JP7157999B2 - Method for culturing microorganisms using vesicles encapsulating microorganisms - Google Patents

Description

The present invention relates to a method for culturing microorganisms using vesicles encapsulating microorganisms.

Microorganisms are very closely related to our lives, and we live by using functions based on the activity of microorganisms and metabolites produced by microorganisms. There are many microorganisms in the natural world that we do not yet know about, and 99% of the microorganisms that exist in the natural world cannot be cultured yet, and unknown useful substances cannot be discovered or acquired. Therefore, development of a new microbial culture method is required.
In recent years, methods using micro-sized (cell-sized) gel droplets and water-in-oil (W/O) droplets have been reported and attracting attention as methods for separating and culturing microorganisms (Non-Patent Document 1). ). As a feature of these methods, each droplet acts as a reactor for reaction and culture, and by adjusting the number of cells, one cell per droplet is possible, enabling single cell analysis and culture. is. Like these micro-sized gel droplets and W/O droplets, liposomes are materials that can serve as reaction and culture sites for bacterial cells. A liposome is a capsule formed of a lipid bilayer membrane, which is the basic structure of a cell membrane. A widely known technique that utilizes the capsule properties of liposomes is a drug delivery system (DDS) in which a drug or the like is encapsulated inside the liposome and delivered to the target site for treatment. However, the size of liposomes used in DDS is about 10 to 100 nm, which limits the size and amount of substances that can be encapsulated, and one liposome cannot be observed with an optical microscope. In recent years, the technology for creating micro-sized liposomes (cell size: about 10 to 100 μm) has evolved, encapsulating the target substance inside, developing artificial cells, and producing substances through internal chemical reactions. It is used as a microreactor and enables real-time direct observation with an optical microscope, and is applied to the development of biology, medicine, and industry.

Techniques using micro-sized gel droplets and W/O droplets are conventional techniques for separating and culturing microorganisms. As for these problems, since the material of the gel droplet is gel, there is no membrane separating the outside and inside, so the microorganisms that grow inside permeate through the mesh of the gel. Can not. Moreover, since this permeability depends on the mesh size, it is difficult to control. On the other hand, in W/O droplets, microorganisms are reliably enclosed inside, and mass transfer between the inside and outside of the droplet through the membrane is almost non-existent. Although it has the advantage of being able to retain the internal nutrients, it is difficult to replenish when the internal nutrients are depleted. In addition, since oil and organic solvents are used, it is difficult to remove 100% of the oils and organic solvents when collecting microorganisms.

Compared to the above two methods, microbial culture in liposomes reliably encloses microbes inside, similar to W/O droplets, and retains useful substances such as microbes and metabolites grown inside. While maintaining the advantage of being able to do so, it is possible to use the semi-permeable effect of the lipid bilayer membrane to allow solvent molecules and small molecules to pass through the membrane, enabling moderate substance and nutrient exchange. In addition, since it is an aqueous solution system even when collecting microorganisms, it is possible to recover the internal microorganisms in a non-invasive manner. Thus, retention of microorganisms in liposomes enables similar or longer long-term culture and high-density culture compared to techniques using gel droplets and W/O droplets.

On the other hand, when culturing microorganisms in liposomes, it is necessary to conduct experiments at a constant optimum temperature (eg, 37°C). A temperature controller or the like can be used to keep the temperature in the solution containing the liposomes encapsulating the microorganisms constant, but the liposomes undergo Brownian motion in the solution due to thermal convection in the solution caused by the heat of the temperature controller. Brownian motion causes obstacles such as collisions between liposomes in solution, friction with a glass substrate, and movement out of the field of view of a camera. In order to prevent this Brownian motion and facilitate long-term observation under a microscope, it was necessary to immobilize the liposomes that culture the microorganisms.

As a means for immobilizing liposomes, a method of immobilizing liposomes on a substrate using biotin-avidin has been reported. For example, Patent Document 1 discloses a method for producing a lipid structure having a lipid nanotube portion. A technique for immobilization on a substrate is disclosed. More specifically, the surface of a slide glass substrate is coated with Biotin-BSA and Casein, in which biotin and BSA are bound, and the lipid Biotin-PEG-DSPE with Biotin-PEG attached is introduced into the lipid membrane of the liposome. Furthermore, they disclose a method of immobilizing nanoparticle-containing liposomes on a slide glass via streptavidin by adding a streptavidin solution to a solution containing liposomes.
Non-Patent Document 2 discloses a three-dimensional microarray using liposomes as an artificial cell membrane, with the goal of constructing an artificial cell membrane array for biochip production. Non-Patent Document 2 describes a method for immobilizing liposomes on a solid substrate by covering the substrate with a supported membrane composed of hydrophobic oligonucleotides and phospholipids and preparing liposomes labeled with hydrophobic oligonucleotides. disclose a technique for immobilizing liposomes on a substrate by interposing oligonucleotides extending from the membrane on the substrate and oligonucleotides extending on the surface of the liposomes via linker oligonucleotides.
In addition, Non-Patent Document 3 discloses a method for producing a liposome encapsulating a microorganism, in which sucrose is added to the solution inside the liposome and glucose is used as the external solution of the liposome in order to observe the liposome under a microscope. to increase the specific gravity of the liposomes and to submerge the liposomes on the substrate.
However, the above literature does not describe the problem of immobilizing liposomes for a long period of time for the purpose of culturing cells such as microorganisms encapsulated in liposomes. There is no report on the method of fixing to the top.

International Publication No. 2012/117587

Minjeong Jang, et al., “Microdroplet-based Cell Culture Models and Their Applications”, BioChip J. (2016) 10(4): 310-317, DOI 10.1007/s13206-016-0407-1 Yoshihiro Sasaki, Kazunari Akiyoshi, “Construction of Artificial Cell Array for Biochip Production”, Annual Reports of the Institute of Biomaterials and Bioengineering (2009) 43, 26-28 Sampreeti Chowdhuri, et al., “Encapsulation of Living Cells within Giant Phospholipid Liposomes Formed by the Inverse-Emulsion Technique”, ChemBioChem (2016) 17, 886－889, DOI: 10.1002/cbic.201500643

The problem to be solved by the present invention is a method for culturing cells on a substrate for a long time using a vesicle composed of a double membrane of amphipathic molecules, in which the cells encapsulated inside the vesicle are stably retained. It's about providing a way to do it.

The present inventors have found that vesicles composed of bilayer membranes of amphipathic molecules encapsulating cells do not undergo Brownian motion even at a solution temperature of 37°C, and many vesicles are formed within the same field of view under a microscope. We considered immobilizing vesicles on a slide glass substrate so that we could observe for a long time at a time.
First, in order to observe a large number of vesicles in the same field of view of the microscope at one time, dextran was added to the solution in the vesicles to increase the specific gravity of the liposomes, and the liposomes were precipitated on a glass substrate. When the specific gravity was increased, the number of vesicles aligned on the substrate increased without Brownian motion even at a solution temperature of 37°C. However, due to factors such as the presence of dextran only in the vesicle, the difference in osmotic pressure between the inside and outside of the vesicle and the direct contact of the vesicle with the glass substrate, it is difficult to adhere to the substrate and Phenomena such as rupture, contraction and fluctuation of the vesicles were observed after 2 hours, and the cells could not withstand long-term culture.

Next, a mixed solution of LB medium and sucrose was used inside the vesicles, and a mixed solution of LB medium and glucose was used as the solution containing the vesicles (external liquid of the liposomes) to increase the specific gravity of the vesicles. By making the concentrations of sucrose and glucose the same, we improved the osmotic pressure difference between the inside and outside of the vesicle. In addition, the slide glass substrate was coated with BSA to prevent the vesicles from directly contacting the glass, and the vesicles were sedimented using the specific gravity due to the difference in molecular weight between sucrose and glucose. However, the contents of the vesicles adhered to the substrate were exposed after only about 10 minutes, and long-term observation was not possible.
Therefore, the present inventors refer to Z. Nourin et al. Angrew. Chem. We attempted to immobilize vesicles on a glass substrate using a PEG-attached lipid (Biotin-PEG-lipid). For the vesicles, an LB medium mixed with sucrose inside and an LB medium mixed with glucose in the external solution were used to give a difference in specific gravity in the same manner as before. By increasing the specific gravity inside the vesicles, it was possible to shorten the time required for immobilization, to arrange many vesicles in the same field of view in an inverted microscope, and to facilitate simultaneous observation. As a result, it was confirmed that 80% or more of the liposomes remained on the substrate even 2 hours after immobilization. However, even with this method, the shape of the liposomes was distorted after 2 hours, and only about 20% of the liposomes remained after 24 hours, resulting in unsatisfactory results.
Therefore, the present inventors covered a slide glass with a planar lipid bilayer membrane composed of amphipathic molecules, and used Biotin-PEG, Neutravidin, and Biotin-PEG-lipid extending from the bilayer membrane. We came up with a method to immobilize vesicles. As a result of intensive studies on this method, it was found that 70% or more of the vesicles can be maintained in a spherical state without distortion such as deformation even after 24 hours. The present invention has been completed based on this finding.

That is, the present invention includes the following aspects:
In one aspect of the present invention, [1] a method for culturing cells using a vesicle composed of a double membrane of amphiphilic molecules, comprising:
creating the vesicle encapsulating the cell;
a step of immobilizing the vesicles via immobilization means onto a substrate whose surface is covered with a planar double membrane composed of amphipathic molecules;
and culturing the cell in the vesicle.
Here, in one embodiment of the culture method of the present invention,
[2] The culture method according to [1] above,
The vesicle is characterized in that the inner aqueous phase of the vesicle is prepared to have a higher specific gravity than the outer aqueous phase.
In one embodiment, the culture method of the present invention includes
[3] The culture method according to [1] or [2] above,
The vesicle is characterized by including those having a diameter in the range of 1 to 100 μm.
In one embodiment, the culture method of the present invention includes
[4] The culture method according to any one of [1] to [3] above,
The vesicle is immobilized on the substrate using a combination of biotin-binding protein and avidin-binding protein, or a combination of complementary oligonucleotides.
In one embodiment, the culture method of the present invention includes
[5] The culture method according to any one of [1] to [4] above,
The vesicle is characterized by containing the cell in units of one cell.
In one embodiment, the culture method of the present invention includes
[6] The culture method according to any one of [1] to [5] above,
The immobilization of the vesicle onto the substrate is characterized in that it is not immobilized via a hydrophobic oligonucleotide and a linker oligonucleotide having complementarity to the hydrophobic oligonucleotide.
In one embodiment, the culture method of the present invention includes
[7] The culture method according to any one of [1] to [6] above,
The means for immobilizing the vesicle on the substrate is biotin-binding protein and avidin-binding protein.
In one embodiment, the culture method of the present invention includes
[8] The culture method according to any one of [1] to [6] above,
The vesicle is immobilized on the substrate via biotin on the planar double membrane, biotin on the double membrane constituting the vesicle, and avidin that binds to both of the two biotins. characterized by
In one embodiment, the culture method of the present invention includes
[9] The culture method according to any one of [1] to [8] above,
The culturing step includes culturing while changing the environment of the internal aqueous phase and/or external aqueous phase of the vesicle.
In one embodiment, the culture method of the present invention includes
[10] The culture method according to any one of [1] to [9] above,
The culturing method, wherein the culturing step is a step of culturing the cells while observing proliferation of the cells in real time, and culturing the cells until a desired state is achieved.
In one embodiment, the culture method of the present invention includes
[11] The culture method according to any one of [8] to [10] above,
The cells are characterized by being pre-cultured prior to encapsulation in vesicles.
In one embodiment, the culture method of the present invention includes
[12] The culture method according to any one of [8] to [11] above,
The step of culturing the cells is a step of culturing for 1 hour or more.
In another aspect of the culture method of the present invention,
[13] A kit for use in the culture method according to any one of [1] to [12] above,
(1) with amphipathic molecules for constructing vesicles and/or planar bilayer membranes
(2) means for immobilizing the vesicle on the planar bilayer membrane.

According to the present invention, it is possible to immobilize a vesicle composed of a double membrane of amphiphilic molecules on a substrate and culture cells on the substrate for a long period of time.

FIG. 1A shows a schematic diagram of a method for immobilizing liposomes on a glass substrate and an observation method thereof, which are shown in Example 1 below. 1B and 1C show the results of stability analysis after microliposome immobilization shown in Example 1 below. More specifically, FIG. 1B shows a microscopic image of liposomes at the start of observation after immobilization of microliposomes, and a microscopic image of liposomes 24 hours after immobilization of microliposomes. FIG. 1C shows the residual rate of liposomes for each elapsed time. FIG. 2A shows a schematic diagram of preparation of E. coli-encapsulated liposomes by the interfacial passage method using E. coli-encapsulated W/O droplets. FIG. 2B shows microscopic images of E. coli-encapsulated liposomes (left: fluorescence image, right: phase-contrast image). FIG. 3A shows fluorescent images of E. coli in bulk solution stained with SYTO ^™ 9 and PI. FIG. 3B shows fluorescence images of E. coli encapsulated in the liposomes prepared in Example 2 and stained with SYTO ^™ 9 and PI. FIG. 3C is a graph showing the viability of E. coli in bulk solution and liposomes. FIG. 4 is a photograph of liposomes obtained by immobilizing liposomes encapsulating E. coli on a glass substrate via biotin and neutravidin and culturing the liposomes at 37°C.

In one aspect, the present invention relates to a method for culturing cells using a vesicle composed of a bilayer membrane of amphiphilic molecules, the method comprising the steps of producing a vesicle enclosing a microorganism; The method includes a step of fixing vesicles via fixing means onto a substrate whose surface is covered with a planar bilayer membrane composed of molecules, and a step of culturing cells in the vesicles.

Here, in the present specification, the term “vesicle composed of a double membrane of amphipathic molecules” refers to a spherical capsule composed of a double membrane of amphipathic molecules and having a membrane structure closed in the form of a spherical shell. and contains a liquid layer inside. Such vesicles include, but are not limited to, liposomes and polymersomes.

The size of the "vesicles" used in the present invention is not limited as long as cells can be encapsulated therein and cells can be stably cultured for a long period of time. , more preferably 5 to 100 μm, still more preferably 5 to 50 μm) or nanosize (for example, the diameter of the vesicle is 1 to 1000 nm, more preferably 10 to 1000 nm, still more preferably 100 to 1000 nm). In a more preferred embodiment, the vesicles are micro-sized.

The "amphiphilic molecule" that constitutes the double membrane of the vesicle is not limited as long as it can constitute a vesicle capable of encapsulating cells and culturing for a long period of time. For example, a polymer prepared by As such amphiphilic molecules, naturally occurring substances, artificially synthesized substances, and animal/plant cell extracts can be used. Specifically, but not limited to, phosphocholine (PC), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, etc. sphingolipids such as ceramide, sphingomyelin, glycolipids GM1, GM3; lipids having a sterol skeleton such as cholesterol and its derivatives and oxides; positively charged lipids such as DOTAP; fluorescently labeled lipids ;Artificially created lipids such as lipids labeled with polymers such as PEG: Polymersomes such as Poly(styrene)-block-(poly(ehtylene glycol), Poly(styrene)-block-poly(acrylic acid) can be mentioned.

Specific examples of phospholipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl- Phosphocholines such as sn-glycero-3-phosphocholine (DPPC), L-α-phosphatidylcholine (Egg PC) or their heads are phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), those substituted with the head of Cardiolipin. Phospholipids can also be used for analysis and immobilization on substrates by fluorescently modified phospholipids (e.g., fluorescently modified phospholipid Rhodamine B-1,2-Dihexadecanoyl-sn-glycero-3-Phosphoethanolamine (Rhodamine-DHPE ), biotin-PEG-attached phospholipids (biotin-polyethylene glycol-modified phospholipids 1,2-distaroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (Biotin-PEG- DSPE), etc. In addition, known phospholipids constituting liposomes can also be used.

The composition ratio of the phospholipid can be appropriately adjusted according to the lipid used and the purpose. For example, as an example of preparation of micro-sized liposomes, a mixture of three kinds of lipids POPC: Rhodamine-DHPE: Biotin-PEG2000-DSPE at a ratio of 1 mM:0.001 mM:0.002 mM can be used.
As the polymer constituting the polymersome, for example, Poly(styrene)-block-(poly(ehtylene glycol), Poly(styrene)-block-poly(acrylic acid), etc. can be preferably used. In addition, known polymers constituting polymersomes (for example, polymers disclosed in JP-A-2011-183309) can be used.

Cells applicable to the method of the present invention are not particularly limited as long as they can grow or proliferate in vesicles, and examples thereof include Escherichia coli, Bacillus subtilis, actinomycetes, and rhizobia. When microorganisms are used as cells to be encapsulated in vesicles, they may be environment-derived microorganisms or artificially produced microorganisms such as recombinant microorganisms that have undergone gene transfer.

In one embodiment of the culture method of the present invention, the cells encapsulated in the vesicles can be environmental microorganisms. The method of the present invention can target, for example, a group of microorganisms contained in an environment such as soil. By encapsulating a group of microorganisms contained in the environment in vesicles, it is possible to observe the growth according to the properties of each microorganism contained in each vesicle, and it is possible to analyze and fractionate vesicles that exhibit different growth abilities. In particular, by encapsulating a microorganism in a vesicle for each cell, it becomes possible to isolate each vesicle containing microorganisms exhibiting different growth and proliferative abilities.

In one embodiment of the culture method of the present invention, the cells to be encapsulated in the vesicles can be pre-cultured prior to encapsulation. As used herein, the term "pre-culture" refers to culturing cells to the stationary phase in order to make the cell concentration uniform to some extent. Pre-culture conditions vary depending on the cells to be used and the desired culture. Culture is preferred. By performing pre-culture, a cell suspension having approximately the same concentration can be obtained each time, which is preferable.

The culture method of the present invention includes a step of producing vesicles encapsulating cells.
Here, the method for producing liposomes is known, for example, the interfacial passage method using the centrifugal force of the centrifuge used in the following examples, and other methods, such as forming a lipid film on a glass substrate and hydrating/swelling it. The static hydration method, in which liposomes are formed from a lipid film by applying voltage, the electroformation method, in which a lipid bilayer is prepared in advance, a nozzle is brought close to it, and a jet of water is blown to create a soap bubble. It can be produced by a pulse jet method, a method of producing using a microchannel, or the like.

A method for producing liposomes using the above interface permeation method using centrifugal force will be specifically described as an example.
An aqueous solution (solution outside the liposomes (external aqueous phase)) was first added to a 1.5 ml microtube, and then the phospholipid-dissolved oil was layered to form a lipid monolayer (lipid monolayer) at the interface between oil and water. oil-water interface) is formed. Next, another tube is prepared, oil in which the phospholipid is dissolved is added to the tube, and an aqueous solution (solution inside the liposome (internal aqueous phase): solution encapsulating E. coli) is added to the oil. After that, tapping is performed to prepare a micro-sized emulsion (water-in-oil). The prepared water-in-oil droplets are put entirely into the microtube in which the oil-water interface was previously prepared, set in a centrifuge, and centrifuged. Centrifugal force penetrates the lipid monolayer to form micro-sized liposomes.
Methods for producing polymersomes are also known, and can be carried out by using, for example, methods similar to those for producing liposomes. Further, a method for producing polymersomes can be carried out with reference to, for example, JP-A-2011-183309.

A method for encapsulating cells in vesicles such as liposomes and polymersomes can be accomplished, for example, by suspending cells in a solution that serves as the internal aqueous phase of the vesicles and using the solution to prepare the liposomes and polymersomes.
The number of cells to be encapsulated in the vesicle may be one cell unit, or two or more cell units. When a plurality of cell units of two or more cells are encapsulated, the cells may be derived from one species, or two or more types of cells may be encapsulated.
In one embodiment of the present invention, vesicles containing two or more types of cells (eg, microorganisms) may be produced. By including two or more kinds of microorganisms in one vesicle, it becomes possible to verify the influence of interaction between the microorganisms encapsulated in one vesicle.
Also, in one embodiment of the culture method of the present invention, the vesicle can contain a single unit of cells. Methods for encapsulating cells in vesicles on a cell-by-cell basis are known. For example, when vesicles are produced by the interfacial permeation method using centrifugal force, each W/O droplet containing cells is contained one cell at a time when the W/O droplets containing cells are produced. A W/O droplet can be prepared as follows. Methods for preparing such W/O droplets are known (see, for example, Boedicker JQ et al., “Detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug-based microfluidics” Lab on a Chip , 2008; 8, 1265-1272). Specifically, although not limited to the following, droplets containing one cell can be produced by adjusting the number of cells present in the aqueous phase and the number of W/O droplets according to the Poisson distribution. can. Microorganisms contained within such droplets are of single cell origin. In particular, when the purpose is to observe and isolate a specific microbial species from a sample containing multiple types of microorganisms, it is preferable that the vesicle contain the microorganisms on a cell-by-cell basis. Therefore, when a microbial group containing a plurality of microbial species is used as a sample, it is possible to contribute to the analysis and scale-up of each microorganism by selectively fractionating droplets that exhibit different growth abilities.
Also, when two or more types of microorganisms are encapsulated in the droplet, each type of microorganism can be encapsulated in the droplet on a cell-by-cell basis.

Moreover, as described above, in one embodiment of the culture method of the present invention, the vesicle can contain two or more cells. By encapsulating a plurality of cells in each vesicle, it is possible to shorten the culture time required for growth to the desired cell number and state, which is preferable. In addition, when microorganisms having a symbiotic relationship are encapsulated in the same vesicle, an effect is expected that the proliferation of the microorganisms is promoted due to the interaction of the microorganisms.

The aqueous phase in the vesicle is not limited as long as it is an environment in which cells to be encapsulated can grow and proliferate. As a solvent that constitutes the aqueous phase in the vesicle, for example, a culture solution suitable for each cell can be used. Specifically, although not limited to the following, for example, LB medium, NB medium, SCD medium, MS medium, etc. can be used.
The aqueous phase outside the vesicle is not limited as long as the structure of the vesicle can be maintained and the cells inside the vesicle can be cultured. Basically, it is preferable to use the same solution as the aqueous phase inside the vesicle. In addition, the vesicle outer aqueous phase can contain other components for the purpose of providing nutrients to the cells in the vesicles and for the purpose of changing the culture conditions.

Further, in one embodiment of the culture method of the present invention, the internal aqueous phase of the vesicle is prepared to have a higher specific gravity than the external aqueous phase. By applying a difference in specific gravity between the inner aqueous phase and the outer aqueous phase of the vesicle, the immobilization time of the vesicle on the substrate can be shortened and the immobilization rate can be increased. As long as such an effect can be obtained, the method of making a difference in specific gravity is not limited, but for example, a specific gravity agent can be used to make a difference in specific gravity between the inner water phase and the outer water phase of the vesicle. The specific gravity agent to be used is preferably a water-soluble substance that does not adversely affect the formation and stability of vesicles. For example, in addition to sucrose and dextran used in the examples below, potassium chloride, polyethylene glycol (PEG) and the like can be used. In actual use, if osmotic pressure is applied to the vesicles, the vesicles will be deformed or broken, so the salt concentration inside and outside the lipid membrane must be uniform. Therefore, as in the following examples, a combination of using sucrose (disaccharide) in the inner water phase and glucose (monosaccharide) in the outer water phase, or using potassium chloride (divalent) in the inner water phase and can be used in combination such as using sodium chloride (monovalent) in As long as the structure of the vesicle is not deformed by the osmotic pressure difference between the inner water phase and the outer water phase, the combination of specific gravity agents is not limited to the above.

In order to increase the specific gravity of the inner water phase of the vesicle, the specific gravity agent added to the inner water phase must have a higher molecular weight than the specific gravity agent added to the outer water phase. The molecular weight of the specific gravity agent added to the inner water phase is not particularly limited, but is preferably about 1.5 to 3 times, more preferably 1.8 to 2 times the molecular weight of the specific gravity agent added to the outer water phase. degree.
Also, the molar concentration of the specific gravity agent added to the inner water phase and the molar concentration of the specific gravity agent added to the outer water phase are substantially the same so that there is no osmotic pressure difference. Here, "substantially the same" means that they are the same or different, but the difference is small enough not to adversely affect the formation and stability of vesicles. The molar concentration of the specific gravity agent to be added is 90% to 110%, more preferably 95% to 105%, most preferably 100% (same molar concentration) of the molar concentration of the specific gravity agent added to the external aqueous phase.
When sucrose is used as a specific gravity agent in the internal aqueous phase and glucose is used in the external aqueous phase, the concentration of the specific gravity agent can be 50 mM to 1 M, more preferably 100 mM to 500 mM, and still more preferably 200 mM to 400 mM. When other specific gravity agents are used, those skilled in the art can appropriately adjust the concentration of the specific gravity agents.

In the culture method of the present invention, after the step of producing vesicles in which cells are encapsulated, the vesicles are placed on a substrate whose surface is covered with a planar double membrane composed of amphipathic molecules via immobilization means. Including the step of fixing.

As used herein, the term “planar double membrane composed of amphipathic molecules” refers to a double membrane that covers a substrate, and the vesicles are in direct contact with the substrate under long-term culture conditions. Used to prevent collapse.
The amphipathic molecules constituting the planar double membrane are not limited as long as they can prevent the vesicles from directly contacting the substrate under long-term culture conditions, and include liposomes and polymersomes such as those listed above. Lipids and macromolecules that form Even when a plurality of lipids or polymers are used in combination, it is possible to appropriately adjust the composition ratio of the constituent components according to the production of vesicles.

The "substrate" that can be used in the present invention is not limited as long as it can be used for cell culture, and is preferably transparent enough to allow observation of cells in culture under a microscope. For example, a glass substrate, PDMS resin, transparent acrylic plate, or the like can be used. Commercially available glass substrates (for example, Matsunami Glass Co., Ltd., cover glass 30 × 40, thickness No. 3 (0.25 mm to 0.35 mm) can be used. It also serves as a lid during culture or microscopic observation. A commercially available cover glass (Matsunami Glass Industry Co., Ltd., cover glass 18×18, thickness No. 1 (0.12 mm to 0.17 mm) can be used.

A method for forming a planar double film on a substrate will be specifically described. A method using nano-sized liposomes as a raw material for a planar bilayer membrane composed of amphipathic molecules is exemplified below. In addition, the method of forming a planar double film on a substrate is not limited to the following.
First, a nano-sized liposome solution is prepared, and the nano-sized liposome solution is spread on a glass substrate. Allow to stand for 30 minutes while taking care not to dry the nano-sized liposome solution. Upon standing, the liposomes come into contact with the glass substrate and break, spontaneously forming a lipid bilayer on the glass substrate.

Methods for producing nano-sized liposomes are known, and can be produced, for example, as follows.
A lipid solution in which lipid components (amphipathic molecules forming a planar bilayer membrane) are dissolved in a chloroform/methanol mixed solution (2/1 v/v) is added to a glass vial tube (Durham tube). It evaporates the organic solvent and forms a lipid film on the glass wall. After that, it is placed in a vacuum desiccator for about 1 hour in order to completely remove the chloroform/methanol. After 1 hour, an appropriate amount of solution (mixed solution of LB medium and glucose) is added, and sonication is performed for 1 hour with an ultrasonic cleaner to dissolve the lipid components into the solution. Nano-sized liposomes are produced using a solution in which lipid components are dissolved and an extruder.
The planar double membrane covering the substrate preferably covers the entire substrate, but partially covers the substrate as long as it is possible to prevent the vesicles from directly contacting the substrate and collapsing under long-term culture conditions. It may be in a form that covers the

A vesicle is immobilized via immobilization means on a substrate whose surface is covered with a planar double membrane composed of amphiphilic molecules prepared as described above.
As used herein, the term "immobilization means" refers to means for immobilizing vesicles in culture on a planar double membrane on a substrate so that the vesicles are not moved by convection or the like. The immobilizing means used in the present invention is not limited as long as it can immobilize the planar double membrane on the substrate and the vesicles encapsulating cells and does not inhibit cell culture.
Examples of such immobilization means include, but are not limited to, a combination of a Biotin protein and an Avidin protein, a combination of a hydrophobized oligonucleotide having a cholesteryl group and a linker oligonucleotide having a sequence complementary to the oligonucleotide, and the like. can be mentioned.
In one preferred embodiment, the immobilization means is a combination of Biotin and Avidin proteins. When Biotin protein and Avidin protein are used as immobilization means, they can be immobilized, for example, as follows. First, preparation of vesicles encapsulating cells using lipids and polymers with biotin added to the components constituting the planar bilayer membrane on the vesicle and substrate, and formation of the planar bilayer membrane on the substrate. I do. A solution containing vesicles in which cells are encapsulated is added to the substrate covered with the planar double membrane. Avidin binds, and Biotin extending from the planar double membrane on the substrate binds to Avidin in the solution, so that the vesicle is fixed on the substrate (more specifically, on the planar double membrane on the substrate). . When Biotin protein and Avidin protein are used as immobilization means, biotin-introduced phospholipids can be used as amphipathic molecules constituting vesicles and planar bilayer membranes. -sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (Biotin-PEG-DSPE) and the like can be used. As the Avidin protein, commercially available Neutravidin (Thermo Fisher Scientific, 31000) can be used.

In addition, as a method for immobilizing vesicles on a substrate through a combination of a hydrophobic oligonucleotide having a cholesteryl group and a linker oligonucleotide having a sequence complementary to the oligonucleotide, for example, an oligonucleotide and a phospholipid The substrate is covered with a supported membrane composed of The liposomes can be immobilized on the substrate through the

When introducing an amphipathic molecule to which a biotin protein or an oligonucleotide is added into the lipid bilayer membrane that constitutes a liposome, the ratio to other phospholipids can be appropriately set by the immobilization means used. , preferably in the range of 0.1 mol % to 100 mol %, more preferably in the range of 0.2 mol % to 5 mol %.
In addition, when introducing an immobilizing means such as an amphipathic molecule having a biotin-binding protein or a hydrophobic oligonucleotide into the planar bilayer membrane on the substrate, the ratio to other phospholipids can be appropriately set. For example, it is preferably introduced in the range of 0.1 mol % to 100 mol %, more preferably in the range of 0.2 mol % to 5 mol %.

In one embodiment of the culture method of the present invention, vesicles are immobilized on a substrate via a hydrophobized oligonucleotide having a cholesteryl group and a linker oligonucleotide complementary to the hydrophobized oligonucleotide. Does not include fixed items.

The culturing method of the present invention includes a step of culturing cells in the vesicles after immobilizing the vesicles on a substrate.
Culture conditions for culturing cells in vesicles immobilized on a substrate are not particularly limited as long as the cells contained in the vesicles can grow and proliferate. Preferable culture conditions can be appropriately set according to the target microorganism. For example, but not limited to, the temperature condition can be 4 to 95°C. The culturing time is also not particularly limited, and can be carried out for at least 1 hour or longer, for example, preferably 24 hours or longer.

In one embodiment of the culturing method of the present invention, the culturing step includes culturing while changing the environment of the internal aqueous phase and/or external aqueous phase of the vesicle.
Here, changes in the environment of the internal water phase and/or the external water phase include, for example, changes in pH, changes in temperature, addition of growth activating factors, addition of growth inhibitors such as antibiotics, and the like. can. It is possible to observe the effects on the growth of cells in culture by giving such environmental changes.

In the culture process, cells in the vesicle can be observed under a microscope. This makes it possible to analyze and evaluate the behavior of cells in culture in real time.
That is, in one embodiment of the culturing method of the present invention, the culturing step is a step of culturing cells while observing cell growth in real time, and culturing the cells until a desired state is achieved. be able to.
Here, the "desired state" includes the cell density of cultured cells, cell shape, and the like. As described above, the culture method of the present embodiment is a technique that enables observation and control of cell conditions such as cell density by real-time monitoring of cells cultured in liposomes.

One aspect of the present invention provides a kit for use in a method of culturing cells using a vesicle composed of a double membrane of amphipathic molecules.
The kit of the present invention comprises (1) amphipathic molecules for constructing vesicles and/or planar bilayer membranes, and (2) means for immobilizing vesicles on planar bilayer membranes.
(1) amphipathic molecules for constructing vesicles and/or planar double membranes, and (2) means for immobilizing vesicles on planar double membranes are disclosed above as the culture method of the present invention. Including things. The kit of the present invention may also contain components other than those described above, such as a specific gravity agent, a culture solution, a culture dish, and the like.

EXAMPLES The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these. Also, the contents of the documents cited herein are incorporated herein by reference.
In the following examples, liposomes are used as vesicles composed of bilayer membranes of amphipathic molecules, and E. coli cells are used as cells to be encapsulated in the liposomes.

(Example 1: Immobilization of liposomes on a glass substrate)
Slide glass (Matsunami glass industry, cover glass 30 × 40, thickness No. 3 (0.25 mm to 0.35 mm) glass substrate, double-sided tape (thickness 1 mm) with holes (hole diameter: about 7 mm) A 100 nm size nano-sized liposome solution (mixed solution of LB medium and 200 mM glucose solution) prepared using an extruder (Avanti polar lipids, 610000) was added to the hole and left to stand for about 30 minutes. By standing still, the nanosized liposomes come into contact with the glass substrate, and the liposomes collapse to form a planar lipid bilayer on the substrate, which constitutes the lipid bilayer membrane of the nanosized liposomes. The lipid composition is POPC: Biotin-PEG2000-DSPE=1 mM:0.002 mM After standing for 30 minutes, the glass substrate was washed twice with a mixed solution of LB medium and 200 mM glucose solution, and then Neutravidin was applied. Solution (Thermo Fisher Scientific, 31000) (using a mixed solution of LB medium and 200 mM glucose solution as the solvent) was added and allowed to stand for about 30 minutes, then washed twice with a mixed solution of LB medium and 200 mM glucose solution. Finally, micro-sized liposomes were added and observation was started.Micro-sized liposomes were prepared by the interfacial permeation method using the centrifugal force of a tabletop centrifuge.Constructing the lipid bilayer membrane of micro-sized liposomes. The lipid composition used was POPC: Rhodamine-DHPE: Biotin-PEG2000-DSPE at 1 mM: 0.001 mM: 0.002 mM.
As a result, 70% or more of the microliposomes could be observed without breaking even after 24 hours from the start of observation. This is probably because the glass substrate was covered with a planar lipid bilayer membrane, which prevented the liposomes from collapsing due to direct contact with the glass substrate. In addition, by fixing the microliposomes on the glass substrate, even if heat is applied on the heat stage, the movement of the liposomes due to Brownian motion due to convection can be suppressed, and direct contact with the substrate can also be prevented. That's what I think.

(Example 2: Encapsulation of E. coli in liposomes)
E. coli to be encapsulated in liposomes was pre-cultured (cultivated for about 2 hours) in LB medium until OD600 was 1.1-1.5. The pre-cultured E. coli was diluted to 1/100 and used in the experiment. Escherichia coli-encapsulating liposomes were produced using the interfacial passage method using the centrifugal force of a desktop centrifuge.
First, W/O droplets encapsulating E. coli were prepared. The preparation method is to put mineral oil (Nacalai Tesque, 23334-85) in which lipids (POPC: Rhodamine-DHPE: Biotin-PEG2000-DSPE are 1mM: 0.001mM: 0.002mM) dissolved in a 0.6mL microtube. , was prepared by adding a 1/100-fold diluted solution of E. coli (diluted solution is a mixed solution of LB medium and 200 mM sucrose solution) and tapping. The diameter of the W/O droplet was about 1 to 100 μm. The solution used as the aqueous phase of the W/O droplets was LB medium containing E. coli diluted above and sucrose (final concentration: 200 mM). The oil phase used was POPC: Rhodamine-DHPE: Biotin-PEG2000-DSPE with a composition ratio of 1 mM:0.001 mM:0.002 mM. After preparing a W/O droplet in which E. coli is encapsulated, the W/O droplet is passed through the interface of the lipid-covered oil and aqueous solution (LB medium containing 200 mM glucose) to obtain the lipid in which E. coli is encapsulated. Micro-sized liposomes with bilayer membranes were produced (Fig. 2A). In this example, lipid membranes were stained with Rhodamine-DHPE (red) and Escherichia coli with SYTO™ 9 (green), and fluorescence images were obtained. Staining of Escherichia coli was performed by adding SYTO ^™ 9 to the solution when preparing a 1/100-fold diluted solution of Escherichia coli and allowing the solution to stand for about 20 minutes. In addition, in the present example, at the same time, phase-contrast images were also used to observe whether E. coli was encapsulated. A micrograph of micro-sized liposomes encapsulating E. coli is shown in FIG. 2B. As shown in FIG. 2B, it was observed that a single liposome encapsulated from about two to an uncountable number of E. coli. The size of the micro-sized liposome encapsulating E. coli was about 20 µm.

(Example 3: Escherichia coli viability analysis in liposomes)
In the process of producing the E. coli-encapsulated micro-sized liposome described in Example 2, the survival rate of E. coli encapsulated in the micro-sized liposome was analyzed. The analysis method used a staining agent called SYTO ^TM 9, which stains both live and dead bacteria, and a staining agent called propidium iodide (PI), which stains only dead bacteria. Specifically, Escherichia coli was stained with the staining agent, irradiated with light having a wavelength that excites each fluorescence, and the viability was analyzed from the color of fluorescence emitted by Escherichia coli. Live E. coli appear green, while dead E. coli appear red or yellow (red and green superimposed). When the survival rate of E. coli was analyzed, 100% of E. coli was observed in the culture medium before encapsulation in micro-sized liposomes, but when encapsulated in micro-sized liposomes, it was about 93%. I found out. It was shown that E. coli can be encapsulated in liposomes almost non-invasively, although some E. coli die.

(Example 4: High-density culture of E. coli in liposomes)
A culture experiment of Escherichia coli encapsulated in micro-sized liposomes was performed. E. coli-encapsulated micro-sized liposomes prepared by the method described in Example 2 were used. In addition, the E. coli-encapsulated micro-sized liposomes were immobilized on a slide glass to which double-sided adhesive tape with holes was attached in the same manner as in Example 1. That is, the glass substrate used had a lipid bilayer membrane formed on its surface. In addition, micro-sized liposomes are placed on a glass substrate (more specifically, lipid bilayers on a glass substrate) via biotin on the lipid bilayer, Neutravidin, and biotin on the lipid bilayer that constitutes the micro-sized liposome. on a molecular membrane). At the time of culture, a double-sided tape was covered with a cover glass, and the stage temperature of the microscope was set at 37°C. During culturing, a camera was used to confirm the degree of growth of E. coli in one liposome by photographing.
Fig. 4 shows the photographs taken. As shown in FIG. 4, at the start of the culture, a countable number of Escherichia coli was enclosed, but as time passed, the Escherichia coli proliferated to the point where it was difficult to count. As described above, it was clarified that Escherichia coli can be cultured in microsized liposomes.

According to the culture method of the present invention, it becomes possible to analyze in real time the response of cells to long-term culture and environmental changes while the cells are encapsulated in vesicles. It is also possible to perform high-density culture in a micro-sized closed space covered with a membrane having moderate permeability.
Nano-sized (about 10 to 100 nm) vesicles, which have been used for DDS, have not been able to enclose and culture cells. However, according to the method of the present invention, it is possible to culture cells in micro-sized (approximately 10 to 100 μm) vesicles, and in the future, it will be possible to enclose and culture microorganisms that are considered to be good for health. It is expected that the development of applications to pharmaceuticals and liposome formulations by realization, and the development of applications to bioengineering and industry by realizing encapsulation and cultivation of unknown microorganisms.

Claims (13)

A method for culturing cells using a vesicle composed of a bilayer membrane of amphiphilic molecules,
creating the vesicle encapsulating the cell;
a step of immobilizing the vesicles via immobilization means onto a substrate whose surface is covered with a planar bilayer membrane composed of amphipathic molecules;
and culturing the cells in the vesicle. The culture method according to claim 1,
A culture method, wherein the inner aqueous phase of the vesicle is prepared to have a higher specific gravity than the outer aqueous phase. The culture method according to claim 1 or 2,
The culture method, wherein the vesicles have a diameter in the range of 1 to 100 μm. The culture method according to any one of claims 1 to 3,
The culture method, wherein the means for immobilizing the vesicle on the substrate is a combination of a biotin-binding protein and an avidin-binding protein, or a combination of complementary oligonucleotides. The culture method according to any one of claims 1 to 4,
A culture method, wherein the vesicles contain the cells on a cell-by-cell basis. The culture method according to any one of claims 1 to 5,
The culture method, wherein the immobilization of the vesicle onto the substrate is not via a hydrophobized oligonucleotide and a linker oligonucleotide having complementarity to the hydrophobized oligonucleotide. The culture method according to any one of claims 1 to 6,
The culture method, wherein the means for immobilizing the vesicle on the substrate is a biotin-binding protein and an avidin-binding protein. The culture method according to any one of claims 1 to 6,
The vesicle is immobilized on the substrate via biotin on the planar bilayer membrane, biotin on the bilayer membrane constituting the vesicle, and avidin that binds to both of the two biotins. culture method. The culture method according to any one of claims 1 to 8,
The culturing method, wherein the culturing step includes culturing while changing the environment of the internal aqueous phase and/or the external aqueous phase of the vesicle. The culture method according to any one of claims 1 to 9,
The culturing method, wherein the culturing step is a step of culturing the cells while observing proliferation of the cells in real time, and culturing the cells until a desired state is achieved. The culture method according to any one of claims 1 to 10,
A culture method, wherein the cells are pre-cultured prior to encapsulation in the vesicle. The culture method according to any one of claims 1 to 11,
The culture method, wherein the step of culturing the cells is a step of culturing for 1 hour or more. A kit for use in the culture method according to any one of claims 1 to 12,
A kit comprising (1) an amphipathic molecule for constructing a vesicle and/or a planar bilayer membrane, and (2) means for immobilizing the vesicle on the planar bilayer membrane.

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