WO2019115549A1 - Method for screening sequences of culture conditions - Google Patents

Abstract

The present invention relates to a method for screening a sequence of culture conditions of interest to obtain a characteristic of interest.

Description

 Method for screening sequences of culture conditions

The present invention relates to a method for screening sequences of culture conditions of interest for bioprocesses.

When designing a method for producing molecules of interest by living cells such as microorganisms (here called bio-process), great importance is generally given to the choice of the microorganism, having regard to its yield, the quality of its production, etc. For this, various screening methods have been developed (Moutel et al., (2016) Process Biochemistry 51: 1855-1865), including methods using microfluidic techniques (Kim et al., (2015) Sci., Rep. 621 155).

 Nevertheless, many other parameters can influence the yield: pH, composition of the medium, temperature, moment of application of a physiological stress, intensity of said stress, cycle application / absence (or release) of stress. Thus, to determine the optimal conditions of production of a bio-process requires to screen a number of very important cultivation conditions and in particular a number of sequences of conditions of culture even more important.

 However, to date, there are few techniques for screening sequences of culture conditions. Kim et al. (2014) Lab Chip 14: 1415 describes a screening method in which microalgae are trapped in a microfluidic chip and in which the lighting conditions are modified at will, allowing to study their impact on the physiological microalgae and lipid production. However, no change in culture conditions other than the lighting conditions could be tested.

 There is therefore today an important need for techniques for screening a large number of sequences of culture conditions, in particular techniques allowing changes in culture media, and which can be used with any type of culture. cells, in particular non-adherent cells.

 The present invention results from the unexpected discovery of the inventors that it was possible to apply the so-called "split-pool" concept to capsules comprising living cells that can be cultured under different culture conditions that may affect the cells contained in the capsules, and which can be specifically labeled with different types of markers to identify them.

Combicult ^® technology, marketed by Plasticell, uses beads (ie solid / matrix particles) and not capsules, which limits the type of cells that can be cultured and does not protect the cells from external stress (unlike capsules that make it possible to grow fragile cells).

 The process developed by the inventors thus takes place schematically in 4 steps:

 1) encapsulation of cells and combinatorial screening,

 2) test to identify a characteristic of interest in the capsules,

 3) sorting the relevant capsules, and

 4) determination of the sequences of culture conditions to which the relevant capsules have been subjected.

 In particular, the inventors have shown that it is possible to "code" specifically and uniquely the sequences of culture conditions to which capsules are subjected by associating these sequences with a unique combination of markers.

 In addition, the use of capsules makes it possible to isolate the cultured cells from the markers used to identify the culture conditions to which they have been subjected, thus minimizing the risk of potentially deleterious interactions between the markers and the cells and also widening the spectrum. usable markers.

 The present invention thus relates to a method for screening a sequence of culture conditions of interest for obtaining a characteristic of interest, said method comprising the following steps:

 (a) providing a plurality of capsules, each of said capsules comprising:

 a heart comprising an internal phase comprising at least one living cell, said living cell not being in particular a human embryonic stem cell obtained by destruction of an embryo and

 an envelope comprising an external phase completely encapsulating the core at its periphery;

 (b) randomly separating the plurality of capsules provided in step (a) into a plurality of subassemblies,

 (c) culturing the plurality of subsets of capsules in a plurality of different culture conditions,

 said subsets of capsules being cultured in a plurality of external culture media, each external culture medium comprising a unique marker different from the markers present in the other external culture media,

said capsules being cultured for a period appropriate to allow said markers to adsorb to said capsules, (d) recovering and grouping the plurality of capsule subsets followed by randomly separating the plurality of capsules thus grouped into a plurality of new subsets,

 (e) culturing the plurality of new subsets of capsules in a plurality of different culture conditions,

 said novel subsets of capsules being cultured in a plurality of external culture media, each external culture medium comprising a unique marker different from the markers present in the other external culture media, said capsules being cultured for a suitable period of time to allow said markers to adsorb on said capsules,

 (f) optionally repeating steps (b) to (e) iteratively,

 (g) identifying and selecting, within the plurality of capsules, capsules of interest having, following the implementation of steps (b) to (f), at least one characteristic of interest, and

 (h) identifying the sequence (s) of culture conditions at which the capsules of interest identified in step (g) were exposed, by means of the markers adsorbed on said capsules, the (said) sequence (s) of culture conditions thus identified being a sequence (s) of culture conditions of interest for obtaining said characteristic of interest.

 FIGS. 1 and 2 illustrate such a method for screening a sequence of culture conditions of interest for obtaining a characteristic of interest according to the invention (ie split-pool concept), the objective being to see the effects of 3 different culture media and their sequence, with two successive incubations, in other words the effect of incubation sequences in two culture media, the two culture media being selected from three different culture media.

 In FIG. 1, a population of capsules is separated into three lots, each batch being incubated in a culture medium chosen from culture media B, R and G. The media each contain a specific marker, which is adsorbed on the capsules. .

 In Figure 2, the capsules are then collected, mixed and the population is randomly separated into three new batches. Each batch is then incubated in a new culture medium selected from culture media B, R and G. The media contain a specific marker, different from those used in the previous step.

The capsules are then collected. Nine subpopulations are distinguishable, each having a different "story" and being unequivocally marked, requiring only two tagging steps and six different markers. Detailed description of the invention

 cells

 The living cells present in the capsules of the invention may be of any type.

 These include prokaryotic cells, archaea, eukaryotic cells, and mixtures thereof. Thus, according to one embodiment, the living cell or cells are chosen from prokaryotic cells, archaea or eukaryotic cells, preferably from prokaryotic cells and eukaryotic cells, and mixtures thereof.

 Preferably, the living cell is not a human embryonic stem cell obtained by destruction of an embryo.

 Among the prokaryotic cells, mention may in particular be made of bacteria, in particular probiotic bacteria.

 Among the eukaryotic cells, mention may in particular be made of animal cells, plant cells, yeasts, fungi and their mixtures.

 According to one embodiment, the living cell or cells are animal cells, in particular animal cells selected from mammalian cells or insect cells. The living cell or cells used in the context of the invention may in particular be stem cells.

 According to another embodiment, the living cell or cells are plant cells, in particular plant cells chosen from algae and microalgae.

 The living cell (s) may also be genetically modified cells.

 In the context of the invention, the living cell is preferably selected from the group consisting of prokaryotic cells, such as bacteria; arches; eukaryotic cells such as animal cells, in particular mammalian cells, stem cells or insect cells, plant cells, yeasts or fungi.

 The living cells present in the capsules of the invention may be adherent or non-adherent cells.

In the context of the invention, the cells present in the capsules are in a living form. They are therefore not cells in a lyophilized state, or in the form of lysate, dead or inactivated. In one embodiment, the living cells present in the capsules of the invention are non-adherent cells suspended in the internal phase of the capsule.

 The method according to the invention has the advantage of being able to work on non-adherent cells or cells in need of a three-dimensional environment to survive, which is not the case with the technologies of the invention. state of the art.

 In another embodiment, the living cells present in the capsules of the invention are adherent cells.

capsules

 Step a) of the screening method according to the invention comprises providing a plurality of caspules, each capsule comprising at least one living cell as defined in the section "Cells" above.

 The capsules used in the context of the invention comprise:

 a heart comprising an internal phase comprising at least one living cell, and

 an envelope comprising an external phase completely encapsulating the core at its periphery.

 Preferably, the envelope comprises a gelled external phase, said external phase comprising at least one gelled polyelectrolyte.

 According to one embodiment, the heart of the capsules is (i) monophasic or (ii) comprises an intermediate drop of an intermediate phase, the intermediate phase being placed in contact with the gelled outer shell, and at least one, preferably a single internal drop of an internal phase disposed in the intermediate drop, the living cells being present in the inner phase and / or the intermediate phase.

 According to one embodiment, the capsule of the invention is a so-called "simple" capsule, meaning that the heart consists of a single phase, the internal phase. A "simple" capsule is for example a capsule as described in WO2010 / 063937 or WO2016062836.

 According to one embodiment, when the heart of the capsules is monophasic, the living cell or cells are present in the internal phase of the heart of the capsules.

According to another embodiment, when the capsules comprise an intermediate phase, the living cell (s) are present in the internal phase and / or in the the intermediate phase. Such a "complex" capsule is for example a capsule as described in WO2013 / 1 13855.

 According to one embodiment, the size of the capsules is less than 5 mm, preferably between 50 μm and 3 mm. For optimum diffusion of the nutrients present in the external culture medium to the center of the capsules, the size of the capsules is advantageously of a few hundred microns, for example between 50 μm and 1000 μm, and better still between 50 μm and 500 μm. .

Internal phase

 The internal phase generally consists of an aqueous composition, liquid or viscous, comprising at least one living cell.

 For the purposes of the present invention, the term "liquid" means a phase, optionally a gel, which may flow under its own weight at ambient temperature and atmospheric pressure.

 According to one embodiment, a phase comprising the living cells according to the invention has a viscosity ranging from 1 mPa.s to 500,000 mPa.s, preferably from 10 mPa.s to 300,000 mPa.s, better than 400 mPa.s to 200,000 mPa.s, and more particularly from 2,000 mPa.s to 150,000 mPa.s, as measured at 25 ° C.

 The viscosity is measured at ambient temperature, for example T = 25 ° C. ± 2 ° C., and at ambient pressure, for example 1013 hPa, by the method described in WO2016 / 096995.

The internal phase may comprise several living cells, of the same type or of different types.

 Encapsulated living cells are what is also called encapsulated biomass.

 The internal phase is typically capable of survival or proliferation of the living cell or cells included in said internal phase.

 Preferably, the internal phase comprises a buffer solution adapted to the survival of living cells.

 As a usable buffer, any buffer known per se can be used to be adapted to the survival of living cells.

 As is well known to those skilled in the art, such a buffer will depend on the type of living cells present in the internal phase (prokaryotic, eukaryotic, animal or plant cell, stem cell, etc.).

The internal phase preferably has a pH of from 5 to 10, more preferably from 6 to 9. According to one embodiment that is particularly suitable for culturing living cells, the internal phase comprises nutrients capable of proliferating the living cell or cells. In particular, according to one embodiment, the internal phase comprises an internal culture medium, identical or different from the external culture medium described below, adapted to the proliferation of the living cell or cells.

 By "culture medium" is meant a solution comprising nutrients capable of proliferation of the living cell (s) and acting as a pH buffer.

 The osmolarity of the internal culture medium is preferably between 10 mOsm and 1000 mOsm.

 As is well known to those skilled in the art, such a culture medium will depend on the type of living cells present in the internal phase (prokaryotic, eukaryotic, animal or plant cell, stem cell, etc.).

 Examples of suitable internal culture medium for the inner phase of the capsule include Parker culture medium, Ham, Dulbecco, Grace, MEM, Erdschreiber culture medium, F / 2 medium, TAP medium, and the like. reconstituted seawater, DM (diatom) medium, Minimum medium, and any of their mixtures. Such culture media are described in more detail below.

During the encapsulation of living cells (ie before any living cell culture method), the internal phase typically comprises from 10 ³ to 10 ⁹ , preferably from 10 ⁴ to 10 ⁸ , more preferably from 10 ⁵ to 10 ⁸ , for example from 10 ⁶ to 5.10 ⁶ living cells, per milliliter of internal phase.

Depending on the size of the capsules, the internal phase typically comprises from 1 to 10 ⁷ , preferably from 5 to 10 ⁶ , from 30 to ⁵ × 10 ⁵ , from 50 to 10 ⁵ , from 75 to 5 × 10 ⁴ , from 100 to 10 ⁴ , from 150 to 10 ⁴ , even from 200 to 10 ³ living cells, per capsule.

 The counting of the living cells of the internal phase is preferably carried out before encapsulation, or after the opening of the capsules.

Counting living cells can be done by the Malassez counting method. Malassez's cell is a glass slide that counts the number of cells in suspension in a solution. On this glass slide, a grid of 25 rectangles has been engraved, containing 20 small squares themselves. To count the plant cells, one deposits on the cell of Malassez between 10 μl and 15 μl of internal phase comprising plant cells in suspension. After sedimentation, we count the number of plant cells in 10 rectangles (squared). The volume of a grid rectangle being 0.01 pL, this number is multiplied by 10 000 to obtain the number of plant cells per milliliter of internal phase. Alternatively, counting of living cells can be done by absorbance measurement. According to the Beer-Lambert law, for a given wavelength, the absorbance of a solution is proportional to its concentration and to the length of the optical path (distance over which the light passes through the solution). The living cell concentration of the inner phase can therefore be measured based on an absorbance measurement method (also known as optical density). To do this, it suffices to measure the optical densities of internal phases containing a known quantity of living cells, which makes it possible to construct a standard curve as a function of the cell concentration.

 Advantageously, the living cells present in the inner phase of the capsules are suspended in the internal phase.

 By "suspended" in the internal phase, it is meant that the living cells do not adhere to the gelled membrane of the capsules, and are not in prolonged contact with said membrane. The living cells are thus completely immersed in the medium constituting the internal phase and are free to move in three dimensions.

 Those skilled in the art are able to verify that the living cells are effectively suspended in the capsules, typically by observing the capsules by microscopy and demonstrating a differentiated movement between the capsule and the living cells it contains. For example, in the particular case of flagellated microalgae, we can observe their swimming, due to the action of their flagella.

 According to one embodiment, the internal phase further comprises at least one viscosity agent, preferably biocompatible, typically selected from cellulose ethers.

 The presence of a viscosity agent makes it possible to facilitate the preparation of the capsules by reducing the difference in viscosity between the internal phase and the external phase, which advantageously comprises a polyelectrolyte in solution.

By "viscosity agent" is meant a product soluble in the internal phase capable of modulating its viscosity. It may especially be a natural polymer, such as glycosaminoglycans (hyaluronic acid, chitosan, heparan sulfate, etc.), starch, plant proteins, welan gum, or any other natural gum; of a semi-synthetic polymer, such as decomposed starches and their derivatives, cellulose ethers, such as hydroxypropyl methyl cellulose (HPMC), hydroxy ethyl cellulose (HEC), carboxymethylcellulose (CMC) and 2 ethylcellulose; or else a synthetic polymer, such as polyethers (polyethylene glycol), polyacrylamides, polyvinyls, and mixtures thereof. Preferably, the inner phase comprises a cellulose ether such as 2-ethylcellulose or 2-hydroxyethylcellulose. The internal phase may comprise a mass concentration of from 0.01% to 5% of viscosity agent (s), preferably from 0.1% to 1%, relative to the total mass of the internal phase.

External phase

 According to the invention, the envelope of the capsules may be of any type known to those skilled in the art, provided that its composition is not detrimental to the survival / growth of the encapsulated living cells and does not prevent the adsorption of the cells. markers.

 According to the invention, the outer phase of the envelope of the capsules typically comprises at least one polyelectrolyte in the gelled state, also called gelled polyelectrolyte. This outer envelope may also be called "gelled envelope".

 The term "gelled envelope" means an external phase at least partially surrounding an internal phase, and comprising a compound in the gelled state or in gel form. Preferably, the gelled envelope is an aqueous phase, and typically a hydrogel of a polyelectrolyte in the gelled state. The gelled envelope may also be referred to as "membrane" or "bark".

 Preferably, the gelled envelope has a thickness of less than 500 μm, advantageously greater than 10 μm. The gelled envelope is generally formed by a monolayer of a homogeneous material.

polyelectrolyte

 The gelled envelope preferably comprises a gel containing water and a polyelectrolyte advantageously chosen from proteins, natural polysaccharides, polyelectrolytes reactive with multivalent ions, and mixtures thereof.

 For the purposes of the present invention, the term "polyelectrolyte reactive with polyvalent ions" means a polyelectrolyte capable of passing from a liquid state in an aqueous solution to a gelled state under the effect of contact with a gelling solution containing multivalent ions such as alkaline earth metal ions chosen for example from calcium ions, barium ions, magnesium, aluminum or iron ions.

In the liquid state, the individual polyelectrolyte chains are substantially free to flow relative to one another. An aqueous solution of 2% by weight of polyelectrolyte then exhibits a purely viscous behavior at the shear gradients characteristic of the forming process. The viscosity of this solution with no shear is between 50 mPa.s and 10,000 mPa.s advantageously between 3,000 mPa.s and 7,000 mPa.s. This viscosity at shear gradients characteristic of the flows involved during the manufacture of the capsules is for example measured using a stress-strain rheometer, or deformation, imposed at the manufacturing temperature, 25 ° C for example. For measurements, a cone-plane geometry with a diameter of 10 to 50 mm and a cone angle of 2 ° maximum will be used.

 The individual polyelectrolyte chains in the liquid state advantageously have a molar mass greater than 65,000 g / mol.

The gelling solution is, for example, an aqueous solution of a salt of formula

X is selected from the group consisting of halide ions (chloride, bromide, iodide and fluoride) and tartrate, lactate and carbonate ions,

M is selected from the group consisting of Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Al ³⁺ and Fe ³⁺ cations, and

 n and m are greater than or equal to 1.

The concentration of salt X _n M _m in the gelling solution is advantageously from 1% to 20% by weight, preferably from 5% to 20% by weight.

 In the gelled state, the individual polyelectrolyte chains together with the multivalent ions form a coherent three-dimensional network which retains the internal phase and prevents its flow. The individual chains are held together and can not flow freely relative to each other.

 The polyelectrolyte is preferably a biocompatible polymer, it is for example produced biologically.

 Advantageously, it is chosen from polysaccharides, synthetic polyelectrolytes based on acrylates (sodium, lithium, potassium or ammonium polyacrylate, or polyacrylamide), or synthetic polyelectrolytes based on sulfonates (polystyrene sulfonate). sodium, for example).

 More particularly, the polyelectrolyte is chosen from alkaline alginates, such as sodium alginate or potassium alginate, gelans and pectins.

 In the case where the polyelectrolyte is a sodium alginate (NaAlg), and where the reagent is calcium chloride, the reaction that occurs during gelation is as follows:

2NaAlg + CaCI ₂ -> Ca (Alg) ₂ + 2NaCI

Alginates are produced from brown algae called "laminar", referred to as "sea weed". Preferably, the polyelectrolyte is an alkali metal alginate advantageously having an α-guluronate block content of greater than 50%, especially greater than 55%, or even greater than 60%.

 The polyelectrolyte is, for example, an alginate, in particular a sodium alginate.

 The polyelectrolyte in the gelled state is typically a calcium alginate.

 According to a preferred embodiment, the total weight percentage of polyelectrolyte (s) in the gelled external phase is from 0.5% to 5%, preferably less than 3%.

 The total weight percentage of polyelectrolyte (s) in the gelled external phase is for example between 0.5% and 3%, preferably between 1% and 2%.

surfactant

 The gelled envelope may further contain at least one surfactant.

 The presence of a surfactant in the external phase makes it easier to prepare the capsules of the invention by increasing the resistance of the liquid external phase during the impact of the double drop with the gelling solution.

 The surfactant is preferably an anionic surfactant, a nonionic surfactant, a cationic surfactant, or any mixture thereof. The molecular weight of the surfactant is typically between 150 g / mol and 10,000 g / mol, preferably between 250 g / mol and 1500 g / mol.

 In general, the mass content of surfactant (s) in the external phase is typically less than or equal to 2%, preferably less than 1%, relative to the total weight of the capsule.

 The mass content of surfactant (s) in the envelope is preferably greater than 0.001% and is advantageously greater than 0.1%.

Intermediate phase

 According to one embodiment, the capsules according to the invention comprise an intermediate phase between the internal phase and the external phase.

 This intermediate phase forms an aqueous intermediate envelope, generally biocompatible, which completely encapsulates the internal phase and is completely encapsulated by the external phase.

This makes it possible to create complex structures, closer to biological reality than cell culture dishes. Thus, and for illustrative purposes, capsules containing a coculture of keratinocytes and human fibroblasts have been created, as shown in WO20131 13855 where the intermediate phase consists of collagen, and hosts the fibroblasts; the keratinocytes grow on the surface of the intermediate phase and give on the liquid heart.

 When a capsule comprises an intermediate phase, the living cells are present in the internal phase and / or in the intermediate phase.

The intermediate phase may comprise at least one living cell, which may be the same as or different from living cells present in the internal phase.

 The intermediate phase, when present and comprising living cells, is preferably capable of survival of said living cells. It advantageously comprises a culture medium capable of culturing said cells, typically a culture medium as defined above for the internal phase. The culture medium present in the intermediate phase may be identical or different from the culture medium of the internal phase.

The intermediate phase, when present and comprising living cells, typically comprises from 1 to 10 ⁷ , preferentially from 5 to 10 ⁶ , from 30 to ⁵ × 10 ⁵ , from 50 to 10 ⁵ , from 75 to 5 × 10 ^4. , from 100 to 10 ⁴ , from 150 to 10 ⁴ , even from 200 to 10 ³ living cells, per capsule.

According to one embodiment, a capsule according to the invention is a capsule which comprises a liquid core or at least partially gelled or at least partly thixotropic and an outer envelope, in particular gelled, completely encapsulating said liquid core, said core being monophasic .

 Such a type of particles then corresponds to a simple capsule comprising two distinct phases, an internal liquid phase or at least partially gelled or at least partially thixotropic and an external phase, especially in the gelled state, surrounding the internal phase. Advantageously, the ratio of the volume of the core to the volume of the outer envelope is between 1 and 50, preferably between 1 and 10, and better between 1 and 2.

According to another particular embodiment, a capsule according to the invention is a capsule which comprises a liquid core or at least partially gelled or at least partially thixotropic and an outer envelope, in particular gelled, totally encapsulating said heart, said heart comprising an intermediate drop of an intermediate phase, the intermediate phase being placed in contact with the outer envelope, and at least one, preferably a single, internal drop of an internal phase disposed in the intermediate drop. Advantageously, the ratio of the volume of the core to the volume of the external envelope is greater than 2, advantageously less than 50, and preferably is between 5 and 10. The intermediate phase is for example made based on an aqueous solution.

 Such a type of capsule then corresponds to a complex capsule signifying that the liquid core, viscous or thixotropic, comprises a single intermediate drop of an intermediate phase, the intermediate phase being placed in contact with the outer gelled envelope, and at least one , preferably a single, internal drop of an inner phase disposed in the intermediate drop.

 According to one variant, the core comprises a continuous intermediate phase within which a plurality of internal phase drops (s) are located.

 According to one embodiment, a capsule according to the invention comprises a liquid core or at least partially gelled or at least partially thixotropic and an outer envelope, in particular gelled, totally encapsulating said heart, said heart having an intermediate drop of a aqueous phase, the aqueous phase being placed in contact with the outer shell, and at least one, preferably a single, internal drop of an oily phase disposed in the intermediate drop. For obvious reasons, in this embodiment above, the living cells are necessarily located in the heart at the level of the aqueous phase, therefore of the intermediate drop.

 Advantageously, the intermediate phase further comprises at least one viscosity agent (or gelling agent), especially as defined below. In particular, the viscosity agent contributes to improving the suspension of the internal drop (s) disposed in the intermediate drop of the capsules of the invention according to this embodiment. In other words, the viscosity agent makes it possible to prevent / avoid the phenomena of creaming or sedimentation of the internal drop (s) arranged in the intermediate drop of the capsules of the invention according to the invention. this embodiment.

 When the viscosity agent is in the aqueous phase comprising living cells, those skilled in the art will be able to adjust the nature and / or amount of agent (s) gelling (s) so as to ensure the desired suspensivity without prejudicing survival or growth of said living cells.

When the capsule shell comprises at least one gelled polyelectrolyte, the capsules of the invention are typically prepared by a process comprising the following steps:

(II) contacting a first solution, in particular a liquid solution, comprising at least one living cell and a second liquid solution comprising at least one polyelectrolyte in the liquid state, (ii1) forming a double drop comprising an inner phase formed of the first solution and an external liquid phase formed of the second liquid solution,

(iii) immersing the double drop in a gelling solution containing a reagent capable of gelling the polyelectrolyte of the external liquid phase (or second solution), whereby the gelled external phase is obtained, and

 (iv) recovery of formed capsules.

 Step (II) may be characterized by the separate conveying in a double jacket of the first solution, in particular liquid solution, and of the second liquid solution containing a liquid polyelectrolyte suitable for gelling, the step (ii1) forming at the outlet double envelope.

 In the context of the present description, the term "double drop" a drop consisting of an internal phase and a liquid external phase, completely encapsulating said inner phase at its periphery. The production of this type of drop is generally carried out by concentric coextrusion of two solutions, according to a hydrodynamic mode of dripping or jetting, as described in applications WO 2010/063937 and FR2964017.

 When the double drop comes into contact with the gelling solution, the reagent capable of gelling the polyelectrolyte present in the gelling solution then forms bonds between the various polyelectrolyte chains present in the liquid external phase. The polyelectrolyte in the liquid state then passes to the gelled state, thus causing the gelation of the liquid external phase.

 Without wishing to be bound to a particular theory, during the transition to the gelled state of the polyelectrolyte, the individual polyelectrolyte chains present in the liquid external phase are connected to each other to form a crosslinked network, also called a hydrogel, which traps the water contained in the external phase.

 A gelled external phase, suitable for retaining the internal phase of the first solution, is thus formed. This gelled external phase has a proper mechanical strength, that is to say that it is capable of completely surrounding the internal phase and retain the living cell or cells present in this internal phase to retain them in the heart of the capsule gelled.

 The capsules according to the invention remain in the gelling solution until the outer phase is completely gelled. They are then collected and optionally immersed in an aqueous rinsing solution, generally consisting essentially of water and / or culture medium.

The size of the different phases initially forming the double drops, and ultimately the capsules, is generally controlled by the use of two syringe pumps independent (at laboratory scale) or two pumps (on an industrial scale), which provide respectively the first solution and the second liquid solution mentioned above.

 The flow rate Qi of the syringe pump associated with the first solution controls the diameter of the internal phase of the final capsule obtained.

The flow rate Q ₀ of the syringe pump associated with the second liquid solution controls the thickness of the gelled external phase of the final capsule obtained.

The relative and independent adjustment of the flow rates Q 1 and Q ₀ makes it possible to control the thickness of the gelled external phase, independently of the external diameter of the capsule, and to modulate the volume ratio between the internal phase and the external phase.

In another embodiment, when the capsule shell comprises at least one gelled polyelectrolyte, the capsules of the invention, in particular comprising an intermediate phase, are typically prepared by a process comprising the following steps:

 (I2) contacting a first solution and a second solution, at least one of the first and second solutions comprising at least one living cell, and a third liquid solution comprising at least one polyelectrolyte; liquid state,

 (N2) forming a triple drop comprising an inner phase formed of the first solution, an intermediate phase formed of the second solution and an external liquid phase formed of the third liquid solution,

 (iii) immersing the triple drop in a gelling solution containing a reagent capable of gelling the polyelectrolyte of the liquid external phase (or third solution), whereby the gelled external phase is obtained, and

 (iv) recovery of formed capsules.

 Step (i2) may be characterized by the separate conveying in a triple envelope of the first solution, in particular liquid solution, of the second solution, in particular liquid solution, and of the third liquid solution containing a liquid polyelectrolyte suitable for gelling, the step (N2) forming at the exit of the triple envelope.

Thus, the capsules comprising an intermediate phase are generally obtained by concentric coextrusion of three solutions, by means of a triple envelope: a first stream constitutes the internal phase, a second stream constitutes the intermediate phase and a third stream constitutes the phase external. The production of such capsules, called "complex", is described in particular in the international application WO 2012/089820. At the exit of the triple envelope, the three flows come into contact and then form a multi-component drop, which is then gelled when immersed in a gelling solution, in the same way as in the process for preparing capsules "Simple" described above.

Thus, in a particular embodiment, the screening method according to the invention comprises a pre-step (a) for preparing the capsules provided in step (a) comprising the following substeps:

 (M) contacting a first solution comprising at least one living cell and a second liquid solution comprising at least one polyelectrolyte in the liquid state,

 (111) forming a double drop comprising an inner phase formed of the first solution and an external liquid phase formed of the second liquid solution,

 or

 (I2) contacting a first solution and a second solution, at least one of the first and second solutions comprising at least one living cell, and a third liquid solution comprising at least one polyelectrolyte; liquid state,

 (112) forming a triple drop comprising an inner phase formed of the first solution, an intermediate phase formed of the second solution and an external liquid phase formed of the third liquid solution,

(iii) immersing the double drop or the triple drop in a gelling solution containing a reagent capable of gelling the polyelectrolyte of the external liquid phase, whereby the gelled external phase is obtained, and

 (iv) recovery of formed capsules.

Preferably, all the capsules of the plurality of capsules provided in step a) of the screening method according to the invention comprise the same type and / or the same number of living cells.

 Preferably, all the capsules of the plurality of capsules provided in step a) of the screening method according to the invention comprise a core and a shell of the same type.

More preferably, all the capsules of the plurality of capsules provided in step a) of the screening method according to the invention are identical. Separation of capsules

 Step b) of the screening method according to the invention comprising randomly separating the plurality of capsules provided in step a) into a plurality of subassemblies.

 Each subset formed in step b) comprises at least one capsule, preferably at least two capsules, better still at least five capsules, or even at least ten capsules.

 Preferably, the plurality of capsules provided in step a) is separated into at least two subsets.

 In a particular embodiment, the plurality of capsules provided in step a) are separated into n subsets, n being an integer greater than or equal to 2. More preferably, each subset comprises at least capsules, p being an integer greater than or equal to 2, preferably greater than or equal to 5, and better still greater than or equal to 10.

 The plurality of capsules provided in step a) can be subassembled by any technique well known to those skilled in the art.

 Typically, the capsules can be recovered using a sieve, and separated by being taken one by one, or with a spoon-type container that can contain only a fixed number of capsules.

 It is known that the culture of living cells within capsules according to the invention allows access to higher cell concentrations than in bulk processes, as described in WO2016062836. The encapsulation of living cells and the capsule culture of these living cells also has the following advantages over conventional methods:

 encapsulated living cells are protected from mechanical stresses, such as shearing, thus decreasing cell death during the cell culture process;

 the encapsulated living cells are partially protected from the external medium, because the membrane of the capsule has a selective permeability (in particular, the bacteria can not penetrate into the capsule), which advantageously makes it possible to avoid possible contaminations and therefore to reduce cell death;

the size of the capsules and their mechanical properties make them easier to handle than living cells as such, in particular for changes in the culture medium or during harvesting; and - Post-growth treatment and possibly licicitation can be used to waterproof the capsules, thus isolating their contents to facilitate preservation until their use.

The mechanical properties of the capsule membrane are also advantageous for the following reasons:

 The hydrogel network in fact confers semi-solid mechanical properties to the capsules of the invention, which are much greater than those of cells as such. In practice, this is of significant interest for the culture of living cells, as this makes it possible to confer a significant mechanical resistance to living cells, which are classically fragile. While the mechanical resistance of these living cells rests natively on the properties of the lipid bilayer constituting their membrane, when they are encapsulated in a capsule according to the invention, they are now protected by a macromolecular network of several micrometers to several tens of micrometers thick (ie the hydrogel membrane of the capsules). In the case of living cells having a low mechanical resistance, a strength gain of a factor greater than 10, or even between 100 and 100,000, is obtained, which facilitates handling.

 In this way, "solid" objects that are easier to handle are obtained, on the scale of industrial processes such as mixing, stirring, filtering, washing and decanting.

 It is also easier to manipulate these objects, and therefore the biomass they encapsulate, to facilitate the differential characterization of the samples. This allows the facilitated and economical implementation of large scale studies of culture conditions and / or elicitation of compounds of interest. Following a characterization of each of the capsules by physicochemical (absorbance, fluorescence) and / or biological (ELISA), for example, a statistical treatment or sorting of different populations is carried out depending on the response obtained.

The hydrogel membrane of the capsules of the invention also has a semi-permeability, based in particular on the porous nature of the polyelectrolyte chain structure. This network is characterized by an average pore size of between 5 nm and 25 nm, and therefore has a cut-off size of the order of 20 nm to 25 nm. This porosity allows the free passage of dissolved gases, minerals, nutrients necessary for the proliferation of living cells, such as small biomolecules (amino acids and peptides), and small macromolecules with a molecular weight of less than 1 MDa. The membrane retains, on the other hand, any element of characteristic size greater than the cutting size, ie macromolecules or biomolecules of molecular weight greater than 1 MDa, or cell organisms such as living cells, for example algae, or bacteria and fungi.

 In practice, this is of significant interest for the culture of living cells by controlling the exchanges between the outside and the inside of the capsule.

 It is thus possible to provide nutrients, oxygen or light to the encapsulated biomass by transit through the external phase. It is also possible to allow the release of molecules from the inside of the capsule to the external culture medium, in particular for the removal of consecutive waste metabolism / catabolism of the encapsulated biomass. These exchanges can be further facilitated by a mixing of the external culture medium, without this threatening living cells as is the case in bulk processes.

 It is also possible to limit the passage of cellular organisms through the membrane. It is therefore possible to encapsulate living cells with all endogenous bacterial flora capable of proliferation, while avoiding contamination by exogenous bacteria after encapsulation. For example, it is possible to wash the capsules containing the living cells after exogenous bacterial contamination, where the same contamination would destroy all the cells in a bulk process.

 Finally, this semi-permeability also makes it possible to control the release of encapsulated elements, such as compounds of interest produced by living cells.

The production of living cells in encapsulated form according to the invention also makes it possible to limit the quorum sensing effects. During their growth, organisms usually release inhibitory molecules, which limit the proliferation of surrounding organisms. This biological effect allows populations to limit their density in order to avoid the effects of starvation and cell death associated with overcrowding. Encapsulation continuously drains these inhibitory molecules, without causing loss of biomass.

 Washing is also useful for eliminating metabolic or catabolic waste, and thus orienting the activity of biomass. This approach proves to be a method of elicitation in itself.

Moreover, one of the limiting factors of living cell culture is the contribution of light. In a bulk process, the light flux is reduced by "fouling", that is to say the bonding of biomolecules and microorganisms on the walls of the bioreactor. This layer absorbs light, which leads to a decrease in the luminous flux captured by living cells. The accumulation of this deposit induces increasing pressure losses and increases production costs.

 Now, the culture of living cells in capsules according to the invention makes it possible:

 limit the deposition of the biomolecules, these being partly retained inside the capsules, moreover these biomolecules can be recovered at the end of the process, and

prevent the deposit of living cells on the walls of the flasks in which the capsules are cultured or stored (anti-fouling property), said cells being in fact encapsulated.

 Finally, this washing can also be envisaged to eliminate possible contaminants from the biomass culture, without direct manipulation thereof. These contaminants can be of chemical origin (molecules of the type heavy metals or other chemical releases), physical (of particulate type), or biological (dead cells, exogenous bacteria ...). This method can therefore limit operating losses.

A screening method according to the invention therefore offers very broad possibilities in terms of usable cell types and culture conditions. In particular, it makes it possible to work with non-adherent cells or cells that need a 3-dimensional environment to survive. This method, through the use of encapsulation, thus provides a significant added value, either generally or for bioprocesses based on living cells, especially microalgae.

Culture of capsules

 Steps c) and e) of the screening method according to the invention each comprise culturing the plurality of subsets of capsules in a plurality of different culture conditions, said subsets of capsules being grown in a plurality of external culture media, each external culture medium comprising a unique marker different from the markers present in the other external culture media, said capsules being cultured for a period appropriate to allow said markers to adsorb to said capsules.

Culture conditions

By "culture condition" is meant here the environment in which the cells are placed or to which the cells are exposed. Thus, the term "culture condition" can refer to a particular medium, a particular temperature, particular atmospheric conditions, a particular substrate, particular agitation conditions, particular lighting conditions, to particular incorporated agents. in the culture medium, a particular pH, a particular salinity, etc., which may affect cell growth and / or cell differentiation and / or cell properties and / or compound production by cells .

 In a step of culturing the plurality of subsets in a plurality of different culture conditions, preferably, each culture condition of the plurality of different culture conditions is different from another culture condition of that plurality. different growing conditions.

 A culture condition of the plurality of culture conditions of a given culture step (e.g., step c)) may be identical to a culture condition of the plurality of culture conditions of another culture step (for example step d)). In other words, during the implementation of the screening method according to the invention, a given capsule can be cultured under the same culture conditions at two different culture stages. Similarly, during the implementation of the screening method according to the invention, a given capsule can be cultured in a culture condition at a given culture step, a culture condition in which another capsule is grown to another stage of culture.

Culture centre

 By "culture medium" is meant here a support, in particular liquid, for cell culture. A culture medium typically contains mineral salts, energy sources, amino acids, vitamins, growth factors.

 As is well known to those skilled in the art, the culture medium used will depend on the type of cells present in the cultured cells.

 Typically, the culture medium used may be selected from Parker, Ham, Dulbecco, Grace, MEM medium, Erdschreiber culture medium, F / 2 medium, TAP medium, reconstituted seawater, DM (diatom) medium, the Minimum medium, and any of their mixtures.

The Erdschreiber culture medium is a solution comprising NaCl (11.4 g / L), Tris (5.90 g / L), NH ₄ CI (2.92 g / L), KOI (0.73 g). / L), K ₂ HPO ₄ .2H ₂ O (72 mg / L), FeSO ₄ H ₂ O (1.9 mg / L), H ₂ SO ₄ (0.04 mg / L), MgSO ₄ ( 7.65 g / L), CaCl ₂ (1.43 g / L), NaNO ₃ (2 mg / L), Na ₂ HPO ₄ (0.2 mg / L), a soil extract (24.36 ml). / L) and water (QSP 1 L). A soil extract refers to the filtrate obtained by filtration of a mixture of soil and water.

The F / 2 medium, commercially available (in particular at the School of Biological Sciences of the University of Texas, or at Varicon Aqua), is a solution comprising NaNO ₃ (8.82 × 10 ⁴ mol / L), NaH ₂ PO ₄ 0.H ₂ O (3.62 × 10 ⁵ mol / L), Na ₂ SiO ₃ .9H ₂ O (1, 06.10 ⁴⁾ mol / L), FeCl ₃ .6H ₂ O (1, 17 × 10 ⁵ mol / L), Na ₂ EDTA ₂ H ₂ O (1, ₁₇ × 10 ⁵ mol / L), CuSO ₄ .5H ₂ O (3.93 × 10 ⁸ mol) / L), Na ₂ MoO ₄ .2H ₂ O (2.60 × 10 ⁸ mol / L), ZnSO ₄ .7H ₂ O (7.65 × 10 ⁸ mol / L), COCl ₂ .6H ₂ O (4.20 × 10 ⁸ mol) / L), MnCl ₂ .4H ₂ O (9.10 × 10 ⁷ mol / L), thiamine HCl (2.96 × 10 ⁷ mol / L), biotin (2.05 × 10 ⁹ mol / L), cyanocobalamin (3.69 × ¹⁰ ) mol / L) and water (QSP 1 L).

 TAP medium, commercially available, (including LifeTech), is a mixture of Beijerincks buffer (2x) (50 mL), 1 M phosphate buffer pH 7 (1 mL), trace solution (1 mL), acid acetic acid (1 mL) and water (QSP 1 L). The compositions of the Beijerincks (2x) and 1 M phosphate pH 7 buffers and of the trace solution are described in the examples below. The pH of the TAP medium is 7.3. The osmolarity of the TAP medium is 60 mOsm.

The reconstituted seawater is a solution comprising NaCl (11.7 g / L), Tris (6.05 g / L), NH ₄ CI (3.00 g / L), KOI (0.75 g / L), L), K ₂ HPO ₄ .2H ₂ O (74.4 mg / L), FeSO ₄ H ₂ O (2 mg / L), H ₂ SO ₄ (0.05 mg / L), MgSO ₄ (7). 85 g / L), CaCl ₂ (1.47 g / L) and water (QSP 1 L). The pH of reconstituted seawater varies from 7.5 to 8.4. The osmolarity of reconstituted seawater is 676 mOsm.

DM (diatom) medium is a solution comprising Ca (NO ₃ ) ₂ (20 g / L), KH ₂ PO ₄ (12.4 g / L), MgSO ₄ .7H ₂ O (25 g / L), NaHCO 3. ₃ (15.9 g / L), FeNaEDTA (2.25 g / L), Na ₂ EDTA (2.25 g / L), H ₃ B0 ₃ (2.48 g / L), MnCl ₂ .4H ₂ 0 (1.39 g / L), (NH ₄ ) ₆ Mo ₇ 0 ₂₄ .4H ₂ O (1 g / L), cyanocobalamin (0.04 g / L), thiamine HCl (0.04 g / L) , biotin (0.04 g / L), NaSiO ₃ .9H ₂ O (57 g / L) and water (QSP 1 L).

 The Minimum medium is a mixture of Beijerincks buffer (2x) (50 mL), phosphate buffer (2x) (50 mL), trace solution (1 mL) and water (QSP 1 L). The compositions of Beijerincks (2x) and phosphate (2x) buffers and Trace Solution are described in the examples below. The osmolarity of the Minimum medium is 37 mOsm.

 The culture medium likely to be present in the heart of the capsules is called "internal culture medium" and the culture medium in which the capsules are cultured is called "external culture medium". The internal culture medium may be identical or different from the external culture medium.

When the culture condition applied is a particular temperature, particular atmospheric conditions, particular stirring conditions, particular lighting conditions, the external culture medium used will preferably be a basic culture medium allowing the survival of the cells in question. culture.

When the applied culture condition is a particular substrate, particular agents incorporated in the external culture medium, which may affect growth cells and / or the differentiation of the cells and / or the properties of the cells and / or the production of compounds by the cells, the external culture medium used will preferably be a basic culture medium allowing the survival of the cells in culture comprising in besides the substrate or the particular agents tested.

 In the context of the invention, each external culture medium comprises a unique marker different from the markers present in the other external culture media. In particular, during a step of culturing the plurality of subassemblies in a plurality of external culture media, each culture medium of the plurality of external culture media comprises a marker different from those contained in the other media of culture. external culture of said plurality of external culture media. In addition, the marker included in an external culture medium of the plurality of external culture media of a given culture step (for example step c)) is also different from the markers contained in the external culture media of the plurality of external culture media of another / other culture step (s) (e.g., step d)). In other words, during the implementation of the screening method according to the invention, a given capsule can not be placed in the presence of the same marker at two different culture stages. Similarly, during the implementation of the screening method according to the invention, two capsules of two given subsets can not be placed in the presence of the same marker at a given culture step.

markers

 By "marker" is meant here a means of uniquely identifying a culture condition to which a cell in a capsule has been exposed at a given time of a sequence of culture conditions. The markers that can be used in the context of the invention are thus, for example, molecules with a sequence, structure or single mass, molecules or objects (such as beads) that fluoresce.

 Any marker may be used as long as it can adsorb to the surface of the capsules, preferably readily and permanently adsorb to the envelope of said capsules, and may be detected on the surface of the capsule. these capsules, such as peptide markers, colored or fluorescent compounds, secondary amines, halocarbons, stable isotope mixtures, oligonucleotides.

Preferably, a marker used in the context of the invention is physically stable and non-toxic for living cells encapsulated and / or not able to penetrate into the heart of the capsules. Those skilled in the art will be able to adjust the nature and / or the amount of markers present in the external culture media according to the aforementioned prerequisites, but also the nature of the external culture medium and / or culture conditions considered.

 In a particular embodiment, the marker is selected from the group consisting of an oligonucleotide, a fluorophore, or a labeled protein. In particular, the markers used in the context of the invention may be fluorophores encapsulated in or grafted onto vehicles of polymers or silica or conjugated to proteins, quantum dots, colloids, in particular magnetic beads, at the The surface of which are grafted oligonucleotides such as DNA probes or RNA probes, or proteins labeled or labeled with a detectable "tag".

 Quantum dots are semiconductor nanocrystals characterized by a broad excitation spectrum, fine emission spectrum, adjustable emission peaks, long photoluminescence lifetime and negligible photobleaching, and are biocompatible. These particles are typically about ten nanometers in diameter.

 The quantum dots can be chosen from the so-called "core" quantum dots, the so-called "core-shell" quantum dots, and their mixtures.

 The so-called "core" quantum dots are composed solely of the semiconductor core and a layer of carboxyl groups.

 As quantum dots "core", there may be mentioned the following QDs in Sigma: 777951, whose maximum emission is at 610 nm; 777978, whose maximum emission is at 710 nm.

 The so-called "core-shell" quantum dots have a core protected by a protective layer, typically in ZnS.

 As quantum dots "core-shell", there may be mentioned the following QDs from OceanNanotech: QSH-490 and QSH-620, protected by a layer of ZnS and having carboxyl groups.

 To measure the evolution of the photoluminescence intensity of quantum dots in a culture medium, it is possible in particular to use a TECAN M200 Pro plate reader.

By "protein labeled or labeled with a detectable tag" is meant here a protein at least one of the amino acids is modified (for example by an amino acid comprising a particular isotope), or is conjugated or fused with a molecule or another protein detectable by standard detection techniques, such as a fluorophore, a GST, Histidine or Flag tag. Such labeled proteins or labeled with a detectable tag may thus typically be detected by immunoprecipitation; immunofluorescence; Western Blot with colorimetric, chemiluminescence, autoradiography or fluorescence detection; flow cytometry; or by fluorescence microscopy.

 The grafting of DNA or RNA probes on the surface of colloids, in particular magnetic beads, can be implemented by any technique well known to those skilled in the art. It is indeed quite possible to establish covalent bonds between a DNA or RNA probe and a magnetic ball. Several methods are well known to those skilled in the art, such as the use of N-hydroxysuccinimide PEG or N-oxysuccinimide, the l-Linker or the enzymatic route.

In a particular embodiment, the markers used in the context of the invention are colloidal particles of polymer or silica encapsulating or on which are grafted a fluorophore or an oligonucleotide, or proteins labeled or labeled with a detectable tag. In a particular embodiment, the labels used in the context of the invention are colloidal particles of polymer or silica encapsulating or on which are grafted a fluorophore or an oligonucleotide.

 In the context of the invention, the markers used mark the capsule by adsorption on said capsule, in particular via electrostatic bonds. In other words, the present invention has the advantage of not requiring additional encapsulation of the cell at the time of marking.

 In order for the markers to electrostatically adsorb to the capsules, it is necessary that the markers and the surface of the capsules have an opposite charge.

 Also, in some embodiments, when the charge of the markers used is the same as the charge of the capsules provided in step a), the capsule charge is inverted before the first culture step c).

 The charge inversion techniques of a capsule are well known to those skilled in the art. Thus, when the charge of the capsules provided in step a) is negative, for example when the polyelectrolyte used to form the outer envelope is an alginate, the charge of the capsules can be reversed by adsorption with a positively charged polymer, such as polylysine.

In other words, in a particular embodiment, the marker (s) is / are adsorbed on the envelope of the capsules by means of weak or non-covalent binding, electrostatic preferences, optionally by means of a intermediate, for example represented by at least one positively charged polymer, especially polylysine.

Recovery and regrouping of capsules and repetition

Step d) of the screening method according to the invention comprises recovering and grouping the plurality of capsule subsets followed by the random separation of the plurality of capsules thus grouped into a plurality of new subsets.

 Recovery and grouping of subsets of capsules can be implemented by any technique well known to those skilled in the art.

 The recovery (or harvesting) of the capsules is typically by removal of the external culture medium by filtration of the capsules, or by any other capsule recovery technique. In order to filter the capsules, a sieve having an opening size smaller than the average diameter of the capsules of the invention, which are substantially spherical, is typically used. When the markers adsorbed on the capsules comprise magnetic beads, the capsules can also be recovered with a magnet.

 In a particular embodiment, all subsets grown in the preceding culture step are grouped together. In another particular embodiment, only a portion of the subsets grown in the preceding culture step are grouped together.

 The separation of the capsules into new subsets can be carried out by any technique well known to those skilled in the art such as those described in the section "Separation of capsules" above.

 Each new subset formed in step d) comprises at least one capsule, preferably at least two capsules, more preferably at least five capsules, or even at least ten capsules. Capsules forming a new subset in step d) could or could not be part of the same subset in the previous culture step.

 Preferably, the plurality of capsules grouped in step d) is separated into at least two subsets.

In a particular embodiment, the plurality of capsules grouped in step d) is separated into m subsets, m being an integer greater than or equal to 2. More preferably, each subset comprises at least p capsules, p being an integer greater than or equal to 2, preferably greater than or equal to 5, and more preferably greater than or equal to 10. The number of subsets formed in step d) may be the same as or different from the number of subsets formed in the previous separation step.

Steps b) to e) of the screening method according to the invention can be repeated iteratively.

 As will be clear to those skilled in the art, the number of repetitions will depend on the number of culture conditions constituting the sequences of culture conditions to be studied.

Identification and selection of capsules of interest

 Step g) of the screening method according to the present invention comprises the identification and selection, within the plurality of capsules, of capsules of interest having, following the implementation of steps (b) to ( f), at least one characteristic of interest.

 By "identification of a capsule of interest" is meant here the fact of showing, by a detection technique, that a given capsule, among a plurality of capsules, has a desired characteristic.

 As will be clear to those skilled in the art, the detection technique used will depend on the desired characteristic or characteristic of interest.

 Preferably, the detection technique does not require destroying the capsule.

Many features of interest can be searched.

 Thus, the characteristic of interest may be chosen from the expression of at least one molecule of interest by the living cells present in the capsule, a level of biomass of interest present in the capsule, the presence in the capsule of a cell or a set of cells having a morphology of interest and the presence in the capsule of cells having a function of interest.

 By "molecule of interest" is meant here any compound, amino acid, protein, metabolite produced by a living cell following the cultivation of said cell under particular culture conditions. Said molecule of interest may be intracellular or secreted by the cell in the internal phase of the capsule containing it.

 Thus, in this particular embodiment, the sequence (s) of culture conditions of interest (s) corresponds (ent) preferably to the optimal elicitation conditions (s).

In the context of the present description, "elicitation" is understood to mean the stimulation of the production of molecules of interest by a living cell, said stimulation being caused by the setting in particular conditions, whether physicochemical, resulting from modulation of temperature, pressure or illumination, or that they are based on the presence of a particular molecule, called "elicitant molecule". Artificial production of molecules of interest by the encapsulated living cells is thus artificially induced.

 The capsules according to the invention can be cultured under eliciting conditions well known to those skilled in the art, such as by adding salicylic acid, ethylene, jasmonate or chitosan to the external culture medium (cf. for example WO2003 / 077881).

 The molecules of interest produced can typically be subjected to one or more treatments, such as purification, concentration, drying, sterilization and / or extraction. These molecules can then be intended to be incorporated in a cosmetic, food or pharmaceutical composition.

 As a molecule of interest, the capsules of the invention may in particular make it possible to produce lipids of interest in cosmetics, for example fatty acids, such as linoleic acid, alpha-linoleic acid, linoleic gamma, palmitic acid, stearic acid, eicosapentaenoic acid, docosahexanoic acid, arachidonic acid; fatty acid derivatives, such as ceramides; or sterols, such as brassicasterol, campesterol, stigmasterol and sitosterol.

Depending on the nature of the molecule (s) of interest and that of the living cells, a person skilled in the art is able to determine the treatment (s) necessary for the recovery and purification of said molecule of interest.

 According to one embodiment, the recovery of the molecule of interest is carried out by treatment of the external culture medium in which the capsules are in culture, by conventional purification methods, such as liquid / liquid extraction (phase separation organic / aqueous), acid-base washing, solvent concentration and / or purification by chromatography, among others.

 This embodiment is particularly suitable for cases where the molecule of interest is excreted by living cells and then diffuses out of the capsules. This embodiment has the advantage of keeping capsules and cells intact.

 According to another embodiment, the recovery of the molecule of interest is effected by opening the capsules, then opening the membrane of the cells, and then treating the resulting mixture with conventional purification methods.

The opening of the capsules can be of chemical or mechanical type. A mode of chemical opening is for example the depolymerization of the membrane, typically by contacting with a solution of citrate ions. A mechanical opening mode is typically the grinding of the capsules.

 The opening of the cell membrane is typically performed by grinding the cells.

 This embodiment is particularly suitable for cases where the molecule of interest remains confined within living cells.

 By "biomass level of interest" is meant here the amount of cells present in the capsule, following the proliferation of cells of the capsule because of the culture of said cells under particular culture conditions.

 By "set of cells having a morphology of interest" is meant here at least one cell present in the capsule having morphological characteristics, typically due to a specific differentiation, it did not present at the beginning of the screening method and which are due to the culture of the cell under particular culture conditions.

 By "cells having a function of interest" is meant here cells having, for example, particular enzymatic activities, secretion capacities of specific molecules, etc., that they did not exhibit at the beginning of the screening method. and which are due to cell culture under particular culture conditions.

 By "selection of a capsule of interest" is meant here the separation of capsules having a characteristic of interest, as defined above, capsules not having this characteristic of interest.

 The selection of the capsule of interest can be implemented by any technique well known to those skilled in the art, such as for example by cytometry, by the COPAS Flow Pilot, or the Millidrop Analyzer sorting tool developed by MilliDrop. , automatic or manual pipetting.

Identification of sequences of culture conditions of interest

Step h) of the screening method according to the invention comprises the identification of the sequence (s) of culture conditions in which the capsules of interest identified in step (g) ) have been exposed, by means of the markers adsorbed on said capsules, the said sequence (s) of culture conditions thus identified being a sequence (s) of culture conditions of interest for the obtaining said characteristic of interest. The fact of using a single marker associated with each culture condition tested at each culture step makes it possible to associate with each sequence of culture conditions a specific and unique combination of markers.

 The present invention is based on this association between a specific and unique combination of markers and a specific sequence of culture conditions. Detecting the combination of single and specific markers adsorbed on a capsule thus makes it possible to identify the specific sequence of culture conditions to which this capsule has been subjected.

 The detection of the combination of markers present on the capsules of interest identified and selected in step g) thus makes it possible to identify the sequence of culture conditions of interest which made it possible to obtain the characteristic of interest sought.

 As will be clearly apparent to those skilled in the art, the detection of markers will depend on the type of markers used.

 Thus, when the marker is a fluorophore, the detection can be carried out by cytometry; when the marker is oligonucleotide, the detection can be carried out by PCR or by DNA chip; when the marker is a specific isotope, the detection can in particular be carried out by mass spectrometry.

In view of the above, and as illustrated by the examples below, it appears that the split-pool concept used in the context of the present invention is particularly effective for screening sequences of culture conditions with capsules comprising living cells.

 For that, it suffices to move the bath capsules in a bath, thus exposing the encapsulated living cells to different culture conditions. The specific and unequivocal labeling of the capsules at each stay in an external culture medium makes it possible to reveal their respective history a posteriori.

 The expressions "between ... and ...", "from ... to ..." and "from ... to ..." must be understood as inclusive, unless otherwise specified.

The examples below illustrate the invention without limiting its scope. EXAMPLES

 Example 1 Preparation of Capsules Used in the Context of the Invention

 1. Preparation of solutions for the manufacture of capsules

 All the material (tubes, connectors, crystallizer, etc.) used is autoclaved. For elements of the fluidic device not resistant to high temperatures, rinsing with 70% ethanol is carried out. The encapsulation process described below is carried out under sterile conditions, under a laminar flow hood. All liquids are also sterilized. Autoclaving is a simple method of sterilizing liquids. The calcium bath is autoclaved. For polymer solutions (alginate and cellulose), if these are brought to 120 ° C for 20 min, their rheological properties are altered to a point that makes encapsulation impossible. Their sterilization is thus effected by filtration at 0.2 μm or by pasteurization at 65 ° C. for 60 minutes (longer but lower temperature than autoclaving, which less modifies the rheological properties of the polymers).

- BF calcium bath (gelling solution): CaCl ₂ (90 mM) and a few drops of a solution of Tween 20 10% w / v and osmosis water.

 - OF external phase (membrane): alginate solution (1.7%) and 0.5 mM SDS in HEPES (50 mM) and osmosis water.

 IF internal phase (core): 50/50 mixture of a 1% solution of cellulose + 50 mM HEPES + water and culture medium MCCM + microalgae Chlamydomonas reinhardtii

 - External culture medium (MCCM): see table 1

 Table 1: Composition of the MCCM Medium

Toutes les phases subissent préalablement à leur utilisation une étape de filtration à 5 pm (sauf milieu de culture MCCM). Cette étape de filtration permet d’éviter la présence de particules ou d’agrégats solides conduisant au bouchage des buses utilisées pour la production, mais est aussi utilisée pour stériliser les phases.

All phases undergo prior to their use a filtration step at 5 pm (except MCCM culture medium). This filtration step avoids the presence of particles or solid aggregates leading to the clogging of the nozzles used for production, but is also used to sterilize the phases.

2. Manufacture of capsules

 The method of manufacturing capsules is based on the concentric coextrusion of two solutions, in particular described in WO 2010/063937 and FR2964017, to form double drops.

 IF flow rate: 130 ml_ / h.

 OF flow: 70 ml_ / h.

 A piezoelectric device (Preloaded piezo actuator, 15 μm, from PI) is then energized, and the excitation frequency is adjusted (typically around 1.6 kHz). An electrode is energized, typically at 1000 V to load the drops. The induced repulsions cause the drops to move away, which transforms the jet into a cone and reduces coalescence in flight. This allows to obtain a population of drops, and therefore capsules, very monodisperse. The capsules then end their fall in the calcium bath.

 Once the collection is complete, the capsules are left for a few minutes in the calcium bath and then are filtered through a cell strainer (100 μm mesh). The capsules are then washed with the MCCM culture medium.

This system makes it possible to encapsulate microalgae without deleterious effect, and to cultivate them in this vehicle. It is possible to reach very high concentrations (3460 million cells per mL in the capsule), without major impact on the viability of the culture.

Example 2: Marker Adsorption Test

The markers used in this example 2 are magnetic beads, represented by particles composed of a paramagnetic core encapsulated in a polymer shell (Ademtech, Carboxyl-Adembeads 0212) or silica (Ademtech, Silica-Adembeads 0252). These beads are 200 nm in diameter. The first carry on the surface carboxyl groups, the second silanols. Thus, these balls carry a negative surface charge. Their Zeta potentials are measured with Zetasizer Nano ZS, and are respectively -9.4 mV and -33.4 mV.

 These particles remain individualized in solution thanks to the electrostatic repulsions (electrostatic and steric in the case of Carboxyl-Adembeads) that they exert on one another.

The inventors verified whether these particles are stable in the MCCM culture medium described in Table 1 above.

 For this, the inventors have dispersed said markers in MCCM, waited about twenty minutes and viewed the state of the suspension under a microscope.

 For comparison, the inventors performed the same experiment in 100 mM calcium chloride.

 The inventors have shown that these markers do not aggregate in the MCCM, and that they are therefore usable in the context of the present invention.

The inventors then verified whether it was possible to adsorb these markers on the capsules.

 Given the negative charges of the envelope of the capsules and the markers, this adsorption step requires a prior step of positively charging the envelope of the capsules of Example 1. Thus, the inventors have incubated a hundred capsules according to Example 1 in 200 μl of MCCM supplemented with 0.001% of a positively charged polymer, polylysine PLL (DKSH), for about twenty minutes. The capsules are then washed with MCCM. Then, 5 μL of a marker suspension is added, and all is allowed to incubate for 30 minutes.

 Control is carried out with capsules according to Example 1 which has not been in the presence of PLL.

 The capsules are then visualized. The inventors have observed that the use of a positively charged polymer makes it possible to reverse the surface charge of the capsules, which allows the adsorption of the negatively charged markers on the surface of said capsules.

The same process is performed by allowing several days to elapse between the treatment with the PLL and the addition of the markers. The inventors did not observe a difference with the incubation of 30 min, which ensures that the PLL, and therefore the markers, remain (s) well adsorbed (s) on the surface of the capsules. Example 3: robustness of the adsorption of markers on the capsules

 It is important to know if the markers desorb easily from the surface of the capsules, or if on the contrary they remain adsorbed when the capsules are agitated or manipulated. The inventors have therefore studied the impact of manipulations and rinses on these capsules.

 Study 1

 The inventors checked whether the markers desorb the capsules according to Example 2. For this, they placed these capsules in a petri dish and visualized the capsules once, then a second time after 12 hours. The absorbance, or optical density (OD), of the capsules is calculated from the images obtained.

 The inventors have thus observed that the markers desorb little from the capsule over time, with a very low desorption of less than 9%.

Study 2

 On the basis of Examples 1 and 2 above, the inventors have covered capsules in markers according to Example 2. These capsules are separated into two lots. One is poured into a petri dish, which is placed on an orbital shaker. The other is placed in an Eppendorf tube which is placed on a roller shaker (20 rotations / min). 12 hours later, the capsules are imaged, and the OD is calculated. The inventors have thus shown that more than 50% of the markers remain adsorbed on the capsules despite these agitations.

 The adsorption of markers on capsules according to the invention is therefore effective and robust. In addition, this approach is generic and can be transposed to other types of markers, particularly colloids.

Example 4 Use of Fluorescent Silica Particle Markers

Fluorophores are grafted onto the surface of silica spheres after synthesis of said spheres. They can also be grafted throughout the volume, which significantly increases the fluorescence signal. The inventors used particles (Sicastar-F 1 μm, Micromod).

1) Stability

The inventors began by ensuring the stability of these particles in the medium. They verified that these particles do not aggregate in the MCCM, using the same protocol as previously in example 2. This non-aggregation has thus been confirmed. In addition, the Zeta potential of these particles is measured and is -37.6 mV.

 The inventors then checked the stability of the fluorescence in the media, and in the presence of PLL.

They measured the fluorescence emission of a suspension of Sicastar Green and Red in MCCM supplemented with ³ % PLL. They observed that the fluorescence intensity of Sicastar Green and Red increases respectively by 20.2 and 8.3%, which can be attributed to the sedimentation of the beads in the well. In any case, no major impact is to be noted on the fluorescence intensity.

2) Adsorption of fluorescent silica particles and color coding

 The inventors adsorbed these 1 μm fluorescent particles (= markers) on the capsules previously coated with PLL as described in Example 2.

For this, 2 Eppendorf tubes containing 500 μl of HEPES-Ca and 290 capsules previously coated with PLL were prepared. Then, 2.4x10 ¹⁰ markers are added, each tube corresponding to one color. The excitation / emission wavelengths are respectively 485/510 nm and 569/585 nm for Sicastar-F Green and Red. After 10 minutes of incubation, the capsules are washed (with HEPES-Ca) and visualized under a fluorescence microscope.

 The inventors have thus observed that the markers adsorb on the capsules. In addition, the fluorescence of the markers in red or green makes it possible to mark the capsules uniquely.

The inventors have verified that these markers do not move from one capsule to another. For this, two populations of capsules are marked with respectively Sicastar-F Green and Sicastar-F Red. The two populations are then mixed and the whole is placed on a fluorescence microscope. Snapshots are taken at the initial time and 12 hours later.

 The inventors have shown that the fluorescent beads remain permanently adsorbed on the capsules, without switching from one capsule to another.

Finally, the inventors have validated the fact that it is possible to adsorb two different markers on one capsule. For this, a population of capsules is first marked with Micromod Green, the whole is rinsed then the capsules are exposed to Micromod Red. The capsules are then imaged. The inventors have thus shown that two different markers can be absorbed consecutively on a capsule.

This confirms the possibility of resorting to the labeling of capsules comprising living cells to rapidly and efficiently screen sequences of culture conditions of interest for obtaining a characteristic of interest, and thus the relevance and significant advantages of the method according to the invention.

Claims

A method of screening a sequence of culture conditions of interest for obtaining a characteristic of interest, said method comprising the steps of:  (a) providing a plurality of capsules, each of said capsules comprising:  a heart comprising an internal phase comprising at least one living cell, said living cell not being a human embryonic stem cell obtained by destruction of an embryo, and  an envelope comprising an external phase, totally encapsulating the core at its periphery;  (b) randomly separating the plurality of capsules provided in step (a) into a plurality of subassemblies,  (c) culturing the plurality of subsets of capsules in a plurality of different culture conditions,  said subsets of capsules being cultured in a plurality of external culture media, each external culture medium comprising a unique marker different from the markers present in the other external culture media,  said capsules being cultured for a period appropriate to allow said markers to adsorb to said capsules,  (d) recovering and grouping the plurality of capsule subsets followed by randomly separating the plurality of capsules thus grouped into a plurality of new subsets,  (e) culturing the plurality of new subsets of capsules in a plurality of different culture conditions,  said novel subsets of capsules being cultured in a plurality of external culture media, each external culture medium comprising a unique marker different from the markers present in the other external culture media, said capsules being cultured for a suitable period of time to allow said markers to adsorb on said capsules,  (f) optionally, repeating steps (b) through (e) iteratively,  (g) identifying and selecting, within the plurality of capsules, capsules of interest having, following the implementation of steps (b) to (f), at least one characteristic of interest, and (h) identifying the sequence (s) of culture conditions at which the capsules of interest identified in step (g) have been exposed, by means of markers adsorbed on said capsules, the so-called sequence (s) of culture conditions thus identified being a sequence (s) of culture conditions of interest for obtaining said characteristic of interest . 2. Screening method according to claim 1, wherein the envelope comprises at least one polyelectrolyte in the gelled state, preferably the polyelectrolyte being a multivalent ion reactive polyelectrolyte, in particular an alginate. A screening method according to any one of claims 1 or 2, wherein the living cell is selected from the group consisting of prokaryotic cells, such as bacteria; arches; eukaryotic cells in particular chosen from animal cells, in particular mammalian cells, stem cells and insect cells, plant cells, yeasts or fungi; and their mixtures. 4. Screening method according to any one of claims 1 to 3, wherein the living cell is a non-adherent cell suspended in the internal phase of the capsule. 5. Screening method according to any one of claims 1 to 3, wherein the living cell is an adherent cell. 6. Screening method according to any one of claims 1 to 5, wherein the heart of the capsules is monophasic, or comprises an intermediate drop of an intermediate phase, the intermediate phase being placed in contact with the external phase, and the at least one, preferably a single, internal drop of an internal phase disposed in the intermediate drop, the living cells being present in the inner phase and / or the intermediate phase. The screening method according to any one of claims 1 to 6, wherein the label is a colloidal particle of polymer or silica encapsulating or on which is grafted a fluorophore or an oligonucleotide, or proteins labeled or tagged with a tag detectable. The screening method according to any one of claims 1 to 7, wherein the marker is selected from the group consisting of magnetic beads on the surface. from which are grafted DNA probes or RNA probes, fluorophores encapsulated in polymer or silica vehicles, quantum dots, and proteins labeled or labeled with a detectable "tag". 9. Screening method according to any one of claims 1 to 8, wherein the characteristic of interest is chosen from the expression of a molecule of interest by the living cells present in the capsule, a level of biomass of interest present in the capsule, the presence in the capsule of a cell or a set of cells having a morphology of interest and the presence in the capsule of cells having a function of interest. The screening method according to any one of claims 2 to 9, comprising a pre-step (a) for preparing the capsules provided in step (a) comprising the following sub-steps:  (M) contacting a first solution comprising at least one living cell and a second liquid solution comprising at least one polyelectrolyte in the liquid state,  (111) forming a double drop comprising an inner phase formed of the first solution and an external liquid phase formed of the second liquid solution,  or  (I2) contacting a first solution and a second solution, at least one of the first and second solutions comprising at least one living cell, and a third liquid solution comprising at least one polyelectrolyte; liquid state,  (112) forming a triple drop comprising an inner phase formed of the first solution, an intermediate phase formed of the second solution and an external liquid phase formed of the third liquid solution, (iii) immersing the double drop or the triple drop in a gelling solution containing a reagent capable of gelling the polyelectrolyte of the external liquid phase, whereby the gelled external phase is obtained, and  (iv) recovery of formed capsules. 11. Screening method according to any one of claims 1 to 10, wherein the marker (s) is / are adsorbed (s) on the envelope of the capsules by means of binding non-covalent, preferably electrostatic, optionally by means of an intermediate, for example figured by at least one positively charged polymer, especially polylysine.