EP3206791B1 - Verfahren zur handhabung von mikrotropfen mit proben - Google Patents

Verfahren zur handhabung von mikrotropfen mit proben Download PDF

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Publication number
EP3206791B1
EP3206791B1 EP14796828.3A EP14796828A EP3206791B1 EP 3206791 B1 EP3206791 B1 EP 3206791B1 EP 14796828 A EP14796828 A EP 14796828A EP 3206791 B1 EP3206791 B1 EP 3206791B1
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EP
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Prior art keywords
microdroplets
trapping
cells
oil
microdrops
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French (fr)
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EP3206791A1 (de
Inventor
Charles Baroud
Gabriel AMSELEM
Sébastien SART
Raphaël TOMASI
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Centre National de la Recherche Scientifique CNRS
Ecole Polytechnique
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Centre National de la Recherche Scientifique CNRS
Ecole Polytechnique
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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
    • B01L3/502769—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 multiphase flow arrangements
    • B01L3/502784—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 multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • 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
    • B01L3/502746—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 the means for controlling flow resistance, e.g. flow controllers, baffles
    • 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
    • B01L2200/0642—Filling fluids into wells by specific techniques
    • 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
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • 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
    • 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
    • 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
    • B01L2400/088—Passive control of flow resistance by specific surface properties

Definitions

  • the present invention relates to a microfluidic method for handling samples, in particular biological samples, in hydrogel microdrops.
  • the invention also relates to a device for implementing such a method and to a sample product obtained by implementing such a method.
  • microfluidics for high-throughput biological assays
  • Lab. Chip. 12 2012
  • drops in microfluidic systems can be used to contain chemical or biological reactions.
  • the content of these drops can be tested by observing the fluorescence of the drop as it passes a focused laser.
  • these systems do not make it possible to observe the evolution over time of the contents of these drops without extracting them from the microfluidic device.
  • hydrogel microbeads including cells are made in a first microfluidic system. They are then collected and washed in a bath, before being injected into a second microfluidic system comprising traps making it possible to fix the microdrops.
  • Such a method is however complex, which requires two separate microfluidic systems and three devices in total. Furthermore, it does not allow the samples to be observed continuously. In particular, it does not make it possible to observe the initial moments between the formation of the drops and their capture.
  • the invention provides a method of manipulation in a microfluidic system of microdrops including samples according to claim 1 or claim 2.
  • the microdrops which contain samples of interest - these microdrops are first of all trapped in surface tension traps (or capillary), then some of the microdrops or some of the oil around them are gelled.
  • the gelation of the microdrops or the oil that surrounds them facilitates sorting by increasing the trapping force of the microdrops in the traps. In other words, the gelation step makes it possible to prevent the microdrops of interest from being lost.
  • this gelation makes it possible to prevent microdrops from being able to merge, which would cause the samples of these microdrops to mix.
  • surface tension trap means a trap a zone of the microfluidic system whose geometry, with the interfacial tension of the microdrop, allows the microdrop to be held in position.
  • microfluidic system is understood to mean a system whose parts are manufactured using microfabrication processes. Such a system has conduits, at least one dimension of which is typically less than a millimeter.
  • the shape of the microdrop can be controlled. This control of the shape of the microdrop can be combined with the control of the instant of gelation of the microdrop or of a part of the oil surrounding it, to provide access to different applications, in particular on the manipulation of cells. .
  • cells By cells, is meant eukaryotic cells (for example plant cells, fungi, yeasts, mammalian cells) and prokaryotic cells (for example bacteria).
  • eukaryotic cells for example plant cells, fungi, yeasts, mammalian cells
  • prokaryotic cells for example bacteria.
  • anchor-independent cells e.g. certain blood line cells and highly transformed tumor cells
  • anchor-dependent cells the majority of other cell types
  • spheroids is understood to mean multicellular structures organized in the form of micro-tissues the functionalities of which are similar to those of tissues derived from organs.
  • the invention relates to a device according to claim 15 or claim 16.
  • the gelation means may comprise a device for injecting a chemical agent into the trapping zone.
  • the device may further comprise means for thawing at least some of the gelled hydrogel microdrops or part of the gelled oil, if applicable.
  • the invention also relates to a product of gelled microdrops according to claim 17.
  • This product includes a microdrop trapping zone, in particular a microfluidic chip, and gelled microdrops each including a sample, trapped in the trapping zone, the gelled microdrops being cryo-preserved.
  • the biochemical solution may contain cryo-protection agents (DMSO, glycerol, trehalose etc.) to allow the cryo-preservation of the samples.
  • cryo-protection agents DMSO, glycerol, trehalose etc.
  • the gelled microdrops can also be bathed in a fluid, preferably in an aqueous solution or in an oil, the fluid and the microdrops preferably being cryo-preserved.
  • the invention also relates to a microdrop product according to claim 18.
  • This product comprises a trapping zone, in particular a microfluidic chip, and microdrops each including a sample, trapped in the trapping zone, the microdrops bathed in a gelled oil, the microdrops and the gelled oil preferably being cryo-preserved.
  • the samples can be mammalian cells, preferably mammalian cells excluding human cells, bacteria, yeasts or other cells used in bioprocesses, molecules, beads trapping particles. molecules on the surface.
  • the invention relates to a method of handling hydrogel microdrops including test samples.
  • the aqueous solution is a hydrogel solution
  • the oil not comprising a gelling agent and where the last step above consists in gelling at least part of the microdrops. trapped, without the oil is not gelled.
  • the method can be continued by implementing different steps, depending on the test that one wishes to implement, in particular.
  • the method can in particular be continued with a step consisting in replacing the oil around the gelled microdrops, with an aqueous solution, without displacing the microdrops from the surface tension traps.
  • the aqueous solution may contain a biochemical solution with at least one of nutrients, growth factors, antibodies, drug molecules, and pH and / or salinity buffers.
  • the method allows the control of the three-dimensional shape of hydrogel beads in a microfluidic channel and / or in surface tension traps, with the primary application of the encapsulation of cells in these microdrops.
  • the encapsulation of cells in hydrogel allows their culture or analysis, while perfusing them with biochemical solutions, or by applying physical stimuli such as heat or light, for example.
  • gel is understood to mean a medium composed of a majority of liquid and containing molecules or particles which can be organized to give it a solid appearance, such as, for example, the absence of flow in its stable state.
  • This solution can be handled in a liquid state and can then be "gelled” by chemical or physical means. Gelation can be reversible in some cases. When the liquid is water, we talk about hydrogel.
  • the proposed microfluidic process comprises a first step of forming hydrogel microdrops containing biological cells in an oil.
  • the microdrops (or microspheres) have a diameter of the order of one micrometer, in particular a diameter of between 10 and 1000 micrometers.
  • the hydrogel is for example an aqueous solution comprising a gelling agent.
  • the gelling agent is chosen by the user depending on the application.
  • An example of a gelling agent which can be gelled in a physical manner is agarose, which is liquid at room temperature and which gels at low temperature.
  • a gelling agent which can be chemically gelled is, for example, alginate, liquid in solution and which gels when calcium ions Ca 2+ are supplied.
  • the biochemical and biomechanical properties of the hydrogel can allow cells sensitive to the anchorage to establish specific interactions with the matrix thus formed. These interactions are essential for the survival of anchor-dependent mammalian cells and participate in the regulation of their phenotype.
  • the nature of the matrix may, for example, make it possible to observe cell migration or proteolysis (digestion of the matrix by the cells).
  • Particularly conclusive experiments have been carried out with agarose, alginate, PEG-DA (Polyethylene glycol Diacrylate), but also gelatin, type I collagen or Matrigel ®.
  • hydrogels containing various proteins, glycoaminoglycans and other components of the extracellular matrix e.g.
  • type I collagen, gelatin or Matrigel® have been shown to be able to maintain viability, support proliferation and the ability to migration, as well as to maintain the phenotype of certain populations of anchorage dependent cells. It should be noted here that the gels can be combined, by adding successive microdrops, for example. Each of the hydrogels mentioned has a specific gelation procedure.
  • hydrogels such as PEG-DA
  • PEG-DA can also be functionalized to allow the survival and / or development of cells, by incorporating peptidomimetics (for example hydrogels can be functionalized with RGD-type consensus sequences on which certain types of cells mammals can establish specific interactions or even PRCG [V / N] PD or HEXGHXXGXXH consensus sequences specific to metalloproteases) or the sensor of specific molecules via the incorporation of antibodies or aptamers, for example the in situ capture of cytokines secreted by encapsulated lymphocytes.
  • the mechanical properties of these hydrogels can also be modulated for different applications, for example by varying their degree of crosslinking and / or their concentration.
  • the cells are mixed with the hydrogel, a priori before the formation of the microdrops.
  • the mixing of the hydrogel and the cells can be carried out directly in the microfluidic device, before the formation of the microdrops.
  • the microdrops are conveyed from the zone where they were formed to the trapping zone by microchannels, carried along by an oil flow and / or by slopes or rails. This routing has been found to aid the formation of spheroids in the microdrops.
  • the microdrops are then trapped by surface tension traps placed in the trapping zone, in particular in a microfluidic chip.
  • the trapping zone (or the microfluidic chip 10) is treated by a hydrophobic surface treatment, and filled with an oil containing a surfactant.
  • the use of surfactant allows the stabilization of the microdrops and the reproducibility of their formation.
  • the surfactants also make it possible to prevent the coalescence of the microdrops in the event of contact during their transport from the production device to the traps in the trapping zone.
  • the microfluidic chip 10, as illustrated on figure 1 is composed of a culture chamber, possibly several square centimeters, containing numerous surface tension traps organized in a table or matrix.
  • the tension traps of surface 12 can have various shapes. For example, in the case of cylindrical traps, their diameter can range from a few tens of microns to several hundreds depending on the desired application. For the encapsulation of single or individualized cells in the microdrops, the diameter of the traps may be, for example, 50 microns, which corresponds to a density of about 5000 traps per square centimeter. For the study of large cell aggregates or spheroids, this diameter can increase to 250 microns, which then corresponds to a trap density of the order of 250 traps per square centimeter.
  • the microdrops 14 including the biological cells 16, formed outside the microfluidic chip 10 are entrained in the latter, for example by means of an oil flow illustrated by the arrow 18 so that some of these microdrops are trapped in the surface tension traps 12.
  • microdrops of hydrogels containing biological cells in an oil without precise control of the flow of hydrogel containing the biological cells in the oil. Indeed, only the microdrops having adequate dimensions are subsequently trapped in the trapping zone, so that the latter is occupied by microdrops finally having a great homogeneity of size, shape and concentration of biological cells.
  • the trapping zone in particular a microfluidic chip 10 contains a hydrogel solution 20 containing biological cells 16.
  • the oil is then injected into the trapping zone (the injection is shown diagrammatically by arrow 18), which pushes the hydrogel solution 20 containing biological cells 16 towards an exit from the trapping zone.
  • the microdrops are then formed directly at the level of the surface tension traps 12, by trapping the hydrogel in these traps of the microfluidic chip, this until a configuration substantially identical to that illustrated on the diagram is obtained. figure 2 .
  • the microdrops are thus formed by spontaneous division (or breakage) of the hydrogel solution containing the biological cells, on the surface tension traps.
  • the traps can be of very different shapes, in particular as a function of the desired application, that is to say in particular as a function of the shape of the trapped microdrops sought.
  • the cavity forming a trap can also be found indifferently on the upper wall, lower wall or one of the side walls of the trapping zone, in particular of the microfluidic chip.
  • the figures 7 to 12 illustrate possible shapes of the surface tension traps 12 of the microfluidic chip 10 and the shape of the microdrops 14 which can be obtained using these surface tension traps 12.
  • the shape of the trap 12 makes it possible to control the shape of the trapped microdrops, according to the geometric parameters of the microfluidic channel in which the trap 12 is formed, and the volume of the trapped microdrops.
  • the figure 7 schematically illustrates the parameters to be taken into account to determine the profile of the microdrop, namely the radius R of the microdrop confined in a channel containing a trap 12, the height h of this channel, less than the radius R of the microdrop in the channel , and the diameter d and the depth p of the trap 12.
  • the microdrop 14 enters as much as possible into the trap 12.
  • the microdrop 14 may or may not have a hemispherical cap, and may or may not have a flat part, confined by the walls. of the canal. So on the figure 8 , the volume of the microdrop 14 is greater than the volume of the trap 12. In this case, the microdrop 14 almost completely fills the trap 12 and has a shape flattened against the walls of the channel and of the trap.
  • the micro-drop 14 has a volume slightly smaller than that of the trap 12, then the micro-drop 14 has two hemispherical caps and only grazes the walls of the channel. Finally, if the microdrop 14 has a volume which is significantly smaller than the volume of the trap 12, as illustrated in figure 10 , then the microdrop 14 (or even several microdrops 14) are fully received in the trap 12.
  • the trap has a diameter d less than twice the height h of the channel.
  • the microdrop 14 remains essentially confined in the channel and only has a small hemispherical cap in the trap 12.
  • the trap 12 is conical and has a diameter d greater than twice the height h of the channel.
  • the microdrop 14 then follows the shape of the wall of the trap 12 to form a hemispherical cap in the trap 12.
  • the cells 16 sediment and settle statistically uniformly at the bottom of the microdrop 14. The cells can then be observed individually, and do not aggregate.
  • the microdrop 14 trapped in a trap 12 has a non-flat bottom, in particular convex, as illustrated in the figures 14 and 15 , then the cells 16 meet the interface of the microdrop 12 during their sedimentation, and find themselves obliged to slide along this interface. The cells 16 thus concentrate at the bottom of the microdrop 14, and can possibly aggregate and form spheroids in the case of certain cells dependent on the anchorage.
  • the microfluidic process proposed here comprises, after the trapping of the microdrops, a step of gelation of these microdrops.
  • the hydrogel contains, preferably is agarose.
  • the gelation of the microdrops is then carried out by cooling the microfluidic chip.
  • the hydrogel contains or, preferably, is alginate, it is possible to bring calcium Ca 2+ ions into the oil in which the microdrops bathe, or else to pre-mix limestone particles with the alginate and saturate the oil in which the microdrops bathe with CO 2 .
  • the alginate is thus acidified and calcium ions are released.
  • other gelling agents can be used, other gelling means can be implemented.
  • this gelation step can be implemented at different times of the handling process.
  • gelation can take place immediately after trapping so as to freeze the cells in place, in the microdrop, and not allow them to sediment. We can then observe the cells independently of each other.
  • gelation is carried out after the cells have sedimented to form spheroids. This makes it possible to observe the behavior of cells that have formed a spheroid.
  • the gelation of the microdrops is implemented only after manipulations of the cells in liquid medium, for example to extract certain cells - those in microdrops. ungelled - selectively. This can be useful for cells such as bacteria or erythrocytes and leukocytes, which are independent of the anchor.
  • aqueous solution containing in particular a biochemical solution comprising biochemical components such as nutrients, growth factors, antibodies, drugs or drug molecules, for example.
  • biochemical components diffuse in the gel and reach the cells. It is thus possible to study the reaction of cells, independent or in the form of spheroids, to these stimuli.
  • the hydrogel thus makes it possible to maintain the cells in a precise location, while allowing their perfusion by an aqueous phase and having previously compartmentalised the biological sample during the encapsulation of the cells in microdrops.
  • the surfactant is preferable to drive the surfactant from the interfaces of the microdrops.
  • the shell formed by the surfactants at the interface of the microdrops can indeed be so effective that it prevents the aqueous phase, which is injected to replace the oil, from filling the microfluidic chip, while retaining the gelled microdrops. in their respective traps.
  • the arrival of the interface of the aqueous phase at the level of a trap results in a force applied to the gelled microdrop which can be driven from the trap if the hydrogel which composes it is sufficiently compressible. This is why it is preferable to promote coalescence by reducing the concentration of surfactant at the level of the interface.
  • the microfluidic chip is perfused before injection of the aqueous phase with oil which, unlike the oil used previously, does not contain any surfactant.
  • concentration of surfactant in the oil of the microfluidic chip decreases, which makes it possible to shift the surfactant adsorption balance at the interface towards desorption.
  • concentrations of surfactant for example of the order of a few percent by mass, it is preferable to perfuse the microfluidic chip with a quantity of oil equivalent to 50 times the volume of the microfluidic chip. This ratio depends on the nature of the surfactant (s) and its / their affinity for the two phases.
  • the shape of the traps can also be optimized to ensure that the gelled microdrops are held in position in the traps.
  • the entry microdrop in the trap will be minimal, resulting in low trapping efficiency. Consequently, there is a limiting speed of the external flow beyond which the microdrops are driven out of the traps.
  • the microdrop when the height of the channel is smaller than the radius of the trap, the microdrop, provided it is large enough, penetrates strongly into the cavity of the trap, resulting in high trapping efficiency. The microdrops remain in place regardless of the speed of the external flow.
  • the shape of the microdrop is very close to its shape in the channel, while in the second case, it locally adopts the shape of the trap.
  • microdrops 14 are gelled agarose microdrops, for example. These agarose microdrops 14 are thawed one by one by heating them locally (the heating being illustrated by the flashes 21), in particular using an infrared laser or electrodes. Heat liquefies the agarose. When the phase surrounding the microdrops 14 is aqueous, the content 16 of the degelled agarose mixes with the aqueous phase.
  • the shape and size of the trap are preferably chosen to allow extraction of the cells. For example, in the case where the cells must remain viable, the trap is sized sufficiently large so that the heating of the hydrogel does not induce the mechanisms of cell death and does not induce the mechanisms of cell death.
  • the shape and the force of the trap are preferably dimensioned so as to allow the extraction of the selected microdrop only, and not of the others. This dimensioning depends in particular on the value of the surface tension between the aqueous phase and the oil, as well as on the rigidity of the gel microdrops and their shape inside the trap.
  • Another alternative is to keep the liquid microdrops for handling cells in suspension, eg bacteria. Gelation is then implemented only for the selective extraction of the microdrops. In this case, we can either gel all the microdrops before extraction and apply the protocol described above, or on the contrary only gel the microdrops which one wishes to keep in the traps.
  • the process presented makes it possible, thanks to slight modifications, to be interested in very varied biological applications.
  • the device can of course also be modified by adjusting the height of the channel in the microfluidic chip and the geometry of the traps.
  • the cells can however be kept encapsulated for a long time in the liquid phase before gelling.
  • a low concentration of cells then makes it possible, for example, to study cells in suspension, independent of the anchoring, such as lymphocytes.
  • the method may also allow the formation of spheroids directly in the chip in a controlled manner.
  • the volume of the microdrops created can be controlled by the microdrop forming device upstream of the microfluidic chip. This volume is preferably adjusted so that the microdrops, once trapped, have a diameter equal to that of the trap and a spherical shape. To do this, the depth of the trap is preferably at least equal to its diameter. The fact that the diameter of the trapped microdrop coincides with that of the trap, makes it possible to ensure high trapping efficiency.
  • the spherical shape favors the formation of spheroids.
  • the microdrops preferably contain cells in suspension in an aqueous phase comprising or consisting of culture medium and hydrogel.
  • the external oil flow is stopped, which stops the recirculations. within the microdrops and promotes cell sedimentation.
  • the spherical shape of the microdrops in the traps then induces a concentration of the cells at their lowest points, until they come into contact. Keeping the chip at rest, in conditions favorable to the survival and proper metabolic functioning of the cells, in particular in temperature, for a period ranging from several hours to several days, allows the reorganization of the cells concentrated at the bottom of the trapped microdrop, in a spheroid.
  • the time required for the formation of spheroids may in particular depend on the cell type used and on the composition of the hydrogel. For H4IIEC3 rat liver cells in a 1% by mass agarose solution diluted in culture medium, it was found that this period is less than 24 hours. Of course, the hydrogel is kept liquid during the time of formation of the spheroids.
  • the size of the spheroids is given by the number of cells encapsulated in each microdrop and therefore by the concentration of the solution of cells injected into the microfluidic chip.
  • the distribution of the number of cells per microdrop, and therefore that of the size of the spheroids formed, is very homogeneous provided that the cells are sufficiently individualized at the time of injection.
  • 98% of the traps were filled with a microdrop of liquid agarose which after 24 hours of incubation contained a well reorganized spheroid.
  • the spheroids obtained in the microfluidic chip can be kept in culture for several days.
  • spheroids of H4IIEC3 cells encapsulated in agarose can be cultured in the chip for a week without significant alteration in their viability while retaining strong functionality (in this example strong and continuous secretion of albumin).
  • the method presented here and the microfluidic chip obtained by implementing it, constitute an excellent drug screening tool. For example, it is possible to create spheroids from cancer cells and observe whether their viability decreases over time as a function of exposure to a molecule tested. By adding a device to the chip that makes it possible to establish a concentration gradient within the chamber, or by placing parallel chips in place, a whole range of concentrations can be tested in a same system. The high efficiency of the process for forming these spheroids also makes it possible to create a large number of them from very limited samples. 500 spheroids of approximately 70 ⁇ m in diameter can thus be formed with only 100,000 cells.
  • the cells that make up the spheroids can also be of different types to address co-culture themes. These cell types can be homogeneously mixed in solution before injection into the chip or be arranged according to a certain structural organization, in several successive hydrogel layers or simply by adhesion to the hydrogel after having been infused into the aqueous phase. external.
  • fibroblasts and epithelial cells can be combined to form a skin model and test the toxicity of cosmetic products, neurons and astrocytes to model the brain, or even endothelial cells and smooth muscle cells such as in the wall of blood vessel.
  • the method presented here makes it possible to achieve very advanced control of the microenvironment of cells in culture, it is also an excellent tool for the study of stem cell differentiation.
  • the encapsulated cells can in fact be subjected to a whole range of concentrations of differentiation factors and, potentially at the same time, to a whole range of rigidity of the matrix, for example by varying the concentration of the hydrogel.
  • the method can be used to observe embryonic development over time in interaction with physico-chemical factors of the external environment.
  • this method makes it possible to make a medical diagnosis based on the response of the cells to certain markers.
  • cells are captured with a very low rate of loss.
  • the cells can then be subjected to known diagnostic tests for certain diseases, such as characterization of a cancer biopsy.
  • diagnostic tests for certain diseases such as characterization of a cancer biopsy.
  • PCR polymerase chain reactions
  • FISH FISH
  • the method described above offers the possibility of carrying out all the steps of analysis and of cell culture in a microfluidic chip, thanks to the gelation of the cells. microdrops as a result of their trapping in the chip. This makes it possible to use much smaller volumes of reagents than for tests carried out in multi-well plates or in culture dishes. This also makes it possible to follow the responses of cells over time, following different stimuli.
  • the gelling means comprise, for example, a device for injecting a chemical agent into the trapping zone and / or means for regulating the temperature, for example for cooling the microfluidic chip.
  • the device may also include means for thawing at least some of the gelled hydrogel microdrops, for example a laser.
  • the method described above also makes it possible to produce a product of gelled microdrops, comprising a microdrop trapping zone, in particular a microfluidic chip, and gelled microdrops each including one or more cells, trapped in the trapping zone, the microdrops.
  • gelled being cryo-preserved.
  • Cells can be aggregated as clusters or spheroids.
  • the gelled microdrops can be bathed in a fluid, preferably in an aqueous solution or in an oil, the fluid and the microdrops preferably being cryo-preserved. This cryo-preservation makes it possible in particular to maintain the cells under stable conditions for a long period, with a view to transporting them or storing them for a subsequent analysis.
  • the biological cells encapsulated in the microdrops can be bacteria, yeasts, eukaryotic cells, mammalian cells, preferably mammalian cells excluding human cells, more preferably rat cells or other mammals. , or human cells isolated from their natural environment.
  • samples used in addition to cells, can in particular also be molecules, plastic beads functionalized by coupling them to molecules.
  • the trapped microdrops can be merged with other microdrops provided by the flow of aqueous solution.
  • the aqueous solution of which the microdrops are made may contain a biochemical solution, the biochemical solution preferably comprising at least one of lipids (fatty acids, etc.), carbohydrates (in monomeric form or of polysaccharides, etc. ), amino acids and proteins (growth factors, cytokines, antibodies, antigens, etc.), as well as salinity and / or pH buffers.
  • lipids fatty acids, etc.
  • carbohydrates in monomeric form or of polysaccharides, etc.
  • amino acids and proteins growth factors, cytokines, antibodies, antigens, etc.
  • the oil (or oily phase) surrounding the microdrops may contain fluorinated oils (type FC40) or else photo-crosslinkable solutions immiscible with water (type Norland Optical Adhesive), which, once polymerized allow the oil to gel and thus physically and selectively isolate the microdrops. It is thus possible to partition, to isolate in a more robust manner, the microdrops from one another. This is to prevent two microdrops from merging, causing the samples they contain to mix. This also makes it possible to store the samples in a durable manner, the risks of evaporation, in particular, of the microdrops being greatly reduced due to the partitioning of the microdrops by the gelled oil, which forms a solid compartment around the microdrops.
  • fluorinated oils type FC40
  • photo-crosslinkable solutions immiscible with water type Norland Optical Adhesive
  • the microdrops around which the oil has not gelled can be pushed out of the trapping zone. To do this, it is possible to implement a flow of oil or of another fluid in the trapping zone, the flow being strong enough to entrain the microdrops. It is thus possible to keep in the trapping zone only the microdrops around which the oil has been gelled.
  • microdrops can be gelled even in the case where the oil is gelled.
  • the process can of course include, in this case where part of the oil is gelled, a subsequent step of degelling the gelled oil.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Colloid Chemistry (AREA)
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Claims (18)

  1. Verfahren zur Handhabung von Mikrotröpfchen (14), die Proben (16) enthalten, in einem Mikrofluidiksystem, bei dem man in den folgenden Schritten:
    i) in einem Öl Mikrotröpfchen (14) einer eine Probe (16) enthaltenden wässrigen Lösung bildet, wobei die eine Probe (16) enthaltende wässrige Lösung ein Geliermittel umfasst,
    ii) die Mikrotröpfchen (14) mithilfe von in einer Einfangzone (10) vorgesehenen Oberflächenspannungsfallen (12) einfängt, und
    iii) mindestens einen Teil der eingefangenen Mikrotröpfchen (14) geliert,
    wobei die Schritte des Verfahrens in einem einzigen Mikrofluidiksystem durchgeführt werden.
  2. Verfahren zur Handhabung von Mikrotröpfchen (14), die Proben (16) enthalten, in einem Mikrofluidiksystem, bei dem man in den folgenden Schritten:
    i) in einem Öl Mikrotröpfchen (14) einer eine Probe (16) enthaltenden wässrigen Lösung bildet, wobei das Öl ein Geliermittel umfasst,
    ii) die Mikrotröpfchen (14) mithilfe von in einer Einfangzone (10) vorgesehenen Oberflächenspannungsfallen (12) einfängt, und
    iii) mindestens einen Teil des Öls in der Einfangzone (10) geliert.
  3. Verfahren nach Anspruch 1, bei dem in Schritt iii) mindestens ein Teil der Mikrotröpfchen geliert wird, wobei das Verfahren außerdem den folgenden Schritt umfasst:
    iv) Ersetzen des Öls, das die gelierten Mikrotröpfchen (14) umgibt, durch eine wässrige Lösung, wobei die das Öl ersetzende wässrige Lösung vorzugsweise eine biochemische Lösung enthält, wobei die biochemische Lösung vorzugsweise mindestens eines aus einem oder mehreren pH- oder Salzgehaltpuffern, einem oder mehreren Nährstoffen, einem oder mehreren Wachstumsfaktoren, Cytokinen, einem oder mehreren Antikörpern, einem oder mehreren Antigenen, einem oder mehreren Molekülen, insbesondere Arzneistoffmolekülen, einer oder mehreren Zellen, Lipiden, Kohlenhydraten, insbesondere in monomerer oder Polysaccharidform, Aminosäuren und/oder Proteinen umfasst.
  4. Verfahren nach Anspruch 1 oder 3, umfassend einen Schritt v) nach Schritt iii) und vorzugsweise nach Schritt iv), bei dem man mindestens einige der in Schritt iii) gelierten Mikrotröpfchen (14) degeliert, wobei das Verfahren vorzugsweise einen Schritt vi) nach Schritt v) umfasst, bei dem man die degelierten Mikrotröpfchen (14) und/oder die in diesen degelierten Mikrotröpfchen (14) enthaltenen Proben (16) aus der Einfangzone (10) ausleitet.
  5. Verfahren nach Anspruch 2, umfassend einen Schritt nach Schritt iii), bei dem man die Mikrotröpfchen, um die das Öl nicht geliert ist, aus der Einfangzone herausschiebt.
  6. Verfahren nach einem der vorhergehenden Ansprüche, wobei es sich bei der Probe (16) um eines aus einer oder mehreren Zellen, insbesondere ein Zellensphäroid, einem oder mehreren Moleküle einfangenden Kügelchen, wobei die Kügelchen insbesondere aus Kunststoffmaterial sind, einem oder mehreren Molekülen handelt.
  7. Verfahren nach einem der vorhergehenden Ansprüche, wobei Schritt iii) nach einer Sedimentation der Proben (16), insbesondere der Zellen, in den eingefangenen Mikrotröpfchen (14), insbesondere nach Bildung von Sphäroiden, durchgeführt wird oder wobei Schritt iii) vor einer Sedimentation der Proben (16) in den eingefangenen Mikrotröpfchen (14) durchgeführt wird.
  8. Verfahren nach einem der vorhergehenden Ansprüche, wobei die Einfangzone von einem Mikrofluidik-Chip (10), der die Oberflächenspannungsfallen (12) umfasst, gebildet wird.
  9. Verfahren nach einem der vorhergehenden Ansprüche, wobei man in Schritt i):
    a) in eine Zone stromaufwärts der Einfangzone (10) eine Proben (16) und gegebenenfalls ein Geliermittel enthaltende wässrige Lösung injiziert,
    b) ein gegebenenfalls ein Geliermittel enthaltendes Öl in die Zone stromaufwärts der Einfangzone injiziert, um die Proben (16) enthaltende wässrige Lösung zu einem Auslass der Einfangzone (10) zu schieben, wobei die Ölinjektion derart durchgeführt wird, dass sich Proben (16) enthaltende Mikrotröpfchen (14) bilden, und
    c) die Mikrotröpfchen (14) bis zur Einfangzone (10) verschoben werden.
  10. Verfahren nach einem der Ansprüche 1 bis 5, wobei die Schritte i) und ii) gleichzeitig in der Einfangzone (10) durchgeführt werden, indem man Folgendes durchführt:
    - Füllen der Einfangzone (10) mit Proben (16) und gegebenenfalls ein Geliermittel enthaltender wässriger Lösung, und
    - Injizieren von Öl, das gegebenenfalls ein Geliermittel enthält, in die Einfangzone (10), um die Proben (16) enthaltende wässrige Lösung zu einem Auslass der Einfangzone (10) zu schieben, wobei die Oberflächenspannungsfallen (12) dafür ausgelegt sind, das Zerbrechen der Proben (16) enthaltenden Mikrotröpfchen (14) an den Oberflächenspannungsfallen (12) zu ermöglichen.
  11. Verfahren nach einem der vorhergehenden Ansprüche, wobei man in Schritt iii) mindestens eines der Folgenden durchführt:
    - Abkühlen oder Erhitzen der Mikrotröpfchen (14) und/oder des Öls,
    - Injizieren einer ein chemisches Geliermittel enthaltenden Lösung in die Einfangzone,
    - Belichten der Mikrotröpfchen (14) und/oder des Öls mit Licht, das die Gelbildung hervorruft, insbesondere UV-Licht.
  12. Verfahren nach einem der vorhergehenden Ansprüche, bei dem das Öl ein Tensid enthält, wobei das Verfahren vorzugsweise einen Tensidwaschschritt vor Schritt iv) umfasst.
  13. Verfahren nach einem der vorhergehenden Ansprüche, umfassend vor Schritt i) einen Schritt der Auswahl der Form der Oberflächenspannungsfallen (12) in Abhängigkeit von der gewünschten Form der Mikrotröpfchen (14), wobei vorzugsweise die Einfangzone und die Fallen derart gewählt werden, dass:
    - eingefangene Mikrotröpfchen (14) mit flachem Boden gebildet werden, oder
    - eingefangene Mikrotröpfchen (14) mit nichtflachem, insbesondere gerundetem, vorzugsweise konvexem Boden gebildet werden.
  14. Verfahren nach einem der vorhergehenden Ansprüche, umfassend einen Schritt der Anwendung eines Stimulus auf die Proben (16), die in mindestens einem Teil der eingefangenen, gelierten oder ungelierten Mikrotröpfchen (14) enthalten sind.
  15. Vorrichtung zur Durchführung eines Verfahrens nach einem der Ansprüche 1, 3 und 4 oder einem der Ansprüche 6 bis 13 in Verbindung mit mindestens Anspruch 1, umfassend:
    - eine Proben (16) enthaltende Hydrogel-Lösung,
    - Mittel zum Bilden von Mikrotröpfchen (14) der die Proben (16) enthaltenden Hydrogel-Lösung,
    - eine Einfangzone, insbesondere einen Mikrofluidik-Chip, zum Einfangen der Mikrotröpfchen (14) an festgelegten Stellen,
    - Mittel zum Gelieren mindestens eines Teils der eingefangenen Mikrotröpfchen (14), wobei die Mittel zum Gelieren vorzugsweise eine Vorrichtung zum Injizieren eines chemischen Mittels in die Einfangzone (10) umfassen, und
    - optional Mittel zum Degelieren mindestens einiger der gelierten Hydrogel-Mikrotröpfchen (14).
  16. Vorrichtung zur Durchführung eines Verfahrens nach einem der Ansprüche 1 und 5 oder einem der Ansprüche 6 bis 13 in Verbindung mit mindestens Anspruch 2, umfassend:
    - Mittel zum Bilden von Proben (16) enthaltenden Mikrotröpfchen (14),
    - eine Einfangzone, insbesondere einen Mikrofluidik-Chip, zum Einfangen der Mikrotröpfchen (14) an festgelegten Stellen, wobei die Einfangzone ein Öl enthält, das ein Geliermittel umfasst,
    - Mittel zum Gelieren des Öls, wobei die Mittel zum Gelieren vorzugsweise eine Vorrichtung zum Injizieren eines chemischen Mittels in die Einfangzone (10) umfassen, und
    - optional Mittel zum Degelieren mindestens eines Teils des gelierten Öls.
  17. Gelierte-Mikrotröpfchen-Produkt, umfassend eine Mikrotröpfcheneinfangzone, insbesondere einen Mikrofluidik-Chip, (10) und in der Einfangzone (10) eingefangene, gelierte Mikrotröpfchen (14), die jeweils eine Probe (16) enthalten,
    wobei die gelierten Mikrotröpfchen (14) kryokonserviert sind, es sich bei den Proben (16) vorzugsweise um Säugetierzellen, zum Beispiel Säugetierzellen mit Ausnahme menschlicher Zellen, Bakterien, Hefen oder andere in Bioprozessen verwendete Zellen, Moleküle, Kügelchen mit eingefangenen Molekülen auf der Oberfläche handelt.
  18. Mikrotröpfchen-Produkt, umfassend eine Mikrotröpfcheneinfangzone mit einer Mehrzahl von Oberflächenspannungsfallen (12), insbesondere einen Mikrofluidik-Chip, (10) und in den Oberflächenspannungsfallen (10) eingefangene Mikrotröpfchen (14), die jeweils eine Probe (16) enthalten,
    wobei die Mikrotröpfchen (14) in einem gelierten Öl schwimmen, die Mikrotröpfchen (14) und das Öl vorzugsweise kryokonserviert sind,
    wobei es sich bei den Proben (16) vorzugsweise um Säugetierzellen, zum Beispiel Säugetierzellen mit Ausnahme menschlicher Zellen, Bakterien, Hefen oder andere in Bioprozessen verwendete Zellen, Moleküle, Kügelchen mit eingefangenen Molekülen auf der Oberfläche handelt.
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