Method and device for sorting organoid drops by acoustic actuation

Abstract

Method and device for sorting organoid drops by acoustic actuation The present invention relates to a method for sorting organoid drops according to a predefined parameter by acoustic actuation. The present application also relates to a device for sorting organoid drops.

Description

Title of the invention: Method and device for sorting organoid drops by acoustic actuation Technical field

[0001] The present invention relates to the technical field of organoids. It relates more particularly to the field of organoid sorting. Technological background

[0002] Cell sorting is a key operation in a number of biotechnological processes. Microfluidics has emerged in recent years as a technology enabling the miniaturization and automation of very high-throughput biological assays, at size scales that allow the analysis of individual cells.

[0003] In recent years, cellular models that are increasingly close to biological reality have been developed, such as organs on chips (OoCs), organoids and spheroids. These structures make it possible, in particular, from human cells, immortalized, primary or derived from stem cells, to establish biological models, designed to reproduce certain physiological and pathophysiological characteristics and functions of human tissues. Organoids and organs on chips reproduce the functionalities of organs and the cells constituting them self-organize according to the 3D environment in which they are immersed.These new models could ultimately contribute to advancing our understanding of the human body, revolutionizing pharmaceutical research by supporting the development of drug candidates, testing the toxicity and efficacy of candidates, improving the success rate of clinical trials while limiting animal testing, and reducing the costs and time taken to develop candidate molecules for marketing.

[0004] Organoids, spheroids, or organs-on-chips are on the order of 10 to 1000 times larger than individual cells. Techniques that have been developed and optimized for the manipulation and use of individual cells cannot be simply transposed to organoids and spheroids without considering the scaling challenge. In particular, microfluidic systems, which are widely used and optimized for the manipulation of individual cells, are not optimized to operate reliably, robustly, and rapidly at larger scales.

[0005] In general, objects such as organoids or spheroids are analyzed and / or sorted by hand, or possibly using pipetting robots. These methods are efficient but limited by the time required for their implementation and reproduction. tibility. The recent development of high-throughput methods for manufacturing spheroids and organoids also requires the development of faster, more automated analysis and / or sorting techniques, preferably operating in a more reproducible manner.

[0006] As described above, the techniques conventionally used for sorting cells such as flow cytometry analysis with FACS (Fluorescence Activated Cell Sorting), cannot be simply transposed to larger objects such as organoids without taking into account the change in scale.

[0007] The publication Freyer et al. Cytometry 1987, 8:427-436 describes a flow cytometry method for sorting spheroids from 41 to 96 micrometers in diameter using a modified commercial device. However, such systems, and in particular the nozzles, are not really suitable for such sizes. Furthermore, this technique is not usable for larger objects, in particular because of the clogging of the channels and the sedimentation of the objects in the channels.

[0008] Furthermore, organoids produced by the same process may vary in terms of physical properties, in particular their size or shape. The need for analysis and sorting therefore exists during production, and the organoids must be able to be re-cultured after the sorting and / or analysis step. This is why it would be useful to be able to use techniques that do not involve their labeling, for example with fluorescent markers or magnetic particles. Similarly, sorting techniques involving the use of an electric field require the generation of a dielectric contrast which is obtained by encapsulating the cell aggregates in drops of water in oil. The presence of oil, or other cytotoxic agents, is incompatible with the re-culture of the organoids after the sorting step.

[0009] Certain forces that can be used for sorting organoids, such as high-intensity electric fields or shear forces, can further impact cell proliferation, which can also pose a problem for re-culturing organoids after sorting.

[0010] It would therefore be useful to have a method for sorting organoids according to predefined parameters, suitable for a scale range from tens of microns to millimeters, involving no labeling and no cytotoxic agent. Advantageously, this method should be applicable for sorting organoids according to different parameters.

[0011] In this context, the inventors have surprisingly demonstrated that it is possible to efficiently and simply sort drops containing organoids according to one or more predefined parameters using acoustic waves. The method thus makes it possible to recover from different containers drops of continuous phase containing organoids having a predefined value of at least one predefined parameter. This method is versatile and can be implemented to sort the organoids according to a wide variety of parameters, by simply adapting the detection and / or analysis device. In addition, the method according to the invention does not affect the properties of the organoids or cell viability, which allows the re-culture of the organoids after the sorting step. Summary of the invention

[0012] Thus, a first object of the present invention is a method for sorting drops comprising an aqueous continuous phase comprising organoids, comprising, for each drop, the following steps: a. Generation of a drop of the aqueous continuous phase containing organoids, b. Determination of a P value of at least one drop parameter, c. Comparison of the P value determined in step b. to a reference value, d. Application to the drop of an acoustic field whose intensity and / or phase depends on the result of the comparison of step c., and e. Collecting the drop in a container.

[0013] In certain embodiments, the acoustic field applied in step d. is of zero intensity if the value P is less than the reference value, and of non-zero intensity if the value P is greater than or equal to the reference value.

[0014] In certain embodiments, the intensity and / or the phase of the acoustic field applied in step d. can take n different values, n being an integer greater than or equal to 2, and the method makes it possible to sort the drops into n different containers according to the n intensity and / or phase values ​​of the acoustic field.

[0015] In certain embodiments, the drop is generated in step a. by conveying the aqueous continuous phase comprising organoids into a glass capillary, in particular a glass capillary of square section, at the outlet of which they are encapsulated in drops of water.

[0016] In certain embodiments, the parameter whose value is determined in step b. is chosen from the group consisting of the presence of an organoid in the drop, the shape of an organoid present in the drop, the size of an organoid present in the drop, the thickness of an organoid present in the drop, the sphericity of an organoid present in the drop, the number of organoids present in the drop, the nature of the cells of an organoid present in the drop, the fluorescence of an organoid and / or of the capsule of an organoid present in the drop and the light intensity transmitted or absorbed by an organoid present in the drop.

[0017] In some embodiments, the organoids are formed in an alginate shell.

[0018] In some embodiments, the diameter of the organoids is between 50 micrometers and 4 mm, preferably between 300 micrometers and 400 micrometers.

[0019] In some embodiments, the device for determining the value P of the parameter in step b. comprises a sensor, such as a camera, and an image analysis device such as image analysis software.

[0020] A second subject of the invention is a drop sorting device for implementing the drop sorting method according to the invention, said device comprising a system for generating drops of an aqueous continuous phase comprising organoids, a system for generating an acoustic field, a device for measuring and / or analyzing at least one parameter, and at least two drop recovery containers.

[0021] In certain embodiments, the device for measuring and / or analyzing at least one parameter comprises a camera associated with image analysis software. Brief description of the drawings

[0022] [Fig. 1] is a diagram showing a device for sorting drops by acoustic actuation according to the invention. The aqueous continuous phase comprising organoids is introduced at the end A of the capillary. The drops are formed at the end B of the capillary. The sensors C and D make it possible to measure at least one parameter of the drops and / or organoids in the capillary just before the formation of the drop. The acoustic transducers located on hemispherical matrices E generate an acoustic field making it possible to deflect the drops and recover them in the containers F and G.

[0023] [Fig.2] shows the results of sorting spheroids according to their size using a method according to the invention. B represents the population sorted positively for the criterion R < 0.8xR0 where R is the radius of the spheroid and Ro the average radius of the spheroids, and C represents the population sorted negatively for this criterion. Detailed description

[0024] A first object of the invention is a method for sorting drops comprising an aqueous continuous phase comprising organoids, comprising, for each drop, the following steps: a. Generation of a drop of the aqueous continuous phase containing organoids, b. Determination of a P value of at least one drop parameter, c. Comparison of the P value determined in step b. to a reference value, d. Application to the drop of an acoustic field whose intensity and / or phase depends on the result of the comparison of step c., and e. Collecting the drop in a container.

[0025] By "drop parameter" is meant either a drop parameter as such as is formed in step a., such as for example the number of organoids in the drop, or a parameter of an organoid which is present in the drop after its generation, in particular in the case where the drop comprises exactly one organoid.

[0026] Steps a. to c. of the method according to the invention may be implemented in any order, provided that step c. of comparison is implemented after step b. of determination. In particular, step b. of determining the value P of at least one parameter may be implemented after generation of the drop (step a.). However, in certain preferred embodiments, step b. and step c. are implemented before step a. of generation of the drop. In such embodiments, the value P of at least one parameter is determined and optionally compared to a reference value when the organoid is in the aqueous continuous phase, before generation of the drop. The value P of at least one parameter is assigned to the drop after generation thereof.

[0027] The drop sorting method comprising a continuous aqueous phase comprising organoids according to the invention allows rapid and efficient sorting without requiring expensive equipment. In addition, the device for implementing this method is compact, which allows, for example, it to be installed under the flow of a microbiological safety station.

[0028] Definitions

[0029] An "organoid" is an aggregate of cells growing three-dimensionally in suspension and developing complex interactions between cells or with a three-dimensional matrix. It is thus a cohesive 3D structure. Their primary interest is to reproduce an architecture similar to that of the original tissue which will generate within it different partial gas pressure gradients. Organoids, most often close to the spherical shape for reasons of surface tension, can however take other forms. Organoids have a characteristic size which can range from ten microns to millimeters.

[0030] In the present application, the term "organoid" is used in a broad sense and can include both organoids in the strict sense and spheroids. Organoids are complex clusters of cells specific to an organ. They consist of stem cells or progenitor cells and self-assemble when subjected to an extracellular scaffolding environment, such as Corning® Matrigel® matrix or collagen. When this occurs, they transform into microscopic versions of viable parent organs for 3D study. Although organoids are widely used in research on pathologies such as cancers, the cells present in organoids are not necessarily pathological cells.

[0031] "Spheroids" are simpler 3D structures prepared from immortalized cell lines, including immortalized cancer cell lines.

[0032] A "drop" is a very small quantity of liquid dispersed in a distinct external medium such as air and which detaches in a more or less spherical form. The volume of a drop may in particular be between 10 microliters and 100 microliters, preferably between 15 microliters and 30 microliters.

[0033] An “aqueous continuous phase containing organoids” is a solution or suspension of organoids in an aqueous medium. The concentration of organoids in the aqueous continuous phase is preferably between 1 and 50 organoids per mL, preferably it is approximately 5 organoids per mL. The concentration of organoids in the aqueous continuous phase can be adjusted by a person skilled in the art, in particular depending on the volume of the drops and / or the number of organoids desired per drop. In certain embodiments, the concentration of organoids in the aqueous continuous phase is adjusted so as to avoid having more than one organoid per drop, for example so as to have approximately one organoid every 10 drops.

[0034] Drops

[0035] Step a. of the method is the generation of a drop of aqueous continuous phase containing organoids.

[0036] Each drop of aqueous continuous phase comprising organoids may contain either aqueous continuous phase without organoids, or aqueous continuous phase and one or more organoids due in particular to the random distribution of the dispersion of the organoids in the continuous phase. In a preferred embodiment, each drop contains either no organoids or exactly one organoid.

[0037] The aqueous continuous phase comprising organoids may be a suspension of organoids in an aqueous medium. The aqueous medium may be any suitable aqueous medium, including water, a culture medium, or an aqueous buffer solution. In certain embodiments, particularly in the case where the organoids comprise living cells, the aqueous medium is preferably an aqueous solution that avoids osmotic shock, calcium shock, or shock generating oxidative stress. It may include a culture medium optimized for cell culture, preferably devoid of growth factors or other hormones. For example, the aqueous medium may be selected from the group consisting of Minimum Essential Medium (MEM), Dulbecco's modified Eagle medium (DMEM), RPMI (Roswell Park Memorial Institute medium), and saline solutions buffered around pH 7 such as phosphate buffered saline (PBS) and HEPES buffer.

[0038] The generation of the drops can be done by any suitable means, in particular using a microfluidic device. In certain embodiments, in particular when the determination of the value P of the at least one parameter is implemented before the generation of the drop, the drop generation device, in particular the microfluidic device, must allow the measurement of the value P. Thus, in the case where the measurement technique is image capture, the drop generation device must allow image capture. It is therefore preferably a drop generation device of which at least part of the wall is transparent.

[0039] The radius of each drop may vary depending on the nature of the aqueous phase, the concentration of organoids in the aqueous continuous phase, the drop generation device used, and the flow rate of the aqueous continuous phase comprising organoids in particular. In certain embodiments, the radius of the drops is between 120 micrometers and 5 millimeters, preferably between 300 micrometers and 5 millimeters, preferably between 500 micrometers and 2 mm, in particular it is approximately 1.5 mm. In certain embodiments, in particular in the case where the organoids do not comprise living cells, the radius of the drops may be modulated by other factors such as the presence of at least one surfactant in the aqueous continuous phase.

[0040] Analysis and sorting are generally facilitated in the case where each drop comprises at most one organoid. Thus, the use of smaller drops makes it possible to increase the concentration of organoids in the continuous aqueous phase comprising organoids.

[0041] In some embodiments, an electric field is applied to the aqueous continuous phase comprising organoids at the location where the drops are generated. This makes it possible in particular to reduce the size of the drops.

[0042] In some embodiments, the drops are generated as a continuous flow, including a continuous flow of the order of 200 microliters per second.

[0043] The drops can be generated in any suitable medium, in particular under an inert atmosphere or in air, at atmospheric pressure, at reduced pressure or in hyperbaric conditions, in particular depending on the nature of the organoids and the purpose of the sorting.

[0044] In some embodiments, the generated drops are water drops that are generated in air at atmospheric pressure.

[0045] The drops can be generated in any direction and in any suitable sense depending on the characteristics of the process, in particular depending on the measuring or analysis device(s) used to determine the parameters.

[0046] In a preferred embodiment, the drops are generated in a vertical or approximately vertical direction, and downward, under the action of gravity. Preferably, the drops are in free fall between their generation and their entry into the acoustic field, and between their exit from the acoustic field and their recovery in a container.

[0047] In certain embodiments, the organoids are conveyed in the form of an aqueous solution or suspension into a glass capillary, in particular a glass capillary of rectangular or square section, at the outlet of which they are encapsulated in water drops. The side of the capillary is preferably of the order of 500 micrometers to 1 millimeter. Flow in the capillary can be achieved using a pressure control unit or a syringe pump system. In such an embodiment, the maximum flow rate at the capillary outlet is of the order of 1 mL / second. Indeed, the flow rate used must be compatible with the formation of drops, avoiding the generation of continuous jets.

[0048] Organoids

[0049] The organoids present in the continuous phase drops sorted according to the invention can be of any size and any shape.

[0050] Each drop subjected to the method according to the invention may comprise one or more organoids; alternatively, it may not comprise any organoids. Preferably, each drop subjected to the method according to the invention comprises zero or exactly one organoid.

[0051] In one embodiment, the organoids have a diameter of between 50 micrometers and 4 mm, preferably between 50 micrometers and 700 micrometers, in particular between 200 micrometers and 400 micrometers.

[0052] In one embodiment, the organoids have an approximately spherical shape. In some embodiments, the organoids are in solid form, such as balls for example. In other embodiments, the organoids are in the form of cysts, that is to say they have an envelope of cells, which surrounds an “empty” core, which does not include cells.

[0053] In one embodiment, the organoids are formed in a shell such as a hydrogel shell, in particular an alginate shell. In such a case, what is referred to as the size of the organoid in the present invention is the size of the shell, i.e. counting the thickness of the shell, in particular the alginate shell, in addition to the size of the organoid as such. In such an embodiment, the parameter P of the organoid (or droplet) that is determined in the method according to the invention preferably does not take into account the shell, i.e. if the parameter of which a measurement P is determined is a dimension of the organoid, it is preferably the dimension without the shell.

[0054] The organoids may be produced by any suitable method known in the art. In some embodiments, the organoids are produced by a high-throughput technique, such as those described in M. Trossbach, al., High-throughput cell spheroid production and assembly analysis by microfluidics and deep learning, SLAS Technology 2023, 28(6), 423-432, K. Alessandri et al., Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro , PNAS 2013, 110(37), 14843-14848 or N. Brandenberg et al., High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays, Nat Biomed Eng. 2020;4(9):863-874.

[0055] The method according to the invention can be implemented with organoids comprising any type of cells, preferably mammalian cells.

[0056] Parameters

[0057] The method according to the invention makes it possible to sort drops of aqueous continuous phase comprising organoids according to the value of at least one parameter. Step b) of the method according to the invention is the determination of a value P of the at least one parameter of the drop, and step c) of the method is the comparison of this value P with a reference value.

[0058] Among the parameters that can be determined for a drop and / or for an organoid by the method according to the invention, there may be mentioned in particular: a. The presence of an organoid in the drop, b. The shape of an organoid present in the drop, c. The size of an organoid present in the drop, d. The thickness of an organoid present in the drop, e. The sphericity of an organoid present in the drop, f. The number of organoids present in the drop, g. The nature of the cells of at least one organoid present in the drop, h. The fluorescence of at least one organoid and / or the capsule of at least one organoid present in the drop, and i. The light intensity transmitted or absorbed by at least one organoid present in the drop.

[0059] Other parameters can be determined, provided that there is a device for measuring and / or analyzing said parameter making it possible to determine a value of the parameter and / or to compare this value with a reference value.

[0060] In some embodiments, the parameter is not optical absorbance, nor fluorescence.

[0061] In some embodiments, the detected parameter is the presence of an organoid in the drop, the value P is equal to 0 if no organoid is present in the drop, and the value P is equal to 1 if an organoid is present in the drop. In this case, the reference value may be the value 1. Thus, if no organoid is present in the drop (P=0), the intensity of the acoustic field applied in step d. may be zero. If an organoid is present in the drop (P=1), the intensity of the acoustic field applied in step d. may be non-zero.

[0062] Thus, the method according to the invention comprises in certain embodiments the following steps: a'. Generation of a drop of the aqueous continuous phase containing organoids, b'. Determination of the presence or absence of at least one organoid in the drop, c'. Application of an acoustic field to the drop of non-zero intensity if the presence of at least one organoid in the drop is determined in step b', and of zero intensity if the absence of organoids in the drop is determined in step b', and d'. Recovery of the drop in a container, preferably a different container depending on whether the absence or presence of organoids in the drop is detected.

[0063] Step a'. may be carried out before or after step b'. Preferably, it is carried out after step b'., i.e. the presence or absence of organoids in the volume of continuous aqueous solution corresponding to a drop is determined before the generation of the drop.

[0064] The particular embodiments are described by assigning respectively zero and non-zero intensity values ​​to the acoustic field as a function of the value P of the detected parameter, but the method can be implemented with any intensities and / or phases of the acoustic field from the moment when different intensities and / or phases are applied for different values ​​of the detected parameter.

[0065] Preferably, the intensities and / or phases of the acoustic field differ sufficiently to allow deflection of the drops allowing their recovery in containers positioned at distinct locations.

[0066] A person skilled in the art can easily determine, depending on the conditions of implementation of the method, the intensities and / or phases of the acoustic field to be applied for each determined value of the parameter and for each result of comparison of the latter with a reference value.

[0067] In some embodiments, the detected parameter is the shape of an organoid in the drop, the value P is equal to 0 if the shape of the organoid does not correspond to a predefined shape, and the value P is equal to 1 if the shape of the organoid corresponds to a predefined shape. In this case, the reference value may be the value 1. Thus, if the organoid does not have the predefined shape, the intensity of the acoustic field applied in step d. may be zero. If the organoid has the predefined shape, the intensity of the acoustic field applied in step d. may be non-zero.

[0068] In certain embodiments, the detected parameter is the light intensity transmitted by an organoid. The detection of this parameter can in particular make it possible to separate organoids which are full from those which are in the form of a cyst.

[0069] Determination of parameters

[0070] The parameters may be determined by the use of any measuring and / or analyzing device suitable for the parameters, or any suitable combination of such devices.

[0071] The measuring and / or analysis device is preferably located at the drop formation zone. Thus, in the case where the drops of solution or suspension aqueous organoids are formed at the outlet of a capillary, the measuring and / or analysis device is preferably located at the outlet of the capillary, preferably to measure the parameter inside the capillary.

[0072] The frequency of determining the parameters must be fast enough so that the parameters are determined for each drop, in particular when the drops form a continuous flow.

[0073] In some embodiments, the determination of the parameter(s) is performed by image analysis.

[0074] The measuring and / or analysis device may in particular comprise a camera, in particular a high-speed camera, in particular a camera capable of acquiring at least 200 images per second, for example 500 images per second.

[0075] In certain embodiments, the measuring and / or analysis device comprises on the one hand a sensor, such as a camera, and on the other hand an analysis device, for example image analysis or recognition software which can be based on a script written in Python.

[0076] In some embodiments, the image recognition software involves deep learning.

[0077] In certain embodiments, the measuring and / or analyzing device comprises a user interface making it possible to define the determined parameter(s), and / or the associated reference value(s).

[0078] Acoustic flesh

[0079] The acoustic field used according to the present invention is in particular a stationary ultrasonic field.

[0080] The frequency of the acoustic field may in particular be between 20 and 100 kHz. A person skilled in the art is able to adjust the frequency depending in particular on the size of the drops. Indeed, for a given frequency f, drops with a maximum diameter of c / 2f, where c is the speed of sound in air, can be deflected. Preferably, the frequency is of the order of 40 kHz, thus making it possible to deflect drops of a size of up to approximately 4 mm.

[0081] The acoustic field makes it possible to deflect the drop from its initial direction as a function of the value of at least one parameter determined in step b. Thus, the drops can be collected in different containers as a function of the value of said parameter(s). The acoustic radiation force derived from the ultrasound field can in particular deflect the freely falling drop in a direction orthogonal to its falling axis. The intensity and phase of the acoustic field applied to the drop as a function of the value of the parameter are adapted so that the drop falls into the appropriate container. Preferably, the drops fall along the field lines of the acoustic field.

[0082] In some embodiments, the acoustic field is focused by arranging the ultrasonic transducers on two matrices, in particular two spherical or hemispherical matrices. The matrices can be obtained simply and at low cost, for example by 3D printing.

[0083] Method

[0084] In some embodiments, the method is performed in a sterile environment.

[0085] The drop sorting method according to the invention is implemented for each drop that it is desired to sort. In a preferred embodiment, the method according to the invention is implemented successively for all the drops of aqueous continuous phase generated. In other embodiments, the method is implemented only on some of the drops generated. For example, the method can be implemented only on the drops having a particular characteristic, or only on one drop out of two, or one drop out of three. The selection of the drops on which the method is implemented, which correspond to the drops that it is desired to sort, is within the scope of a person skilled in the art depending in particular on the conditions in which the method is implemented and the sorting that it is sought to carry out.

[0086] The drop sorting method according to the invention can be implemented several times in a row on the same volume of aqueous solution or suspension of organoids, in particular if it is sought to sort the organoids and / or the drops containing them according to several parameters. Thus, in one embodiment, the drop sorting method according to the invention is implemented a first time on each drop of a volume of aqueous solution or suspension of organoids, then the contents of one of the containers in which certain drops have been recovered is again subjected to the method according to the invention. Preferably, the sorting parameter(s) or their value(s) when the method is implemented for the second time is / are different from those when the method is implemented for the first time.

[0087] In certain embodiments, the sorting parameter(s) or their value(s) when the method is implemented for the second time is / are identical to those when the method is implemented for the first time. This makes it possible in particular to increase the purity of the sorted sample.

[0088] In some embodiments, the acoustic field applied in step d. of the method varies not only in terms of intensity, but also in terms of phase, which affects the direction of deviation of the drops. This makes it possible to recover the drops, depending on the value of the analyzed parameter, in more than two different containers.

[0089] Device

[0090] A second object of the invention is a device suitable for implementing the method according to the present invention.

[0091] The device according to the invention comprises: a. A system for generating drops of an aqueous solution or suspension of organoids, b. A system for generating an acoustic field, c. A device for measuring and / or analyzing at least one parameter, and d. At least two drip collection containers.

[0092] Each element of the device is as described in the method section. The device may also comprise other elements, said elements being adapted to implement each of the steps described in the method section. Examples

[0093] Example 1: Implementation of a method according to the invention

[0094] A suspension of 252 spheroids of varying sizes was sorted according to the invention with a device similar to that shown in [Fig.l]. The drops were generated at the outlet of a capillary. The parameter detected was the radius of the spheroids R, the reference value Ro to which the measured value was compared being 140 micrometers, or 0.8 times the average radius in the sample (175 micrometers). The threshold intensity chosen for detection was 160 on a gray scale from 0 to 255.

[0095] The suspension consisted of 252 spheroids of 293T cells in 50 milliliters of transparent DMEM culture medium, i.e. 1 spheroid per 10 volume units corresponding to 1 drop (20 microliters).

[0096] The suspension was set in motion in a capillary with a square cross-section and a side of 800 μm with a constant flow rate of 60 microliters per second, imposed by a pressure controller. The flask containing the suspension was continuously agitated during the experiment to maintain the spheroids in homogeneous suspension.

[0097] The drops containing the spheroids were collected in 2 different containers depending on whether the measured radius of the spheroids was less than 0.8 x Ro, or greater than or equal to 0.8 x Ro. The drops not containing spheroids were collected in the same container as the drops containing spheroids whose measured radius was less than 0.8 x Ro.

[0098] The images were acquired at a rate of 400 images per second and processed in real time with a Python script allowing the size of the objects present in the images to be measured.

[0099] The drops were deflected by an acoustic field generated by 72 transducers operating at 40 kHz and each producing a field of 120 dB, facing each other and two at two and separated by 8.4 millimeters (corresponding to 10 wavelengths at 24 degrees Celsius). The transducers were arranged on two 3D-printed hemispherical surfaces.

[0100] The field intensity was zero (0 dB) when no spheroid with a diameter greater than or equal to 0.8 x Ro was identified, and non-zero otherwise. The field intensity and phase were adapted to allow the recovery of drops in both containers.

[0101] [Fig.2] shows the size distribution in the spheroid population before sorting, and the size distributions in the positively and negatively sorted populations. The method allows spheroids to be sorted efficiently according to their size.

Claims

Claims

1. A method for sorting drops comprising an aqueous continuous phase comprising organoids, comprising, for each drop, the following steps: a. Generation of a drop of the aqueous continuous phase containing organoids, b. Determination of a value P of at least one parameter of the drop, c. Comparison of the value P determined in step b. with a reference value, d. Application to the drop of an acoustic field whose intensity and / or phase depends on the result of the comparison of step c., and e. Recovery of the drop in a container.

2. A drop sorting method according to claim 1, wherein the acoustic field applied in step d. is of zero intensity if the value P is less than the reference value, and of non-zero intensity if the value P is greater than or equal to the reference value.

3. A method of sorting drops according to claim 1 or claim 2, wherein the intensity and / or phase of the acoustic field applied in step d. can take n different values, n being an integer greater than or equal to 2, and wherein the method makes it possible to sort the drops into n different containers according to the n intensity and / or phase values ​​of the acoustic field.

4. A method of sorting drops according to any one of claims 1 to 3, wherein the drop is generated in step a. by conveying the aqueous continuous phase comprising organoids into a glass capillary, in particular a glass capillary of square section, at the outlet of which they are encapsulated in drops of water.

5. A method for sorting drops according to any one of claims 1 to 4, wherein the parameter whose value is determined in step b. is selected from the group consisting of the presence of an organoid in the drop, the shape of an organoid present in the drop, the size of an organoid present in the drop, the thickness of an organoid present in the drop, the sphericity of an organoid present in the drop, the number of organoids present in the drop, the nature of the cells of an organoid present in the drop, the fluorescence of an organoid and / or of the capsule of an organoid present in the drop and the light intensity transmitted or absorbed by an organoid present in the drop.

6. A droplet sorting method according to any one of claims 1 to 5, wherein the organoids are formed in an alginate shell.

7. A method of sorting drops according to any one of claims 1 to 6, wherein the diameter of the organoids is between 50 micrometers and 4 mm, preferably between 300 micrometers and 400 micrometers.

8. A method of sorting drops according to any one of claims 1 to 7, wherein the device for determining the value P of the parameter in step b. comprises a sensor, such as a camera, and an image analysis device such as image analysis software.

9. Drop sorting device for implementing the drop sorting method according to any one of claims 1 to 8, said device comprising a system for generating drops of an aqueous continuous phase comprising organoids, a system for generating an acoustic field, a device for measuring and / or analyzing at least one parameter, and at least two drop recovery containers.

10. Device according to claim 9, in which the device for measuring and / or analyzing at least one parameter comprises a camera associated with image analysis software.

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