Single cell processing device

Abstract

The present field relates generally to devices for the treatment of single cells or small cell masses, in particular for the treatment of single mammalian cells or mammalian cell masses, methods of making the same, and methods of using the same. The device is generally a single cell treatment device for treating a single cell during a microinjection procedure, comprising: a base vessel having an upper surface and a lower surface; an array of micro-pores comprising one or more micro-pores, the one or more micro-pores comprising: at least one micro-pore wall defining an upper micro-pore opening and a central pore, the at least one micro-pore wall having two protruding cell-stabilizing protrusions formed therein, each cell-stabilizing protrusion defining cavities positioned opposite each other on a common horizontal plane; and the groove is used for maintaining fluid therein, is positioned on the upper surface of the base vessel and extends around the periphery of the micro-pore array.

Description

Technical Field

[0001] The present invention relates generally to devices for processing single cells or small cell clusters, and in particular to devices for processing single mammalian cells or mammalian cell clusters, methods for making the same, and methods for using the same. The devices are generally applicable to the field of infertility treatment and assisted reproduction; their uses may include in vitro fertilization (or "IVF") techniques or more particularly intracytoplasmic sperm injection (or "ICSI"). However, the single cell processing devices described herein are also applicable to mammalian cell culture, stem cell differentiation, cell array assays, and other infertility treatments. Background Art

[0002] Advances in microscale tissue culture technologies have enabled the creation of new therapies, products, and processes, many of which are still in their infancy. The potential benefits of improved cell culture and proliferation at the microscale are particularly important within the field of reproductive medicine. The nature of this field offers few alternatives to culturing and manipulating single autologous cells in vitro, which must then be harvested, manipulated, and reintroduced into the patient.

[0003] Reproductive assistance with the aid of in vitro fertilisation ("IVF") has become more accessible and has improved recently. As a result, more and more patients are using it. Data released by the Human Fertilisation and Embryology Authority show that, overall, women who start IVF treatment are more likely to have a child than before. However, the success rates of IVF clinics observed between individual clinics vary widely, with some clinics achieving success rates as high as 46% and others as low as 10%.

[0004] The likelihood of success varies at several stages during the IVF process: during oocyte harvest, oocyte fertilization, early embryo development, embryo vitrification, and embryo transfer. Currently, the success of each process requiring oocyte manipulation depends largely on the skill of the embryologist.

[0005] Intracytoplasmic sperm injection (or “ICSI”) is an assisted reproductive technology procedure performed in infertility clinics, primarily to treat male factor infertility (Boulet et al., 2015). It is an alternative procedure to IVF procedures where sperm fertilize an oocyte.

[0006] In IVF, a sperm solution is incubated with the oocyte and fertilization is achieved by one sperm out of thousands of sperm that binds to and penetrates the oocyte. The ICSI procedure differs in that a single sperm is injected directly into the cytoplasm of the oocyte.

[0007] The procedure, first described by Palermo et al. (1992), offers a treatment option for male factor infertility, particularly men with oligozoospermia and azoospermia, in which the sperm count in the ejaculated semen sample is too low to provide an optimal concentration of coincubated motile sperm to maximize the chances of fertilization during IVF.

[0008] ICSI surgery relies on tools to manipulate the positioning of sperm and oocytes, and then sperm is injected into oocytes. These tools include two micromanipulators, one for positioning oocytes, held in place by a finely stretched and heat-polished pipette commonly referred to as a "holding pipette", and the other for positioning a finely stretched "injection" pipette containing sperm. Both pipettes are attached to a finely controlled syringe-type "injector". The first micromanipulator controls the degree of negative and positive pressure to hold, position and release oocytes with a pipette. The second micromanipulator is used to control the capture and injection of sperm in the injection pipette. This process and equipment are well known in the art and are described in detail by Joris et al. (1998).

[0009] ICSI and other IVF procedures carry the risk of injury due to the physical manipulation required to introduce the sperm (whether this is intracytoplasmic or not). The risk of injury or shock due to manipulation and handling, and therefore the success rate of ICSI and other IVF procedures, is greatly influenced by the following factors:

[0010] a. The skill and care of the embryologist,

[0011] b. the precision of the tools and equipment available to the embryologist, and

[0012] c. Environmental conditions created in the laboratory under the influence of the embryologist (e.g. sterility, temperature, protection against any form of contamination, including volatile organic compounds).

[0013] The present disclosure addresses the second problem discussed above, namely the lack of precision in the tools and equipment available to embryologists.

[0014] To compensate for the lack of precision of single-cell handling devices, holding pipettes and injection pipettes with positive and negative pressure are required to pick up, manipulate, and place oocytes.

[0015] In use, the control of the amount of pressure used to create a vacuum seal between the oocyte and the holding pipette cannot be quantified by the embryologist. This results in the amount of negative pressure applied to the oocyte during ICSI surgery varying between surgeries and between embryologists. Typically, the deformation of the oocyte is caused by the suction applied by the pipette, causing the oothelial cell in the pipette to move.

[0016] Negative pressure treatment of the oocyte often causes invagination of the zona pellucida within the aspiration micropipette, causing stress or damage to the cell. Invagination of the oolemma (the plasma membrane of the oocyte) refers to the degree of cytoplasmic deformity caused by the injection pipette being initially pushed through the zona pellucida and then through the oolemma. Shear stress is actually caused by squeezing the oocyte and is the main cause of cellular stress during the ICSI procedure.

[0017] Oocytes are usually "protected" by a layer of "nursery cells" (i.e., cumulus ovum). It is well known that a two-way communication system applies to the cumulus ovum and oocyte, which determines the quality of the oocyte (Gilchrist et al., 2008). It is well known that removing the cumulus ovum before fertilization can have a negative impact on the health of the oocyte (Gilchrist et al., 2008). In order to achieve unimpeded microscopic visualization of the oocyte, ICSI surgery requires the removal of the cumulus ovum layer. In standard IVF treatments, cumulus cells are usually removed after the fertilization event.

[0018] During ICSI, the oocyte is placed in an environment without a protective cell layer prior to fertilization. The oocyte is then exposed to many manipulations it does not experience in IVF, whether it is exposure to microscopic light, negative pressure applied by a holding pipette to maintain its position, point pressure of the injection pipette against the holding pipette to cause the oocyte to invaginate, withdrawal of cytoplasm into the injection pipette to assess complete insertion, and injection of a synthetic polymer (PVP) used to "slow down" the sperm to capture it in the injection pipette.

[0019] According to Latham (2016), cellular stress often mediates cellular responses characterized by the activation of the endoplasmic reticulum stress response, which causes protein unfolding, leading to apoptosis and necrosis.

[0020] Although the functional control of the micromanipulator and syringe has improved, the actual workflow of the surgery remains the same as when it was first developed in 1992. Furthermore, the procedure requires extensive training to master (Tiegs and Scott, 2020).

[0021] Improvements to the ICSI procedure have not yet emerged that would reduce the technical skills and time spent performing the procedure. Attempts to improve ICSI surgical outcomes have primarily focused on modifying the holding pipette design (Fernandez et al., 2020; Ma et al., 2020) or introducing injection systems that rely on piezoelectric motor function (Zander-Fox et al., 2021).

[0022] Zander-Fox et al. (2021) developed a PIEZO-ICSI technique that uses a piezoelectric actuator to generate high-speed movement of the injection pipette, effectively causing precise microdrilling of the zona pellucida. This technique reduces the need for physical pressure on the oocyte membrane and eliminates the need for cytoplasmic agitation. It is therefore potentially a gentler form of microinjection, but does not improve the handling of oocytes.

[0023] While advances have been made in microinjection devices and visualization devices used in IVF therapy, little progress has been made toward the development of devices for handling the cells involved when they must be manipulated or when they are contained in a manner that minimizes excessive handling or manipulation.

[0024] US2021 / 0230525 (Alcaide, FV2021) describes a vacuum pipette for retaining oocytes and a method for intracytoplasmic sperm injection, which is based on an elongated and narrow cylindrical body and a truncated conical narrowing portion for retaining oocytes under vacuum pressure. The vacuum pipette eliminates the need to aspirate the cytoplasm before microinjection, however, vacuum pressure can cause deformation of the cell and does not prevent invagination of the egg membrane at the microinjection point.

[0025] Similarly, Ma et al. (2020) proposed a trumpet-shaped variant of the traditional holding pipette for use in human reproductive technology. This modification also did not address the problem of cell deformation during microinjection and invagination of the egg membrane at the microinjection site.

[0026] A simpler, more precise oocyte and / or sperm cell processing device is needed to remove the need for and alleviate the problems of pressurized processing devices. Such a simple, precise oocyte and / or sperm cell processing device should preferably be compatible with precise microinjection techniques (such as PIEZO-ICSI techniques).

[0027] Precision tools and equipment reduce the likelihood that an embryologist will make a mistake that could result in damage or shock to the sperm, oocyte, or embryo. Further, tools or equipment that reduce or eliminate physical intervention or manipulation by the embryologist reduce the likelihood that an incident that could occur that causes physical shock to the sperm, oocyte, or embryo could occur.

[0028] A cell handling device is described herein that optionally maintains the physical stability of sperm, oocytes, and / or embryos within a dedicated culture environment. Embryologists can more easily and / or accurately handle or manipulate sperm, oocytes, or embryos without the use of pressurized pipetting devices, thereby minimizing the risk of physical shock and / or biochemical stress to the cells. The stability of cells during culture may improve the success rate of ICSI procedures.

[0029] Additionally, oocytes are sensitive to any changes in environmental factors such as temperature, osmotic pressure, oxygen concentration, nutrient restriction, hyperglycemic conditions, and pH (Latham, 2016). The cell processing device described herein can optionally reduce changes in the optimized ex vivo environment, thereby further reducing biochemical stress on sperm, oocytes, or embryos.

[0030] The cost benefits of eliminating the need for vacuum equipment and streamlining the cell handling process come from reduced labor costs, reduced capital expenditures, and reduced training time (Yagoub et al., 2022). This not only improves clinic profitability, but also increases the accessibility of ICSI technology to less affluent communities. For new clinics, improved cell handling processes reduce the cost burden of traditional equipment required to perform ICSI, potentially benefiting both clinics and patients.

[0031] The cell handling device described in this article can not only solve the difficulties in the successful fertilization and culture of developing embryos, but can also be applied to the culture of other cell lines that are sensitive to changing culture environments during gradient processes. Summary of the invention

[0032] In a broad aspect, embodiments described herein relate to a single cell processing device for processing single cells during a microinjection procedure, comprising: a substrate having an upper surface and a lower surface; a microwell array comprising one or more microwells, the one or more microwells comprising: at least one microwell wall defining an upper microwell opening and a central hole, the at least one microwell wall having two protruding cell-stabilizing protrusions formed therein, each cell-stabilizing protrusion defining a cavity positioned relative to each other on a common horizontal plane, the cell-stabilizing protrusions comprising a first cell-stabilizing protrusion defining a channel opening at the periphery of an entry channel extending outward through the microwell wall, and a second cell-stabilizing protrusion defining a ventilation opening, the entry channel having a channel base aligned in a horizontal plane with a center point of the second cell-stabilizing protrusion; and a groove for maintaining fluid therein, located on the upper surface of the substrate and extending around the periphery of the microwell array.

[0033] The groove of the single-cell processing device preferably exchanges fluid with one or more micropores via an entry channel, the groove having an inner wall on the proximal side of the microarray and an outer wall on the distal side of the microarray, the outer wall vertically extending to a height higher than the channel base of the entry channel, and the ventilation opening is positioned as a whole higher than the channel base of the entry channel.

[0034] The first cell stabilizing protrusion and the second cell stabilizing protrusion are preferably elliptical in a horizontal plane. Preferably, the diameter of the first cell stabilizing protrusion and the second cell stabilizing protrusion is in the range of 10 microns to 500 microns. Alternatively, the diameter of the first cell stabilizing protrusion and the second cell stabilizing protrusion is in the range of 100 microns to 200 microns. The protrusion size can be adapted to accommodate the size range of various cells or cell clusters.

[0035] Preferably, at least one microwell wall is cylindrical in shape and one or more microwells comprise a microwell base, wherein the at least one cylindrical microwell wall and the microwell base define a central aperture.

[0036] In other aspects, at least one cylindrical micropore wall has a diameter greater than the diameter of the cell stabilizing protrusion. In particular, at least one cylindrical micropore wall has a diameter in the range of 0.1 micrometers to 1000 micrometers; alternatively, at least one cylindrical micropore wall has a diameter in the range of 200 micrometers to 300 micrometers.

[0037] Preferably, the entry channel comprises two parallel channel walls, a channel base and an upper opening.

[0038] A preferred single cell processing device includes a cover and one or more stacking connectors located at the upper surface and the lower surface to connect multiple substrates when stacked.

[0039] In various embodiments, the sample support platform at the upper surface of the base plate has a sampling edge proximal to the entry channel, wherein the sampling edge is stepped or ramped at a gradient to align with a horizontal plane of the channel base.

[0040] The method for using the single cell processing device is preferably used to perform intracytoplasmic sperm injection surgery, comprising the following steps:

[0041] Obtaining the single cell processing device according to claim 1,

[0042] Add culture medium to the grooves of the single cell processing device.

[0043] Obtain oocyte and sperm samples,

[0044] Add the oocyte to the central hole of the microwell.

[0045] Place the sperm sample on the sample support platform.

[0046] Access to microinjection equipment,

[0047] Sperm are aspirated from the sperm sample through the tip of the microinjection device.

[0048] Place the tip through the entry channel of the microwell,

[0049] Use the tip to move the oocyte to the second cell stabilizing protrusion,

[0050] Microinjection of sperm into oocytes,

[0051] The microinjection tip is withdrawn by holding the oocyte in the first cell stabilizing protrusion while the microinjection tip is removed separately.

[0052] A preferred method of manufacturing a single cell processing device may include making a substrate and making the microarray and grooves thereon. An alternative method of manufacturing may include making the microarray and grooves within the substrate.

[0053] The preferred method of fabrication involves a two-part fabrication consisting of a two-photon polymerization printed part inserted into a polystyrene injection mold.

[0054] In a broad aspect, various embodiments are directed to a single cell processing device comprising: a microwell formed by at least one microwell wall having two opposing access orifices positioned through the microwell wall.

[0055] The single cell processing device is preferably configured to be formed at a microscale. Therefore, it is also preferably formed of a material suitable for microscale production and non-toxic to cells.

[0056] Preferably, the micropore wall comprises an outer wall surface, and the outer wall surface has two or more micropore hemispheres formed therein. In an alternative form, the outer wall surface can form a single micropore hemisphere of the circumference of the micropore wall therein. Preferred micropore hemispheres are shaped to provide optimal mechanical support to the cells maintained therein. Thus, the micropore hemispheres can form various structures and sizes. For example, the hemisphere can have a circular hemispherical shape (such as a hemispherical cylinder), an elliptical hemispherical shape (such as a sphere), a profile with a conical cross section or a parabolic profile (such as an elliptical parabola). The profile of the hemisphere can be graded to increase the hemispherical groove formed along the outer wall surface of the micropore.

[0057] Preferably, the diameter of the microwell hemisphere is sized to be suitable for the size of the cells maintained therein. For example, the oocyte including the zona pellucida is approximately between 165 microns and 150 microns; therefore, preferably, for all mammalian oocytes (including oocytes of non-human origin), the size of the microhemisphere is set to be between about 50 microns and 300 microns, especially for human oocytes, the size of the microhemisphere is set to be between about 100 microns and 200 microns, or for embryos and other cells, the size of the microhemisphere is set to be between about 10 microns and 500 microns; all of which have a configuration that can accommodate oocytes within the range of 165 microns to 150 microns.

[0058] Preferably, two opposing access orifices are formed through the microporous wall within the microporous hemisphere. Thus, in certain embodiments, the two microporous hemispheres may be positioned opposite each other. Alternatively, at least one microporous hemisphere may span a sufficient length around the circumference of the microporous wall so that two access orifices can be formed through the microporous hemisphere and also positioned opposite each other.

[0059] The design choices used to form the microwell hemisphere will focus on designs that provide the most support to the oocyte during injection and retrieval of the injection pipette during the ISCI procedure.

[0060] The access opening can form a passage between the inner surface of the microporous wall and the outer surface of the microporous wall in the microporous wall. Preferred channel configuration and size can be designed and selected based on the benefit provided to the embryologist when handling and operating the injection pipette. Preferred passage can have a closed profile or an open profile. They can have a simple profile, such as a circular hole or an oval groove. Or, they can have a complicated profile, such as a T-shaped profile or a cross-hair profile.

[0061] The channels may be further shaped to aid in the removal of air pockets or bubbles when the embryologist loads the microwells with culture medium.

[0062] Alternatively, the channel can be shaped to assist in the movement of the culture medium for balancing fluid pressure, for example, to avoid back pressure behind the cell when it is manipulated. Avoiding back pressure behind the oocyte is particularly important when the embryologist is performing the ICSI procedure.

[0063] Preferably, the microwell is open from above to enable access to the microwell for adding culture medium, or to access and retrieve oocytes. Further, one or more inner surfaces of the microwell are preferably curved.

[0064] In a further broad aspect, embodiments described herein relate to cell processing arrays comprising one or more microwells.

[0065] Preferred cell processing arrays include two or more microwells as described herein. Preferably, the two or more microwells are located at fixed positions relative to each other to facilitate operator navigation of the array. The microwells can be positioned within the array to form a variety of configurations, or to form different spacings of fixed relative positions. For example, the microwells can form a linear array, a circular array, a grid array, other shapes, or any or other shapes and combinations of configurations.

[0066] In a further preferred form, the single cell processing device or cell processing microarray is located in the base hole. The base hole is preferably formed by at least one side wall and at least one base wall. At least one side wall and at least one base wall form a container to accommodate the single cell processing device or cell processing microarray and fluid culture medium therein. The single cell processing device or cell processing microarray may preferably be further located in a larger hole for additionally accommodating culture medium and / or oil used in performing the ICSI procedure.

[0067] The preferred larger wells may be specifically adapted for use with the single cell processing devices or cell processing microarrays described herein. Alternatively, they may be "off-the-shelf" cell culture products, which may or may not be specifically designed for performing ICSI procedures.

[0068] The preferred base well includes one or more sperm platforms, more preferably, two sperm platforms. Preferably, the single cell processing device is arranged in a cell processing microarray located on the base wall of the base well, and the two linear sperm platforms are also located on the base wall of the base well, roughly parallel to and adjacent to the cell processing microarray.

[0069] The preferred sperm platform comprises an uppermost surface configured in a stepped arrangement. The preferred configuration of the stepped arrangement is designed and / or selected to facilitate gravity-induced motile sperm selection. However, the sperm platform(s) may be configured in many shapes and sizes.

[0070] Preferably, the uppermost surface of the sperm platform and the single cell handling device are generally positioned relative to each other at a central focal plane. For efficient collection of sperm and oocytes during ICSI procedures, co-localization across a generally common plane is preferred. The sperm platform is preferably configured with a platform surface positioned relative to the single cell handling device to position and collect sperm.

[0071] The sperm platform itself can be configured in many shapes and sizes. Also, each step of the stepped platform can be configured in many shapes and sizes. Any number of steps can be selected to construct the stepped platform.

[0072] Preferably, the cell processing array may include an unlimited number of single cell processing devices, each of which is adapted to engage with other single cell processing devices. Preferably, the cell processing array will be a linear array in a horizontal plane, but may alternatively be stacked in a vertical plane or a mixture of both. Preferably, the single cell processing devices will be clamped together in the horizontal plane and stacked in the vertical plane, or alternatively may be slidably engageable with each other in either the horizontal plane or the vertical plane.

[0073] In a preferred form, the cell culture substrate is adapted to be connected to another cell culture substrate or a cover.The substrate is preferably configured to physically stabilize the cell processing array when placed on a surface or connected to another component or device.

[0074] Preferably, the basic well described herein is included in a culture system, which culture system further comprises a support platform and a culture dish. Preferably, the basic well described herein is formed in a culture dish and / or a support platform.

[0075] Additional features may be provided in the culture systems described herein. For example, an identification tag may be associated with each microwell to allow individual identification of the microwell and the contents contained therein. Such measures provide traceability when the culture system is in use, for example, they may provide traceability of oocytes when performing an ICSI procedure.

[0076] Preferred identification tags may provide numbers, letters or other markings visible at the central focal plane of the sperm platform and single cell processing device. This enables the identification tag to be seen when performing an ICSI procedure.

[0077] Preferably, the identification tag can be configured so as to be clearly discernible at different angles or orientations. For example, a double mark can be provided for viewing different marks at 0° and 180°.

[0078] In a further broad aspect, embodiments described herein relate to methods for performing an intracytoplasmic sperm injection (ICSI) procedure.

[0079] A preferred method for performing ICSI comprises the following steps:

[0080] ·Prepare culture dishes with culture medium, sperm samples and oocyte samples,

[0081] Cover the petri dish with oil,

[0082] Separation of sperm samples and oocyte samples,

[0083] Insert the injection pipette into the micromanipulator,

[0084] Fix a sample of motile, morphologically normal sperm from a sperm sample,

[0085] Collect normal sperm samples using an injection pipette,

[0086] ·Transport the collected normal sperm sample to the oocyte sample,

[0087] · Select the oocyte to be injected and aspirate a small amount of oocyte cytoplasm into the injection pipette,

[0088] Injection of oocyte cytoplasm and normal sperm sample into the selected oocyte,

[0089] Withdraw the injection pipette.

[0090] In a further broad aspect, embodiments described herein relate to methods for making a single cell processing device or a cell processing array.

[0091] Preferably, the single cell processing devices or cell processing arrays described herein are fabricated as a two-part construction, comprising a two-photon polymerization [2PP] printed part inserted into a polystyrene injection molded (or alternative transparent and non-cytotoxic material).

[0092] Preferably, such a method comprises the step of mechanically mating the two parts.Such a method accommodates mechanical mating of the uncured parts followed by post-curing assembly.

[0093] In alternative configurations, the components may be glued, clip assembled, or a combination of both.

[0094] The method described herein for making a cell culture system includes making a base well and a cover having four sides and a top and configured to fit over an uppermost surface of the base well.

[0095] The method used to manufacture the device is designed to support variations in the base well and cover and can be customized to fit either off-the-shelf dish designs or custom designs of base well and cover.

[0096] The method for manufacturing the device may permit separation of component manufacturing and final assembly.

[0097] Methods for manufacturing the device may include features to support assembly automation and quality control inspection.

[0098] As used herein, the term "cell" will be understood to be interchangeable with the term "cellular material" and shall refer to the cell, cell population, tissue or organoid that is the subject of the invention described herein.

[0099] As used herein, the term "cell culture" shall describe any instrument or process for isolating and maintaining cellular material under controlled conditions for testing, growth, observation, experimentation, harvesting culture, or other bioscience processes.

[0100] As used herein, the term "device" shall describe articles produced on a micrometer scale (eg, between 0.1 micrometer and 1000 micrometers). Device shall encompass both static devices and mechanical devices, as well as devices in the field of microfluidics.

[0101] As used herein, the term "maintaining cells" shall refer to any process of storing cellular material in a controlled environment so as to produce the conditions necessary for its viability. The term "culturing cells" shall refer to the process of culturing cells.

[0102] As used herein, the terms "cryopreservation" or "associated cryopreservation" shall refer to vitrification or freezing in an interchangeable manner.

[0103] As used herein, the term "processing" and derivative terms such as "processing", "processed", etc. will be understood to include all forms of physical manipulation, including but not limited to collecting, picking up, holding, grasping, holding, nudging, rolling, touching and moving, etc.

[0104] As used herein, the term "stable" and derivative terms such as "stabilizing," "stabilized," and the like will be understood to include maintaining a relatively stable, firm, or firm physical state of an object relative to a state in which no stabilizing intervention exists.

[0105] As used herein, the term "groove" is used to define a closed loop channel or trench that can receive and maintain a fluid therein. The term "groove" should not be construed to imply any scale limitation, but can indeed be formed at a microscale.

[0106] A broad range of embodiments of the present invention will now be described with reference to the accompanying drawings together with the examples and preferred embodiments disclosed in the detailed description. The present invention may be embodied in many different forms and should not be construed as being limited to the embodiments described herein. These embodiments are provided by way of illustration only so that this disclosure will be thorough and complete and will convey the full scope and breadth of the present invention.

[0107] Broad embodiments of the present invention include cell processing devices for holding oocytes, and may optionally include cell processing devices for holding sperm. DETAILED DESCRIPTION BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 A top view of a single cell processing device according to an embodiment of a raised microwell of the present invention is provided.

[0109] Figure 2 A side perspective view of a single cell processing device according to a raised microwell embodiment of the present invention is provided.

[0110] Figure 3 A side view of a single cell processing device according to a raised microwell embodiment of the present invention is provided.

[0111] Figure 4 A cross-sectional view of a single cell processing device according to an embodiment of a raised microwell of the present invention is provided.

[0112] Figure 5 A top view of a microwell array and a sperm platform of a single cell processing device according to an embodiment of a raised microwell of the present invention is provided.

[0113] Figure 6 A side perspective view of a microwell array and sperm platform of a single cell processing device according to a raised microwell embodiment of the present invention is provided.

[0114] Figure 7 A top view of a sperm platform according to a raised microwell embodiment of the present invention is provided.

[0115] Figure 8 A top transparent view of a microwell according to an embodiment of a raised microwell of the present invention is provided.

[0116] Fig. 9 A front cross-sectional view of a sperm platform according to a raised microwell embodiment of the present invention is provided.

[0117] Fig.10 A front cross-sectional view of a microwell according to an embodiment of a raised microwell of the present invention is provided.

[0118] Fig.11 A top view of a single cell processing device and device cover according to a raised microwell embodiment of the present invention is provided.

[0119] Fig.12 A side perspective view of a single cell processing device and device cover according to a raised microwell embodiment of the present invention is provided.

[0120] Fig.13 A side view of a single cell processing device according to a raised microwell embodiment of the present invention is provided.

[0121] Fig.14 A side cross-sectional view of a single cell processing device and device cover according to a raised microwell embodiment of the present invention is provided.

[0122] Figure 1 5 provides a top view of a device cover according to a raised microwell embodiment of the present invention.

[0123] Fig.16 A side perspective view of a device cover according to a raised microwell embodiment of the present invention is provided.

[0124] Fig.17 A side view of a single cell processing device according to a raised microwell embodiment of the present invention is provided.

[0125] Fig.18 A side cross-sectional view of a single cell processing device according to a raised microwell embodiment of the present invention is provided.

[0126] Fig.19 A top view of a single cell processing device according to an embedded microwell embodiment of the present invention is provided.

[0127] Fig. 20 A top view of a microarray of a single cell processing device according to an embedded microwell embodiment of the present invention is provided.

[0128] Fig.21 A side cross-sectional view of a single cell processing device according to an embedded microwell embodiment of the present invention as viewed from the microarray side of a substrate is provided.

[0129] Fig. 22 A side cross-sectional view of a single cell processing device according to an embedded microwell embodiment of the present invention as viewed from the sperm platform side of a substrate is provided.

[0130] Fig.23 A side cross-sectional view of a microwell through opposing trench walls of a single cell processing device according to an embedded microwell embodiment of the present invention is provided.

[0131] Fig.24 A top view of a microwell of a single cell processing device according to an embedded microwell embodiment of the present invention is provided.

[0132] Fig.25 A side perspective view of a base dish and a cover of a single cell processing device according to an embedded microwell embodiment of the present invention is provided.

[0133] Fig.26 A side perspective view of two stacked substrates of a single cell processing device according to an embedded microwell embodiment of the present invention is provided.

[0134] Fig. 27 A side view of a substrate of a single cell processing device according to an embedded microwell embodiment of the present invention is provided.

[0135] Fig.28 A side perspective view of a cover of a single cell processing device according to an embedded microwell embodiment of the present invention is provided.

[0136] Fig.29 A comparative study evaluating the setup efficiency between conventional ICSI and the embedded microvia embodiment of the present invention is provided, including Fig.29 (a) Equipment handling, Fig.29 (b) Pipette settings, Fig.29 (c) Oocyte processing and Fig.29 (d) Comparison of oocyte unloading time.

[0137] Fig.30A comparative study evaluating the procedural efficiency between conventional ICSI and the embedded microport embodiment of the present invention is provided, including Fig.30 (a) Oocyte injection treatment, Fig.30 (b) injection time, Fig.30 (c) Polar body orientation and Fig.30 (d) Comparison of injection time.

[0138] Fig.31 A comparative study evaluating the number of manual adjustments during oocyte injection between conventional ICSI and an embedded microport embodiment of the present invention is provided.

[0139] Fig.32 A comparative study evaluating the level of invagination during oocyte injection between conventional ICSI and the embedded microwell embodiments of the present invention is provided.

[0140] Fig.33 A comparative study evaluating the development rate between conventional ICSI and the embedded microporous embodiment of the present invention is provided, including Fig.33 (a) the fragmentation rate, Fig.33 (b) survival rate, Fig.33 (c) The cleavage rate and Fig.33 (d) Comparison of blastocyst rates.

[0141] FIG. 34 provides a comparative study of human oocytes between conventional ICSI and an embedded microwell embodiment of the present invention, including a comparison of injection rates in FIG. 34( a ) and the number of manual adjustments in FIG. 34( b ).

[0142] Several embodiments of the invention are described in the following examples.

[0143] Example

[0144] Examples involve two-photon polymerization (2PP) 3D printing fabrication of single-cell handling devices designed with either a raised microwell design or an embedded microwell design. The fabrication method was enhanced by creating a single-piece printed device that holds up to twenty oocytes at a time. Functional and workflow differences between a novel ICSI technique (microICSI) and conventional ICSI (C-ICSI) employed in the single-cell handling device were compared, and procedural efficiency and embryo development outcomes were evaluated. Finally, a pilot study is described that demonstrates the utility of the device for microinjection of human oocytes.

[0145] The single cell handling device described herein is suitable for holding oocytes and sperm during ICSI surgery. An exemplary device includes a base dish and a cap or cover. The base dish combines the functionality of a conventional ICSI cell culture dish with improved features for positioning oocytes to eliminate the need for a holding pipette. The oocyte can be positioned without vacuum manipulation of the cell. The configuration of the device is optimized to provide maximum support to the oocyte to minimize deformation and invagination of the oocyte, thereby improving the quality of the fertilized egg produced and / or increasing the fertilization rate.

[0146] Positioning the oocyte in a fixed position within the dish eliminates the need for a vacuum pipette and allows the embryologist to easily position the oocyte to perform ICSI, thereby increasing the efficiency of performing ICSI.

[0147] Universal substrate and microwell design

[0148] The exemplary substrate is designed to support the oocyte within a hemispherical structure (or cup) during ICSI surgery to remove the need to hold a pipette and suction. During microinjection, the oocyte is cupped within a hemisphere surrounded by micropores. The channel allows the injection pipette to maneuver the oocyte into position within the hemisphere or cup before picking up sperm, where the oocyte can be handled and stabilized for microinjection.

[0149] Figures 1 to 18 A raised microwell design is illustrated, in which the microwell array is fabricated on a substrate platform in a raised manner. Figures 19 to 28 A variation of the fabrication method based on an embedded microvia design is illustrated.

[0150] Raised micro-hole design

[0151] refer to Figure 1 to Figure 1 8. Figure 1 A top view of a device substrate 210 according to an embodiment of the present invention is shown, which has a single cell processing device located thereon. The substrate 210 consists of a 65 mm square dish, which is equipped for patient identification. The substrate 210 is located in a base hole 220. The base hole 220 allows culture medium to be distributed in the substrate 210 and covered with oil.

[0152] Centrally positioned within base well 220 is microwell array 230. Microwell array 230 is comprised of a plurality of individual wells that provide individual locations for holding and maintaining individual oocytes from a single patient therein. Sperm platforms 280 are positioned adjacently on either side of the microwell array.

[0153] Figure 2A side perspective view of substrate dish 210 is shown. The substrate dish 210 is located above the support platform 310 and is stepped upward from the support platform 310. The substrate dish 210 and the support platform 310 are each approximately 4.5 mm high. The depth of the base hole 220 spans the height of the support platform 310 and the substrate dish 210, thereby providing a depth of approximately 8.5 mm for the base hole 220.

[0154] Figure 3 A side view of the single cell processing apparatus is provided, showing an upward step between the support platform 310 and the substrate 210, which are of approximately equal height.

[0155] Figure 4 A side cross-sectional view of a single cell processing device is provided, taken through the middle of the base well 220 and longitudinally through the microwell array 230. Figure 4 As shown, substrate 210 is offset above support platform 310. Figure 4 The stepped profile of the device at the edge of the microwell array is shown. Figure 4 The depth of the base hole 220 is shown, extending through the height of the base dish 210 and the support platform 310. The side walls of the base hole 220 are sloped to facilitate injection molding manufacturing.

[0156] In use, the base well 210 is filled with cell culture medium to immerse the microwell array 230 in the culture medium. The total capacity of the base well 210 is approximately 7.8 ml to allow for oil coverage.

[0157] Figure 5 A detailed view of a microwell array 230 and a sperm platform 280 is provided. A series of microwells 240 are positioned in a row longitudinally to provide a microwell array 230. Each well provides a separate single cell handling device for holding and manipulating a single oocyte in each well. Each microwell array 230 contains a series of oocytes from a single patient. The array is compact and linear for efficient visualization, repositioning, and manipulation by embryologists.

[0158] Figure 6 A side perspective view of a microwell array 230 and a sperm platform 280 is shown. Each individual microwell 240 within the microwell array 230 is printed with its adjacent wells to form the microwell array 230 as a single, unitary structure. Each microwell opening is chamfered to enable the embryologist's injection pipette to be positioned to place an oocyte within the microwell 240 and retrieve a fertilized egg formed therein.

[0159] Figure 7A partial top view of a sperm platform 280 is shown. Two sperm platforms 280 are positioned adjacent to both sides of the microwell array 230. The sperm platform 280 is at the same height as the center plane of the oocyte. The alignment of the sperm platform and the oocyte microwell allows for picking up sperm, positioning and manipulating the oocyte to perform ICSI surgery in the same nominal focal plane. Thus, the embryologist needs to make minimal adjustments to ensure that the visualization of the surgery is in focus.

[0160] The sperm platform 280 is positioned approximately 2 mm from the microwell array 230. This separates the sperm from the oocyte to avoid contact that could result in uncontrolled fertilization (IVF).

[0161] By separating the sperm platform 280 from the microwell array 230, this also provides clearance for any angle positioning of the ICSI pipette when the embryologist is positioning the oocyte and performing ICSI.

[0162] The sperm platform 280 extends the full length of the microwell array 230. This enables sperm pickup to be performed by the embryologist in close proximity to the oocyte. This minimizes handling so that the embryologist can complete the ICSI procedure efficiently.

[0163] By providing two sperm platforms 280 on either side of the microwell array 230, embryologists can perform ICSI procedures based on their preference for left-handed or right-handed manipulation of the ICSI pipette.

[0164] Sperm platform 280 comprises a series of vertical sperm platform steps 290. Sperm platform steps 290 enable embryologists to carry out gravity-induced active sperm selection. In such an operation, sperm can be located at the highest step in sperm platform steps 290, while dead and inactive sperm remain at the lowest point of the dish.

[0165] Go to Figure 8 Adjacent to the microwell 240 on either side are two opposing microwell hemispheres 250. Each microwell hemisphere 250 has a hemispherical depression formed therein that extends circumferentially around the perimeter of the microwell wall, such that each microwell 240 has two opposing hemispherical depressions formed in its wall. The oocyte is positioned within the depressions, which provides optimal support for the oocyte during the ICSI procedure.

[0166] refer to Fig.10 , hemisphere 250 is positioned tangentially at the base of microwell 240. This allows an oocyte that has been placed at the base of a microwell to be easily moved by an ICSI pipette (via access channel 260) to the opening of the opposing hemisphere and then positioned into the full depth of the hemispherical cup.

[0167] Hemisphere 250 is formed as a depression within the wall thickness of microwell 240. An oocyte positioned at the opening is partially supported at its circumference, allowing the oocyte to be oriented with an ICSI pipette (via access channel 260) prior to full insertion into the hemispherical cup, where it is fully supported and prevented from rolling.

[0168] During the ICSI operation, the oocyte is positioned in a hemispherical depression in two possible hemispherical depressions. The embryologist can choose which of the two hemispherical depressions of his preference to allow his preference for left-hand or right-hand manipulation. The embryologist can enter the oocyte by entering the passage 260 with an injection pipette. During the ICSI operation, the oocyte is supported in the hemispherical depression, thereby minimizing the invagination of the ovum during the injection, and sperm is directly deposited in the ovum without the need to withdraw the cytoplasm into the injection pipette. Similarly, the micropore hemisphere 250 provides support for the oocyte during the withdrawal of the injection pipette. The diameter of the micropore hemisphere 250 is approximately between 100 microns and 200 microns, to support the expected range of the oocyte size including the zona pellucida.

[0169] refer to Fig. 9 , which provides a front cross-sectional view of the sperm platform 280, clearly showing the sperm platform step 290. The sperm platform step 290 allows active sperm to be located at the highest step in the sperm platform steps 290, while dead and inactive sperm remain at the lowest point.

[0170] Fig.10 A front cross-sectional view of micropores 240 is shown. Each micropore 240 includes two opposing micropore hemispheres 250 and two opposing access channels 260. The access channels 240 allow embryologists to insert an injection pipette laterally into the hole to perform an ICSI procedure. The size of the channel is set to be as small as possible to maximize the surface contact of the hemispheres for optimal support of the oocyte. The access channels 240 have chamfered openings formed therein to allow the injection pipette to enter the micropores 240 and micropore hemispheres 250 and to allow the micropipette to be angled to adjust the positioning of the oocyte prior to injection.

[0171] While the opposing access channels 260 primarily provide options for the embryologist's preference for left-handed or right-handed manipulation, the symmetry also has additional functionality. The second access channel facilitates the removal of air bubbles when the microwell 240 is filled with culture medium, and additionally, it prevents back pressure from building up behind the oocyte when the embryologist is performing an ICSI procedure.

[0172] During 2PP printing of the insert, identification of the microwell array 230 and individual microwells 240 is provided by local overpolymerization. This allows numbering to be created by changing the material refractive index. It is positioned at the center plane of the oocyte to allow identification by the embryologist when performing the ICSI procedure.

[0173] Fig.11 A top view of a substrate cover 320 is provided, which is shaped to cover the entire substrate 210 and remain supported on the top surface of the support platform 310. Fig.12 A side perspective view of a substrate cover 320 covering the substrate 210 is provided, showing the substrate cover 320 stepped upward on the support platform 310.

[0174] Figure 1 3 provides a side view of the substrate cover 320 and the support platform 310. Fig.14 A side cross-sectional view is provided showing the mating of substrate cover 320 onto substrate 210 to form a tight fit thereon.

[0175] Fig.15 A top view of the substrate cover 320 is provided and Fig.16 A side perspective view of substrate cover 310 is provided to illustrate the shape of substrate cover 320. Two rounded corners are provided in substrate cover 320, which are opposite to two beveled corners formed into substrate cover 320.

[0176] Fig.17 and Fig.18 The shape of the base dish cover 320 is also illustrated; Fig.17 is a side view, and Fig.18 It is a side cross-sectional view.

[0177] Embedded micro-hole design

[0178] Go to Figures 19 to 28 , Figure 1 9 shows a top view of a device substrate 400 having embedded microwells therein. Fig.19 As shown, the embedded microwell design includes an asymmetric placement of the microwell array 470 to allow sufficient functional space for the sperm preparation area 420. The embedded microwell design is simplified by providing a single planar sperm preparation area 420 embedded in the raised platform surface 430 (instead of the elliptical channel sperm platform of the raised design). The cover recess 440 provides a physical guide for the placement of the cover. Two opposite parallel sides of the cover recess 440 extend through the raised platform surface 430 and through two opposite base dish walls 450a and 450b. The cover recess 440 is angled at two recess corners 460a and 460b to provide guidance for accurate assembly of the cover.

[0179] The microwell array 470 is disposed within the perimeter of a recessed groove 480 formed within the raised platform surface 430. The groove 480 includes a base 490 and a wall 500 having four sides 500a, 500b, 500c and 500d, all of which are formed by the space created within the substrate 400. The base 490 and sides of the groove 480 are designed to contain a large volume of microwell culture medium for bathing the cellular contents of the microwells, the volume of which is sufficient to dilute any byproducts of cellular activity from the cells contained within the microarray.

[0180] The microwell array 470 includes spaced-apart microwell groups 510, each group having five microwells 520, such as Fig. 20 As shown. This configuration prevents the formation of bubbles in the culture medium contained in the groove 480. The groove 480 is defined by a multi-stage perimeter 530 to facilitate the seamless transition of the needle across the dish to each micropore 520. The extended end 540 provides a reference point to allow easy alignment and assembly. A label area 550 is provided on the base dish to allow customized identification of the base dish. Partially knurled edges 560a, 560b, 560c and 560c are provided on the four sides of the base dish to allow easy handling of the dish during transportation.

[0181] Fig.21 and Fig. 22 Provides a Fig.21 ) and sperm platform side ( Fig. 22 ) Cross-sectional side view of the embedded single-cell processing device observed. Fig.21 An embedded design of microwells 520, microwell groupings 510, microwell arrays 470, and grooves 480 is illustrated. The embedded fabrication is achieved by fabricating the substrate 400 so that the raised platform surface 430 is stepped downward at approximately 93° to form a cover recess 440 formed as a shoulder to maintain a cover (not shown) thereon in the same plane as the raised platform surface 430. Further steps downward form grooves 480 continuous with the cover recess 440 and the raised platform surface 430, the grooves 480 being fabricated in the substrate 400 and having walls formed at 90° to the cover recess 440.

[0182] Fig. 22 The provided view shows the sperm preparation area 420 formed on the same plane as the microwells 520. The cover 580 occupies the space above the cover recess 440 and forms a cover upper surface 590 that is in the same plane as the raised platform surface 430.

[0183] Fig.23A side cross-sectional view of the micropore 520 through the groove wall 500a and 500c is provided. The wall is made to add chamfers 590a and 590b to the base of the groove 480. When the dish is in use, this serves as a visual reminder for the user to indicate that the user's needle has entered the groove area. The groove 480 is recessed into the dish to ensure that the raised platform surface 430 is aligned with the focal position of the micropore. The base dish 400 includes a micropore support portion 600, which is located below each micropore 520 along the length of the micropore array 470 and is fully positioned within the periphery of the groove 480. The edges 602a and 602b of the micropore support portion 600 allow the base dish 400 to have good thermal contact with a heating table (not shown) commonly used during ISCI surgery.

[0184] Fig.24 The configuration of micropore 520 is illustrated in more detail. Each micropore is formed by a single curved outer wall 610 and a flat micropore base 620. Each micropore is opened from above to allow larger objects including oocytes or pipettes for transferring liquids directly to the micropores to enter the micropores. A circular cavity 630 is centrally arranged in the micropore and is flanked by two circular oocyte holding cups 640a and 640b on opposite sides. The diameter of the cavity 630 is approximately 250 microns to support an expected range of oocyte sizes of 100 microns to 200 microns (including the zona pellucida).

[0185] The wall forming the bore 630 includes two openings to form a continuous shape with the wall forming two oocyte holding cups. The inner holding cup 640a is oriented on the near side of the sperm preparation area and is formed by the inner holding cup wall 650a, which has a channel opening 660, and the channel opening 660 extends to the syringe channel 670 through the inner holding cup wall 650a. The syringe channel 670 is formed by a channel wall 680, which is formed through the single curved outer wall 610 of the micropore 520 to allow smaller objects such as microinjection equipment to enter the micropore. The shape of the channel wall 680 provides guidance for placing the microinjection needle to align with the center of the outer holding cup 640b. It is shaped to also allow the user to use a microinjector to direct the polar body of the oocyte before ICSI surgery.

[0186] The outer holding cup 640b is located at the far side of the sperm preparation area. The outer holding cup wall 650b is symmetrically formed with the inner holding cup wall 650a, and both are roughly in the shape of a hemispherical cylinder. The outer holding cup wall 650b has a discharge opening 680 formed through it. The discharge port 690 is located at the discharge opening 680 to ensure that the oocyte can be placed in the cavity formed in the outer holding cup 640b without generating the fluid resistance that affects the oocyte. And the cavity formed in the inner holding cup 640a maintains the oocyte therein, so as to be easy to remove the microinjector from the oocyte after microinjection.

[0187] The inner surface of the microwell 520 has been designed to achieve high quality visualization. The microwell base 620 adopts a flat shape to facilitate visualization of the oocyte during the microinjection procedure. The outer holding cup 640b adopts a semi-cylindrical cell stabilizing protrusion to facilitate visualization. The flat visualization lens 700 extends across the top opening 710 of the microwell array 520 to allow visualization of the microwell identification information during use. The numbers (not shown) embossed in the cutout grooves on each microwell array allow identification and tracking of the sample.

[0188] Fig.25 , Fig.26 , Fig. 27 and Fig.28 The exterior of the substrate 400 and the cover 720 are illustrated. Fig.25 The fit of the cover 720 over the cover recess 440 formed in the raised platform surface 430 and through two opposing base walls 450a and 450b is illustrated. The cover 720 includes two angled cutouts 730a and 730b at two corners of the cover 720. These cutouts correspond to the two recessed corners 460a and 460b to provide an accurate fit of the cover.

[0189] Fig.26 The ability to stack multiple substrates is illustrated, each substrate having a separate cover 720 mating therewith. An upper stacking lug 740 is located at each corner of the raised platform surface 430 to provide mating with the lower side surface 750 of the substrate. A lower stacking recess 760 is provided in the lower side surface 750 of the substrate to correspond to the shape of the upper stacking lug 740 to facilitate secure stacking of multiple substrates.

[0190] Fig. 27 An underside surface 750 of the substrate is illustrated that is shaped to facilitate thermal contact with a heating table during an ICSI procedure. Fig.28 Spacer pins 780 are shown located on the underside of the cover 720 to allow gas exchange during incubation.

[0191] Device production

[0192] The microarrays were printed using a NanoOne 1000 high-resolution 2PP 3D printer and a dedicated acrylate-based 2PP resin (“UpFlow”, UpNano GmbH, Vienna, Austria). After modeling the 3D parts in a CAD software (“Solidworks”, DassaultSystèmes Solidworks Corp., Waltham, Massachusetts, USA), the exported STL files were imported into Think3D software (version 1.7.3, UpNano GmbH). The parts were printed using a 10x objective operating in coarse mode or “voxel slicing mode” with a layer thickness of 5 μm, a line distance of 4 μm, and a power setting of 430 mW.

[0193] The microarrays were post-processed by washing in a propylene glycol monomethyl ether acetate (PGMEA ≥ 99.5%) bath for 3x10 minutes (min), 1x3 min and 1x2 min in a 2-propanol (99.9%) bath before being removed from the printed substrate with a razor blade and subsequently air-dried. The dried parts were cured for 5 min at 60% power in an ODS Cure Box (One Digital System, Incheon, Korea) prior to plasma treatment. The microarrays were treated with oxygen plasma for 60 seconds (sec) at a pressure of 780-820 mTorr using a PDC-002-HP chamber (Harrick Plasma, NY, USA). A small amount (~25-30123 mg) of UpFlow resin was used to adhere the plasma-treated arrays to commercial polystyrene culture dishes (Product No.: 16006, Vitrolife Pty. Ltd., Sydney, Australia), followed by a 2x5 min curing treatment in an ODS Cure Box.

[0194] After solidification, the dish was washed twice in 3 mL 96% ethanol (Chem-Supply Pty Ltd, 125 Gillman, SA, Australia) and rinsed twice in filtered (pore size 0.22 micron; Millipore, Bedford, MA, USA) 5% 7X detergent (MP Biomedicals Australasia, Seven Hills, NSW, Australia). The dish was then rinsed three times in Milli-Q water to remove residual detergent and left to dry overnight on a hot plate set at 37.5°C (Ratek Instruments Pty Ltd, VIC, Australia). In each washing step, the wash solution was pipetted onto the microarray using a P1000 micropipette to rinse the wells.

[0195] For human oocytes, the size of the hemispheres was adjusted to accommodate the larger size of oocytes before printing the microarrays. Thereafter, printing, post-processing, and washing were performed as described above.

[0196] Previous toxicity testing of 2PP parts was shown to be non-toxic using a mouse embryo assay (MEA) (IVF VET Solutions, SA, Australia) including negative and positive controls (Yagoub SH et al. (2022), Yagoub S.H. et al. (2014) and McLennan HJ et al. (2023)). This assay was repeated in the current study and no toxicity was detected. The lid was made following the same parameters and cleaning protocol detailed above.

[0197] Method for using single cell processing device

[0198] The following method can be easily adapted to either embedded or raised microarray designs.

[0199] Prepare the substrate dishes by labeling them and obtaining PVP, culture oil, p10 and p20 pipettes, pipette tips, serological pipettes, and serological pipettes and working under a laminar flow hood.

[0200] Add approximately 10 microliters of PVP from the 2PP part in a strip parallel to the microarray on the available side. Add 30 microliters of culture medium to the groove on the injection well side of the microarray.

[0201] The substrate dish is positioned so that the syringe groove mark faces the preferred syringe side of the user. Then remove the cap. About 2 millimeters (mm) from the 2PP microarray, 10 microliters of PVP are added to the substrate dish with a p10 pipette. The PVP drop is stretched along the length of the microwell array. 20 microliters of culture medium are slowly added to the syringe side of the groove with a p20 pipette. Repeat this step until a total of 30 microliters of culture medium are added to the dish. The culture medium should be diffused around the entire groove of the microwell array.

[0202] 6 mL of oil was placed on the section of the dish away from the PVP and microwell array. The cap was placed back on the dish. The dish was then equilibrated by placing it in an incubator.

[0203] The cryopreserved sperm is warmed and then prepared by centrifugation (washing) to remove semen and other preservatives. The washed sperm is loaded onto the substrate using a 300 microliter processing pipette, a balanced dish, and a dissecting microscope with a heating table and / or a heating bed. Work under a dissecting microscope with a heating table and / or in a heating bed. Gently stir the sperm sample by inversion to achieve uniform dispersion of the sperm in the solution. Use a 300 microliter processing pipette to collect sperm aliquots under sterile conditions. Then, by pressing the processing pipette evenly along the PVP stretch, the sperm sample is slowly distributed onto the substrate PVP strip so that the sperm is evenly distributed.

[0204] By obtaining the culture dish with the oocyte after the cumulus (denuded), the oocyte is loaded into the micropore of the base dish. Under the dissecting microscope with a heating stage and / or in a heating bed, the magnification is set to 3x to amplify, so as to observe five holes at a time. Before collecting the oocyte, the surface bubbles must be removed first. Use a processing pipette to aspirate five oocytes and move them to the base dish so that a single oocyte is deposited in each micropore. Repeat this step until all oocytes are loaded into the micropore. Then, the base dish that can be loaded with the oocyte is moved to the microscope stage for performing ICSI surgery.

[0205] If desired, a mock injection with debris or blue beads can be performed at this stage. Obtain an injection pipette and tilt the syringe slightly for holding the sperm. Set the syringe to position 1 (if the workstation allows) by focusing the syringe with the meniscus of the sperm and PVP droplet. Raise the syringe slightly and move the stage to the microport and focus on the microport and egg. Bring the syringe into the same focal plane as the egg and place it parallel to the injection slot of the microport. Push the syringe to slide through the injection slot and gently move the oocyte into the hemisphere with the polar body correctly oriented. Pull the syringe back and slide it toward the syringe slot while keeping it parallel to the slot. Gently slide the syringe out of the oocyte and then gently push it back into the microport with the syringe tip (to aid unloading later).

[0206] Move the stage to shift the focus to the next microwell. All polar bodies are oriented in all microwells. The syringe must be positioned above the PVP strip and changed to position 1 or adjusted to be aligned with the sperm. Select, secure and gently aspirate a single sperm. After raising the syringe, move the microscope stage to focus on the middle of the oocyte in the first microwell. Inject the sperm into the oocyte. Repeat these steps until all oocytes are injected.

[0207] As a variation, the injection pipette can be loaded with more than one sperm (nominally three or so), and a continuous injection sequence can occur, which further reduces the time. This "sequential injection" process is known in the art as "shotgun injection" (shotguninjection).

[0208] Raise the arm of the syringe to provide space for the dish to be removed. Remove the dish to the dissecting microscope for unloading the post-operative oocytes. Rinse the oocyte handling pipette tip in the wash dish. Aspirate five post-operative oocytes from the base dish by holding the pipette at 80 °. Pipette these post-operative oocytes around the dish until clean. Aspirate the oocytes and place in the culture dish. Repeat these steps for each oocyte.

[0209] Results of validation tests and pilot studies

[0210] Oocyte collection and maturation

[0211] Porcine ovaries from circulating gilts and sows were collected from a local slaughterhouse and transported to the laboratory in phosphate buffered saline (PBS) in a thermos at 30°C to 35°C. The ovaries were cleaned with warm saline and placed in a beaker in a water bath set to 37°C. Transparent antral follicles between 3mm and 6mm were aspirated into a 9mL Greiner No Additive Vacuette tube (Interpath Services, Somerton, VIC, Australia) with a 21-gauge needle at constant suction (1 L / min). Cumulus-oocyte complexes (COCs) with several layers of cumulus cells were isolated from the aspirate. Once pooled, COCs were washed three times and placed in groups of 50 in 4-well NUNC dishes (Thermo Fisher) in 500 L of IVM medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 IU / mL equine chorionic gonadotropin (Folligon MSD, Australia), 10 IU / mL human chorionic gonadotropin (hCG, Chorulon MSD, Australia), 5 μg / mL insulin, 10 ng / mL epidermal growth factor (EGF), 1 mM cysteamine, 100 μg / mL sodium pyruvate, 75 μg / mL penicillin-G, 50 μg / mL streptomycin sulfate, and 10% filtered sow follicular fluid under mineral oil (Origio, Sydney, NSW, Australia). The cells were incubated at 38.5°C in 5% CO in air. 2 The oocytes were allowed to mature for approximately 40 hours in a humidified environment.

[0212] At the end of the maturation period, morphologically normal COCs were isolated using a small-bore glass pipette (Rowe Scientific, Lonsdale, SA, Australia). Groups of 50-60 COCs were placed into individual 5 mL round-bottom tubes containing IVM medium without any hormones for transport in an incubator (CryoLogic, Blackburn, VIC, Australia) at 37°C until the time of de-cumulus removal, approximately 1 hour after maturation had ceased.

[0213] Intracytoplasmic sperm injection (ICSI)

[0214] For C-ICSI, a 5 microliter drop of 7% polyvinylpyrrolidone (PVP; OrigioAustralasia Pty Ltd, CooperSurgical, Denmark) was used to print an injection pipette, and seven 5 microliter drops of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered NCSU23 (ART Lab Solutions Pty Ltd, Adelaide, SA, Australia) were placed into a Vitrolife ICSI dish in a 3x3 droplet grid. 5 microliters of HEPES-buffered NCSU23 were added to the central PVP droplet to make a 3.5% PVP solution to partially slow the motility of sperm. The dish containing the microarray differed in the arrangement of the culture medium, including two 5 microliter strips of 7% PVP placed on the left side of the microarray, with 5 microliters of HEPES-buffered NCSU23 added to the PVP droplet closest to the array for picking up sperm. 20 microliters of HEPES-buffered NCSU23 were pipetted over and around the microarray grooves. All dishes were covered with 5 ml of MEA test paraffin oil (Merck Group, Macquarie Park, NSW, Australia) and thermally equilibrated to 38.5°C for a minimum of 3 hours.

[0215] Freshly collected and diluted semen was purchased from a local boar farm (Sabor, Clare, SA, Australia). Semen was balanced to room temperature from storage temperature (18°C) and centrifuged at a speed of 300g for five minutes. The supernatant was discarded, and the remaining precipitate was resuspended in 10mL of warm sperm cleaning medium (Medium 199, supplemented with 0.1mg / mL sodium pyruvate, 0.9mg / mL calcium lactate, 0.075mg / mL penicillin G, 0.05mg / mL streptomycin sulfate and 10% fetal bovine serum (FBS)). Then, the resuspended precipitate was centrifuged and further suspended in the sperm cleaning medium. Sperm was further diluted with warm HEPES-buffered NCSU23 to obtain a final concentration of ~5x106 sperm / mL.

[0216] Mature COCs were treated with 0.1 mg / mL hyaluronidase in HEPES-182-buffered NCSU23 for one minute and then gently pipetted using a Flexipet pipette tip with an inner diameter (ID) of 130 μm to 140 μm and a Cook Adjustable Handle (Cook Medical Pty Ltd, Eight Mile Plains, QLD, Australia) to remove cumulus cells. After cumulus removal, oocytes were washed three times in HEPES-buffered NCSU23 and assessed for suitability and the presence of a single polar body for selection for ICSI surgery.

[0217] Pipette setup for conventional ICSI surgery vs. microarray surgery was timed because microarray requires only one injection pipette, whereas conventional ICSI requires a holding pipette and an injection pipette. This includes the time spent positioning the pipette on the medium drop and the pipette in the oil-submerged medium. Two ICSI workstations were utilized to allow operators to perform experiments simultaneously and to avoid oocyte aging. The Nikon ICSI station utilized a Nikon TE2000 Eclipse inverted microscope (Nikon Corporation, Tokyo, Japan), combined with an Eppendorf micromanipulator (Eppendorf, Hamburg, Germany) and an air microinjector (Eppendorf) for a 25 μm ID holding pipette, and an oil microinjector (Eppendorf) for a 6 μm ID injection pipette, both from ICSION (Thebarton, SA, Australia). The second Olympus station included an Olympus IX83 inverted microscope (Evident Corporation, Tokyo, Japan) and a Narishige ON4 micromanipulator (Tokyo, Japan) and Narishige syringes (IN-21 for oil and IN-9B for air). The order of treatments was randomized for each operator for each replicate. The operators used a combination of a 10× objective and a 2× magnifier on the Olympus microscope and a 20× objective on the Nikon microscope to visualize sperm and perform ICSI procedures.

[0218] For conventional ICSI, 10-20 mature oocytes were transferred to a 5 microliter droplet of HEPES medium using a 170 micron flexible pipette in a custom-made modified heated crib set to 38.5°C before the ICSI procedure. Approximately 3 microliters of diluted sperm were pipetted into a 3.5% PVP droplet using a flexible pipette. The dish was carefully transferred to either an inverted microscope on a heated stage set to 38.5°C. For each injection, a single active sperm with good morphology was selected and fixed in a 3.5% PVP sperm droplet. The oocyte was held in position with a holding pipette, where the polar body was rotated to the 6 o'clock or 12 o'clock position using an injection pipette. The injection pipette with the sperm at the tip was introduced into the oocyte at the 9 o'clock position, and the ooplasm was aspirated into it to ensure that the egg membrane was broken. The injection time was recorded from the time the oocyte was focused and the holding pipette entered the droplet to the time the oocyte was released from the holding pipette.

[0219] Invagination was scored based on previous literature from Danfour et al. (2021) by estimating the grade of oocyte invagination when the injection pipette entered the oocyte at the 9 o'clock position for both treatments, with 1 being the lowest and 3 being the highest. The number of manual adjustments between controllers required for each single oocyte injection was quantified by video recording. An adjustment was defined as the operator's hand leaving one controller and then using a separate controller.

[0220] For microarray testing, 10-20 mature oocytes were transferred to microarray microwells containing 20 microliters of HEPES medium in the grooves with a 1 70 micron ID flexible pipette in a heated bed. All oocytes were manipulated into the injection hemisphere in each microwell using an injection pipette and oriented to allow the 224 polar body to be located at the 6 o'clock or 12 o'clock position. The time taken to orient 10 oocytes was recorded and the average was calculated for each oocyte. Sperm was loaded into a syringe according to the above conventional ICSI, the syringe entered the microwell through the injection groove, the injection was performed on the correctly oriented oocyte, and the injection pipette was pulled back through the injection groove, thereby gently removing the pipette from the oocyte before the oocyte was gently pushed back to the middle of the hole with the pipette. The time taken for the injection to focus on the injection pipette from the oocyte and the injection pipette in the pipette groove until the injection pipette was removed from the oocyte during the microarray surgery was recorded. If injections were unsuccessful for either conventional ICSI or microarray ICSI, the oocytes were excluded from culture.

[0221] Sham injections were performed using microarray ICSI to assess parthenogenetic activation and subsequent embryonic development. For sham injections, oocytes were injected with 7% PVP without sperm injection. Uninjected oocytes were also placed in culture alone as a control.

[0222] Embryo culture

[0223] The fertilized eggs, sham injections and controls are grouped in 5 groups, and cultured to the 4th day in the humidified incubator of 38.5 ℃ (6%CO2, 7%O2, balanced with N2) in the 50 microliters of modified NCSU-23 droplet in the 60mm culture dish (Falcon product number: 351007) covered with ~8mL MEA test paraffin oil. On the 4th day of cultivation, the cleavage situation is scored, as well as the survival rate after injection and the fragmentation degree of the embryo without cleavage and the embryo with cleavage. When the perivitelline space greater than 30% is occupied by small, membrane-bound vesicle bodies, fragmentation is just classified as having occurred. All surviving embryos are moved to the dish after balance, wherein there is modified blastocyst NCSU-23 (ART Lab Solutions), supplemented with 10%FBS and covered with paraffin oil. Blastocysts are scored on the 6th day of cultivation.

[0224] Differential staining

[0225] Immediately after scoring of blastocysts on day 6, blastocysts generated from four replicates were collected for staining. Blastocysts were washed in warmed (37°C) HEPES-buffered NCSU23 for differential staining. The zona pellucida was removed by incubation with 0.5% pronase dissolved in PBS for approximately 2 minutes, followed by washing in HEPES-MEM-PVA (minimum essential medium and 1 mg / mL polyvinyl alcohol (PVA)), followed by further incubation in 10 mM trinitrobenzene sulfonic acid (TNBS) in HEPES-MEM-PVA on ice for 1 5 minutes. Blastocysts were then removed and further washed in HEPES-MEM-PVA, followed by incubation of blastocysts with 0.2 mg / mL anti-dinitrophenol BSA in HEPES-MEM-PVA for 10 minutes at 38°C. The blastocysts were then washed again in HEPES-MEM-PVA and incubated for 10 minutes in HEPES MEM-PVA containing ~10% guinea pig complement serum, 0.01 mg / mL propidium iodide and 0.05 mM bisbenzimide (Hoechst 33258). After washing, as a post-incubation step, the blastocysts were transferred to HEPES-MEM-PVA, then dehydrated in 100% ethanol (Chem-Supply Pty Ltd, Gillman, SA, Australia) and mounted on a microscope slide in 2 microliters of glycerol under a coverslip. Fluorescent images of the nuclei of the blastocysts were captured using an epifluorescence inverted microscope (Eclipse TS100; Nikon Corporation; excitation filter 330-380 nm and barrier filter 420 nm). The number of blue inner cell mass (ICM) and pink to red trophectoderm (TD) cells was counted after imaging by researchers who did not know the treatment, a total of 265.

[0226] Human microarray ICSI validation

[0227] Vitrified human oocytes (n=18, from 2 patients) (University of Adelaide Ethics Approval No. H-2023-082) consenting to the study were obtained from a local IVF clinic and warmed in the laboratory according to the SAGE vitrification warming kit instructions (OrigioAustralasia Pty Ltd). For C-ICSI, a 5 microliter droplet of 7% PVP (OrigioAustralasia Pty Ltd) was placed in the center of the Vitrolife ICSI dish, surrounded by eight 5 microliter drops of GMOPS PLUS (Vitrolife Pty Ltd). The microarray dish included a 5 microliter strip of 7% PVP, which was placed along the left side of the microarray, with 20 microliters of GMOPS PLUS being pipetted above and around the microarray grooves. All ICSI dishes were covered with 5 mL paraffin oil (Merck Group) and equilibrated at 37°C for a minimum of 3 hours.

[0228] Oocytes were loaded into conventional ICSI dishes or microarrays using 170 micron Flexipet pipette tips, as described for porcine oocytes. 4 micron Tetraspeck microspheres (Thermofisher) centrifuged and washed in GMOPS PLUS culture medium were loaded into 7% PVP droplets in each culture dish instead of sperm as a visual substitute for sperm injection into human oocytes. Single beads were aspirated from the PVP droplets into a 5 micron ID injection pipette (OrigioAustraliaasia Pty Ltd). The injection process was performed according to conventional porcine ICSI and microarray ICSI. The time of bead injection and the number of manual adjustments were recorded, as for porcine ICSI. After successful injection, the cleavage of the oocyte was observed after 90 minutes of culture. If not cleaved, it was reinjected to provide further timing and manual adjustment data. After obtaining the reinjection data, the oocyte was immediately discarded as a biohazard material.

[0229] Statistical analysis

[0230] All statistical analyses were performed using GraphPad Prism v9.5.1. All data were tested for normality to determine whether parametric or nonparametric analysis was applied. All binomial data were arcsine transformed to make the data continuous for statistical comparisons, but were plotted as binomial data. Paired comparisons were used to account for repeated effects where appropriate. The statistical tests used, number of replicates, sample size, and p-values ​​are reported separately in each legend. Data are expressed as Mean / -SEM, and statistical significance was accepted at P<0.05.

[0231] Study Results

[0232] Impact on preparation time

[0233] Fig.29 (a) shows that dish preparation time for conventional ICSI and microarray ICSI is comparable. Fig.29 (b) By removing the holding pipette, the pipette setup time is cut in half. Loading the microwell increases the processing time for loading and unloading oocytes by 15 to 20 seconds ( Fig.29 (c) and Fig.29 (d)), but this is much smaller than the increase in time taken for pipette setup (>100 seconds).

[0234] Impact on ICSI surgery

[0235] like Fig.30 As shown in (a), the average surgical time spent on handling and injecting each oocyte for microarray ICSI was reduced compared to conventional ICSI. The time spent on the injection procedure for each oocyte was reduced by more than half ( Fig.30 (b) ), and even when the average polar body orientation time per oocyte was taken into account, the reduction in time for microarray ICSI was still significant ( Fig.30 (c) and Fig.30 (d)). Because the holding pipette was removed, the manual adjustments required to perform microarray ICSI were reduced by two-thirds, which can be tracked by the number of manual adjustments between different controllers ( Fig.31 The levels of invagination between conventional ICSI and microarray ICSI were comparable ( Fig.32 ).

[0236] Effect on development speed

[0237] The rate of embryonic development remained consistent between treatments, and the fragmentation, survival, and cleavage rates were comparable between the two ICSI techniques ( Fig.33 (a)- Fig.33 (c)). However, the blastocyst rate was significantly increased in the microarray ICSI group compared with conventional ICSI ( Fig.33 (d)). The cell numbers of ICM and TD were comparable between blastocysts produced by conventional ICSI and microarray ICS, as shown in Table 1 below.

[0238] Table 1: Comparison of blastocyst cell numbers between conventional ICSI and microarray ICSI

[0239]

[0240] Validation of translation into human oocytes

[0241] After adjusting the hemisphere size, the microarray ICSI device is also suitable for performing microinjection on human oocytes. No immediate cleavage was observed after the first round of bead injection, and in 1 8 oocytes, one oocyte was excluded from re-injection due to cleavage after 90 minutes. Consistent with the results of pigs, compared to conventional ICSI, the injection time and manual adjustment of microarray ICSI are reduced (Figure 34 (a) and Figure 34 (b)).

[0242] Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0243] It will be understood that if a person skilled in the art considers the interpretation of the term "fastener" or "fastening", "coupling" or "sealing" to be functionally interchangeable with other terms, then the term can be used interchangeably when used alone or in combination with other terms such as "device". Further, the use of one of the above terms does not hinder the interpretation when another term is included.

[0244] As described herein, various devices and components of the devices can be provided in various sizes and / or dimensions as required. Suitable sizes and / or dimensions will vary according to the specifications or fields of use of the connecting components and can be selected by those skilled in the art.

[0245] It will be appreciated that features, elements and / or characteristics described with respect to one embodiment of the disclosure may be used with other embodiments of the invention as appropriate.

[0246] Although the preferred embodiments of the present disclosure have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the disclosure and the accompanying claims.

[0247] It will be understood that when an element or layer is referred to as being “on” or “in” another element or layer, the element or layer can be directly on or within the other element or layer or on or within intervening elements or layers. In contrast, when an element is referred to as being “directly on” or “directly within” another element or layer, there are no intervening elements or layers present.

[0248] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0249] It will be understood that although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another region, layer or section. Thus, without departing from the teachings of the present disclosure, a first element, component, region, layer or section may be referred to as a second element, component, region, layer or section.

[0250] For ease of description, spatial relative terms such as "lower", "upper", "higher", "higher", "top", "bottom", "left", "right", etc. may be used herein to describe the relationship between an element or feature illustrated in the figure and another (multiple) element or feature. It will be understood that in addition to the orientation depicted in the drawings, spatial relative terms are also intended to cover different orientations of structures in use or operation. For example, if the device in the drawings is turned over, the element described as "lower" relative to other elements or features will be oriented as "upper" relative to other elements or features. Thus, the exemplary term "lower" can cover both upper and lower orientations. The device can be oriented in other ways (rotated 90 degrees or other orientations), and the spatial relative descriptors used herein should be interpreted accordingly.

[0251] The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that when the terms "comprises", "comprises" and / or "comprising" are used in this specification, the presence of the features, integers, steps, operations, elements and / or parts is specified, but the presence or addition of one or more other features, integers, steps, operations, elements, parts and / or groups thereof is not excluded.

[0252] Embodiments of the present specification are described herein with reference to, for example, figures and / or cross-sectional illustrations, which are schematic illustrations of preferred embodiments (and intermediate structures) of the present specification. As such, variations in the shapes illustrated are expected due to, for example, manufacturing techniques and / or tolerances. Thus, the embodiments of the present specification should not be construed as limited to the specific shapes of the components illustrated herein, but should include deviations in shapes due to, for example, manufacturing.

[0253] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as those generally understood by those skilled in the art to which this specification belongs. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0254] Any reference in this specification to "one embodiment," "an embodiment," "an example embodiment," etc. means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the specification. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with any embodiment, it is within the purview of those skilled in the art to implement and / or use such feature, structure, or characteristic in connection with other embodiments.

[0255] The embodiments are also intended to comprise or otherwise encompass methods of using and methods of making any or all of the elements disclosed above.

[0256] Although the present invention has been described above with respect to specific embodiments, it will be understood that the present invention is not limited to these disclosed embodiments. After reading the teachings of the present disclosure, many modifications and other embodiments of the present invention will occur to those skilled in the art to which the present invention belongs, and these modifications and other embodiments are intended to and are covered by the present disclosure and the appended claims.

[0257] All publications mentioned in this specification are incorporated herein by reference. Any discussion of documents, acts, materials, devices, articles and the like contained in this specification is intended solely to give a context to the present invention. This is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

[0258] Rather, the scope of the present invention should be determined by proper interpretation and construction of the appended claims and their legal equivalents, as understood by those skilled in the art in light of the disclosure in the specification and drawings.

[0259] Citation List

[0260] Data on IVF clinics show wide variation in success rate,BMJ 2002;325doi:https: / / doi.org / 10.1136 / bmj.325.7362.460 / e(Published 31 August 2002).

[0261] Boulet,S.L.,Meht,A.,Kissin,D.M.,Warner,L,Kawwass,J.F.and Jamieson,D.J.(2015)Trends in use of and reproductive outcomes associated withIntracytoplasmic Sperm Injection.Obstet Gynecol Surv.70:325-6.

[0262] Palermo,G.,Joris,H.,Devroey,P.and Van Steirteghem,A.C.(1992)Pregnancies after intracytoplasmic injection of single spermatozoon into anoocyte.Lancet 340:17-18.

[0263] Joris,H.,Nagy,Z.,Van de Velde,H.,De Vos,A.and Van Steirteghem,A.C.(1998)Intracytoplasmic sperm injection:laboratory set-up and injectionprocedure.Human Reproduction 13Suppl.1:76-86.

[0264] Latham,K.E.Stress signaling in mammalian oocytes and embryos:A basisfor intervention and improvement of outcomes.Cell Tissue Res.2016,363,159-167.

[0265] Gilchrist,R.B.,Lane,M.and Thompson,J.G.(2008)Oocyte-secretedfactors:regulators of cumulus cell function and oocyte quality Human ReproductionUpdate 1 4:159-1 77.

[0266] Tiegs,A.W.and Scott,R.T.(2020)Evaluation of fertilization,usableblastocyst development and sustained implantation rates according tointracytoplasmic sperm injection operator experience.Reprod Biomed Online.41:19-27.

[0267] Fernandez,J.,Pedrosa,C.,Vergara,F.,Nieto,A.I.,Quintas,A.,et al.(2020)A new oocyte-holding pipette for intracytoplasmic sperm injection withoutcytoplasmic aspiration:An experimental study in mouse oocytes.Reprod Biol 20:584-588.

[0268] Ma,S.,Wang,P.,Zhou,W.,Chu,D.Zhao,S.,Fu,l.and Li,F.(2020)A modifiedholding pipette for mouse oocyte fertilization.Theriogenology 141:142-145.

[0269] Zander-Fox,D.,Lam,K.,Pacella-Ince,L,Tully,C.,Hamilton,H.,Hiraoka,K.,et al.(2021)PIEZO-IC SI increases fertilization rates compared with standardICSI:a prospective cohort study.Reprod Biomed Online.43:404-12.

[0270] Danfour M.A.,Elmahaishi M.S.,(2010)Human oocyte oolemmacharacteristic is positively related to embryo developmental competence arterICSI procedure.Middle East Fertility Society Joumal.2010;15:269-73.

[0271] Yagoub,S.H.,Thompson,J.G.,Orth,A.,Dholakia,K.,Gibson,B.C.,andDunning,K.R.(2022)Fabrication on the microscale:a two-photon polymerizeddevice for oocyte microinjection.J Assist Reprod Genet.2022;39:1503-13.

[0272] Yagoub,S.H.,Lim,M.,Tan,T.C.Y.,Chow,D.J.X,Dholakia,K,Gibson,B.C.,Thompson,J.G.,and Dunning,K.R.(2014)Vitrification within a nanoliter volume:oocyte and embryo cryopreservation within a 3D photopolymerized device.JAssist Reprod Genet.2022;39:1997-2014.

[0273] McLennan,H.J.,Blanch,A.J.,Wallace,S.J.,Ritter,L.J.,Heinrich,S.L.,Gardner,D.K.,Dunning,K.R.,Gauvin,M.J.,Love,A.K.,and Thompson,J.G.Nano-literperfusion microfluidic device made entirely by two-photon polymerization fordynamic cell culture with easy cell recovery.Sci Rep.2023;13:562.

Claims

1. A single cell processing device for processing a single cell during a microinjection procedure, the single cell processing device include: A substrate having an upper surface and a lower surface; A microwell array, the microwell array comprising one or more microwells, the one or more microwells comprising: at least one micropore wall, the at least one micropore wall defining an upper micropore opening and a central aperture, the at least one micropore wall having two raised cell stabilizing protrusions formed therein, each of the cell stabilizing protrusions defining a cavity positioned relative to one another in a common horizontal plane, the cell stabilizing protrusions comprising a first cell stabilizing protrusion defining a channel opening at a periphery of an entry channel extending outwardly through the micropore wall, and a second cell stabilizing protrusion defining a vent opening, the entry channel having a channel base aligned in a horizontal plane with a center point of the second cell stabilizing protrusion; as well as A groove, for maintaining a fluid therein, is located on the upper surface of the substrate and extends around the periphery of the microwell array.

2. The single cell processing device according to claim 1, in, The groove exchanges fluid with the one or more micropores via the entry channel, the groove having an inner wall on the proximal side of the microarray and an outer wall on the distal side of the microarray, the outer wall vertically extending to a height higher than the channel base of the entry channel, and wherein the ventilation opening is positioned as a whole higher than the channel base of the entry channel.

3. The single cell processing device according to claim 1, in, The first cell stabilizing protrusions and the second cell stabilizing protrusions are elliptical in a horizontal plane.

4. The single cell processing device according to claim 3, in, The diameter of the first cell stabilizing protrusion and the diameter of the second cell stabilizing protrusion are in the range of 10 micrometers to 500 micrometers.

5. The single cell processing device according to claim 3, in, The diameter of the first cell stabilizing protrusion and the diameter of the second cell stabilizing protrusion are in the range of 100 microns to 200 microns.

6. The single cell processing device according to claim 1, in, The at least one microwell wall is cylindrical in shape and the one or more microwells include a microwell base, wherein the at least one cylindrical microwell wall and the microwell base define the central pore.

7. The single cell processing device according to claim 6, in, The at least one cylindrical micropore wall has a diameter that is greater than a diameter of the cell stabilizing protrusions.

8. The single cell processing device according to claim 6, in, The at least one cylindrical micropore wall has a diameter in the range of 0.1 micrometers to 1000 micrometers.

9. The single cell processing device according to claim 6, in, The at least one cylindrical micropore wall has a diameter in the range of 200 microns to 300 microns.

10. The single cell processing device according to claim 1, in, The entry channel includes two parallel channel walls, a channel base and an upper opening.

11. The single cell processing device according to claim 1, comprising a cover and one or more stacking connectors, wherein the one or more stacking connectors are located at the upper surface and the lower surface so as to connect multiple substrates when stacked.

12. The single cell processing device according to claim 6, comprising a sample support platform having a sampling edge at the upper surface of the base plate on the proximal side of the entry channel, in, The sampling edge is stepped or sloped with a gradient to align with the horizontal plane of the channel base.

13. A method for using the single cell processing device according to claim 12, for performing intracytoplasmic sperm injection surgery, The following steps are involved: a. obtaining the single cell processing device according to claim 1, b. adding culture medium to the groove of the single cell processing device, c. Obtain oocyte and sperm samples, d. adding the oocyte to the central hole of the microwell, e. placing the sperm sample on the sample support platform, f. Obtaining microinjection equipment, g. aspirating sperm from the sperm sample through the tip of the microinjection device, h. placing the tip through the entry channel of the micropore, i. moving the oocyte to the second cell stabilizing protrusion with the tip, j. microinjecting the oocyte with the sperm, k. Withdraw the microinjection tip by cradling the oocyte in the first cell-stabilizing protrusion while removing the microinjection tip separately.

14. A method for manufacturing the single cell processing device according to claim 1, comprising manufacturing the substrate and manufacturing the microarray and the grooves thereon.

15. A method for manufacturing a single cell processing device according to claim 1, comprising fabricating the microarray and the grooves in the substrate.

16. A method of manufacture according to claim 14 or 15 comprising a two-part production comprising a two-photon polymerisation printed part inserted into a polystyrene injection moulding.

Images