FIELD OF THE INVENTION

The invention presented here relates to novel approaches (format and protocol) for culturing and manipulating (mammalian) embryos during their pre- implantation period, i.e. directly after their fertilization until the blastocyst stage, at which point they are placed back in the uterus of a female mammal. Such culture devices are to be employed in the field of assisted reproductive technologies (ART) for human embryos or for cattle reproduction. Furthermore, the invention belongs to a novel class of miniaturized and integrated devices for medical applications. BACKGROUND OF THE INVENTION

A typical and conventional approach for culturing in vitro mammalian embryos when assisted reproduction technologies are employed consists of using 5-50 droplets of growth medium in which embryos are kept during their pre-implantation development. The droplets are covered with mineral oil to limit evaporation phenomena and to physically separate the droplets. Embryos are mostly cultured as groups of 10-20 embryos together in one droplet. If medium is to be refreshed or its composition changed (e.g., when multiple medium culture protocols are employed), the embryos are manually displaced with the help of a glass pipette from one droplet of "old" medium to another droplet of fresh medium, which implied extensive manipulation of the embryos.

Alternatively, microwells with a larger capacity (400 μί) are employed; in this case, no oil is used as the microwells are equipped with a lid. Alternative culture formats have been reported, but the golden standard remains the droplet-based approach.

Microfluidics is now acknowledged as a potentially interesting format for the culture of embryos due to the numerous advantages microfluidics brings in terms of (i) control on the volume of solutions, (ii) control on flow patterns, (iii) control on embryo microenvironment, and (iv) integrated approach for embryo culture. To date, attempts to employ microfluidic for embryo culture have provided mitigated results, and they have not yet fully explored the capability of this technology.

In Raty, S., et al, Embryonic development in the mouse is enhanced via microchannel culture., Lab On A Chip, 2004, 4, 186-190., First, static culture conditions have been employed in a straight channel equipped with a dam structure to trap embryos. There, mouse embryos are cultured in groups, and the culture volume is not well-defined.

In Hickman, D.L. et al., Comparison of Static and Dynamic Medium Environments for Culturing of Pre-implantation Mouse Embryos, Comparative

Medicine, 2002, 52, 122-126, it was reported that dynamic culture in the same microfluidic structures, still using groups of mouse embryos; and continuous pumping of liquids at 0.1-0.5 μΕ/h, resulted in a loss of embryo viability.

In Melin, J., et al., In Vitro Embryo Culture in Defined, Sub-micro liter Volumes. Developmental Dynamics, 2009, 238, 950-955, a closed 100-nL microfluidic chamber was designed that was equipped with two valves, where mouse embryos are cultured in groups. The group size was reduced to 2 embryos, and the embryo

development was assessed in terms of blastocyst rates (pre-implantation development).

In Kim, M.S., et al, A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos, Electrophoresis, 2009, 30, 3276-3282, an original microfluidic device was disclosed that consisted of a long microchannel equipped with constrictions, mimicking thereby the geometry of the female oviduct. The device is placed on a tilted support to promote the displacement of the embryos in the channel, and every time an embryo passes a constriction, it undergoes a mild mechanical stimulation. Bovine embryos are cultured under "semi-dynamic" conditions, but again, their development is impaired compared to embryos cultured in conventional droplets.

In Han, C. et al. Integration of single oocyte trapping, in vitro fertilization and embryo culture in a microwell-structured microfluidic device, Lab Chip, 2010, 10, 2848-2854, it was proposed to integrate a miniaturized microwell array in a

"microfluidic device" where embryos are generated by in-device fertilization; here, the embryos are cultured until the blastocyst stage is reached. However, this device is more a miniaturized approach for culture rather than a truly microfluidic device.

In Heo, Y.S., et al., Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates, Human Reproduction, 2010, 25, 613-622, a semi- micro fluidic device was reported in which embryos are cultured in groups in a 10 droplet-like volume of liquid covered with mineral oil, connected to a microfluidic circuit providing regularly fresh nutrients to the embryos. This device gave comparable pre- implantation rates for mouse embryos as conventional droplets, and it was as well successfully tested for the full-term development of mouse embryos. At the end of the pre-implantation development, embryo quality is assessed using apparent criteria such as their morphology or their development rate (cell division rate) throughout the pre-implantation period. Again, other approaches appear in the field, but they have not fully proved their suitability, and the results obtained with certain parameters are even still controversial, to reliably identify competent embryos yet; subsequently, the morphology-based assessment is employed in most of the clinics and in vitro fertilization (IVF) centers.

SUMMARY OF THE INVENTION

The conventional culture protocol employed for in vitro pre-implantation growth is not optimized. This overall culture protocol has been developed based on simple observations and using an empirical approach. More interestingly, this protocol has partly been derived from the culture protocol employed for somatic cells, which are very different to embryos. As a consequence, ~ 50% of the embryos placed in culture are seen to arrest development during this in vitro culture period (before day 4).

The current invention seeks to at least partially obviate at least part of these problems.

The invention thus provides a microfluidic device comprising a chamber for holding at least one embryo, said chamber enclosing a volume and comprising an inlet and an outlet, dimensioned for allowing said embryo to enter and leave said chamber and said chamber adapted for holding a culture solution when said microfluidic device is in operation, said chamber further comprising at least one sensor integrated in said chamber for in situ measuring of at least one selected from a chemical, physical and biochemical parameter of said culture solution when said microfluidic device is in operation.

The origin for the embryo impairment in the prior art is found to be caused by various reasons.

It was found that at least some or the problems of the art originated from a lack of knowledge on the precise physiological needs of embryos during the first days of their life.

The inventors further concluded that the culture conditions found in a droplet are static (see figure 1) while dynamic conditions are found in vivo and the embryos do undergo slight mechanical stimulation as they travel along oviduct during this developmental period. The inventors also found out that the conventional protocol implies extensive manipulation of the embryos between various droplets of medium: the preparation droplet, the fertilization droplet, 2-3 culture droplets depending on how long the culture is performed in vitro.

The format of the culture is very different to what is found in vivo. The embryos are cultured in relatively large volumes of medium (compared to that of an oviduct), and the medium is "saturated" with nutrients. This is a consequence of the lack on basic knowledge on the embryo daily requirements; the trend is to saturate the embryo microenvironment so that the embryos don't suffer from a lack of food.

The inventors realized that most of the time the culture protocol is applied for groups of embryos while, depending on the species (e.g., human embryos), embryos develop singly in vivo. Furthermore, there is a potentially negative influence of "bad" embryos on good embryos.

Furthermore, inventors found that embryos do secrete factors that reflect their state, and these factors are likely to influence the development of their fellow embryos. Some factors have a positive effect on the growth of the other embryos (growth factors) while unhealthy and impaired embryos may have a negative influence on the development of neighboring embryos. Moreover, the group embryo approach prevents from accessing data at the single embryo level, on their individual needs but also on their individual growth. Consequently, it is not possible to follow single embryo development by other means than simple observation, i.e. using subjective criteria (cleavage rate, morphology). Still it is difficult to follow individual embryos if they are not physically separated from each other.

The inventors also found that when medium droplets are covered with oil, as illustrated in figure 1 , this oil can be a possible source for contamination as this hydrophobic milieu is prone to accumulation of toxic hydrophobic compounds (PAHs).

The inventors also found that using the droplet approach, there is no possible control on the precise microenvironment of embryos. The Petri dishes with the droplets is placed in an incubator, where all physical parameters (gas tension, temperature, ...) are regulated. This system does not measure precisely parameters in the close vicinity of the embryos but in the bulk of the incubator. This culture approach has provided little valuable information on the embryo needs and basic research in this direction is still mandatory. Consequently, there is a need for novel tools to monitor single and multiple embryo growth in a reliable and precise way using non- invasive means.

At the end of the pre-implantation culture period, embryos are scored upon "superficial/apparent" criteria such as their morphology and cleavage rate. However, these parameters are not only subjective but they are also acknowledged not to fully reflect the embryo developmental competence, as "ugly" embryos have been seen to develop into viable and healthy "babies" while "nice-looking" embryos failed in their full-term development. Furthermore, different scoring systems co-exist showing the lack of rationality of this approach.

Alternatively, embryos have been scored using genetic parameters, after gene analysis of one cell retrieved from the embryo. This approach is invasive, and is now recognized not to be a reliable way to measure embryo viability.

We address here these various issues found in the in vitro culture protocol of embryos employed in the field of ART by developing an integrated sensing micro fluidic platform as detailed below.

The sensor may be selected from a sensor for glucose, lactate and/or pyruvate, for metabolic parameters, and/or for physical parameters like temperature and/or flow rate of medium.

In an embodiment, the device comprises an oxygen sensor. In an embodiment, the oxygen sensor is of the type that measures oxygen amperometrically. Typically, the voltage applied for this reaction is -0.5 V to - 0.3 V. In an embodiment, the sensor comprises an array of ultramicorelectrodes. Such a sensor is more sensitive than larger electrodes and consumes less oxygen. Both aspects being of importance in a low volume like in a micro fluidic device where biological material must survive.

Other potential electrochemical sensors comprise a sensor for measuring glucose, lactate and pyruvate amounts. For other cell culture, sensors can be built to measure the amount of stress in cells, by looking at their reactive ion species (ROS) production or NO can also be measured in the vicinity of cells. NO is a measure for angiogenesis-blood capillary formation. NO and ROS can directly be measured using electrochemical reactions, while the three other substrates are detected using an enzymatic reactions. Using specific enzymes, oxidases (e.e., glucose oxidase for glucose), H ₂ 0 ₂ is produced during the enzymatic degradation of the substrates, which is detected on the electrodes electrochemically. In an embodiment, the micro fluidic device is for pre-implantation culturing of mammalian embryos, wherein said chamber comprises at least one trapping structure for trapping an embryo at a location in said chamber, and said at least one sensor comprises an oxygen sensor positioned at the location of said trapping structure.

In an embodiment, the at least one sensor comprises an electrochemical sensor. This allows measurements in the vicinity of am embryo, en even without influencing the embryo. It ma even allow monitoring.

In an embodiment, the sensor comprises an oxygen sensor.

In an embodiment, the sensor allows in situ measuring of an embryo microenvironment in spatial and/or temporal way, in particular monitoring of an embryo microenvironment, more in particular monitoring at least one parameter selected from gas tension and temperature.

In an embodiment, the micro fluidic device further comprises a controller to control an embryo microenvironment.

In an embodiment, the chamber defines a volume of liquid, in particular said chamber has a volume of 1 nL - 2 microliter, more in particular a volume of 10 nL - 500 nL.

In an embodiment, the sensor is positioned in a bottom of said chamber, in particular integrated in a bottom of said chamber. In microdevice production, this proved to provide a production method that allows large-scale production.

In an embodiment, the micro fluidic devicefurther comprising a trapping structure for maintaining an embryo at a position in said chamber.

In an embodiment, the trapping structure comprises at least two trapping elements extending from a bottom of the chamber around and enclosing a trapping area of the chamber, said trapping elements providing a first opening to said trapping area and having dimensions allowing an embryo to pass said first opening to said trapping area, and at least one second opening having dimensions for allowing said culture solution to leave said trapping structure but preventing said embryo from passing said second opening.

In an embodiment, the trapping structure fences off said trapping area, and wherein said first opening is directed towards said inlet.

In an embodiment, the first opening has a diameter of 20 micron smaller than an embryo for which said device is used, in particular said diameter is smaller than 150 microns, more in particular smeller than 130 micron, more in particular smaller than 80 microns, more in particular smaller than 60 micron.

In an embodiment, the first opening has a diameter wider than 30 microns, more in particular wider than 50 micron.

In an embodiment, the trapping area is at least 80 micron x 80 micron and has a surface area of at least 5000 microns ² , in particular the trapping area surface area less than 40,000 microns ² .

In an embodiment, the second opening has a diameter of less than 30 % of an embryo diameter, in particular said diameter is less than 45 micron, more in particular said diameter is less than 24 micron.

In an embodiment, the sensor is positioned at said trapping structure, in particular positioned under said trapping structure, allowing sensing near a trapped embryo when said microfluidic device is in operation.

In an embodiment, the chamber comprises multiple trapping structures in said chamber.

In an embodiment, each trapping structure comprises at least one sensor, in particular at least one sensor positions for measuring locally at said trapping structure, specifically positioned below said trapping structure, in particular position in the bottom of said chamber below the trapping structure.

In an embodiment, the chamber comprises an array of trapping structures and an array of sensors, the layout of trapping structures matching the layout of sensors for in situ measuring of chemical parameters at the locations of said trapping structures.

In an embodiment, the inlet comprises a retention structure to retain embryo in chamber.

In an embodiment, the microfluidic device comprises an inlet channel in fluid connection with said inlet, said inlet channel comprising said retention structure for retaining said embryo in said chamber, which retention structure comprises a tapered channel diameter having a first smallest passage diameter at its chamber end and a largest passage diameter at its opposite end, wherein a diameter of said smallest passage is less than 150 micron. In an embodiment, the diameter is less than 130. In another

embodiment, in particular for instance for mouse embryos, the diameter is less than 80 micron. In yet a more particular embodiment, the diameter is less than 60 micron.

In an embodiment, the microfluidic device comprises a multiplex structure comprising a series of chambers. The microfluidic device further comprises a sensor having its sensing surface divided over the bottom of said chamber for measuring said chemical parameter as an average value in said chamber.

The invention further pertains to a method of cultivation cells, in particular mammalian embryo's, comprising introducing at least one embryo in a microfluidic device described above.

In an embodiment, the at least one embryo is cultivated in said microfluidic device until the end of its morula stage and before it reaches its blastocyst stage.

In an embodiment, during the cultivation of said at least one embryo, using said sensor an oxygen tension is measured in situ, in particular in the vicinity of said embryo

In an embodiment, of the method said sensor monitors the oxygen tension in said chamber.

The invention further pertains to an oxygen sensing microfluidic device for the culture of (mammalian) embryos during their pre-implantation period, comprising at least one sensor to control the embryo microenvironment during their in vitro culture by monitoring the oxygen tension, or as a means to assess embryo quality before their implantation by measuring their metabolic rate by monitoring the oxygen tension.

In an embodiment, the microfluidic device comprises at least one chamber with a capacity between 1 microliter and down to 10 nL, in which embryos are cultured during their pre-implantation development, wherein the chamber is equipped with dedicated structures (i) for embryo insertion in and retrieval out of the chamber as well as (ii) for preventing the embryo from escaping out of the chamber.

In an embodiment, the microfluidic is equipped with an oxygen-sensing capability which is either integrated at the bottom of the chamber under the embryos, in operation, or external and inserted in the chamber, for instance through the lid of the microfluidic device.

A first aspect is a microfluidic device containing a chamber with a well- defined capacity for embryo culture. The capacity of the chamber can be varied from < 1 nL up to 2 μΐ,. Preferably, it is varied between 10 nL and 500 nL for embryo culture, while for other applications (cell culture) it can be made smaller. The chamber is connected to two inlet and outlet channels for flushing of solutions and insertion of the embryos. The use of micro fluidics as a new format for the culture of embryos provides higher control on the embryo microenvironment on a spatial and temporal way (laminar flow profile). Figure 2 corresponds to a schematic view of such a device including a microchamber for embryo culture.

A micro fluidic device containing a chamber with a well-defined capacity for embryo culture where the culture conditions (temperature, gas concentrations, nutrient distribution, flow and shear stress exerted on the cells) and the embryo microenvironment are highly controlled in a spatial and temporal way.

A micro fluidic device containing a chamber with a well-defined capacity for embryo culture as a tool for varying and screening various culture parameters (flow- rate, gradients, temperature, gas concentrations, medium composition...).

A micro fluidic device containing a chamber with a well-defined capacity for embryo culture. The microfluidic chamber consists of an "open" space in the sense that the accesses to the inlet and outlet channels are not closed by valves: this configuration enables diffusion-based delivery of new nutrients from reservoirs to the microchamber to a certain extent. Alternatively, if a complete confinement is to be created around the environment, valves can be added at the inlet and outlet of the chamber.

A microfluidic device containing a chamber with a well-defined capacity for embryo culture and comprising dedicated structures for insertion of an embryo, possible release and preventing him/her from escaping out of the chamber. Figure 2 illustrates the herein proposed microfluidic device whose chamber comprises the hereby described microstructures, and in figure 3 pictures of embryos cultured in the

microfluidic device as groups or individually are included.

A microfluidic device containing a chamber with a well-defined capacity for embryo culture where medium can easily be refreshed at given time points during the embryo development. Medium is for instance pumped in the chamber using a pipette or using droplets of liquid (i.e. the passive pumping technique, Walker et al, Lab Chip, 2002, 131).

A microfluidic device containing a chamber with a well-defined capacity for culture of groups of embryos.

The device has been shown to support viable development of mouse embryos cultured as groups (groups of 5 or 20) during pre-implantation period, as shown in figure 4. The pre- implantation development rate varies between 90 and 100% at 4.5 days, against 66-73% in droplets (groups of 5 and 20 embryos, respectively). The device also provides viable full-term development of mouse embryos (groups of 5 or 20) with rates comparable to what is obtained with a conventional culture approach, while the precise rate values depend on group size and microchamber capacity.

A micro fluidic device containing a chamber with a well-defined capacity for culture of single embryos. The device has been shown to support viable development of mouse embryos cultured singly (94-97% development rate at 4.5 day) against 30%> for a droplet-based culture. Furthermore, development of single embryo delayed as no blastocyst observed before 4 dpc. Singly cultured embryos were seen to develop into viable pups, providing higher birth rates (29-33 %) than a conventional droplet-based culture approach (20%>) for single embryo culture. These data are collected in figure 4.

A micro fluidic device containing a chamber with a well-defined capacity for embryo culture and including trapping structure(s) for precise localization of the embryo(s) in the microfluidic chamber. Figure 6 illustrates possible structures for trapping embryos in well-defined locations in a microchamber.

A microfluidic device containing a series of chambers with a well-defined capacity for parallel culture of individual embryos. Figure 5 shows an example of a multiplexed system for parallel culture of individual embryos.

A microfluidic device containing a series of chambers with a well-defined capacity for parallel culture of groups of embryos.

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability, as detailed below:

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability in the form of an integrated sensor fabricated at the bottom substrate of the microfluidic system and placed under the group of embryos. Figure 7 shows a possible design for the integrated sensing structures for oxygen measurement. Here, an electrochemical sensor -which is one option to realize an integrated oxygen sensor- is represented, and oxygen is detected through its reduction by the sensor. In this precise case, the sensor is based on an array of ultra-microelectrode, as previously developed in the BIOS group (Krommenhoek et al; 2008; Biotechnol.

Bioeng.).

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability in the form of an integrated sensor fabricated in the bottom substrate of the microfluidic system and placed under a single embryo below trapping structures in the microchamber. Figure 7 illustrates a local oxygen sensor with a similar geometry and working principle as described above; here the size of the sensor is decreased for local measurements.

A micro fluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability in the form of individual integrated sensors fabricated in the bottom substrate of the microfluidic system and placed under a series of individual embryos.

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability in the form of an external electrode inserted in the microchamber in the vicinity of the embryos. Figure 8 is a schematic representation of such a platform comprising of a microfluidic culture device and an external electrode placed in the chamber for in situ oxygen measurements.

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability in the form of an external electrode inserted in the microchamber in the vicinity of a single embryo whose localization in the chamber is controlled using trapping structures.

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability in the form of individual external electrodes inserted in the microchamber in the vicinity each embryo of a series whose localizations in the chamber are controlled using trapping structures.

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability for controlling the oxygen tension in the cell culture microchamber.

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability for monitoring the oxygen tension in a culture microchamber, for better control on the embryo microenvironment.

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability for monitoring the oxygen tension in a culture microchamber for better control on the embryo microenvironment and measuring the impact of the oxygen tension on the embryo development. This will for instance enable precise comparison between a "low" oxygen tension (5%) and an atmospheric oxygen tension (20-21%).

A microfluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability for monitoring the oxygen tension in a culture microchamber, for better control on the microenvironment of somatic cells. A micro fluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability for determining the consumption of oxygen by cells.

A micro fluidic device containing a chamber with a well-defined capacity and equipped with a local oxygen sensing capability for determining the consumption of oxygen by embryos, as a measure of their oxidative metabolic rate and, indirectly, of their viability.

A micro fluidic device containing a chamber with a well-defined capacity where culture parameters are varied, and equipped with a local oxygen sensing capability to study embryo viability, and ultimately determine the impact of various culture parameters on embryo viability.

A oxygen sensing microfluidic device is presented here for the culture of (mammalian) embryos during their pre-implantation period. Such an integrated platform is to be used as a means to control the embryo microenvironment during their in vitro culture by monitoring the oxygen tension, or as a means to assess embryo quality before their implantation by measuring their metabolic rate.

The microfluidic platform presents a microfluidic chamber -or a series of individual chambers- with a low (and down to 10 nL) capacity in which embryos are cultured during their pre-implantation development. The chamber is equipped with dedicated structures (i) for embryo insertion in and retrieval out of the chamber as well as (ii) for preventing the embryo from escaping out of the chamber. Furthermore, the device is equipped with an oxygen-sensing capability which is either integrated at the bottom of the microchamber under the embryos, or external and inserted in the chamber, e.g., through the lid of the microfluidic device.

The terms "upstream" and "downstream" relate to an arrangement of items or features relative to the propagation of a fluid entering the chamber of the microfluidic device or leaving said chamber.

The term "substantially" herein, such as in "substantially consists", will be understood by the person skilled in the art. The term "substantially" may also include embodiments with "entirely", "completely", "all", etc. Hence, in embodiments the adjective substantially may also be removed. Where applicable, the term "substantially" may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%). The term "comprise" includes also embodiments wherein the term "comprises" means "consists of. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The devices or apparatus herein are amongst others described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation or devices in operation.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention further applies to an apparatus or device comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterising features described in the description and/or shown in the attached drawings.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Furthermore, some of the features can form the basis for one or more divisional applications.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

Figure 1 shows a conventional approach using droplets of growth medium covered with an oil layer and placed in a standard Petri dish;

Figure 2 shows on the left a schematic drawing of an embodiment of a micro fluidic device for the culture of embryos and on the right above one another two details as indicated in the leftmost drawing;

Figure 3 shows three photographs of the device of figure 2 with 5, 20 and a single embryo;

Figures 4A and 4B show bar graphs of test results of the apparatus of figures 1 and 2 using mouse embryos;

Figure 5 a schematic shows an example of a multiplexed microfluidic device holding several devices of figure 2;

Figure 6 schematically shows two embodiments of trapping structures in the chamber of a microfluidic device of figure 2, with a detail of a trapping structure in the center;

Figure 7 shows left and right schematically two embodiments of a sensor in the chamber of a microfluidic device of figure 2;

Figure 8 schematically shows a microfluidic device of figure 2 with a sensor inserted through the lid of the device, and

Figures 9A, 9B and 9C schematically show some embodiments of a multiplexed microfluidic device.

The drawings are not necessarily on scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In a conventional approach, schematically depicted in Figure 1, embryos are cultured in droplets of liquid (2) which are placed in a Petri dish (1). As the volume of the droplets is in the 5-50 range, all droplets are covered with a layer of mineral oil (3) whose role is to limit medium evaporation in the droplets and to physically separate the droplets, and thereby the embryos (shown as black dots).

Figure 2 shows a microfluidic device (10) that has been developed as an alternative format for the culture of embryos. The device contains a microchamber (12) in which embryos can be cultured in a well-defined volume of liquid. This chamber (12) is connected to two channels, an inlet channel (13) and an outlet channel (13') leading to corresponding inlet and outlet reservoirs (4 and 4', respectively). Details of figure 2, depicted in the two enlargements to the right of the drawing, show that the microchamber (12) includes dedicated retaining structures (5, 6) to retain the embryos in the chamber. At the inlet of the chamber (upper enlargement), a V-shape structure (5) helps guiding the embryo(s) in the microchamber (12), and the spacing (7) in the V-shape structure, the opening (7) at the chamber end, is chosen large enough to enable embryo retrieval out of the chamber (12) with the help of a mild pressure applied from the outlet reservoir (4') or lowered pressure in inlet reservoir (4). Usually, the chamber-end (7) of the retaining structure (5) will have dimensions smaller than the size of an embryo. For mouse embryos, this means for instance smaller than about 80 microns, and for human embryos, this means smaller than about 150 microns. It was found that if the opening has dimensions of 20-30 micron smaller than an embryo it is applied for, the embryo will not leave the chamber without assistance. Thus, for mouse embryos, the dimensions will be between 50 and 60 microns. For human embryos, the dimensions will be 120-130 micron. The first opening diameter in an embodiment is more than 50% of the embryo diameter. At its opposite end, the retaining structure (5) will have dimensions at least the dimensions of an embryo for which the device is applied. Thus, form mouse embryos the dimensions will be at least 80 micron, and for human embryos at least 150 microns. Thus, an embryo can relatively easy pass the retaining structure (5) when entering the chamber (12), but will not leave the chamber (12) unless some urging force is applied, for instance a relatively strong flow of cultivation medium out of the chamber, or a suction force. In the embodiment shown, the retaining structure (5) has two wall parts (18, 19). These wall parts extend from the bottom of channel (13). The walls (18, 19) are positioned at an angle with resect to the sidewalls of the channel (13).

At the outlet of the chamber, in this embodiment grids (6) are placed that act as a barrier for the embryo(s). Usually, the height of the chamber is at least 2 times the embryo diameter. The outlet retaining structure (6) can be for instance pillars that extend from the bottom of the outlet channel (13'). The height can be such that the channel height minus the height of the retaining structure (6), the remaining space, is less that the thickness of an embryo. Often, the remaining space is less than 80 microns. In other embodiments, the height of the retaining structure equals the channel height.

Figure 3 shows three subsequent photographs of a micro fluidic device (10) in which mouse embryos (14) have been successfully cultured in 30-nL micro fluidic chambers (12) made from PDMS, in groups of 5 embryos or 20 embryos, or, more interestingly, individually during their pre-implantation development. Here, embryos (14) have been cultured for 3.5 days, and most of them have reached the blastocyst stage. Often, this is the end-point of their pre-implantation development.

Figures 4A and 4B shows bar graphs showing development rates of mouse embryos cultured in microfluidic chambers (see figure 2) or in 5-μΙ, droplets of medium (see figure 1). Development rates are determined at two end-points: at the end of the pre- implantation period (day 4.5) in terms of blastocyct rates (figure 4A), and after birth in terms of birth rates (figure 4B). For the latter experiments, embryos cultured in microfluidic devices or in droplets have been transferred in pseudo-pregnant mice at day 3.5. Several microfluidic conditions are tested here: 30-nL and 270-nL microchambers (12), different embryo group size (1, 5, 20). For one set of experiments (5 embryos), medium is refreshed at day 3 in the microchamber (white bar in figure 4B).

Figure 5 shows a multiplexed device (100) for parallel and simultaneous culture of several groups of embryos of several individual embryos. The device includes a number of individual chambers (12, 12', 12" ... 12n) as described in figure 2 which are arranged in a single device (100). Here, 12 microchambers (12, 12', ...12 ⁿ ) are represented, but this amount is easily scalable. For ART applications, in particular for human embryos, The microfluidic device would hold up to 12 culturing units (10, 10'). This allows a clinician to use one device per patient.

Figure 6 shows on the left, A, a microfluidic device (10) as described in figure 2 whose microchamber (12) includes a micro fabricated structure (23) or trapping structure (23) for trapping of a single embryo. The trapping structure (23), shown enlarged on the right of the drawing, consists here of two parts (24 and 24') which are separated by a narrow spacing (25), added to enable embryo retrieval out of the trapping structure with the help of a mild back flow. The dimension of the spacing (25) is chosen carefully not to let embryos go through. The diameter of the inlet opening (26) of the trapping structure (23) usually is smaller than 150 micron. For smaller embryos, for instance mouse embryos, the inlet opening (26) is smaller than 80 micron. Usually, the dimensions of the inlet opening or first opening (26) of the trapping structure (23) depends upon the size of the embryo. In an embodiment, the inlet opening (26) of the trapping structure (23) is 20-30 micron smaller that the embryo diameter. In an embodiment, the diameter of the first opening is larger than 50% of the embryo diameter. For human embryos, this means that the diameter is more than 75 micron. For mouse embryos, the diameter is more than 40 micron.

In an embodiment the outlet opening or second opening (25) of the trapping structure (23) will be smaller than the embryo diameter. In an embodiment, the outlet opening (25) is less than 30 % of the embryo diameter. In an embodiment the trapping structure (23) comprises a series of pillars or wall parts provided with several openings for allowing fluid to pass.

The role of the trapping structure (23) is to control the position of the embryo in the microchamber (12). On the right, B, figure 6 shows alternatively the microchamber (12) of the micro fluidic device (10) as described in figure 2 which can contain an array of identical trapping structures (23, 23', 23", ..., 23 ⁿ ) when a group of embryos is to be cultured in the chamber (12). The role of the trapping structures (23, 23', 23", ..., 23 ⁿ ) is to control the position of the embryos in the microchamber (12). This allows measuring at the location or in close proximity of an embryo. Thus, parameters of an embryo microenvironment can be measured.

Figure 7 shows two embodiments of a microfluidic device (10, 10') as described in figure 1 whose microchamber (12, 12') includes an integrated sensor (33, 33') to monitor the oxygen concentration in the vicinity of the embryo(s). The sensor is fabricated in the substrate that forms the bottom of the microchamber (12, 12'). On the left, A, figure 7 shows an embodiment in which the sensor (33) covers the whole surface area of the microchamber (12) for "global" sensing in the whole microchamber (12). This enables to monitor for instance in real-time the environment of the embryo(s) culture in the microchamber (12). On the right, B, figure 7 shows an embodiment in which the sensor (33') represents a limited part of the microchamber (12') bottom for local measurement of the oxygen concentration in the device (10'). This local approach is to be combined with the use of trapping structures (23) as illustrated in figure 6, and the sensor (33') will be implemented just under trapping structures (23) (see figure 6 parts A and B). In that case, the microchamber (12') can include one local sensor (33') or an array of identical sensors (not represented here). In an embodiment, the sensor (33) measures a surface area of less than 200 micron x 200 micron. This means that in an embodiment, the various surfaces of a sensor (33) cover a part of the bottom of the chamber that measures 200 micron x 200 micron. In an embodiment, for instance in the use of the device for human embryos, the sensor (33) would cover less than 150 micron xl50 micron. In an embodiment, the sensor (33) would cover an area less than 100 micron x 100 micron. For embryos like mouse embryos, the sensor (33) may even cover less than 80 micron x 80 micron.

Figure 8 shows schematically again a micro fluidic device (1) as described in figure 1 whose microchamber (12) includes an oxygen sensor (33) to monitor the oxygen concentration in the vicinity of the embryo(s). The sensor (33) is external here and is inserted from the e.g., the lid of the microdevice (10) in the microchamber (12). As for integrated sensors (see figure 7), this sensor (33) can be employed in a global manner for the whole chamber (12) or in a local manner when it is placed in close vicinity to one embryo which is for instance trapped using dedicated structures as described in figure 6. Alternatively, several external sensors can be connected to the microchamber (12) for parallel and simultaneous measurements on several embryos.

Figures 9A, 9B and 9C schematically show some embodiments of a multiplexed device of the current invention. In some of these embodiments, separate cultivation chambers and sensing chambers can be provided. These embodiments can, of course, also be combined.

In figure 9 A, the concept is to provide sensors (33) in the cultivation chambers (12), i.e., the chambers (12) in which the embryos are grown. In this embodiment, there is a single reservoir (4) for providing embryos to the microfluidic device. Furthermore, each chamber (12) has its individual reservoir (4') in which one ore more embryos for a chamber (12) can be retrieved. This microfluidic device can be relatively compact, as only a minimal amount of chambers is needed. Furthermore, the design allows prevention of cross-contamination or mutually influencing of embryos.

In figure 9B, an alternative layout of a microfluidic device is shown schematically. In this embodiment, a set of cultivation chambers (12) is provided, and each cultivation chamber (12) is coupled to its own measuring/sensing chamber (12'). In this embodiment, again all the cultivation chambers (12) are coupled to an embryo inlet reservoir (4). The cultivation chambers (12) as well as the measuring/sensing chambers (12') are provided with an individual reservoir (4, 4"). These reservoirs (4, 4") can be used to flush each individual chamber (12). In that way, one or more embryos in a chamber (12) can be moved back and forth between chambers (12, 12').

In figure 9C, yet another alternative layout of a microfluidic device is shown. In this embodiment, several cultivation chambers (12) are coupled to one measuring/sensing chamber (12'). Again, all the chambers (12, 12') are coupled to a reservoir (4, 4'). All the cultivation chambers (12) are coupled to an embryo inlet reservoir (4). In this embodiment, for measuring an embryo it is flushed to the measuring/sensing chamber (12'), measurements are done, and the embryo may be flushed back to its cultivation chamber (12). This action is repeated for each cultivation chamber (12). In this way, a relative simple and cheap micro fluidic device can be provided with only a limited amount of sensors (33). In fact, when measurement intervals of relatively large, this layout may be sufficient.