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WO2013004644A1 - Dispositif microfluidique doté de capteurs intégrés pour la culture de cellules - Google Patents

Dispositif microfluidique doté de capteurs intégrés pour la culture de cellules Download PDF

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Publication number
WO2013004644A1
WO2013004644A1 PCT/EP2012/062789 EP2012062789W WO2013004644A1 WO 2013004644 A1 WO2013004644 A1 WO 2013004644A1 EP 2012062789 W EP2012062789 W EP 2012062789W WO 2013004644 A1 WO2013004644 A1 WO 2013004644A1
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WIPO (PCT)
Prior art keywords
chamber
embryo
microfluidic device
trapping
sensor
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PCT/EP2012/062789
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WO2013004644A8 (fr
Inventor
Severine Le Gac
Van Den Albert Berg
Telma Cristina ESTEVES
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Twente Universiteit
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Twente Universiteit
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Anticipated expiration legal-status Critical
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0663—Whole sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions

Definitions

  • 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.
  • ART assisted reproductive technologies
  • the invention belongs to a novel class of miniaturized and integrated devices for medical applications.
  • 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.
  • microwells with a larger capacity 400 ⁇
  • 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.
  • control on the volume of solutions e.g., a volume of solutions
  • control on flow patterns e.g., a flow pattern
  • iii e.g., iv
  • integrated approach for embryo culture e.g., iv
  • microfluidic device where embryos are generated by in-device fertilization; here, the embryos are cultured until the blastocyst stage is reached.
  • this device is more a miniaturized approach for culture rather than a truly microfluidic device.
  • 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.
  • 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.
  • 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.
  • growth factors 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.
  • 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 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.
  • embryos are scored upon "superficial/apparent” criteria such as their morphology and cleavage rate.
  • 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.
  • different scoring systems co-exist showing the lack of rationality of this approach.
  • 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.
  • 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.
  • the device comprises an oxygen sensor.
  • the oxygen sensor is of the type that measures oxygen amperometrically.
  • the voltage applied for this reaction is -0.5 V to - 0.3 V.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the senor comprises an oxygen sensor.
  • 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.
  • the micro fluidic device further comprises a controller to control an embryo microenvironment.
  • 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.
  • the senor 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.
  • the micro fluidic devicefurther comprising a trapping structure for maintaining an embryo at a position in said chamber.
  • 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.
  • the trapping structure fences off said trapping area, and wherein said first opening is directed towards said inlet.
  • 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.
  • the first opening has a diameter wider than 30 microns, more in particular wider than 50 micron.
  • 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 .
  • 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.
  • the senor 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.
  • the chamber comprises multiple trapping structures in said chamber.
  • 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.
  • 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.
  • the inlet comprises a retention structure to retain embryo in chamber.
  • 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
  • the diameter in particular for instance for mouse embryos, is less than 80 micron. In yet a more particular embodiment, the diameter is less than 60 micron.
  • 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.
  • 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.
  • an oxygen tension is measured in situ, in particular in the vicinity of said embryo
  • 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.
  • 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.
  • 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 ⁇ ,. 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 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.
  • valves can be added at the inlet and outlet 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.
  • 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.
  • Figure 6 illustrates possible structures for trapping embryos in well-defined locations in a microchamber.
  • 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:
  • Figure 7 shows a possible design for the integrated sensing structures for oxygen measurement.
  • 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.
  • the sensor is based on an array of ultra-microelectrode, as previously developed in the BIOS group (Krommenhoek et al; 2008; Biotechnol.
  • 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.
  • 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 for controlling the oxygen tension in the cell culture microchamber.
  • 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.
  • 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.
  • 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 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.
  • 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
  • Figures 9A, 9B and 9C schematically show some embodiments of a multiplexed microfluidic device.
  • embryos are cultured in droplets of liquid (2) which are placed in a Petri dish (1).
  • 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.
  • 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).
  • 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.
  • 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.
  • the dimensions will be between 50 and 60 microns.
  • the dimensions will be 120-130 micron.
  • the first opening diameter in an embodiment is more than 50% of the embryo diameter.
  • the retaining structure (5) will have dimensions at least the dimensions of an embryo for which the device is applied.
  • the dimensions will be at least 80 micron, and for human embryos at least 150 microns.
  • 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).
  • grids (6) are placed that act as a barrier for the embryo(s).
  • 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.
  • 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- ⁇ , 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).
  • 12 microchambers (12, 12', ...12 n ) are represented, but this amount is easily scalable.
  • 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.
  • the dimensions of the inlet opening or first opening (26) of the trapping structure (23) depends upon the size of the embryo.
  • the inlet opening (26) of the trapping structure (23) is 20-30 micron smaller that the embryo diameter.
  • 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.
  • 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).
  • 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').
  • FIG 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).
  • 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).
  • the microchamber (12') can include one local sensor (33') or an array of identical sensors (not represented here).
  • 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.
  • the senor (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).
  • 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.
  • 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.
  • separate cultivation chambers and sensing chambers can be provided.
  • These embodiments can, of course, also be combined.
  • the concept is to provide sensors (33) in the cultivation chambers (12), i.e., the chambers (12) in which the embryos are grown.
  • the cultivation chambers (12) i.e., the chambers (12) in which the embryos are grown.
  • 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.
  • the design allows prevention of cross-contamination or mutually influencing of embryos.
  • FIG 9B an alternative layout of a microfluidic device is shown schematically.
  • a set of cultivation chambers (12) is provided, and each cultivation chamber (12) is coupled to its own measuring/sensing chamber (12').
  • 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').
  • FIG 9C yet another alternative layout of a microfluidic device is shown.
  • 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).
  • 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).
  • 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.

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Abstract

L'invention porte sur un dispositif microfluidique (10) comprenant une chambre (12) destinée à contenir au moins un embryon (14), ladite chambre (12) délimitant un volume et comprenant une entrée (13) et une sortie (13'), dimensionnées pour permettre audit embryon (14) d'entrer et de sortir de ladite chambre (12) et ladite chambre (12) étant conçue pour contenir une solution de culture lorsque le ledit dispositif microfluidique (10) est en fonctionnement, ladite chambre (12) comprenant en outre au moins un capteur (33, 33') intégré dans ladite chambre (12) pour la mesure in situ d'au moins un paramètre choisi parmi les paramètres chimiques, physiques et biochimiques de ladite solution de culture lorsque ledit dispositif microfluidique (10) est en fonctionnement.
PCT/EP2012/062789 2011-07-01 2012-07-01 Dispositif microfluidique doté de capteurs intégrés pour la culture de cellules Ceased WO2013004644A1 (fr)

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EP3259342A4 (fr) * 2015-02-17 2018-10-17 Genea IP Holdings Pty Limited Procédé et appareil pour la culture dynamique d'un échantillon biologique
WO2020084324A1 (fr) * 2018-10-25 2020-04-30 Montanari, Bernard Capteur d'environnement pour incubateur ivf de mammifère
CN117660183A (zh) * 2024-01-31 2024-03-08 山东大学 胚胎微流控动态培养皿及其培养方法
EP4144829A4 (fr) * 2020-04-30 2025-04-09 Xingyue Peng Procédé de construction d'une niche cellulaire artificielle microcyclique lente et dispositif correspondant
WO2025179341A1 (fr) * 2024-02-28 2025-09-04 Cell Tech Holding Pty Ltd Dispositifs et procédés de micromanipulation

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015001397A1 (fr) * 2013-07-05 2015-01-08 At Medical Ltd. Dispositif de culture d'un matériel biologique
EP3259342A4 (fr) * 2015-02-17 2018-10-17 Genea IP Holdings Pty Limited Procédé et appareil pour la culture dynamique d'un échantillon biologique
WO2020084324A1 (fr) * 2018-10-25 2020-04-30 Montanari, Bernard Capteur d'environnement pour incubateur ivf de mammifère
EP4144829A4 (fr) * 2020-04-30 2025-04-09 Xingyue Peng Procédé de construction d'une niche cellulaire artificielle microcyclique lente et dispositif correspondant
CN117660183A (zh) * 2024-01-31 2024-03-08 山东大学 胚胎微流控动态培养皿及其培养方法
CN117660183B (zh) * 2024-01-31 2024-04-16 山东大学 胚胎微流控动态培养皿及其培养方法
WO2025179341A1 (fr) * 2024-02-28 2025-09-04 Cell Tech Holding Pty Ltd Dispositifs et procédés de micromanipulation

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