Classifications

• • 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
  • B01L3/502753—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 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • 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
• • 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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
• • 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
  • B01L2200/02—Adapting objects or devices to another
  • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
  • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
• • 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
• • 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
  • B01L2200/0652—Sorting or classification of particles or molecules
• • 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
• • 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
  • B01L2300/0681—Filter
• • 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
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0848—Specific forms of parts of containers
• • 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
  • B01L2300/0848—Specific forms of parts of containers
  • B01L2300/0851—Bottom walls
• • 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
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/087—Multiple sequential chambers
• • 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
  • B01L2300/16—Surface properties and coatings
  • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
  • B01L2300/163—Biocompatibility
• • 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
  • B01L3/502715—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 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• • 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

Definitions

• the present invention relates to the field of microfluidics.
• Microfluidics implements systems of micrometric dimensions, the size of which is generally between a few tens and a few hundred microns.
• control can concern the quantity of molecules interacting with the cancer cells, the concentration profile of the molecules to which the cancer cells are subjected, the evolution over time of the quantity of these molecules and / or the concentration profile of these molecules applied. to cancer cells, etc.
• microfluidic systems can be used for toxicity tests of certain molecules on living cells and / or cellular tissues.
• the control of the amount of molecules, possibly toxic, administered to the cells and the way in which these molecules are administered is necessary to determine the threshold of toxicity.
• An example of a microfluidic system widely used to stimulate living cells is presented in document US Pat. No. 7,374,906. This microfluidic system makes it possible, in particular, to subject living cells to a concentration gradient of molecules whose profile is linear and stable over time. .
• a major disadvantage of this type of microfluidic system is that living cells are subjected to a flow generating disturbing shear forces. This shearing effect is particularly troublesome when one seeks to study the chemotactic response of the growth cone of nerve cells. Indeed, the flow generates shear stresses that modify the response of the target cells in the best case, or even cause the death or tearing of the cells.
• a microfluidic system for applying a concentration gradient of molecules capable of stimulating living cells is for example presented in the article "Generating steep, shear-free gradients of small molecules for cell culture", Taesung Kim, Mikhail Pinelis and Michel M. Maharbiz, Biomed Microdevices (2009), vol. 11, pp. 65-73.
• This microfluidic system comprises a microfluidic device 10 and means (not shown) for supplying the device with fluids.
• FIG. 1 The microfluidic system disclosed herein is shown in FIG. 1 in an exploded perspective view.
• a base 11 made of polydimethylsiloxane (PDMS) comprising a central zone 12, of substantially square shape, connected to channels 13a, 13b, 13c and 13d arranged in the shape of a cross with respect to the central zone 12. It also comprises a membrane microporous 14 in polyester, covering the central zone 12 of the base 11 in PDMS. It finally comprises a PDMS cover 15, covering both the polyester membrane 14, the PDMS base 11 and the channels 13a, 13b, 13c, 13d (the cover 15 is truncated in FIG. 1).
• PDMS polydimethylsiloxane
• the microfluidic system 10 is thus separated into two parts by the membrane 14 made of polyester.
• a first part forms a channel in which moving fluids can circulate, this channel being closed in its upper part by the membrane 14, the lower face of the membrane thus forming a wall of this channel subjected to a flow.
• a second part is formed by the upper face of the membrane 14, opposite the channel, and on which are located the living cells (CV) in culture.
• the fluid supply means are not shown. It should however be noted that a first fluid is introduced into the base 11 of the microfluidic system 10 via the inlet E1 and a second fluid is introduced into this base 11 of the microfluidic system 10 via the inlet E2, opposite to the E1 input. At least one of these fluids comprises molecules intended to stimulate living cells, passing through the membrane 14 made of polyester.
• the fluids entering through the inlets E1, E2 thus circulate in the base 11 of the microfluidic system 10, are brought into face-to-face contact, which creates a mixing zone at the interface of these two fluids, and then comes out of this base 11 by the outputs S.
• microfluidic system 10 To control the culture of living cells in time as well as in space, it is possible, with the microfluidic system 10, to adjust the flow rates of the fluids to establish a predetermined concentration profile of molecules on the membrane 14 of polyester.
• the regulation of the flow of each of the two fluids makes it possible to create a very particular mixing zone at the interface between the two fluids, that is, to create a very specific concentration profile of molecules intended to stimulate living cells.
• This mixing zone in which a concentration gradient is generated according to a given profile extends substantially along an axis A, shown in FIG. 1, passing through the two outputs S of the base 11.
• the respective flow rates of the fluids from the inputs E1, E2 must be controlled very precisely to achieve a stable molecule concentration profile on the underside of the polyester membrane 14.
• This control of the fluid flow rates is effected upstream of the microfluidic system 10, namely at the level of the fluid supply means themselves, the gradient being generated in the base 11, at the interface between both fluids.
• the concentration profile applied to the living cells substantially corresponds to the profile applied to the lower face of the membrane. This is even more true that the membrane 14 has a low thickness of 10pm.
• the slope of the concentration profile obtained at the living cell level depends on the fluid velocity in the microfluidic channel and the position of the interface between the two fluids from the inputs E1, E2.
• the slope of the profile is very difficult to control.
• the microfluidic system 10 implements a membrane 14 made of polyester, bonded by its underside on the edges of the base 11 in PDMS, of square shape, and glued, by its upper face, to the lid 15 made of PDMS.
• a membrane 14 made of polyester bonded by its underside on the edges of the base 11 in PDMS, of square shape, and glued, by its upper face, to the lid 15 made of PDMS.
• These materials are chosen because they allow in particular a bonding of the membrane 14, the base 11 and the lid 15 together according to a method specified in this document.
• the presence of the cover 15 on the membrane 14 and the channels 13a, 13b, 13c, 13d makes it possible to reinforce the mechanical strength and the seal between the membrane 14 and the base 11, the bonding between the membrane 14 and the base 11 does not occur. effecting only on the edges of the base 11.
• the bonding of the membrane 14 is performed using a prepolymer deposit of the PDMS, which allows an irreversible manufacture of the device by heating under pressure mechanical.
• the microfluidic system To seal between the channel and the membrane 14, the microfluidic system must be closed by the lid. It is then necessary to culture the cells within the microfluidic system 10. This is not very practical for complex cell cultures, such as primary cultures of neurons, explants or tissue slices.
• a PDMS lid 15 does not allow or makes it difficult to visualize the response of living cells to stimulation. This is all the more critical that a PDMS cover must have a certain thickness to allow its handling, this material having a low elastic modulus. A large thickness further decreases the optical qualities of this material. It is therefore very difficult to observe, by suitable optical means, the response of living cells arranged on the membrane.
• An object of the invention is to overcome at least one of these disadvantages.
• the invention proposes a microfluidic system for controlling a concentration profile of molecules capable of stimulating a target, for example formed by a set of living cells, the system comprising:
• a microfluidic device comprising at least one microfluidic channel provided with at least one inlet orifice and at least one outlet orifice for at least one fluid;
• At least one chamber or another microfluidic channel comprising a base intended to receive the target
• At least one microporous membrane separating the chamber or the other microfluidic channel from the microfluidic channel
• microporous membrane being disposed away from the base so that when the supply means provides the microfluidic channel said at least one fluid flowing in a laminar regime in contact with the microporous membrane, the molecules capable of stimulating the target then diffuse, after passing through the microporous membrane, through the chamber or said other microfluidic channel to finally form a stable concentration profile in this chamber or other microfluidic channel.
• the system may provide other technical features, taken alone or in combination:
• the microfluidic channel comprises a cover made of a material chosen from: glass or silicon, a non-elastomeric photocrosslinked polymer, a metal, an electrically conductive or semiconductive alloy, a ceramic, quartz, sapphire, an elastomer;
• said at least one inlet orifice and said at least one outlet orifice for the fluids are formed in the lid;
• the microfluidic channel comprises at least one photocured and / or thermoset resin wall
• the microporous membrane extends transversely on the side wall of the microfluidic channel to close said channel in its lower part; the microfluidic channel is organized in several levels, each level comprising at least one inlet for at least one fluid;
• the base of the chamber or of said other microfluidic channel is made of an optically transparent material
• the chamber or said other microfluidic channel comprises side walls made of photocured and / or thermoset resin
• the microporous membrane extends transversely between the side walls of the chamber or of said other microfluidic channel to close said chamber or said other microfluidic channel in its upper part;
• the microporous membrane is made of a material chosen from: glass, polycarbonate, polyester, polyethylene terephthalate, quartz, silicon, silica or silicon carbide;
• the microporous membrane comprises pores whose density is between 10 3 and 10 10 pores / cm 2 ;
• the pores have a hydraulic diameter of between ⁇ , ⁇ and 12 ⁇ , preferably between ⁇ , ⁇ and 3 ⁇ :
• optical display means it comprises an optical display means
• the optical means uses a photoactivation localization microscopy technique or a stimulated emission depletion microscopy technique.
• FIG. 2 is a diagram of a microfluidic device according to the invention, in a partly cut perspective view;
• FIGS. 3 (a) to 3 (d) represent, as the case may be, steps of a process for manufacturing the microfluidic device represented in FIG. 3 or intermediate structures obtained at the end of certain steps of this process;
• FIGS. 4 (a) to 4 (c) show intermediate structures obtained during the manufacture of an assembly formed by a base and side walls of the device, said assembly being intended to form a part of the microfluidic device of FIG. 2;
• FIG. 5 (a) represents a microfluidic channel of the microfluidic device according to the invention
• FIG. 5 (b) represents fluids flowing in this first channel according to the invention
• FIG. 5 (c) represents a profile. concentration obtained in a chamber of the device according to the invention
• FIGS. 6 (a) to 6 (c) all represent the microfluidic device according to the invention for cutting, for which it is possible to observe successively different stages of the stabilization, over time, of a concentration profile of molecules. for stimulating living cells in the chamber of the device;
• FIG. 7 (a) represents another microfluidic channel of the microfluidic device according to the invention
• FIG. 7 (b) represents fluids flowing in this first channel
• FIG. 7 (c) represents a concentration profile obtained. in a chamber of the device according to the invention
• FIG. 8 represents the evolution over time for establishing, by diffusion, a steady state of molecules intended to stimulate living cells, and this for different solutions;
• FIG. 9 is a diagram, in a sectional view, of an alternative embodiment of the microfluidic device according to the invention, for generating more complex concentration profiles with the microfluidic device shown schematically in Figure 2;
• FIG. 10 represents a spatially periodic concentration profile that can be obtained in the chamber of the microfluidic device according to FIG. 9;
• FIGS. 11 (a) to 11 (c) show several intermediate structures obtained during a manufacturing process of the microfluidic device shown in FIG. 9.
• the invention relates to a microfluidic system for controlling a concentration profile of molecules capable of stimulating a target, for example formed by a set of living cells, the system comprising a microfluidic device and at least one means for feeding this device with less a fluid comprising molecules capable of stimulating this target.
• microfluidic device is described in support of FIG. 2 and a method of manufacturing this device is described in support of FIGS. 3 (a) to 3 (d) and 4 (a) to 4 (c).
• FIGS. 3 (a) to 3 (d) and 4 (a) to 4 (c) will then describe, as non-limiting examples, particular forms of microfluidic channel that can be used within this device, in support of Figures 5 (a) and 6 (a).
• FIG 2 there is shown a microfluidic device 1 according to the invention, in a partially cut perspective view.
• This microfluidic device 1 comprises a lid 2, advantageously rigid, provided with two orifices 21, 22, a side wall 3 advantageously made of photocured and / or thermoset resin,
• the side wall 3 of the device 1 is made of a single layer of photocured resin and / or thermoset.
• the microfluidic device 1 also comprises, in its lower part, an opening 47 covered by a microporous membrane 5 extending transversely to the base of the side wall 3.
• the side wall 3, the lid 2 and the microporous membrane 5 make it possible to define a microfluidic channel 4 whose inlet and outlet are constituted by said orifices 21, 22.
• the microporous membrane 5 prevents the fluid intended to flow in the microfluidic channel 4 from passing on the other side of this membrane, the latter nevertheless allowing to spread the molecules capable of stimulating the target transported by the fluid in the microfluidic channel 4.
• the microfluidic device 1 also comprises a base 6, advantageously rigid and transparent, and sidewalls 7a, 7b, advantageously made of photocured resin and / or thermoset. These sidewalls 7a, 7b, the base 6 and the microporous membrane form a chamber 8, constituting a culture chamber for the target cells. To form the chamber 8, four side walls are provided, these walls can actually be assimilated to a single contour, because the manufacturing method advantageously performs these walls in one piece.
• the bottom of the chamber 8 is formed by the upper face 61 of the base 6, which is intended to receive the target, for example formed of living cells.
• the living cells are therefore not intended to be placed on the microporous membrane 5, but away from it, on the base 6 of the chamber 8. They can thus be cultured under standard conditions, separately from the microfluidic device. 4.
• the microporous membrane 5 thus separates the device into two distinct microfluidic channels 4, 8.
• the microfluidic channel 4 makes it possible to circulate a fluid comprising molecules capable of stimulating the target. This is done, as will be explained in more detail in the following description, by diffusion through the microporous membrane 5 to the chamber 8, then by diffusion through the chamber 8 (culture chamber) at the bottom of which find, for example, living cells (CV) that we try to stimulate.
• CV living cells
• the base 6 is made of an optically transparent material, for example glass. This is interesting because it is then possible to have an optical display means outside the device to visualize, for example, the response to a stimulation of the living cells arranged at the bottom of the chamber 8.
• the cover 2 may be made of a material chosen from: glass or silicon, a non-elastomeric photocrosslinked polymer, a metal, an electrically conductive or semi-conductive alloy, a ceramic, quartz, sapphire, an elastomer.
• the pore size of the microporous membrane 5 is chosen to avoid any fluid passage between the first mircrofluidic channel 4 and the chamber 8. If the pores are cylindrical, this dimension is comparable to the pore diameter. More generally, the size of a pore can be likened to the hydraulic diameter thereof.
• the microporous membrane 5 can not actually be completely sealed to a fluid passage. Also, it can be considered that the cells located in the bottom of the chamber 8 are subjected to no flow if the fluid flow rate through the microporous membrane 5 is less than a limit value.
• this limit speed is of the order ⁇ / s.
• the shear stresses applied to the cells are negligible, even for a chamber 8 having a low height h ', for example 20 ⁇ ).
• the speed in the microfluidic channel 4 will generally be between ⁇ / s and ⁇ / s.
• the hydraulic resistance Rh.membrane of the microporous membrane 5 must be, according to the speed of the fluid in the channel 4, from 100 to 1000 times greater than the hydraulic resistance Rh, channel of the microfluidic channel 4.
• ⁇ must be less than 10 "9 m to respect the relation (R3).
• the pore surface density p should be less than 10 6 pores / cm 2 .
• R3 can of course be generalized as a function of the considered value of the limit velocity of the fluid passing through the microporous membrane 5, on the one hand, and of the flow velocity of this fluid in the microfluidic channel 4d. 'somewhere else.
• the microporous membrane 5 may have pores whose hydraulic diameter is between 0.05 ⁇ ⁇ 12 ⁇ . In particular, if the pore is cylindrical then, the hydraulic diameter of the pore corresponds to its diameter.
• this hydraulic diameter will however be between 0.05 ⁇ and 3 ⁇ . Indeed, it should be noted that the use of a membrane with pores whose hydraulic diameter is less than 3 ⁇ will avoid any passage of flow in the chamber 8, for most conditions of use likely to be encountered.
• the pores may therefore have hydraulic diameters advantageously between 0.2 ⁇ and 3 ⁇ .
• there is theoretically no limit lower for the hydraulic diameter of the pores which is why it is possible to implement pores whose hydraulic diameter reaches ⁇ , ⁇ .
• microfluidic device is however more delicate (for example in the choice of flow rates in the microfluidic channel 4) to ensure that the fluid does not cross the microporous membrane. 5.
• the pore density can be between 10 3 and 10 10 pores / cm 2 .
• the height of the pores can be between 50 nm and 100 ⁇ m.
• microporous membrane 5 can be made in various materials such as glass, quartz, silicon, silica or silicon carbide or polymers of the same nature as the polymers that can be used for the rest of the microfiuidic device. It is thus possible to use polycarbonate, polyester or polyethylene terephthalate.
• a microporous membrane 5 made of polycarbonate the hydraulic pore diameter of which is between 0.2 ⁇ and 1 ⁇ , for example of the cyclopore type from Whatman (Whatman Cyclopore TM).
• a microporous membrane 5 of polyester whose hydraulic diameter of the pores is between 0,4 ⁇ and 3 ⁇ , e.g., Transwell type Corning (Corning ® Transwell ®).
• a microporous membrane 5 of polyethylene terephthalate the hydraulic pore diameter of which is between 0.4 ⁇ and ⁇ , for example of the "Track-Etched" type from Becton Dickinson.
• microporous membranes have the advantage of being compatible with a method of manufacturing the microfiuidic device 1, which is described hereinafter with reference to FIGS. 3 (a) to 3 (d). They present also the advantage of being biocompatible and functionalized to be specifically permeable to various molecules. Functionalizable means that the microporous membrane 5 can be chemically modified to fulfill a particular function (retention of certain species, chemical reactions, etc.).
• the device may have the following dimensions.
• the height h of the microfluidic channel can be between 1 ⁇ and ⁇ ⁇ , advantageously between 10 ⁇ and 200 ⁇ . Its width (not shown) can be between 10 ⁇ and 2mm.
• the height h 'of the chamber 8 may be 10 ⁇ and ⁇ , advantageously between 50 ⁇ and 20 ⁇ .
• the distance between the entrance E and the exit S is a few centimeters.
• An exemplary method of manufacturing the microfluidic device 1 according to the invention is a method which comprises at least the steps of:
• step (a) This process is based on the method disclosed in WO 2008/009803.
• the operation performed in step (a) is shown in Fig. 3 (a).
• the stamp T used in step (a) may be made of elastomeric material such as PDMS. It comprises a profile used as a mold complementary to that of the microfluidic device 1 that is to be produced.
• the stamp T thus comprises a protuberance Ta corresponding to the channel 4 of the microfluidic device 1 that it is desired to obtain. It also comprises a hollow zone Tb surrounding the protrusion Ta, zone in which said first side wall 3 of the microfluidic device 1 is intended to be formed.
• the support 2 'can also be made of PDMS and has a planar profile.
• the microporous membrane 5 is previously arranged on the support 2 ', then the stamp T is pressed against the support 2'. The stamp T wedges the membrane 5 against the support 2 'via the protuberance a.
• the photocurable and / or photopolymerizable resin in liquid form RL fills the volume located between the patch T and the support 2 'in an appropriate quantity, in particular in the hollow zone Tb of the patch T. This filling does not modify the position of the membrane microporous 5 because it is wedged between the stamp T and the support 2 '.
• the photocurable and / or photopolymerizable resin is a solution or a dispersion based on monomers and / or prepolymers. Photocure and / or photopolymerizable resins commonly used as adhesives, adhesives or surface coatings are used in the process of the invention.
• adhesives, adhesives or surface coatings usually used in the optical field are chosen.
• Such resins when irradiated and photocrosslinked and / or photopolymerized, become solid.
• the solid thus formed is transparent, free of bubbles or any other irregularity.
• Such resins are generally based on monomers / comonomers / pre-polymers of the epoxy, epoxysilane, acrylate, methacrylate, acrylic acid or methacrylic acid type, but there may also be mentioned thiolene, polyurethane and urethane-acrylate resins.
• the resins can be replaced by photocurable aqueous gels such as polyacrylamide gels and are chosen to be liquid at room temperature.
• the resins can also be replaced by polydimethylsiloxane (PDMS).
• PDMS polydimethylsiloxane
• the polymerization and / or the crosslinking of these resins is carried out by photoactivation using any appropriate means, such as irradiation with visible UV radiation, I.R.
• a resin is preferably chosen which, once polymerized and / or crosslinked, is rigid and non-flexible, since the elastomeric resins tend to deform when circulating pressurized fluids in the microfluidic device 1.
• the use of elastomeric photocurable resins is not excluded.
• step (b) After filling with the liquid resin RL of the volume located between the stamp 1 'and the support 2', then a pressure P is applied to the stamp V to expel any excess resin.
• a pressure P is applied to the stamp V to expel any excess resin.
• the projections and in particular the protuberance of the stamp 1 'of elastomer are in contact with the support 2'.
• the liquid resin takes the form of the hollow zones of the V patch.
• step (b) the irradiation of the resin is made in the axis perpendicular to the base of the device, through the stamp 1 '.
• the irradiation must be dosed so, if desired, to leave on the surface of said first side wall 3 resin, active polymerization sites and / or crosslinking.
• the stamp 1 ' is removed from the device.
• the first side wall 3 made of photopolymerized and / or photocrosslinked resin has a profile complementary to that of the hollow zones of the stamp 1 '.
• the profile of the stamp V is adapted so that the photopolymerized and / or photocrosslinked resin defines other patterns. This is particularly the case for the microfluidic device 100 according to the invention which will be described later in support of FIG. 9.
• the printing with a stamp 1 'elastomer in a resin in the liquid state allows to obtain structures of very small sizes with a very good resolution.
• step (c) the cover 2 having at least two orifices 21, 22 on the device is fixed on the side of said first lateral wall 3 previously in contact with the stamp 1 '.
• the support 2 ' can then be removed.
• the structure obtained at the end of step (c) is represented in FIG. 3 (c).
• the removal of the support 2 ' is carried out without the microporous membrane peeling off the photopolymerized resin and / or photo-crosslinked, and without it being torn off or partially torn.
• the cover 2 may be made of glass, silicon, a solid polymer film, a metal, a conductive or semiconductor alloy, a ceramic, quartz, sapphire, an elastomer.
• a glass slide, a polymer film or a silicon slide is chosen.
• the materials used to form the cover 2 are chosen according to the application that will be made of the microfluidic device 1.
• a cover 2 of optically transparent material such as glass
• optically transparent material such as glass
• Another asset of the glass is its very good thermal conductivity which allows a homogeneous heating of the devices.
• the arrangement of the microporous membrane 5 at the bottom of the microfluidic channel 4 makes its use compatible with standard living cell culture protocols. Indeed, it is then conceivable that the base 6 is a glass slide on which a culture of living cells is carried out, this blade then being fixed on the structure obtained at the end of step (c) to form the room 8 (culture room), as explained in the following description.
• the assembly comprising a base 6 and two second lateral walls 7a, 7b can be made from the following process steps:
• step (e- ⁇ ) to ( ⁇ ) The structure obtained at the end of steps (e- ⁇ ) to ( ⁇ ) is represented in FIG. 4 (a), in the case where step ( ⁇ 3) consists of a photo-irradiation of the liquid resin.
• the mold 3 ' such as the stamp 1' and the support 2 ', can be made of an elastomer such as PDMS.
• the photocurable and / or thermosetting liquid resin used to form the walls 7a, 7b may be chosen from the possibilities already described for the liquid resin used in step (a).
• the liquid resins used for steps (a) and (e- ⁇ ) to ( ⁇ 3) are the same.
• photocurable aqueous gels such as those previously described or polydimethylsiloxane (PDMS) could be used.
• the base 6 can be chosen from the materials used for the lid.
• an optically transparent material may be used to facilitate optical visualization by a dedicated device.
• This optically transparent material may in particular be glass, the base 6 thus forming a glass cover usually used for the culture of living cells (CV).
• CV living cells
• the use of glass also makes it possible to take advantage of existing chemical and biological surface treatments for this substrate.
• the mask 4 ' may have orifices 4'a, 4'b for photo-irradiating specific areas of the liquid resin to form said second sidewalls 7a, 7b of the microfluidic device.
• step ( ⁇ ) Once the step ( ⁇ ) is complete, it remains only to remove the mask 4 'and the mold 3' during a step (e 4 ) to leave only the assembly formed by said second side walls 7a , 7b and the base 6. This assembly is shown in Figure 4 (b).
• a step (e 5 ) is then carried out, the latter consisting in rinsing said assembly, for example by an ethanol / acetone mixture in 90/10 volume proportions. This rinsing makes it possible to remove all the non-photo-irradiated or unheated resin that can remain on the base 6.
• this assembly can be strongly photo-irradiated, for example by UV, then perform an energetic rinsing in a neutral solution, such as water for several hours.
• a living cell culture can then be performed on the upper face 61 of the base 6, as shown in Figure 4 (c). This culture is carried out under standard conditions. In particular, this culture can be carried out on a base 6 in the form of a conventional glass slide.
• step (d) can be performed.
• step (d) The operation performed in step (d) is shown in Fig. 3 (d).
• the microfluidic device 1 is ready for use. It comprises in particular living cells on the upper face 61 of the base 6, which is opposite to the microporous membrane 5 within the chamber 8 (culture chamber).
• this microfluidic device To use this microfluidic device 1, it is associated with a fluid supply means (not shown) also belonging to the microfluidic system according to the invention.
• This feeding means makes it possible to feed the microfluidic channel with at least one fluid comprising molecules capable of stimulating living cells.
• This supply means may for example be formed of a set of fluid reservoirs connected to the microfluidic channel 4 by capillaries. This means then makes it possible to carry out dilutions and / or mixtures between the different fluids coming from the different reservoirs, before entering the microfluidic channel.
• microfluidic channel 4 may have a particular shape.
• FIG. 5 (a) An example of a microfluidic channel 4 that can be used is shown schematically in FIG. 5 (a), in a perspective view.
• the fluids F 1 , F 2 differ only in the presence, in one of the two fluids and in low concentration, of stimulation molecules for the target cells.
• the fluids are fed into two branches 42, 43 which join into a common branch 44 having an interface 41 on which the microporous membrane 5 of the microfluidic device 1 is intended to be arranged.
• the supply means provides two fluid sources, for the fluids Fi, F 2 respectively.
• microfluidic channel 4 These fluids Fi, F2 are introduced into the microfluidic channel 4 through the inlets 420, 430. Moreover, the end of the common branch 44 has a common outlet 45 for the two fluids Fi, F 2 .
• the general shape of the microfluidic channel 4 is a Y shape.
• the inputs 420, 430 are to be brought closer to the inlet orifice 21 of FIG. 2 and that the outlet 45 is to be brought closer to the outlet orifice 22 of FIG. 2.
• the microfluidic channel 4 is designed to circulate a single fluid, entering through a single orifice 21 and out through a single orifice 22.
• FIG. 5 (b) schematizes the flow of fluids F 1, F 2 in the various branches of the microfluidic channel 4.
• the fluids F 1, F 2 flow in a laminar flow, one next to the other. Since the flow is laminar, the fluids do not mix hydrodynamically.
• the flow is considered laminar if the Reynolds number of the flow in this common branch 504 is smaller than the critical Reynolds number, which can easily be determined using fluid mechanics manuals.
• the hydraulic diameter depends on the geometry of the common branch 44.
• the flow is laminar for the fluids and flow velocities of these fluids usually used for the applications targeted by the invention.
• the transport of these molecules is carried out first by diffusion through the microporous membrane 5, then by diffusion through the chamber 8, to finally reach the upper face 61 of the base 6 of the chamber 8, face 61 on which living cells are located.
• the concentration profile takes some time to stabilize in the chamber.
• the stabilization time t sta b is of the order of h ' 2 / D where h' is the height of the chamber 8 and D the diffusion coefficient of the molecules intended to stimulate the target cells in the chamber 8. It should be noted that to avoid too high stabilization times, the height of the chamber will generally be limited to 500 ⁇ .
• Figures 6 (a) to 6 (c) show several steps in the scattering phenomenon, considering a "co-flow" type fluid supply.
• the fluid F 2 comprises stimulation molecules for the living cells intended to be placed on the base 61 of the chamber 8.
• the fluid F1 is neutral.
• the power supply fluid arrives at the level of the microporous membrane 5 and the molecules begin to diffuse into the chamber 8.
• it is in a transient state where the concentration profile is in the stabilization phase.
• Figure 6 (c) the concentration profile is stabilized.
• the diffusion is carried out mainly according to the height of the chamber 8 (culture chamber), that is to say a direction which is substantially perpendicular to the flow direction of the fluids Fi, F 2 in the channel microfluidic 4, even if this diffusion in the chamber 8 is also carried out in the other directions,
• FIG. 5 (c) shows, in sectional view, what happens at the interface 41 with the microfluidic device 1.
• the fluorescent solution F2 comprises molecules having a diffusion coefficient similar to that of the molecules usually used for the stimulation of living cells.
• the fluorescent solution used may be fluorescein isothiocyanate, include a fluorescent protein called GFPuv for "Green Fluorescent Protein” according to the English terminology or include a protein associated with fluorescent molecules dextran-70MW-rhodamine. This is the dextran-70 protein coupled to fluorescent molecules of rhodamine-B.
• the concentration profile of the fluorescent solution shown in FIG. 5 (c) is square (as in FIG. 6 (c) moreover ). Indeed, this fluorescent solution occupies only part of the microfluidic channel 4, the other part of said channel 4 being occupied by the neutral solution.
• the device microfluidic material 1 was then observed by optical means 18 belonging to the microfluidic system according to the invention.
• the concentration profile obtained on the upper face 61 of the base 6 is in the form of a curve representative of an "erf" type function. This form is obtained by the diffusion of the neutral and fluorescent solutions through the microporous membrane 5, then through the chamber 8.
• microfluidic channel 4 Another form of microfluidic channel 4 'is shown in Figs. 7 (a) through 7 (c).
• the fluids Fi, F 2 are fed into two branches 42 ', 43' which both end in the same pipe 44 'having three outlets leading to three coils (not referenced).
• the first fluid F 1 passes through a first outlet and goes to a first coil
• the second fluid F 2 passes through a second outlet and goes to a second coil
• a mixture of the two fluids F 1 and F 2 finally passing through a central outlet of the pipeline 44 'and goes to a central coil.
• the fluids meet in a common branch 45 'having an interface 41' with the microporous membrane 5 of the microfluidic device 1 according to the invention.
• the end 46 'of the common branch 45' has an outlet for the fluids F 1 , F 2 .
• Figure 7 (b) schematizes the flow of fluids Fi, F 2 in the various branches of the microfluidic channel 4 '.
• the various fluids flow into the microfluidic channel 4 in a laminar regime.
• FIG. 7 (c) shows, in a sectional view, the behavior of the fluids at the interface 41 '.
• the experimental device used for this purpose is similar to that previously presented for the microfluidic channel 4 of the "co-flow" type.
• FIG. 7 (c) shows in particular that the concentration profile in the microfluidic channel 4 'is in the form of a staircase. It also shows that the concentration profile obtained on the upper face 61 of the base 6 (optically transparent) has a linear central zone, which can be used to stimulate certain target cells,
• microfluidic channel 4 'of the "tri-flow" type it is possible to apply a linear concentration profile in the center of the lid to living cells that are to be stimulated.
• microfluidic system according to the invention is not limited to the use of first microfluidic channels 4, 4 'designed according to the only types "co-flow” or "tri-flow” described above. Also, other types of first microfluidic channels can be envisaged according to the concentration profile that it is desired to apply to living cells.
• microfluidic system thus allows a much easier control of a concentration profile of molecules capable of stimulating a target, such as living cells.
• the design of the microfluidic system according to the invention also has a dynamic behavior making it possible to rapidly perform numerous tests.
• the microporous membrane 5 is a Cyclopore Whatman membrane with pores of 400 nm.
• a first fluid F1 neutral solution
• a second fluid F 2 was circulated in this same channel 4, the fluid F 2 being in this case formed by a fluorescent solution comprising molecules having a diffusion coefficient comparable to the molecules usually used to stimulate living cells.
• FIG. 8 represents the evolution of the normalized intensity of the fluorescent solution measured by an optical means such as a confocal microscope located behind the base 6, as a function of time. The measurement of the microscope takes place at the base 6, which is in this case a glass slide.
• the time of establishment of the concentration profile at this glass slide is between a few tens of seconds and a few minutes, depending on the nature of the fluorescent solution. Logically, the larger the molecules in this solution, the longer the setup time. In general, it is observed that the establishment times are relatively low, comparable to those of a diffusion over a distance of the order of the height h 'of the chamber 8 (chamber of culture) and make it possible to implement an experience quickly, with a stable concentration profile.
• the tests can be done quickly, in a time typically between 1 h and 2 h. Then, we can move on to another test, with another culture.
• the microfluidic system will comprise an optical device for viewing the culture chamber 8 through the base 6.
• the base is advantageously made of an optically transparent material. It is thus easier to follow the response of living cells arranged on this base 6 to a stimulation of certain molecules.
• FIG. 9 there is shown an alternative embodiment of the microfluidic device according to the invention, in a partial sectional view. Indeed, FIG. 9 only shows the upper part of the microfluidic device, an assembly comprising a base (capable, for example, of receiving a culture of living cells) and sidewalls capable of forming a chamber under the microporous membrane, being necessary for that the microfluidic device 100 can be used.
• a base capable, for example, of receiving a culture of living cells
• sidewalls capable of forming a chamber under the microporous membrane
• the microfluidic device 100 has characteristics similar to the microfluidic device 1 described in support of FIG. 2 and can, as such, be used with a microfluidic channel 40 of the "coflow" or "triflow” type.
• the microfluidic channel 40 may be powered by a fluid supply means such as that described above.
• the microfluidic channel 40 is organized in several levels, in this case two levels 40 1 , 40 2 in the example shown in this FIG. 9.
• Each level comprises a fluid inlet corresponding to an orifice 201, 202 formed in the cover 20, the fluid outlet 203 being common.
• An advantage of implementing a microfluidic device comprising a multilevel microfluidic channel is to allow the application of more complex profiles of concentration of molecules capable of stimulating living cells. For example, it is conceivable to implement in the chamber 8 concave, convex or periodic concentration profiles, while maintaining a limited number of inputs and outputs for the fluids.
• FIG. 10 also shows the result of a simulation obtained with a microfluidic device comprising a channel with two levels, making it possible to obtain a spatially periodic concentration profile.
• On the abscissa is represented the width of the chamber 8 and on the ordinate, the normalized concentration of stimulation molecules.
• the different solid curves represent the evolution over time of the simulated concentration profile until stabilization.
• the dashed line corresponds to the experimental data obtained with fluorescein flow.
• first channel 40 could provide more than two levels, depending on the complexity of the concentration profile that it is desired to apply.
• Steps (a) and (b) are thus implemented to achieve the structure 200 shown in Fig. 11 (a).
• the support used during these steps is referenced 20 'and a side wall 30 "closed at its base by the microporous membrane 5.
• the structure 200 'shown in Figure 1 1 (b) is then implemented by implementing steps similar to the steps (a) to (c) mentioned above.
• This fixation can be effected by photo-irradiation or heating, so that the side walls 30 "and 30"'are fixed to one another to form the side wall 30'.
• This step makes it possible to form the second 4U 2 of the microfluidic channel 40.
• step (d) a step similar to step (d) is used for an assembly comprising two second side walls of photocured and / or thermoset resin in order to form the chamber under the microporous membrane.
• a method incorporating the steps (e- ⁇ ) to (e 3 ) previously described in support of Figures 4 (a) to 4 (c) is implemented for this purpose.
• the devices 1, 100 described above comprise a chamber 8 (by nature closed when glued with the microfluidic channel 4, 40), in which the living cells are located.
• This chamber 8 could include a culture gel, although advantageously this will not be the case.
• this chamber can be replaced by a microfluidic channel, thus comprising openings advantageously arranged laterally.
• the microfluidic device will then comprise the microfluidic channel 4, 40 in which the fluid flows and another microfluidic channel in which the living cells are located.
• microfluidic system according to the invention are therefore multiple.
• a concentration gradient of molecules intended to stimulate living cells having the desired shape (the examples cited above show a function-like gradient "erf", or a gradient of linear form in the central portion of the chamber 8 of the device 1, 100) at these living cells.
• the concentration profile is established in the chamber 8, the living cells being disposed on a base of the device located away from the microporous membrane 5, and more precisely, opposite this membrane 5 within the chamber. 8 (this chamber can be replaced by a microfluidic channel).
• the fluid supply means is then simple and provides only fluids whose flow can vary slightly without the concentration profile applied to the living cells substantially changes.
• the control of the concentration profile at the living cell level is therefore easier, and less sensitive to any disturbances outside the microfluidic system.
• the method of manufacturing the microfluidic device according to the invention also makes it possible to use a base of optically transparent material, for example a glass slide on which a cell culture can be carried out according to a standard method.
• a base of optically transparent material for example a glass slide on which a cell culture can be carried out according to a standard method.
• the observation of the behavior of living cells (growth, etc.) can thus be easily performed with an optical display means located behind the glass slide.
• Optical observation can be performed at high spatial resolution because the base of optically transparent material can be very thin.
• high resolution or even super-resolution fluorescence microscopy obtained using techniques such as photoactivated localization microscopy (PALM) can be carried out using techniques such as photoactivation localization microscopy (PALM). or stimulated emission depletion microscopy (STED for "STimulated-Emission-Depletion"), using a base formed with a glass slide 150 ⁇ thick.
• PAM photoactivated localization microscopy
• PAM photoactivation localization microscopy
• STED stimulated emission depletion microscopy
• this means of visualization makes it possible to know the concentration profile of the stimulating molecules applied to living cells. It is therefore much easier to experiment with correlations between the observed behavior of living cells and the concentration profile applied to them.
• the invention finds particular application in the field of biology, for the culture, observation and study of living cells.
• the microfluidic system can also be used for the production of biochips.
• the advantages of the invention may be of interest for other fields of application, for example to determine thresholds of toxicity of certain molecules in cosmetology.

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