JP2021004900A - Method for dealing with micro droplets including sample - Google Patents

Abstract

To provide a method for dealing with micro droplets containing a sample that makes a simple and yet wide testing of the sample possible, and a method for dealing with micro droplets that can effectively select micro droplets.SOLUTION: A method for dealing with micro droplets (14) containing a sample (16) in a micro fluid system includes: a step which forms, in oil, micro droplets (14) of aqueous solution containing the sample (16); a step containing the sample (16) in which oil and/or the aqueous solution contains gelling agents; a step for capturing the micro droplets (14) by the surface tension trap (12) arranged in the capture area (10) in advance; and a step which at least partially gells the oil in the capture area and/or at least partially gells the captured micro droplets (14).SELECTED DRAWING: Figure 1

Description

The present invention relates to microfluidic methods for handling samples within hydrogel microdroplets, especially biological samples. The present invention also relates to devices for performing such methods and to sample products obtained by performing such methods.

Guo, Rotem, Heyman, & Weitz, "Droplet microfluidics for high-throughput bioassays", Lab. Chip. From 12 (2012), it is known that droplets (or "microdroplets") in microfluidic systems may be used to contain chemical or biological reactions. In these systems, the content of these droplets can be assayed by observing the fluorescence of the droplets as they pass in front of the focusing laser. However, in these systems it is not possible to observe changes in the content of these droplets over time without extracting the droplets from the microfluidic device.

Studies of individualized cells in microdroplets have also been made, for example, by Joensson, H. et al. N. & Andersson Svan, H. et al. , "Droplet microfluidics-a tool for single-cell analysis", Angew. Chem. Int. Ed. Engl. It is known from pages 51, 12176 to 12192 (2012). In practice, these microdroplets form well-defined compartments that allow the isolation of biological samples, such as cells, for example. This document specifically teaches that the localization of encapsulated cell populations can be controlled by the accumulation of droplets in culture chambers, elongated channels, or static traps. The literature also teaches that cells can be encapsulated within a functionalized hydrogel surrounded by an oil phase.

However, the supply of nutrients, or more generally biologically interesting molecules, to cells in such devices has been found to be limited and the method used (eg, electrical fusion or pico injection). ) Has turned out to be complicated. As a result, these devices have numerous limitations, especially in terms of time, for studying cell behavior.

Furthermore, L. Yu, M.M. C. W. Chen and K. C. From Cheung, "Droplet-based microfluidic system for multicellular tumor sphere spheroid formation and anticancer drug testing", a method known to handle hydrogels from Lab Chip (2010), a method known to handle hydrogels in a Lab Chip (2010), contains multicellular spheroids. According to this method, cell-containing hydrogel microbeads are produced in the first microfluidic system. The hydrogel microbeads are then recovered, washed in the tank and then injected into a second microfluidic system containing a trap, which allows the microdroplets to be immobilized.

However, such a method is complicated and requires two separate microfluidic systems and three devices as a whole. In addition, this method does not allow continuous observation of samples. In particular, it does not make it possible to observe the first moment between the formation of a droplet and its capture.

FR-A-29581886

Guo, Rotem, Heyman, & Weitz, "Droplet microfluidics for high-throughput bioassays", Lab. Chip. 12 (2012) Joensson, H. et al. N. & Andersson Svan, H. et al. , "Droplet microfluidics-a tool for single-cell analysis", Angew. Chem. Int. Ed. Engl. 51, pp. 12176-12192 (2012) L. Yu, M.M. C. W. Chen and K. C. Cheung, "Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing", Lab Chip (2010) S. L. Anna, N.M. Bontoux and H. et al. A. Stone, "Formation of dispersions using'Flow-Focusing'in microchannels", Apple. Phys. Lett. 82, 364 pages (2003) T. Thorsen, R.M. W. Roberts, F.M. H. Arnold et S. R. Quake, "Dynamic pattern formation in a vesicle-generating microfluidic device", Phys. Rev. Lett. 86, pp. 4163-4166 (2001)

Therefore, there is a need for a method for handling microdroplets containing samples that is simpler and yet allows for a wider range of tests on the sample. There is also a need for methods for handling microdroplets that allow for more effective sorting of microdroplets.

To this end, the present invention is a method for handling microdroplets containing samples in a microfluidic system.
i) A step of forming microdroplets of an aqueous solution containing a sample in oil, wherein at least one of the oil and the aqueous solution contains a gelling agent.
ii) The process of capturing microdroplets by surface tension traps pre-arranged within the capture zone, and
iii) Provided is a method including a step of gelling at least a part of oil in a trapping zone and at least a part of trapped microdroplets.

Therefore, according to the present invention, in order to select the desired microdroplets, in other words, the microdroplets containing the desired sample, these microdroplets are first placed in a surface tension trap (or capillary trap). It captures and then gels some of the microdroplets and / or some of the oil that surrounds the microdroplets. Gelation of the microdroplets and / or the oil surrounding the microdroplets facilitates sorting by increasing the strength of capture of the microdroplets in the trap. In other words, the gelling step makes it possible to prevent the loss of the desired microdroplets.

In addition, this gelation makes it possible to prevent the microdroplets from becoming fusing, which can cause mixing of these microdroplet samples.

The surface tension trap means a zone of a microfluidic system as a trap, and its geometric shape makes it possible to hold the microdroplets in a predetermined position with the interfacial tension of the microdroplets. It becomes.

All steps of this method are performed in a single microfluidic system. The microfluidic system shall mean a system in which a part thereof is manufactured according to a microfabrication process. Such systems have ducts whose at least one dimension is typically less than a millimeter.

The shape of the microdroplets can be controlled. This microdroplet shape control can be combined with control at the time of gelation of the microdroplets or a portion of the oil surrounding the microdroplets to enable a variety of applications, especially in terms of cell manipulation. ..

The cell is meant to mean a eukaryotic cell (eg, a plant, mushroom, yeast or mammalian cell) and a prokaryotic cell (eg, a bacterium). In mammalian cells, scaffold-independent cells (eg, some cells in blood cell lines and highly transformed tumor cells) and some subtypes are scaffold-dependent, which can be organized into spheroids. Distinguish from cells (most of other cell types). Spheroids are meant to be multicellular structures organized in the form of microtissues that are similar in functionality to the functionality of tissues derived from organs.

According to a preferred embodiment, the method according to the invention comprises one or more of the following features alone or in combination.
-Step iii) is to gel at least a portion of the oil in the capture zone, except for microdroplets.
-Step iii) is to gel at least a portion of the microdroplets, except for the oil that surrounds the microdroplets in the capture zone.
-Samples are one or more cells, especially cell spheroids, one or more beads that capture molecules, especially one of plastic beads, or one or more molecules. There is.
-Step iii) is to be performed in the captured microdroplets, especially after cell sedimentation, especially after spheroid formation.
-Step iii) shall be performed prior to sedimentation of the sample within the captured microdroplets.
− This method
iv) Further include a step in replacing the oil surrounding the gelled microdroplets with an aqueous solution.
-The aqueous solution that replaces the oil contains a biochemical solution, which is preferably one or more pH buffers or saline buffers, one or more nutrients, one or more growth factors. , Cytokines, 1 or more antibodies, 1 or more antigens, especially 1 or more molecules of pharmaceuticals, 1 or more cells, lipids, especially monomeric or polysaccharide carbohydrates, amino acids and / or Contain at least one of the proteins.
-The capture zone is formed by a microfluidic chip containing a surface tension trap.
− Step i)
a) An aqueous solution containing a sample and, if necessary, a gelling agent is injected into the zone upstream of the capture zone.
b) If necessary, oil containing a gelling agent to form microdroplets containing the sample in the zone upstream of the capture zone in order to expel the aqueous solution containing the sample toward the outlet of the capture zone. Inject into, then
c) To move the microdroplets to the capture zone and capture the microdroplets in the capture zone.
− Step i) and step ii)
-Fill the capture zone with an aqueous solution containing the sample and, if necessary, a gelling agent, and then
-In order to expel the aqueous solution containing the sample toward the exit of the capture zone, the capture zone is infused with oil containing a gelling agent as needed, and a surface tension trap is provided for the microdroplets containing the sample. Simultaneous in the capture zone by performing certain tasks that crushing is adapted to be possible with surface tension traps.
− Step iii)
-Cooling or heating microdroplets and / or oil,
− Injecting a solution containing a chemical gelling agent,
-Exposure of microdroplets and / or oil to at least one of the light that causes gelation, especially UV light.
-The oil contains a surfactant, and the method preferably comprises the step of cleaning the surfactant before step iv).
− This method includes, prior to step i), the step of selecting the shape of the surface tension trap according to the desired shape of the microdroplets.
− Capture zones and traps
-Selected to form captured microdroplets with a flat bottom, or-to form captured microdroplets with a non-flat, especially curved, preferably convex bottom.
− This method comprises non-gelling at least a portion of the gelled microdroplets in step iii), followed by step iii), preferably step iv).
− This method comprises step vi) in which the non-gelled microdroplets and / or the sample contained in these non-gelled microdroplets is expelled from the capture zone, following step v). thing.
-The method comprises stimulating a sample contained in at least a portion of captured microdroplets, gelled microdroplets or non-gelled microdroplets.
− This method involves expelling non-gelled microdroplets around it from the capture zone simply to hold the microdroplets with oil gelling around them in the capture zone. , Step iii) to be included.

According to another aspect, the present invention is a device for carrying out the method as described above, in all combinations thereof.
-Means for forming microdroplets containing samples,
-A capture zone for capturing microdroplets in place, especially with a microfluidic chip,
-For devices including means for gelling at least a portion of captured microdroplets and / or oil.

The gelling means can include a device for injecting a chemical agent into the capture zone.

The device can also include means for non-gelling at least a portion of the gelled hydrogel microdroplets and / or a portion of the gelled oil.

The present invention is also a product of gelled microdroplets, each containing a zone for capturing microdroplets, particularly a microfluidic chip, each containing a sample and captured in the capture zone, preferably. With respect to the product of gelled microdroplets, including with gelled microdroplets stored cryopreserved.

Therefore, for the purpose of storage and / or distribution of this microdroplet product, the biochemical solution may contain an antifreeze agent (DMSO, glycerol, trehalose, etc.) to allow cryopreservation of the sample. it can.

The gelled microdroplets can also be immersed in a fluid, preferably in an aqueous solution or oil, and the fluid and microdroplets are preferably cryopreserved.

The present invention also relates to a product of microdroplets, including a capture zone, particularly a microfluidic chip, and microdroplets, each containing a sample and captured in the capture zone. The microdroplets are immersed in gelled oil, and the microdroplets and gelled oil are preferably cryopreserved.

The sample may be a mammalian cell, preferably a bead that surface captures cells from a mammal other than human cells, bacteria, yeast, or other cells, molecules, or molecules used in a bioprocess.

The invention may be further understood by reading the following description of exemplary embodiments of the invention in light of the accompanying drawings.

It is a figure which shows schematicly the microfluidic chip. FIG. 5 is a diagram schematically showing a microfluidic chip from FIG. 1, in which some traps are occupied by hydrogel microdroplets containing a sample. FIG. 5 is a schematic representation of a microfluidic chip from FIG. 1 containing a mixture of a hydrogel and a sample to be tested. It is a figure which shows typically the means for discharging a part of the sample contained in the hydrogel microdroplet trapped in a microfluidic chip from this microfluidic chip. It is a figure which shows typically the means for discharging a part of the sample contained in the hydrogel microdroplet trapped in a microfluidic chip from this microfluidic chip. It is a figure which shows typically the means for discharging a part of the sample contained in the hydrogel microdroplet trapped in a microfluidic chip from this microfluidic chip. FIG. 5 is a diagram schematically showing an example of the geometric shape of a surface tension trap and the shape of a microdroplet that can be obtained thereby. FIG. 5 is a diagram schematically showing an example of the geometric shape of a surface tension trap and the shape of a microdroplet that can be obtained thereby. FIG. 5 is a diagram schematically showing an example of the geometric shape of a surface tension trap and the shape of a microdroplet that can be obtained thereby. FIG. 5 is a diagram schematically showing an example of the geometric shape of a surface tension trap and the shape of a microdroplet that can be obtained thereby. FIG. 5 is a diagram schematically showing an example of the geometric shape of a surface tension trap and the shape of a microdroplet that can be obtained thereby. FIG. 5 is a diagram schematically showing an example of the geometric shape of a surface tension trap and the shape of a microdroplet that can be obtained thereby. It is a figure which shows typically the example of the sedimentation of a sample in a hydrogel microdroplet. It is a figure which shows typically the example of the sedimentation of a sample in a hydrogel microdroplet. It is a figure which shows typically the example of the sedimentation of a sample in a hydrogel microdroplet.

The present invention relates to a method for handling hydrogel microdroplets containing a sample to be tested.

The following documents are particularly relevant for samples in the form of cells, but it goes without saying that other types of samples can be used.

This method basically involves three steps, all of which are carried out in a single microfluidic system, and these three steps are
− A step of forming microdroplets of a liquid aqueous solution containing one or more cells in an oil, wherein the oil and / or the aqueous solution contains a gelling agent.
-The process of capturing liquid microdroplets with a surface tension trap pre-arranged within the capture zone, and-the step of gelling at least one of the oil and the captured microdroplets.

The document below states that the aqueous solution is a hydrogel solution, the oil does not contain a gelling agent, and the last step above gels at least a portion of the captured microdroplets without the oil itself gelling. It is especially relevant when there is something to do. In this case, this method can be continued by performing different steps following the above three steps, especially depending on the test desired to be performed.

This method can be particularly continued by the step of replacing the oil around the gelled microdroplets with an aqueous solution without removing the microdroplets from the surface tension trap. The aqueous solution can contain nutrients, growth factors, antibodies, drug molecules and biochemical solutions having at least one of pH and / or saline buffer.

According to another aspect, by this method, the three-dimensional shape of the hydrogel beads in the microfluidic channel and / or in the surface tension trap, the main application of which is the encapsulation of cells in these microdroplets. It becomes possible to control. Thus, depending on the shape of the microdroplets and the concentration of cells per microdroplet, by encapsulating the cells in a hydrogel, while injecting the biochemical solution into the cells, or physically, for example, heat or light. It is possible to culture or analyze the cells while giving the stimulus to the cells.

The gel is intended to mean a medium containing molecules or particles that are composed primarily of liquids and can be organized to give a solid appearance, for example, do not flow in its stable state. The solution can be handled in a liquid state and then "gelled" by chemical or physical means. In some cases, gelation may be reversible. When the liquid is water, it refers to hydrogel.

As mentioned above, the proposed microfluidic method comprises a first step of forming a hydrogel microfluidic containing living cells in oil.

In this case, the diameter of the microdroplets (or microbeads) is on the order of micrometers, especially between 10 and 1000 micrometers.

The hydrogel is, for example, an aqueous solution containing a gelling agent. The gelling agent is selected by the user according to the application. An example of a gelling agent that can be physically gelled is agarose, which is a liquid at room temperature but gels at low temperatures. A gelling agent that can be chemically gelled is, for example, alginate, which is a liquid in solution but gels when the calcium ion Ca ²⁺ is supplied.

At the biological level, the biochemical and biomechanical properties of hydrogels can allow scaffold-sensitive cells to establish specific interactions with the matrix thus formed. These interactions are essential for the survival of scaffold-dependent mammalian cells and are involved in their phenotypic control. The nature of the matrix makes it possible, for example, to observe cell migration or proteolysis (digestion of the matrix by cells). Particularly definitive experiments were performed using not only agarose, alginate and PEG-DA (polyethylene glycol diacrylate), but also gelatin, type I collagen or Matrigel®. Maintaining the viability of hydrogels, for example, with hydrogels containing various proteins, glycosaminoglycans, and other components of the extracellular matrix (eg, type I collagen, gelatin or Matrigel®). The ability to assist in proliferation, to assist in proliferation, and to migrate, as well as to maintain the phenotype of a particular scaffold-dependent cell population, has also been demonstrated. Note that gels can be combined, for example, by supplying continuous microdroplets. Each of the above hydrogels has a specific corresponding gelation procedure. Some hydrogels, such as PEG-DA, can also be functionalized to allow cell survival and / or development by incorporating peptide mimetics (eg, some mammalian cell types are specific). Hydrogels can be functionalized by RGD-type consensus sequences capable of constructing interactions, or PRCG [V / N] PD or HEXGHXXXGXXH consensus sequences specific for metalloprotease), or antibodies or aptamers. Incorporation can also be functionalized, for example, to allow in-situ capture of cytokines secreted by encapsulated lymphocytes to enable sensing of specific molecules. The mechanical properties of these hydrogels can also be modified for different uses, for example by varying the degree of cross-linking and / or concentration of the hydrogels. All of these physicochemical properties may vary depending on the trap in the capture zone. Setting a stiffness gradient within the captured hydrogel microdroplets allows, for example, to control stem cell differentiation into different cell types. Finally, after several layers are mixed or continuously formed around the gelled core in the trap, several hydrogels can coexist in the same microdroplet.

A priori, cells are mixed with Hydegel prior to the formation of microdroplets. However, the hydrogel and cells can be mixed directly within the microfluidic device prior to the formation of microdroplets.

A large number of methods have already been proposed for forming such microdroplets in mobile phases such as oil. For example, the following method example can be mentioned.
-For example, S. cerevisiae, the contents of which are incorporated herein by reference. L. Anna, N.M. Bontoux and H. et al. A. Stone, "Formation of dispersions using'Flow-Focusing' in microchannels", Apple. Phys. Lett. 82, 364 (2003), a method referred to as "flow-focusing",
-For example, T.I., whose contents are incorporated herein by reference. Thorsen, R.M. W. Roberts, F.M. H. Arnold et S. R. Quake, "Dynamic pattern formation in a vesicle-generatoring microfluidic device", Phys. Rev. Lett. 86, 4163-4166 (2001), the "T-junction" method, or-for example, the application FR-A-29581886, the contents of which are incorporated herein by reference. The "confinement gradient" method used.

These methods make it possible to form microdroplets of substantially the same size.

After the formation of these microdroplets, the microdroplets are transported from the zone in which they were formed to the capture zone by the microchannel, but by a stream of oil and / or by a slope or rail. This transport has been observed to aid in spheroid formation within microdroplets. The microdroplets are then captured, among other things, by surface tension traps arranged within the capture zone within the microfluidic chip. The capture zone (or microfluidic chip 10) is treated by a hydrophobic surface treatment and filled with an oil containing a surfactant. The use of surfactants allows the stabilization of microdroplets and the reproducibility of their microdroplet formation. Surfactants can also prevent the coalescence of microdroplets in the event of contact while transporting the microdroplets from the production device to the trap in the capture zone.

As shown in FIG. 1, the microfluidic chip 10 is composed of a culture chamber of several square centimeters containing a large number of surface tension traps, sometimes organized in the form of a table or matrix. The surface tension trap 12 can take various shapes. For example, in the case of cylindrical traps, the diameter of these traps can range from tens of microns to hundreds of microns, depending on the desired application. For encapsulation of cells individualized within a single cell or microdroplet, the diameter of the trap may be, for example, 50 microns, which corresponds to a density of about 5000 traps per square centimeter. In studies of large cell aggregates or spheroids, this diameter can be up to 250 microns, which corresponds to a trap density of around 250 traps per square centimeter.

As shown in FIG. 1, the microdroplets 14 containing living cells 16 formed on the outside of the microfluidic chip 10 have a part of these microdroplets on the surface due to, for example, the flow of oil indicated by the arrow 18. It is carried to the latter chip so that it is trapped in the tension trap 12.

However, as a modified form, it is proposed here that hydrogel microdroplets containing living cells in oil are formed without precise control of the flow of hydrogels containing living cells in oil. To do. This is because only microdroplets of appropriate size are later captured in the capture zone, so that the latter capture zone is occupied by microdroplets that actually have living cells of highly homogeneous size, shape and concentration. Because it is possible.

According to another variant shown in FIG. 3, the capture zone, especially the microfluidic chip 10, contains a hydrogel solution 20 containing living cells 16. The capture zone is then infused with oil (this infusion is outlined by arrow 18), which expels the hydrogel solution 20 containing the living cells 16 from the capture zone towards the exit. The microdroplets are then formed directly in these traps by trapping the hydrogel in the surface tension trap 12 of the microfluidic chip until a configuration substantially identical to that shown in FIG. 2 is obtained. Therefore, microdroplets are formed by spontaneously dividing (or crushing) a hydrogel solution containing living cells on a surface tension trap. Again, no precise control of the flow is required, but it is even possible to manually push the syringe without the need for elaborate equipment. In this case, a deep surface tension trap is preferred in order to allow the surface tension trap to crush the microdroplets (ie, form those microdroplets). Subsequently, such a trap will be described.

It should be noted here that the shape of the trap can be very different, especially depending on the desired application, especially depending on the desired shape of the captured microdroplets. A cavity forming a trap may be located on the upper wall, lower wall, or capture zone, especially one of the side walls of the microfluidic chip, to preference.

7 to 12 show the shapes that can be assumed for the surface tension trap 12 of the microfluidic chip 10, and the shape of the microdroplets 14 that can be obtained by these surface tension traps 12.

In particular, the shape of the trap 12 makes it possible to control the shape of the captured microdroplets according to the geometric parameters of the microfluidic channel in which the trap 12 is formed and the volume of the captured microdroplets. FIG. 7 shows the parameters taken into account for determining the profile of the microdroplets, namely the radius R of the microdroplets confined in the channel containing the trap 12, and the radius R of the microdroplets in the channel. , The height h of this channel and the diameter d and depth p of the trap 12 are shown schematically.

As shown in FIGS. 8-10, if the trap 12 is cylindrical and its diameter d is greater than twice the channel h, the microdroplet 14 fits the trap 12 as closely as possible. Depending on the relative volume of the trap 12 and the microdroplet 14, the microdroplet 14 may or may not have a hemispherical dome and may or may not have a flat portion confined by the wall of the channel. May be good. Therefore, in FIG. 8, the volume of the microdroplet 14 is larger than the volume of the trap 12. In this case, the microdroplet 14 fills the trap 12 almost completely and has a flat shape with respect to the channel and the wall of the trap. In FIG. 9, since the volume of the microdroplet 14 is slightly smaller than the volume of the trap 12, the microdroplet 14 has two hemispherical domes and is only slightly in contact with the wall of the channel. Finally, if the volume of the microdroplets 14 is significantly smaller than the volume of the trap 12, as shown in FIG. 10, then the microdroplets 14 (or even a few microdroplets 14) are in the trap 12. Fully contained.

On the other hand, in the case of FIG. 11, the diameter d of the trap is less than half the height h of the channel. As a result, the microdroplet 14 basically remains trapped in the channel and only has a small hemispherical dome in the trap 12.

Finally, in the case of FIG. 12, the trap 12 is conical and its diameter d is twice as large as the channel height h. Therefore, the microdroplets 14 fit snugly into the shape of the wall of the trap 12 to form a hemispherical dome within the trap 12.

Furthermore, as shown in FIG. 13, when the microdroplets 14 trapped in the trap 12 have a flat bottom, the cells 16 settle and the bottom of the microdroplets 14 is statistically uniform. The cells themselves deposit in. At that time, the cells can be observed individually, but they do not aggregate. On the other hand, if the microdroplets 14 trapped in the trap 12 have an uneven bottom, especially a protrusion, as shown in FIGS. 14 and 15, the cells 16 will have the microdroplets 12 upon settling. It comes into contact with the interface, but it has to slide along this interface. Thus, cells 16 may aggregate at the bottom of microdroplets 14 and, in the case of some scaffold-dependent cells, optionally aggregate to form spheroids.

The microfluidic method proposed here includes the steps of capturing these microdroplets and then gelling the microdroplets.

This step can be carried out in various ways, among other things, depending on the hydrogelling agent used. Therefore, according to the first example, the hydrogel contains agarose, preferably the hydrogel is agarose. The microdroplets are then gelled by cooling the microfluidic chip. If the hydrogel contains agarose, or preferably the hydrogel is alginate, it provides calcium ion Ca ²⁺ in the oil in which the microdroplets are immersed, or actually alginates the calcareous particles. It is possible to premix with and saturate the oil in which the microdroplets are immersed in CO ₂ . Therefore, alginate is acidified and calcium ions are released. It goes without saying that other gelling means can also be used because other gelling agents can be used.

Furthermore, this gelling step can be performed at different time points between handling methods, depending on the application sought. In particular, gelation can be performed immediately after capture so as to anchor the cells in place within the microdroplets and prevent them from settling. At that time, it is possible to observe the cells independently of each other. Alternatively, gelation is performed after the cells have settled to form spheroids. This makes it possible to observe the behavior of cells that have formed spheroids. According to another alternative form, the microdroplets are, for example, after work to handle cells in a liquid medium to selectively extract certain cells, cells in non-gelled microdroplets. It is only gelled. It may be useful for scaffold-independent cells such as bacteria or erythrocytes and leukocytes.

After gelation, it is possible to replace the oil in which the microdroplets are immersed with an aqueous solution that specifically contains a biochemical solution containing biochemical components such as nutrients, growth factors, antibodies, drugs, drug molecules and the like. .. These biochemical components diffuse through the gel and reach the cells. Therefore, it is possible to study the response of cells in the form of independent or spheroids to these stimuli. Thus, hydrogels can keep cells in place while allowing the injection of cells in the aqueous phase and pre-dividing the biological sample during encapsulation of the cells into microdroplets. It becomes.

In order to carry out this operation successfully, it is preferable to extrude the surfactant from the interface of the microdroplets. This is because the shell that the surfactant forms at the interface of the microdroplets is so effective that the aqueous phase injected to replace the oil fills the microfluidic chips and fills the microfluidic chips with their respective microdroplets. This can prevent it from remaining gelled in the trap. The presence of the surfactant prevents the coalescence, so that the interface of the aqueous phase reaches the trap, resulting in a force applied to the gelled microdroplets, which the microdroplets capture the microdroplets. If the constituent hydrogels are sufficiently compressible, they can be extruded from the trap. This is the reason why it is preferable to promote coalescence by reducing the concentration of surfactant at the interface. For this reason, a surfactant-free oil, unlike previously used oils, is injected into the microfluidic chip prior to the injection of the aqueous phase. The concentration of surfactant in the oil of the microfluidic chip is reduced, which allows the equilibrium to shift from adsorption to desorption of the surfactant at the interface. For example, for a high concentration surfactant of about several mass percent, it is preferable to inject an amount of oil corresponding to 50 times the volume of the microfluidic chip into the microfluidic chip. This ratio depends on the nature of the surfactant and on its / their affinity for the two phases.

The shape of the trap can also be optimized to ensure that the gelled microdroplets are held in place within the trap. Therefore, in the case of a sufficiently deep cylindrical trap, if the channel height is greater than the trap radius, the entry of microdroplets into the trap will be minimal and the resulting capture efficiency will be low. Subsequently, there is a limit to the velocity of the outer flow of microdroplets that are pushed out of the trap when crossed. Conversely, if the channel height is smaller than the trap radius, the microdroplets will significantly enter the trap cavity as long as they are large enough, resulting in higher trapping efficiency. The microdroplets stay in place regardless of the velocity of the outer flow. In the first case, the shape of the microdroplet is very close to its shape in the channel, while in the second case it is locally trapped.

If the gelling agent for the hydrogel selected is reversible, the microdroplets are non-gelled and then the contents of the microdroplets are expelled from the microfluidic chip, as shown in FIGS. 4-6. It is possible. In the case of these FIGS. 4 to 6, for example, the microdroplet 14 is a gelled microdroplet of agarose. These agarose microdroplets 14 are non-gelled one by one, among other things, by locally heating the microdroplets with an infrared laser or electrodes (indicating heating by lightning bolt 21). This heat liquefies agarose. When the phase surrounding the microdroplets 14 is aqueous, the non-gelled agarose content 16 mixes with the aqueous phase. In that case, the contents can be carried using the aqueous phase stream 22 for optional recovery. Thereby, it is also possible to eliminate cells that are considered uninteresting by the microfluidic chip 10. Again, the shape and size of the trap is preferably chosen to allow the extraction of cells. For example, if the cells must remain alive, the size of the trap is large enough for heating the hydrogel so as not to induce cell death mechanisms.

Alternatively, if the phase surrounding the microdroplets is oily, an oil stream can be applied to remove the liquid microdroplets from the trap. In this case, the shape and strength of the trap are preferably sized to allow only the extraction of selected microdroplets and not to extract other microdroplets. This sizing depends, among other things, on the value of surface tension between the aqueous phase and the oil, as well as the stiffness of the gel microdroplets and the shape of those microdroplets in the trap.

Another alternative is to keep the microdroplets in liquid to handle suspended cells, such as bacteria. In that case, only gelation is performed for the selective extraction of microdroplets. In this case, it is possible to gel all the microdroplets prior to extraction and apply the protocol described above, or on the other hand, gel only the microdroplets that are desired to be kept in the trap.

With a few modifications, the process presented allows us to explore a wide variety of biological applications. In either case, it goes without saying that the device can also be modified by adjusting the height of the channels within the microfluidic chip and the geometry of the trap.

Thus, for example, rapid gelation with low concentrations of cells allows individualization of several single cells within each microdroplet while attempting to limit direct cell interactions. These cells may be, for example, bacterial, yeast, or mammalian cells.

Alternatively, high concentrations of cells, also also with rapid gelation, make it possible to obtain a large number of cells that are also individualized but in close proximity to each other. At that time, for example, the interaction between cells can be investigated via paracrine secretion, in some cases during co-culture.

However, cells may be encapsulated and maintained in the liquid phase for extended periods of time prior to gelation. At that time, the low concentration of cells makes it possible to investigate scaffold-independent cells such as suspended lymphocytes. The high concentration of cells, and the shape of the captured microdroplets that allow them to settle, allows them to rally cells that can reorganize themselves into spheroids.

This method also allows control to form spheroids directly within the chip. The volume of microdroplets produced can be controlled by a device for forming microdroplets upstream of the microfluidic chip. This volume is preferably adjusted so that once captured, the diameter of the microdroplets is equal to the diameter of the trap and the shape is spherical. For this reason, the depth of the trap is preferably at least equal to the diameter of the trap. By matching the diameter of the captured microdroplets with the diameter of the trap, it is possible to ensure high capture efficiency. The spherical shape promotes the formation of spheroids. In this particular application, the microdroplets preferably contain suspended cells in an aqueous phase comprising culture medium and hydrogel, or consisting of culture medium and hydrogel. Once the trap is filled with such microdroplets, the external flow of oil is stopped, thereby stopping recirculation within the microdroplets and facilitating cell sedimentation. At that time, if the shape of the microdroplets in the trap is spherical, the assembly at the bottom of the cells is induced until the cells come into contact with each other. In particular, under conditions favorable for cell survival and proper metabolic function in terms of temperature, the chips are then aggregated at the bottom of the captured microdroplets by allowing the chips to stand for a duration ranging from hours to days. It is possible to reorganize the cells into spheroids.

The duration required for spheroid formation may depend, among other things, on the cell type used and on the composition of the hydrogel. It has been observed that this period is less than 24 hours for H4IIEC3 rat hepatocytes in 1% by mass agarose solution diluted in culture medium. It goes without saying that the hydrogel remains liquid during the period of spheroid formation.

This method makes it possible to quickly form a large number of spheroids of very uniform size. In practice, the size of the spheroid is given by the number of cells encapsulated within each microdroplet and thus by the concentration of the cell solution injected into the microfluidic chip. The distribution of the number of cells per microdroplet, and thus the size of the spheroids formed, is very uniform as long as the cells are sufficiently individualized at the time of injection. In experiments conducted by the inventors, an average of 98% of traps were filled with a single microdroplet of liquid agarose containing well-rearranged spheroids after a 24-hour incubation.

The spheroids obtained in the microfluidic chip may be maintained in culture for several days. For example, a week of H4IIEC3 cell spheroids encapsulated in agarose without significantly altering its viability and preserving high functionality (strong and continuous albumin secretion in this example). It can be cultured in chips.

The method presented here and the microfluidic chips obtained by performing the method constitute an excellent tool for screening drugs. For example, it is possible to produce spheroids in cancer cells and observe whether the viability of those spheroids diminishes over time with exposure to the molecule being tested. It is possible to test the entire concentration range in the same system by adding a device to the chip that allows the concentration gradient to be set in the chamber, or by installing a parallel chip. The high efficiency of the methods for forming these spheroids also makes it possible to produce a large number of spheroids from a very limited sample. Therefore, 500 spheroids having a diameter of about 70 μm can be formed with only 100,000 cells.

The cells that make up the spheroid may also be of different types to address the theme of co-culture. These cell types can be mixed uniformly in solution prior to injection into the chip, or after injection into the external aqueous phase, they are adhered to several consecutive hydrogel layers, or simply to the hydrogel. Thereby, it can be arranged according to a specific structural structure. For example, fibroblasts and epithelial cells to form a skin model and test cosmetic toxicity, neurons and astrocytes to model the brain, or endothelial cells as well as vessel walls. It is possible to bind to smooth muscle cells.

This method is also an excellent tool for studying stem cell differentiation, as the method presented here allows for a very high degree of control of the microenvironment of cultured cells. In practice, the encapsulated cells can be provided over the entire concentration range of the differentiation factor, and potentially at the same time, over the entire stiffness range of the matrix, eg, adjusting the hydrogel concentration. Similarly, this method can be used to observe embryogenesis interacting with physicochemical factors from external media over time.

In the case of patient-derived primary cells, this method makes it possible to make a medical diagnosis based on the cell's response to a particular marker. In this case, the loss is very low and the cells are captured. At that time, it is possible to perform known tests on cells for diagnosing a specific disease, such as characterization of cancer biopsy. For example, it is possible to test for the presence of mutations in the genome of encapsulated cells, for example by in-situ polymerase chain reaction (PCR) or by FISH method. It is also possible to detect the expression of a specific protein by the labeling method, for example, by supplying an antibody for immunolabeling or for an in-situ immunoenzymatic method, ELISA (enzyme-linked immunosorbent assay). ..

The method described above offers the possibility of performing all cell analysis and cell culture steps within the chip by gelling the microdroplets following capture of the microdroplets within the microfluidic chip. This allows the use of much smaller amounts of reagents than for tests performed on multi-well plates or culture dishes. This also makes it possible to monitor cellular responses over time after different stimuli.

The above method
-Means for forming hydrogel microdroplets containing cells,
-A capture zone for capturing hydrogel microdroplets in place, especially with a microfluidic chip,
-Can be easily implemented in devices that include means for gelling at least a portion of the captured microdroplets.

The gelling means include, for example, a device for injecting a chemical agent into the capture zone and / or, for example, a temperature control means for cooling the microfluidic chip.

The device can also include means for non-gelling at least a portion of the gelled hydrogel microdroplets, such as a laser.

Gelation, preferably cryopreserved, each comprising a zone for capturing microdroplets, particularly a microfluidic chip, and one or more cells trapped within the trapping zone by the method described above. It is also possible to produce a gelled microdroplet product containing the microdroplets. The cells may aggregate in the form of clusters or spheroids. The gelled microdroplets may be immersed in a fluid, preferably in an aqueous solution or oil, but the fluid and microdroplets are preferably cryopreserved. This cryopreservation allows the cells to be maintained for long periods of time under stable conditions, especially for the purpose of transporting or preserving the cells for later analysis.

Living cells encapsulated in microdroplets are bacterial, yeast, eukaryotic cells, mammalian cells, preferably mammalian cells other than human cells, more preferably cells from rat cells or other mammals, or cells thereof. It may be a human cell isolated from the natural environment.

It goes without saying that the present invention is not limited to the above-mentioned examples, but rather, within the scope of the appended claims, there is room for many modifications that can be understood by those skilled in the art.

In particular, the sample used may be a molecule or, among other things, plastic beads functionalized by binding to a molecule, apart from cells.

Furthermore, the captured microdroplets can be fused with other microdroplets supplied by the flow of aqueous solution.

The aqueous solution constituting the microdroplets can also contain a biochemical solution, which preferably contains lipids (such as fatty acids), carbohydrates (such as monomeric or polysaccharide), amino acids and proteins (growth factors). , Cyclogs, antibodies, antigens, etc.), and at least one of saline and / or pH buffers.

Finally, according to one variant, the oil (or oil phase) surrounding the microdroplets can contain fluorine oil (FC40 type) or photocrosslinkable water immiscible solution (Norland Optical Adaptive type). Once polymerized, they gel the oil, which allows the microdroplets to be physically and selectively isolated. Therefore, it is possible to more reliably separate the microdroplets from each other. This makes it possible to prevent the two microdroplets from fusing and mixing the samples contained in the microdroplets. This also allows the sample to be permanently stored, with the risk of microdroplet evaporation being particularly significantly reduced by segmenting the microdroplets with gelled oil that forms a solid compartment around the microdroplets. It becomes.

Once a portion of the oil has gelled, it is possible to expel microdroplets around which the oil has not gelled from the capture zone. This allows the use of an oil stream or another fluid that is strong enough to carry microdroplets in the capture zone. Therefore, it is possible to retain only the microdroplets of oil gelling around them in the capture zone.

It should be noted here that microdroplets can gel even when the oil is gelling. In addition, it goes without saying that if part of the oil is gelled, this method can include a subsequent step of non-gelling the gelled oil.

10 Capture zone 12 Surface tension trap 14 Microdroplets 16 Living cells 18 Arrow 20 Hydrogel solution 21 Lightning 22 Flow

Claims (26)

A method for handling microdroplets (14) containing sample (16) in a microfluidic system.
i) A step of forming microdroplets (14) of an aqueous solution containing a sample (16) in oil, wherein at least one of the oil and the aqueous solution containing the sample (16) contains a gelling agent. When,
ii) A step of capturing microdroplets (14) by a surface tension trap (12) pre-arranged in the capture zone (10).
iii) A method comprising the step of gelling at least a portion of the oil in the capture zone and at least one portion of the captured microdroplets (14). The method of claim 1, wherein step iii) is to gel at least a portion of the oil in the capture zone. The method of claim 1, wherein step iii) is to gel at least a portion of the microdroplets. Sample (16) is of one or more cells, especially cell spheroids, one or more beads that capture molecules, especially plastic beads, or one or more molecules. The method according to any one of claims 1 to 3, which is one. 13. The aspect of any one of claims 1 to 4, wherein step iii) is performed on the sample (16) in the captured microdroplets (14), especially after cell sedimentation, especially after spheroid formation. Method. The method according to any one of claims 1 to 4, wherein the step iii) is performed before the sample (16) is settled in the captured microdroplets (14). iv) Further comprising the step of replacing the oil surrounding the gelled microdroplets (14) with an aqueous solution.
The method according to any one of claims 1 to 6, which is combined with claim 3. The aqueous solution that replaces the oil contains the biochemical solution, which is preferably one or more pH buffers or saline buffers, one or more nutrients, one or more growth factors, cytokines. One or more antibodies, one or more antigens, especially one or more molecules of pharmaceuticals, one or more cells, lipids, especially monomeric or polysaccharide carbohydrates, amino acids and / or proteins The method of claim 7, comprising at least one of them. The method of any one of claims 1-8, wherein the capture zone is formed by a microfluidic chip (10) that includes a surface tension trap (12). Step (i) is
a) An aqueous solution containing the sample (16) and, if necessary, a gelling agent is injected into the zone upstream of the capture zone (10).
b) In order to expel the aqueous solution containing the sample (16) toward the outlet of the capture zone (10), if necessary, oil containing the gelling agent was placed in the zone upstream of the capture zone (16). Infused to form the contained microdroplets (14) and then
c) The invention according to any one of claims 1 to 9, wherein the microdroplets (14) are moved to the capture zone (10) and the microdroplets (4) are captured in the capture zone (10). Method. Step i) and step ii)
-Fill the capture zone (10) with an aqueous solution containing sample (16) and, if necessary, a gelling agent, followed by
-In order to expel the aqueous solution containing the sample (16) toward the outlet of the capture zone (10), an oil containing a gelling agent is injected into the capture zone (10) as needed, and a surface tension trap (10). The capture zone (10) by performing certain tasks in which 12) is adapted to allow crushing of microdroplets (14) containing sample (16) with a surface tension trap (12). The method according to any one of claims 1 to 9, which is carried out at the same time in. Process iii)
-Cooling or heating microdroplets (14) and / or oil,
− Injecting a solution containing a chemical gelling agent into the capture zone,
-The method of any one of claims 1-11, wherein the microdroplets (14) and / or oil are exposed to at least one of the light that causes gelation, especially UV light. The method according to any one of claims 1 to 12, wherein the oil contains a surfactant and preferably comprises a step of washing the surfactant before step iv). The method according to any one of claims 1 to 13, wherein the step of selecting the shape of the surface tension trap (12) according to the desired shape of the microdroplet (14) is included before the step i). The capture zone and trap
-To form a captured microdroplet (14) with a flat bottom, or-to form a captured microdroplet (14) with a non-flat, especially curved, preferably convex bottom. The method of claim 14, which is selected in. 1. The step v), which is to non-gelling at least a part of the gelled microdroplets (14) in the step iii), is included following the step iii), preferably the step iv). Or the method according to any one of claims 1 to 15, which is combined with 3. Step vi) in which the non-gelled microdroplets (14) and / or the sample (16) contained in these non-gelled microdroplets (14) is expelled from the capture zone (10). 16. The method of claim 16, comprising following step v). Claims 1 to 17, comprising stimulating the sample (16) contained in at least a portion of the captured microdroplets, gelled microdroplets or non-gelled microdroplets (14). The method according to any one item. The step of expelling the non-gelled microdroplets around it from the capture zone is combined with claim 1 or 2, comprising step iii) followed by any one of claims 1-18. The method of description. -Means for forming microdroplets (14) containing sample (16), and
-A capture zone for capturing microdroplets (14) in place, especially with a microfluidic chip,
-A device for performing the method according to any one of claims 1 to 19, comprising means for gelling at least a portion of the captured microdroplets (14) and / or oil. 20. The device of claim 20, wherein the gelling means comprises a device for injecting a chemical agent into the capture zone (10). 21. The device of claim 21, comprising means for non-gelling at least a portion of the gelled hydrogel microdroplets (14) and / or a portion of the gelled oil. A zone for capturing microdroplets, particularly a microfluidic chip (10), and a gelled micro, each containing a sample (16) and captured in the capture zone (10), preferably cryopreserved. The product of gelled microdroplets, including the droplet (14). 23. The product of claim 23, wherein the gelled microdroplets are immersed in a fluid, preferably in an aqueous solution or oil, and the fluid and microdroplets (14) are preferably cryopreserved. A microliquid containing a zone for capturing microdroplets, particularly a microfluidic chip (10) and microdroplets (14), each containing a sample (16) and trapped in the capture zone (10). The product of the droplets, in which the microdroplets (14) are immersed in the gelled oil and the microdroplets (14) and the gelled oil are preferably cryopreserved. Stuff. Claim (16) is a bead that surface captures mammalian cells, preferably cells from mammals other than human cells, bacteria, yeast, or other cells, molecules, or molecules used in a bioprocess. The product according to any one of 23 to 25.

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