GEL CAPSULE COMPRISING A PLANT CELL

Abstract

Disclosed is a capsule including: an internal phase including at least one plant cell; and an external gel phase, that totally encapsulates the internal phase at its periphery, the external phase including at least one surfactant and at least one polyelectrolyte in the gel state.

Claims

1. A capsule comprising : an internal phase comprising at least one plant cell; and an external gel phase, that totally encapsulates said internal phase at its periphery, said external phase comprising at least one surfactant and at least one polyelectrolyte in the gel state.

2. A capsule according to claim 1, in which the internal phase comprises a culture medium.

3. A capsule according to claim 1, in which the plant cell or cells are algae cells.

4. A capsule according to claim 1, in which the plant cell or cells are in suspension in the internal phase.

5. A capsule according to claim 1, in which the polyelectrolyte is a polyelectrolyte reactive to multivalent ions.

6. A capsule according to claim 1, wherein the capsule size is less than 5 mm.

7. A method for culturing plant cells including a step of putting in culture at least one capsule according to claim 1.

8. A method for producing a compound of interest, that includes: a step of putting in culture at least one capsule according to claim, said capsule comprising at least one plant cell that is capable of producing said compound of interest. possibly, a step of elicitation of the plant cells included in said capsule; and a step of recovering the compound of interest.

9. Plant cells produced using the capsule according to one claim 1.

10. A composition comprising at least one capsule according to claim 1.

11. A capsule according to claim 2, in which the plant cell or cells are algae cells.

12. The capsule of claim 3, wherein the algae cells are micro algae cells.

13. A capsule according to claim 2, in which the plant cell or cells are in suspension in the internal phase.

14. A capsule according to claim 3, in which the plant cell or cells are in suspension in the internal phase.

15. A capsule according to claim 1, in which the polyelectrolyte is a polyelectrolyte reactive to multivalent ions.

16. The capsule according to claim 6, wherein the capsule size is in a range of 50 μm to 3 mm.

17. A capsule according to claim 2, wherein the capsule size is less than 5 mm.

18. A capsule according to claim 3, wherein the capsule size is less than 5 mm.

19. A capsule according to claim 4, wherein the capsule size is less than 5 mm.

20. A method for culturing plant cells including a step of putting in culture at least one capsule according to claim 2.

Description

EXAMPLES

Example 1

Preparation of Gel Capsules Comprising Micro-Algae

1. Preparation of the Solutions for the Manufacture of Capsules

External Phase (Membrane)

[0202] A solution containing 1.69% sodium alginate (Protanal LF 200 FTS, FMC Bioploymer) (w/w or weight per weight) and 1 millimolar mM (or 0.0288% by mass) of SDS (Sigma Aldrich) was prepared and then filtered at 5 μm.

[0203] This step of filtration makes it possible to prevent the presence of particles or solid aggregates that result in the clogging of the nozzles used for the production, but is also used so as to sterilise the phases. It is also possible to heat these phases at a temperature that is higher than 60° C. in order to sterilise them.

Internal Phase (Core)

[0204] A solution of micro-algae was prepared with a typical concentration of 1 million cells/mL in the TAP medium (MC1).

[0205] The 2-ethylcellulose (0.5% by mass) was added in order to facilitate the co-extrusion of phases and stabilise the process by avoiding having excessively large differences in viscosity between the internal and external phases.

[0206] The following micro-algae in particular were encapsulated: Chlamydomonas reinhardtii (strains WTS24-, Sta6 and CW15).

2. Manufacture of Capsules

[0207] The manufacture of capsules is based on the concentric co-extrusion of two solutions, notably described in the patent documents WO 2010/063937 and FR2964017, in order to form double drops.

[0208] 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).

[0209] The ratio r.sub.q between the flow rate of the fluid constituting the core and the flow rate of the fluid constituting the membrane was fixed at 1.6. This made it possible to obtain capsules having a ratio of membrane thickness to radius of less than 0.9, which maximises the rate of encapsulation of the internal phase, and thus also of the micro-algae.

[0210] The capsules obtained have a diameter of 300 μm (+/−50 μm).

[0211] The drops thus formed were gelled by using a gelling solution of sterile calcium chloride at 200 mM (minimum concentration 50 mM), to which were added a few drops of a 10% (w/w) sterile solution of Tween 20 (Sigma Aldrich).

[0212] The capsules formed were collected by making use of a screen/sieve, and then emptied into one of the culture media described here below.

Example 2

Production of Micro-Algae in Capsules

1. Preparation of Solutions

Phosphate Buffer (2×)

[0213]

TABLE-US-00001 K.sub.2HPO.sub.4 14.34 g KH.sub.2PO.sub.4 7.26 g pure water 1 L

Phosphate Buffer 1 M pH 7

[0214]

TABLE-US-00002 1 mol/l solution of K.sub.2HPO.sub.4 60 mL 1 mol/l solution of KH.sub.2PO.sub.4 40 mL

Beijerinck's Buffer Solution (2×)

[0215]

TABLE-US-00003 NH.sub.4Cl 8 g CaCl.sub.2 1 g MgSO.sub.4 2 g pure water 1 L

Trace Elements Solution

[0216]

TABLE-US-00004 Solution S1 EDTA 50 g pure water 250 mL Solution S2 ZnSO.sub.4•7H.sub.2O 22 g pure water 100 mL Solution S3 H.sub.3BO.sub.3 11.4 g pure water 200 mL Solution S4 MnCl.sub.2•4H.sub.2O 5.06 g FeSO.sub.4•7H.sub.2O 4.99 g CoCl.sub.2•6H.sub.2O 1.61 g CuSO.sub.4•5H.sub.2O 1.57 g (NH.sub.4).sub.6Mo.sub.7O.sub.2•4H.sub.2O 1.1 g pure water 50 mL

[0217] In order to prepare the Trace Elements Solution, the solutions S2, S3 and S4 were mixed, then the solution S1 was added therein. The mixture was brought to a boil for a few minutes. Then the mixture was agitated strongly keeping the temperature above 70° C. The pH of the mixture was then adjusted to a value of between 6.5 and 6.8 by addition of the necessary amount of a solution of KOH at a concentration of 20% by weight. The volume of the mixture was adjusted to 1 L with the addition of pure water. The mixture was then left to stand without being disturbed for a week, until it took on a pink/violet colour. Finally the solution was filtered.

2. Culture Media (MC2)

TAP (Tris-Acetate-Phosphate) Medium

[0218]

TABLE-US-00005 Tris 2.42 g Beijerinck's solution (2X) 50 mL Phosphate buffer 1M pH 7 1 mL Trace elements solution 1 mL Acetic acid 1 mL Pure water QSF 1 L

Minimum Medium

[0219]

TABLE-US-00006 Beijerinck's solution (2X) 50 mL Phosphate buffer (2X) 50 mL Trace elements solution 1 mL Pure water QSF 1 L

3. Culture and Growth of Encapsulated Micro-Algae According to the Invention and of Micro-Algae in Bulk Mode

[0220] On the one hand, the capsules described in Example 1, containing the micro-algae Chlamydomonas reinhardtii (strain WTS 24-) according to an initial cell concentration adjusted to 1 million cells/mL, were put in culture in the TAP medium or Minimum medium (MC2) (that is to say, about 200 capsules per 20 mL flask, or 10,000 capsules for 1 L of the medium MC2), in flasks that allow for the gas exchanges, under conditions of moderate stirring, at 25° C.

[0221] On the other hand, the same micro-algae Chlamydomonas reinhardtii (strain WTS-24) were put in culture as per bulk growth mode, according to an initial cell concentration adjusted to 1 million cells/mL, under the same conditions.

[0222] With the Minimum medium, a light source of the order of 3400 lumens was used.

[0223] The monitoring of cell growth was carried out in a qualitative manner, by comparing the evolution of the volume occupied by the micro-algae in the capsules, and in a quantitative manner, by means of conventional cell biology experiments. In order to do this, the capsules were opened by being placed in contact with a solution of (sodium) citrate in a concentration of 10% by weight for a few seconds. The citrate anions enable the complexing of the calcium cations, which depolymerises the alginate gel of the membrane.

[0224] The contents of the capsules were analysed by means of flow cytometry and Malassez cell count method.

[0225] In the capsules of the invention, the micro-algae proliferated up to attaining an average concentration in the capsules ranging between 120 and 250 million cells/mL at the end of one week, such as measured by the Malassez cell count method.

[0226] In comparison, with a bulk growth culturing method (cells not encapsulated, in freely suspended form), the micro-algae did not exceed a concentration of 10 million cells/mL, moreover with all other conditions being equal.

4. Evaluation of the Stability of the Capsules in Culture Medium (MC2)

[0227] The stability of the capsules was assessed in the TAP culture medium, in the TAP culture medium with addition of 10 mM of CaCl.sub.2, and the TAP culture medium to which was added 0.1% by weight of EDTA.

[0228] 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 Provided to the Micro-Algae by the Capsules

[0229] The micro-algae Chlamydomonas reinhardtii strain WTS 24—were put in culture at a concentration of 250,000 cells/ml in TAP medium (that is to say, around 10,000 capsules for 1 L of TAP medium), on the one hand in free form (in bulk), and on the other hand, in encapsulated version according to the invention (capsules of Example 1).

[0230] The two samples obtained were imaged in the presence of SYTOX Green, a marker of cell death visible in fluorescence with the Nikon FITC (fluorescein isothiocyanate) filtre, before and after strong agitation.

[0231] Prior to agitation, it was observed that the majority of the micro-algae did not display the green marking that is an indicator of cell death. Thus very few cells had died prior to agitation, the proportion of dead micro-algae corresponds simply to the cell cycle.

[0232] After agitation, it was observed that in the sample of micro-algae grown in bulk, the majority of micro-algae appeared to be coloured green, which indicates that the majority of the micro-algae were dead, under the effect of the strong shear caused by the agitation.

[0233] In contrast thereto, the encapsulated micro-algae displayed a green marking equivalent to the marking observed prior to the agitation. Few cells had died, despite the strong shear imposed. As in the initial suspension of micro-algae, the presence of a small proportion of dead algae is normal and simply comes about as a result of the cell cycle of the micro-algae considered.

[0234] An overall quantification of the fluorescence associated with the cell death was conducted on each sample after agitation and showed that the cell death was significantly higher in the case of the micro-algae grown in bulk, than in the case of the encapsulated micro-algae.

[0235] This experiment served to make evident the mechanical protection conferred to the micro-algae by the capsules.

6. Semi-Permeability of the Membranes: Molecules VS Bacteria

[0236] The capsules according to Example 1 were put in culture in TAP medium (that is to say, around 10,000 capsules for 1 L of TAP medium), to which were added the bacteria Escherichia coli RFP (red fluorescent protein), that is to say, bacteria belonging to the species E. coli that have been genetically modified in order for them to synthesise a fluorescent molecule, visible using the Nikon TRITC (tetramethyl rhodamine isothiocyanate) filtre.

[0237] It was noted that the RFP bacteria were not invading the interior of the capsules.

[0238] On the other hand, the capsules according to Example 1 were placed in the presence of a 1 mM solution of rhodamine (visible in fluorescence using the Nikon TRITC filtre) for a few minutes, and thereafter were transferred into an oil bath and imaged with a microscope. These results showed that, in contrast to the E. coli RFP bacteria, the rhodamine had diffused through the alginate membrane so as to be found within the interior of the capsules.

[0239] The capsules according to the invention are thus semi-permeable: they allow the through-passage of molecules, such as the nutrients from the culture medium MC2, and do not allow penetration by the bacteria, which has the advantage of preventing any bacterial contamination during the process of culturing the micro-algae.

Example 3

Elicitation of Encapsulated Micro-Algae for the Production of Lipids and Organic Selenium

1. Production of Lipids

The Beijerinck's buffer solution 2× N0 was prepared

[0240]

TABLE-US-00007 CaCl.sub.2 1 g MgSO.sub.4 2 g pure water 1 L

The N0 Medium was Prepared

[0241]

TABLE-US-00008 TRIS 2.42 g Beijerinck's solution (2X) N0 50 mL Phosphate buffer 1M pH 7 1 mL Trace elements solution 1 mL Acetic acid 1 mL Pure water QSF 1 L

[0242] The capsules according to Example 1, comprising the micro-algae Chlamydomonas reinhardtii (strain Sta6) were put in culture in the TAP medium for 48 hours (that is to say, about 10,000 capsules for 1 L of TAP medium), then the latter was eliminated and replaced by N0 medium, for a period of 48 hours.

[0243] The micro-algae then produced lipids, in the form of lipid bodies present within the interior of the said micro-algae.

[0244] The capsules were then collected and put in the presence of 25% of DMSO and 1 μM of Nile Red for a period of 10 minutes. The capsules were then imaged under the microscope in the bright field and in fluorescence mode (source: mercury lamp).

[0245] The chloroplast of the micro-algae were revealed in fluorescence with the use of the Nikon UV-1A filtre.

[0246] The Nile Red, serving as evidence of the presence of lipids produced by elicitation, was revealed with the use of the Nikon FITC (fluorescein isothiocyanate) filtre.

2. Production of Organic Selenium

[0247] Certain micro-algae produce molecules containing selenium, such as for example Peridinium cinctum. Such micro-algae can thus produce selenium in organic form, from mineral selenium.

[0248] The micro-algae that produce molecules containing selenium were encapsulated in accordance with the present invention, and then placed in incubation in a medium enriched with mineral selenium, in order to induce a bioaccumulation of organic selenium by these micro-algae.

[0249] The quantification of bio-accumulated Selenium by the encapsulated micro-algae was carried out by extraction of the cells of the capsules, and then by the use of physical methods, such as Atomic Absorption Spectrometry (AAS) (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 Lindstrom (1983), Hydrobiologia: “Selenium as a growth factor for plankton algae in laboratory experiments and in some Swedish lakes”).