液体通过的边界结构36。在优选实施例中，相邻阻塞柱56之间的间隙58在10至100μm之间。同样，微通道38末端的横截面需要近似小，以将选择颗粒17容纳在结合室16中。
阻塞柱56的形状不限于任何特定的几何形状，然而优选能够将精子导向出口储液器18并防止精子重新进入结合室16的形状。合适的横截面形状包括梯形、半圆形、三角形、人字形、半环形和新月形。在图3I中示出了一个新月形横截面形状的示例。
如图2A和2D所示，可以通过连接到室16中的上壳体板26中的入口32将选择(或结合)颗粒17填充到结合室16中，以及从中取出。该装置外侧的结合室的入口32的上孔可以在0.5和5mm之间，而该入口的下孔可以在0.5和5mm之间。入口可以是漏斗形或圆柱形，但优选为漏斗形，更优选也可以是渐变的，以使选择颗粒17能够更好地流入结合室16，并减少填充过程中堵塞的可能性。
装置10中的结合室16使在精子外表膜上外表达预定的、非期望的生物标记的精子能够被结合在结合室16中。这可以通过诸如提供涂覆有配体的选择颗粒17来实现，该配体能够结合和捕获表达这些预定的、非期望的生物标记的精子。这些标记可以在精子解剖结构上的任何点处表达，例如头部、中段(容纳精子的线粒体)和尾部。术语“表面标记”是指，诸如转移到精子质膜外层并与精子的局部环境相互作用的蛋白质。
现有技术文献US‘655中使用的精子分离方法所带来的一个问题是对具有除基于活动力以外的特性的精子缺乏选择性。通过装置10可以解决该问题，装置10包括其中容纳有选择颗粒17的结合室16。选择颗粒17可以为小球状，优选为微球或微珠。微珠配制/制造成能够基于微珠表面上存在的缀合配体对精子细胞进行分子筛选。可以选择包含能够与标记相附着的蛋白质的缀合配体，这些标记选自凝集素(例如花生凝集素和豌豆凝集素)、抗体和膜联蛋白A5(优选膜联蛋白A5。配体也可以是药物，包括咪唑并喹啉胺(imidazoquinolinamine)(例如瑞喹莫德、咪 喹莫特和嘎德莫特)。
这些缀合配体通过化学键，例如通过与精子形成蛋白质-蛋白质相互作用而与表达标记的活动精子相结合。在一个优选的实施例中，选择颗粒17可以用膜联蛋白A5包被，膜联蛋白A5是膜联蛋白组中的细胞蛋白。膜联蛋白A5能够结合在通过该装置的活动精子表面上表达的外膜磷脂酰丝氨酸。在其他实施例中，选择颗粒可以用抗体包被，以便靶向在活动精子表面表达的特定抗原。
在一些实施例中，所使用的选择颗粒17的直径可以在50μm至300μm之间，这也将决定结合室16的径向内部格栅圆形边界结构36中的阻塞柱56之间的间距59，以及提供结合室16的径向外部格栅圆形边界结构34的微通道38的高度/宽度的最大值。优选地，选择颗粒为球形，直径在150μm至200μm之间。柱56之间的间隙59在6μm和100μm之间。这些柱的尺寸和直径是决定它们之间所需距离和所用微珠尺寸的因素。选择颗粒17在结合室16中的形状、尺寸和堆积密度使其基本上不阻碍从结合室16出来时不表现出非期望的标记的活动精子通过，但是需要足够大，以在结合室16中适当地容纳表现出非期望的特征的活动精子。
可以使用在本领域中已知的各种方法将配体“附着”到选择颗粒17的表面上(在注入到装置10中之前)，并且在目前的情况下，优选的形式是通过聚合物包被方法来实现。配体聚合物通常由对蛋白质和羧基化微珠表面具有亲和力的金属络合物组成。在一些实施例中，与供电子的羧基形成的强多价键使得聚合物粘附层能够稳定地留在选择颗粒17的表面上。在一个实施例中，为了固定抗体蛋白，聚合物层利用配体结合Ab Fc结构域。
通常将选择颗粒17引入到结合室16并悬浮在溶液(载液)中，该溶液适合于锚定到选择粒子17表面的一个或多个分子实体。通常，结合室16设计成能容纳10至1000μl的溶液，包括微珠17。结合室16的径向外部格栅圆形边界结构34和径向内部格栅圆形边界结构36之间的距离可以在0.1和2mm之间。
下面对精子出口储液器20进行说明，其位于结合室16的径向内部格栅圆形边界结构36的径向内部。本质上，它是壳体25中心的圆柱形空隙，由圆柱形阵列的柱56包围，具有穿过其材料形成的入口30，板状上壳体部分26形成于沿着扁平圆柱形壳体25的中心轴线上。下壳体部分22的上表面提供了储液器20的底面。优选地，通过入口30将化学引诱物引入出口储液器20中，例如黄体酮、RANTES、新铃兰醛、对叔丁基苯丙醛、心房利钠肽、透明质酸或其组合，这有助于将未结合到选择颗粒17上的活动精子吸引出结合室16。
在图2和图3所示的实施例中，多个(在该例子中为九个)独立的卵母细胞保存结构24在精子出口室18的中心区域内以3×3的正方形阵列共同定位，每个卵母细胞保存结构24都适于接收和保存卵母细胞，从而利用积累在出口室18中的活动精子与卵母细胞结合进行受精。在制造下壳体部分22的过程中有利地用3D打印制造保存结构18。可以说，保存结构24共同限定了卵母细胞保存室20。
每个保存结构24由八个弧形的壁部分或柱形成，这些壁部分或柱以圆形、圆柱形方式排列，它们之间具有小间距，但该间距又足够大以允许活动精子进入卵母细胞保存室的内部。虽然在该实施例中示出的是八个弧形壁部分，但是在另一个优选实施例中，卵母细胞保存室包括六个柱。同样，选择壁部分之间合适的间距以限制容纳在其中的卵母细胞不会穿过间隙。通常，间距为10μm到200μm就足够了，但最好为30μm到50μm。应注意，卵母细胞保存结构位于入口30的下方，卵母细胞将通过该入口插入该结构，并且该结构的高度可以与微通道的高度相同或更大。然而，在优选实施例中，保存结构24的高度以及延伸的精子出口室本身的高度在0.5至5mm之间。卵母细胞保存室的数量可以变化，这取决于临床样本的质量以及在整个体外受精(IVF)周期中存放卵母细胞所需的时间。卵母细胞保存结构24之间的距离通常在0.1mm至1mm之间。
因此，还应注意到，精子出口室18和卵母细胞保存结构24/卵母细胞保存室20是流体连通的，由此除了化学引诱物之外，出口室18还填充有合适的、具有适当粘度的缓冲液体(引导结构14的微通道38和精液保存室12也是如此)，以产生实际上无流动的停滞液体，使精子从精液保存室12经引导结构14和结合室16，穿过装置10内的 各种分离和筛选区域，游入精子保存室18和卵母细胞保存结构24。优选地，精子缓冲液是羟乙基哌嗪乙硫磺酸(HEPES)基的缓冲液或精子冷冻保护剂介质。
在开始描述使用上述装置10从精液样本中分离和筛选精子的优选方法以及使用该装置进行体外受精的方法之前，将参考图9中包括的一系列附图非常简要地描述这种装置100的另一个实施例。
图9A至9E示意性且部分详细地示出了根据本发明的用于精子分离和筛选的装置100的第二实施例。与图2A至3J的装置相比，装置100由三个堆叠的壳体部件126、123和122组成，其也采用如前所述的3D打印制造。
装置100具有多个同心堆叠的微通道层114(类似于洋葱中的层，见图9E和9F，而不是具有单个径向延伸的微通道的平面层)来提供精子引导结构14，该微通道层114在圆柱形中间壳体部分123中以抛物面弯曲的模式延伸。层114在环形精液收集储液器112和环形结合室116之间延伸，环形精液收集储液器112位于/限定在装置100的上部区域或高度上，环形精液收集储液器112设置在上部圆形板状壳体部分126处，环形结合室116被分成四个独立的弧形区域，形成在圆柱形中间壳体部分123的下部区域或高度上(见图9F)。圆柱形精子出口保存室118也形成在结合室下方的中间壳体部分123中。类似于图2C所示的底部圆形基板(底部壳体部分)122承载卵母细胞保存结构(仅在图9C中以124示意性示出)，其位于精子出口室118中(因此具有前面提到的双重功能)，并提供装置100的底部，在使用时卵母细胞保存结构位于该底部上。
图9D以仰视图示出了顶部壳体部分126，以示出径向外部环形精子接收储液器或室112，其具有两个相邻的入口128，入口128由连续的腹板129分隔开，腹板129与围绕圆形端口130的径向内部环形腹板131连接，圆形端口130又与管状导管132连通，管状导管132延伸穿过装置100的壳体125的中部123的高度，如图9A和9B的半透明部分以及图9E的平面图所示。导管132与精子出口室连通，并允许在需要时从该室取出活动精子。
在图9A和9C中，以沿着中间壳体部分123的高度延伸的虚线轮廓示出了六层的结构114，每层包括多个周向相邻的微通道138，而图9E及其放大的等比例详图和图9F的放大的详细侧视图更清楚地示出了弯曲微通道层114的几何形状和布局。应当理解，六个微通道层114的上端都落在环形精液样本接收室112的轮廓(或界限)内，使得分配在室112内的精子可以进入各个微通道138，使其朝装置100的较低高度处的结合室116行进。
微通道138本身的构造类似于前面参照图2和3描述的微通道38、38’、38”。
装置100的特征在于，不同的功能单元/结构112、114、116和118/120位于装置壳体125内的不同高度(也称为区域)。如图所示，装置100在其圆柱形壳体125内具有包括入口储液器112的第一区别区域、包括引导结构114的微通道的第二区域、包括结合室116的第三区域、包括出口储液器118的第四区域和包括卵母细胞保存室的第五区域120，注意第三区域、第四区域和第五区域可以优选地配置在共同的区域中，这意味着卵母细胞保存室将位于出口储液器的范围内，并且出口储液器116(如图2和3所示实施例中的情况)可以处于相同的高度，但是以环形方式径向向外围绕出口室118，并具有与前述类似的边界结构。例如，当出口储液器设计成基本为圆柱形的圆盘室时，一个或多个独立的壁结构可以存在于该室内，每个壁结构都用于接收卵母细胞，且活动精子能够通过以接触卵母细胞。
还应当理解，虽然图9A至9F示出了在截头抛物面(或类似)封套内延伸的层114，但是多层3D通道阵列可以由具有直线微通道(而不是在垂直于水平面的平面内的垂直弯曲微通道)的截头圆锥形封套限定，。更贴切地说，如在装置10的第一实施例中，区域(无论是处于相同高度还是处于不同高度)彼此流体连通，缓冲溶液填充各种结构，以使得(a)活动精子能够在微通道或引导结构114内从精液样本中分离出来，(b)已经进入结合室116并具有预定特征的活动精子能够与结合到选择颗粒117并因此保留在结合室116中的活动精子分离，以及(c)未结合的活动精子到达出口储液器118。然后，如此筛选到的(未结合的)精子可以从出口储液器118中取出和/或被允许“游动”到卵母细胞保存室120中，在卵母细胞保存室120中与其中接收的一个或多个卵母细胞结合实现受精。
如已经参照图1所示，本发明还提供了一种从精液样本中分离和/或筛选活动精子的方法，包括以下步骤：提供根据上述第一实施例或第二实施例的装置10、100；用精子缓冲溶液填充引导结构14、114的微通道38、138以及该装置的精液样本入口储液器12、112和活动精子出口储液器18、118，以在该装置的各种功能区/结构内产生基本上无流动的液体环境，供活动精子在其中游动；将精液样本引入入口储液器12、112；等待一段预定的时间；以及取出积累在出口储液器118、118中的精子，用于进一步处理或使用。
在将精子缓冲液或精液引入装置之前，将包含选择颗粒17的溶液经专用入口注入结合室16、116。通过连续缓慢的注入方式将包含选择颗粒17的溶液引入到装置中，并且通过这种注入方式将储液器加注到期望的百分比。
可以将装置本身抽真空，然后预充入缓冲溶液(以维持精子的活动力和生命力)，该缓冲溶液可以被预热到期望的温度，例如室温或近似核心体温，以模拟体内环境。
在进行精子分离和筛选过程中，可以将该装置置于室温下受控的气氛条件下。例如，在注射精液样本后，可以将氛围中氧气和二氧化碳的水平以及湿度预设为最佳水平，以使精子通过微通道向收集室出口行进。
该方法还可以包括在将精液样本注入到环形保存室之前，用封闭件盖住精子出口和选择颗粒(微珠)入口，并且在放置卵母细胞之前仅露出出口储液器。例如，封闭件可以是盖子、胶带、专用塞子或卡扣式盖子。
在另一个广泛的方面，在本文描述的至少一个实施例中，提供了一种利用从精液样本中获得的分离和筛选到的活动精子进行卵母细胞体外受精的方法，包括以下步骤：提供根据本发明的装置；用精子缓冲溶液填充微通道或引导结构，(视情况而定)以及装置的入口储液器和出口储液器；用选择颗粒填充结合室，选择颗粒具有与活动精子结合的选择性，该活动精子具有与卵母细胞受精相关的预定的非期望的特征，例如凋亡相关标记的表达、性别选择性相关标记的表达或具有渗透性膜，从而将与颗粒结合的活动精子限制在结合室中，而不会阻碍未与选择颗粒结合的活动精子的通过；将卵母细胞引入卵母细胞保存室；将精液样本引入入口储液器；等待一段时间，使筛选到的活动精子积累在出口储液器中；将活动精子从出口储液器导入卵母细胞室；以及允许所筛选的活动精子有足够的时间使容纳在卵母细胞保存室中的卵母细胞受精。
在一些实施例中，用移液器将裸卵母细胞直接吸取到装置10、100的卵母细胞保存室/保存结构24、124中。
卵母细胞的放置优选在装置充满包含选择颗粒(如果使用)、精子缓冲液和精液样本的溶液后进行。在卵母细胞放置后，可以任选地用矿物油密封出口储液器并覆盖卵母细胞保存室。
如前所述，化学引诱物可辅助用于将未结合的精子引导出选择颗粒容纳/结合室16、116，并进入装置的卵母细胞保存区。化学引诱物可以在卵母细胞放置之前或之后作为浓缩滴液加到出口的中间。这可以使化学引诱物向外梯度扩散，从而为精子提供跟随其行进的化学梯度。
有许多方法可用于制造根据本发明的装置，图4示意性地示出了一种使用3D打印技术的潜在的制造方法。该过程包括3D打印、清洗树脂材料、固化树脂材料以及在施加压力时通过双面胶带(如果希望装置能够拆卸)或者用永久粘合剂将装置10、100的相关壳体部件22、26、122、123、126进行粘合。
图5示出了用作选择颗粒17的蛋白质包被的微珠的生产过程的示例。如图5所示，在第一微流体混合模块中，将微珠(在这种情况下为聚苯乙烯芯)与聚合物溶液混合。在随后的洗涤步骤之后，在第二混合模块中将缀合的微珠与抗体或蛋白质溶液一起添加到第二模块组件中，以产生完全包被的微珠。缀合抗体的微珠此时能够结合外部表达的表面标记，例如在精子上发现的标记。如图5B所示，精子细胞通过蛋白质与蛋白质之间的相互作用牢固地附着在缀合了蛋白质的微珠上。
图6示意性地示出了用于将微珠(选择颗粒)装入到装置10的结合室(储液器)16中的示例性方法。简而言之，该方法包括一些步骤，例如重新悬浮微珠溶液、用移液器吸取溶液并将溶液注入结合室入口(以一种平稳的方式)。可以定制微珠储液器的容积以适应不同的溶液体积。例如，可以注入200至300μl溶液。此外，可以调节储 液器的容积，以确保微珠充分填充该室的区域。微珠溶液中使用的微珠浓度取决于微珠的大小。溶液浓度和体积可以根据所需的样本和尺寸进行调整。该过程的一个示例包括将高浓度的微珠注入到该室中，以完全充满该室或储液器。流程图的末尾还示出了装载有蛋白质包被微珠17的微珠储液器16的清晰图像以及参考图2和3描述的边界结构34和36。
图7的示意性流程图示出了使用上述装置10执行分离精子和使卵母细胞受精的方法的过程。这包括在真空室中用精子缓冲液填充装置10，将精液样本装入精液储液器12中，将化学引诱物滴加到中心出口18(其作用参见所附插图)，最后将卵母细胞放置到位于由环形精子出口室18包围的卵母细胞保存室20内的保存结构24中。如前所述，加入化学引诱物可以通过化学梯度增强精子向卵母细胞保存室20行进的导向作用，促使精子根据浓度的变化重新定向。
图8A、8B和8C示出了根据本发明的示例性实施例，使用根据图2和3的图示制造的装置10时的性能测量结果(精子回收量、活动力和生命力方面的性能)。从该装置的精子收集室中回收的精子的浓度通常是时间，从精液保存室通过引导结构、结合室最后进入精子收集(或出口)室的精子行进路径的长度，以及精液样本的起始浓度的函数。在引导结构中，精子主要基于活动力进行分离，在结合室中，通过结合和保留结合室中的“有缺陷的”或“非期望的”精子来进行负筛选。
精子回收的平均浓度(mL)与原始精液的起始浓度直接相关。即使来自同一人的不同样本，人原始精液的浓度也可能有很大差异。精子回收量随着装置内样本孵育时间的增加而增加，并导致精子浓度高于常规体外受精所需的浓度。
如图8B所示，在所有变体中，精子在出口处的活动力均高于95％，而生命力接近100％，但长时间放置精子会失去能量。这表明与原始精液相比，回收的精子群的活动力和生命力有了显著提高；在多次测试中，活动力超过95％且始终接近100％。由于采用混合的筛选机制，只有具有高度活动力和健康的精子细胞才能从装置中回收。随着时间推移，整个装置中的活精子细胞百分比一直很高，这也表明该实施例中使用的3D打印材料具有生物相容性。
图8D示出了未经处理的原始精子与使用根据本发明的装置筛选的精子相比的DNA碎片化的结果。结果显示，与未处理的精子相比，通过该装置筛选的精子表现出较低的DFI，这表明该装置能够筛选到具有更大受孕机会的质量更高的精子。
上述实施例仅旨在通过示例而并非限制性的方式进行描述。例如，虽然所示的装置结合了一个或两个入口储液器，但是也可以结合更多的入口储液器、多个不同的卵母细胞保存室以及堆叠结构内不同数量的层。此外，虽然给出了用于分离精子的装置和方法的各种实施例，但是这些实施例也可以用于其他具有自主游动能力或活动力的真核细胞或原核细胞。
还必须注意的是，如说明书和权利要求书中所使用的，单数形式的“一个”、“所述”和“该”也包括复数对象，除非另有说明。因此，例如，“通道入口”可以包括一个以上的通道入口。
在整个说明书中，术语“包括”或“包含”或其语法变体的使用应视为指明存在所陈述的特征、整体、步骤或组件，但不排除存在或还具有未具体提及的一个或多个其他特征、整体、步骤、组件或其组合。
除非特别说明或从上下文中显而易见，否则本文所用的术语“约”应理解为在本领域的正常公差范围内，例如在平均值的两个标准偏差内。“大约”一般可以理解为测量值或规定值的典型％以内。除非上下文中另有说明，否则说明书和权利要求书中提供的所有数值都可以用术语“大约”来修饰。
本文提供的范围应理解为该范围内所有值的简写。例如，范围1到50应理解为包括从1、2、3、4等至48、49或50的组中的任何数字、数字组合或子范围。
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Claims (21)

  1. 一种用于从精液样本中分离活动精子的装置，所述装置包括：
    入口储液器，用于接收精液样本；
    出口储液器，用于收集从所述样本中分离的活动精子；以及
    至少一个微通道，设置成与所述入口储液器和所述出口储液器流体连通并位于所述入口储液器和所述出口储液器之间，所述微通道包括至少一个壁，所述壁限定了边界表面；
    其中所述至少一个壁具有至少一个表面结构，所述表面结构具有一个或多个凹部和凸部，所述凹部和凸部在微通道的至少一部分长度上延伸，并提供至少一个额外的边界表面，活动精子沿着所述边界表面向所述出口储液器行进。
  2. 一种用于从精液样本中筛选具有预定特征的活动精子的装置，所述装置包括：
    入口储液器，用于接收精液样本；
    结合室，设置成经由引导结构与入口室在下游流体连通，所述引导结构设计成将活动精子从所述精液样本导向所述结合室；以及
    出口储液器，与所述结合室在下游流体连通，并且设计成接收已经穿过所述结合室的活动精子；
    其中所述结合室布置成用于容纳选择颗粒，所述选择颗粒具有与以下活动精子结合的选择性，所述活动精子(i)已经穿过所述引导结构，并且(ii)具有预定的、非期望的标记，所述结合室和/或所述选择颗粒设计成将与所述选择颗粒结合的精子限制在所述结合室中。
  3. 根据权利要求2所述的装置，其中所述引导结构包括至少一个设置在所述入口储液器和所述结合室之间的微通道，所述微通道包括至少一个壁，所述至少一个壁限定了边界表面。
  4. 根据权利要求3所述的装置，其中所述微通道的至少一个所述壁具有一个或多个表面结构，所述表面结构具有一个或多个凹部和凸部，所述凹部和凸部在微通道的至少一部分长度上延伸，并提供额外的边界表面。
  5. 根据权利要求1至4中任一项所述的装置，其中所述凹部包括至少一个凹槽，所述凹槽沿着所述微通道的部分或整个长度沿着一个或多个轴向直的、锯齿形、波浪形或其组合的路径延伸，其中所述凹槽的深度是恒定的或可变的，并且所述凹槽的横截面限定了至少一个尖锐或圆形的边缘，所述表面结构设置在所述壁中并沿着其延伸。
  6. 根据权利要求1至4中任一项所述的装置，其中所述凸部包括至少一个肋，所述肋沿着所述微通道的部分或整个长度沿着一个或多个轴向直、锯齿形、波浪形或其组合的路径延伸，其中所述肋的高度是恒定的或可变的，并且所述肋的横截面限定了至少一个尖锐或圆形的边缘，所述表面结构设置在所述壁中并沿着其延伸。
  7. 根据权利要求5或6所述的装置，其中所述至少一个壁包括所述凹槽和所述肋中的一个或两个，所述凹槽和所述肋以沿着微通道的长度或宽度，呈包括矩形、半圆形或阶梯形侧壁构造的方式布置。
  8. 根据权利要求3至7中任一项所述的装置，其中所述结合室在入口侧由所述多个微通道的下游端界定，在出口侧由多个阻塞柱界定，以限定笼形结构。
  9. 根据权利要求8所述的装置，其中在所述出口储液器或所述结合室处的所述微通道的末端成形为抑制活动精子运动回到所述微通道中，并且/或者其中所述多个阻塞柱相对于彼此成形和/或布置成抑制已离开所述结合室的未与所述颗粒结合活动精子回到所述结合室中。
  10. 根据权利要求2至9中任一项所述的装置，其中所述选择颗粒容纳在所述结合室中，所述选择颗粒包括与配体缀合的小球，所述配体包括附着于标记的蛋白质，所述标记选自：凝集素、抗CRISP、膜联蛋白A5、抗趋化因子受体抗体和抗CD63、抗CD9、ALIX或TSG101；或包括药物，包括瑞喹莫德、咪喹莫特和嘎德莫特。
  11. 根据权利要求8至10中任一项所述的装置，其中相邻的所述阻塞柱之间的间隙足够宽以使活动精子能够通过，并且足够小以防止与所述颗粒结合的精子和/或选择颗粒流出。
  12. 根据权利要求1和3至11中任一项所述的装置，其中所述多个微通道的宽度在10μm至5000μm之间，并且高度在10μm至5000μm之间。
  13. 根据权利要求1和3至12中任一项所述的装置，其中所述微通道的长度在0.1mm至15mm之间。
  14. 根据权利要求1和3至13中任一项所述的装置，其中两个或多个所述微通道从所述入口储液器开始，到出口储液器或结合室处合并成一个单独的微通道。
  15. 根据权利要求1和3至14中任一项所述的装置，包括5至5000个所述微通道，并以单层或多层堆叠的形式布置。
  16. 根据权利要求1和3至15中任一项所述的装置，其中所述微通道在所述入口储液器和所述出口储液器之间以径向、平行、螺旋、蛇形或其组合的方式延伸。
  17. 根据前述权利要求中任一项所述的装置，其中将一种或多种化学引诱物施加到所述出口储液器上，或者将足量缓冲液至少施加到所述微通道上。
  18. 一种用于从精液样本中筛选和/或分离活动精子以及使卵母细胞体外受精的装置，包括根据权利要求1至16中任一项所述的装置，还包括卵母细胞保存室，所述卵母细胞保存室与所述出口储液器流体连通，并设计成从所述出口储液器接收所筛选和/或分离的活动精子。
  19. 根据权利要求2至18中任一项所述的装置，具有包括所述入口储液器的第一区域、包括所述微通道或所述引导结构的第二区域、包括所述结合室的第三区域、包括所述出口储液器的第四区域和包括所述卵母细胞保存室的第五区域，并且其中所述第一区域至第五区域中的一个或多个位于不同于所述第一区域至第五区域中的另一个或多个的高度上，上述区域流体连通以使得(a)当活动精子接收在所述入口储液器中时，活动精子能够通过所述微通道或所述引导结构从所述精液样本中分离，(b)具有预定特征的活动精子能够与结合到所述选择颗粒并包含在所述结合室中的活动精子分离，(c)未结合到所述选择颗粒的活动精子能够到达所述出口储液器，以及(d)筛选的精子能够从所述出口储液器中取出和/或与容纳在所述卵母细胞保存室中的卵母细胞接触，并且可以从所述出口储液器和/或所述卵母细胞保存室中除去未结合的精子。
  20. 一种从精液样本中筛选和/或分离活动精子的方法，包括以下步骤：
    提供根据权利要求1至17中任一项所述的装置；
    用精子缓冲溶液视情况填充所述微通道或所述引导结构，以及装置的所述入口储液器和所述出口储液器；
    将精液样本引入所述入口储液器；
    等待一段预定的时间；以及
    回收所述出口储液器中积累的活动精子。
  21. 一种利用从精液样本中分离并筛选的活动精子使卵母细胞体外受精的方法，包括以下步骤：
    提供根据权利要求18或19所述的装置；
    用精子缓冲溶液视情况填充所述微通道或所述引导结构，以及所述装置的所述入口储液器和所述出口储液器；
    用所述选择颗粒填充所述结合室，所述选择颗粒具有与后述活动精子结合的选择性，所述活动精子具有与卵母细胞受精相关的预定的非期望的特征；
    将卵母细胞引入所述卵母细胞保存室；
    将所述精液样本引入所述入口储液器；
    等待一段时间，以允许所筛选的活动精子积累在所述出口储液器和所述卵母细胞保存室中；以及
    允许所筛选的活动精子有足够的时间使容纳在所述卵母细胞保存室中的卵母细胞受精。
PCT/CN2021/118151 2020-10-16 2021-09-14 一种从精液样本中分离精子的装置和方法 WO2022078144A1 (zh)

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