Micromanipulation devices and methods

Abstract

The present disclosure generally relates to micromanipulation devices and methods for the manipulation of mammalian sub-cellular structures. Uses include intra cytoplasmic sperm injection procedures, embryo biopsy procedures, trophectoderm biopsy procedures, mitochondrial transfer procedures, pronuclear transfer procedures, maternal spindle transfer procedures, cumulus cell biopsy procedures and other sub-cellular manipulation procedures. Devices comprise a cellular manipulation dish further comprising multiple sub- cellular manipulation units having a microwell and a cell deposit well wherein the cell deposit opening is disposed adjacent to the microwell opening.

Description

TITLE

[0001] Micromanipulation devices and methodsTECHNICAL FIELD

[0002] The technical field generally relates to micromanipulation devices and methods for use in intra cytoplasmic sperm injection procedures, embryo biopsy procedures, trophectoderm biopsy procedures, mitochondrial transfer procedures, pronuclear transfer procedures, maternal spindle transfer procedures, cumulus cell biopsy procedures and other sub-cellular manipulation procedures.BACKGROUND

[0003] The successful manipulation of individual cells or small cell clusters typically requires highly specialised equipment and precision operation to avoid cellular stress and consequently cellular damage or death.

[0004] Use of many manipulation techniques during cellular growth and development allows for both changes to the cellular microenvironment and direct intervention in cellular physiology, for example, by altering the genetic makeup of cells and / or proteins and the molecular profile of cells or their surrounding environments.

[0005] In the area of assisted reproductive technology, micromanipulation of cells, such as oocytes, sperm and embryos, use instruments that enable a skilled person, normally working with a microscope, to sample one or more cells, inject sperm or other materials (including genetic and biological materials such as cellular mitochondria) and / or sample the microenvironment adjacent to cells.

[0006] For example, Preimplantation Genetic Testing (PGT) is used to test embryos for a specific known single gene condition or chromosomal variation. It requires cells to be extracted from the embryo for DNA testing through a procedure called blastomere (specificfor cleavage stage embryos) and trophectoderm biopsy (specific for blastocyst stage embryos). The procedure has become increasingly popular over the past 20-30 years due to improvements in the sensitivity and specificity of testing arising from more accurate molecular analysis techniques, as well as the ability to biopsy not only cleavage stage embryos (at the 4-8 cell stage) but increasingly blastocyst stage embryos (100+ cells).

[0007] Trophectoderm biopsy involves removal of 5-10 cells of the trophectoderm (outer) layer of the blastocyst stage embryo (at day 5-6 post fertilisation in humans). The technique involves a complex workflow that includes the use of two micromanipulators with control joysticks, two microinjectors, on an inverted microscope. The coarse and fine focus knobs are used to focus on the embryo, and stage controls allow the operator to navigate to individual embryos within a culture droplet.

[0008] Generally, embryos that are deemed suitable for trophectoderm biopsy have either a natural herniation of the zona pellucida (the glycoprotein coat surrounding the embryo), a morphological feature described as embryo hatching, or have been subject to laser treatment to thin the zona pellucida (or, if a laser is not available, an alternate method may be used to create a partial thinning of the zona at a suitable position) to accelerate the herniation of trophectoderm cells. The embryo is held onto a holding pipette attached to one of the micromanipulators with suction. A biopsy pipette is then placed adjacent to the herniated cells of the embryo and with negative pressure, and preferably between 5-10 cells are aspirated into the pipette and either a laser is used to separate the herniated cell sample from the embryo, which is then pulled away, or a "flick" method may be used.

[0009] In the pulling method, blastocysts are held firmly with the holding pipette and the biopsy needle is used to pull trophectoderm cells away from the blastocyst, while laser pulses can be applied. In the flicking method, laser pulses, light suction or careful manipulation allows trophectoderm cells to be drawn inside the biopsy pipette and subsequently the trophectoderm cells are excised with a quick movement of the biopsy pipette against the holding pipette or other hard surface.

[0010] Once separated, the cells are deposited in a medium droplet where they can be pipetted for cell washing and tubing. The tube containing the biopsied cells is sent for genetic analysis.

[0011] These biopsy techniques, however, require highly skilled staff with micromanipulation skills that take years to master. Furthermore, there is a need to reduce the difficulty of micromanipulation by eliminating the need for so many handling apparatus.

[0012] Other sub-cell extraction techniques require similar equipment as embryo biopsy techniques. There are several pathologies that are associated with the application of these micromanipulations. Usually these are done in conjunction with sourced donor oocytes. In practice, there are similar aspects; the oocyte is stabilised with a holding pipette whilst enucleation of the metaphase II plate of the matured but unfertilized oocyte is performed, or transfer of pronuclei or metaphase II plate or mitochondria are performed. These include pronuclear transfer (PNT) of the zygote, maternal spindle transfer (MST) of the oocyte, and mitochondrial transfer.

[0013] By way of further example, the prevalence of mitochondrial disease (mtDNA) in Australia is 1 in 250 people with several hundred families already diagnosed. IVF-based assisted reproductive technology involving mitochondrial donation is a viable preventative measure for debilitating mitochondrial disease. The technique allows an IVF clinic to implant an embryo containing nuclear DNA from both biological parents and donor mitochondrial DNA.

[0014] The procedure requires similar handling equipment as embryo biopsy techniques, whereby the oocyte / embryo is stabilised with a holding pipette whilst enucleation of unfertilized oocyte or pronuclear extraction of the embryo is performed.

[0015] Performing highly complex procedures such as mitochondrial transfer and enucleation of oocytes or embryos requires a highly experienced and a skilled technician with organization skills, planning skills and specialized equipment. The traceability ofrecipient and donor eggs is also paramount to the success of the procedure, thus, dish setup and labelling is critical.

[0016] Performing highly complex procedures such as biopsy, mitochondrial transfer and enucleation of oocytes or embryos requires micromanipulation equipment and skills.

[0017] Micromanipulation typically consists of an input joystick, a mechanism for reducing the range of movement and an output section with a means of holding a microtool to hold, inject, cut or otherwise manipulate the object as required. Micromanipulation further requires a mechanism for reducing movement, which usually requires significant suction forces on the cells. These procedures require a high level of traceability and technical skill to perform.

[0018] The shortcomings of these instruments and the use of them in such procedures includes:• Holding pipette adds suction forces that are frequently detrimental to the cell.• Holding pipette control requires extensive training to master.• Dishes have no uniform structure for set-up and arrangement of oocyte location.• Few dishes have a specifically designed numbering system for traceability (particularly important for separation of donor and recipient oocytes for enucleation and mitochondrial transfer).• Procedures are complex and take extensive practice to reproduce proficiently.• More experienced embryologists deliver more successful results (2).• Traceability is low, unless one dish per embryo is used.• Flick biopsy methods have a higher risk of damaging embryos (3).• Use of laser generates localised heat that can be detrimental to cell integrity.• Increased exposure to environmental and / or temperature fluctuations during handling and manipulation.

[0019] Within the laboratory there is a need to bridge the gap between a lack of skilled staff for highly technical procedures. Micromanipulation is required for procedures bringingadded complexity that causes more time-consuming procedures and a reduced likelihood of success. More experienced laboratory staff performing these types of procedures have been linked to improved outcomes for procedures like ICSI (1)(2), however, it is envisaged that reducing the complexity of procedure can mitigate the need for highly experienced staff who are in high demand.

[0020] Here we describe improved devices and methods of single cell and cell cluster micromanipulation, in particular, for assisted reproduction which ameliorates the need for complex apparatus that are inherently difficult for laboratory staff to operate; making these procedures more consistent and easier to perform, in turn, reducing the chance of errors leading to the loss of viable cells during workflow procedures.SUMMARY

[0021] In a first broad form, the present disclosure relates to a micromanipulation device for the manipulation of a sub-cellular structure of a mammalian cell cluster.

[0022] More particularly, the present disclosure relates to a micromanipulation device for the manipulation of a sub-cellular structure of a mammalian cell cluster comprising; a cellular manipulation dish further comprising; multiple sub-cellular manipulation units, each sub-cellular manipulation unit comprising; a microwell formed from at least one discontinuous microwell wall having at least one upper microwell wall edge defining a microwell access opening, at least one microwell wall lateral edge defining a microwell side opening and a lower cell support, and a cell deposit well formed from a least one discontinuous cell deposit wall having at least one upper cell deposit wall edge defining a cell deposit well access opening, and at least one cell deposit wall lateral edge defining a cell deposit side opening, wherein the cell deposit opening is disposed adjacent to the microwell opening.

[0023] Preferred embodiments of the present disclosure relate to micromanipulation devices wherein; the at least one discontinuous cell deposit wall has at least a first cell deposit wall lateral edge and a second cell deposit wall lateral edge defining a cell depositside opening, and at least a one lower cell deposit wall edge, the cell deposit well comprising a cell deposit base wherein the at least a one lower cell deposit wall edge is joined to the cell deposit base, and the lower cell support and the cell deposit base are disposed in a single plane.

[0024] In preferred embodiments of the present disclosure, the micromanipulation device are characterised wherein; the at least one discontinuous microwell wall comprises at least two lateral edges defining a microwell side opening, the at least two lateral edges projecting outwardly to form two parallel channel walls defining a channel to guide the passage of fluid therethrough, the channel walls terminating in two lateral channel edges terminating at an oblique lateral angle to form a cutting edge, wherein the first cell deposit wall lateral edge and a second cell deposit wall lateral edge abut against the parallel channel walls of the microwell wall.

[0025] Preferably, each sub-cellular manipulation unit of micromanipulation device of the present disclosure comprise; a cellular washing well, a unique identifier, and a media reservoir containing the microwell and cell deposit well at least partially therein.

[0026] In a preferred form, each sub-cellular manipulation unit preferably comprises a media reservoir configured to maintain the cell deposit well entirely therein, and configured to maintain the microwell partially therein.

[0027] In a preferred form, the cell deposit well comprises at least one cell deposit wall wherein the cell deposit walls have a height lower than the height of the media reservoir.

[0028] A preferred micromanipulation device is disclosed wherein the at least one discontinuous cell deposit wall has a height lower than the at least one discontinuous microwell wall, and wherein the cell deposit well is contained entirely within the media reservoir, to permit the flow of fluid from the media reservoir to the cell deposit well, and from the cell deposit well to the microwell through the channel.

[0029] In use, the media reservoir is preferably filled with media to a volume having a level higher than the cell deposit well and lower than the height of the microwell, such that media is exchanged with the cell deposit well over the cell deposit wall and with the microwell from the cell deposit well through the discontinuous portion of the microwell.

[0030] Further, in use, a cell is placed in the cell deposit well prior to micromanipulation and is transferred to the microwell through the discontinuous portion of the microwell wall where it is maintained in place during a micromanipulation procedure. The relative configuration of the media reservoir, the cell deposit well and the microwell alleviates any "media shock" experienced by a cell during a micromanipulation procedure.

[0031] Preferred sub-cellular manipulation units comprise a rinse well for containing media therein. Such units are preferably external to the media reservoir.

[0032] In use, the rinse well is typically filled with the same media as the sub-cellular manipulation unit media reservoir. The rinse well is typically located in close proximity to the media reservoir. The rinse well is provided to allow the user to rinse a pipette tip or other instrument prior to its use in performing a micromanipulation procedure.

[0033] Preferably, the discontinuous microwell side wall forms a channel continuous with the cell deposit wall to permit the exchange of fluid therethrough. Preferably, at least one edge of the discontinuous microwell side wall is sharp and comprises a cutting edge.

[0034] Preferably, the internal diameter of the microwell is greater at an upper opening of the microwell than a lowermost point of the microwell. Preferably, the internal diameter of the microwell is between 1000pm and 100p.m. More preferably, the internal diameter of the microwell is between 600pm and 200pm.

[0035] In a preferred embodiment of the present disclosure, the microwell of a micromanipulation device comprises an internal diameter of less than 800pm.

[0036] Preferably, the lower cell support of the microwell is tapered in shape.

[0037] The internal surface of the microwell wall is preferably rounded circumferentially and tapers centrally toward a lowermost point of the microwell. Preferably, the internal surface of the microwell wall forms a conic shape.

[0038] Preferably, the internal diameter of the microwell tapers by 25% to 50% from an upper opening of the microwell to a lowermost point of the microwell. Preferably, the internal diameter of the microwell tapers by 35% to 45% from an upper opening of the microwell to a lowermost point of the microwell.

[0039] Preferably, the internal diameter of the microwell tapers from 500pm an upper opening of the microwell to 300pm at a lowermost point of the microwell.

[0040] In preferred embodiments of the present disclosure, the cellular manipulation dish of the micromanipulation device further comprises; a dish base for supporting multiple sub- cellular manipulation units thereon, and a dish frame positioned upon the dish base circumferentially surrounding the multiple sub-cellular manipulation units.

[0041] Preferably, each sub-cellular manipulation unit of the multiple sub-cellular manipulation units is substantially identical and is arranged in an array.

[0042] Micromanipulation devices of the disclosure preferably comprise a dish base. The dish base is preferably configured (i.e. sized and shaped) to engage with imaging apparatus. The dish base preferably comprises a recess on its upper surface for positioning a sub- cellular manipulation array therein.

[0043] Preferably, the sub-cellular manipulation array is positioned within the recess of the support base to improve the focal plane of the sub-cellular manipulation unit.

[0044] Preferably, the dish base comprises a width greater than 50mm and a length of greater than 50mm, and the multiple sub-cellular manipulation units comprise a width of less than 1000pm and a length of less than 1000p.m.

[0045] A preferred cellular manipulation dish may comprise one or more sub-cellular manipulation unit arrays, comprising multiple sub-cellular manipulation units.

[0046] Micromanipulation devices and methods are described herein for use in substitution of known holding pipettes. Elimination of the holding pipette ameliorates any need for suction forces to hold cells or cell clusters in position for any procedure.

[0047] Additional benefits arise from the simplification of micromanipulation tools, which are traditionally very complex. Consequently, users experience increased efficiency, increased traceability and decreased complexity in any micromanipulation technique.

[0048] Sharp instruments and lasers are typically deployed in traditional micromanipulation procedures, however, systems involving a combination of the sub-cellular manipulation dish and a simple pipette, or similar instrument, provide an alternative to the use of scalpels and lasers at the sub-cellular scale.

[0049] The use of micromanipulation devices and systems reduces stress on oocytes and embryos improving the accessibility of micromanipulation techniques and success rates from their use.

[0050] Preferred micromanipulation devices comprise a surface of the two parallel channel walls or a portion thereof which is coated or treated to be non-polar.

[0051] In particular, a preferred surface of the two parallel channel walls or a portion thereof is coated with non-polar groups selected from the group consisting of; alkyl groups, silicones having the functional group — [Si(CH3 )2 O]-, fluorocarbons having the functional group -Cn F2n+1, any composite thereof or any other coating that renders the surface nonwettable.

[0052] In particular, a preferred surface of the two parallel channel walls or a portion thereof is coated or treated to be functionalised with a silane compound, a silane conjugate or any composite thereof.

[0053] Preferably, micromanipulation devices of the present disclosure are used in a procedure selected from the group consisting of; intra cytoplasmic sperm injection procedures, embryo biopsy procedures, trophectoderm biopsy procedures, mitochondrial transfer procedures, pronuclear transfer procedures, maternal spindle transfer procedures, cumulus cell biopsy, assisted hatching, high magnification sperm selection (IMSI), and assisted collapse of blastocyst before vitrification.

[0054] In a second broad form, the present disclosure relates to a method for diagnosis of a medical condition comprising the steps of; obtaining the micromanipulation device of the disclosure, obtaining a patient sample, performing a manipulation of a sub-cellular structure the patient sample, and assessing a result of a manipulation of a patient sample.

[0055] Preferred methods comprise the additional step of performing a procedure selected from the group consisting of; intra cytoplasmic sperm injection procedures, embryo biopsy procedures, trophectoderm biopsy procedures, mitochondrial transfer procedures, pronuclear transfer procedures, maternal spindle transfer procedures, cumulus cell biopsy, assisted hatching, high magnification sperm selection (IMSI), and assisted collapse of blastocyst before vitrification.

[0056] Preferred methods comprise the additional step of manoeuvring the patient sample against at least one of the cutting edges of at least one of the two parallel channel walls of a sub-cellular manipulation unit.

[0057] In a second broad form, the present disclosure relates to a method of performing a sub-cellular manipulation of a cell comprising the steps of; obtaining a micromanipulation device or system according to the first or second broad forms, obtaining a cell sample, and manipulating the cell sample.

[0058] A preferred method of the disclosure may include the additional steps of; obtaining a sub-cellular manipulation dish, placing the cell sample in a cell deposit well, transferring the cell sample to a microwell, or performing a manipulation of the cell within the microwell.

[0059] Used in these procedures, micromanipulation devices and methods are likely to have a positive impact on live birth rates due to a reduction in the stress of these procedures on oocytes and embryos.

[0060] Micromanipulation devices and systems offer improvements in known devices, systems and their uses as they offer:• Simplification of complex procedures that require extensive training to be considered proficient.• High levels of traceability of sample materials.• Design features that allow cellular and subcellular washing within the device.• Design that simplifies the 'flick biopsy' technique (speeding up the procedure, reducing risk of damage to blastocyst and reducing training overheads).• Cellular and subcellular cradling, which reduces mechanical stress and localises the sample.• Improved collection of the desired number of cellular or subcellular samples which reduces the percentage of re-biopsies required.• Standardisation of the cellular and subcellular biopsy process (4)(5).• Standardisation of the pronuclear transfer or maternal spindle transfer techniques.• Increased traceability and uniformity of layout with designated labelled areas for sample materials.

[0061] As used herein the term "sub-cellular" and any pluralisations and derivatives thereof, define the nature of an article described as "sub-cellular" as applicable to uses at an intracellular scale. While the term "sub-cellular" defines a scale of possible uses, it is not to be understood to imply a limitation to intracellular uses only. For example, cumulus cell cutting occurs at a sub-cellular scale but is performed on the cell surface. Similarly, other cell surface treatments may be described as "sub-cellular" due their scale whilst occurring outside the cell.

[0062] Broad embodiments of the invention now will be described with reference to the accompanying drawings together with the Examples and the preferred embodiments disclosed in the detailed description. The invention may be embodied in many different forms and should not be construed as limited to the embodiments described herein. These embodiments are provided by way of illustration only such that this disclosure will be thorough, complete and will convey the full scope and breadth of the invention.DETAILED DESCRIPTION OF EMBODIMENTSBRIEF DESCRIPTION OF THE FIGURES

[0063] Figure 1 provides a side perspective view of a micromanipulation device according to an embodiment of the disclosure.

[0064] Figure 2 provides an enlarged portion of a sub-cellular manipulation array shown at portion A of the micromanipulation device illustrated in Figure 1.

[0065] Figure 3 provides a side sectional view through section A-A of a sub-cellular manipulation unit illustrated in Figure 2.

[0066] Figure 4 provides an enlarged top view of a media reservoir of portion B of a sub- cellular manipulation unit illustrated in Figure 2.

[0067] Several embodiments of the invention are described in the following examples.EXAMPLESExample 1 - Micromanipulation device

[0068] Figure 1 provides a macro-scale view of micromanipulation device 100, which takes the general form of a cellular manipulation dish 105 constructed of a square, planar, injection moulded, polystyrene dish base 102 having rounded edges and having, projectingfrom the upper surface of dish base 102, a smaller, rectangular dish frame 104 formed thereon.

[0069] The scale of dish base 102 is of a macro scale to enable an operator to directly handle and move micromanipulation device 100 as needed, for example to transfer micromanipulation device 100 from or to an incubator, a laminar flow unit, a microscope stage and the like.

[0070] Dish base 102 is approximately 175mm in length, 75mm in width and 5mm in height to fit within the dimensions required of a microscope stage, however, it is anticipated that these dimensions may be adjusted anywhere between approximately 1mm to 100mm in height or width to meet the dimensions required of a visualisation apparatus desired by an operator. Dish base 102 may be square or rectangular or other planar shape to meet the needs of the user or the geometry of a desired visualisation apparatus.

[0071] The rounded edges of dish base 102 provide a more comfortable grip to the user and prevent inadvertent chipping of the corners of cellular manipulation dish 105 during handling and manipulation.

[0072] Positioned within the perimeter of dish frame 104 and positioned upon dish base 102 at a generally centralised position is a polymer-based, injection moulded, or micron 3D printed, sub-cellular manipulation array 101. Sub-cellular manipulation array 101 is adhered to the upper surface of dish base 102 with an inert, biocompatible adhesive.

[0073] The height of dish base 102 has been selected to position sub-cellular manipulation array 101 within the focal plane of a microscope when sub-cellular manipulation array 101 is positioned upon the upper surface of dish base 102, however sub-cellular manipulation array 101 may also be embedded within the upper surface of dish base 102 (not shown) to bring the contents of sub-cellular manipulation array 101 into a desired focal plane according to the choice of visualisation apparatus desired by a user.

[0074] Dish frame 104 is of a smaller length and width that the dish base 102 and is raised from the upper surface of dish base 102. Dish frame 102 provides a lip upon which a cover (not shown) may be placed and held in position to protect the contents of sub-cellular manipulation array 101, maintain humidity or contain gases in the environment of sub- cellular manipulation array 101, or prevent evaporation from sub-cellular manipulation array 101.

[0075] Dish frame 104 provides a lip upon which additional micromanipulation devices may be stacked without making contact with sub-cellular manipulation array 101. The lower surface of dish base 102 may have grooves formed therein (not shown) which correspond with the dimensions of dish frame 104 to enable the fitting of one micromanipulation device 100 upon another when stacked.

[0076] Dish frame 104, formed to surround sub-cellular extraction dish 101, may hold a large volume of media therein, such that sub-cellular extraction dish 101 may be submerged in a large volume of media, as required by the user. The height of dish frame 104 may be raised or lowered to modulate the volume or media required to maintain culture conditions within sub-cellular extraction dish 101. The height of dish frame 104 may be raised or lowered as required to fit within a groove formed within the lower surface of dish base 102.

[0077] Dish frame 104 frames a platform on which sub-cellular procedures may be performed with the aid of sub-cellular manipulation array 101. Additionally, one or more internal portions of the upper surface of dish base 102, within dish frame 104, may be occupied by devices for performing additional micro-scale procedures. For example, an internal portion of the upper surface of dish base 102 may be designated for the pickup of sperm, and / or another internal portion may be modified for the performance of an ICSI procedure. Advantages of such approaches occur where a series of micro scale procedures are required to be performed in a single workflow, or where devices for performing the additional micro-scale procedures may be fabricated by the fabrication methods described herein.

[0078] Sub-cellular manipulation array 101 is of a micro scale, measuring approximately 5000 ,m in length, approximately 5000 .m in width and approximately 500 .m in height. Sub-cellular manipulation array 101 is shaped to enable cells to be gently held therein whilst a procedure is being performed on a mammalian cell or cell cluster.

[0079] It will be appreciated that the width of sub-cellular manipulation array 101 is determined by the number of sub-cellular manipulation units 203 within the array. While the five unit, sub-cellular manipulation array 101 is generally square, the width of sub- cellular manipulation array 101 will increase where more than five sub-cellular manipulation units are present in the array. The dimensions of the array will also vary depending on the shape of the array, for example, the provision of sub-cellular manipulation units 203 in a circular array would alter the total size of sub-cellular manipulation array 101.

[0080] Figure 2 provides an enlarged view of the portion of Figure 1 indicated by "A", showing further detail of sub-cellular manipulation array 101. Sub-cellular manipulation array 101 is formed from five sub-cellular manipulation units 203, with each sub-cellular manipulation unit 203 comprising a microwell 201 in fluid communication with a cell deposit well 209. Each microwell 201 is formed from a generally circular, microwell wall providing an upper access opening to microwell 201 from above, shaped in a generally conical form at the lowermost point of microwell 201 providing a lower cell support.

[0081] The generally circular microwell wall of microwell 201 is broken by a slotted, vertical opening in the microwell wall to provide guided access to the internal space within microwell 201 from a horizontal plane. The slotted, vertical opening in microwell wall is flanked by a pair of parallel, channel walls 204 having a similar height to the microwell wall and forming a channel therebetween. Channel walls 204 terminate within the internal space of cell deposit well 209, to channel fluid or guide apparatus between the internal space of cell deposit well 209 and microwell 201.

[0082] Cell deposit well 209 is formed from a generally circular cell deposit wall providing an upper access opening to cell deposit well 209 from above, and a planar, circular cell deposit base. The generally circular wall of cell deposit well 209 is broken by a slotted, verticalopening in the wall, terminating in a pair of parallel, generally vertical edges each abutting against the exterior surface of each parallel channel wall 204 in a sealed join.

[0083] Microwell 201 and cell deposit well 209 are formed upon reservoir base 205 which, in turn, is contained within media reservoir 208. The cell deposit base of cell deposit well 209 and lower cell support of microwell 201 rest upon reservoir base 205, positioned at a common or single horizontal plane.

[0084] The height of cell deposit well 209 is lower than the height of media reservoir 208 to permit the flow of fluid from media reservoir 208 over the wall of cell deposit well 209. The channel formed by channel walls 204 permits the ingress of any such fluid into microwell 201. The height of microwell 201 is greater than the height of cell deposit well 209 and media reservoir 208 to prevent the flow of fluid to into microwell 201 other than through the channel or upper access opening.

[0085] In addition to media reservoir 208, each sub-cellular manipulation unit 203 further comprises a unique identifier provided by numbering 206 and a wash station 207. Each sub- cellular manipulation unit 203 measures approximately 800p.m in length, approximately 400p.m in width and approximately 400p.m in height. The relationship between each sub- cellular manipulation unit 203 and each cellular sample is one to one.

[0086] Each sub-cellular manipulation unit 203 of sub-cellular manipulation array 101 is aligned in parallel to form a series of microwells 201 positioned in a linear array, each microwell 201 identified by unique numbering 206. The layout of the cell units within the dish improves traceability of autologous cells with numbering and specific areas and locations for each cell sample and individual reagents.

[0087] Sub-cellular manipulation array 101 comprises a dry frame 202 for cell washing.Each wash station 207 is formed within dry frame 202 providing a pipette rinsing reservoir containing 20p.l of culture media.

[0088] Media reservoir 208 is contained by four vertical reservoir walls and reservoir base 205 to contain media therein and prevent media exchange with adjacent cell samples. Cell deposit well 209 provides a designated place to deposit cells separated from the cell sample; in the present example a blastocyst.

[0089] Each cell deposit well 209 provides an outlined area for cells to be easily located after all biopsies are completed thereby providing an easier tubing process once the dish is moved back to a dissecting microscope, or similar apparatus. The walls of each cell deposit well 209 also prevent cells drifting in the dish, eliminating any risk of mixing up genetic profiles of each blastocyst.

[0090] Frame 202 defines an area over which an oil overlay may be applied to cover any fluids contained in media reservoir 208 and cell deposit portion 209 to prevent evaporation and minimise environmental fluctuations.

[0091] Sub-cellular manipulation array 101 is designed with two functional sections, one section for performing extraction or other procedures and the other section for cell washing and tubing from the same dish.

[0092] Tubing of cells is typically performed without an oil overlay to prevent contamination.

[0093] Sub-cellular manipulation array 101 provides a surface for pipetting cells from a phosphate buffered solution (or similar) to a separate tube. Both sections of sub-cellular manipulation array 101 are provided in each sub-cellular manipulation unit 203, such that each sub-cellular manipulation unit 203 contains its own reservoir of culture media in microwell 201 and cell deposit well 209.

[0094] By maintaining each sub-cellular manipulation unit 203 section together with an individual wash station 207 (also uniquely numbered 206), any risk of cross contamination and compromised results is significantly reduced.

[0095] A section through microwell 201, cell deposit well 209 and wash station 207 is provided at Figure 3 as indicated at section A-A in Figure 2. The side sectional view provided at Figure 3 shows the shape of microwell 201. Microwell 201 is shaped to accommodate the varying sizes of cell clusters.

[0096] Microwell 201 is designed to hold a blastocyst and is therefore shaped such that the uppermost edge of the internal surface of the microwell wall is approximately 400pm in diameter and the lowermost edge of internal surface of the microwell wall is 300pm in diameter, which ensures that no blastocyst is too big or too small for the microwell. The shape alleviates the need for a holding pipette to maintain the position of the blastocyst (or other cell) for procedures involving sub-cellular manipulation.

[0097] Conic section profile 301 illustrates the inverted conic shape of the internal surface of the microwell wall (e.g. a rotated parabolic profile) which, as described above, is shaped to contain a range of cell cluster sizes, from approximately 300pm to approximately 500pm, therein.

[0098] The profiles of wash station 207, cell deposit well 209 and channel wall 204 are also illustrated in the side sectional profile illustrated in Figure 3. Wash station 207 is generally hemispherical in shape and, while it remains in close proximity to cell deposit well 209, it is fluidically separated from cell deposit well 209 and microwell 201. The depth of wash station 207 is greater than the depth of cell deposit well 209 to contain the required volume of fluid for washing cells.

[0099] The side section view shown in Figure 3 shows the placement of the cell deposit base 302, channel walls 204 and lower cell support 303 in a single, horizontal plane to permit the flow of fluid between them. The channel walls 204 are a height approximately equal to the microwell to provide a guiding channel for users manipulating apparatus within microwell 201.

[0100] Figure 4 provides an enlarged top view of a portion of a sub-cellular manipulation unit showing microwell 201, channel 210, channel walls 204 and cell deposit well 209 in further detail.

[0101] Figure 4 shows the formation of pipette slot 401 extending from cell deposit base 302 to the lowest point of lower cell support 303 of microwell 201. Pipette slot 401 guides the placement of cells or cell clusters transferred from cell deposit well 209 to lower cell support 303.

[0102] Pipette slot 401 is also formed by parallel channel walls 204 project outwardly from microwell 201 which also define channel 210. Microwell wall 403 can be seen to be contiguous with channel walls 204 which terminate within the space formed within cell deposit wall 406. Cell deposit wall 406 abuts against and is joined to the exterior surface of channel walls 204 to seal the structures and contain fluid within cell deposit well 209 from the fluid within reservoir base 205.

[0103] Cell deposit well 209 forms a sealed basin for containing a cell and media mixture therein, formed from a generally circular, vertical cell deposit wall 406 terminating at a lower horizontal edge with the reservoir base 205. The rounded vertical wall terminates at two vertical edges with the exterior surface of channel walls 204 to provide fluid communication between the microwell 201 and the cell deposit well 209 through channel 201.

[0104] The channel edges 404 of channel walls 204 are shaped at a lateral oblique angle to form a pair of cutting edges 402. Cutting edges 402 are angled at a opposite oblique lateral angles. The outer edge of channel edges 404 are curved to prevent undesired cutting or damage to cells or cell clusters.

[0105] Cutting edges 404 are positioned and shaped to enable a user to manipulate a cell upon one or both cutting edges 404, for example by manoeuvring the cell or cell cluster across the surface of the cutting edge 404, to remove exterior cells or surface molecules.

[0106] To avoid surface interactions that may impede or interfere with the manipulation of a cell or cell clusters, the internal or external surfaces of channel walls 204, cutting edges 402, channel edges 404 or pipette slot 401 are selectively surface treated to provide a nonpolar surface. In the present example, each of these surfaces is silane functionalised.

[0107] Examples of use of the micromanipulation device described herein in the performance of a trophectoderm biopsy procedure and a mitochondrial transfer procedure are further described below. These examples are illustrative of many procedures in which the micromanipulation device may be used.Example 2 -Trophectoderm Biopsy Procedure

[0108] Trophectoderm biopsies are performed during procedures for genetic testing of embryos, involving the removal of 5-10 blastocyst cells from a viable embryo. At the blastocyst stage, the embryo comprises trophectoderm cells (TE) and inner cell mass cells (ICM), which must remain intact to maintain the viability of the blastocyst.

[0109] Five blastocysts are each placed in one of five microwells 201 of a sub-cellular manipulation array 101 and are each individually numbered. A biopsy pipette is place through channel 210 guided by pipette slot 401. A small amount of suction is applied to the herniating cells of the blastocyst which enables the user to manoeuvre the blastocyst nearest to pipette slot 401.

[0110] The blastocyst is effectively trapped in the rotated conic section shape of microwell 201 wherein the cells can be swiped cleanly away using the flick method, well known to persons skilled in the art, against cutting edges 402.

[0111] The compact and linear array of microwells 201 aid visualisation of both blastocyst and cells after extraction from the blastocyst. The visual markers and numbering on the sub- cellular manipulation array 101 enables the safe retrieval of cells on a lower power magnification.

[0112] Cells are washed using one of several possible protocols. Depending on clinical protocol this is likely to be 3 x 20 .l phosphate buffered saline (PBS) and polyvinylpyrrolidone (PVP) droplets; the cells are washed through each drop then pipetted into the tube.

[0113] To avoid cell contamination and eliminate biopsy pipette changes between embryos, pipette rinsing is performed at individual wash stations 207 which are neatly positioned next to each microwell 201. This increases the accuracy and speed of the procedure. Cells are finally aspirated and moved to individual tubes for genetic analysis.

[0114] Traditional trophectoderm biopsy procedures involve a complex workflow which includes the use of two micromanipulators with control joysticks and two microinjectors on an inverted microscope. Embryos are held onto a holding pipette attached to one of the micromanipulators with suction.

[0115] By eliminating the need for a holding pipette, the procedure becomes technically simpler for the user, as several micromanipulation tools are no longer required. Not only does it reduce the number of micromanipulation tools but also offers an improved flick biopsy technique with sharp edges 402 of microwell 201 to flick against.

[0116] The traditional practice for collecting the sample from the embryo involves making a hole with a laser in the zona pellucida and either using a laser to separate the cell sample from the embryo or using the flick method. As a laser is not required by using sub-cellular manipulation array 101, its use is likely to improve embryo and cell viability for improved implantation rates and successful genetic diagnosis, respectively.

[0117] As the only tool required in addition to sub-cellular manipulation array 101 is the biopsy pipette, gentle aspiration is the only force applied to cells to cleanly shear them off from the blastocyst for genetic testing. This means many more operators may be comfortable completing a biopsy, and a gentler biopsy for all users may result in higher implantation rates after embryos are vitrified and warmed for embryo transfer.

[0118] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0119] It will be understood that the terms 'fastener' or 'fastening', 'coupling' or 'sealing' when used alone or together with other terms such as 'means' or others, may be used interchangeably where interpretation of the term would be deemed by persons skilled in the art to be functionally interchangeable with another. Further, the use of one of the aforementioned terms does not preclude an interpretation when another term is included.

[0120] The various apparatuses and components of the apparatuses, as described herein, may be provided in various sizes and / or dimensions, as desired. Suitable sizes and / or dimensions will vary depending on the specifications of connecting components or the field of use, which may be selected by persons skilled in the art.

[0121] It will be appreciated that features, elements and / or characteristics described with respect to one embodiment of the disclosure may be used with other embodiments of the invention, as desired.

[0122] Although the preferred embodiments of the present disclosure have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the disclosure and accompanying claims.

[0123] It will be understood that when an element or layer is referred to as being "on" or "within" another element or layer, the element or layer can be directly on or within another element or layer or intervening elements or layers. In contrast, when an element is referred to as being "directly on" or "directly within" another element or layer, there are no intervening elements or layers present.

[0124] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0125] It will be understood that, although the terms first, second, third, etcetera, may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another region, layer or section. Thus, a first element, component, region, layer or section could be termed a second element, component, region, layer or section without departing from the teachings of the present disclosure.

[0126] Spatially relative terms, such as "lower", "upper", "lowest", uppermost", "top", "bottom", "left", "right", "horizontal", "vertical" and the like, may be used herein for ease of description to describe the relationship of one element or feature to another element(s) or feature(s) as illustrated in the figures. It will be understood that spatially relative terms are intended to encompass different orientations of structures in use or operation, in addition to the orientation depicted in the drawing figures. For example, if a device in the drawing figures is turned over, elements described as "lower" or "lowest" relative to other elements or features would then be oriented "upper" or "uppermost" relative the other elements or features. Thus, the exemplary term "lower" or "lowest" 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.

[0127] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of 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 indicates otherwise. It will be further understood that the terms "including," "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0128] Embodiments of the description are described herein with reference to diagrams and / or cross-section illustrations, for example, that are schematic illustrations of preferred embodiments (and intermediate structures) of the description. As such, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and / or tolerances, are to be expected. Thus, embodiments of the description should not be construed as limited to the particular shapes of components illustrated herein but are to include deviations in shapes that result, for example, from manufacturing.

[0129] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this description belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealised or overly formal sense unless expressly so defined herein.

[0130] Any reference in this specification to "one embodiment," "an embodiment," "example embodiment," etc., means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the description. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with any embodiment, it is within the purview of one skilled in the art to effect and / or use such feature, structure, or characteristic in connection with other ones of the embodiments.

[0131] Embodiments are also intended to include or otherwise cover methods of using and methods of manufacturing any or all of the elements disclosed above.

[0132] While the invention has been described above in terms of specific embodiments, it is to be understood that the invention is not limited to these disclosed embodiments. Upon reading the teachings of this disclosure many modifications and other embodiments of the invention will come to the mind of those skilled in the art to which this invention pertains,and which are intended to be and are covered by both this disclosure and the appended claims.

[0133] All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

[0134] It is indeed intended that the scope of the invention should be determined by proper interpretation and construction of the appended claims and their legal equivalents, as understood by those skilled in the art relying upon the disclosure in this specification and the attached drawings.CITATION LIST(1) Tiegs, A.W. & Scott, R.T. "Evaluation of fertilization, usable blastocyst development and sustained implantation rate according to intracytoplasmic sperm injection operator experience" Reproductive BioMedicine Online, 2020, 41, 19-27 DOI: 10.1016 / j.rbmo.2020.03.008(2) Swain, J. E. "Controversies in ART: considerations and risks for uninterrupted embryo culture." Reproductive BioMedicine Online, 2019, 39, 1, 19-26DOI :https: / / pubmed. ncbi.nlm.nih.gov / 32466993 / (3) 3 Xue, S., Y. Gao, R. Wang, D. Yang, Q. Peng and D. Li, "An innovative design for trophectoderm biopsy without laser pulses: a step-by-step demonstration." Fertil Steri. Video, 2023 DOI: 10.1016 / j.fertnstert.2023.07.010(4) 4 Sachdev, N. M., L. Ribustello, E. Liu, D. H. McCulloh, J. Grifo and S. Munne (2016). "The rate of mosaic embryos from donor egg as detected by next generation sequencing (NGS) varies by IVF laboratory." Fertility and Sterility 106(3, Supplement): el56-el57(5) 5 Wells, D., S. Alfarawati, S. Taylor, N. Kubikova, K. Spath, K. Turner, C. Hickman and E. Fragouli (2016). "Evidence that differences between embryology laboratories can influence the rate of mitotic errors, leading to increased chromosomal mosaicism, with significant implications for IVF success rates." Human Reproduction 31: 25-26.(6) Guerrero, C.A, Rodriguez, J. A, Skora, D, Chilvers, R.A, Patel, S.S and Goldstein, J.S,"Excessive trimming of cumulus cells is associated with lower fertilisation rates after conventional insemination", Fertility and Sterility, 2020, DOI:

Claims

CLAIMS1. A micromanipulation device for the manipulation of a sub-cellular structure of a mammalian cell cluster comprising; a cellular manipulation dish further comprising; multiple sub-cellular manipulation units, each sub-cellular manipulation unit comprising; a microwell formed from at least one discontinuous microwell wall having at least one upper microwell wall edge defining a microwell access opening, at least one microwell wall lateral edge defining a microwell side opening and a lower cell support, and a cell deposit well formed from a least one discontinuous cell deposit wall having at least one upper cell deposit wall edge defining a cell deposit well access opening, and at least one cell deposit wall lateral edge defining a cell deposit side opening, wherein the cell deposit opening is disposed adjacent to the microwell opening.

2. The micromanipulation device of claim 1, wherein; the at least one discontinuous cell deposit wall has at least a first cell deposit wall lateral edge and a second cell deposit wall lateral edge defining a cell deposit side opening, and at least a one lower cell deposit wall edge, the cell deposit well comprising a cell deposit base wherein the at least a one lower cell deposit wall edge is joined to the cell deposit base, and the lower cell support and the cell deposit base are disposed in a single plane.

3. The micromanipulation device of claim 2, wherein; the at least one discontinuous microwell wall comprises at least two lateral edges defining a microwell side opening, the at least two lateral edges projecting outwardly to form two parallel channel walls defining a channel to guide the passage of fluid therethrough, the channel walls terminating in two lateral channel edges terminating at an oblique lateral angle to form a cutting edge,wherein the first cell deposit wall lateral edge and a second cell deposit wall lateral edge abut against the parallel channel walls of the microwell wall.

4. The micromanipulation device of claim 1 wherein each sub-cellular manipulation unit comprises; a cellular washing well, a unique identifier, and a media reservoir containing the microwell and cell deposit well at least partially therein.

5. The micromanipulation device of claim 4, wherein the at least one discontinuous cell deposit wall has a height lower than the at least one discontinuous microwell wall, and wherein the cell deposit well is contained entirely within the media reservoir, to permit the flow of fluid from the media reservoir to the cell deposit well, and from the cell deposit well to the microwell through the channel.

6. The micromanipulation device of claim 1, wherein the microwell comprises an internal diameter of less than 800p.m.

7. The micromanipulation device of claim 1, wherein the cellular manipulation dish further comprises; a dish base for supporting multiple sub-cellular manipulation units thereon, and a dish frame positioned upon the dish base circumferentially surrounding the multiple sub-cellular manipulation units.

8. The micromanipulation device of claim 1, wherein the dish base comprises a width greater than 50mm and a length greater than 50mm, and the multiple sub-cellular manipulation units comprise a width of less than 1000pm and a length of less than 1000pm.

9. The micromanipulation device of claim 1, wherein each sub-cellular manipulation unit of the multiple sub-cellular manipulation units is substantially identical and is arranged in an array.

10. The micromanipulation device of claim 1, wherein the lower cell support of the microwell is tapered in shape.

11. The micromanipulation device of claim 3 wherein a surface of the two parallel channel walls or a portion thereof is coated or treated to be non-polar.

12. The micromanipulation device of claim 3 wherein a surface of the two parallel channel walls or a portion thereof is coated with non-polar groups selected from the group consisting of; alkyl groups, silicones having the functional group — [Si(CH 3 )2 O]-, fluorocarbons having the functional group -Cn F2n+1, any composite thereof or any other coating that renders the surface non-wettable.

13. The micromanipulation device of claim 1 for use in a procedure selected from the group consisting of; intra cytoplasmic sperm injection procedures, embryo biopsy procedures, trophectoderm biopsy procedures, mitochondrial transfer procedures, pronuclear transfer procedures, maternal spindle transfer procedures, cumulus cell biopsy, assisted hatching, high magnification sperm selection (I MSI ), and assisted collapse of blastocyst before vitrification.

14. A method for diagnosis of a medical condition comprising the steps of; obtaining the micromanipulation device of claim 1, obtaining a patient sample performing a manipulation of a sub-cellular structure the patient sample, and assessing a result of a manipulation of a patient sample.

15. The method of claim 14 comprising the additional step of performing a procedure selected from the group consisting of; intra cytoplasmic sperm injection procedures, embryo biopsy procedures, trophectoderm biopsy procedures, mitochondrial transferprocedures, pronuclear transfer procedures, maternal spindle transfer procedures, cumulus cell biopsy, assisted hatching, high magnification sperm selection (IMSI), and assisted collapse of blastocyst before vitrification.

16. The method of claim 14 comprising the additional step of manoeuvring the patient sample against at least one of the cutting edges of at least one of the two parallel channel walls of a sub-cellular manipulation unit.

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