Microfluidic device and methods

Abstract

The present disclosure relates generally to microfluidic systems for isolating embryonic cells from donor embryos, and methods of multiplying donor embryos comprising use of such systems. The present disclosure also relates to the use of such methods in animal breeding.

Description

[0001] "MICROFLUIDIC DEVICE AND METHODS "

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the right of priority to Australian Provisional No. 2024903954, filed 29 November 2024, the complete contents of which is incorporated by reference herein in its entirety.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates generally to microfluidic systems for isolating embryonic cells from donor embryos, and methods of isolating embryonic cells from donor embryos and / or multiplying donor embryos comprising use of such systems. The present disclosure also relates to the use of such methods in animal breeding.

[0006] BACKGROUND

[0007] Assisted reproductive technologies (ART) have made tremendous advances, particularly during the past few decades. Artificial insemination (Al) remains the most (cost) effective method for achieving genetic gain in cattle populations and is widely used in the dairy industry. In this regard, the global market remains strong for frozen semen and embryos, with millions of cattle bred by Al, and more than a million embryos transferred annually worldwide. Most of the top sires in the dairy industry that provide semen for Al are derived from embryo transfer (ET), and improvements in methods of controlling the oestrous cycle and ovulation have resulted in more effective programs for Al, superovulation of donor cows, and the management of ET recipients. Notwithstanding these advanced in ART, uptake by producers of reproductive technologies like multiple ovulation and embryo transfer (MOET) remains limited owing to the expense associated with the production of each embryo. Unlike conventional Al, MOET is therefore unlikely to be used by producers as a conventional reproductive method.

[0008] More recently, approaches for producing genetically identical monozygotic twins by embryo bisection, as well as from blastomeres separated from cleavage stage embryos, have been reported. Whilst this new addition to the ART “toolbox” is exciting and would enable producers to more effectively capture and select for female (dam) genetics (in addition to sire genetics), the widespread adoption of embryo twinning, like other ET- and IVF-based approaches, at a commercial level is likely to be hampered by the prohibitive cost to the producer, as well as by the challenges associated with scaling of the technology. Accordingly, there is a need for improved approaches for embryo multiplication to address one or more of these limitations and assist with uptake by industry.

[0009] SUMMARY

[0010] The present disclosure is broadly directed to microfluidic systems and devices for isolating embryonic cells from donor embryos. Specifically, the inventors have designed a microfluidic system comprising three distinct regions (z.e., an unzipping region, a dissociation region, and a trapping region) that utilise hydrodynamic traps and precise fluidic manipulation to mimic current manual laboratory processes used for embryo “unzipping” and isolation of embryonic cell isolation from zona pellucida (ZP)-free embryos (z.e., unzipped embryos).

[0011] Using the microfluidics system of the disclosure, the inventors have shown for the first time that embryos in culture medium can be perfused through a microfluidics system, trapped and held in place in a first region (z.e., the unzipping region) using a hydrodynamic trap, thereby allowing reagents (such as culture medium containing an enzyme) to be flowed sequentially over the trapped embryo to remove the zona pellucida. In doing so, the inventors have shown that it is possible to produce a zona pellucida (ZP)-free embryo using a microfluidics system rather than labour-intensive and technical laboratory processes. Through the use of precise fluidic manipulation within the system, the inventors have then shown that it is possible to eject the ZP-free embryo from the hydrodynamic trap, and flow the ZP-free embryo downstream from the first region to a second region (i.e., the dissociation region), where embryonic cells (or blastomeres) can be dissociated from the ZP-free embryo by exposing the embryo to shear force capable of breaking cell-to-cell interactions without damaging the cells themselves. This shear force was achieved by flowing the ZP-free embryos through a constriction or ‘stenosis’ within the second region (z.e., the ‘shear applicator’), establishing a large gradient in flow rate of fluid across the narrow area within the constriction, which applies shear to the ZP-free embryo as it passes therethrough. The shear applicator is suitable to dissociate ZP-free embryo into individual embryonic cells or aggregates of embryonic cells. Once dissociated from one another, the blastomeres are flowed through to a third region comprising a series of smaller hydrodynamic traps capable of capturing the individual blastomeres or blastomere aggregates. These individual blastomeres or blastomere aggregates may then be harvested for downstream applications e.g., embryo multiplication. Thus, the inventors have shown for the first time that embryos can be flowed (e.g., perfused) through the different regions of the microfluidics system of the disclosure and, in so doing, mimic the current manual laboratory processes used to isolate blastomeres from embryos using the “unzipping” method. Advantageously, the microfluidic system disclosed herein can be fully automated, thus providing an opportunity for scaled-up and cost-effective isolation of blastomeres from donor embryos, with the potential to increase efficiency and lower the cost of downstream embryo multiplication steps.

[0012] Accordingly, the present disclosure provides a microfluidic system comprising:

[0013] (i) a first region comprising:

[0014] (a) an inlet;

[0015] (b) a first outlet; and

[0016] (c) a first channel disposed between the inlet and first outlet and comprising a first trap; wherein the first region is configured to receive an intact embryo, comprising two or more embryonic cells, via the inlet in fluid communication with the first channel, the first trap is configured to receive and trap the intact embryo as the intact embryo is flowed through the first channel, and the first channel is configured to flow a reagent therethrough for removal of the intact embryo’s zona pellucida (ZP);

[0017] (ii) a second region in fluid communication with the first region and configured to receive a zona pellucida (ZP)-free embryo from the first trap, the second region comprising:

[0018] (a) a second outlet; and

[0019] (b) a second channel in fluid communication with the first channel and the second outlet, wherein the second channel comprises a shear applicator configured to apply shear force to the ZP-free embryo when flowed through the second channel to thereby dissociate one or more embryonic cells from the ZP-free embryo; and

[0020] (iii) a third region in fluid communication with the second region and configured to receive one or more embryonic cells dissociated from the ZP-free embryo, the third region comprising:

[0021] (a) a third outlet; and

[0022] (b) a third channel in fluid communication with the second channel and the third outlet, wherein the third channel comprises a plurality of second traps, each second trap configured to trap one or more embryonic cells dissociated from the ZP-free embryo.

[0023] In some examples, the first region, second region, and third region of the microfluidics system may be provided as separate system components that may be connected to one another such that they are in fluid communication. However, it is also contemplated that the first region, second region, and third region of the microfluidics system may be provided as an integrated microfluidics device. Accordingly, each and every example describing a feature or aspect of the microfluidics system of the disclosure may be applied mutatis mutandis equivalent features or aspects of the microfluidics device of the present disclosure, and vice versa, unless the context clearly states or requires otherwise. Accordingly, unless an example requires (explicitly or otherwise) that the first region, second region, and third region of the microfluidics system are to be provided as separate system components, reference to the microfluidics system of the disclosure shall be understood to also be a reference to the microfluidics device of the disclosure.

[0024] In one example, the inlet of the first region comprises an inlet reservoir. In one example, the first channel is in fluid communication with the inlet reservoir and the first outlet. In one example, the first trap is a hydrodynamic trap. In one example, the first trap comprises a chamber within a wall of the first channel, wherein the first trap comprises a trap outlet in fluid communication with the first outlet such that the first trap forms a bypass channel between the inlet and the first outlet, wherein the bypass channel provides a lower fluid resistance than the first channel. In one example, the trap outlet of the first trap is configured to inhibit passage of the intact embryo therethrough.

[0025] In one example, the substance for removal of the zona pellucida comprises an enzyme. In one example, the reagent for removal of the zona pronase pellucida comprises pronase. In one example, the reagent for removal of the zona pronase pellucida comprises a calcium chelator.

[0026] As described herein, the second region comprises the second channel in fluid communication between the first channel and the second outlet, and the second channel comprises the shear applicator. In one example, the shear applicator comprises a region of constriction (or stenosis) within the second channel. In one example, the second channel comprises a dilated region (e.g., a channel expansion or widening) upstream and / or downstream of the region of constriction, wherein the or each dilated region of the second channel has an average hydraulic diameter which is equal to or greater than that of the first channel. In one example, the second channel comprises a dilated region (e.g., a channel expansion) upstream and downstream of the region of constriction. In other examples, the second channel comprises a dilated region (e.g., a channel expansion or widening) upstream of the region of constriction only. In yet other examples, the second channel comprises a dilated region (e.g., a channel expansion or widening) downstream of the region of constriction only. In each of the foregoing examples describing one or more dilated regions of the second channel flanking the shear applicator, the or each dilated region may have a width which is greater than the width of the first channel, provided that the dilated region has an average hydraulic diameter which is equal to or greater than that of the first channel. In one example, the region of constriction is of sufficient dimension to allow the ZP-free embryo to pass therethrough. The region of constriction is of sufficient dimensions such that as the ZP- free embryo passes therethrough, the flow of fluid is transiently accelerated as the fluid is forced through the region of constriction. In one example, the region of constriction is of a size sufficient to establish a gradient flow rate within the region of constriction in the second channel.

[0027] As described herein, the first and second channels of the first and second regions respectively are of a size and depth (i.e., dimension) sufficient to accommodate an intact embryo, for example an intact bovine embryo, such as an intact bovine embryo comprising from about 2 to about 32 embryonic cells. For example, the first and second channels may each have an average depth of greater than 100 pm, or greater than 150 pm, or greater than 200 pm, or greater than 250 pm, or greater than 300 pm, or greater than 350 pm, or greater than 400 pm. In one example, one or both of the first and second channels in the first and second regions have an average depth of between about 150 pm and about 250 pm. For example, both the first and second channels may have an average depth of between about 150 pm and about 250 pm. In one example, one or both of the first and second channels in the first and second regions each have an average depth of between about 180 pm and about 220 pm. For example, both the first and second channels may have an average depth of between about 180 pm and about 220 pm. In one example, one or both of the first and second channels in the first and second regions have an average depth of between about 190 pm and about 210 pm. For example, both the first and second channels may have an average depth of between about 190 pm and about 210 pm. In one example, one or both of the first and second channels in the first and second regions have an average depth of between about 195 pm and about 205 pm. For example, both the first and second channels may have an average depth of between about 195 pm and about 205 pm. In one example, one or both of the first and second channels in the first and second regions have an average depth of about 200 pm. For example, both the first and second channels may have an average depth of about 200 pm.

[0028] In each of the foregoing examples, the third region comprises a third channel in fluid communication between the second channel and a third outlet. In one example, the third channel has an average hydraulic diameter which is less than that of the second channel. For example, the third channel may be narrower than the second channel. In some examples, the third channel may have an average depth of about 35 pm to about 80 pm. For example, the third channel may have an average depth of about 40 pm to about 70 pm. For example, the third channel may have an average depth of about 45 pm to about 60 pm. In one example, the third channel has an average depth of about 50 pm.

[0029] The person skilled in the art will appreciate that the third region may be configured to comprise any number of traps (each of which is referred to herein as a second trap), each configured to trap one or more dissociated embryonic cells (e.g., individual embryonic cells or aggregates of embryonic cells, i.e., where 2 or more cells are isolated together) that flow through the third channel. In one example, the third region comprises between about 5 and 50 second traps. In one example, the third region comprises between about 5 and 25 second traps. In one example, the third region comprises between about 8 and 12 second traps. For example, the third region may comprise 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 traps.

[0030] In some examples, each of the second traps within the third channel is a hydrodynamic trap. In accordance with this example, each second trap comprises a chamber having a trap outlet in fluid communication with a bypass channel, wherein the bypass channel is in fluid communication with the third outlet, and the bypass channel provides a lower fluid resistance than the third channel when in direct fluid communication with the third outlet. In some examples, the second traps of the third region have a higher aspect ratio than that of the first trap of the first region.

[0031] The plurality of second traps in the third region will be of a size sufficient to accommodate at least a single embryonic cell, (i.e., a blastomere), or small cell aggregates (e.g. cell pairs, cell triples, cell quads, cell quints or cell sexts). For example, one or more of the second traps of the third region may have a maximum width greater than about 10 pm, or greater than about 20 pm, or greater than about 30 pm, or greater than about 40 pm, or greater than about 50 pm, or greater than about 60 pm, or greater than about 70 pm, or greater than about 80 pm. Alternatively, or in addition, one or more of the second traps of the third region may have a maximum depth of greater than about 30 pm, or greater than about 40 pm, or greater than about 50 pm, or greater than about 60 pm, or greater than about 70 pm. In one example, one or more of the second traps in the third region has a maximum width of between about 20 pm and about 40 pm, and / or a maximum depth of between about 40 pm and about 60 pm, preferably a maximum width of between about 20 pm and about 40 pm and a maximum depth of between about 40 pm and about 60 pm. In accordance with one example in which one or more of the second traps of the third region is configured to receive one or more individual embryonic cells, the one or more second traps has a maximum width of about 30 pm and / or a maximum depth of about 50 pm, preferably a maximum width of about 30 pm and a maximum depth of about 50 pm. In accordance with an example in which one or more of the second traps of the third region is configured to receive aggregates of embryonic cells (i.e., an aggregate of 2 or more embryonic cells, such as pairs or quads), the one or more second traps has a maximum width of about 50 pm and / or a maximum depth of about 70 pm, preferably a maximum width of about 50 pm and a maximum depth of about 70 pm.

[0032] In another example, the plurality of second traps of the third region have an average width greater than about 10 pm, or greater than about 20 pm, or greater than about 30 pm, or greater than about 40 pm, or greater than about 50 pm, or greater than about 60 pm, or greater than about 70 pm, or greater than about 80 gm. Alternatively, or in addition, the plurality of second traps of the third region have an average depth of greater than about 30 gm, or greater than about 40 gm, or greater than about 50 gm, or greater than about 60 gm, or greater than about 70 gm. In one example, the plurality of second traps in the third region have an average width of between about 20 pm and about 40 pm, and / or an average depth of between about 40 gm and about 60 gm, preferably an average width of between about 20 gm and about 40 gm, and an average depth of between about 40 gm and about 60 gm. In one example, the plurality of second traps of the third region have an average width of about 30 gm and / or an average depth of about 50 gm, preferably an average width of about 30 gm and an average depth of about 50 gm.

[0033] In another example, the plurality of second traps of the third region are varying sizes such that one or more of the second traps in the plurality is of sufficient size to accommodate a single embryonic cell and one or more of the second traps in the plurality is of sufficient size to accommodate aggregates of embryonic cells (e.g., cell pairs, cell triples, cell quads, cell quints or cell sexts). For example, one or more of the second traps of the third region may have a maximum width of greater than about 10 gm, or greater than about 20 gm, or greater than about 30 gm, or greater than about 40 gm and one or more of the second traps in the third region may have a maximum width greater than about 50 gm, or greater than about 60 gm, or greater than about 70 gm, or greater than about 80 gm.

[0034] Microfluidic systems of the disclosure can be made of any suitable material. In one example, the material is glass, silicone, hydrogel, or a polymer. In one example, the material is glass.

[0035] The system can be of any suitable dimension, and dimensions may be adapted to the end use or application. In one example, the system is of a dimension that is suitable for microscopic imaging. In one example, the system is contained within a 75 mm x 50 mm glass slide.

[0036] In one example, the system is controllable with a displacement pipette.

[0037] In one example, the system is controllable with a syringe, e.g., a handheld syringe. In one example, the system is controllable with a programmable syringe pump. The present disclosure also provides a method of isolating one or more embryonic cells from a donor embryo, the method comprising:

[0038] (i) introducing an intact donor embryo comprising two of more embryonic cell to the microfluidic system of as described herein above via the inlet of the first region;

[0039] (ii) flowing the intact embryo in media through the first channel under conditions suitable to trap the intact donor embryo in the first trap comprised within the first region; (iii) removing the zona pellucida (ZP) of the intact embryo trapped within the first trap by flowing a reagent capable of degrading the ZP through the first channel of the first region for a time and under conditions sufficient to remove the ZP from the embryo, thereby producing a ZP-free embryo;

[0040] (iv) ejecting the ZP-free embryo from the first trap and flowing the ZP-free embryo to the second region, and then dissociate one or more embryonic cells from the ZP-free embryo, by flowing the ZP-free embryo through the shear applicator within the second channel under conditions suitable to apply shear forces to the ZP-free embryo and thereby dissociate one or more embryonic cells therefrom; and

[0041] (v) flowing the one or more embryonic cells from the second channel to the third region of the system, and then flowing the one or more embryonic cells through the third channel under conditions suitable to trap one or more of the embryonic cells in the second traps comprised with the third region.

[0042] In one example, the donor embryo is loaded into the inlet reservoir and drawn through the first region using a fixed flow rate. In one example, the flow rate is about 2 pL / min. Accordingly, in some examples, the donor embryo is drawn through the first region and to the first trap at a flow rate of about 2 pL / min.

[0043] In one example, the reagent capable of degrading the ZP from the intact embryo is a medium containing one or more of pronase, hyaluronidase, acrosin, acidified Tyrode’s solution and / or collagenase. In one example, the reagent capable of degrading the ZP from the intact embryo is a medium containing pronase. In some examples, the medium may further contains a substance for disrupting adhesive cell junctions. For example, the medium may further contain a calcium chelator. However, in other examples, the substance for disrupting adhesive cell junctions (e.g., a medium containing a calcium chelator) may be routed through the second region of the microfluidics system separately to the reagent capable of degrading the ZP from the intact embryo.

[0044] In one example, the method comprises performing one or more wash steps between steps (iii) and (iv) to remove residual amounts of the reagent capable of degrading the ZP from the microfluidics system. In one example, the one or more wash steps comprises media exchange. Accordingly, in certain examples, the method comprises removal of the ZP from the intact embryo trapped within the first trap by: (i) flowing the reagent capable of degrading the ZP (e.g., a medium comprising pronase) through the first region in contact with the intact embryo; (ii) flowing a wash solution through the first region in contact with the embryo, and (iii) flowing a reagent comprising a substance for disrupting adhesive cell junctions (e.g., calcium chelator) in contact with the one or more embryos. In one example, the calcium chelator is routed through the second region prior to flowing the ZP-free embryo to the second region at (iv).

[0045] Reagents are flowed at a flow rate and duration suitable to remove the zona pellucida at step (iii), whist at the same time retaining the donor embryo within the first trap. For example, the duration that the intact embryo is exposed to the substance capable of degrading the ZP (e.g., the medium comprising pronase) may be at least about 1 minute, or at least about 2 minutes, or at least about 3 minutes, or at least about 4 minutes, or at least about 5 minutes, or greater than 5 minutes. In one example, the medium is flowed (e.g. perfused) through the first region at a flow rate of about 2 pL / min.

[0046] As described herein, following ejection of the ZP-free embryo from the first trap, the ZP-free embryo is flowed from the first region to the second region, where embryonic cells are dissociated from the ZP-free embryo by flowing the embryo through the shear applicator within the second channel. In one example, the zona pellucida-free embryo is flowed through the shear applicator using pulsatile or cyclic flow. In one example, the pulsatile or cyclic flow comprises a volume of between about 0.5 and about 2 pL and a flow rate of between about 200 and about 400 pL / min. In one example, the pulsatile or cyclic flow comprises a volume of about 1 pL and a flow rate of about 300 pL / min. In one example, the ZP-free embryo is exposed to multiple pulses or cycles to move the embryo back and forth across (i.e., through) the shear applicator (i.e., the region of constriction orstenosis). For example, the ZP-free embryo may be exposed to at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 pulses or cycles. In one example, the ZP-free embryo is exposed to at least 4 pulses or cycles.

[0047] As described herein, dissociated embryonic cells are flowed from the second channel to the third region of the system, where they are flowed through the third channel under conditions suitable to trap one or more of the embryonic cells in the second traps of the third region. In some examples, the conditions suitable trap the one or more embryonic cells in the second traps comprises a flow rate of about 1 pL / min to about 1.5pL / min through the third channel. In one particular example, the one or more embryonic cells are flowed through the third channel at a flow rate of about 1.5 pL / min.

[0048] The distribution of embryonic cells trapped within the second traps may differ for each donor embryo processed in the microfluidics device. In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of at least 2 embryonic cells. For example, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 2 embryonic cells. For example, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 3 embryonic cells. For example, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 4 embryonic cells. For example, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 5 embryonic cells. For example, a proportion of the embryonic cells trapped within the second traps are present as aggregates of

[0049] 6 embryonic cells. For example, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 7 embryonic cells. For example, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 8 embryonic cells.

[0050] In another example, a proportion of the embryonic cells trapped within the second traps may be present as individual embryonic cells and a proportion of the embryonic cells within the second traps may be present as aggregates of at least 2 embryonic cells, for example, 2 embryonic cells, 3 embryonic cells, 4 embryonic cells, 5 embryonic cells, 6 embryonic cells,

[0051] 7 embryonic cells, and / or 8 embryonic cells. In some examples, the majority of embryonic cells trapped within the second traps may be present as individual embryonic cells. In other examples, substantially all, or all, of embryonic cells trapped within the second traps may be present as individual embryonic cells. In some examples, the majority of embryonic cells trapped within the second traps may be present as embryonic cell aggregates (e.g., 2 or 3 or 4 or 5 or 6 or 7 or 8 embryonic cells). In other examples, substantially all, or all, of embryonic cells trapped within the second traps may be present as embryonic cell aggregates (e.g. 2 or 3 or 4 or 5 or 6 or 7 or 8 embryonic cells). In one example, the majority of embryonic cells trapped within the second traps are present as pairs or quads of embryonic cells.

[0052] In order to use the isolated embryonic cells in downstream applications (e.g., such as embryo multiplication), the method may further comprise aspirating the one or more embryonic cells from the second traps in the third region of the microfluidics system and collecting the one or more embryonic cells from the microfluidics system.

[0053] The present disclosure also provides a method of multiplying a donor embryo, the method comprising:

[0054] (i) introducing one or more intact donor embryos to a microfluidic system of the disclosure and isolating one or more embryonic cells therefrom by performing the method of isolating one or more embryonic cells from a donor embryo as described herein, thereby obtaining a plurality of isolated embryonic cells;

[0055] (ii) expanding the isolated embryonic cells in vitro under conditions suitable to produce a plurality of conceptuses from the donor embryo, wherein at least a portion of the embryonic cells are expanded as one or more cell aggregates, each aggregate comprising 2 or more embryonic cells; and

[0056] (iii) culturing the plurality of conceptuses under conditions suitable to produce a plurality of blastocysts. In one example, substantially all of the obtained embryonic cells are expanded at (ii) as cell aggregates. For example, all of the obtained embryonic cells may be expanded at (ii) within cell aggregates. The embryonic cell aggregates may comprise between about 2-8 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 2-8 (e.g., 2-4) embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 2 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 3 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 4 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 5 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 6 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 7 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 8 embryonic cells prior to expansion. In some examples, a portion of the trapped embryonic cells are obtained as an existing aggregate (e.g., as pairs, triples, quads, quints or sexts of embryonic cells). In other examples, substantially all of the trapped embryonic cells are obtained within existing aggregates (e.g., as pairs, triples, quads, quints or sexts). Alternatively, or in addition, a portion of the trapped embryonic cells are aggregated after being obtained at (i). In some examples, the embryonic cells which are aggregated are derived from the same donor embryo (i.e., the embryonic cells are monozygotic). In some examples, the embryonic cells which are aggregated are derived from more than one donor embryo (e.g., such as from more than one donor embryo from the same animal). In accordance with some examples in which aggregated embryonic cells are derived from more than one donor embryo, the embryonic cells may be monozygotic (e.g., such as may be the case where the respective donor embryos were obtained from a previous embryo multiplication or “twinning” process). However, it is also contemplated that embryonic cells which are aggregated are derived from multiple donor embryos which are not monozygotic. In one example, embryonic cells which are aggregated may be derived from multiple donor embryos which share a common sire and dam (i.e., full sibs). In one example, embryonic cells which are aggregated may be derived from multiple donor embryos which share either a common sire or a common dam (i.e., half sibs). In other examples, embryonic cells which are aggregated may be derived from multiple donor embryos which share neither a common sire or a common dam.

[0057] In one example, the embryonic cells or conceptuses may be cultured in the presence of one or more factors capable of promoting embryogenesis. For examples, the embryonic cells may be cultured in the presence of one or more factors capable of promoting embryogenesis in order to form and expand the embryos. In another example, the embryonic cells or embryos comprising same may be cultured in the presence of one or more factors capable of maintaining totipotency and / or inhibiting or preventing embryogenesis. For example, the embryonic cells or embryos comprising same may be cultured in the presence of one or more factors capable of maintaining clearance of maternal mRNAs.

[0058] In some examples, one or more intact donor embryos in step (i) of a method of the disclosure is / are produced by in vivo fertilisation and flushed from the oviduct of a donor animal. In other examples, one or more donor embryos in step (i) of a method according to the disclosure is / are produced by in vitro fertilisation (IVF).

[0059] In each of the foregoing examples, the method comprises expanding the plurality of embryos in vitro to form blastocysts. For example, the method may comprise expanding the embryos in vitro to form mature blastocysts which are ready for implantation.

[0060] The method may further comprise harvesting the plurality of embryos produced by the method. For example, the method may comprise harvesting the embryos once they mature to the blastocyst stage.

[0061] In each of the foregoing examples, the donor embryos may be obtained from a vertebrate animal.

[0062] In one example, the vertebrate animal is a mammalian species.

[0063] In one example, the mammalian species may be a livestock species.

[0064] In one example, the livestock species may be a ruminant species. For example, the livestock species may be a bovine species. For example, the livestock species may be an ovine species (z.e., sheep). For example, the livestock species may be a caprine species (z.e., goat). For example, the livestock species may be a cervid species (z.e., deer). For example, the livestock species may be a camelid species (e.g., camel or alpaca).

[0065] In one example, the livestock species may be a porcine species (z.e., pig).

[0066] In one example, the livestock species may be an equine species (z.e., horse).

[0067] In some examples, methods of the disclosure further comprises transferring one or more of the embryos produced by the method to the oviduct(s) or uterus of one of more recipient females.

[0068] The present disclosure also provides one or more embryos produced by the method of the disclosure.

[0069] In one example, the embryo is from a mammalian species (e.g., a non-human mammalian species). In one example, the non-human mammalian species is a livestock species (e.g., a ruminant species). For example, the livestock species may be a bovine species. For example, the livestock species may be an ovine species (z.e., sheep). For example, the livestock species may be a porcine species (z.e., pig). For example, the livestock species may be an equine species (z.e., horse). For example, the livestock species may be a caprine species (z.e., goat). For example, the livestock species may be a cervid species (z.e., deer). For example, the livestock species may be a camelid species (e.g., camel or alpaca).

[0070] The present disclosure also provides a method of breeding an animal, comprising:

[0071] (i) transferring one or more of the embryos produced by the method of the disclosure to the oviduct(s) or uterus of one of more recipient females to establish a pregnancy;

[0072] (ii) producing the animal from the pregnant recipient female by parturition.

[0073] In one example, the animal is a vertebrate animal. For example, the vertebrate animal may be a mammal, an amphibian, a reptile, a fish or a bird.

[0074] In one particular example, the animal is a mammal (e.g., a non-human mammal). Exemplary non-human mammals which may be produced using the method include livestock species (e.g., cattle, buffalo, pigs, sheep, goats, camelid, deer, horses etc.), companion animals (e.g., dogs, cats etc.), laboratory animals (e.g., rats, mice, hamsters, guinea pigs, rabbits, etc.), non-human primates (macaque and marmoset etc.) and wildlife species (e.g., marsupials, cats, rhino, giant panda, etc.). In one particular example, the method of the disclosure may be used to breed a ruminant livestock species. For example, the method of the disclosure may be used to breed cattle. For example, the method of the disclosure may be used to breed sheep. For example, the method of the disclosure may be used to breed goats. For example, the method of the disclosure may be used to breed cervids. For example, the method of the disclosure may be used to breed camelids. In another example, the method of the disclosure may be used to breed pigs. In yet another example, the method of the disclosure may be used to breed horses.

[0075] BRIEF DECRIPTION OF THE DRAWINGS

[0076] Figure 1. Schematic representation of the three separate components of the microfluidic prototype. (1A) shows the design for the unzipping region, (IB) shows the design for the dissociation region, (1C) shows the design for the trapping region.

[0077] Figure 2. Schematic representation of the microfluidic prototype with integrated components. Figure 3. Image of microfluidic prototype (left image), representative image of initial dye experiment result (middle image), and representative image of bead handling experiment result (right image).

[0078] Figure 4. Representative live cell image of an embryo undergoing the unzipping protocol in the unzipping region of the microfluidic device. 4A shows the embryo arriving within the trap, 4B shows the zona pellucida (ZP) being partially removed, 4C shows the embryo with ZP fully removal and wash complete, 4D shows embryo loading onto the dissociation region. Figure 5. Representative live cell image of an embryo undergoing the dissociation protocol in the dissociation region of the microfluidic device. As shown, ZP-free embryos were dissociated into single cells or small cell clusters by flowing the embryo through the stenosis. Figure 6. Representative live cell image of a blastomeres trapped in the series of hydrodynamic traps in the trapping region.

[0079] DETAILED DESCRIPTION

[0080] General Techniques and Definitions

[0081] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in animal nutrition, feed formulation, microbiology, livestock management).

[0082] As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise.

[0083] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0084] 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.

[0085] The term “about” is used herein to mean approximately. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the recited numerical values. In general, the term “about” is used herein to modify a numerical value above and below the stated value by 10%, up or down (higher or lower).

[0086] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Thus, each feature of any particular aspect or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment of the present disclosure.

[0087] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0088] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0089] Furthermore, any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification 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 disclosure as it existed before the priority date of each of the appended claims.

[0090] Specific definitions

[0091] The term “trap”, as used herein in the context of microfluidics, refers to a mechanism that uses fluid flow patterns to capture, hold and / or isolate small particles, cells or droplets in specific locations within a microfluidic device or system. In an example, the trap is a hydrodynamic trap. In the context of microfluidics, the term “hydrodynamic trap” shall be understood to mean a trapping mechanism which relies on passive hydrodynamic forces, channel geometries, and other structural obstacles (such as micropost arrays, microfilters microwells and chambers) to manipulate, separate and entrap small particles, cells or droplets within a microfluidics system.

[0092] As used herein, the term “stenosis” refers to a narrowing or constriction within a channel or flow path. Accordingly, the terms “stenosis” and “constrictions” may be used interchangeably and shall be understood to have the same meaning in the context of the microfluidics system of the disclosure.

[0093] The term “dissociate”, also called “disaggregation”, as used herein in the context of embryonic cells, refers to the breaking down, detaching or separating of a cell mass comprising a plurality of embryonic cells (such as a zona pellucida-free embryo) to acquire individual cells or smaller groups of cells (e.g. pairs, triplets, quadruplets, quintuplets or sextuplets).

[0094] The term “conceptus” typically refers to an embryo from fertilisation (i.e., the zygote that is formed when two haploid gametic cells (e.g., an unfertilized oocyte and a sperm cell) unite to form a diploid totipotent cell (e.g., a fertilized ovum)), until the appearance of the primitive streak, at which time the entity is then referred to as an “embryo”. However, in some instances, the terms “embryo” and “conceptus” are used interchangeably during the time prior to the appearance of the primitive streak. For example, the term “embryo” may also be used to refer to the zygote that is formed at fertilisation, as well as to the embryo that results from the subsequent cell divisions (i.e. embryonic cleavage), including the morula stage (i.e. when the embryo is compacting or has compacted) and blastocyst stage with differentiated trophectoderm and inner cell mass.

[0095] As used herein, the term “morula” refers to a stage of embryonic development. The morula is an early stage embryo that consists of a ball of cells (called blastomeres) contained within a glycoprotein membrane called a zona pellucida. The morula is produced from the single-celled zygote through a series of cleavage events (which is illustrated in Figure 1 for bovine). A key event prior to morula formation is "compaction", where the embryo containing about 32-64 cells (depending on species) undergoes changes in cell morphology and cell-cell adhesion that initiates the formation of this solid ball of cells. A “morula” typically comprises around 32-64 cells (depending on species), and resembles a mulberry, hence the name morula (Latin, morus: mulberry). In the context of bovine species, the process of compaction typically takes place after the 16-cell stage and the developing embryo reaches the early morula stage at the 32-cell stage, at which time cell-to-cell adhesion between the embryonic cells (or blastomeres) is progressed.

[0096] As used herein, the term “intact embryo”, “intact donor embryo” and similar, shall be understood to mean an embryo that comprises between 2-64 embryonic cells (or blastomeres) contained within a glycoprotein membrane called a zona pellucida. By contrast, a “zona pellucida-free embryo” or “ZP-free embryo” shall be understood to be an embryo (comprising between 2-64 embryonic cells) which does not comprise a zona pellucida (ZP), either because the ZP-free embryo has been produced from an in-tact embryo by disrupting or “unzipping” the zona pellucida, or because the ZP-free embryo has been expanded from an isolated embryonic cell or aggregate of 2 or more embryonic cells.

[0097] Through a process involving cellular differentiation and cavitation, the morula gives rise to a blastocyst. As used herein, the term “blastocyst” shall be understood to refer to an embryo which possesses an inner cell mass (ICM), or embryoblast comprising pluripotent embryonic stem cells, and an outer layer of cells, or trophectoderm comprising trophoblasts, which later forms the placenta. The trophectoderm surrounds the inner cell mass and a fluid- filled blastocyst cavity known as the blastocoel. A blastocyst typically comprises between 70- 300 embryonic cells (which may vary depending on species and maturity of the embryo). In some examples, a blastocyst may comprise about 64 to about 128 cells. In some examples, a blastocyst may comprise between about 128 to about 256 cells. In some examples, a blastocyst may comprise between 150-256 cells. In some examples, a blastocyst may comprise between about 256-300 cells.

[0098] As used herein, the terms “embryonic cell” or “embryonic cells” is intended to encompass all totipotent or pluripotent cells within the developing embryo from the zygote to the blastocyst stage. For example, embryonic cells obtained from within the developing embryo from zygote to morula stage (also referred to as “blastomeres”) are totipotent or pluripotent embryonic cells (depending on the stage of embryogenesis). Likewise, embryonic cells obtained from the inner cell mass of a blastocyst may be pluripotent.

[0099] As used herein, the term “totipotent” is used to describe a cell that is capable of giving rise to any cell type. For example, in the context of embryonic cells, a “totipotent” cell is one that can give rise to all of the cell types in an embryo, and ultimately differentiate into any one of the specialised cells required for different tissues in the body (e.g., skin, bone, marrow and muscle etc.}. The term “totipotent” is to be distinguished from the term “pluripotent”, the latter referring to cells that differentiate into specific subpopulations of cells within a developing cell mass but which may not give rise to any and all cell types.

[0100] A “pluripotent” cell is one that is capable of differentiating to generate primitive ectoderm, which is then able to differentiate during gastrulation into cells of all three germ layers: ectoderm (giving rise to skin and nervous system), endoderm (forming the gastrointestinal and respiratory tracts, endocrine glands, liver, and pancreas) and mesoderm forming bone, cartilage, most of the circulatory system, muscles, connective tissue, and more. A pluripotent cell is also one that is able to self-renew, thereby creating new copies of itself.

[0101] As used herein, the term “monozygotic embryos” shall be understood to mean two or more embryos formed or derived from a single zygote.

[0102] The term “demi-embryo” as used herein shall be understood to mean a portion of an embryo after it has been separated from the donor embryo. For example, an embryo which is split or otherwise separated into two portions of embryonic cells may produce two demiembryos, each comprising embryonic cells. Likewise, an embryo that is split into three portions or more, each comprising embryonic cells, may give rise to three or more demiembryos. A skilled person will appreciate that embryonic cells may be separated from a ZP- free donor embryo using the microfluidics system or method of the disclosure by the shear applicator within the second region of the microfluidics system.

[0103] As used herein, the term “animal” shall be understood to include all vertebrate animals, such as mammals (z.e., non-human mammals), amphibians, reptile, fish and birds. In one example, the animal is a mammal. Exemplary mammals for which the method of the disclosure may be useful include livestock (e.g., cattle, buffalo, pigs, sheep, goats, camelid, deer, horses etc.}, companion animals (e.g., dogs, cats, horses etc.}, laboratory animals (e.g., rats, mice, hamsters, guinea pigs, rabbits, etc.}, non-human primates (macaque and marmoset etc.} and wildlife species (e.g., marsupials, large cats, rhino, giant panda etc.}. In one example, the method of the disclosure may be useful in ruminant livestock species (e.g., cattle, buffalo, sheep, goats, camelid, deer etc). In one particular example, the method of the disclosure may be useful in a bovine species.

[0104] Microfluidic systems and devices

[0105] An exemplary microfluidic system of the disclosure is shown in Figure 2. As illustrated, the microfluidic system of the disclosure comprises three interconnected regions: a first region for trapping an intact embryo and facilitating removal of the zona pellucida (also referred to as “unzipping” of the embryo); a second region comprising a shear applicator for dissociating embryonic cells from the zona pellucida-free embryo; and a third region for trapping the dissociated embryonic cells (e.g., individual embryonic cells or aggregates of embryonic cells, i.e., where 2 or more cells are isolated together).

[0106] As discussed hereinabove, the first region, second region, and third region of the microfluidics system may be provided as separate system components that are interconnected to, and in fluid communication with, one another. However, it is also contemplated that the first region, second region, and third region of the microfluidics system may be provided as an integrated microfluidics device. Accordingly, each and every example describing a feature or aspect of the microfluidics system of the disclosure may be applied mutatis mutandis equivalent features or aspects of the microfluidics device of the present disclosure, and vice versa, unless the context clearly states or requires otherwise. Accordingly, unless an example requires (explicitly or otherwise) that the first region, second region, and third region of the microfluidics system are to be provided as separate system components, reference to the microfluidics system of the disclosure shall be understood to also be a reference to the microfluidics device of the disclosure.

[0107] As disclosed herein, the inventors have designed a microfluidic system comprising a first (“unzipping”) region that utilises a trap, preferably a hydrodynamic trap, and which is adapted for precise fluidic manipulation to mimic current manual laboratory processes used for embryo isolation and “unzipping” to produce ZP-free embryos (z.e., unzipped embryos). The first region of the microfluidic system comprises a first channel which is in fluid communication between an inlet reservoir and a first outlet. A fluidic interface is also provided in connection with the first outlet. The first channel will preferably have a channel depth sufficient to accommodate an intact embryo, such as intact embryo from a bovine animal or other livestock species. For example, the first channel may have an average channel depth of between about 100 pm and 400 pm (e.g., about 100 pm, or about 150 pm, or about 200 pm, or about 250 pm, or about 300 pm, or about 350 pm, or about 400 pm, or greater. In one particular example, the first channel has an average channel depth of about 200 pm. The first channel may include a fluidic resistor to assist with control of flow rate through the first channel. For example, the first channel may include serpentine channels or an alternative channel formation configured to increase fluidic resistance. The first channel will also comprise a first trap. As illustrated in Figures 1 and 2, an exemplary first trap for inclusion in a microfluidics system of the disclosure is a hydrodynamic trap. A hydrodynamic trap relies on passive hydrodynamic forces, channel geometries, and other structural obstacles (such as micropost arrays, microfilters microwells and chambers) to manipulate, separate and entrap small particles, cells or droplets. As shown in Figures 1 and 2, the first trap may be a hydrodynamic trap which comprises a chamber patterned into a wall of the first channel of the first region, wherein the chamber is in fluid communication with the inlet reservoir. The first trap will also include a trap outlet which in fluid communication between the chamber and the first outlet. The first trap thus forms a bypass channel, to bypass the fluidic resistor of the first channel. The fluid resistance within the bypass channel is lower than that within the first channel (due to the presence of the fluidic resistor in the first channel), such that the flow rate of fluid through the hydrodynamic trap is higher than the flow rate of fluid through the fluidic resistor of the first channel. As such, fluid flows preferentially through the bypass channel formed by the hydrodynamic trap and “short circuits” the fluid resistor of the first channel. The main body of the first trap chamber will be configured to have a size and shape which is suitable to receive an intact embryo therein. For example, where the microfluidics system is designed for manipulation of bovine embryos, the chamber of the first trap will be configured to receive an intact bovine embryo. The first trap outlet will also be configured to inhibit passage of the one or more intact embryos therethrough, and thereby trap the intact embryo within the chamber. The first trap outlet may also be configured to inhibit passage of zona pellucida-free embryos therethrough. For example, the first trap outlet may comprise a portion having a narrowed width (or constriction) relative to a width of the chamber body, as shown in Figure 1 A. The first region therefore allows particles (e.g. intact embryos) flowing through the first channel to flow into the chamber of the first trap, partially blocking the trap outlet. Since the trapped particles act as partial plugs, flow resistance in the bypass channel increases and subsequent fluids flow along the first channel, bypassing the first trap which is occupied.

[0108] The second region comprises a second channel in fluid communication between the first channel and a second outlet. A fluidic interface is also provided in connection with the second outlet. The second channel will comprise a shear applicator configured to apply shear force to a ZP-free embryo when flowed through the second channel on order to dissociate one or more embryonic cells from the ZP-free embryo. In some examples, the shear applicator may be in the form of a ‘stenosis’ or constriction within the second channel. In this regard, a person skilled in the art will appreciate that stenosis structures are commonly used to apply shear in fluidic systems. During use, flow may be driven via the fluidic interface in connection with the second outlet in a pulsed or cyclic manner, such that fluid within the second channel flows back and forth across the stenosis constriction. In some examples, the flow may be driven by a syringe pump in fluid connection with the fluidic interface of the second outlet. However, any device or instrument known to be useful for driving flow of a liquid and which is capable of coupling with the fluidic interface may be used. In accordance with that example, a displacement pipette is coupled with the fluidic interface to control the flow of liquid through the microfluidic system. In another example, a syringe is coupled with the fluidic interface to control the flow of liquid through the microfluidic system. For example, a handheld syringe may be used. In another example, a controllable syringe pump may be used. In accordance with an example in which a syringe pump is used, the syringe pump may be a computer- controlled syringe pump. The computer-controlled syringe pump may be configured to rapidly pulse a defined volume of fluid at a given flow rate in a cyclic manner back and forward over the stenosis (or constriction). In one example, flow is controlled through the second channel via a syringe pump configured to rapidly pulse or cycle a volume of fluid between about 0.5 and about 2 pL, at a flow rate of between about 200 pL / min and about 400 pL / min. In one example, flow is controlled through the second channel via a syringe pump configured to rapidly pulse or cycle about 1 pL of fluid at a flow rate of about 300 pL / min. Fluid forced through the constriction accelerates within the narrow passage, establishing a large gradient in flow rate across a small area. Fluid flow at the walls is close to stagnant, while the centre of the channel experiences high transient flow rates. This flow gradient applies shear force to the ZP-embryo or embryonic cell aggregates passing through the stenosis. The skilled person will appreciate that the stenosis or region of constriction will be of sufficient dimension to allow a ZP-free embryo to pass therethrough e.g., to avoid physical damage to the embryo, and yet sufficiently narrow relative to the adjacent regions of the second channel such that the rate of flow is transiently accelerated as the fluid is forced through the region of constriction. In some examples, the region of constriction has an average width and / or depth of between about 100pm and about 160pm. The second channel further comprises a dilated region (e.g., a channel expansion or widening) upstream and / or downstream of the region of constriction (or ‘stenosis’). The or each dilated region of the second channel has an average hydraulic diameter which is greater than that of the first channel. In one example, the width of the or each dilated region of the second channel is greater than the width of the first channel. In one example, the second channel comprises a dilated region (e.g., a channel expansion) upstream and downstream of the region of constriction. In other examples, the second channel comprises a dilated region (c.g, a channel expansion) upstream of the region of constriction only. In yet other examples, the second channel comprises a dilated region (e.g., a channel expansion) downstream of the region of constriction only. In each of the foregoing examples, the channel expansion(s) may be configured to locally reduce flow rates and retain the dissociated blastomeres in the region(s) adjacent the stenosis, which may be defined as an imaging area.

[0109] As described herein, the first and second channels of the first and second regions respectively are of a size and depth (i.e., dimensions) sufficient to accommodate an intact embryo, for example an intact bovine embryo, such as an intact bovine embryo comprising from about 2 to about 32 embryonic cells. For example, the first and second channels may each have an average depth of greater than 100 pm (e.g., greater than 150 pm, or greater than 200 pm, or greater than 250 pm, or greater than 300 pm, or greater than 350 pm, or greater than 400 pm). In one example, one or both of the first and second channels have an average depth of between about 150 pm and about 250 pm (e.g., between about 180 pm and about 220 pm, or between about 190 pm and about 210 pm, or between about 195 pm and about 205 pm). In one example, one or both of the first and second channels in the first and second regions have an average depth of about 200 pm. In some examples, the first and second channels have substantially the same size and depth. However, in other examples, the first and second channels have different size and depth e.g., to modify the flow rates therethrough.

[0110] The third region comprises a third channel in fluid communication between the second channel and a third outlet. A fluidic interface is also provided in connection with the third outlet. The third region comprises a plurality of second traps in fluid connection with the third channel. Each of these second traps is configured to trap one or more embryonic cells dissociated from a ZP-free embryo as they flow through the third region. A person skilled in the art will appreciate that the third region may be configured to comprise any number of trap according to the needs of the user, each configured to trap one or more dissociated embryonic cells that flow through the third channel. For example, the third region may comprises between about 5 and 50 second traps, such as between about 5 and 25 second traps, or between about 8 and 12 second traps. For example, the third region may comprise 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 traps. The second traps may each comprise or be hydrodynamic traps, similar in structure and principle of operation to the first trap in the first region. Accordingly, each second trap may comprise a chamber patterned or otherwise formed into a wall of the third channel and in fluid communication with the second channel. The chamber of each second trap will further comprise a trap outlet in fluid communication with a bypass channel, wherein the bypass channel is in fluid communication with the third outlet, and the bypass channel provides a lower fluid resistance than the third channel when in direct fluid communication with the third outlet. The third channel may include a fluidic resistor to assist with control of flow rate through the third channel. For example, the third channel may include serpentine channels or an alternative channel formation configured to increase fluidic resistance. The fluid resistance provided by the bypass channel is lower than the fluid resistance provided by the fluidic resistor of the third channel, such that a fluid flow rate through the second traps is higher than a fluid flow rate through the fluidic resistor of the third channel. As such, fluid flows preferentially through the bypass channels and “short circuits” the fluid resistor of the third channel.

[0111] The second traps may have a higher aspect ratio (i.e. channel width:depth) compared to that of the first trap in the first region. Each second trap may be of a size sufficient to accommodate at least a single embryonic cell (i.e., a blastomere), or multiple single embryonic cells, or a small cell aggregate comprised of multiple embryonic cells (e.g. cell pairs, cell triples, cell quads, cell quints or cell sexts). In one example, one or more second traps may be a sufficient size to accommodate an embryonic cell pair (i.e., aggregate of 2 embryonic cells). In one example, one or more second trap may be a sufficient size to accommodate an embryonic cell triple (i.e., aggregate of 3 embryonic cells). In one example, one or more second trap may be a sufficient size to accommodate an embryonic cell quad (i.e., aggregate of 4 embryonic cells). In one example, one or more second trap may be a sufficient size to accommodate an embryonic cell quint (i.e., aggregate of 5 embryonic cells). In one example, one or more second trap may be a sufficient size to accommodate an embryonic cell sext (i.e., aggregate of 6 embryonic cells). For example, one or more of the second traps has a maximum width greater than about 10 pm (e.g., about 20 pm, or about 30 pm, or about 40 pm, or about 50 pm, or about 60 pm, or about 70 pm, or about 80 pm or greater). Alternatively, or in addition, one or more of the second traps has a maximum depth of greater than about 30 pm (e.g., about 40 pm, or about 50 pm, or about 60 pm, or about 70 pm or greater). In one example, one or more of the second traps has a maximum width of between about 20 pm and about 40 pm, and / or a maximum depth of between about 40 pm and about 60 pm, preferably a maximum width of between about 20 pm and about 40 pm and a maximum depth of between about 40 pm and about 60 pm. In one example, one or more of the second traps has a maximum width of about 30 pm and / or a maximum depth of about 50 pm, preferably a maximum width of about 30 pm and a maximum depth of about 50 pm. For example, the second traps may each have a chamber width of 30 pm and channel depth of 50. pm. In another example, one or more of the second traps has a maximum width of about 50 pm and / or a maximum depth of about 70 pm, preferably a maximum width of about 50 pm and a maximum depth of about 70 pm. For example, the second traps may each have a chamber width of 50 pm and channel depth of 70 pm. The second traps may be provided in parallel. The second traps may be configured to load sequentially (e.g. in sequence from the trap closest to the outlet to the trap furthest from the outlet). A fluidic interface may be provided in connection with each of the first, second, and third outlets respectively, thereby permitting each of the outlets to connect or couple with a tube and / or instrument. In some examples, one or more of the fluidic interfaces is a Luer connector or adapter (e.g., a male or female Luer connector or adapter). In some examples, one or more of the fluidic interfaces is a pipette adapter. In some examples, one or more of the fluidic interfaces is a syringe adapter.

[0112] Use of the microfluidics system or device

[0113] In use, it is envisioned that an embryo can be deposited into the inlet reservoir of the system or device, and drawn through the first channel by flowing a buffer or other suitable medium through the first channel e.g., at a flow rate of about 2 pL / min. The fluid will preferentially flow through the bypass channel owing to its lower fluid resistance, thereby drawing the embryo into the chamber of the first trap as shown in Figure 4A. Whilst the embryo is being retained in the first trap, media and / or other reagents capable of degrading the ZP are then added to the inlet reservoir and flowed through the first channel at a suitable flow rate so that the reagents contact the embryo for a time sufficient to remove the zona pellucida, as shown in Figures 4B and 4C. The reagent capable of degrading the ZP from the intact embryo may be a medium containing one or more of pronase hyaluronidase, acrosin, acidified Tyrode’s solution and / or collagenase. In one example, the reagent capable of degrading the ZP from the intact embryo is a medium containing pronase. In some examples, the medium may further contain a substance for disrupting adhesive cell junctions. For example, the medium may further contain a calcium chelator. However, in other examples, the substance for disrupting adhesive cell junctions (e.g., a medium containing a calcium chelator) may be routed through the second region of the microfluidics system separately to the reagent capable of degrading the ZP from the intact embryo.

[0114] The duration that the intact embryo is exposed to the substance for degrading the ZP (e.g., the medium comprising pronase) may be varied based on the choice of reagent, concentration and flow rate, for example. In some examples, the intact embryo is exposed to the substance for degrading the ZP for at least about 1 minute, or at least about 2 minutes, or at least about 3 minutes, or at least about 4 minutes, or at least about 5 minutes, or greater than 5 minutes. In one example, the medium is flowed (e.g., perfused) through the first region at a flow rate of about 2 pL / min.

[0115] Following degradation of the ZP from the embryo, one or more wash steps may be performed to remove residual amounts of the reagent capable of degrading the ZP from the microfluidics system. For example, fresh media may be introduced into the inlet reservoir and drawn through the first region to the first outlet, where it is removed. In certain examples, the method comprises removal of the ZP from the intact embryo trapped within the first trap by: (i) flowing the reagent capable of degrading the ZP (e.g., a medium comprising pronase) through the first region in contact with the intact embryo and removing the reagent via the first outlet; (ii) flowing a wash solution (e.g., fresh media) through the first region in contact with the embryo and removing the wash solution via the first outlet; and (iii) flowing a further reagent for disrupting adhesive cell junctions (e.g., calcium chelator) through the first region in contact with the one or more embryos and removing the reagent via the first outlet.

[0116] Once the ZP is removed from the embryo (producing a ZP-free embryo), the ZP-free embryo is ejected from the first trap (e.g., by reversing the direction of flow) as shown in figure 4D, after which a forward flow is resumed to draw the ZP-free embryo into the second region and through the second channel to the shear applicator (e.g., a ‘stenosis’ or constriction in second channel) as shown in Figure 5. Pulsatile or cyclic flow may be used to move the ZP-free embryo back and forth through the stenosis to dissociate the embryonic cells therefrom. As discussed above, a displacement pipette, a handheld syringe or a computer- controlled syringe pump may be configured to pulse the fluid for a predetermined number of cycles, for example to achieve 4 passages of the embryo through the stenosis, and at a predetermined flow rate. In accordance with examples in which the zona pellucida-free embryo is flowed through the shear applicator using pulsatile or cyclic flow, the pulsatile or cyclic flow may comprise a pulsatile volumes of between about 0.5 and about 2 pL and a flow rate of between about 200 pL / min and about 400 pL / min. In one example, the pulsatile or cyclic flow comprises a volume of about 1 pL and a flow rate of about 300 pL / min. In one example, the ZP-free embryo is exposed to multiple pulses or cycles to move the embryo back and forth across (i.e., through) the shear applicator (i.e., the region of constriction or stenosis). For example, the ZP-free embryo may be exposed to at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 pulses or cycles. In one example, the ZP-free embryo is exposed to at least 4 pulses or cycles.

[0117] Once embryonic cells are dissociated from the ZP-free embryo, the dissociated cells are then drawn or flowed through the second outlet and into the third region. As the dissociated cells move through the third channel, they enter the second traps. In some examples, the flow conditions suitable trap the one or more embryonic cells present as one or more individual embryonic cells or one or more embryonic cell aggregates (e.g., cell pairs, cell triples, cell quads, cell quints or cell sexts) in the second traps comprises a flow rate of about 1 pL / min to about 1.5 pL / min through the third channel. In one particular example, the one or more embryonic cells are flowed through the third channel at a flow rate of about 1.5pL / min. As described hereinabove, the second traps may be configured (e.g., by configuring a length dimension of the second traps) to allow capture of single cells, sets of stacked single cells, as well as pre-existing pairs (or other aggregates). The traps also allow for the vertical stacking of captured cells. Figure 6 shows an example array of second traps with single and multiple cell captures. The skilled person will appreciate that the distribution of embryonic cells trapped within the second traps may differ for each donor embryo processed in the microfluidics device. In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of at least 2 embryonic cells (e.g., aggregates of 2 embryonic cells, aggregates of 3 embryonic cells, aggregates of 4 embryonic cells, aggregates of 5 embryonic cells, aggregates of 6 embryonic cells, aggregates of 7 embryonic cells, and / or aggregates of 8 embryonic cells). In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of at least 2 embryonic cells. In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 2 embryonic cells. In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 3 embryonic cells. In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 4 embryonic cells. In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 5 embryonic cells. In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 6 embryonic cells. In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 7 embryonic cells. In some examples, a proportion of the embryonic cells trapped within the second traps are present as aggregates of 8 embryonic cells. For example, a proportion of the embryonic cells trapped within the second traps may be present as individual embryonic cells and a proportion of the embryonic cells within the second traps may be present as aggregates of at least 2 embryonic cells (e.g., aggregates of 2 embryonic cells, aggregates of 3 embryonic cells, aggregates of 4 embryonic cells, aggregates of 5 embryonic cells, aggregates of 6 embryonic cells, aggregates of 7 embryonic cells, aggregates of 8 embryonic cells or more). In other examples, the majority of embryonic cells trapped within the second traps may be present as individual embryonic cells. In other examples, substantially all, or all, of embryonic cells trapped within the second traps may be present as individual embryonic cells. In yet another example, the majority of embryonic cells trapped within the second traps may be present as embryonic cell aggregates (e.g., cell pairs, cell triples, cell quads, cell quints or cell sexts). In other examples, substantially all, or all, of embryonic cells trapped within the second traps may be present as embryonic cell aggregates (e.g., cell pairs, cell triples, cell quads, cell quints or cell sexts). In accordance with an example in which the majority or all embryonic cells are present as embryonic cell aggregates, the embryonic cell aggregates may comprise aggregates of 4 embryonic cells. The isolated embryonic cells captured in the second traps may then be ejected from the second traps and drawn into the third outlet where they can be collected for downstream application.

[0118] An exemplary microfluidic system of the disclosure is depicted in Figure 2. The person skilled in the art will appreciate that a variety of configurations and conformations of the system could be used, and that the parameters are not particularly limited provided that the chambers, channels, and traps are of a size sufficient for embryos and embryonic cells.

[0119] Microfluidic systems of the disclosure can be made using any suitable fabrication material. In a preferred example, the material is glass.

[0120] In one example, flow of liquid through the microfluidic system may be driven by any suitable means known in the art. For example, flow of liquid may be driven by a displacement pipette or a syringe which is in fluid connection with an outlet of the system. The syringe may be a handheld syringe or driven by a pump. In one example, microfluidic systems of the disclosure can be driven by a computer controlled programmable syringe pump. For example, the system can contain one port or multiple ports. In one example, the system contains multiple ports with only one port being is connected at any given time. This configuration means that regions can be isolated, if desirable.

[0121] The microfluidic devices and systems of the disclosure can be employed in methods of multiplying embryos, also referred to herein as “twinning” (z.e., producing multiple monozygotic embryos from a donor embryo), and in particular, to methods capable of producing a plurality of embryos from one or more initial donor embryos.

[0122] In one example, a method of the disclosure possesses the following general method steps:

[0123] (i) introducing one or more intact donor embryos to the microfluidic system of the disclosure and isolating one or more embryonic cells therefrom by performing the method of isolating one or more embryonic cells from a donor embryo as described herein, thereby obtaining a plurality of isolated embryonic cells;

[0124] (ii) expanding the isolated embryonic cells in vitro under conditions suitable to produce a plurality of conceptuses from the donor embryo, wherein at least a portion of the embryonic cells are expanded as one or more cell aggregates, each aggregate comprising 2 or more embryonic cells; and

[0125] (iii) culturing the plurality of conceptuses under conditions suitable to produce a plurality of blastocysts.

[0126] In this way, each isolated embryonic cell, or each aggregate of embryonic cells (z.e., where two or more cells are isolated together) is expanded to become an embryo (e.g., a ZP- free embryo). As described herein, at least a portion of the embryonic cells which are isolated from each donor embryo are expanded as one or more cell aggregates, wherein each aggregate comprising 2 or more embryonic cells. For example, one or more or each cell aggregate may comprise 2 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 3 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 4 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 5 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 6 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 7 embryonic cells prior to expansion. For example, one or more or each cell aggregate may comprise 8 embryonic cells prior to expansion. In some examples, a portion of the isolated embryonic cells are obtained as an existing aggregate (e.g., as pairs, triples, quads, quints or sexts of embryonic cells). In other examples, substantially all of the isolated embryonic cells are obtained within existing aggregates (e.g., as pairs, triples, quads, quints or sexts). In yet another example, a portion of the trapped individual embryonic cells may be aggregated to form one or more small cell aggregates (e.g., aggregates of 2 embryonic cells, aggregates of 3 embryonic cells, aggregates of 4 embryonic cells, aggregates of 5 embryonic cells, aggregates of 6 embryonic cells, aggregates of 7 embryonic cells, and / or aggregates of 8 embryonic cells) prior to expansion. In one example, the portion of the trapped individual embryonic cells may be aggregated to form an aggregate of 2 embryonic cells prior to expansion. In one example, the portion of the trapped individual embryonic cells may be aggregated to form an aggregate of 3 embryonic cells to expansion. In one example, the portion of the trapped individual embryonic cells may be aggregated to form an aggregate of 4 embryonic cells prior to expansion. In one example, the portion of the trapped individual embryonic cells may be aggregated to form an aggregate of 5 embryonic cells prior to expansion. In one example, the portion of the trapped individual embryonic cells may be aggregated to form an aggregate of 6 embryonic cells prior to expansion. In one example, the portion of the trapped individual embryonic cells may be aggregated to form an aggregate of 7 embryonic cells prior to expansion. In one example, the portion of the trapped individual embryonic cells may be aggregated to form an aggregate of 8 embryonic cells prior to expansion. The expansion of embryonic cells as aggregates may assist in improving the efficiency of embryo multiplication.

[0127] In some examples, the embryonic cells which are aggregated are derived from the same donor embryo (i.e., the embryonic cells are monozygotic). In some examples, the embryonic cells which are aggregated are derived from more than one donor embryo (e.g., such as from more than one donor embryo from the same animal). In accordance with some examples in which aggregated embryonic cells are derived from more than one donor embryo, the embryonic cells may be monozygotic (e.g., such as may be the case where the respective donor embryos were obtained from a previous embryo multiplication or “twinning” process). However, it is also contemplated that embryonic cells which are aggregated are derived from multiple donor embryos which are not monozygotic. In one example, embryonic cells which are aggregated may be derived from multiple donor embryos which share a common sire and dam (z.e., full sibs). In one example, embryonic cells which are aggregated may be derived from multiple donor embryos which share either a common sire or a common dam (z.e., half sibs). In other examples, embryonic cells which are aggregated may be derived from multiple donor embryos which share neither a common sire nor a common dam.

[0128] In some examples, the embryo multiplication process can be repeated in a serial fashion using the newly produced embryos as the donor embryos. The process can be repeated ‘N’ number of times (each referred to as a cycle), using the embryos produced from the previous cycle as donors embryos for the subsequent cycle. The number of cycles to be performed using the “unzip” method will depend on various factors including the number of embryos to be produced, the number of starting donor embryos, whether or not any embryos are harvested from the method during intervening cycles, and the number of embryonic cells in the donor embryos, the latter determining the upper limit of how many embryos can be produced from any one donor embryo.

[0129] Donor embryos for embryonic cell isolation and / or embryo multiplication using the methods of the disclosure can include embryos comprising one or more embryonic cells that are developmentally equivalent to embryonic cells from a 16-cell embryo or pre-compacted morula. In one example, the donor embryo may comprise 9 or more embryonic cells, wherein at least one of the embryonic cells is developmentally equivalent to an embryonic cell from a 16-cell embryo or pre-compacted morula. For example, the donor embryo may comprise between 9-64 embryonic cells (e.g., 9-60 embryonic cells), with the proviso that the embryo has not yet progressed to a blastocyst. For example, the donor embryo may comprise between 16-64 embryonic cells (e.g., such as 16-60 embryonic cells), with the proviso that the embryo has not yet progressed to blastocyst. In some examples, it may be advantageous to select a donor embryo for multiplication which has a greater number of embryonic cells which are totipotent e.g., a pre-compaction morula comprising about 32-64 embryonic cells (e.g., such as 32-60 embryonic cells), with the proviso that the embryo has not yet progressed to a blastocyst. For example, the donor embryo may comprise one or more embryonic cells which are developmentally equivalent to embryonic cells from a 32-cell embryo. In other examples, the donor embryo may be a pre-morula stage embryo comprising between 16-32 embryonic cells. In each case, a skilled person will appreciate that the number of embryonic cells within the developing embryo at each stage may vary between species. In one example, the or each donor embryo comprises 9 or more embryonic cells, wherein at least one of the embryonic cells is developmentally equivalent to an embryonic cell from a 16-cell embryo or pre-compacted morula, and at least a portion of the embryonic cells separated therefrom are expanded in cell aggregate comprising 2 or more embryonic cells (e.g., such as about 2-6 cells per aggregate).

[0130] In another example, the or each donor embryo comprises 16-64 embryonic cells (e.g., about 16-60 embryonic cells), wherein at least one of the embryonic cells is developmentally equivalent to an embryonic cell from a 16-cell embryo or pre-compacted morula, and at least a portion of the embryonic cells separated therefrom are expanded in cell aggregate comprising 2 or more embryonic cells (e.g., such as about 2-8 cells per aggregate). For example, the method may comprise separating a plurality of embryonic cells from a donor embryo comprising about 16 cells, and expanding the embryonic cells in vitro under conditions suitable to produce a plurality of conceptuses from the donor embryo, wherein at least a portion (or all) of the embryonic cells are expanded as part of one or more cell aggregates comprising about 2 embryonic cells per aggregate prior to expansion. For example, the method may comprise separating a plurality of embryonic cells from a donor embryo comprising about 32 cells, and expanding the embryonic cells in vitro under conditions suitable to produce a plurality of conceptuses from the donor embryo, wherein at least a portion (or all) of the embryonic cells are expanded as part of one or more cell aggregates comprising about 4 embryonic cells per aggregate prior to expansion.

[0131] The skilled person will appreciate that the number of conceptuses which are cultured for each donor embryo at step (iii) to produce a plurality of blastocysts will depend on the developmental stage of the donor embryo (i.e., number of cells), as well as the number of cell aggregates separated from the donor. However, it is contemplated that at least about 4 (e.g., such as 5 or 6 or 7 or 8 or 9 or 10 or more) conceptuses will be cultured for each donor embryo at step (iii) to produce a plurality of blastocysts.

[0132] In some examples, the embryo multiplication method of the disclosure may be used in conjunction with a serial twinning approach previously developed by the Applicant. For example, prior to culturing the plurality of conceptuses at (iii) to produce the plurality of blastocysts, the method may further comprise the steps of:

[0133] (iv) isolating one or more of the plurality of conceptuses produced to be used as donor embryos in subsequent multiplications, wherein each donor embryo isolated for subsequent multiplications comprises at least two embryonic cells;

[0134] (v) separating one or more of the embryonic cells from the one or more donor embryos;

[0135] (vi) expanding the embryonic cells in vitro under conditions suitable to produce a plurality of conceptuses, each comprising at least two embryonic cells; and (viii) optionally repeating steps (iv)-(vi) ‘N’ times.

[0136] Following the desired number of serial multiplications, the plurality of conceptuses may then be cultured under conditions suitable to produce a plurality of blastocysts, in accordance with step (iii) of the method of the disclosure.

[0137] As described herein, steps (iv)-(vi) of the method may be repeated ‘N’ times in order to produce the desired number of embryos from the original donor embryos. Depending on (1) the number of embryonic cells in the initial donor embryos, (2) the technique(s) used to separate the embryonic cells therefrom and (3) unless stated otherwise, the number of repetitions of steps (iv)-(vi) to be performed z.e., ‘N’, may vary. In this regard, and unless stated otherwise, ‘N’ may be >1, e.g., 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9 or 10 or more.

[0138] As described herein, embryonic cells or demi-embryos comprising embryonic cells which have been separated from donor embryos using the method of the disclosure are cultured in vitro and expanded to produce a plurality of embryos (e.g., monozygotic embryos).

[0139] Methodologies for culturing embryos in vitro at various stages of development are known in the art and contemplated herein. A skilled person will appreciate that the culture conditions are important for growing the developing embryo to a blastocyst stage of development and may be varied / tailored according to the stage of embryonic development, as well as to control rate of embryonic development (e.g., cleavage) to provide sufficient windows of time to perform the multiplications steps of the method of the disclosure. For example, during embryo culture, variables such as temperature and CO2 levels can be controlled to optimise growth of the developing embryo. For example, the optimum temperature for the development of an embryo is from about 32°C to about 40°C, preferably from about 35°C to 39°C, with a temperature of about 37°C to about 39°C (e.g., 38.5°C) being particularly preferred. The optimum CO2 levels in the culturing environment for the development of an embryo is from about 1% CO2 to about 10% CO2, preferably from about 3% CO2 to about 8% CO2, and even more preferably about 5% CO2.

[0140] Suitable media for culturing and expanding embryonic cells and embryos are known in the art. For example, culture media that allow embryos to mature to blastocysts at rates comparable with those that occur in vivo are described in Summers and Biggers (2003) Human Reprod Update, 9:557-582. Many of these culture media are based loosely on the concentrations of ions, amino acids, and sugars found in the reproductive tract of the female at the time of egg release, fertilization, and development (Gardner and Lane (1998) Hum Reprod 13: 148-160). Typically, culture media containing a phosphate buffer or HEPES organic buffer are used for procedures that involve handling of gametes outside of the incubator, flushing of follicles and micromanipulation. Most culture media utilize a bicarbonate / CCh buffer system to keep pH in a suitable range e.g., pH 7.2-7.4. The osmolarity of the culture medium is typically in the range of 275-290 mosmol / kg. Embryos may also be cultured under paraffin oil (or alternative oil which is not toxic to embryos) to prevent evaporation of the medium preserving a constant osmolarity. The oil also minimizes fluctuations of pH and temperature when embryos are taken out of the incubator for microscopic assessment.

[0141] Suitable culture medium also typically contains a protein source, such as albumin or synthetic serum that is added at a concentration of about 5 to 20% (w / v or v / v, respectively). Salts may also be added to the medium, such as NaCl, KC1, KH2PO4, CaCh2H2O, MgSO47H2O, or NaHCOs. Culture medium also typically contains a carbohydrate source (e.g., glucose) and monocarboxylates (e.g., pyruvate and lactate), since carbohydrates and monocarboxylates are present in the female reproductive tract. Together, they are the main energy source for the developing embryo. Culture media that support the development of zygotes up to 8-cells contain pyruvate and lactate. Some commercial media are glucose free, while others add a very low concentration of glucose to supply the needs of the sperm during conventional insemination. Media that support the development of 8-cell embryos up to the blastocyst stage contain pyruvate and lactate in low concentrations and a higher concentration of glucose. Supplement of the culture medium with amino acids may also be desirable for embryo development. Media that support the development of zygotes up to 8-cells are typically supplemented with non-essential amino acids such as proline, serine, alanine, asparagine, aspartate, glycine, and glutamate. Media that support the development of 8-cell embryos up to the blastocyst stage are also typically supplemented with essential amino acids, such as cysteine, histidine, isoleucine, leucine, lysine, methionine, valine, argentine, glutamine, phenylalanine, threonine, tryptophan. The culture medium may also contain vitamins.

[0142] The culture medium may also contain antibiotics. The majority of ART laboratories use culture media containing antibiotics to minimize the risks of microbial growth. The most commonly used antibiotics being Penicillin (P-lactam for Gram-positive bacteria; disturbs cell wall integrity) and Streptomycin (Aminoglycoside for Gram-negative bacteria; disturbs protein synthesis).

[0143] Three examples of sequential media for embryo development which may be useful in culturing embryos in the methods of the disclosure are: G1 / G2 (Gardner et al, (1998) Hum. Reprod. 13:3434); Universal IVF Medium / MS (Bertheussen et al., (1997); and PI / Blastocyst Medium (Behr et al., (1998) Am. Soc. Rep. Med. 0-262). Media for culturing embryo at different stages of development are commercially available from a range of sources. In some examples, the embryonic cells and / or developing embryos are cultured in the presence of one or more factors capable of maintaining totipotency of the embryonic cells and / or inhibiting or preventing embryogenesis. Such factors may be added to culture media in order to prevent or slow down embryogenesis and thereby provide further opportunity to perform additional cycles of steps (i)-(iii) before cellular differentiation starts to occur. Factors which maintain totipotency of embryonic cells and / or which inhibit or prevent embryogenesis are known in the art and contemplated for use herein. For example, factors that maintain totipotency of embryonic cells and / or which inhibit or prevent embryogenesis include anti-miRs and / or ribozymes that block miRNA stability or activity produced by the early embryo. Exemplary anti-miRs may target miRNAs expressed by the embryo which promote clearance of maternal mRNAs (e.g., anti-miRs that target the miR-30 family).

[0144] The skilled person will appreciate that the culture conditions may also contribute to maintaining the state of totipotency of the embryonic cells. Accordingly, during culture of the embryonic cells, embryos or demi-embryos, variables such as cell or embryo density, temperature, CO2 levels and O2 levels can be controlled to moderate / control the rate of development of the cultured embryos.

[0145] As described herein, the method of the disclosure also comprises culturing and expanding the embryos in vitro to form blastocysts, which can then be harvested (e.g., for storage and / or implantation into a recipient female). Accordingly, at some stages of the method, the embryonic cells and / or developing embryos may be cultured in the presence of one or more factors capable of promoting embryogenesis. For example, factors capable of promoting embryogenesis (z.e., embryogenic factors) may be added to culture media used to culture embryos through to blastocyst stage for harvest. Factors which promote embryogenesis are known in the art and contemplated herein.

[0146] The skilled person will also appreciate that the culture conditions; e.g., embryo density, temperature and CO2 levels, can be varied and / or optimised in order to promote embryogenesis.

[0147] As described herein, the species of animal from which the donor embryo(s) is / are obtained may be any vertebrate animal, including a species of mammal, a species of amphibian, a species of reptile, a species of fish and a species of bird (e.g., poultry).

[0148] In one example, the animal is a mammal (e.g., a non-human mammal). Exemplary nonhuman mammals for which the method of the disclosure may be useful include livestock species (e.g., cattle, buffalo, pigs, sheep, goats, cam elid, deer, horses, etc.), companion animals (e.g., dogs, cats, horses etc.), laboratory animals (e.g., rats, mice, hamsters, guinea pigs, rabbits, etc.), non-human primates (macaque and marmoset, etc.) and wildlife species (e.g., marsupials, cats, rhino, giant panda, etc.). In one particular example, the method of the disclosure may be used to produce a plurality of embryos (e.g, monozygotic embryos) in a ruminant livestock species. For example, the livestock species may be a bovine species. For example, the livestock species may be an ovine species. For example, the livestock species may be a caprine species. For example, the livestock species may be a cervine species. For example, the livestock species may be a camelid species.

[0149] In another example, the method of the disclosure may be used to produce a plurality of embryos (e.g., monozygotic embryos) from pigs. In another example, the method of the disclosure may be used to produce a plurality of embryos (e.g., monozygotic embryos) from goats. In another example, the method of the disclosure may be used to produce a plurality of embryos (e.g., monozygotic embryos) from horses.

[0150] Donor embryo used in the method of the disclosure may be prepared in vivo (e.g., by conventionally flushing embryos from a pregnant animal) or by in vitro fertilisation (IVF) methods.

[0151] In one example, the donor embryo used in the method is prepared by in vivo methods. For example, an oocyte may be fertilised in vivo (e.g., following copulation or by artificial insemination) and subsequent embryos retrieved from the pregnant female by conventional embryo flushing. In one example, the donor embryos are produced by multiple ovulation embryo transfer (MOET), whereby the donor female is administered hormones prior fertilisation, primarily follicle stimulating hormone (FSH), to stimulate the ovaries of the cycling female animal to induce multiple ovulations.

[0152] In another example, the donor embryo used in the method is prepared by in vitro methodologies (z.e., IVF). Methods for producing embryos using IVF are well known in the art. IVF generally involves the production of oocytes from donor animals by follicle aspiration, following by in vitro maturation, fertilisation and culture until the resulting embryos have reached a desired developmental stage. Conveniently, this approach permits the repeated production of embryos from live animals of particular value under controlled conditions. Methods for IVF production of embryos are described in Berlinguer F. “ Embryo Production”, In: Animals Production in Livestock, Encyclopedia of Life Support Systems (EOLSS), the full content of which is incorporated herein.

[0153] It is also contemplated that the donor embryo used in the method, whether produced by in vivo or in vitro means, may be fresh, stored or thawed (i.e., a thawed cryopreserved embryo). In one example, the donor embryo is fresh. In one example, the donor embryo has been stored in embryo holding media (e.g, at about 4°C). In another example, the donor embryo is a thawed cryopreserved embryo. Donor embryos useful in the method of the disclosure may also have undergone genetic modifications. For example, embryonic cells within the donor embryo may be genetically modified prior to performance of the method such that all embryos produced from the donor carry the genetic modification. In one example, the donor embryo is genetically modified by introducing an exogenous nucleic acid to the genome of the embryonic cells comprised therein. The exogenous nucleic acid may be an alternative allele for a gene or loci associated with a trait of interest. Alternatively, the exogenous nucleic acid may be a transgene. In another example, the donor embryo may be genetically modified by editing the genome of the embryonic cells comprised therein (z.e., genome editing). The genome edit may be selected from the group consisting of an insertion, deletion, substitution, inversion or translocation. For example, the genome edit may be an insertion, deletion and / or substitution of a nucleic acid sequence, or one or more nucleotide positions therein, in order to replace an existing allele of a gene or loci associated with a trait of interest with an alternative allele.

[0154] Genome editing may also be employed to introduce one or more genetic modifications (e.g., nucleotide substitutions) which, considered alone or in combination, provide a unique genetic profile or fingerprint in the developing embryo. This unique genetic profile or fingerprint can then be used to identify and / or trace embryos produced from the donor embryo (and animals produced therefrom). For example, one or more conservative nucleotide substitutions within safe harbour regions of genome may be made to embryonic cells within the donor embryo in order to generate unique genetic profiles or fingerprints.

[0155] Preferably, the genetic modification or editing occurs at the single cell stage such that all subsequent cells in the developing embryo derived from the modified cell (and animal resulting therefrom) comprise the modification. If, however, the genetic modification event occurs after one or more cell divisions, and not all embryonic cells within the donor embryo are modified, then the donor embryo may be a mosaic for the modification / edit event, in that it will have some cells derived from the modified / edited cell and some cells derived from unmodified / unedited cells.

[0156] A number of methods for genetically modifying genomes of a cell using targeted nucleases are described in the art. These include but are not limited to (1) clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) or other Cas systems, (2) transcription activator-like effector nucleases (TALENs), (3) zinc-finger nucleases (ZFNs), and (4) homing endonucleases or meganucleases. These are other methods for genetically modifying cells are contemplated for use in the method of the disclosure in order to genetically modify donor embryos.

[0157] The methods of the disclosure may further comprise one or more steps to assist with selection of donor embryos to be multiplied using the method. For example, the method may comprise selecting the donor embryo prior to step (i) on the basis of one or more genetic screening criteria, genetic diagnoses and / or one or more morphological criteria.

[0158] In one example, genetic screening criteria may be determined by screening for the presence or absence of one or more genetic markers (e.g., SNP alleles or haplotype) associated with a (favourable variant of) phenotypic trait of interest; e.g., a commercially-important production trait as may be the case for a livestock species. Exemplary phenotypic traits of interest include, but are not limited to, production traits (e.g., growth rate, fecundity, feed conversion efficiency, etc.), drug resistance, susceptibility to pests and / or parasites, and sex (i.e., male or female). In this way, donor embryos for multiplication using the method of the disclosure can be obtained from elite animals.

[0159] Alternatively, or in addition, the donor embryo may be selected on the basis of a genetic diagnosis for one or more conditions, diseases or predisposition thereto. In this regard, preimplantation genetic diagnosis (PGD) or preimplantation genetic testing (PGT) of embryos has become more common place in the field of IVF. PGD tests have largely focused on two methodologies: fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). However, a number of techniques for PGD / PGT are known in the art and one or more of these techniques may be used in the method of the disclosure to select donor embryos. These include, but are not limited to, methods which rely on polymerase PCR, FISH, single strand conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP), primed in situ labelling (PRINS), comparative genomic hybridisation (CGH), COMET analysis (single cell gel electrophoresis), heteroduplex analysis, Southern analysis, and denaturing gradient gel electrophoresis (DGGE) analysis.

[0160] Alternatively, or in addition, the donor embryo may be selected on the basis of one or more morphological characteristics, such as morphological characteristics which are indicative of embryo health.

[0161] As described herein, once a desired number of embryos (e.g., monozygotic embryos) are produced using the method of the disclosure, those embryos may be matured to a desired embryonic developmental stage in vitro (e.g., preimplantation blastocyst) and harvested from the culture media. Harvested embryos may then be stored in an appropriate embryo holding or transfer media until they are transferred to recipient females and / or until such a time as the embryo is cryopreserved. Any commercially available embryo holding and transfer media is contemplated for use herein. In accordance with examples in which the embryos are to be placed in short term storage prior to transfer to a recipient female (such as during transport), the harvested embryos may be stored at between about 2°C to about 8°C depending on the specifications of the particular holding or transfer media. In some preferred examples, the harvested embryos are stored at about 4°C. Harvested embryos may also be cryopreserved for storage. The main techniques used in the art for embryo cryopreservation are vitrification and slow programmable freezing (SPF), both of which are contemplated herein. In accordance with this example, the harvested embryos can be transferred to an appropriate cryopreservation media (e.g., containing ethylene glycol freeze media or similar), cryopreserved, and maintained at about -180°C to about -196°C until they are thawed for use and / or they are shipped. For example, the cryopreserved embryos may be stored in liquid nitrogen at about -196°C.

[0162] In addition to the application of the method of the disclosure in commercial livestock breeding, it is also contemplated that the method of the disclosure may have applications in the area of animal conservation and management. For example, embryos produced from donor embryos obtained from endangered or threatened species (including wildlife and domesticated species) using the method of the disclosure may be deposited with biobanks and / or disseminated for breeding programs. This may assist with breeding programs and management of populations of endangered or threatened species. Accordingly, in some examples, the method may further comprise depositing one or more cryopreserved embryos prepared by the method of the disclosure with a biobank.

[0163] In accordance with embodiments in which the harvested embryos are transferred fresh to recipient females, the method of the disclosure may further comprise transferring one or more of the embryos to the oviduct(s) or uterus of one of more recipient females. Whether the embryo is transferred to the uterus or oviduct will depend on the developmental stage of the embryo. Methods for embryo transfer are known in the art. For example, embryos may be manually transferred using a catheter or other means.

[0164] Animal breeding

[0165] The present disclosure also provides a method of breeding an animal, comprising:

[0166] (i) transferring one or more of the plurality of embryos produced by the method described herein to the oviduct(s) or uterus of one of more recipient females to establish a pregnancy; and

[0167] (ii) producing the animal from the pregnant recipient female by parturition.

[0168] As described herein, the animal may be any vertebrate animal, including a species of mammal, a species of amphibian, a species of reptile, a species of fish and a species of bird (e.g., poultry). In one particular example, the animal is a mammal (e.g., a non-human mammal). Exemplary non-human mammals for which the method of the disclosure may be useful include livestock species (e.g., cattle, buffalo, pigs, sheep, goats, cam elid, deer, horses etc.), companion animals (e.g., dogs, cats etc.), laboratory animals (e.g., rats, mice, hamsters, guinea pigs, rabbits, etc.), non-human primates (macaque and marmoset etc.) and wildlife species (e.g., marsupials, cats, rhino, giant panda etc.). In one particular example, the method of the disclosure may be used to breed cattle. In another example, the method of the disclosure may be used to breed sheep. In another example, the method of the disclosure may be used to breed pigs. In another example, the method of the disclosure may be used to breed goats. In another example, the method of the disclosure may be used to breed horses. In another example, the method of the disclosure may be used to breed camelids (e.g., alpacas).

[0169] EXAMPLES

[0170] EXAMPLE 1: Microfluidic prototype for embryo twinning process

[0171] Embryo twinning is the process of producing multiple embryos from a donor embryo. The approach employed by the Applicant generally involves isolating totipotent blastomeres from an embryo (z.e., the donor embryo), either as single blastomeres or small aggregates of blastomeres (e.g., 2-4 blastomeres), and then expanding those isolated blastomeres (e.g., as aggregates of 2-4 cells) to produce multiple monozygotic blastocysts. In this regard, each blastomere in a 32-cell bovine embryo is totipotent and can, in theory, give rise to an entire animal. With aggregates of 2 or 4 of these blastomeres, one 32-cell embryo can give rise to 16 or 8 new embryos, respectively. This increased number of embryos can then be cultured to a point in development where they are suitable for transfer to a recipient. The process of isolating blastomeres from an intact donor embryo involves 3 main steps: “unzipping”, which involves disrupting and removing the zona pellucida (ZP); “dissociation” which involves separation of individual blastomeres from the embryo cell mass; and “twinning” which is the process of selecting blastomeres and combining them to be cultured into a viable embryo. Embryo twinning processes are currently performed manually, which is time, labour, and cost intensive. Accordingly, microfluidic systems were strategically designed to mimic current manual processes, thus providing an on-chip scaled up twinning strategy.

[0172] Unzipping

[0173] The embryo unzipping process involves removal of the zona pellucida through exposure to a pronase, washing, exposure to calcium chelator to “unzip” adhesive junctions between cells, washing, and pipetting into fresh media. Current processes require 5 manual transfers of the embryo into oil immersed droplets of five reagents with a mouth pipette. It was hypothesised that it should be possible to perform the current laboratory process within a microfluidic system through use of a hydrodynamic trap and precise fluidic manipulation. Cell traps have been described previously in the literature (Luan et al., Biomicrofluidics 2020; 14:031502; Tan and Takeuchi, Proc Natl Acad Sci 2007; 104: 1146-51). It was hypothesised that if similar traps were adapted to the scale required for a bovine embryo, it should be possible to trap and hold an embryo, allowing reagents to flow sequentially over the trapped embryo.

[0174] Targeted outcomes were to determine if it is possible to hold an embryo in place for a long duration, if it was possible to perfuse media thus exposing the embryos to different reagents, and release and collect the embryos after a defined duration.

[0175] Dissociation

[0176] Dissociation involves separation of the embryo into its individual constituent blastomere cells. Current laboratory processes involve aspirating the embryo into a mouth pipette with tip of around 20-30pm - the embryo is disassociated through mechanical agitation, with cyclic aspiration and dispensing of the cell mass until it has broken up sufficiently for subsequent twinning steps.

[0177] It was hypothesised that it should be possible to disassociate the embryo within a microfluidic system with application of precisely controlled shear stress. Shear stress is commonly utilised within microfluidic systems to apply force to cells (Chen et al., TrAC Trends Anal Chem 2019;117: 186-99). Application of shear stress has seen use in dissociation of cell clusters (Mutlu et al., Lab a Chip 2020;20: 1612-20; Qiu et al., Lab a Chip 2014;15:339-50). A suitably designed microfluidic shear applicator utilising principals drawn from the literature (Mutlu et al., Lab a Chip 2020;20: 1612-20; Qiu et al., Lab a Chip 2014;15:339-50) should be able to disassociate an embryo. Targeted outcomes here are to disassociate the embryos in a manner similar to the mouth pipette method, but within a fluidic channel.

[0178] Twinning

[0179] Twinning is the process of selecting blastomeres and combining them to be cultured into a viable embryo. Twinning is currently performed in the laboratory by a skilled operator manually positioning blastomeres in intimate proximity. When transferred to a round bottom well as a set, they will naturally adhere and grow into an embryo. Blastomere manipulation is currently achieved in the lab through use of a mouth pipette with a glass tip drawn to approximately 20-30pm in diameter. This tip is used to “capture” individual cells by drawing them into the ~30um opening and holding them there while transporting them to a new location to be released in proximity to partners (to be paired in twinning process). When a disparity in cell size is apparent, triplets and quadruplets are built in an effort to homogenise output set size.

[0180] The capacity to trap and hold individual blastomeres is a pre-requisite to implementing an on-chip twinning strategy. To capture and hold blastomeres from the dissociation process an array of hydrodynamic traps, similar to those employed in the unzipping process, were adapted for the scale of the individual blastomeres (25, 15, or 7 pm). Hydrodynamic traps are routinely utilised for cells in this size range (Narayanamurthy et al., Anal Methods 2017;9:3751-72). If the lateral trap opening dimension is set to ~30um, it was hypothesised that it should be possible to capture blastomeres in a similar manner to the mouth pipette method. The vertical dimensions were set to a multiple of the blastomere size to enable the capture of sets of blastomeres (doubles, triplets, perhaps quadruplets) trapped within the same chamber. This will achieve a version of twinning, albeit without control over blastocyst selection.

[0181] Microfluidic design

[0182] Microfluidic systems were designed with three subsystems designed to mimic current processes for unzipping, dissociation, and twinning. The intent was to explore the main embryo handling questions - specifically, can an embryo be drawn from a reservoir into a microfluidic system, trapped and the unzipping protocol performed? Following unzipping can the embryo be disassociated into its individual blastomere components? Finally, following dissociation, is it possible to capture and hold the individual blastomeres within defined areas? These subsystems were validated sequentially with bovine embryos.

[0183] Basic components

[0184] The design of the unzipping region (embryo trap) is shown in Fig. 1 A. It comprises a small hydrodynamic trap which allows holding of a target cell within a defined area. The traps consist of a chamber patterned into the wall of a channel, short circuiting a fluidic resistor. The resistance of the trap is lower than the resistor so that particles flowing through the channel will flow into the trap, partially blocking the trap outlet.

[0185] The design of the dissociation region (shear applicator) is shown in Fig. IB. This region comprises a constriction, or stenosis - commonly used to apply shear in fluidic systems. Fluid forced through the constriction accelerates within the narrow passage, establishing a large gradient in flow rate across a small area - the walls are stagnant and the centre of the channel experiences high transient flow rates, this applies shear to particles passing through a channel expansion up and downstream should locally reduce speed and retain the blastomeres within an imaging area.

[0186] The design of the trapping region (blastomere trap) is shown in Fig. 1C. This region comprises a small set (9 in this example) of hydrodynamic traps, similar in principle to the embryo trap, however with a much higher aspect ratio. These traps are within the 50 pm deep area of the chip - with a trap width of 30 pm and depth of 50 pm, it should be possible to trap multiple blastomeres within the same trap well. These should load sequentially, i.e. as the trap closest to the outlet fills, the next should load.

[0187] Basic component integration

[0188] The three component fluidic systems described above were integrated on a 75 x 50mm glass slide and interconnected to allow passing of the embryo from system to system as required. In accordance with this embodiment, the system is intended to be driven by a computer controlled programmable syringe pump with multiple ports, only one is connected at any given time, providing isolation to the other connected lines.

[0189] A schematic of the integrated prototype is shown in Fig. 2. The unzipping region comprises sections C7, C8 and C9. The on-chip reservoir is located at C7 (inlet reservoir). The reservoir is loaded with the embryo, as well as reagents. The embryo trap is located at C8. The fluidic interface required to drive the system is located at C9.

[0190] The dissociation region comprises sections D7, D8 and D9. This region is connected to the embryo trap. The embryo can be drawn into the region through the fluidic interface at D9, where the final step of the unzipping process will occur. Dissociation, using the shear applicator, will occur at D8.

[0191] The trapping region comprises sections E7, E8 and E9. This region is patterned at 50 pm. It is connected to the outlet of the dissociation system. It will draw blastomeres from this system by withdrawing from the interface at E9. Trapping will occur at E8.

[0192] Within the majority of the chip (unzipping and dissociation regions), channels are 200 pm deep to accommodate bovine embryos. As noted above, the channels in the trapping region are 50 pm deep to allow for patterning of the fine features required from trapping small blastomeres while also retaining the depth required to capture multiple blastomeres in each trap.

[0193] EXAMPLE 2: Embryo twinning with a microfluidics device

[0194] Experimental validation

[0195] Initial dye experiments were performed to characterise the system and program the pump. Bead handling experiments were conducted with beads approximating the size of a bovine embryo and blastomere (shown in Fig. 3). All subsystems performed as expected, both traps functioned as expected and routing was possible between them. Following preliminary validation, experiments were performed assessing the functionality of each subsystem with bovine embryos- each experiment was repeated 3 times and results were consistent.

[0196] Unzipping protocol To asses unzipping, the system was primed with buffer and an embryo deposited within the inlet of the reservoir with a mouth pipette, then buffer was drawn through the embryo trap to outlet #1. The embryo was drawn into the trap easily at low controllable velocity and held in the trap chamber with maintained flow of 2 pL / min (Fig. 4A.). The embryo was held in place during zona pellucida (ZP) removal through exposure to pronase in the media, as well as the subsequent wash step (Fig. 4B and Fig. 4C). Media exchange was done by pausing flow, emptying the reservoir, refilling with the next reagent and resuming flow. After ZP removal, calcium chelator was routed through the dissociation system without disturbing the trapped embryo. The embryo was successfully ejected the embryo from the trap, moving it to the start of the dissociation system, where it was exposed to the calcium chelator.

[0197] Dissociation protocol

[0198] To assess dissociation, the unzipped embryos from the previous unzipping experiment were positioned immediately prior to the stenosis. The computer-controlled syringe pump was then set to rapidly pulse a defined volume at a given flow rate in a cyclic manner back and forward over the stenosis. After sweeping the flow rate and volume, it was found that dissociation occurred reproducibly and quickly when displacing 1 pL at 300 pL / min. Spatial distribution of the blastomeres was comparable to the pipette method - the overall distance travelled was low, and the blastomeres end up scattered mostly close to the stenosis (Fig. 5). Approximately 4 passages were sufficient to break the embryo into mostly single cells, with a few existing twins remaining. This is consistent with the manual pipette process, and comparing the output with an example pipette-based dissociation shows similar distribution of singles and doublets.

[0199] Trapping (partial twinning) protocol

[0200] It was hypothesized that it should be possible to capture and hold blastomeres utilising an array of similar hydrodynamic traps employed in the unzipping process, adapted for the scale of the individual blastomeres. The horizontal trap opening was set to ~30 pm, which should be able to capture blastomeres in a similar manner to the mouth pipette method. Additionally, the vertical dimensions were set to a multiple of the blastomere size to enable the capture of sets of blastomeres stacked vertically within the same chamber.

[0201] To test this, the cohort of blastomeres unzipped and dissociated in the previous experiments were moved as close as possible to the outlet of the dissociation system. The trap array was aspirated at low rate through outlet #3 and the results observed (Fig. 6.).

[0202] The observed trapping efficiency was very high. Nearly all blastomeres that made it into the trapping array were trapped, with only two cells passing without trapping throughout the three replicate experiments. The traps captured single blastomeres, as well as pre-existing pairs. Many instances of pairing within the traps were observed, where incoming blastomeres fill partially occupied traps before starting new traps. Accordingly, the system is able to reproducibly trap blastomeres with a high efficiency, including pre-existing twins, and instances were observed where blastomeres stacked vertically within the traps.

[0203] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A microfluidic system comprising:(i) a first region comprising:(a) an inlet;(b) a first outlet; and(c) a first channel disposed between the inlet and first outlet and comprising a first trap; wherein the first region is configured to receive an intact embryo, comprising two or more embryonic cells, via the inlet in fluid communication with the first channel, the first trap is configured to receive and trap the intact embryo as the intact embryo is flowed through the first channel, and the first channel is configured to flow a reagent therethrough for removal of the intact embryo’s zona pellucida (ZP);(ii) a second region in fluid communication with the first region and configured to receive a zona pellucida (ZP)-free embryo from the first trap, the second region comprising:(a) a second outlet; and(b) a second channel in fluid communication with the first channel and the second outlet, wherein the second channel comprises a shear applicator configured to apply shear force to the ZP-free embryo when flowed through the second channel to thereby dissociate one or more embryonic cells from the ZP-free embryo; and(iii) a third region in fluid communication with the second region and configured to receive one or more embryonic cells dissociated from the ZP-free embryo, the third region comprising:(a) a third outlet; and(b) a third channel in fluid communication with the second channel and the third outlet, wherein the third channel comprises a plurality of second traps, each second trap configured to trap one or more embryonic cells dissociated from the ZP-free embryo.

2. The microfluidic system of claim 1, wherein the inlet of the first region comprises an inlet reservoir.

3. The microfluidic system of claim 2, wherein the first channel is in fluid communication with the inlet reservoir and the first outlet.

4. The microfluidic system of any one of claims 1 to 3, wherein the first trap is a hydrodynamic trap.

5. The microfluidic system of claim 4, wherein the first trap comprises a chamber within a wall of the first channel, wherein the first trap comprises a trap outlet in fluid communication with the first outlet such that the first trap forms a bypass channel between the inlet and the first outlet, wherein the bypass channel provides a lower fluid resistance than the first channel.

6. The microfluidic system of claim 5, wherein the trap outlet of the first trap is configured to inhibit passage of the intact embryo therethrough.

7. The microfluidic system of any one of claims 1 to 6, wherein the shear applicator comprises a region of constriction within the second channel.

8. The microfluidic system of claim 7, wherein the second channel comprises a dilated region upstream and / or downstream of the region of constriction, wherein the or each dilated region of the second channel has an average hydraulic diameter which is equal to or greater than that of the first channel, optionally wherein the region of constriction is of sufficient dimension to allow the ZP-free embryo to pass therethrough and transiently accelerate flow of fluid in the second channel as fluid is forced through the region of constriction.

9. The microfluidic system of any one of claims 1 to 8, wherein the first and second channels each have an average depth of about 200 pm.

10. The microfluidic system of any one of claims 1 to 9, wherein the third region comprises a third channel in fluid communication with the second channel and a third outlet.

11. The microfluidic system of claim 10, wherein the third channel has an average hydraulic diameter which is less than that of the second channel, optionally wherein the third channel has an average depth of about 50 pm.

12. The microfluidic system of any one of claims 1 to 11, wherein the third channel comprises a plurality of second traps, each configured to receive and trap one or more dissociated embryonic cells, optionally wherein the third region comprises between about 5 and about 50 second traps, optionally between about 5 and about 25 second traps.

13. The microfluidic system of claim 12, wherein each second trap is a hydrodynamic trap.

14. The microfluidic system of any one of claims 12 or 13, wherein each second trap comprises a chamber having a trap outlet in fluid communication with a bypass channel, wherein the bypass channel is in fluid communication with the third outlet, and the bypass channel provides a lower fluid resistance than the third channel when in direct fluid communication with the third outlet.

15. The microfluidic system of any one of claims 12 to 14, wherein the second traps of the third region have a higher aspect ratio than that of the first trap of the first region, optionally wherein the second traps of the third region each have a maximum width of about 30 pm and / or a maximum depth of about 50 pm, optionally a maximum width of about 30 pm and a maximum depth of about 50 pm.

16. The microfluidic system of any one of claims 1 to 15, wherein:(a) the microfluidic system is made of a material selected from the group including: glass, silicone, hydrogel, or a polymer; preferably wherein the material is glass; and / or(b) the microfluidic system is configured to be controllable with a programmable syringe pump and / or a handheld syringe.

17. A method of isolating one or more embryonic cells from a donor embryo, the method comprising(i) introducing an intact donor embryo comprising two of more embryonic cell to the microfluidic system of any one of claims 1 to 16 via the inlet of the first region;(ii) flowing the intact embryo in media through the first channel under conditions suitable to trap the intact donor embryo in the first trap comprised within the first region;(iii) removing the zona pellucida (ZP) of the intact embryo trapped within the first trap by flowing a reagent capable of degrading the ZP through the first channel of the first region for a time and under conditions sufficient to remove the ZP from the embryo, thereby producing a ZP-free embryo;(iv) ejecting the ZP-free embryo from the first trap and flowing the ZP-free embryo to the second region, and then dissociate one or more embryonic cells from the ZP-free embryo, by flowing the ZP-free embryo through the shear applicator within the second channel under conditions suitable to apply shear forces to the ZP-free embryo and thereby dissociate one or more embryonic cells therefrom; and(v) flowing the one or more embryonic cells from the second channel to the third region of the system, and then flowing the one or more embryonic cells through the third channel under conditions suitable to trap one or more of the embryonic cells in the second traps comprised with the third region.

18. The method of claim 17, wherein:(a) step (ii) comprises flowing the intact embryo in media at a maintain flow rate of about 2pL / min;(b) step (iv) comprises flowing the ZP-free embryo through the shear applicator at a flow rate which displaces about IpL at about 300pL / min; and / or(c) step (v) comprises a flow rate of about 1 pL / min to about 1 ,5pL / min within the third region, optionally about 1.5pL / min.

19. The method of claim 17 or 18, wherein:(a) the reagent capable of degrading the ZP from the intact embryo is a medium containing one or more of pronase, hyaluronidase, acrosin, acidified Tyrode’s solution and / or collagenase; and / or(b) the reagent capable of degrading the ZP from the intact embryo is a medium containing pronase.

20. The method of any one of claims 17 to 19, comprising performing one or more wash steps between steps (iii) and (iv) to remove residual amounts of the reagent capable of degrading the ZP from the microfluidics system, optionally wherein the one or more wash steps comprises media exchange.

21. The method of any one of claims 17 to 20, wherein a calcium chelator is routed through the second region prior to flowing the ZP-free embryo to the second region at (iv).

22. The method of any one of claims 17 to 21, wherein a proportion of embryonic cells trapped within the second traps are present as aggregates of at least 2 embryonic cells.

23. The method of any one of claims 17 to 22, further comprising aspirating and / or collecting the one or more embryonic cells from the third region of the microfluidics system.

24. A method of multiplying a donor embryo, the method comprising(i) performing the method of any one of claims 17 to 23 with a donor embryo to obtain a plurality of isolated embryonic cells;(ii) expanding the isolated embryonic cells in vitro under conditions suitable to produce a plurality of conceptuses from the donor embryo, wherein at least a portion of the embryonic cells are expanded as one or more cell aggregates, each aggregate comprising 2 or more embryonic cells; and(iii) culturing the plurality of conceptuses under conditions suitable to produce a plurality of blastocysts.

25. The method of claim 24, further comprising harvesting the embryos produced by the method.

26. The method of claim 24 or 25, further comprising transferring one or more of the embryos produced by the method to the uterus or oviduct(s) of one of more recipient females, optionally wherein the one or more donor embryos is / are produced by in vivo fertilisation or in vitro fertilisation.

27. The method of any one of claims 17 to 26, wherein the donor embryos are from a mammalian species, optionally wherein the mammalian species is:(i) a livestock species;(ii) a ruminant species; and / or(iii) a bovine, ovine or caprine species.

28. One or more embryos produced by the method of any one of one of claim 17 to 27.

29. A method of breeding an animal, comprising:(i) transferring one or more of the embryos produced by the method of any one of claims 17 to 27 to the uterus or oviduct(s) of one of more recipient females to establish a pregnancy; and(ii) producing the animal from the pregnant recipient female by parturition.

30. The method of claim 29, wherein:(i) the animal is a mammalian species;(ii) the mammalian species is a livestock species;(iii) the livestock species is a ruminant species; and / or(iv) the livestock species is a bovine, ovine or caprine species.

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