Single cell handling device.

Abstract

The present invention relates generally to apparatus for handling single cells or small cell clusters, and particularly to devices for handling single mammalian cells or mammalian cell clusters, as well as methods for making and using such devices. The device generally relates to a single cell handling device for handling single cells during a microinjection procedure, comprising: a base dish having an upper surface and a lower surface; and a microwell array including one or more microwells, the one or more microwells having at least one microwell wall defining an upper microwell opening and a central well, the at least one microwell wall having two cell stabilizing projections formed thereon, positioned opposite each other on a common horizontal plane and each defining a cavity; and a fluid-retaining moat disposed on the upper surface of the base dish and extending around the periphery of the microwell array.

Description

[Technical Field]

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

[0002] Advances in microscale tissue culture technology are enabling new therapies, products, and processes, many of which are still in their infancy. The potential benefits of improved cell culture and growth at the microscale are particularly significant in the field of reproductive medicine, where, due to the nature of the field, there are few alternatives to the in vitro culture and manipulation of individual autologous cells, which must be harvested, manipulated, and reintroduced into the patient.

[0003] Assisted reproductive technology through in vitro fertilization ("IVF") has become more accessible and has improved in recent years, resulting in an increasing number of patients taking advantage of it. Data published by the Human Fertilization and Embryology Authority (HFEA) shows that, overall, women who begin IVF treatment are more likely to result in a live birth than in the past. However, the success rates of IVF clinics vary widely between individual clinics, with some clinics achieving success rates as high as 46% and others achieving success rates as low as 10%.

[0004] The chances of success vary at each stage of the IVF process: oocyte retrieval, oocyte fertilization, early embryo development, embryo vitrification, and embryo transfer. The success of processes that require oocyte manipulation currently depends heavily on the skill of the embryologist.

[0005] Intracytoplasmic sperm injection (or "ICSI") is an assisted reproductive technology procedure performed in fertility clinics, primarily for the treatment of male factor infertility (Boulet et al. 2015). It is an alternative technique for fertilization of oocytes with sperm, replacing IVF procedures.

[0006] Unlike IVF, in which a sperm solution is incubated with an oocyte and one of the thousands of sperm binds to and penetrates the oocyte to fertilize it, the ICSI procedure differs in that a single sperm is injected directly into the oocyte's cytoplasm.

[0007] This technique, first described by Palermo et al. (1992), is used to treat male factor infertility, particularly in men with oligospermia and azoospermia, in which the number of sperm in the ejaculated semen sample is so low that the optimal concentration of motile sperm required for co-incubation with oocytes in IVF to maximize the chances of fertilization is not achieved.

[0008] The ICSI procedure relies on tools to manipulate the position of both the sperm and the oocyte and to inject the sperm into the oocyte. These tools include two micromanipulators: one used to position the oocyte and hold it in place with a finely elongated, heat-polished pipette, commonly referred to as the "holding pipette." The other is used to position a finely elongated "injection" pipette containing the sperm. Both pipettes are attached to a precisely controlled syringe-like "injector." The first controls the amount of negative and positive pressure to hold, position, and release the oocyte in the holding pipette. The second is used to control the capture and injection of the sperm in the injection pipette. This process and equipment are well known in the art and are described in detail in Joris et al. (1998).

[0009] In ICSI and other IVF processes, there is a risk of damage from the physical manipulation required for the introduction of sperm (whether intracytoplasmic or not). The risk of damage or impact from manipulation and handling, and therefore the success of ICSI and other IVF procedures, depends primarily on the following factors: a. The skill and discretion of the embryologist; b. The accuracy of the tools and equipment available to the embryologist; and c. Environmental conditions maintained in the laboratory as influenced by the embryologist (e.g., sterility, temperature, prevention of any type of contamination, including volatile organic compounds).

[0010] The present disclosure addresses the second problem mentioned above, namely the lack of precision in the tools and equipment available to embryologists.

[0011] In the absence of precise single-cell handling devices, holding and injection pipettes that utilize positive and negative pressure are required to hold, manipulate, and position oocytes.

[0012] In use, the control of the pressure used to form a vacuum seal between the oocyte and the holding pipette is not quantified by the embryologist. This leads to variability in the amount of negative pressure exerted on the oocyte during the ICSI procedure from procedure to procedure and from embryologist to embryologist. Oocyte deformation is usually caused by the suction exerted by the holding pipette, which displaces the oocyte cytoplasm within the pipette.

[0013] When handling oocytes with negative pressure, the zona pellucida often invaginates within the suction micropipette, causing stress and damage to the cell. Oocyte membrane invagination refers to the degree of cytoplasmic deformation that occurs when the injection pipette penetrates first the zona pellucida and then the oocyte membrane. Shear stress, which literally crushes the oocyte, is the main source of cellular stress in ICSI procedures.

[0014] Oocytes are normally "protected" by a layer of "nurse cells," or cumulus cells. It is well known that two-way communication occurs between the cumulus cells and the oocyte, which determines the quality of the oocyte (Gilchrist et al. 2008). It is widely known that removing cumulus cells before fertilization has a negative impact on the health of the oocyte (Gilchrist et al. 2008). In the ICSI procedure, the cumulus layer must be removed to allow unobstructed microscopic observation of the oocyte. This removal of cumulus cells is usually performed after fertilization in standard IVF treatments.

[0015] In ICSI, oocytes are positioned without a protective cell layer before fertilization. They then undergo various manipulations not performed in IVF, including exposure to microscope light, negative pressure applied by the holding pipette to maintain position, oocyte invagination caused by the injection pipette exerting point pressure against the holding pipette, aspiration of cytoplasm into the injection pipette to confirm complete insertion, and injection of a synthetic polymer (PVP) used to "delay" and trap sperm in the injection pipette.

[0016] According to Latham (2016), cellular stress often mediates a cellular response characterized by the activation of the unfolded protein response, which leads to protein unfolding and consequently cell apoptosis or necrosis.

[0017] Although functional control of the micromanipulator and injector has improved, the actual workflow steps remain the same as those originally developed in 1992, and extensive training is required to master the steps (Tiegs and Scott, 2020).

[0018] No improvements have been made to reduce the required technical skills or procedure time during ICSI. Attempts to improve ICSI outcomes have focused on either modifying the structure of the holding pipette (Fernandez et al. 2020; Ma et al. 2020) or introducing injection systems that rely on piezo motor functionality (Zander-Fox et al. 2021).

[0019] Zander-Fox et al. (2021) developed a PIEZO-ICSI technique that utilizes a piezoelectric actuator to achieve high-speed movement of the injection pipette, effectively enabling precise microdrilling of the zona pellucida. This technique reduces the need for applying physical pressure to the oocyte membrane and eliminates the need for agitation of the cytoplasm. Therefore, it may be a gentler form of microinjection, but it does not improve oocyte handling.

[0020] While there have been advances in the microinjection and visualization devices used in IVF treatments, there has been little progress in developing devices used to manipulate the cells or to hold them in a way that limits excessive manipulation or handling.

[0021] US Patent Application Publication No. 2021 / 0230525 (Alcaide, FV2021) describes a vacuum pipette for holding an oocyte and a method for intracytoplasmic sperm injection based on a long, narrow cylindrical body and a truncated cone-shaped constricted portion for holding the oocyte under vacuum pressure. While this vacuum pipette eliminates the need to aspirate the cytoplasm before microinjection, the vacuum pressure deforms the cell and does not prevent invagination of the oolemma during microinjection.

[0022] Similarly, Ma et al. (2020) proposed a trumpet-shaped modification of the traditional holding pipette used in human reproductive technology, which also does not address the issues of cell deformation during microinjection or invagination of the oolemma at the time of microinjection.

[0023] There is a need for a simpler, more precise oocyte and / or sperm cell handling device that eliminates the need for pressurized handling devices and ameliorates the problems associated with pressurized handling devices. Preferably, such a simple, precise oocyte and / or sperm cell handling device would be compatible with precision microinjection techniques such as PIEZO-ICSI.

[0024] Precision tools and equipment reduce the likelihood of an embryologist making an error that damages or shocks the sperm, oocytes, or embryos. Additionally, tools and equipment that reduce or eliminate physical intervention or manipulation by the embryologist reduce the likelihood of an event causing physical shock to the sperm, oocytes, or embryos.

[0025] The cell handling devices described herein maintain the physical stability of sperm, oocytes, and / or embryos within a dedicated culture environment, as needed. Embryologists can more easily and / or accurately handle sperm, oocytes, or embryos without the need for a pressure pipette device, thereby minimizing the risk of physical shock and / or biochemical stress to the cells. Stabilizing cells in culture can improve the success rate of ICSI procedures.

[0026] Additionally, oocytes are sensitive to changes in environmental factors such as temperature, osmolality, oxygen concentration, nutrient limitation, hyperglycemic conditions, and pH (Latham, 2016). The cell handling devices described herein may further reduce biochemical stress on sperm, oocytes, or embryos by reducing fluctuations in the in vitro environment, which is optimized as needed.

[0027] Eliminating the need for vacuum equipment and simplifying the cell handling process also offers cost benefits by reducing labor and capital expenditures and training time (Yagoub et al. 2022). This not only improves clinic profitability but also makes ICSI technology more accessible to less affluent communities. In new clinics, the improved cell handling process reduces the cost burden of traditional equipment required to perform ICSI, benefiting both clinics and patients.

[0028] The cell handling device described herein not only overcomes the challenges associated with successful fertilization and culture of developing embryos, but may also be applicable to the culture of other cell lines that are sensitive to gradual changes in the culture environment. Summary of the Invention

[0029] In broad aspects, embodiments described herein feature a single cell handling device for handling single cells during a microinjection procedure, the single cell handling device comprising: a base dish having an upper surface and a lower surface; and a microwell array including one or more microwells, the one or more microwells comprising at least one microwell wall defining an upper microwell opening and a central well, the at least one microwell wall having two cell stabilization protrusions formed thereon, the two cell stabilization protrusions positioned opposite each other in a common horizontal plane and each defining a cavity, the cell stabilization protrusions including a first cell stabilization protrusion defining a channel opening at a periphery of an access channel extending outwardly through the microwell wall and a second cell stabilization protrusion defining an exhaust opening, the access channel having a channel base aligned with a center point of the second cell stabilization protrusion in a horizontal plane, and comprising a fluid-retaining moat disposed on the upper surface of the base dish and extending around the periphery of the microwell array.

[0030] Preferably, the moat of the single cell handling device is in fluid communication with one or more microwells via the access channel, the moat having an inner wall proximal to the microarray and an outer wall distal to the microarray, the outer wall extending vertically to a position higher than the channel base of the access channel, and the exhaust opening is entirely positioned higher than the channel base of the access channel.

[0031] Preferably, the first and second cell stabilization protrusions have an elliptical shape in the horizontal plane. Preferably, the diameter of the first and second cell stabilization protrusions is in the range of 10 microns to 500 microns. Alternatively, the diameter of the first and second cell stabilization protrusions is in the range of 100 microns to 200 microns. The size of the protrusions can be adapted to accommodate a range of sizes of various cells or cell clusters.

[0032] Preferably, the at least one microwell wall is cylindrical and one or more microwells comprise a microwell base, the at least one cylindrical microwell wall and the microwell base defining the central well.

[0033] In further embodiments, the at least one cylindrical microwell wall has a diameter greater than the diameter of the cell stabilization projections. In particular, the at least one cylindrical microwell wall has a diameter in the range of 0.1 micrometers to 1000 micrometers. Alternatively, the at least one cylindrical microwell wall has a diameter in the range of 200 micrometers to 300 micrometers.

[0034] Preferably, the access channel comprises two channel walls, a channel base, and an upper opening.

[0035] Preferably, the single cell handling device comprises a cover and one or more stack connectors arranged on the upper and lower surfaces to connect multiple base dishes together when the base dishes are stacked.

[0036] In various embodiments, the sample support includes a sample support platform on the upper surface of the base plate having a sampling edge proximal to the access channel, the sampling edge being stepped or sloped to align with the horizontal surface of the channel base.

[0037] Preferably, the method of using said single cell handling device in performing an intracytoplasmic sperm injection procedure comprises: Obtaining the single cell handling device of claim 1; adding culture medium to the moat of the single cell handling device; Obtaining oocyte and sperm samples; adding the oocyte to the central well of the microwell; placing the sperm sample on the sample support; Obtaining a microinjection device; aspirating sperm from the sperm sample through the tip of the microinjection device; placing a tip through the access channel of the microwell; shifting the oocyte with the tip onto the second cell stabilizing protrusion; microinjecting the sperm into the oocyte; and withdrawing the micro-injection tip by removing only the micro-injection tip while the oocyte is held by the first cell stabilization projection.

[0038] A preferred method of manufacturing a single cell handling device may include creating the base dish and fabricating the microarray and the moat thereon, or an alternative manufacturing method may include fabricating the microarray and the moat within the base dish.

[0039] The preferred manufacturing method involves a two-part construction in which a two-photon polymerized printed part is inserted into a polystyrene injection molded part.

[0040] In a broad aspect, a single cell handling device according to various embodiments comprises a microwell formed from at least one microwell wall having two opposing access openings therethrough.

[0041] The single cell handling device is preferably fabricated on a microscale and is therefore preferably fabricated from materials that are non-toxic to cells and suitable for microscale production.

[0042] Preferably, the microwell wall includes two or more microwell hemispheres formed on its outer wall surface. In another embodiment, the outer wall surface may have a single microwell hemisphere formed therein that spans the circumference of the microwell wall. Preferred microwell hemispheres are shaped to provide optimal mechanical support for cells retained therein. Thus, microwell hemispheres may be formed in a variety of configurations and sizes. For example, the hemispheres may have a circular hemispherical shape (e.g., a hemispherical cylinder), an elliptical hemispherical shape (e.g., an ellipsoid), a conical cross-section shape, or a parabolic shape (e.g., an elliptical paraboloid). The hemispherical shape may be formed with an increasing slope along a hemispherical groove formed on the outer wall surface of the microwell.

[0043] Preferably, the diameter of the microwell hemisphere is appropriate for the size of the cells to be held therein. For example, an oocyte including the zona pellucida is approximately 165 microns to 150 microns. Therefore, the microwell hemisphere is preferably approximately 50 microns to 300 microns for all mammalian oocytes (including those of non-human origin), approximately 100 microns to 200 microns for human oocytes, and approximately 10 microns to 500 microns for embryos and other cells, all of which are configured to accommodate oocytes with diameters ranging from 165 microns to 150 microns.

[0044] Preferably, two opposing access openings are formed through the microwell wall within the microwell hemispheres. Thus, in some embodiments, the two microwell hemispheres may be positioned opposite one another. Alternatively, at least one microwell hemisphere may extend a sufficient length around the periphery of the microwell wall such that two access openings can be formed within the microwell hemisphere and are positioned opposite one another.

[0045] Design choices regarding the formation of the microwell hemisphere are directed towards maximal support of the oocyte during injection and withdrawal of the injection pipette during the ISCI procedure.

[0046] The access opening may form a channel within the microwell wall, between the inner surface of the microwell wall and the outer surface of the microwell wall. A preferred channel configuration and size may be designed and selected based on the benefits of facilitating handling and manipulation of the injection pipette by the embryologist. A preferred channel may have a closed or open profile. A channel may have a simple profile, such as a circular hole or an oval slot. Alternatively, a channel may have a complex profile, such as a T-shaped or cross-shaped profile.

[0047] The channels may also be shaped to help remove air pockets or bubbles when the embryologist is filling the microwells with medium.

[0048] Alternatively, the channels may be configured to assist the flow of medium to balance fluid pressure, for example to avoid back pressure behind the cells as they are being manipulated, which is particularly important when embryologists perform ICSI procedures to avoid back pressure behind the oocyte.

[0049] Preferably, the microwells are open at the top to allow for the addition of media to the microwells and for access and recovery of the oocytes, and one or more of the interior surfaces of the microwells are preferably curved.

[0050] In a broader aspect, the embodiments described herein relate to cell handling arrays that include one or more microwells.

[0051] Preferably, the cell handling array comprises two or more microwells as described herein. Preferably, the two or more microwells are arranged in fixed positions relative to one another so that the array can be easily manipulated by an operator. The microwells can be arranged in the array to form various configurations or with varying pitches of fixed relative positions. For example, the microwells can form a linear array, a circular array, a grid-like array, other shapes, or combinations of these shapes and configurations.

[0052] In a further preferred embodiment, the single cell handling device or cell handling microarray is disposed within a base well. Preferably, the base well is formed with at least one side wall and at least one base wall. The at least one side wall and the at least one base wall form a container for containing the single cell handling device or cell handling microarray and a fluid medium therein. More preferably, the single cell handling device or cell handling microarray may be disposed within a larger well to additionally hold medium and / or oil used in the ICSI procedure.

[0053] Preferred large wells may be specially adapted for use with the single cell handling devices or cell handling microarrays described herein, or they may be commercially available cell culture products that may or may not be specifically designed for performing ICSI procedures.

[0054] The preferred base well comprises one or more sperm platforms, more preferably two sperm platforms. Preferably, the single cell handling device is arranged within a cell handling microarray disposed on the base wall of the base well, and two linear sperm platforms are also disposed on the base wall of the base well substantially parallel to and adjacent to the cell handling microarray.

[0055] The preferred sperm platform has a stepped top surface, the preferred configuration of which is designed and / or selected to allow for gravity-based sorting of motile sperm, although the sperm platform may be configured in a variety of shapes and sizes.

[0056] Preferably, the top surface of the sperm platform and the single cell handling device are positioned generally in a central focal plane relative to each other, and are preferably positioned substantially in a common plane for efficient sperm and oocyte retrieval when performing an ICSI procedure. The sperm platform is preferably configured as a localized platform surface relative to the single cell handling device for positioning and retrieving sperm.

[0057] The sperm platform itself may be configured in various shapes and sizes. Furthermore, each step of the stepped platform may be configured in various shapes and sizes. Any number of steps may be selected to construct the stepped platform.

[0058] Preferably, the cell handling array may include, without limitation, a plurality of single cell handling devices that are engageable with one another. Preferably, the cell handling array is a linear array in a horizontal plane, but may alternatively be stacked vertically, or a combination of both. Preferably, the single cell handling devices are clip-connected in a horizontal plane, stacked vertically, or slide-engaged with one another in a horizontal or vertical plane.

[0059] In a preferred embodiment, the cell culture base is configured to be connectable to other cell culture bases or covers, and is preferably configured to physically stabilize the cell handling array when placed on a surface or when connected to other components or devices.

[0060] Preferably, the base well described herein is included in a culture system further including a support base and a culture dish. Preferably, the base well described herein is formed in the culture dish and / or the support base.

[0061] The culture systems described herein may be provided with additional features. For example, each microwell may be associated with an identification label, allowing the microwell and the contents contained therein to be individually identified. In this way, traceability is ensured during use of the culture system, for example, allowing traceability of oocytes during an ICSI procedure.

[0062] A preferred identification label may provide a number, letter, or other symbol that is visible in the central focal plane of the sperm platform and the single cell handling device, allowing the identification label to be viewed during the performance of an ICSI procedure.

[0063] Preferably, the identification label may be configured to be readable from different angles or directions, for example, double labeling may be provided so that different labels are visible at 0° and 180°.

[0064] In a broader aspect, embodiments described herein relate to methods of performing an intracytoplasmic sperm injection (ICSI) procedure.

[0065] The preferred method for performing ICSI is providing a culture dish with a culture medium, a sperm sample, and an oocyte sample; covering the culture dish with oil; Separating the sperm sample and the oocyte sample; Inserting the injection pipette into a micromanipulator; Immobilizing a group of motile and morphologically normal sperm samples from the sperm sample; collecting said normal sperm sample with an injection pipette; Transporting the collected normal sperm sample to the oocyte sample; selecting an oocyte, injecting the oocyte, and aspirating the oocyte cytoplasm into a small injection pipette; injecting the aspirated oocyte cytoplasm and normal sperm sample into a selected oocyte; withdrawing the injection pipette.

[0066] In a broader aspect, embodiments described herein relate to methods for manufacturing single cell handling devices or cell handling arrays.

[0067] Preferably, the single cell handling devices or cell handling arrays described herein are fabricated as two-part components consisting of two-photon polymerization [2PP] printed components inserted into polystyrene injection molded parts (or alternative transparent and non-toxic to cells).

[0068] Preferably, such a method includes mechanically mating two components, which allows for the mechanical mating of uncured components, followed by a subsequent curing process before assembly.

[0069] In alternative constructions, the components may be glued together, assembled with clips, or a combination of both.

[0070] The method of manufacturing the cell culture system described herein includes fabricating a base well and a cover having four sides and a top surface and configured to fit onto the top surface of the base well.

[0071] The manufacturing method for the device is intended to accommodate a wide variety of base well and cover variations and is customizable to accommodate commercially available dish configurations or custom base well and cover configurations.

[0072] The method of manufacturing the device may allow for separation of component manufacturing and final assembly.

[0073] The device manufacturing method may include features to support automated assembly and quality control inspection.

[0074] As used herein, the term "cell" shall be construed synonymously with the term "cellular material" and shall refer to any cell, group of cells, tissue, or organoid that is the subject of the invention described herein.

[0075] As used herein, the term "cell culture" is intended to refer to any tool or process by which cellular material is isolated and maintained under controlled conditions for testing, growth, observation, experimentation, collection of culture medium, or other biological processing.

[0076] As used herein, the term "device" refers to a microscale structure fabricated, for example, in the range of 0.1 microns to 1000 microns. Devices are intended to encompass both static and mechanical devices, as well as devices in the field of microfluidics.

[0077] As used herein, the term "maintaining cells" refers to any process of storing cellular material in a controlled environment to create the conditions necessary for viability. The term "culturing cells" refers to the process of cell culture.

[0078] As used herein, the terms "cryopreservation" or "related cryopreservation" refer interchangeably to vitrification or freezing.

[0079] As used herein, the term "handle" and derivative terms such as "handling," "handled," etc. are understood to include any form of physical manipulation, including, but not limited to, collecting, picking up, holding, grasping, grasping, stroking, rolling, touching, and moving.

[0080] As used herein, the term "stabilize" and derivative terms such as "stabilize" and "stabilized" are understood to include the physical condition of holding an object in a relatively stable, rigid, or immobile state, the term referring to a state relative to that in the absence of the stabilizing intervention.

[0081] The term "moat" is used herein to define a closed-loop channel or groove that can receive and retain a fluid. The term "moat" does not imply any limitation on scale and may indeed be formed on a microscale.

[0082] The broad embodiments of the present invention will now be described with reference to the accompanying drawings and 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 limited to the embodiments set forth herein. These embodiments are provided by way of example only, and are intended to ensure that this disclosure will be thorough and complete, conveying the full scope and breadth of the invention.

[0083] Broad embodiments of the present invention include a cell handling device for holding oocytes, and may optionally include a cell handling device for holding sperm. Detailed Description of the Embodiments [Brief explanation of the drawings]

[0084] [Figure 1] FIG. 1 is a top view of a single cell handling device according to an embodiment of the raised microwell of the present invention.

[0085] [Figure 2] FIG. 2 is a side perspective view of a single cell handling device according to an embodiment of the raised microwell of the present invention.

[0086] [Figure 3] FIG. 3 is a side view of a single cell handling device according to an embodiment of the raised microwell of the present invention.

[0087] [Figure 4] FIG. 4 is a cross-sectional view of a single cell handling device according to an embodiment of the raised microwell of the present invention.

[0088] [Figure 5] FIG. 5 is a top view of a microwell array and sperm platform of a single cell handling device according to an embodiment of the raised microwells of the present invention.

[0089] [Figure 6] FIG. 6 is a side perspective view of a microwell array and sperm platform of a single cell handling device according to an embodiment of the raised microwells of the present invention.

[0090] [Figure 7] FIG. 7 is a top view of a sperm platform according to an embodiment of the raised microwells of the present invention.

[0091] [Figure 8] FIG. 8 is a top perspective view of a microwell according to an embodiment of a raised microwell of the present invention.

[0092] [Figure 9] FIG. 9 is a front cross-sectional view of a sperm platform according to an embodiment of the raised microwells of the present invention.

[0093] [Figure 10]FIG. 10 is a front cross-sectional view of a microwell according to an embodiment of a raised microwell of the present invention.

[0094] [Figure 11] FIG. 11 is a top view of a single cell handling device and device cover according to an embodiment of the raised microwells of the present invention.

[0095] [Figure 12] FIG. 12 is a side perspective view of a single cell handling device and device cover according to a raised microwell embodiment of the present invention.

[0096] [Figure 13] FIG. 13 is a side view of a single cell handling device according to an embodiment of the raised microwell of the present invention.

[0097] [Figure 14] FIG. 14 is a side cross-sectional view of a single cell handling device and device cover according to an embodiment of the raised microwells of the present invention.

[0098] [Figure 15] FIG. 15 is a top view of a device cover according to an embodiment of the raised microwells of the present invention.

[0099] [Figure 16] FIG. 16 is a side perspective view of a device cover according to an embodiment of the raised microwells of the present invention.

[0100] [Figure 17] FIG. 17 is a side view of a single cell handling device according to an embodiment of the raised microwell of the present invention.

[0101] [Figure 18] FIG. 18 is a cross-sectional side view of a single cell handling device according to an embodiment of the raised microwells of the present invention.

[0102] [Figure 19] FIG. 19 is a top view of a single cell handling device according to an embodiment of the recessed microwell of the present invention.

[0103] [Figure 20] FIG. 20 is a top view of a microarray of a single cell handling device according to an embodiment of the recessed microwell of the present invention.

[0104] [Figure 21] FIG. 21 is a cross-sectional side view of a single-cell handling device according to an embodiment of the embedded microwell of the present invention, viewed from the microarray side of the base dish.

[0105] [Figure 22] FIG. 22 is a cross-sectional side view of a single cell handling device according to an embodiment of the embedded microwell of the present invention, viewed from the sperm platform side of the base dish.

[0106] [Figure 23] FIG. 23 is a cross-sectional side view of a microwell through opposing peripheral moat walls of a single cell handling device according to an embodiment of the recessed microwell of the present invention.

[0107] [Figure 24] FIG. 24 is a top view of a microwell of a single cell handling device according to an embodiment of the recessed microwell of the present invention.

[0108] [Figure 25] FIG. 25 is a side perspective view of the base dish and cover of a single cell handling device according to an embodiment of the recessed microwell of the present invention.

[0109] [Figure 26]FIG. 26 is a side perspective view of two stacked base dishes of a single cell handling device according to an embodiment of the recessed microwell of the present invention.

[0110] [Figure 27] FIG. 27 is a side view of a base dish of a single cell handling device according to an embodiment of the recessed microwell of the present invention.

[0111] [Figure 28] FIG. 28 is a side perspective view of a cover for a single cell handling device according to an embodiment of the recessed microwell of the present invention.

[0112] [Figure 29] FIG. 29 shows a comparative study evaluating the efficiency of preparation tasks between traditional ICSI and the implantable microwell embodiment of the present invention, including a comparison of (a) instrument operation, (b) pipette preparation, (c) oocyte handling, and (d) oocyte retrieval time.

[0113] [Figure 30] FIG. 30 shows a comparative study evaluating treatment efficiency between traditional ICSI and an embodiment of the implantable microwell of the present invention, including a comparison of (a) oocyte injection procedure, (b) injection time, (c) polar body alignment, and (d) injection time.

[0114] [Figure 31] FIG. 31 shows a comparative study evaluating the number of hand adjustments during oocyte injection between conventional ICSI and an implantable microwell embodiment of the present invention.

[0115] [Figure 32] FIG. 32 shows a comparative study evaluating the degree of invagination during oocyte injection between conventional ICSI and an embodiment of the implantable microwell of the present invention.

[0116] [Figure 33]FIG. 33 shows a comparative study evaluating embryo development rates between traditional ICSI and implantable microwell embodiments of the present invention, including a comparison of (a) fragmentation rate, (b) survival rate, (c) cleavage rate, and (d) blastocyst rate.

[0117] [Figure 34] FIG. 34 shows a comparative study on human oocytes between traditional ICSI and an implantable microwell embodiment of the present invention, including a comparison of (a) injection rate and (b) number of hand adjustments.

[0118] Some embodiments of the present invention are described in the examples set forth below. [Example]

[0119] This example describes the fabrication of single-cell handling devices designed with raised or recessed microwell structures using two-photon polymerization (2PP) 3D printing. The fabrication method was improved by creating a single-piece printed device capable of holding up to 20 oocytes at a time. Functional and workflow differences between the new ICSI technique employed in the single-cell handling device (microICSI) and conventional ICSI (C-ICSI) were compared, and both procedural efficiency and embryo developmental outcomes were evaluated. Finally, a pilot study demonstrating the device's utility for human oocyte microinjection is described.

[0120] The single-cell handling device described herein is suitable for holding oocytes and sperm during ICSI procedures. An exemplary device includes a base dish and a lid or cover. The base dish combines the functionality of a conventional ICSI cell culture dish with improved functionality that allows oocytes to be placed without a holding pipette. Oocytes can be placed without the need for vacuum manipulation of the cells. The device configuration is optimized to provide maximum support to the oocyte, minimizing oocyte deformation and invagination, thereby improving the quality of the resulting zygotes and / or increasing fertilization rates.

[0121] Placing the oocytes in a fixed position within the dish eliminates the need for a vacuum pipette, allowing the embryologist to easily position the oocytes for ICSI, improving the efficiency of performing ICSI. General base dish and microwell structure

[0122] An exemplary base dish is designed to support oocytes within a hemispherical structure (or cup) during ICSI procedures, eliminating the need for a holding pipette and suction. Oocytes are held encased during microinjection within the hemisphere, surrounded by microwells. Channels allow an injection pipette to move the oocyte into position within the hemisphere or cup, where it can be handled and stabilized for microinjection before sperm pickup.

[0123] Figures 1 to 18 show a raised microwell structure in which a microwell array is formed in a raised shape on a base platform, while Figures 19 to 28 show a modified manufacturing method based on a recessed microwell structure. Raised microwell structure

[0124] 1-18, FIG. 1 is a top view of a base dish 210 of a single cell handling device according to an embodiment of the present invention, on which the device is placed. The base dish 210 is a square dish measuring 65 mm on each side, with space for a patient label. The base dish 210 is located within a base well 220. The base well 220 allows media to be dispensed into the base dish 210 and oil to be layered on top of it.

[0125] A microwell array 230 is centrally located within the base well 220. The microarray 230 includes multiple individual wells for individually holding and maintaining oocytes from a single patient. Sperm platforms 280 are located adjacent to both sides of the microwell array.

[0126] 2 is a side perspective view of base dish 210. Base dish 210 is positioned on support platform 310 and is one step higher than support platform 310. Base dish 210 and support platform 310 each have a height of approximately 4.5 millimeters. The depth of base well 220 spans the height of support platform 310 and base dish 210, and is approximately 8.5 millimeters.

[0127] FIG. 3 is a side view of the single cell handling device, showing the step between the support platform 310 and the base dish 210, which are approximately equal in height.

[0128] Figure 4 is a side cross-sectional view of the single-cell handling device, taken through the middle of the base well 220 and cutting the microwell array 230 longitudinally. As shown in Figure 4, the base dish 210 is offset above the support platform 310. Figure 4 shows the stepped shape of the device at the edge of the microwell array. Figure 4 shows the depth of the base well 220 across the height of the base dish 210 and support platform 310. The sidewalls of the base well 220 are sloped to facilitate manufacturing by injection molding.

[0129] In use, the base well 210 is filled with cell culture medium and the microwell array 230 is immersed in the medium. The total volume of the base well 210 is approximately 7.8 milliliters to allow for coverage with an oil layer.

[0130] Figure 5 shows details of the microwell array 230 and sperm platform 280. A series of microwells 240 are arranged vertically to form the microwell array 230. Each well functions as an individual single-cell handling device for holding and manipulating a single oocyte within each well. Each microwell array 230 contains a set of oocytes from a single patient. The array is organized in a compact, linear manner for efficient visualization, repositioning, and manipulation by the embryologist.

[0131] 6 shows a side perspective view of the microwell array 230 and sperm platform 280. Individual microwells 240 within the microwell array 230 are printed into adjacent wells, forming the microwell array 230 as a single unit structure. The opening of each microwell is beveled to allow placement of an embryologist's injection pipette to deposit oocytes into the microwells 240 and remove zygotes formed therein.

[0132] 7 is a partial top view of a sperm platform 280. Two sperm platforms 280 are positioned adjacent to each other on either side of the microwell array 230. The sperm platforms 280 are positioned at the same height as the midplane of the oocytes. The alignment of the sperm platforms and oocyte microwells allows for sperm pickup, oocyte positioning, and manipulation to perform the ICSI procedure within the same nominal focal plane. This minimizes the adjustments that the embryologist must make to ensure visualization of the procedure is in focus.

[0133] The sperm platform 280 is positioned approximately 2 mm from the microwell array 230. This separates the sperm from the oocytes, preventing contact that could result in unintended fertilization (IVF).

[0134] Separating the sperm platform 280 from the microwell array 230 provides clearance for the embryologist to position the ICSI pipette at any angle when placing the oocytes and performing ICSI.

[0135] The sperm platform 280 extends the entire length of the microwell array 230. This allows the embryologist to perform sperm pickup in close proximity to the oocytes, minimizing handling for the embryologist to efficiently complete the ICSI procedure.

[0136] By providing two sperm platforms 280 on either side of the microwell array 230, the embryologist can operate the ICSI pipette with either the left or right hand of their choice to perform the ICSI procedure.

[0137] Sperm platform 280 includes a series of vertical sperm platform steps 290. Sperm platform steps 290 allow the embryologist to use gravity to select motile sperm. In this procedure, motile sperm are located on the highest steps of sperm platform steps 290, while dead and non-motile sperm remain at the lowest level of the dish.

[0138] Referring to Figure 8, a microwell 240 is flanked by two opposing microwell hemispheres 250. Each microwell hemisphere 250 defines a hemispherical recess extending circumferentially around the microwell wall, thereby defining two opposing hemispherical recesses in the wall of each microwell 240. Oocytes are positioned within the recesses to provide optimal support for the oocyte during the ICSI procedure.

[0139] 10, hemisphere 250 is positioned tangentially to the base of microwell 240. This allows an oocyte placed at the base of the microwell to be easily moved by the ICSI pipette (via access channel 260) to the opening of the opposing hemisphere before being placed in the innermost part of the hemispherical cup.

[0140] The hemisphere 250 is formed as a recess within the wall thickness of the microwell 240. An oocyte placed in the opening is partially supported around its periphery and can be oriented using the ICSI pipette (via the access channel 260) before being fully inserted into the hemispherical cup, where the entire oocyte is supported and prevented from rotating.

[0141] During the ICSI procedure, the oocyte is placed in one of two possible hemispherical recesses. The embryologist may choose the preferred hemispherical recess depending on their preference for left- or right-handed operation. The embryologist can access the oocyte using an injection pipette through the access channel 260. During the ICSI procedure, the oocyte is supported within the hemispherical recess, allowing sperm to be introduced directly into the oocyte without aspirating the oocyte with the injection pipette, minimizing cytoplasmic invagination during injection. Similarly, the microwell hemisphere 250 supports the oocyte during injection pipette withdrawal. The microwell hemisphere 250 is approximately 100 to 200 microns in diameter and supports the expected size range of oocytes, including the zona pellucida.

[0142] 9, which shows a front cross-sectional view of the sperm platform 280, clearly shows the sperm platform step 290. In the sperm platform step 290, the motile sperm are located at the highest step of the sperm platform step 290, while the dead and non-motile sperm remain at the lowest.

[0143] FIG. 10 shows a front cross-sectional view of a microwell 240. Each microwell 240 contains two opposing microwell hemispheres 250 and two opposing access channels 260. The access channels 240 allow the embryologist to insert an injection pipette laterally into the well to perform the ICSI procedure. The channels are designed to be as small as possible to maximize surface contact with the hemispheres for optimal oocyte support. The access channels 240 have internal beveled openings that allow the injection pipette to access the microwell 240 and microwell hemispheres 250 and allow the micropipette to be angled to adjust oocyte positioning before injection.

[0144] While the opposing access channels 260 primarily provide the embryologist with the option of left- or right-handed operation, this symmetrical structure also has another function: the second access channel facilitates the removal of air bubbles as the microwells 240 are filled with medium, and also prevents backpressure from building up behind the oocyte as the embryologist performs the ICSI procedure.

[0145] The microwell array 230 and individual microwells 240 are identified by localized overpolymerization of resin during 2PP printing of the insert. This allows for numbering by changing the refractive index of the material. This allows for identification by the embryologist during the ICSI procedure, positioned at the mid-plane of the oocyte.

[0146] Fig. 11 is a top view of base dish cover 320, which is molded to cover the entire base dish 210 and be supported on the upper surface of support stand 310. Fig. 12 shows a side perspective view of base dish cover 320 covering base dish 210, showing how base dish cover 320 forms a step on support stand 310.

[0147] Figure 13 shows a side view of the base dish cover 320 and support base 310. Figure 14 shows a side cross-sectional view showing how the base dish cover 320 fits snugly onto the base dish 210.

[0148] A top view of base dish cover 320 shown in Figure 15 and a side perspective view of base dish cover 310 shown in Figure 16 illustrate the shape of base dish cover 320. Base dish cover 320 has two rounded corners that oppose two angled corners also formed on base dish cover 320.

[0149] 17 and 18 similarly show the shape of the base dish cover 320, with FIG. 17 showing a side view and FIG. 18 showing a side cross-sectional view. Recessed microwell structure

[0150] 19-28, FIG. 19 is a top view of the base dish 400 of the device with recessed microwells. As shown in FIG. 19, the recessed microwell structure asymmetrically positions the microwell array 470 to ensure sufficient functional space for the sperm preparation area 420. The recessed microwell structure is simplified by providing a single planar sperm preparation area 420 embedded in a raised platform surface 430 (instead of the channeled oval sperm platform of the raised structure). The cover recess 440 serves as a physical guide for cover placement. Two opposing parallel sides of the cover recess 440 extend through the raised platform surface 430 and onto two opposing base dish walls 450a and 450b. The cover recess 440 is angled at two concave corners 460a and 460b to provide guides for accurate installation of the cover.

[0151] The microwell array 470 is disposed within the periphery of a recessed moat 480 formed in the raised platform surface 430. The moat 480 comprises a base 490 and a wall 500 having four sides 500a, 500b, 500c, and 500d, all of which are formed by spaces within the base dish 400. The base 490 and the side walls of the moat 480 are designed to accommodate a large volume of microwell medium for immersing cellular components within the microwells, sufficient to dilute by-products of cellular activity generated by the cells contained within the microarray.

[0152] The microwell array 470, as shown in FIG. 20, includes microwell groups 510, each spaced apart and consisting of five microwells 520. This configuration inhibits air bubble formation from the media contained within the moat 480. The moat 480 is defined by a multi-step perimeter 530, allowing a needle to seamlessly move across the dish to each microwell 520. Extended ends 540 serve as reference points for easy alignment and assembly. A label area 550 is provided on the base dish to allow for custom identification of the base dish. The four sides of the base dish feature partially knurled edges 560a, 560b, 560c, 560d, 560e to facilitate handling of the dish during transport.

[0153] 21 and 22 are cross-sectional side views of the implantable single-cell handling device from the microarray side (FIG. 21) and the sperm platform side (FIG. 22). FIG. 21 shows the implantable structure of the microwells 520, microwell clusters 510, microwell array 470, and moat 480. The embedding is achieved by processing the base dish 400, where the raised platform surface 430 steps down at an angle of approximately 93° to form a shelf-shaped cover recess 440. This cover recess 440 holds a cover (not shown) thereon and keeps it flush with the raised platform surface 430. A further step down forms a moat 480, which is continuous with the cover recess 440 and raised platform surface 430 and is located within the base dish 400, with walls rising at an angle of 90° from the cover recess 440.

[0154] 22 shows the sperm preparation area 420 fabricated in the same plane as the microwells 520. The cover 580 occupies the space above the cover recess 440, forming a cover upper surface 590 in the same plane as the raised platform surface 430.

[0155] FIG. 23 shows a side cross-sectional view of the microwell 520 across the moat walls 500a and 500c. The walls are machined to form chamfers 590a and 590b at the base of the moat 480. This serves as a visual cue to the user when their needle has entered the moat during use of the dish. The moat 480 is recessed into the dish so that the raised platform surface 430 is aligned with the focal plane of the microwell. The base dish 400 includes a microwell support 600 positioned below each microwell 520 along the length of the microwell array 470 and located entirely within the periphery of the moat 480. The edges 602a and 602b of the microwell support 600 allow the base dish 400 to make good thermal contact with a heating stage (not shown), typically used during ISCI procedures.

[0156] The configuration of the microwells 520 is shown in more detail in Figure 24. Each microwell is formed by a single curved outer wall 610 and a flattened microwell base 620. Each microwell is open at the top to allow for the insertion of a pipette to insert or remove large objects, including oocytes, into the microwell or to transfer fluids directly into the microwell. A rounded well cavity 630 is centrally located within the microwell, flanked by rounded oocyte-retaining cups 640a and 640b. The well cavity 630 is approximately 250 microns in diameter and will support the expected size range of oocytes, including the zona pellucida, between 100 microns and 200 microns.

[0157] The wall forming the well cavity 630 has two openings and is continuous with the walls forming the two oocyte-retaining cups. The inner retaining cup 640a is oriented proximal to the sperm preparation area and is formed by an inner retaining cup wall 650a. This inner retaining cup wall 650a has a channel opening 660 that extends through the inner retaining cup wall 650a to an injector channel 670. The injector channel 670 is formed by a channel wall 680 formed through the single curved outer wall 610 of the microwell 520, allowing small objects such as a microinjection device to access the microwell. The shape of the channel wall 680 provides a guide for aligning a microinjection needle with the center of the outer retaining cup 640b. The shape also allows the user to adjust the orientation of the oocyte's polar body using a microinjector prior to the ICSI procedure.

[0158] The outer retaining cup 640b is located distal to the sperm preparation area. The outer retaining cup wall 650b is symmetrical to the inner retaining cup wall 650a, and both have a generally hemispheric-cylindrical shape. The outer retaining cup wall 650b has an exhaust port 680 extending therethrough. The exhaust port 680 includes an exhaust port 690, which ensures that the oocyte is positioned in the cavity formed within the outer retaining cup 640b without experiencing any fluid resistance. Meanwhile, the cavity formed within the inner retaining cup 640a holds the oocyte in place and allows for easy removal of the microinjector from the oocyte after microinjection.

[0159] The inner surfaces of the microwells 520 are designed to allow for high-quality visualization. The flattened shape of the microwell base 620 is employed to facilitate visualization of oocytes during the microinjection procedure. The outer retaining cup 640b employs semi-cylindrical cell-stabilizing protrusions to facilitate visualization. A flat visualization lens 700 extends across the top opening 710 of the microwell array 520, allowing visualization of microwell identification information during use. Numbers embossed into the cutout slots of each microwell array (not shown) allow for sample identification and traceability.

[0160] Figures 25, 26, 27, and 28 show exterior views of base dish 400 and cover 720. Figure 25 shows how cover 720 fits over cover recess 440 formed in raised platform surface 430 and extending across two opposing base dish walls 450a and 450b. Cover 720 has two angled cutouts 730a and 730b at either corner of cover 720, which correspond to two recessed corners 460a and 460b for precise mounting of the cover.

[0161] 26 illustrates the ability to stack multiple base trays, with each base tray having an individual cover 720 attached. Upper stacking projections 740 are located at each corner of the raised platform surface 430 to mate with the lower surface 750 of a base tray. Lower stacking recesses 760 are provided in the lower surface 750 of the base tray to correspond to the shape of the upper stacking projections 740 and aid in the safe stacking of multiple base trays.

[0162] Figure 27 shows the underside of the base dish 750, which is shaped to promote thermal contact with the heating stage during the ICSI procedure, and Figure 28 shows spacer pins 780 located on the underside of the cover 720 to allow for gas exchange during incubation. Device fabrication

[0163] Microarrays were printed using a NanoOne 1000 high-resolution 2PP 3D printer and the proprietary acrylate-based 2PP resin "UpFlow" (UpNano GmbH, Vienna, Austria). After modeling the 3D parts in CAD software "Solidworks" (Dassault Systèmes Solidworks, Waltham, MA, USA), the exported STL file was imported into Think3D software (version 1.7.3, UpNano GmbH). Parts were printed using a 10x objective operating in coarse or "voxel slice" mode with a layer thickness of 5 microns, a line spacing of 4 microns, and a power setting of 430 mW.

[0164] Microarrays were detached from the printed circuit board with a razor blade and washed three times for 10 minutes with propylene glycol monomethyl ether acetate (PGMEA ≥ 99.5%), one time for 3 minutes, and one time for 2 minutes with 2-propanol (99.9%) before air-drying. The dried parts were cured for 5 minutes at 60% power in an ODS Cure Box (One Digital System, Incheon, Korea) before plasma treatment. Microarrays were treated with oxygen plasma for 60 seconds at 780–820 mTorr pressure using a PDC-002-HP chamber (Harrick Plasma, New York, USA). The plasma-treated arrays were bonded to commercially available polystyrene dishes (product number 16006, Vitrolife Pty. Ltd., Sydney, Australia) using a small amount (approximately 25–30 mg) of UpFlow resin, followed by two 5-minute cure cycles in the ODS Cure Box.

[0165] After curing, the dishes were washed twice with 3 mL of 96% ethanol (Chem-Supply Pty Ltd, 125 Gilman, South Australia, Australia) and then rinsed twice with filtered (0.22 micron pore size; Millipore, Bedford, MA, USA) 5% 7X detergent (MP Biomedicals Australasia, Seven Hills, New South Wales, Australia). The dishes were then rinsed three times with Milli-Q water to remove any residual detergent and dried overnight on a heating plate (Ratek Instruments Pty Ltd, Victoria, Australia) set at 37.5°C. During each wash, a P1000 micropipette was used to dispense a drop of washing solution onto the microarray, thereby rinsing each well.

[0166] For human oocytes, the hemisphere size was adjusted to accommodate the larger oocyte size before printing the microarray, which was then printed, post-processed, and washed as described above.

[0167] Previous toxicity testing of 2PP components using a mouse embryo assay (MEA) (IVF VET Solutions, South Australia, Australia), including both negative and positive controls, showed them to be non-toxic (Yagoub SH et al. (2022), Yagoub SH et al. (2014), McLennan HJ et al. (2023)). This was repeated in the present study, and no toxicity was detected. Cover fabrication was performed following the same parameters and cleaning protocols as described above. How to use the single cell handling device

[0168] The following method is readily applicable to either recessed or raised microarray structures.

[0169] The base dishes are prepared by labeling the base dishes, preparing the PVP, medium oil, p10 and p20 pipettes, pipette tips, serological pipettes and serological pipettes, and working under a laminar flow hood.

[0170] On the available side, approximately 10 microliters of PVP is applied from the 2PP part in a long strip parallel to the microarray, and 30 microliters of medium is applied to the trench on the injection slot side of the microarray.

[0171] The base dish is placed with the syringe slot marker facing the user's preferred syringe side. The lid is then removed. Using a p10 pipette, 10 microliters of PVP is added to the base dish approximately 2 mm away from the 2PP microarray. The PVP droplet is stretched along the length of the microwell array. 20 microliters of medium is slowly added to the groove on the syringe side of the microarray with a p20 pipette. This step is repeated until a total of 30 microliters of medium has been added to the dish. The medium should be spread across the entire groove of the microwell array.

[0172] 6 mL of oil is placed in the portion of the dish away from the PVP and microwell array. The lid is placed back on the dish. The dish is then placed in an incubator to allow it to equilibrate.

[0173] Cryopreserved sperm are prepared by warming and centrifuging (washing) to remove semen and other preservatives. The washed sperm are loaded into a base dish using a 300-microliter handling pipette under an equilibrated dish and a dissecting microscope with a heated stage and / or in a heated crib. This procedure is performed under a dissecting microscope with a heated stage and / or in a heated crib. The sperm sample is gently agitated by inverting the container to ensure the sperm are evenly dispersed throughout the solution. An aliquot of sperm is aseptically collected using a 300-microliter handling pipette. The sperm sample is then slowly dispensed onto the PVP extension on the base dish by pressing the handling pipette evenly down along the PVP extension to evenly distribute the sperm.

[0174] Oocytes are loaded into the microwells of the base dish by obtaining a culture dish with detached oocytes. Five wells can be observed at once under a dissecting microscope with a heated stage and / or in a heated crib at 3x magnification. Air bubbles on the surface must first be removed before oocyte collection. Five oocytes are aspirated using a handling pipette and transferred to the base dish, with a single oocyte placed in each microwell. This step is repeated until all oocytes have been loaded into the microwells. The oocyte-loaded base dish can then be transferred to the microscope stage for ICSI.

[0175] If necessary, a mock injection can be performed at this stage using debris or blue beads. Prepare the injection pipette and tilt the syringe slightly to immobilize the sperm. Set the syringe to position 1 (if the workstation allows) by focusing the syringe on the meniscus of the sperm and PVP droplet. Slightly raise the syringe and move the stage to the microwell to focus on the microwell and oocyte. The syringe is aligned in the same focal plane as the oocyte and parallel to the injection slot of the microwell. Push the syringe through the injection slot and gently move the oocyte into the hemisphere, maintaining the polar body orientation. Pull back the syringe and, while keeping it parallel to the injection slot, slide it toward the injection slot. Gently slide the syringe away from the oocyte, then gently push the tip of the syringe back into the microwell (to assist with subsequent removal).

[0176] The stage is moved to shift the focus to the next microwell. All polar bodies are aligned in all microwells. The injector must be placed on the PVP strip and switched to position 1 or adjusted to match the sperm position. A single sperm is selected, immobilized, and gently aspirated. After raising the injector, the microscope stage is moved to focus on the center of the oocyte in the first microwell. The sperm is injected into the oocyte. These steps are repeated until all oocytes have been injected.

[0177] Alternatively, multiple sperm (nominally about three) can be aspirated into the injection pipette to perform a sequential injection sequence, further reducing the time required for the procedure. This "sequential injection" process is known in the art as "shotgun injection."

[0178] The syringe arm is raised to allow time for the dish to be removed. The dish is transferred to a dissecting microscope for post-procedure oocyte removal. The oocyte handling pipette tip is rinsed in the wash dish. Holding the pipette at 80°, five post-procedure oocytes are aspirated from the base dish. These oocytes are pipetted in the dish until washed. The oocytes are aspirated and placed in a culture dish. These steps are repeated for each oocyte. Validation and pilot test results Oocyte collection and maturation

[0179] Ovaries were collected from cycling gilts and sows at a local abattoir and transported to the laboratory in phosphate-buffered saline (PBS) in a thermos maintained at 30–35°C. The ovaries were washed with warm saline and placed in a beaker in a water bath set at 37°C. 3–6 mm clear antral follicles were aspirated into a 9 mL Greiner No Additive Vacuette tube (Interpath Services, Somerton, Victoria, Australia) using a 21-gauge needle at constant suction (1 L / min). Cumulus-oocyte complexes (COCs), containing multiple layers of cumulus cells, were isolated from the aspirate. The COCs were pooled, washed three times, and placed in groups of 50 in 500 μL of IVM medium. The IVM medium was prepared by adding 10 IU / mL equine chorionic gonadotropin (Folligon, MSD Animal Health, Australia), 10 IU / mL human chorionic gonadotropin (hCG, Chorulon, MSD Animal Health, 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 porcine follicular fluid to medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The medium was cultured in a 4-well NUNC dish (Thermo Fisher Scientific) under mineral oil (Origio, Sydney, NSW, Australia). Oocytes were matured at 38.5°C for approximately 40 hours in a humidified atmosphere of 5% CO2 in air.

[0180] At the end of the maturation period, morphologically normal COCs were isolated using a fine-bore glass pipette (Rowe Scientific, Lonsdale, South Australia, Australia). Groups of 50–60 COCs were placed in individual 5 mL round-bottom tubes with hormone-free IVM medium and transported at 37°C in a transport incubator (CryoLogic, Blackburn, Victoria, Australia) until oophorus removal, approximately 1 h after the end of maturation. Intracytoplasmic sperm injection (ICSI)

[0181] For C-ICSI, a 5-microliter drop of 7% polyvinylpyrrolidone (PVP, Origio Australasia Pty Ltd, a CooperSurgical subsidiary, Denmark) was used to prime the injection pipette, and seven 5-microliter drops of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered NCSU23 (ART Lab Solutions Pty Ltd, Adelaide, South Australia, Australia) were arranged in a 3 × 3 grid in a Vitrolife ICSI dish. Five microliters of HEPES-buffered NCSU23 was added to the central PVP drop to create a 3.5% PVP solution, partially reducing sperm motility. The dish containing the microarray had a different media configuration: two 5-microliter drops of 7% PVP were placed in a strip to the left of the microarray, and the PVP drop closest to the array received 5 microliters of HEPES-buffered NCSU23 for sperm pickup. Twenty microliters of HEPES-buffered NCSU23 was dispensed onto and around the microarray moat. All dishes were covered with 5 milliliters of MEA-tested paraffin oil (Merck Group, Macquarie Park, New South Wales, Australia) and allowed to equilibrate at 38.5°C for a minimum of 3 hours.

[0182] Freshly collected, diluted semen was purchased from a local boar stud (Sabor, Clare, South Australia, Australia). Semen was equilibrated from storage temperature (18°C) to room temperature and centrifuged at 300 g for 5 minutes. The supernatant was discarded, and the remaining pellet was suspended in 10 mL of warm sperm wash medium (199 medium supplemented with 0.1 mg / mL sodium pyruvate, 0.9 mg / mL calcium lactate, 0.075 mg / mL penicillin G, 0.05 mg / mL streptomycin sulfate, and 10% fetal bovine serum (FBS)). The resuspended pellet was centrifuged and further resuspended in sperm wash medium. Sperm were further diluted with warm HEPES-buffered NCSU23 to a final concentration of approximately 5 x 10 sperm / mL.

[0183] Mature COCs were treated with 0.1 mg / mL hyaluronidase in HEPES-182-buffered NCSU23 for 1 minute and gently pipetted using a 130-140 micron inner diameter (ID) Flexipet pipette tip and a Cook Adjustable Handle (Cook Medical Pty Ltd, Eight Mile Plains, Queensland, Australia) to remove cumulus cells. After cumulus removal, oocytes were washed three times with HEPES-buffered NCSU23 and assessed for fitness and the presence of a single polar body for selection as candidates for the ICSI procedure.

[0184] Because conventional ICSI requires both a holding pipette and an injection pipette, whereas microarraying requires only a single injection pipette, we measured the pipette setup time for both the conventional ICSI and microarraying procedures. This included the time required to focus the medium drop and the pipette while positioning the pipette in the medium under oil. To avoid oocyte aging and allow multiple operators to perform experiments simultaneously, two ICSI workstations were used. The Nikon ICSI station consisted of a Nikon TE2000 Eclipse inverted microscope (Nikon Corporation, Tokyo, Japan) combined with an Eppendorf (Hamburg, Germany) micromanipulator, an air microinjector for a 25-micron inner diameter holding pipette (Eppendorf), and an oil microinjector for a 6-micron inner diameter injection pipette (Eppendorf), all purchased from ICSION (Severton, South Australia, Australia). The second Olympus station consisted of an Olympus IX83 inverted microscope (Evident Co., Ltd., Tokyo, Japan) and a Narishige ON4 micromanipulator equipped with Narishige injectors (IN-21 for oil and IN-9B for air). The order of treatments per operator was randomized within each replicate. Operators used a 10x objective lens combined with a 2x magnifier on the Olympus microscope and a 20x objective lens on the Nikon microscope to visualize sperm and perform ICSI procedures.

[0185] For conventional ICSI, 10–20 mature oocytes were transferred into a 5-microliter drop of HEPES medium using a 170-micron flexipipet immediately before the ICSI procedure in a custom-made heated infant crib set at 38.5°C. Approximately 3 microliters of diluted sperm were then dispensed into the 3.5% PVP drop using a flexipipet. The dish was carefully transferred to an inverted microscope and placed on a heated stage set at 38.5°C. For each injection, a single motile sperm with good morphology was selected and immobilized in a 3.5% PVP sperm drop. The oocyte was held in place with a holding pipette, and the polar body was rotated to the 6 or 12 o'clock position with an injection pipette. The injection pipette with the sperm at its tip was introduced into the oocyte at the 9 o'clock position, and the ooplasm was aspirated to confirm rupture of the oolemma. The injection time was recorded from the time the oocyte came into focus and the holding pipette entered the droplet to the time the oocyte was released from the holding pipette.

[0186] Invagination was based on the previous study by Danfour et al. (2021) and was scored based on the degree of invagination of the oolemma when the injection pipette was inserted into the oocyte from the 9 o'clock position in both treatments. Scores were calculated from 1 (minimal) to 3 (maximal). The number of hand adjustments between the controllers required for injection into each oocyte was quantified using video recordings. An adjustment was defined as the operator's hand moving away from one controller to operate another.

[0187] For microarray experiments, 10–20 mature oocytes were transferred into microarray microwells containing 20 μL of HEPES medium in the groove using a 170 μm inner diameter flexipette in a heated crib. All oocytes were transferred to the injection hemisphere within each microwell using the injection pipette and oriented so that the polar bodies were at the 6 or 12 o'clock position. The time required to orient 10 oocytes was recorded and averaged for each oocyte. Sperm were aspirated into the syringe as in conventional ICSI described above, and the syringe was inserted into the microwell through the injection slot to inject the correctly oriented oocyte. The injection pipette was then retracted from the slot to gently separate it from the oocyte, and the pipette was then gently pushed back into the center of the well. The injection time during the microarray procedure was recorded from the time the oocyte and the injection pipette in the pipette slot came into focus until the injection pipette was removed from the oocyte. If injection failed during either conventional or microarray ICSI, the oocytes were excluded from culture.

[0188] To assess parthenogenetic activation and subsequent embryonic development, sham injections were performed using microarray ICSI. In sham injections, oocytes were injected with 7% PVP but no sperm were injected. As a control, uninjected oocytes were also cultured separately. embryo culture

[0189] Presumptive zygotes, sham-injected zygotes, and controls were placed in 50-microliter drops of modified NCSU-23 medium in 60-mm Petri dishes (Falcon, product number 351007) in groups of five. Covered with approximately 8 mL of MEA-tested paraffin oil, they were cultured in a humidified incubator at 38.5°C (equilibrated with 6% CO2, 7% O2, and N2) until day 4. On day 4, embryos were scored for cleavage, post-injection viability, and the degree of fragmentation of uncleaved and cleaved embryos. Fragmentation was defined as occurring when 30% or more of the perivitelline space was occupied by small membrane-enclosed vesicle-like structures. All surviving embryos were transferred to equilibrated dishes containing NCSU-23 medium (ART Lab Solutions) adjusted for blastocyst culture, supplemented with 10% FBS and covered with paraffin oil. Blastocysts were scored on day 6. differential staining

[0190] Blastocysts from four replicates were harvested and stained immediately after blastocyst scoring on day 6 of culture. Blastocysts were washed with HEPES-buffered NCSU23 prewarmed to 37°C for differential staining. The zona pellucida was removed by incubation with 0.5% pronase in PBS for approximately 2 minutes, followed by washing with HEPES-MEM-PVA (minimum essential medium and 1 mg / mL polyvinyl alcohol (PVA)), and then by incubation with 10 mM trinitrobenzenesulfonic acid (TNBS) in HEPES-MEM-PVA for 15 minutes on ice. Blastocysts were then removed, washed again with HEPES-MEM-PVA, and incubated with 0.2 mg / mL anti-dinitrophenol BSA in HEPES-MEM-PVA for 10 minutes at 38°C. Blastocysts were then washed again with HEPES-MEM-PVA and cultured 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, blastocysts were transferred to HEPES-MEM-PVA for post-culture treatment, then dehydrated in 100% ethanol (Chem-Supply Pty Ltd, Gilman, South Australia, Australia) and mounted on microscope slides under coverslips in 2 microliters of glycerol. Fluorescent images of blastocyst nuclei were captured using an epifluorescence inverted microscope (Eclipse TS100, Nikon Corporation, excitation filter: 330–380 nm, barrier filter: 420 nm). The numbers of blue inner cell mass (ICM) and pink-red trophectoderm (TD) cells were counted after imaging by a single investigator blinded to treatment. Human microarray ICSI validation

[0191] Vitrified human oocytes (n = 18, from two patients; University of Adelaide Ethics Approval Number H-2023-082) that consented to the study were obtained from a local IVF clinic and warmed in the laboratory according to the instructions of the SAGE vitrification and warming kit (Origio Australasia Pty Ltd). For C-ICSI, one 5-microliter microdrop of 7% PVP (Origio Australasia Pty Ltd) was placed in the center of a Vitrolife ICSI dish, surrounded by eight 5-microliter drops of GMOPS PLUS (Vitrolife Pty Ltd). The microarray dish consisted of a 5-microliter strip of 7% PVP on the left side of the microarray, and 20-microliter drops of GMOPS PLUS on and around the microarray moat. All ICSI dishes were covered with 5 mL of paraffin oil (Merck Group) and equilibrated at 37°C for a minimum of 3 h.

[0192] As with porcine oocytes, oocytes were dropped into conventional ICSI dishes or microarray dishes using a 170-micron Flexipet pipette tip. Instead of sperm, 4-micron Tetraspeck microspheres (Thermo Fisher), centrifuged and washed with GMOPS PLUS medium, were dropped into a 7% PVP droplet in each dish as a visual surrogate for sperm injection into human oocytes. From the PVP droplet, one bead was aspirated into a 5-micron internal diameter injection pipette (Origio Australasia Pty Ltd). The injection process was similar to conventional and microarray ICSI in porcine oocytes. As with porcine ICSI, the bead injection time and number of hand adjustments were recorded. After successful injection, the oocytes were incubated for 90 minutes and observed for lysis. If lysis was not detected, they were re-injected to obtain additional data on timing and hand adjustments. Immediately after obtaining the re-injection data, the oocytes were discarded as biohazardous material. statistical analysis

[0193] All statistical analyses were performed using GraphPad Prism v9.5.1. All data were tested for normality to determine whether parametric or nonparametric analyses should be applied. All binomial data were arcsine transformed to convert them to continuous data for statistical comparisons but displayed as binomial data on the graphs. Where appropriate, paired comparisons were used to account for the effect of replication. The statistical tests used, number of replicates, sample size, and p-values ​​are listed in the legends of each figure. Data are presented as mean ± SEM, and statistical significance was accepted at P < 0.05. Research results Impact on preparation time

[0194] Figure 29(a) shows that the dish preparation times for conventional ICSI and microarray ICSI are comparable. However, in Figure 29(b), the pipette setup time is reduced by half by eliminating the holding pipette. The oocyte transfer procedure into the microwells increased the handling time by 15–20 s for both oocyte introduction and retrieval (Figures 29(c) and (d)), but this was much smaller than the reduction in pipette setup time (>100 s). Impact on ICSI procedures

[0195] As shown in Figure 30(a), microarray ICSI reduced the average procedural time spent manipulating and injecting each oocyte compared to conventional ICSI. The injection process reduced the time per oocyte by more than half (Figure 30(b)), and even when considering the average polar body orientation time per oocyte, the time savings were still evident with microarray ICSI (Figures 30(c) and (d)). Eliminating the holding pipette reduced the number of manual adjustments required to perform microarray ICSI by two-thirds, as tracked based on the number of manual adjustments between different controllers (Figure 31). The level of invagination was comparable between conventional and microarray ICSI (Figure 32). Impact on development rate

[0196] Embryo development rates were consistent across treatments, with fragmentation, survival, and cleavage rates comparable across ICSI procedures (Figure 33(a)-(c)). However, the blastocyst rate was significantly increased in the microarray ICSI group compared to conventional ICSI (Figure 33(d)). As shown in Table 1 below, ICM and TD cell numbers were comparable between blastocysts obtained with conventional and microarray ICSI. [Table 1] Verification of application to human oocytes

[0197] The microarray ICSI device was also suitable for performing microinjections into human oocytes by adjusting the hemisphere size. After the first round of bead injection, no immediate lysis was observed, and of 18 oocytes, one oocyte lysed after 90 minutes and was excluded from reinjection. Consistent with the pig results, microarray ICSI reduced injection time and manual coordination compared to conventional ICSI (Figure 34(a) and Figure 34(b)).

[0198] Throughout this specification, the words "comprise" or variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated component, element, or step, or group thereof, but not the exclusion of other components, elements, or steps, or groups thereof.

[0199] It is understood that terms such as "fastener" or "fastening," "coupling," or "sealing," whether used alone or in combination with other terms such as "means," may be interpreted as interchangeable with other terms that those skilled in the art would consider to be functionally equivalent. Furthermore, the use of any of the aforementioned terms does not exclude the inclusion of other terms.

[0200] Each device and each device component described herein may be provided in a variety of sizes and / or dimensions as desired, with the appropriate size and / or dimensions varying depending on the specifications of the components being connected or the field of use, and may be selected by one skilled in the art.

[0201] It is to be understood that features, elements and / or characteristics described in one embodiment with respect to this disclosure can be used in other embodiments of the invention, as desired.

[0202] While preferred embodiments of the present disclosure have been disclosed for purposes of illustration, those skilled in the art will recognize that various modifications, additions, and substitutions may be made thereto without departing from the spirit and scope of the disclosure and the appended claims.

[0203] When an element or layer is described as being "on" or "in" another element or layer, it is understood to include not only the element or layer directly on or directly within the other element or layer, but also the element or layer when there are intervening elements or layers. In contrast, when an element is described as being "directly on" or "directly within" another element or layer, it means that there are no intervening elements or layers.

[0204] As used herein, the term "and / or" is intended to include any and all combinations of one or more of the associated listed items.

[0205] In this specification, terms such as first, second, and third may be used to describe various elements, components, regions, layers, and / or sections, but these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are used merely to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, a first element, component, region, layer, or section could be referred to as a second element, component, region, layer, or section without departing from the teachings of the present disclosure.

[0206] For ease of description, spatially relative terms such as "below," "above," "higher," "taller," "top," "bottom," "left," and "right" may be used herein to describe the relationship of one element or feature to another, as shown in the figures. It will be understood that spatially relative terms are intended to encompass different orientations of the structures in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures were inverted, an element described as "below" would be oriented "upper" relative to the other elements. Thus, the exemplary term "below" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein should be interpreted accordingly.

[0207] The terms used herein are for the purpose of describing particular embodiments and are not intended to limit the disclosure. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, the terms "comprise," "include," and "comprises" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0208] The embodiments described herein are illustrated with reference to figures and cross-sections and / or are schematic illustrations of preferred embodiments (and intermediate structures) of the present disclosure. As such, variations from the shapes of the illustrations as a result of manufacturing techniques and / or tolerances are to be expected. Therefore, the embodiments herein should not be limited to the particular shapes of the components illustrated herein, but should include deviations in shape that result, for example, from manufacturing.

[0209] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by a person skilled in the art to which the description belongs. Furthermore, terms defined in commonly used dictionaries should be interpreted in a meaning consistent with the context in the art, and should not be interpreted in an idealized or overly formal sense unless explicitly defined in this specification.

[0210] References herein to "one embodiment," "embodiment," "exemplary embodiment," etc. mean that a particular attribute, structure, or characteristic described in connection with this embodiment is included in at least one of the described embodiments. The appearances of such phrases in various places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, when a particular feature, structure, or characteristic is described in connection with any embodiment, one of ordinary skill in the art can implement and / or use that feature, structure, or characteristic in connection with any other embodiment.

[0211] The embodiments described herein also encompass or are directed to methods of using and making all of the elements disclosed above.

[0212] While the present invention has been described above in terms of specific embodiments, it should be understood that the invention is not limited thereto. Numerous modifications and other embodiments of the invention will occur to those skilled in the art upon reading the teachings herein, which are intended to be encompassed and protected by this disclosure and the appended claims.

[0213] All publications mentioned in this specification are incorporated herein by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in this specification is solely for the purpose of providing a context for the present invention. No admission should be taken as an admission that any or all of such matters form part of the prior art base or were general general knowledge in the fields relevant to this invention existing in Australia or elsewhere prior to the priority date of each claim in this application.

[0214] It is indeed intended that the scope of the present invention should be determined by the proper construction and interpretation of the appended claims and their legal equivalents, as understood by one of ordinary skill in the art in reliance on the disclosure herein and the accompanying drawings.

[0215] Data on IVF clinics show wide variation in success rate. BMJ 2002; 325 doi: https: / / doi.org / 10.1136 / bmj.325.7362.460 / e (Published 31 August 2002).

[0216] Boulet, SL, Meht, A., Kissin, DM, Warner, L., Kawwass, JF and Jamieson, DJ (2015) Trends in use of and reproductive outcomes associated with intracytoplasmic sperm injection. Obstet Gynecol Surv. 70:325-6.

[0217] Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, AC (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340:17-18.

[0218] Joris, H., Nagy, Z., Van de Velde, H., De Vos, A. and Van Steirteghem, AC (1998) Intracytoplasmic sperm injection: laboratory set-up and injection procedure. Human Reproduction 13 Suppl. 1:76-86.

[0219] Latham, K. E. Stress signaling in mammalian oocytes and embryos: A basis for intervention and improvement of outcomes. Cell Tissue Res. 2016, 363, 159-167.

[0220] Gilchrist, RB, Lane, M. and Thompson, JG (2008) Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Human Reproduction Update 14: 159-177.

[0221] Tiegs, AW and Scott, RT (2020) Evaluation of fertilization, usable blastocyst development, and sustained implantation rates according to intracytoplasmic sperm injection operator experience. Reprod Biomed Online. 41:19-27.

[0222] Fernandez, J., Pedrosa, C., Vergara, F., Nieto, AI, Quintas, A., et al. (2020) A new oocyte-holding pipette for intracytoplasmic sperm injection without cytoplasmic aspiration: An experimental study in mouse oocytes. Reprod Biol 20: 584-588.

[0223] Ma, S., Wang, P., Zhou, W., Chu, D., Zhao, S., Fu, I. and Li, F. (2020) A modified holding pipette for mouse oocyte fertilization. Theriogenology 141: 142-145.

[0224] Zander-Fox, D., Lam, K., Pacella-Ince, L., Tully, C., Hamilton, H., Hiraoka, K., et al. (2021) PIEZO-ICSI increases fertilization rates compared with standard ICSI: a prospective cohort study. Reprod Biomed Online. 43:404-12.

[0225] Danfour MA, Elmahaishi MS, (2010) Human oocyte oolemma characteristic is positively related to embryo developmental competence after ICSI procedure. Middle East Fertility Society Journal. 2010;15:269-73.

[0226] Yagoub, SH, Thompson, JG, Orth, A., Dholakia, K., Gibson, BC, and Dunning, KR (2022) Fabrication on the microscale: a two-photon polymerized device for oocyte microinjection. J Assist Reprod Genet. 2022;39:1503-13.

[0227] Yagoub, SH, Lim, M., Tan, TCY, Chow, DJX, Dholakia, K, Gibson, BC, Thompson, JG, and Dunning, KR (2014) Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device. J Assist Reprod Genet. 2022;39:1997-2014.

[0228] McLennan, HJ, Blanch, AJ, Wallace, SJ, Ritter, LJ, Heinrich, SL, Gardner, DK, Dunning, KR, Gauvin, MJ, Love, AK, and Thompson, JG. Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery. Sci Rep. 2023;13:562.

Claims

1. 1. A single cell handling device for handling single cells during a microinjection procedure, comprising: a base plate having an upper surface and a lower surface; and a microwell array comprising one or more microwells, wherein the one or more microwells comprise: a microwell wall defining an upper microwell opening and a central well, the at least one microwell wall having two cell stabilization projections formed thereon, the two cell stabilization projections being positioned opposite each other on a common horizontal plane and each defining a cavity, the cell stabilization projections including a first cell stabilization projection defining a channel opening at a periphery of an access channel extending outwardly through the microwell wall, and a second cell stabilization projection defining an exhaust opening, the access channel having a channel base aligned with a center point of the second cell stabilization projection in the horizontal plane; The microwell array may further include a fluid-retaining moat disposed on the top surface of the base dish and extending around the microwell array.

2. 2. The single-cell handling device of claim 1, wherein the moat is in fluid communication with one or more microwells via the access channel, the moat having an inner wall proximal to the microarray and an outer wall distal to the microarray, the outer wall extending vertically to a position higher than a channel base of the access channel, and the exhaust opening is entirely positioned higher than the channel base of the access channel.

3. 2. The single cell handling device according to claim 1, wherein the first cell stabilizing protrusion and the second cell stabilizing protrusion have an elliptical shape in a horizontal plane.

4. 4. The single cell handling device according to claim 3, wherein the diameter of the first cell stabilization protrusion and the diameter of the second cell stabilization protrusion are in the range of 10 microns to 500 microns.

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

6. 10. The single cell handling device of claim 1, wherein the at least one microwell wall is cylindrical and one or more microwells comprise a microwell base, and the at least one cylindrical microwell wall and the microwell base define the central well.

7. 7. The single cell handling device of claim 6, wherein the at least one cylindrical microwell wall has a diameter greater than the diameter of the cell stabilization protrusion.

8. 7. The single cell handling device of claim 6, wherein the at least one cylindrical microwell wall has a diameter in the range of 0.1 micrometers to 1000 micrometers.

9. 7. The single cell handling device according to claim 6, wherein the at least one cylindrical microwell wall has a diameter in the range of 200 micrometers to 300 micrometers.

10. 2. The single cell handling device of claim 1, wherein the access channel comprises two channel walls, a channel base, and an upper opening.

11. The single cell handling device described in claim 1 is characterized in that it comprises a cover and one or more stack connectors arranged on the upper and lower surfaces so as to connect multiple base dishes together when the base dishes are stacked.

12. A single cell handling device as described in claim 6, characterized in that it comprises a sample support stage on the upper surface of a base plate having a sampling edge proximal to the access channel, the sampling edge being formed in a stepped or sloped shape so as to align with the horizontal surface of the channel base.

13. 13. A method of using the single cell handling device of claim 12 in performing an intracytoplasmic sperm injection procedure, comprising: a. Obtaining the single cell handling device of claim 1; b. adding culture medium to the moat of the single cell handling device; c. Obtaining oocyte and sperm samples; d. adding the oocyte to the central well of the microwell; e. placing the sperm sample on the sample support; f. Obtaining a microinjection device; g. Aspirating sperm from the sperm sample through the tip of the microinjection device; h. placing a tip through the access channel of the microwell; i. shifting the oocyte onto the second cell stabilizing protrusion with the tip; j. Microinjecting the sperm into the oocyte; k) withdrawing the microinjection tip by removing only the microinjection tip while the oocyte is held by the first cell stabilization projection.

14. 2. A method for manufacturing a single cell handling device according to claim 1, comprising: preparing the base dish; and processing the microarray and the moat thereon.

15. 2. A method for manufacturing a single cell handling device according to claim 1, comprising processing the microarray and the moat in the base dish.

16. 16. The manufacturing method according to claim 14 or 15, characterized in that it includes a two-component structure in which a two-photon polymerized printed part is inserted into a polystyrene injection molded part.

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