• 专利附录
  • 专利说明
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    • 专利摘要:
    The present invention relates to a capsule comprising: an internal phase comprising at least one plant cell, and a gelled external phase, totally encapsulating said internal phase at its periphery, said external phase comprising at least one surfactant and at least one polyelectrolyte at least one gelled state.
    公开号:FR3027608A1
    申请号:FR1460169
    申请日:2014-10-22
    公开日:2016-04-29
    发明作者:Edouard Duliege；Thomas Delmas；Sebastien Bardon；Jerome Bibette；Nicolas Bremond；Hugo Domejean
    申请人:Capsum SAS；
    IPC主号:
    专利说明:

    [0001] The present invention relates to a gelled capsule comprising a plant cell, and a method for culturing said plant cell. Currently, macroscopic algae (or macro-algae) are either harvested in the wild or farmed at sea. Micro-algae are mainly grown in open-pond (open ponds). ) or bioreactor.
    [0002] The open-lay process has the advantage of being inexpensive, but does not produce algae in high concentration. On the other hand, it requires cultivation of extremophilic organisms in order to avoid contamination (by the external environment, since, by definition, no physical barrier exists between the culture medium and the outside). Among these contaminations, mention may be made of the result of animal droppings or the death of insects and / or animals. The bio-reactor process makes it possible to isolate the algae culture from the outside world and to concentrate the algae. In this process, the plant cells are in free suspension in a culture medium, it is also called bulk process. This process, however, induces a significant production cost, mainly related to the need to continuously brew the crop and bring in the gases and light necessary for the growth of organisms. On the other hand, harvesting methods are not easy and expensive (centrifugation, tangential filtration, etc.). Finally, the mixing of the culture medium makes it difficult to cultivate fragile algae, because of the large cell death caused by this mixing.
    [0003] There is also a method of three-dimensional culture of mammalian cells, wherein said cells are encapsulated and grow in contact with the inner membrane of the capsules. Nevertheless, this type of three-dimensional culture method is not adapted to the culture of plant cells, in particular of unicellular organisms.
    [0004] There is therefore a need for a process for improving the production of plant cells, in particular algae, by advantageously making it possible to increase the concentration of plant cells. The object of the present invention is therefore to provide a means for the proliferation (here also referred to as growth) and, optionally, the elicitation of at least one plant cell, preferably at least one algal cell.
    [0005] The present invention relates to a capsule comprising: an internal phase comprising at least one plant cell, and a gelled external phase, totally encapsulating said internal phase at its periphery, said external phase comprising at least one surfactant and at least one polyelectrolyte at least one gelled state. The subject of the present invention is also a method for culturing plant cells, as well as a method for producing compounds of interest produced by said plant cells, if necessary after elicitation.
    [0006] In the context of the present description, the inner phase also refers to the heart of a capsule, and the gelled outer phase also refers to its gelled envelope, also called membrane. The capsules of the invention are also known as gelled capsules.
    [0007] According to one embodiment, the capsule of the invention is a capsule called "simple", meaning that the heart consists of a single phase. A "simple" capsule is for example a capsule as described in the international application WO 2010/063937.
    [0008] The present invention also relates to a method for preparing the capsules according to the invention. The capsules of the invention are typically prepared by a process comprising the steps of: contacting a first solution comprising at least one plant cell and a second liquid solution comprising at least one surfactant and at least one polyelectrolyte with the liquid state, the formation of a double drop comprising an internal phase formed of the first solution and an external liquid phase formed of the second liquid solution, the immersion of the double drop in a gelling solution containing a reagent suitable for gelation the polyelectrolyte of the liquid external phase, whereby the gelled external phase is obtained, and the recovery of the capsules formed. 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 clean mechanical strength, that is to say that it is capable of completely surrounding the internal phase and retain the plant 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 independent syringe pumps (at the laboratory scale) or two pumps (on an industrial scale), which respectively provide the first solution and the second liquid solution mentioned above. The flow IQ of the syringe pump associated with the first solution controls the diameter of the internal phase of the final capsule obtained.
    [0009] The flow rate 00 of the syringe pump associated with the second liquid solution controls the thickness of the gelled outer phase of the final capsule obtained. The relative and independent adjustment of the flow rates QI and 00 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. The invention generally has an average size of less than 5 mm, and more generally of 50 lm to 3 mm, advantageously 100 lm to 1 mm. According to an advantageous embodiment, the gelled outer phase has an average thickness of from 5 μm to 500 μm, preferably from 7 μm to 100 μm. According to an advantageous embodiment, the volume ratio between the internal phase and the external phase is greater than 1, and preferably less than 50. Internal phase The internal phase, also called the heart of the capsules, is generally composed of an aqueous composition liquid or viscous composition comprising at least one plant cell. By "at least one plant cell" is meant at least one cell of a plant cell line.
    [0010] The internal phase may comprise several plant cells, of the same line or of different lines. Encapsulated plant cells are what is also called encapsulated biomass. The plant cells are differentiated from animal cells, among others, by the presence of a pectocellulosic wall and the presence of plastids, including chloroplasts that allow photosynthesis. According to one embodiment, the plant cells included in the internal phase are unicellular plant cells. According to one embodiment, the capsules of the invention comprise at least one algal cell (also called algal cell), preferably a microalgae cell. According to one variant, the capsules of the invention comprise several cells of algae, of the same species or of different species. Algae are living beings capable of photosynthesis whose life cycle generally takes place in the aquatic environment. In algae, there are prokaryotes (cyanobacteria) or eukaryotes (several very diverse sets). The term "micro-algae" refers to microscopic algae. They are undifferentiated, photosynthetic, eukaryotic or prokaryotic unicellular or multicellular beings.
    [0011] As algae that can be used in the capsules of the invention, mention may be made of a green alga, a red alga or a brown alga.
    [0012] According to one embodiment, it is a prokaryotic algae. According to another embodiment, it is a eukaryotic algae. The alga is preferably of the genus Chlamydomonas, such as Chlamydomonas reinhardtii, or of the genus Peridinium, such as Peridinium cinctum.
    [0013] Other algae suitable for the implementation of the invention may be chosen from the group consisting of Alexandrium minutum, Amphiprora hyalina, Anabaena cylindrica, Arthrospira platensis, Chattonella verruculosa, Chlorella vulgaris, Chlorella protothecoides, Chysochromulina breviturrita, Chrysochromulina kappa, Dunaliella Sauna, Dunaliella minuta, Emiliania huxleyi (Haptophyta), Gymnodinium catenatum, Gymnodinium nagasakiense, Haematococcus pluvialis, lsochrysis galbana, Noctiluca scintillans, Odontella aurita, Oryza sativa, Ostreococcus lucimarinus, Pavlova utheri, Porphyridium cruentum, Spirodela oligorrhiza, Spirulina maxima, Tetraselmis tetrathele and Thalassiosira pseudonana.
    [0014] The internal phase is typically suitable for the survival of the plant cell or cells included in said internal phase. Preferably, the internal phase comprises a buffer solution capable of survival of the plant cells. As usable buffer, any buffer known per se can be used to be adapted to the survival of plant cells. The internal phase preferably has a pH of between 5 and 10, more preferably between 6 and 9. According to one embodiment that is particularly suitable for plant cell culture, the internal phase comprises nutrients capable of proliferating the plant cells. Preferably, the internal phase comprises a culture medium called MC1 in the context of the present invention. By "culture medium" is meant a solution comprising nutrients suitable for the proliferation of the plant cell or cells and acting as a pH buffer.
    [0015] The osmolarity of the culture medium MC1 is preferably between 10 mOsm and 1000 mOsm. The MC1 culture medium is for example selected from Erdschreiber culture medium, F / 2 medium, TAP medium, reconstituted seawater, DM (diatom) medium, Minimum medium, and any of their mixtures.
    [0016] The Erdschreiber culture medium is a solution comprising NaCl (11.4 g / L), Tris (5.90 g / L), NH4Cl (2.92 g / L), KCl (0.73 g / L) , K2HPO4.2H2O (72 mg / L), Fe504.2H2O (1.9 mg / L), H2SO4 (0.04 mg / L), MgSO4 (7.65 g / L), CaCl2 (1.43 g / L), L), NaNO3 (2 mg / L), Na2HPO4 (0.2 mg / L), 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. 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. 3.62 × 10 -5 mol / L), Na 2 O 3 · 3H 2 O (1.06 × 10 -4 mol / L), FeCl 3 · 6H 2 O (1.17 × 10 -5 mol / L), Na 2 EDTA 2H 2 O (1.17 × 10 -5 mol / L) ), Cu504.5H2O (3.93.10-8 mol / L), Na2MoO4.2H2O (2.60.10-8 mol / L), Zn504.7H2O (7.65 × 10 -8 mol / L), CoCl2 · 6H2O (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 , 69.10-10 mol / L) and water (QSP 1 L). TAP medium, commercially available (including LifeTech), is a mixture of Beijerincks buffer (2x) (50 mL), 1M phosphate buffer pH 7 (1 mL), trace solution (1 mL), acetic acid (1 mL) and water (QSP 1 L). The Beijerincks (2x) and phosphate 1M pH 7 buffer and trace solution compositions 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), NH4Cl (3.00 g / L), KCl (0.75 g / L), K2HPO4.2H2O (74.4 mg / L), FeSO4.2H2O (2 mg / L), H2SO4 (0.05 mg / L), MgSO4 (7.85 g / L), CaCl2 (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 (NO3) 2 (20 g / L), KH2PO4 (12.4 g / L), MgSO4.7H2O (25 g / L), NaHCO3 (15.9 g / L) ), FeNaEDTA (2.25 g / L), Na2EDTA (2.25 g / L), H3B03 (2.48 g / L), MnCl2.4H2O (1.39 g / L), (NH4) 6Mo7024.4H2O (1 g / L), cyanocobalamin (0.04 g / L), thiamine HCl (0.04 g / L), biotin (0.04 g / L), NaSiO3.9H2O (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. During the encapsulation of the plant cells (ie before any plant cell culture process), the internal phase typically comprises from 103 to 109, preferably from 104 to 108, more preferably from 105 to 108, for example from 106 to 5.106. plant cells, per milliliter of internal phase.
    [0017] Depending on the size of the capsules, the internal phase typically comprises from 1 to 107, preferably from 5 to 106, from 30 to 5 × 10 5, from 50 to 105, from 75 to 5 × 10 4, from 100 to 104, from 150 to 104, and even from 200 to 103 plant cells, per capsule. The counting of the plant cells of the internal phase is preferably carried out before encapsulation, or after the opening of the capsules. The counting of plant cells can be done by the counting method Malassez. 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. In order to count the plant cells, Malassez cell is deposited between 10 μl and 1.1 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.
    [0018] Alternatively, counting of plant cells can be done by absorbance measurement. According to the Beer-Lambert law, for a given wavelength λ, 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 concentration of plant cells in the inner phase can therefore be measured on the basis of an absorbance measurement method (also known as optical density). It suffices to measure the optical densities of internal phases containing a known quantity of plant cells, which makes it possible to construct a standard curve as a function of the cell concentration.
    [0019] By way of example, the internal phase comprises one million cells per milliliter of internal phase before any culture method, which corresponds to approximately 60 cells per capsule of 500 μm in diameter. After a culture method as described below, the internal phase typically comprises from 50 million to 150 million plant cells, per milliliter of internal phase. Advantageously, the plant cells present in the inner phase of the capsules are suspended in the internal phase. By "suspended" in the internal phase is meant that the plant cells do not adhere to the gelled membrane of the capsules, and are not in prolonged contact with said membrane. The plant 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 plant cells are effectively suspended in the capsules, typically by observing the capsules by microscopy and demonstrating a differentiated movement between the capsule and the plant cells it contains. For example, in the particular case of flagellated micro-algae, we can observe their swimming, due to the action of their flagella. According to one embodiment, the internal phase comprises at least one viscosity agent, preferably biocompatible, typically chosen from cellulose ethers.
    [0020] 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 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 a synthetic polymer, such as polyethers (polyethylene glycol), polyacrylamides, and polyvinyls. Preferably, the inner phase comprises a cellulose ether, such as 2-ethylcellulose. When present, the viscosity agent is present in the internal phase in a mass concentration of 0.01% to 5%, preferably 0.1% to 1%, relative to the total mass of the internal phase. . External Phase The external phase comprises at least one gelled polyelectrolyte, also called gelled polyelectrolyte, and at least one surfactant.
    [0021] Polyelectrolyte Preferably, the polyelectrolyte is chosen from polyelectrolytes reactive with multivalent ions. In the context of the present description, the term "polyelectrolyte reactive with multivalent ions" 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 multivalent cations of calcium, barium, magnesium, aluminum or iron. 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 zero shear solution is between 50 mPa.s and 10,000 mPa.s, preferably between 1000 mPa.s and 7000 mPa.s.
    [0022] 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, M ,, in which: X is chosen 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 X, M salt in the gelling solution is advantageously 1 % to 20% by weight, preferably 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.
    [0023] 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 (poly (styrene) sulfonate), for example).
    [0024] 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 + CaCl2 Ca (Alfa) 2 + 2NaCl 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-L-guluronate block content of greater than 50%, especially greater than 55%, or even greater than 60%. The polyelectrolyte is, for example, sodium alginate. The polyelectrolyte in the gelled state is typically a calcium alginate. According to a preferred embodiment, the total weight percentage of polyelectrolyte in the gelled external phase is from 0.5% to 5%, preferably less than 3%. The total weight percentage of polyelectrolyte in the gelled external phase is for example between 0.5% and 3%, preferably between 1% and 2%. 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 in the external phase is typically less than or equal to 2%, preferably less than 1%, relative to the total weight of the capsule.
    [0025] In the case where the surfactant is an anionic surfactant, it is, for example, chosen from alkyl sulphates, alkyl sulphonates, alkyl aryl sulphonates, alkaline alkyl phosphates, dialkyl sulphosuccinates and alkaline earth salts of saturated or unsaturated fatty acids. These surfactants advantageously have at least one hydrophobic hydrocarbon chain having a number of carbons greater than 5 or even 10 and at least one hydrophilic anionic group, such as a sulphate, a sulphonate or a carboxylate linked to one end of the hydrophobic chain. An anionic surfactant particularly suitable for the implementation of the invention is sodium dodecyl sulphate (SDS).
    [0026] When the surfactant is an anionic surfactant, the mass content of surfactant in the external phase is typically from 0.001% to 0.5%, preferably from 0.001% to 0.05%, based on the total weight of the capsule. In this case, the mass content of surfactant in the external phase is preferably less than or equal to 0.025%, preferably less than or equal to 0.010%, or even less than or equal to 0.005%, relative to the total weight of the capsule. In the case where the surfactant is a cationic surfactant, it is for example chosen from alkylpyridium or alkylammonium halide salts such as n-ethyldodecylammonium chloride or bromide, cetylammonium chloride or bromide (CAB) . These surfactants advantageously have at least one hydrophobic hydrocarbon chain having a number of carbon atoms greater than 5 or even 10 and at least one hydrophilic cationic group, such as a quaternary ammonium cation. When the surfactant is a cationic surfactant, the mass content of surfactant in the external phase is typically from 0.001% to 0.5%, preferably from 0.001% to 0.05%, based on the total weight of the capsule. In this case, the mass content of surfactant in the external phase is preferably less than or equal to 0.025%, preferably less than or equal to 0.010%, or even less than or equal to 0.005%, relative to the total weight of the capsule.
    [0027] In the case where the surfactant is a nonionic surfactant, it is for example chosen from polyoxyethylenated and / or polyoxypropylenated derivatives of fatty alcohols, fatty acids, or alkylphenols, arylphenols, or from alkylglucosides, polysorbates and cocamides .
    [0028] A nonionic surfactant particularly suitable for the implementation of the invention is polysorbate 20 (Tween 20). When the surfactant is a nonionic surfactant, the mass content of surfactant in the external phase is typically from 0.01% to 2%, preferably from 0.1% to 1%, relative to the total weight of the capsule Intermediate phase According to one embodiment, the capsules according to the invention comprise an intermediate phase between the internal phase and the gelled external phase. This intermediate phase forms an aqueous or, where appropriate, oily, generally biocompatible, intermediate envelope which completely encapsulates the internal phase and is completely encapsulated by the gelled external phase. Such capsules 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 external phase. 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. The intermediate phase may comprise at least one plant cell, preferably an algal cell, which may be identical or different from the plant cells present in the internal phase. The intermediate phase may also comprise at least one viscosity agent as described above. The intermediate phase, when present and comprising plant cells, is preferably suitable for the survival of said plant cells. It advantageously comprises a culture medium capable of culturing said cells, typically one of the MC1 culture media mentioned above for the internal phase.
    [0029] The intermediate phase, when present and comprising plant cells, typically comprises from 1 to 107, preferably from 5 to 106, from 30 to 5 × 10 5, from 50 to 105, from 75 to 5 × 10 4, from 100 to 104 from 150 to 104, or even 200 to 103 plant cells, per capsule.
    [0030] Culture Method The subject of the present invention is also a method for culturing plant cells comprising a step of culturing at least one capsule according to the invention. By "culturing" is meant the action of placing the capsules of the invention in a culture medium called MC2 in the context of the present invention, typically suitable for the cultivation of encapsulated plant cells, under temperature conditions. and brightness adapted to the culture of said plant cells, for a time necessary to obtain the desired plant cell concentration within the capsules. Depending on the cultured plant cells, those skilled in the art are able to select the appropriate MC2 culture medium, as well as the temperature and light conditions appropriate for the proliferation of plant cells. The culture medium MC2 is, for example, chosen from Erdschreiber culture medium, F / 2 medium, TAP medium, reconstituted seawater, DM (diatom) medium, Minimum medium, and any of their mixtures.
    [0031] The osmolarity of the culture medium MC2 is preferably between 10 mOsm and 1000 mOsm. It is preferable that the ratio (osmolarity of MC1) / (osmolarity of MC2) is between 1/50 and 50, preferably between 1/10 and 10. According to one embodiment, the culture medium MC2 is identical to the medium of MC1 culture. According to another embodiment, the culture medium MC2 is different from the culture medium MC1. It is advantageous to choose different culture media to cause the production of compounds of interest within the cells simultaneously with culturing.
    [0032] Typically, for culturing, from 10,000 to 10,000,000 capsules according to the invention are placed per liter of MC2 culture medium. The capsules are typically cultured at a temperature of 10 ° C to 40 ° C, preferably 15 ° C to 25 ° C.
    [0033] The capsules are typically cultured under light conditions ranging from total black to 500pE.rn-2.s-1. The capsules are typically cultured for a period of one hour to one month, usually 24 hours to a week.
    [0034] At the end of the culture process, capsule harvesting is typically by removal of the MC2 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.
    [0035] The inventors have discovered, surprisingly, that the cultivation of plant cells, in particular of algae, within the capsules according to the invention makes it possible to access higher plant cell concentrations than in the bulk processes mentioned in FIG. introduction. For example, cell concentrations of 50 million to 150 million cells per ml are obtained in the capsules, 5 to 15 times more than those obtained in bulk processes. The encapsulation of plant cells and the capsule culture of these plant cells, in particular algae such as microalgae, also has the following advantages over conventional methods: the encapsulated plant cells are protected from mechanical stresses, such as shearing, thereby decreasing cell death during the cell culture process; the encapsulated plant cells are partially protected from the external environment, because the capsule membrane has a selective permeability (in particular, the bacteria can not enter the capsule); the size of the capsules (several hundred micrometers) and their mechanical properties make them easier to handle than the algae, in particular for changes in MC2 culture medium or during harvesting; and post-growth treatment and possibly elicitation can be implemented to waterproof the capsules, thus isolating their contents to facilitate preservation until use. Some advantages of the invention due to the mechanical properties of the hydrogel forming the capsule membrane will now be detailed.
    [0036] The hydrogel network confers indeed semisolid mechanical properties to the capsules of the invention, much higher than those of plant cells. In practice, this is of significant interest for the cultivation of plant cells, such as algae, as this makes it possible to confer a high degree of mechanical resistance on plant cells, which are classically fragile. While the mechanical resistance of these plant cells rests natively on the properties of the lipid bilayer (3 nm of phospholipids) constituting their membrane, when they are encapsulated in a capsule according to the invention, they are now protected by a network. macromolecular from several micrometers to several tens of micrometers thick (ie the hydrogel membrane of the capsules). In the case of plant 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.
    [0037] 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 screening 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 on the porous nature of the structure of the chain network of the polyelectrolyte. 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 plant cells, such as small biomolecules (amino acids and peptides), and small macromolecules with a molecular weight of less than 1 MDa. On the other hand, the membrane retains any element with a characteristic size greater than the cutting size, ie macromolecules or biomolecules with a molecular weight greater than 1 MDa, or cellular organisms such as plant cells, for example algae, or even bacteria. and mushrooms. In practice, this is of significant interest for the cultivation of plant cells, such as algae, by controlling the exchanges between the outside and the inside of the capsule, that is to say between the culture medium MC2 and encapsulated biomass. 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 MC2 culture medium, in particular for the removal of the consecutive waste metabolism / catabolism of the encapsulated biomass. These exchanges can be further facilitated by mixing the MC2 culture medium, without this threatening the plant 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 the plant cells with all the 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 plant cells after exogenous bacterial contamination, where the same contamination would destroy all the cells in a bulk process.
    [0038] Finally, this semi-permeability also makes it possible to control the release of encapsulated element, such as compounds of interest produced by the plant cells. The production of plant cells, in particular of algae, 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.
    [0039] In addition, one of the limiting factors of algae 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 induces a decrease in the luminous flux captured by the plant cells. The accumulation of this deposit induces increasing pressure losses and increases production costs. However, the culture of plant cells in capsules according to the invention makes it possible: to limit the deposition of the biomolecules, these being partially retained inside the capsules, moreover these biomolecules can be recovered at the end of the process, and to prevent depositing the plant cells on the walls of the flasks in which the capsules are cultured or stored (anti-fouling property), said cells being indeed 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.
    [0040] The present invention also relates to a process for producing a compound of interest, comprising: a step of culturing at least one capsule according to the invention, said capsule comprising at least one plant cell capable of producing said compound of interest, optionally a step of elicitation of the plant cells included in said capsule, and a step of recovering the compound of interest.
    [0041] The step of culturing the above production method generally corresponds to the step of culturing the culture process of the invention. The culture medium MC2 may be identical or different from the culture medium MC1 of the capsule core.
    [0042] In the context of the present description, "elicitation" is understood to mean the stimulation of the production of compounds of interest by a plant cell, said stimulation being caused by the setting in particular conditions, whether physicochemical, resulting from a modulation of temperature, pressure or illumination, or that they rely on the presence of a particular molecule, called "elicitant molecule". Artificial production of compounds of interest by the encapsulated plant cells is thus artificially induced. According to one embodiment, the elicitation step takes place during the culturing step.
    [0043] This embodiment is for example implemented by carrying out the step of culturing under eliciting conditions, for example by carrying out the step of culturing in a culture medium MC2 different from the culture medium MC1, said medium MC2 culture containing an elicitant molecule. According to another embodiment, the elicitation step takes place at the end of the culturing step, that is to say once the plant cells have proliferated within the capsules of the invention. This embodiment is for example implemented by performing the culturing step under conventional conditions, that is to say by choosing a culture medium MC2 identical to the culture medium MC1, then, at the resulting from the culturing step, placing itself under eliciting conditions, for example by replacing the culture medium MC2 with another culture medium, different from the culture medium MC1.
    [0044] These two embodiments are facilitated by the encapsulation of plant cells, which makes the manipulation of encapsulated cells more convenient. As described above, because of the encapsulation, it is indeed easier to proliferate the plant cells within the capsules, then optionally to divide the capsules into separate lots, to replace the MC2 culture medium with various mediums. culture and / or to apply various elicitation conditions depending on the batches, in order to test different eliciting conditions and to determine the conditions suitable for the production of the compound of interest that the person skilled in the art wishes to obtain. Thus, after a first step of culturing, the elicitation step of the plant cells typically comprises: a step of culturing the capsules in separate lots under different conditions, a step of detecting and quantifying the compound of interest produced, and - possibly the selection of individuals with the best phenotypes.
    [0045] The elicitation stage of the plant cells consists, for example, in culturing the capsules of the invention in a culture medium MC2 different from the culture medium MC1, or in a culture medium comprising an eliciting molecule, or in the same MC1 medium with a change in culture conditions (temperature, light, etc.).
    [0046] The capsules according to the invention can be cultured under eliciting conditions well known to those skilled in the art, such as by adding to the culture medium MC2 salicylic acid, ethylene, jasmonate or chitosan (cf. for example the international application WO 2003/077881).
    [0047] The compounds of interest produced by the production method of the invention are typically subjected to one or more treatments, such as purification, concentration, drying, sterilization and / or extraction. These compounds are then intended to be incorporated into a cosmetic, agri-food or pharmaceutical composition. As a compound of interest, the capsules of the invention make it possible in particular to produce lipids of interest in cosmetics, such as, for example, fatty acids, such as linoleic acid, alpha-linoleic acid or gamma acid. linoleic, palmitic acid, stearic acid, eicosapentaenoic acid, docosahexanoic acid, arachidonic acid; fatty acid derivatives, such as ceramides; or else sterols, such as brassicasterol, campesterol, stigmasterol and sitosterol. The capsules of the invention also make it possible to produce organic selenium, an essential trace mineral. This chemical element, not synthesized by the human body, is an asset of interest in agri-food and cosmetics, particularly for its antioxidant properties. In its metallic form, it is an essential trace element but many of its compounds are extremely toxic, and are obtained by reprocessing the residues of the electrolysis of lead, arsenic or copper. This is why it is preferable to obtain it in its organic form for biological applications, that is to say as a constituent of biomolecules.
    [0048] Selenium is typically incorporated into amino acids, peptides and proteins, especially in the form of selenomethionine. Depending on the nature of the compound (s) of interest to be recovered and that of the plant cells, one skilled in the art is able to determine the treatment (s) necessary for the recovery and purification of said compound of interest. According to one embodiment, the recovery of the compound of interest is carried out by treatment of the culture medium (MC2) in which the capsules are immersed, 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 compound of interest is excreted by the plant 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 compound 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 contact 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 compound of interest remains confined within the plant cells. The subject of the present invention is also the use of a capsule according to the invention for the production of plant cells and / or the production of molecules of interest. Typically, the production of molecules of interest is obtained by elicitation of plant cells, as described above. Composition The present invention also relates to a composition comprising at least one capsule according to the invention.
    [0049] According to one embodiment, the capsule according to the invention has been treated by a process for producing a compound of interest according to the invention. According to one variant, the capsule according to the invention has, in addition, undergone a post-process treatment for the production of a compound of interest, consisting in forming a second membrane completely encapsulating at its periphery the gelled membrane of said capsule. Such a second membrane may be formed by gelling in the presence of a compound capable of forming electrostatic bonds with the constituents of the gelled membrane, typically in the presence of a polyelectrolyte. This variant has the advantage of protecting the heart of the capsules and to avoid the migration out of the capsules of the compounds of interest contained in said core.
    [0050] The composition according to the invention is typically a cosmetic, pharmaceutical or agri-food composition. The subject of the present invention is also the use of a capsule according to the invention for the preparation of a cosmetic, pharmaceutical or agri-food composition.
    [0051] The present invention also relates to a composition comprising a capsule extract. By "capsule extract" is meant a compound of interest produced by the plant cells, typically by elicitation, and recovered as described above. A plant cell is also meant, originating from a cultivation process of the invention, and possibly from a process for producing a compound of interest according to the invention. Other characteristics and advantages of the invention will emerge more clearly from the examples which follow, given by way of illustration and not limitation.
    [0052] EXAMPLES Example 1 Preparation of Gelled Capsules Comprising Microalgae 1. Preparation of Solutions for the Manufacture of Capsules - External Phase (Membrane) A solution containing 1.69% of sodium alginate (Protanal LF200 FTS, FMC Bioploymer) ( w / w) and 1 mM (ie 0.0288% by weight) of SDS (Sigma Aldrich) was prepared, then filtered at 5 °. This filtration step avoids the presence of solid particles or aggregates leading to clogging of the particles. nozzles used for production, but is also used to sterilize the phases. It is also possible to heat these phases to a temperature above 60 ° C to sterilize them. Internal Phase (Heart) A microalgae solution was prepared at a typical concentration of 1 million cells / ml in TAP (MC1) medium. 2-ethylcellulose (0.5% by weight) was added to facilitate the coextrusion of the phases and to stabilize the process by avoiding excessive viscosity differences between the internal and external phases.
    [0053] The following micro-algae have been encapsulated: Chlamydomonas reinhardtii (strains WTS24-, Sta6 and CW15). 2. Manufacture of Capsules The capsule manufacturing process is based on the concentric coextrusion of two solutions, in particular described in WO 2010/063937 and FR2964017, to form double drops. The size of the internal phase and the thickness of the external phase of the drops formed were controlled by the use of two independent syringe pumps (HA PHD-2000).
    [0054] The ratio rq between the flow rate of the fluid constituting the core and the flow rate of fluid constituting the membrane was set at 1.6. This made it possible to obtain capsules having a membrane thickness-to-radius ratio of less than 0.9, which maximizes the rate of encapsulation of the internal phase, and therefore micro-algae. The capsules obtained have a diameter of 300 μm (+/- 50 μm).
    [0055] The drops formed were gelled with a gelling solution of sterile calcium chloride 200 mM (minimum concentration 50 mM), to which were added a few drops of a sterile solution of Tween 20 (Sigma Aldrich) to 10% (w / w). The formed capsules were harvested using a sieve and then transferred to one of the culture media described below. Example 2 Production of Microalgae in Capsules 1. Preparation of Solutions - Phosphate Buffer (2x): K2HPO4 14.34 g KH2PO4 7.26 g pure water 1 L - 1M phosphate buffer pH 7: 1 mol / L solution K2HPO4 60 mL 1 mol / L solution of KH2PO4 40 mL - Beijerincks buffer (2x): NH4Cl 8g CaCl2 1 g MgSO4 2g pure water 1 L - Trace solution: Solution 51 EDTA 50g pure water 250 mL Solution S2 ZnSO4.7H20 22 g pure water 100 mL Solution S3 H3B03 11.4g pure water 200 mL Solution S4 MnCl2.4H2O 5.06 g Fe504.7H20 4.99 g CoCl2.6H2O 1.61 g Cu504.5H2O 1.57 g (NH4) 6Mo702.4H20 1.1 g pure water 50 mL To prepare the trace solution, solutions S2, S3 and S4 were mixed, then solution 51 was added. The mixture was boiled for a few minutes. Then the mixture was stirred strongly keeping the temperature above 70 ° C. The pH of the mixture was then adjusted to between 6.5 and 6.8 by adding the required amount of a 20% by weight KOH solution. The volume of the mixture was then adjusted to 1 L by addition of pure water. The mixture was then allowed to stand for one week until it turned pink / purple. The solution has finally been filtered. 2. Culture Media (MC2) - TAP Media Tris 2.42 g Beijerincks (2x) 50 mL 1M Phosphate Buffer pH 7 1 mL Trace Solution 1 mL Acetic Acid 1 mL QSP Pure Water 1 L - Minimum Media: Beijerincks (2x) 50 mL phosphate buffer (2x) 50 mL trace solution 1 mL QSP 1 L pure water 3. Culture and growth of micro-algae encapsulated according to the invention and bulk microalgae On the one hand, the capsules of the example 1, containing microalgae Chlamydomonas reinhardtii (strain WTS 24-) according to an initial cell concentration adjusted to 1 million cells / ml, were cultured in TAP medium or Minimum medium (M02) (approximately 200 capsules). 20 ml flask, ie 10,000 capsules per 1 L of M02 medium), in flasks allowing gaseous exchange, with moderate stirring, at 25 ° C. On the other hand, the same microalgae Chlamydomonas reinhardtii (strain WTS 24-) were cultured in bulk, according to an initial cell concentration adjusted to 1 million cells / ml, under the same conditions.
    [0056] In a minimum medium, a light source of the order of 3400 lumens was used.
    [0057] The monitoring of cell growth was done qualitatively, comparing the evolution of the volume occupied by microalgae in the capsules, and quantitatively, by classical cell biology experiments. To do this, the capsules were opened by contacting with a solution of citrate (sodium) at 10% by mass for a few seconds. The citrate anions make it possible to complex the calcium cations, which depolymerizes the alginate gel of the membrane. The contents of the capsules were analyzed by flow cytometry and Malassez counting. In the capsules of the invention, the microalgae proliferated to a mean concentration in the capsules of between 120 and 250 million cells / mL after one week, as measured by Malassez counting. In comparison, with a bulk culture method (non-encapsulated cells, in free form in suspension), the microalgae did not exceed a concentration of 10 million cells / mL, all conditions being equal. 4. Evaluation of the stability of the capsules in culture medium (MC2) The stability of the capsules was evaluated in TAP culture medium, TAP culture medium supplemented with 10 mM CaCl 2 and the TAP culture medium to which was added. 0.1% EDTA by mass.
    [0058] The capsules of Example 1 remained stable for at least 3 weeks in each of these 3 culture media (MC2). 5. Evaluation of the mechanical protection afforded to the microalgae by the capsules Microalgae Chlamydomonas reinhardtii strain WTS 24- were cultured at 250,000 cells / ml in TAP medium (ie about 10,000 capsules per 1 L of medium TAP), firstly in free form (in bulk), and secondly in encapsulated version according to the invention (capsules of Example 1). The two samples obtained were imaged in the presence of SYTOX Green, a fluorescence-visible cell death marker with the Nikon FITC filter, before and after strong stirring. Before shaking, it was observed that most of the microalgae did not show green marking indicating cell death. Few cells were dead before agitation, the proportion of dead micro-algae simply corresponding to the cell cycle.
    [0059] After shaking, it was observed that in the microalgae sample grown in bulk, most of the microalgae appeared green, indicating that most microalgae are dead, under the effect of high shear. caused by agitation. In contrast, the encapsulated microalgae showed a green marking equivalent to the marking observed before stirring. Few cells died, despite the strong shear imposed. As in the initial microalgae suspension, the presence of a small proportion of dead micro-algae is normal and stems simply from the cellular cycle of the microalgae considered. Overall quantification of the fluorescence associated with cell death was performed on each sample after shaking and showed that cell death was considerably higher in the case of bulk microalgae than in the case of encapsulated microalgae. This experiment has demonstrated the mechanical protection conferred by the capsules with micro-algae. 6. Semi-Permeability of Membranes: VS bacteria molecules Capsules according to Example 1 were cultured in TAP medium (approximately 10,000 capsules per 1 L of TAP medium), to which Escherichia coli RFP bacteria were added. that is, E. coli bacteria genetically engineered to synthesize a fluorescent molecule, visible using the Nikon TRITC filter. It was found that the RFP bacteria did not invade the inside of the capsules. On the other hand, capsules according to Example 1 were placed in the presence of a 1 mM solution of rhodamine (visible in fluorescence using the Nikon TRITC filter) for a few minutes, then were transferred to a bath of oil and imaged under the microscope. These results showed that, unlike E. coli RFP bacteria, rhodamine diffused through the alginate membrane to be found inside the capsules. The capsules according to the invention are therefore semi-permeable: they allow the passage of molecules, such as the nutrients of the culture medium MC2, and do not allow the bacteria to penetrate, which has the advantage of preventing any bacterial contamination during the microalgae cultivation process.
    [0060] EXAMPLE 3 Elicitation of Encapsulated Microalgae for the Production of Lipids and Organic Selenium 1. Production of Lipids The Beijerincks 2X NO buffer was prepared: CaCl 2 1 g MgSO 4 2 g pure water 1 L The NO medium was prepared: Tris 2 , 42 g Beijerincks (2x) NO 50 mL 1M phosphate buffer pH 7 1 mL Trace solution 1 mL acetic acid 1 mL QSP 1 L pure water Capsules according to Example 1, including microalgae Chlamydomonas reinhardtii (strain Sta6) were cultured in TAP medium for 48 hours (approximately 10,000 capsules per 1 L of TAP medium), then the latter was removed and replaced with NO medium for 48 h. The microalgae then produced lipids, in the form of lipid bodies present inside said microalgae. Capsules were removed and placed in the presence of 25% DMSO and 1 1M Nile Red for 10 minutes. The capsules were then imaged under bright field microscopy and fluorescence (source: mercury lamp). The chloroplast of the micro-algae was revealed in fluorescence thanks to the Nikon UV-1A filter. The Nile Red, testifying to the presence of lipids produced by elicitation, was revealed thanks to the Nikon FITC filter. 2. Production of organic selenium Some microalgae produce molecules containing selenium, such as Peridinium cinctum. Such microalgae can therefore produce selenium in organic form, from mineral selenium. Microalgae producing selenium-containing molecules have been encapsulated in accordance with the present invention and then incubated in a mineral selenium-enriched medium to induce bioaccumulation of organic selenium by these microalgae. Quantification of bioaccumulated selenium by encapsulated microalgae was performed by extracting capsule cells and then using physical methods, such as ASA (see Niedzielski (2002), Polish Journal of Environmental Studies: "Atomic Absorption Spectrometry in Determination of Arsenic, Antimony and Selenium in Environmental Samples") or by the use of a bioassay (see Lindstreim (1983), Hydrobiologia: "Selenium as a growth factor for plankton algae in laboratory experiments". in some Swedish lakes ").
    权利要求:
    Claims (10)
    [0001]
    REVENDICATIONS1. Capsule comprising: - an internal phase comprising at least one plant cell, and - a gelled external phase, totally encapsulating said internal phase at its periphery, said external phase comprising at least one surfactant and at least one polyelectrolyte in the gelled state.
    [0002]
    The capsule of claim 1, wherein the inner phase comprises a culture medium.
    [0003]
    3. Capsule according to claim 1 or 2, wherein the one or more plant cells are algal cells, preferably microalgae cells. 15
    [0004]
    4. Capsule according to one of claims 1 to 3, wherein the plant cell or cells are suspended in the internal phase.
    [0005]
    5. Capsule according to one of claims 1 to 4, wherein the polyelectrolyte 20 is a multivalent ion reactive polyelectrolyte, preferably an alginate.
    [0006]
    6. Capsule according to one of claims 1 to 4, characterized in that it has a size less than 5 mm, preferably from 50 pm to 3 mm. 25
    [0007]
    7. A method of culturing plant cells comprising a step of culturing at least one capsule according to one of claims 1 to 6.
    [0008]
    8. A process for producing a compound of interest, said process comprising: a step of culturing at least one capsule according to one of claims 1 to 6, said capsule comprising at least one plant cell capable of to produce said compound of interest, - optionally, a step of elicitation of the plant cells included in said capsule, and - a step of recovering the compound of interest.
    [0009]
    9. Use of a capsule according to any one of claims 1 to 6 for the production of plant cells and / or the production of compounds of interest.
    [0010]
    10. Composition comprising at least one capsule according to one of claims 1 to 6.
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    引用文献:
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    FR2969907B1|2010-12-31|2014-03-07|Capsum|SERIES OF CAPSULES COMPRISING AT LEAST ONE INTERNAL PHASE DROP IN AN INTERMEDIATE PHASE DROP AND METHOD OF MANUFACTURING THE SAME|FR3071505A1|2017-09-22|2019-03-29|Capsum|BACTERICID OR BACTERIOSTATIC OR ANTIFUNGAL CAPSULES COMPRISING LIVING CELLS AND USES THEREOF|
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    CN113005041A|2021-03-23|2021-06-22|山东大学|Diamond algae, culture method thereof and application thereof in super-salt oil production|
    法律状态:
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    2016-04-29| PLSC| Publication of the preliminary search report|Effective date: 20160429 |
    2016-08-24| PLFP| Fee payment|Year of fee payment: 3 |
    2017-07-20| PLFP| Fee payment|Year of fee payment: 4 |
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    2021-09-10| PLFP| Fee payment|Year of fee payment: 8 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1460169|2014-10-22|
    FR1460169A|FR3027608B1|2014-10-22|2014-10-22|GELIFIED CAPSULE COMPRISING A PLANT CELL|FR1460169A| FR3027608B1|2014-10-22|2014-10-22|GELIFIED CAPSULE COMPRISING A PLANT CELL|
    EP15786905.8A| EP3209266A1|2014-10-22|2015-10-22|Gel capsule comprising a plant cell|
    EP20174903.3A| EP3718530A1|2014-10-22|2015-10-22|Gel capsule comprising a plant cell|
    US15/520,269| US20170312323A1|2014-10-22|2015-10-22|Gel capsule comprising a plant cell|
    CN201580057823.0A| CN107105737A|2014-10-22|2015-10-22|Gel capsule including plant cell|
    PCT/EP2015/074549| WO2016062836A1|2014-10-22|2015-10-22|Gel capsule comprising a plant cell|
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