KR20030009482A - Microfluidic channel embryo and/or oocyte handling, analysis and biological evaluation - Google Patents

Abstract

Embryonic size microfluidic channels 14 used for handling and placing embryos in accordance with the present invention provide an opportunity to evaluate and process embryos in an improved manner. Fluid flow can be used to move and position the embryo within the microfluidic channel, and the structure of the channel can be used to place the embryo at a specific location. Embryo surface and compliance (deformation) characteristics are evaluated as indicators of survival. The microfluidic channel allows for precise control of the pressure so that various assessments can be made at a force slightly less than that known to cause damage to the embryo. By measuring the distance and / or speed at which the embryo progresses within the microfluidic channel of the same pressure gradient, it can be seen that healthy embryos travel slower and shorter distances, which means that healthy embryos Because it appeared to attach better.

Description

Microfluidic channel embryo and / or oocyte handling, analysis and biological evaluation {MICROFLUIDIC CHANNEL EMBRYO AND / OR OOCYTE HANDLING, ANALYSIS AND BIOLOGICAL EVALUATION}

Assisted reproductive technology, which handles embryos independently from their mammalian source, is of increasing importance and frequency of use. This technology provides a huge direct benefit to those who cannot have babies through non-assisted sexual reproduction. Agriculture is also increasingly dependent on this reproductive technique. To control phenomena such as faster genetic evolution of cattle and from one cow or bull with superior genetic traits to allow the trait to be transferred to a much larger number of offspring than would be possible by non-assisted sexual reproduction Embryo manipulation is used for livestock breeding.

Livestock embryo manipulation is becoming more common due to the development of genetic engineering, cloning and in vitro fertilization (IVF) techniques. The overall purpose of embryo manipulation in livestock is to increase production efficiency, in particular to increase reproduction, milk production or the production of special milk ingredients, low fat dry tissue development and reduced susceptibility to certain diseases. Embryo transplants are also used to introduce or rescue valuable germ plasm and to breed low-reproductive animals such as endangered exotic species.

Cost issues and relatively low success rates impose significant limitations on the application of this assisted reproductive technology to humans and livestock. In the case of human reproduction, in addition to cost and failure rate, ethical and economic problems are added. In addition, protection against failure often results in the birth of unwanted or difficult-to-recover multiple fetuses, as well as additional storage embryos that require maintenance and the difficulty of determining disposal after a period of time. Cost is a major concern for livestock breeding.

In addition to reproductive technology, the failure rate in test techniques and other embryo handling techniques is largely due to the handling and manipulation of many embryos when performing these techniques. Animal breeding techniques have advanced in recent years, but the physical tools used for animal breeding have not changed significantly. Microporous glass pipettes are still one of the basic tools of the embryologist. Procedures such as in vitro maturation of eggs (IVM), in vitro fertilization and embryo culture (EC) require picking up and dispensing individual eggs and embryos several times for each procedure using standard Petri dishes.

This handling and the process of moving from one Petri dish to another Petri dish are likely to be damaged or contaminated. Perhaps more important, however, is that stagnant embryos in Petri dishes do not simulate the corresponding natural biological reproductive conditions. Efforts have also been made to move the embryos in the dishes by stirring the Petri dishes, but this is a reckless method. Cost issues also arise because conventional manual petri dish embryo handling methods require relatively large amounts of biological media. Since bovine embryos are individually handled using a pipette, the manipulation is quite expensive. In the case of human embryos, a large amount of biological medium is used which contains the growth factors required for its cultivation, thus making the in vitro procedure more expensive. Livestock growth factors, for example, cost over $ 200 per 50 μg.

In addition, such a static culture system also has a problem that it is not possible to change the environment of the culture medium as the embryo develops. Current culture systems for flowing medium include small culture chambers on the order of 0.2-0.5 ml. However, the culture volume is larger than necessary and the medium must be refilled quickly. Endogenous growth factors that promote development dilute and disappear. The large amount of media required significantly increases the cost when using expensive growth factors such as IGF-II ($ 200/50 μg). In addition, known systems are unable to detect individual embryos.

Conventional handling techniques provide limited embryo evaluation capabilities. The ability to assess preimplantation embryos, including embryos, pronuclear conjugates and oocytes, will increase the success rate of implantation. Currently, most embryos are evaluated before they are implanted into their recipients. Morphological observation can pick out embryos with large defects but are not very reliable indicators of survival. Chemical monitoring over a period of time is used, but this must be measured several times over a period of time. The result is a much more accurate estimate of survival (approximately 80%), but many embryos do not survive the monitoring process. This also requires the use of additional embryos. This results in multiple births and other complications, and adds labor costs.

In addition, the prior art utilizes a harsh method for removing zona pellucida, which is an important step in the preparation of chimeric embryos. Typically, embryos are transferred by mouse pipetting from one tissue culture dish containing culture medium to a culture dish containing acidic medium. Embryos are placed in the medium for a period of time (tens of seconds) and then transferred by pipetting the mouse into a dish containing fresh culture medium. The embryos are then sucked and flushed several times to allow all acidic media to disperse quickly and to minimize damage to the cell membrane. Since the opening of the mouse pipette is about the same size as the embryo, the outflow through the pipette exerts shear stress on the embryo. Therefore, inaccurate mouse pipetting increases the chance of damage.

Accordingly, there is a need for an improved embryo handling apparatus and method that solves the problems in known embryo handling techniques. Improved embryo handling devices and methods should better simulate the natural state. It should also provide building blocks, such as embryo culture systems, embryo assay systems, embryo storage systems, and similar systems, which may be the basis for larger and / or more powerful and accurate instruments. Improved embryo survival rates are also desired.

Summary of the Invention

These requirements are not met, or more, are provided by current microfluidic embryo handling devices and methods. The present invention simulates biological rotation of the embryo. Embryonic fluid channels move the embryo inserted therein with the fluid, the size of which is equal to the size of the particular type of embryo (s) to be handled. Channel size and fluid transport cause simulated biological rotation of the embryo. In addition, the fluid flowing around the embryo (s) prevents the embryos from stagnation, thereby reducing the likelihood that the embryo (s) will form "bed sores."

The present invention also allows for a gradual change in biological medium fluid, which has significant advantages over the manual and repeated transfer of embryos from one medium to another via pipettes or Petri dishes. Gradual changes prevent shock from sudden changes in the local environment. The microfluidic system of the present invention also allows for co-culture of one embryo and another embryo, co-culture of upstream cells of the embryo (s) and embryo (s), and separate control cultures sharing a common biological medium with the embryo (s) of interest. To ensure that the test embryos face the same environmental conditions as the target embryo (s).

Another aspect of the invention relates to the specific use of a broader range of microfluidic embryo handling principles for manipulation, evaluation and placement of embryos. One aspect relates to using a series of constrictions in stages to remove surrounding material from an embryo. This proved to be able to remove peripheral red cells from oocytes. Several stenosis in the first section cleaves the red cells, and can then exhale the cut red cells from the oocytes in the small stenosis in the back that is large enough to block the passage of oocytes.

Embryo evaluation is also possible according to the basic principles of the present invention. In a preferred assessment, the surface properties and compliance (deformation) properties of the embryo are evaluated. Microfluidic channels provide an opportunity to precisely control pressure so that various assessments can be performed at a force slightly less than what is known to cause damage to the embryo. Measurement of the rotational distance and / or velocity of embryos within microfluidic channels of the same pressure gradient provides information that healthy embryos travel slower or shorter distances due to more adherence to the channel walls. In addition, healthy embryos placed in the stenosis appear to be less deformed than unhealthy embryos that are more easily attracted to the stenosis. In addition, healthy embryos appear to recover their form more easily.

Fluid exiting the microfluidic channel is easily collected downstream without changing the embryo environment, providing a better opportunity for chemical analysis of chemical fluid analysis than conventional manual handling and sampling techniques. In addition, all fluid collected from the microfluidic channel passed past the embryo. This provides better evaluation information than the fluid stagnated around the embryo in the Petri dish.

According to the present invention, the medium can be collected downstream downstream, either continuously or periodically, so that no additional handling is required in the Petri dish technique. Since the fluid can be collected repeatedly in the same manner, the present invention provides a more consistent fluid sample, while the sample taken from the petri dish may vary depending on the position of the pipette to inhale the medium, i.e., how far or close to the embryo it is inhaled. have.

The use of transparent channel compartments enables many types of optical analysis. Colorants or dyes may be added for visual inspection in transparent compartments. Transparent compartments also provide an opportunity to use an image analysis device because the microfluidic channel can be structured to precisely place the embryo in position for analysis by imaging equipment.

Accurate placement of the embryos, and / or flow manipulations using the channels and constrictions of the present invention also allow for improved methods for removing the zona pellucida of mammalian embryos. The embryo is flowed to an exact location where the embryo can be washed with solubilizer to remove the zona pellucida.

The present invention relates generally to the handling of embryos. The invention also relates to the handling of oocytes (primary embryos) and eggs. Thus, the embryo as used herein includes both oocytes and eggs as well as fertilized embryos. More specifically, the present invention relates to microfluidic handling of embryos for culture, manipulation and analysis.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art upon reading the detailed description and the accompanying drawings.

1 shows a cross section of a preferred microfluidic embryo handling device constructed in accordance with the present invention.

2 (a) is a plan view showing a preferred narrow microfluidic channel constriction for embryo placement.

2 (b) is a cross-sectional view of an alternative preferred shallow microfluidic channel constriction for embryo placement.

2 (c) and 2 (d) are schematic diagrams of alternative preferred hydrodynamic constrictions.

2 (e) is a schematic representation of an alternative preferred hydrodynamic constriction.

2 (f) shows an alternative preferred mechanical constriction structure.

FIG. 2 (g) shows a particular flow pattern in the hydrodynamic constriction of FIG. 2 (e) useful, for example, for translucent treatment.

Figure 3 (a) is a perspective view of a preferred gravity flow propulsion microfluidic culture device and test device made in accordance with the present invention.

Figure 3 (b) is a schematic cross-sectional view of the microfluidic channel for the present invention strip removal procedure.

4 is a block diagram of an embryo analysis apparatus manufactured according to the present invention.

5 (a) -5 (c) illustrate preferred embryo microfluidic channel insertion and removal structures in accordance with the present invention.

6 (a) -6 (b) show a preferred culture device constructed in accordance with the present invention.

7 (a) and 7 (b) schematically illustrate the constricted channel structure useful for removing erythrocytes from oocytes.

8 (a) and 8 (b) show the entire prototype device for red cell removal.

8 (c)-(f) illustrate the steps used in the experiment using the prototype device of FIGS. 8 (a) and 8 (b) to remove red cells from oocytes.

9 (a) -9 (d) show modification evaluation of embryos used as an indicator of embryo survival.

The present invention provides a microfluidic embryo handling device that reduces stress on embryos handled outside a natural biological host. These devices and methods recreate simulated biological rotation of the embryo through fluid assisted movement in the channel to facilitate the sliding and rotation of the embryo. As used herein, the term rotation can include both complete rotation or partial rotation. Partial rotation may be referred to as rocking motion.

In FIG. 1 a cross-sectional view of a microfluidic embryo handling device 10 comprising an embryo transport network 12 formed at least in part of a substantially embryonic size channel 14 is shown. The embryo 16 in the channel 14 moves with the fluid flow in the channel 14, and the size of the channel close to the size of the embryo causes the embryo 14 to move in a simulated biological rotational movement. Channels up to 10 times the size of the embryo were used to cause rotation and slippage. In a biological host, embryos that develop during the early stages of development are directed toward the uterus and attached to the uterus by rotational and sliding movements. Microfluidic channel 14 simulates this movement.

Sizing of channels is important for establishing biological rotation. Height is an important dimension, and it has been found that heights up to about three times the diameter of the embryo induce rotation. This ratio can be measured somewhat differently because the fluid flow plays a role, but it has been found that the maximum ratio of 3 to 1 induces rotation. Channel width is considered less important. The width may be arbitrarily selected. Therefore, when arranging embryos in a row, the width should be no more than twice the diameter of the embryo. If you use more embryos, you can use a wider channel.

The network of channels 14 is not only a means for culturing the embryo but also a means for moving and placing the embryo in place. During the early developmental stage, the size of most mammalian embryos remains largely constant during the first few days after fertilization. Thus, the size of the channel 14 does not interfere with the culture of the embryo therein. In order to avoid possible harmful biological effects on the embryo 16, it is advantageous to keep the embryo 16 moving and / or allow continuous or regular fluid flow to pass around it.

A preferred configuration of the device 10 comprising a channel is also shown in FIG. 1. The microfluidic channel 14 may be formed of a suitable material such as silicon wafer 18 by any suitable micromachining method. The material chosen should be sterile and should not pose a biological threat to the embryo. The channel (s) 14 of the device are sealed through the cover 20. Forming a cover of glass or other transparent material facilitates visual monitoring of the embryo in the channel (s) 14. The binder 22 couples the cover 20 to the wafer 18. In addition, the material of the cover can be made to protect the embryo (s) in the channel (s) 14 from harmful radiation.

Unlike other cells, which tend to float in the fluid medium, relatively large and heavy embryos sink to the bottom of the microfluidic channel 14. Typical sizes of preimplantation embryos of important mammals have a sphere diameter of 90-180 μm. In each embryo, a membrane surrounds each cell (a blast), and a zona pellucida (glycoprotein membrane or envelope) surrounds the entire cell mass. Within the same device as the one manufactured based on the principles of the present invention, the cells divide several times in the first few days after fertilization, in which the volume of the embryo remains constant, and the eggs can be fertilized and cultured into blastocysts. Blastocysts are the final stage before embryos implant in the uterus.

The ability to handle individual or few embryos is also important in making such and similar devices. Placing embryos in position, moving them to other positions, and keeping or changing biological conditions around the embryo (s) is a capability provided by the basic principles of the present invention and depends on some or all of these capabilities. It is possible to manufacture a crystal device, a culture device, a test device and other devices. Long channels or loops can be formed to keep the embryos moving throughout the incubation period. Alternatively, the settling of the embryo can take place in a culture station such as shown in FIGS. 6 (a) and 6 (b). In addition, a limited size compartment or channel can be used to vary the flow of fluid to roll the embryo back and forth, as will be further described below with reference to FIGS. 6 (a) and 6 (b).

In the present invention, the individual embryos are accurately positioned using the constriction, a preferred example of which is shown in FIGS. 2 (a) and 2 (b). 2A is a plan view of a cross section of a narrow constriction 24 formed in the microfluidic channel 14. There are many reasons why precise placement in the embryo handling device 10 is required. In the analytical equipment installed on the device, the embryo needs to be precisely placed on the electrode, photodetector, microscope focus or other similar sensing device. Transportation of the embryos to the constriction 24 enables this required placement without depending on the feedback system. Simply backflowing the flow of biological fluid medium 30 frees the embryo 16 from the constriction 24. Even when the embryo 16 is fixed to the constriction, the fluid 30 flows through the constriction 24 and passes through the embryo, so that a biological fluid medium flows around the embryo 16. The above points work advantageously because of the increased likelihood that embryos in stagnant fluid will form "bedsores" (which are suspected to be the cause of the low success rate of embryo handling techniques but are not proven).

Sidewall portions 28a and 28b of microfluidic channel 14 confine the channel at a desired location to prevent passage of the embryo 16. The constriction 24 does not completely close the microfluidic channel 14 so that the biological fluid medium 30 can pass through the embryo 16 located in the constriction 24. 2 (b) shows a lateral cross section of an alternative shallow constriction 26 through which biological fluid medium 30 can similarly pass when the embryo 16 is located in the constriction. Other forms of stenosis are possible. In general, any shape (eg, asymmetric and comb fibers) that does not allow the embryo 16 to pass through the constriction while allowing fluid to flow therethrough is suitable for placing the embryo within the device 10 according to the present invention. can do. The size of the constriction is preferably such that the placement of the embryo is such that it prevents passage of the embryo without increasing pressure from the hydraulic pressure used in the device 10 to move the embryo. Since the constriction of the microfluidic channel has much greater fluid resistance per unit length than the non constriction of the microfluidic channel 14, the length of the constriction should be kept small to avoid fluid control problems.

Embryo placement may also utilize fluid dynamics in the microfluidic channel 14. While the constriction of FIGS. 2 (a) and 2 (b) is a physical constriction, an effective hydrodynamic constriction is realized in FIGS. 2 (c) and 2 (d) without a physical barrier to passage of the embryo. In FIGS. 2 (c) and 2 (d), the microfluidic channel well portion 14a of increased depth is intentionally retained by the regulation of the fluid dynamics (ie flow through the well portion 14a) of the embryo 16. It defines the possible location. In FIG. 2 (c), the high velocity laminar or non-laminar flow causes the embryo 16 to stay in the well portion because flow separation occurs at the leading edge of the well portion 14a. At (lower) flow rates, the fluid flow line conforms to the contour of the microfluidic channel 14 including the well portion 14a to sweep the embryo out of the well portion. The constant flow rate for individually holding and sweeping one embryo can be calculated for a particular well structure and size or can be determined experimentally. 2 (e) shows an alternative embryo 16 placement method. In FIG. 2E, a T-junction 14b is formed at the intersection of the microfluidic channel 14. In a balanced fluid flow condition as shown in FIG. 2 (e), no flow occurs at the embryo's position, allowing the embryo to stay in place. Although not shown, indentation or other small physical forms can increase the stability of the embryo 16 in FIG. 2 (e). Another constriction structure is shown in Fig. 2 (f).

Using the structure and flow of FIGS. 2 (a) -2 (f) it is possible to place the embryo at the correct location within the microfluidic channel network. This provides opportunities for visual examination of embryos, embryo removal, embryo testing, analysis of embryos by imaging or other devices, and treatment of embryos. Accordingly, a number of mechanical embryo manipulations can be performed, and any processing, analysis, or manipulation that benefits from the ability to manipulate and control the exact positioning of the embryo's movements and flow environment would benefit from applying the present invention. Can be.

One specific example of a treatment technique using constriction and microfluidic channel flow to place embryos in a device such as the device of FIGS. 2 (a) -2 (e) is to remove the zona pellucida from the embryo. The zona pellucida is a glycoprotein cytoplasm that surrounds germ cells. Single chimeric animals with two sets of DNA are prepared by removing individual zona pellucida and combining individual germ cells. The zona pellucida thinning or removal is also important for transformation techniques, IVF and cell biopsies. The anchoring position is determined for the removal of the zona pellucida, see Fig. 2 (f) as an example. The plan view of the constriction of FIG. 2 (f) may require a support to prevent collapse due to a low aspect ratio of about 0.01. In a prototype device, this was accomplished with a small PDMS (polydimethylsiloxane) column surrounding the constricted area. An embryo, or a constriction region in which a plurality of embryos stay, can take the form of binding embryos together, which is necessary for chimera formation. A v-shaped resting surface joins the embryo in this manner. Upstream of the constriction as shown in FIG. 2 (f), the microfluidic channel has a structure such that a controlled wash of acidic solution induces flow of the settled embryo (s). For example, the acid inlet may be t-bonded such that the main flow is directed to the anchoring region of FIG. 2 (f). For example, the dissolution plug provides an acidic solution to the main channel near or upstream of the constriction for a short time to effect the removal of the zona pellucida. Using microfluidic channels it is possible to precisely control the flow for the required time. In the prototype device, a syringe was connected to the main microfluidic channel including the constriction and the "T" junction channel to control embryo placement and flow over the embryos placed. Syringes allow precise fluid control, and computer-controlled microsyringes will assist with flow control and timing accuracy.

An alternative mechanical method for removing the zona pellucida is to mechanically damage the zona pellucida. This method is achieved by passing the transparent band through the microfluidic channel of the present invention, which includes a mechanical structure that scratches or cuts the transparent band, for example. Accurate fluid control of the present invention also allows the use of laminar flow methods that "scratch" the zona pellucida. At the "T" junction of Figure 2 (e) two independent streams of two different solutions meet at "T" and enter one channel, which is shown in Figure 2 (g), where the dashed line separates the flow. Indicates. This flow can be kept separated by laminar flow within a single channel. For example, one stream (left) contains an acid for transparence thinning. The propulsion pressure regulates the lateral position of the interface between the two solutions such that the zona pellucida of the embryo 16 is "scratched" by acid.

A culture and test device 31 comprising a constriction as shown in FIG. 2 (a) for placement of the embryo is shown in FIG. 3 (a). The device 31 retains the fluid flow driven by gravity in the network 32 of the microfluidic channel 14 depending on the level of the fluid 30 in the plurality of fluid reservoirs 34. Any suitable means for propelling the fluid 30 (eg, pumping) is deemed to be compatible with the general principles of the present invention, but is preferred because the gravity method shown in FIG. 3 (a) is simple and efficient. . The direction of flow is simply controlled by the level of fluid in the reservoir 34. Thus, for example, the embryo 16 retained in the constriction 24 for incubation or observation by a suitable instrument is first placed by setting the fluid level to induce movement from the inlet 36 to the constriction 24 and the fluid flow is It dissociates when it flows back through the constriction 24. Removal of the embryo 16 is accomplished by inducing fluid flow to move the embryo to the outlet 38.

During movement through the microfluidic channel 14 of the net 32, the embryo (s), as described above, rotate and slide to simulate the natural movement of the embryo towards the uterus in a mammalian host. This preferred mode of migration can be facilitated by suitable surfactants such as BSA (bovine serum albumin). The surfactant will promote some sliding as the embryo rotates.

3 (a) also illustrates a further advantage of the present invention that provides for the combination of additional microfluidic handling and culture apparatus 31a. The further device 31a has a structure similar to that of the device 31 but can have fewer microfluidic channels or a single microfluidic channel. The structure is ideally the same. An important feature of the device 31a is that it shares a common fluid source with the inlet 36 and the outlet 38 of the main device. The embryo (s) handled in the device 31a are biologically isolated from the embryo (s) in the main device 31 but exposed to the same biological conditions by sharing the same fluid source, pressure and / or the same biological medium conditions. do. Thus, in a representative use case, the additional device 31a can form an important control culture that can confirm the suitability or incompatibility of the conditions formed in the main device 31 through the development or lack of development of the test embryo (s).

4 is a block diagram of an embryo analysis apparatus. In the device of FIG. 4, a network 32 of microfluidic channels 14 moves the embryo to one or more assay stations 40a, 40b or 40c. The embryo passes through a constriction similar to that described above and is placed at that assay station. The assay station may include any instrument capable of obtaining information about the embryo, and the constriction is formed to place the embryo at an appropriate sensing point for the particular instrument used in the assay station. Embryos may be moved out of the device through one or more outlets 38a, 38b, or otherwise in a culture station in the form of a fixed area for embryos, microfluidic channels 14 of additional length, or for microscopic movement of embryos in culture. It can be led to a fluid channel loop.

Inlets and outlets used in the device of the present invention may comprise any conventional manner or structure for embryo insertion or removal. However, further preferred structures for insertion and removal are shown in Figures 5 (a) -5 (c). In FIG. 5A, a well 42 in fluid communication with the microfluidic channel 14 is used. The fluid in the well 42 also includes a gravity source that facilitates the promotion of the microfluidic flow in the channel 14. When the embryo 16 is placed in the well 42, the embryo moves to the channel 14 filled with the biological medium, or simply sinks into the channel 14 without the aid of flow if no flow condition is formed. A similar second well 42 can be used to remove the embryo using a pipette 44 or similar device, which can also be used for insertion. In Figure 5 (b) a hanging drop 46 is used at the end of the channel 14 for insertion and removal. Hanging drop 46 is supported by surface tension. Fluid may be added at that point after embryo insertion, or the embryo may be aspirated by fluid flow in the device. Alternatively, the device may be tilted to facilitate movement of the embryo from the hanging drop 46. In FIG. 5C, a funnel-shaped hole 48 used for insertion and removal in direct communication with the channel 14 is used. The funnel shape assists in placing the pipette 44 or similar device. Although the surface tension at the small diameter holes 48 prevents the fluid from leaking, the pressure in the channel 14 should not exceed the point that causes the fluid to leak beyond the surface tension. The inserted embryo sinks into the channel 14, but if the embryo is approaching, the embryo can be removed by sucking fluid from the hole 48. Of course, any of the techniques of FIG. 5 can be combined with conventional techniques for insertion and removal in a given handling device. In addition, the well 42 or hole 48 may be covered with a removable cover or flap as a protector to prevent contamination and / or evaporation.

6 (a) and 6 (b) show an embryo culture apparatus 50 according to the present invention. The fluid medium flow in the culture apparatus 50 is formed in either direction between the medium inlet 52 and the medium outlet 54. The device includes a number of traps or compartments 56. As shown in FIG. 6 (b), the trap 56 includes a deep region separated by a shallow region 58. Fluid between inlet 52 and outlet 54 flows over the shallow region and through the deep region to move the embryo back and forth in deep region compartment 56. The embryo is inserted and removed through the entry hole 60, which can be formed by one of the preferred methods shown in FIGS. 5 (a) -5 (c). Thus, those skilled in the art will appreciate that in device 50 embryos can be moved back and forth within compartment 56 to simulate biological rotation, be exposed to the same media conditions as other embryos in culture, and can be easily removed and inserted. . Figures 6 (a) and 6 (b) show an embodiment of upper loading for placing embryos in compartments, but the device is inverted lower charging substantially as shown in Figures 6 (a) and 6 (b). It can also work in batches. In this lower loading arrangement, the embryo is still held deep but cannot pass through the shallow portion. Another embodiment may include a gap instead of a shallow constriction, wherein the embryo may not pass through the gap but fluid flow may occur between the gaps, and the depth of the gap may be equal to the depth of the embryo retention compartment.

Circular devices similar to those shown in FIG. 3 (a) were made and tested. For the sake of clarity, a conventional prototype is described herein. Those skilled in the art will appreciate that the prototype fabrication scheme may be by any other conventional precision fabrication technique. In addition, those skilled in the art will appreciate that the method of fabricating the device can vary widely, and that the particular numerical size and conditions of the prototype device do not limit the invention to the foregoing ranges.

In a typical circular channel, a pressure gradient of 1 Pa / mm induces media flow at 10 ⁻¹⁰ m ³ / s (100 nl / s) at an average speed of 1-2 mm / s. Under these flow conditions, the embryo rolls along the bottom of the channel, where the rate of travel is 1/3 to 1/2 of the fluid velocity that can be formed in the same region of the channel. By manipulating the pressure in a well connected to the end of the channel, the embryo can be transported to and retained at a specific location including the culture compartment and recovery wells. The embryo fills a significant portion of the channel, which significantly changes the flow of the medium. The flow of medium through the channel is laminar.

Circular microfluidic channel networks were fabricated in an apparatus similar to that shown in FIG. 3 (a) by etching trenches in a 3 inch silicon wafer and then joining the glass covers to form a channel. Devices comprising microfluidic channels were also made of elastomer by micromolding techniques. Other sculptural techniques and techniques, such as injection molding of thermoplastic materials, are also contemplated. Conventional channel networks include several microfluidic channel branches that cross near the center of the device. Branches 1.5-2.5 cm in length are 160-200 μm deep and 250-350 μm wide at the top. The first step in manufacturing the prototype device involves patterning the silicon nitride (SiN) coating using conventional photolithography. The microfluidic channel is anisotropically etched with potassium hydroxide (KOH) solution. Carbide tip augers are typically used or ultrasonic waves are used to drill entry holes in the glass cover. The glass cover is attached to the wafer using a UV curable epoxy (NOA 61, Norland Products, New Brunswick, NJ), or the Pyrex 7740 cover is an anode of the wafer using 500 V at 450 ° C. Attach to. The nitride coating is removed using a buffered oxide etchant (BOE) prior to anode attachment. Epoxy (Quick Stick 5 Minute Epoxy or 5 Hour Set Epoxy Glue; both from GC Electronics, Rockford, Ill.) Or silicone adhesive (RTV 108 and RTV 118, manufactured by General Electric Company, Waterford, NY, or Miami Sylgard, manufactured by Dow Corning Corporation, Midland Brand 184) is used to attach the glass cover to the glass well at the end of each branch of the channel network.

In the prototype device, a constriction as shown in both FIG. 2 (a) ("narrow") and 2 (b) ("shallow") was also fabricated and tested. Channels with “narrow” constrictions as shown in FIG. 2 (a) can be fabricated using one mask and etching operation. Channels with “shallow” constrictions as shown in FIG. 2 (b) require two masks and two etching operations.

Except for 5 Minute Epoxy, all component materials of the prototype device were tested for embryo biocompatibility. Those skilled in the art will appreciate that alternative materials other than those selected for the prototype device may be used in the application of the present invention, but must be pre-data collected and / or tested for biocompatibility. Although many materials are known to be compatible with or toxic to specific cells, little is known about the compatibility of materials used for precision fabrication with embryos. The material selected may also vary depending on the type of mammal taking the particular embryo to be handled.

In prototype testing, two-cell mouse embryos (B6SJL / F2) were randomly allocated on a substrate and in medium M16 (Sigma, St. Louis, MO) containing bovine serum albumin (BSA; 4 mg / ml; Sigma) The medium was incubated with mineral oil (Sigma). All embryos were incubated for 96 hours at 37 ° C. in air containing 5% CO ₂ . The rate of embryo development was examined every 24 hours. The proportion of embryos that reached the blastocyst stage for each material was compared with that of the control group. Mouse embryos that have reached the blastocyst stage (the last possible stage prior to embryo transplantation) are probably considered unhindered in developmental progression. Since embryos are implanted in surrogate mice and not monitored until the birth of the pups, it is not certain that there is no adverse effect, so for practical and economic reasons the test usually concludes at the blastocyst stage. Most materials tested, including silicon wafers, SiN coatings, NOA 61 and RTV 118, have proven to be compatible with very embryonic embryos. Some materials, such as 5-minute epoxy, have not been tested because these materials are used only in the conceptual apparatus for describing the mechanical and fluid components of the present invention and will not be used in production equipment.

Tests were conducted to investigate some aspects of the prototype device. Different tests required devices with different channel structures. In all tests, the channel was illuminated with a halogen bulb using an optical fiber, so the channel was observed under a stereoscopic microscope. The flow velocity was measured using a graduated cylinder and stopwatch. Since the fluid is not compressible, the average fluid velocity in any section of the channel is approximately the flow rate divided by the cross section.

Measurements of the rate of movement of the embryo for a given flow rate were made in a simple straight channel 29 mm long, 162 μm deep and 160-380 (bottom-top) μm wide. Pressure gradients were varied and embryo speed was measured at each setting. The channels were filled with phosphate buffered saline (PBS) with or without BSA. The medium flask was connected to the channel. The height of the flask was adjusted using a micrometer head translation stage to precisely adjust the pressure difference to be within 0.05 Pa. The flasks were piped and connected to each other between each test to bring the pressure head to zero. The precision manufactured prototype device was washed in hydrogen peroxide / ammonium hydroxide / deionized water solution and a new pipette tip was attached using epoxy prior to testing. All tests were performed using PBS containing no BSA before using PBS containing BSA. Once the channel was filled with medium, mouse embryos were placed in the inlet wells at the channel inlet.

Tests were conducted to observe the effect of channel size and shape on embryo transport. For this test, the device was fabricated with one long bypass channel with 11 compartments, each one of 140, 164, 194 and 210 μm in depth. At each depth the channels had two or three different widths. The range of the width | variety measured from the surface of the wafer was 275-480 micrometers. In the narrowest section, the embryos were structurally limited to move over the V-groove, but in other areas the structure was formed to move along the flat bottom channel. Travel speed and rotational properties were observed and compared with those of the other compartments.

Observation of the embryos in the stenosis was carried out on several devices equipped with both narrow and shallow stenosis. The embryo was actually directed to a specific constriction. The pressure gradient in each branch of the channel network was adjusted by varying the height of the medium in each well by adding or subtracting fluid. The pressure head was adjusted to 1-8 mm (10-80 Pa).

As soon as embryos placed in the medium sink to the bottom of the vessel, embryos placed in the microfluidic channel begin to settle to the bottom. In all tests, as the medium flowed, the embryo rotated and slipped along the bottom of the channel in the flow direction. Embryos were sometimes attached to one of the side walls of the channel. Initial tests without any surfactant in the medium (phosphate buffered saline) showed that the embryos did not slide along the bottom of the channel. In subsequent tests containing BSA (4 mg / ml) in the medium, the embryo glides or slides along the bottom.

Testing revealed that the rate of movement of the embryos in the channel depends on the flow rate of the medium. If the flow rate around the embryo is 50 μm / s or less, the embryo is often attached to the bottom of the channel. A pressure gradient of 0.16 Pa / mm in both PBS and PBS / BSA induces an average rate of about 380 μm / s flow through the channel. The embryos were rotated at 187-250 μm / s, 49-66% of 380 μm / s. The faster the medium flowed, the faster the embryos rotated and they slipped as they rotated. Actual travel speeds and attachment trends vary from embryo to embryo. One embryo was found to move 25% faster than the other, even in the same channel at the same time in the same channel. In the observed range 150-1000 μm / s, the velocity is proportional to the pressure gradient.

The results of testing the effects of channel size and shape are consistent with a priori estimates. For constant flow rates, mean fluid velocities and embryo velocities are faster in channels with smaller cross sections. In contrast, for constant pressure gradients, the mean fluid velocity and embryo velocity are faster in channels with larger cross sections. In both cases the embryos travel more slowly on the V-groove than in the flat bottom channel. Embryos were also more prone to clinging to V-grooves than flat bottom channels.

Fluid under electroosmotic flow induced the embryo to rotate through the channel. The embryo was rotated along the channel bottom at about 10 μm / s by pressure-driven trickle. Upon application of voltage, mouse embryos rotated along the bottom of the channel toward the well with the cathode at about 30 μm / s faster, about 30 μm / s. By changing the voltage polarity, the embryos rotated at a rate of about 10 μm / s in the opposite direction. There was little or no slip since no surfactant such as BSA was used. If electrical assistance is used to move the embryo, it must be energized under specially controlled conditions to avoid undesired heating of the medium.

Computational fluid dynamics modeling using Fluent / UNS 4.2 (Fluorent Inc., Lebanon, New Hampshire) and a uniform cross-section with Quickfield (Teraanaxis Inc. Two-dimensional finite element analysis of the microfluidic channel confirmed the observed flow rate and flow pattern. Embryos were modeled as solid spheres. Recall that embryos do not deform under normal conditions. To analyze laminar flow, a square network with 10,000 to 30,000 holes was installed in the 1 mm or 2 mm section of the channel. After verification, computer modeling was used to determine the velocity profile, to design a constriction with lower pressure, to observe the force applied to the embryo retained in the constriction, and to analyze the flow induced in similar channels. However, the analysis of the attachment of the embryo to the channel wall and the deformation of the embryo is much more complicated and has not been attempted.

As described above, in the embryo velocity test in the straight channel, the medium had an average velocity of 380 μm / s at a pressure gradient of 0.16 Pa / mm. Finite component analysis showed that the median velocity under these conditions was 815 μm / s. Embryos were in contact with the floor and one wall when moving the channel. Assume that the diameter of the circle contacting the bottom and one side of the channel is 100 μm. If no embryo is present, the average velocity of the fluid flowing through this circle is 480 μm / s. However, embryos were spun at 187-250 μm / s, 39-52% of their speed in both PBS and PBS / BSA medium. This velocity profile allowed the embryo to rotate forward along the wall, which is confirmed by visual observation. In summary, the embryos rotate at a rate of 1/3 to 1/2 of the rate at which fluid flows in the same cross section.

The constriction significantly increases the fluid resistance in the channel. Standard analytical equations give an approximation of the resistance, but the cross-sectional shape of the constriction varies with location. After analyzing the three-dimensional model of the constriction, a mask was designed and the wafer was etched. The information obtained from the finite element analysis yielded the optimal size of the constriction. Individually sized shallow constrictions for each device structure compromise the minimum flow resistance and demanding fabrication requirements. Typical constrictions have a minimum depth of 20 μm.

Studies of embryo position in the trap have revealed that side forces of 10 ⁻⁸ to 10 ⁻⁷ N move the embryo laterally and diverge the entry way into the shallow constriction.

Testing has revealed some interesting microfluidic transport properties, such as variations in speed between embryos and the tendency to move along the bottom of the channel, often along the sidewalls. However, the test did not reveal some other phenomena. Electroosmotic flow is useful for assisting embryo migration and can assist in the formation of plugs used for embryo processing and fertilization. Because high voltage can harm embryos in many ways, electrical control of fluid flow must be done with care. Even if there is an embryo in the compartment of the channel away from the electric field, the applied energy heats the temperature of the medium above the physiological temperature (joule heating), and the electrolysis product changes the pH. Note that the embryo needs about 0.029 Osmol, ie relatively high conductivity. In addition, EOF degrades in the channel containing the surfactant, but the embryo survives better in the medium containing the surfactant such as BSA.

The microfluidic transport without the help of electricity supplied through gravity applied to a device similar to that shown in FIG. 3 (a), or through a pumped hydraulic device, provides an important advantage. The medium can easily be changed over time to meet the changing requirements of developing embryos. Gradually changing the medium composition avoids the stress induced on the embryo due to the sudden environmental changes often involved during the transfer from the first Petri dish to the second Petri dish containing different media. Microfluidic handling of embryos in accordance with the present invention is physically less severe than delivery with pipettes and certainly less damage than many techniques used in conventional practice, including through the outer membrane.

It is expected that the regulation of fluid flow in a handling device similar to that shown in FIG. 3 (a) and the resulting embryo placement will be handled through a programmed control mechanism for an almost automated device. An alarm and a warning device may be included based on the detected state in the embryo handling device of the present invention. In a similar manner, monitoring of embryos using conventional instruments was applied to the handling device of the present invention. Those skilled in the art will generally understand that microfluidic embryo handling devices form the basic building blocks upon which many useful devices can be based, and that such devices encompass the core of the present invention.

Several special devices for the manipulation, testing, and handling of embryos and oocytes have been disclosed. The specific geometry of the microfluidic channel is schematically shown in Figures 7 (a) and 7 (b), which has proven to be able to remove red cells from oocytes. This structure is in the form of a series of compartments that become narrower in the microfluidic channel, which is located within the curved compartments. 7 (a) and 7 (b) include curved constriction sections, but the constriction sections are not necessarily curved. The inner surface of the stenosis compartment has teeth or serrated protrusions to help cut off the red cells as they pass through. The last section of this series of structures is spaced apart at a distance to prevent damage to the oocytes. When the hydraulic flows oocytes into the stenosis, the front compartment cuts off some of the surrounding red cells. It has progressively spaced apart projections at narrower distances. In Figure 7 (a) a number of constricted compartments (curved compartments) each have projections spaced apart individually, with the downstream compartment of the immediately preceding compartment having projections of smaller spacing. It is also possible to construct a single compartment such that the protrusions in the compartments are located closer to each other in order to achieve the same purpose of cutting deeper red cells at different positions without damaging the embryo. The structures of FIGS. 7 (a) and 7 (b) were used to remove red cells from bovine oocytes. The straight section of the microfluidic channel in the prototype device was 500 microns wide. Five stenosis compartments were used. The first largest compartment contained protrusions that were gradually spaced apart at 300 micron intervals to the fifth stenosis compartment, 50 microns wide. The first four constrictions cleave the erythrocytes by force of fluid flow as the erythrocyte-oocyte complexes pass ("mohawk" cleavage was observed). Half of the red cells were aspirated from the fifth constriction. The flow was reversed several times to rearrange oocytes until the other half of the red cells passed through the last constriction. Thus, embryos and surrounding structures can be placed and conditioned using constrictions and structures of appropriate size.

The entire circular device for erythrocyte removal is shown in Figures 8 (a) and 8 (b), and Figures 8 (c) -8 (f) show Figures 8 (a) and 8 for removing erythrocytes from oocytes and The steps used in the experiment with 8 (b) prototypes are illustrated. The polypropylene well is coupled to an inlet to provide a larger fluid reservoir at the inlet shown in FIG. 8 (a). The acrylic syringe connects the module outlet so that a standard syringe or other accessory can be connected to the device. Syringes were capable of passive pressure regulation, or the syringe pumps acted as accurate flow controllers, allowing the plexiglass and PDMS primitives to set up oocytes in place during testing and embryo placement throughout the channel network. In addition, the prototype device provides complete optical access (important for embryo analysis), rapid prototyping, and easy integration with additional analytical sensors. Loading of oocytes into the device can be simplified using funnel inlet wells (inserted in FIG. 8 (b)). The funnel shape is molded at the inlet of the channel which holds the end of the funnel connected to the channel head. This wide funnel structure allows for easy insertion of oocyte complexes. Oocytes usually sink to the bottom of the funnel. The sloping wall guides the complex to the channel entrance at the bottom of the funnel. This charging method simplifies the handling procedure because it does not require accurate side placement. The complex is passed through two constriction zones to manipulate the red cells to a structure that allows complete red cell removal (FIG. 8 (c)). As shown in Fig. 8 (d), this narrow region divides the red cells into two large masses before and after the oocytes. The two red cell conditioning constricted regions (FIG. 8 (a)) were 200 μm and 150 μm in width, respectively. The erythrocytes are damaged and aggregated in the conditioning area, which then flows to the purge (see FIGS. 8 (d) -8 (f)), which are placed at 90 degrees to each other in the bend in the microfluidic channel of the circular device. Thin channels were included. The erythrocytes are able to enter through these eliminators (Figs. 8 (e) and 8 (f)) but are too small for the oocytes to enter. Using fluid flow control, first pass the first sphere and then pass the remaining spheres to draw out the red cells from the oocytes.

The microfluidic channels of the present invention have also been used to realize new embryonic health assessment methods by analyzing the physical properties of embryos. Specific physical properties that have been demonstrated to distinguish healthy embryos include the surface and deformation properties of the embryo. The tendency to recover its shape after it has been deformed to the point just before permanent damage is considered an indicator of health because the embryo actively maintains its shape. Embryos selectively transport ions through their membranes, and the zona pellucida protein constantly redirects itself to withstand external forces. Transport of ions and other substrates / metabolites into and out of embryonic cells becomes a function of embryo health. Unhealthy embryos have a defect in their ability to transport ions, and therefore their ability to recover their shape after being deformed to the point of avoiding permanent damage. The ability to control the pressure and place the embryo in the constriction provides an opportunity to transform the embryo by adjusting it to prevent permanent deformation of a healthy embryo. The deformation test was performed while maintaining the pressure gradient below 0.1 Pa / m (1 mm water / cm) and the flow rate below several mm / second.

9 (a) -9 (d) show modification evaluation of embryos used as an indicator of embryo survival. The stenosis is large enough to cause deformation of the embryo due to hydraulic pressure during passage of the embryo. In Figure 9 (d) healthy embryos easily recover their shape after passing through the constriction. Alternatively, the embryo may be deformed at a constriction large enough to block passage. After the deformation period in the constriction, the flow is reversed, refluxed and stopped, or stopped so that the embryo recovers its shape.

Because healthy embryos stick better, the precise pressure control provided by the fluid channel allows us to assess the attachment of the embryo to the channel wall. Thus, healthy embryos that move more slowly down the channel than unhealthy embryos can be quantitatively measured. Or, similarly, distances can be compared, where healthy embryos travel shorter distances under the same flow and pressure conditions.

Embryo fluid analysis can be performed more easily with a device comprising the microfluidic channel of the present invention. Fluid is collected from the downstream channel of the microfluidic channel culture apparatus of the present invention. According to the device manufactured according to the present invention, the collected fluid flows past the embryo, because the device of the present invention avoids stagnation and the size of the microfluidic channel allows the fluid in the channel to pass closer to the embryo. Once the downstream fluid is collected, it may be desirable to add a similar amount of upstream fluid to maintain the pressure so that the fluid sample flows through the microfluidic device being recovered. A programmed syringe pump can be used to remove the fluid sample and further insert the upstream fluid. Alternatively, all fluids downstream of the embryo may be collected. This fluid is then analyzed by analysis such as chemical analysis or optical analysis.

The system of the present invention can be used for complete oocyte maturation, fertilization and embryo culture without the use of harsh manipulation techniques. The ability to control fluids and place embryos offers the opportunity to mature oocytes to come into contact with sperm and then simulate biological embryo culture, all of which are included within a single device of the present invention. Maturation (IVM), fertilization (IVF) and culture (EC) require different media, sperm fertility acquisition and washing procedures. The system of the present invention using microfluidic channels allows for rapid changes and precise control of these conditions, altering the composition of the fluid medium from hours to days to simulate the secretion and accumulation of growth factors in the female reproductive tract, Simulate the movement of embryos to different parts of the coffin. The medium can be adjusted to flow slowly through individual embryos (or oocytes) to provide fresh nutrients. Periodic flows, such as once per hour, rather than continuous flows ensure that only useful self-secretory and lateral secretory factors can accumulate while preventing waste from accumulating. Since all current IMF, IVF and EC techniques can be performed in a single device of the present invention, a given oocyte-embryo need not be handled or moved during either sequential IMF, IVF and EC procedures or individual procedures.

The system of the present invention can reduce the sperm fertilization rate and can include a swim-up technique to use fewer sperm and to screen the most motile sperm. In a typical human IVF procedure, eggs are surrounded by 50,000-100,000 sperm in 0.5-1.0 ml of medium. Microfluidic channels can reduce this number because they regulate the flow of sperm and eggs to bind to each other.

In livestock IVF, sperm fertilization rate is high, for example about 40-70% in pigs. According to the present invention the microfluidic channel induces sperm to pass very close to oocytes, eg at a distance of about 30 μm. In addition, the time interval in which sperm stays near oocytes can be controlled by controlling the medium flow rate. Alternatively, the anchored oocytes arranged in accordance with the present invention can be kept very close to the insertion point of the sperm of a relatively small fluid packet, thereby increasing sperm concentration near the oocytes while reducing the chance of fertilization. Individual plugs or rings can deliver only one to several sperm, creating a situation similar to that of an in vivo fallopian tube in which only a few sperm are present at any point in time. This ability allows only one to several sperm to be delivered in the vicinity of the egg to reduce the chance of polysperm fertilization by using a T-junction or other physical or effective stenosis to place the embryo. In addition, embryonic sized channel structures increase the contact between the ovum and the sperm. Sperm "jumping" from the sidewalls increases the effective contact between the sperm and the egg.

Cryopreservation is another technique that can benefit from the ability to place embryos and the ability to precisely control the fluid environment around the embryos in accordance with the present invention. Cryopreservatives can be delivered to embryos placed in or on the device of the present invention, which can later be used to restore the cryopreservation process. Changes in the fluid can then be used to effect maturation, fertilization and incubation as described above.

While various embodiments of the invention have been disclosed and described so far, those skilled in the art will be familiar with other variations, substitutes, and alternatives. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims.

Various features of the invention are set forth in the appended claims.

Claims (15)

Placing the embryo in a fluid channel of approximately embryo size; Causing fluid flow in the fluid channel; And Assessing the characteristics of the embryo while the embryo is located in an embryonic fluid channel Embryonic evaluation method comprising a. The method of claim 1, wherein the step of inducing fluid flow moves the embryo in a fluid channel, and the step of evaluating embryo characteristics comprises measuring the rate at which the embryo moves within the fluid channel. The method of claim 1, wherein the step of inducing fluid flow moves the embryo in a fluid channel and the step of evaluating embryo characteristics comprises measuring the distance the embryo travels in the fluid channel. The method of claim 1, wherein the evaluating step comprises taking a fluid sample from a fluid channel downstream of the embryo in the fluid channel and conducting a chemical analysis of the fluid sample. The method of claim 1, wherein the fluidic channel comprises a constriction of a size capable of deforming the embryo as the embryo passes through the channel, wherein the evaluating step assesses the tendency of the embryo to regain its shape after passing through the constriction. How to be. The method of claim 1, wherein the fluid channel comprises a constriction of a size capable of preventing passage of the embryo, wherein the fluid flow inducing step causes the fluid flow to slightly change its shape by hydraulic pressure for a short time after moving the embryo to the constriction. Wherein the evaluating step evaluates the tendency of the embryo to recover its shape after being deformed for a short period of time. Placing the embryo in a fluid channel of approximately embryo size; Causing fluid flow in the fluid channel; And Processing the embryo such that the characteristics of the embryo change while the embryo is in a fluid channel Embryo treatment method comprising a. 8. The fluidic channel of claim 7, wherein the fluidic channel comprises a series of constrictions with protrusions that become increasingly narrower from each other, the processing step in which the fluidic channel passes through the one or more constrictions to remove red cells. Manipulating the fluid flow in the chamber. The method of claim 8, wherein the last portion of the series of constrictions is sized to prevent passage of the embryo, wherein the processing step involves manipulating the fluid flow in the fluid channel so that the embryo passes through the remaining portions of the series of constrictions. Manipulating the fluid flow to aspirate red cells when positioned at the end of a series of constrictions. The method of claim 7, wherein the fluid channel comprises a constriction sized to prevent passage of the embryo, wherein the processing step comprises changing the fluid in the fluid channel to allow an acidic solution to pass over the embryo located in the constriction to remove the zona pellucida. Which method. The method of claim 10, wherein the treating step changes the fluid in the fluid channel to allow an acidic solution to pass over two embryos located in the constriction to remove the zona pellucida, and then the fluid in the fluid channel to promote culture of chimeras from the two embryos. Used to form chimeras by changing. A microfluidic embryo handling device comprising an embryo transport network (32,50) that retains a biological medium for the movement of an inserted embryo, wherein the transport network delivers fluid to an embryonic fluid channel (14) and transport network of approximately embryo size. And an opening (36,52) for delivery. 13. An apparatus according to claim 12, comprising a t-junction (14b) in the transport network formed at the intersection of the two fluid channels and a second opening for delivering fluid to the transport network at separate fluid channel locations. 14. The well 14a in the fluid channel according to claim 13, wherein the well 14a in the fluid channel is formed to retain the embryo for a period of constant flow rate in the fluid channel and to discharge the embryo during a period of flow rate lower than the constant flow rate in the fluid channel. And further comprising. A microfluidic embryo fertilization device comprising an embryo transport network (32,50) that retains a biological medium for the transfer of embryos inserted therein, said transport network into an embryonic fluid channel (14) and transport network of approximately embryo size. Openings 36 and 52 for delivering fluids; And a T-junction (14b) in the transport network formed at the intersection of the two fluid channels and a second opening for delivering semen to the transport network at separate fluid channel locations.