Simulated cells for verifying cell culture device, simulated cell dispersion for verifying cell culture device, and cell culture device verification method

Abstract

To provide simulated cells for verification of a cell culture device having specific gravity, surface characteristics, and strength similar to those of cells, and a method for verification of the cell culture device.SOLUTION: Provided are simulated cells for verification of a cell culture device. The cell consists of a liposome 100 having a lipid bilayer membrane 1 and an internal aqueous phase 2. The lipid bilayer membrane 1 comprises at least one first pigment 3. The internal aqueous phase 2 comprises at least one second pigment 4 different from the first pigment 3.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a simulated cell for verifying a cell culture device, a simulated cell dispersion for verifying a cell culture device, and a method for verifying a cell culture device. [Background technology]

[0002] In bypass surgery for obstructive diseases of the coronary arteries and peripheral arteries, surgical treatment using artificial blood vessels made of synthetic fibers that are covered with the patient's endothelial cells and are less likely to form clots has been attracting attention. In order to make such artificial blood vessels available to a large number of people, a cell culture device is used that can efficiently culture the various cells that make up blood vessels (Patent Document 1).

[0003] When developing a cell culture device, imitation cells made of synthetic resin beads the size of cells may be used instead of cultured cells. When using cultured cells in the development of a cell culture device, it is necessary to prepare the facilities and equipment necessary for cell culture, as well as the time and personnel with the skills to culture the cells, which is costly and time-consuming. In contrast, when using simulated cells, there is no need to culture cells, which allows for significant savings in costs and time. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4820979 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the imitation cells made of beads made of synthetic resin such as polystyrene that have been conventionally used are significantly different in specific gravity, surface properties, and strength from the cells to be cultured.

[0006] An object of the present invention is to provide a simulated cell for verifying a cell culture device, which has a specific gravity, surface characteristics, and strength similar to those of a cell, a simulated cell dispersion for verifying a cell culture device, and a method for verifying a cell culture device. [Means for solving the problem]

[0007] [1] A liposome having a lipid bilayer and an inner aqueous phase, the lipid bilayer membrane comprises at least one first dye; The internal aqueous phase contains at least one second dye different from the first dye; Simulated cells for verifying cell culture equipment. [2] The simulated cell for verifying a cell culture device according to [1], wherein the liposome has a particle size of 1 to 100 μm. [3] The simulated cell for verifying a cell culture device described in [1] or [2], wherein the first dye and the second dye are both fluorescent dyes. [4] The simulated cell for verifying a cell culture device according to [3], wherein one of the first dye and the second dye has a fluorescent wavelength of 570 to 820 nm, and the other has a fluorescent wavelength of 420 to 540 nm. [5] The simulated cell for verifying a cell culture device according to [3], wherein the excitation wavelength of one of the first dye and the second dye is 550 to 800 nm, and the excitation wavelength of the other is 400 to 520 nm. [6] The simulated cell for verifying a cell culture device according to any one of [1] to [5], wherein the first dye is rhodamine or a rhodamine derivative, and the second dye is FITC or an FITC derivative. [7] The simulated cell for verifying a cell culture device according to any one of [1] to [6], wherein the lipid bilayer is a phospholipid bilayer. [8] The simulated cell for verifying a cell culture device according to any one of [1] to [7], wherein the liposomes are of a multilamellar vesicle type, a small unilamellar vesicle type, a large unilamellar vesicle type or a spiral wound type. [9] A simulated cell dispersion for verifying a cell culture device, comprising a simulated cell for verifying the cell culture device according to any one of [1] to [8] and a dispersion medium.

[10] A method for verifying a cell culture device, in which a simulated cell for verifying the cell culture device according to any one of [1] to [8] is used instead of a cell to be cultured. Effect of the Invention

[0008] According to the present invention, it is possible to provide a simulated cell for verifying a cell culture device, a simulated cell dispersion for verifying a cell culture device, and a method for verifying a cell culture device, which have a specific gravity, surface characteristics, and strength similar to those of a cell. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram showing a schematic configuration of a liposome used in a simulated cell for verifying the cell culture device of this embodiment. [Diagram 2] FIG. 2 is a micrograph showing the results of Example 1. [Diagram 3] FIG. 3 shows the fluorescence spectrum of the fluorescent dye used in Example 1. [Figure 4] FIG. 4 is a micrograph showing the results of Example 2. [Diagram 5] FIG. 5 shows the fluorescence spectrum of the fluorescent dye used in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Below, a form for carrying out the present invention will be described, but the present invention is not limited to the embodiments described below, and various modifications are possible without departing from the gist of the present invention.

[0011] [Mock cells for evaluating cell culture equipment] The simulated cell for verifying the cell culture device of this embodiment will be described in detail below with appropriate reference to the drawings. The simulated cell for verifying the cell culture device according to this embodiment shown in FIG. The lipid constituting the lipid bilayer 1 is not particularly limited as long as it is a lipid capable of constituting the lipid membrane of a liposome and capable of constituting a lipid bilayer. Examples of lipids capable of constituting a lipid bilayer include glyceroglycolipids, sphingoglycolipids, cholesterol, and phospholipids.

[0012] Examples of glyceroglycolipids include sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, glycosyldiglyceride, etc. Examples of sphingoglycolipids include galactosylcerebroside, lactosylcerebroside, and ganglioside.

[0013] Examples of phospholipids include natural or synthetic phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, and hydrogenated phospholipids.

[0014] Examples of phosphatidylcholine include soybean phosphatidylcholine, egg yolk phosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, and distearoylphosphatidylcholine. Examples of the phosphatidylethanolamine include dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, and distearoylphosphatidylethanolamine. Examples of phosphatidylserine include dilauroylphosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, and distearoylphosphatidylserine. Examples of phosphatidylglycerol include dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, and distearoylphosphatidylglycerol. Examples of phosphatidylinositol include dilauroylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol, and distearoylphosphatidylinositol.

[0015] The lipid constituting the lipid bilayer membrane 1 is preferably a phospholipid.

[0016] In addition to lipids, the lipid bilayer membrane 1 may contain a membrane stabilizer, an antioxidant, and the like, as necessary. Examples of the antioxidant include ascorbic acid, uric acid, and tocopherol homologues (e.g., vitamin E). There are four types of tocopherol isomers, α, β, γ, and δ, and any of them can be used in this embodiment.

[0017] The lipid bilayer 1 is composed of an outer membrane 1a and an inner membrane 1b, and the lipids constituting the outer membrane 1a and the lipids constituting the inner membrane 1b may be the same or different. Usually, for ease of production of the liposome 100, the outer membrane 1a and the inner membrane 1b in the lipid bilayer 1 are composed of the same type of lipid.

[0018] The aqueous component (solvent) of the internal aqueous phase is not particularly limited, but examples thereof include HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris-HCl buffer, phosphate buffer, phosphate buffered saline (PBS), aqueous NaCl (sodium chloride) solution, carbonate-bicarbonate buffer, sodium citrate buffer, sodium borate buffer, and aqueous solutions of sugars such as glucose and sucrose. The pH of the internal aqueous phase is not particularly limited, but is preferably pH 5.5 to 8.5, more preferably pH 6.0 to 8.0, and even more preferably pH 6.0 to 7.5. When the pH of the internal aqueous phase is within the above range, the simulated cells of this embodiment are more similar to cultured cells. The osmotic pressure of the internal aqueous phase is not particularly limited, but is preferably 250 to 320 mOsm / L, more preferably 260 to 310 mOsm / L, and even more preferably 270 to 300 mOsm / L. When the osmotic pressure of the internal aqueous phase is within the above range, the simulated cells of this embodiment are more similar to cultured cells.

[0019] The lipid bilayer membrane 1 contains at least one type of first dye, and the internal aqueous phase 2 contains at least one type of second dye different from the first dye. The first dye is not particularly limited as long as it can be contained in a lipid bilayer membrane, but a fat-soluble dye is preferable. The fat-soluble dye may be a fat-soluble dye itself or a fat-soluble compound labeled with the dye. The second dye is not particularly limited as long as it can be contained in the internal aqueous phase, but is preferably a water-soluble dye. The water-soluble dye may be a dye itself that is water-soluble, or may be a water-soluble compound that is labeled with the dye.

[0020] The first dye and the second dye are preferably fluorescent dyes or derivatives thereof, and examples thereof include the following. DyLight 405 (excitation 400 nm, emission 420 nm), DY-405 (excitation 400 nm, emission 420 nm), Alexa Fluor 405 (excitation 401 nm, emission 421 nm), AMCA (excitation 353 nm, emission 440 nm), AMCA-X (excitation 347 nm, emission 447 nm), Pacific Blue (excitation 405 nm, emission 455 nm), DY-415 (excitation 418 nm, emission 467 nm), Royal Blue (excitation 426 nm, emission 480 nm) and ATTO 425 (excitation 436 nm, emission 484 nm);

[0021] Cy2 (excitation 489 nm, emission 505 nm), ATTO 465 (excitation 453 nm, emission 508 nm), DY-475XL (excitation 492 nm, emission 509 nm), NorthernLights 493 (excitation 493 nm, emission 514 nm), DY-490 (excitation 490 nm, emission 516 nm), DyLight 488 (excitation 493 nm, emission 518 nm), Alexa Fluor 488 (excitation 495 nm, emission 519 nm), 5-FITC (excitation 494 nm, emission 519 nm), FAM (5'FAM) (excitation 495 nm, emission 520 nm), DY-495-X5 (excitation 495 nm, emission 520 nm), DY-495 (excitation 493 nm, emission 521 nm), Fluorescein (excitation 494 nm, emission 521 nm), FITC (excitation 498 nm, emission 522 nm), ATTO 488 (excitation 501 nm, emission 523 nm), HiLyte Flour 488 (excitation 499 nm, emission 523 nm), MFP488 (excitation 501 nm, emission 523 nm), ATTO 495 (excitation 495 nm, emission 527 nm), Oyster 500 (excitation 505 nm, emission 530 nm) and Spectrum Green (excitation 497 nm, emission 538 nm);

[0022] ATTO 520 (excitation 525nm, emission 545nm), ATTO 532 (excitation 532nm, emission 553nm), DY-500XL (excitation 505nm, emission 555nm), DY-485XL (excitation 485nm, emission 560nm), Alexa Fluor 555 (excitation 555nm, emission 565nm), HiLyte Plus 555 (excitation 552nm, emission 567nm), DyLight 549 (excitation 550nm, emission 568nm), HiLyte Fluor 555 (excitation 553nm, emission 568nm), Cy3 (excitation 550nm, emission 570nm), DyLight 547 (excitation 557nm, emission 570nm), Rhodamine (excitation 550nm, emission 570nm), TRITC (excitation 550nm, emission 570nm), DY-548 (excitation 558nm, emission 572nm), DY-554 (excitation 551nm, emission 572nm), DY-555 (excitation 547nm, emission 572nm), Alexa Fluor 546 (excitation 556nm, emission 573nm), DY-556 (excitation 548nm, emission 573), NorthernLights 557 (excitation 557nm, emission 574nm), Oyster 550 (excitation 555nm, fluorescence 574nm), 5-TAMRA (excitation 547nm, fluorescence 574nm), DY-505-X5 (excitation 505nm, fluorescence 574nm), DY-547 (excitation 557nm, fluorescence 574nm), Oyster 556 (excitation 562nm, fluorescence 575nm), DY-549 (excitation 560nm, fluorescence 575nm), ATTO 550 (excitation 554nm, fluorescence 576nm), PE (excitation 488nm, fluorescence 578nm), B-PE (excitation 545nm, fluorescence 578nm), R-PE (excitation 566nm, fluorescence 578nm), DY-560 (excitation 559nm, fluorescence 578nm), TAMRA (excitation 555nm, fluorescence 580nm), MFP555 (excitation 560nm, fluorescence 585nm), Spectrum Orange (excitation 559 nm, emission 588 nm) and DY-510XL (excitation 509 nm, emission 590 nm);

[0023] HiLyte Fluor TR (excitation 591nm, fluorescence 622nm), ATTO 590 (excitation 594nm, fluorescence 624nm), MFP590 (excitation 597nm, fluorescence 624nm), DY-610 (excitation 610nm, fluorescence 630nm), DY-480XL (excitation 500nm, fluorescence 630nm), ATTO 610 (excitation 615nm, fluorescence 634nm), DY-615 (excitation 621nm, fluorescence 641nm), C-PC (C-Phycocyanin) (excitation 616nm, fluorescence 647nm), ATTO 620 (excitation 619nm, fluorescence 643nm), Phycocyanin (excitation 620nm, fluorescence 650nm), DY-481XL (excitation 515nm, fluorescence 650nm), ATTO 633 (excitation 629nm, emission 657nm), DY-630 (excitation 636nm, emission 657nm), DY-632 (excitation 637nm, emission 657nm), DY-633 (excitation 637nm, emission 657nm), MFP631 (excitation 633nm, emission 658nm), DyLight 633 (excitation 638nm, emission 658nm), NorthernLights 637 (excitation 637nm, emission 658nm), DY-631 (excitation 637nm, emission 658nm), DY-634 (excitation 635nm, emission 658nm), APC (Allophycocyanin) (excitation 650nm, emission 660nm), APC-XL (excitation 650nm, emission 662nm), DY-520XL (excitation 520nm, emission 664nm), Alexa Fluor 647 (excitation 650nm, fluorescence 665nm), Cy5 (excitation 643nm, fluorescence 667nm), DY-521XL (excitation 523nm, fluorescence 668nm), Oyster 645 (excitation 650nm, fluorescence 669nm), Quantum Red (excitation 488nm, fluorescence 670nm), Tri-Color (excitation 488nm, fluorescence 670nm), DY-635 (excitation 647nm, fluorescence 671nm), DY-636 (excitation 645nm, fluorescence 671nm), DY-647 (excitation 653nm, fluorescence 672nm), DyLight 647 (excitation 652nm, fluorescence 673nm), HiLyte Fluor 647 (excitation 653nm, fluorescence 673nm), DyLight 649 (excitation 646nm, fluorescence 674nm), HiLyte Plus 647 (excitation 649nm, emission 674nm), Oyster650 (excitation 655nm, emission 674nm), DY-648 (excitation 653nm, emission 674nm), DY-650 (excitation 653nm, emission 674nm), PerCP (excitation 488nm, emission 675nm), DY-652 (excitation 654nm, emission 675nm), DY-649 (excitation 655nm, emission 676nm), DY-651 (excitation 656nm, emission 678nm), Oyster 656 (excitation 662nm, emission 679nm), ATTO 655 (excitation 663nm, emission 684nm), Cy5.5 (excitation 675nm, emission 694nm), DY-677 (excitation 673nm, emission 694nm), DY-678 (excitation 674nm, emission 698nm), HiLyte Fluor 680 (excitation 678nm, emission 699nm), DY-675 (excitation 674nm, emission 699nm), DY-676 (excitation 674nm, emission 699nm), IRDye700DX (excitation 689nm, emission 700nm), DY-681 (excitation 691nm, emission 708nm), DY-680 (excitation 690nm, emission 709nm), DY-682 (excitation 690nm, emission 709nm), DyLight 680 (excitation 682nm, emission 715nm), Alexa Fluor 700 (excitation 702nm, emission 723nm), DY-700 (excitation 707nm, emission 730nm) and DY-701 (excitation 706nm, emission 731nm);

[0024] DY-730 (excitation 732nm, fluorescence 758nm), DY-732 (excitation 736nm, fluorescence 759nm), DY-734 (excitation 736nm, fluorescence 759nm), DY-731 (excitation 736nm, fluorescence 760nm), DY-752 (excitation 748nm, fluorescence 772nm), DY-750 (excitation 747nm, fluorescence 776nm), DyLight 750 (excitation 752nm, fluorescence 778nm), HiLyte Fluor 750 (excitation 754nm, fluorescence 778nm), DY-749 (excitation 752nm, fluorescence 778nm), HiLyte Plus 750 (excitation 751nm, fluorescence 779nm), DY-751 (excitation 751nm, fluorescence 779nm), DyLight 800 (excitation 770 nm, emission 794 nm), IRDye800CW (excitation 774 nm, emission 800 nm), DY-780 (excitation 782 nm, emission 800 nm), DY-781 (excitation 783 nm, emission 800 nm), DY-782 (excitation 784 nm, emission 800 nm), DY-776 (excitation 771 nm, emission 801 nm), DY-777 (excitation 771 nm, emission 801 nm) and IRDye800 (excitation 778 nm, emission 806 nm);

[0025] and derivatives thereof.

[0026] Both the first dye and the second dye are preferably fluorescent dyes. Since the first dye and the second dye have different fluorescence wavelengths and are easy to distinguish from each other, the fluorescence wavelength of one of the first dye and the second dye is preferably 570 to 820 nm, and the fluorescence wavelength of the other is preferably 420 to 540 nm. Since the first dye and the second dye have different excitation light wavelengths and can be easily distinguished from each other, the excitation wavelength of one of the first dye and the second dye is preferably 550 to 800 nm, and the excitation wavelength of the other is preferably 400 to 520 nm. The first dye is preferably a rhodamine derivative obtained by hydrophobizing rhodamine, and particularly preferably rhodamine PE (rhodamine B-dioleoylphosphatidylethanolamine) obtained by labeling dioleoylphosphatidylethanolamine with rhodamine B. The second dye is preferably a FITC derivative obtained by hydrophilizing FITC (fluorescein isothiocyanate), and particularly preferably FITC-HSA (FITC-human serum albumin) obtained by labeling human serum albumin with FITC.

[0027] The particle size of the liposome 100 is not particularly limited, but is preferably 1 to 100 μm, more preferably 10 to 100 μm, and even more preferably 50 to 100 μm. When the particle size of the liposome 100 is within this range, the simulated cell of this embodiment is more similar to a cultured cell. Here, the particle size of the liposome is the 50% particle size determined from the volume-based particle size distribution measured using a laser diffraction / scattering particle size distribution measuring device.

[0028] The liposome 100 is a large unilamellar vesicle type, but the liposome in the simulated cell of this embodiment is not limited to this, and may be any of a multilamellar vesicle type, a small unilamellar vesicle type, and a cochleate type.

[0029] The simulated cells of this embodiment are preferably used by dispersing them in a dispersion medium. In this case, the dispersion medium is a substitute for the culture solution used in cell culture. As the dispersion medium, the external phase liquid used in forming the liposomes may be used as it is, or the external phase liquid may be replaced with another appropriate dispersion medium.

[0030] <How to prepare liposomes> The method for producing liposomes used in the simulated cells of this embodiment is not particularly limited, but examples include the thin film method, ultrasonic method, surfactant treatment method, solvent injection method, French press method, reverse phase evaporation method, microencapsulation method, coacervation method, etc.

[0031] The thin film method, also known as the Bangham method, is a method in which an aqueous solution is added to a thin lipid film, which causes the lipids to self-organize and form liposomes. If the compound to be encapsulated is fat-soluble, it can be mixed with the lipid in advance, and if it is water-soluble, it can be dissolved in the aqueous solution to be hydrated, allowing the compound to be encapsulated. The outline of the method for preparing liposomes by the thin film method is as follows. (1) Dissolve lipids in an organic solvent (chloroform, dichloromethane, etc.). (2) The lipid solution is placed in a round-bottom flask, and the organic solvent is removed using a rotary evaporator to form a thin lipid film on the bottom of the flask. (3) An aqueous solution that will become the internal aqueous phase is injected onto the lipid thin film to hydrate (or swell) the lipids and form liposomes. First, in (1), lipids are completely dissolved in an organic solvent with a relatively low boiling point. The lipid concentration should be about several tens of μmol to several tens of mmol. If charged substances (dicetyl phosphate, stearylamine, etc.) and cholesterol are used as additives, they are dissolved uniformly in the low boiling point solvent at this stage together with the lipids. Next, in (2), the relationship between lipid concentration and flask size is important, and it is necessary to set the concentration so that a thin film of phospholipids can be formed at the bottom of the flask. Furthermore, it is necessary to completely remove the solvent, so it is recommended to dry it overnight in a vacuum desiccator. (3) The phase transition temperature (T c The most important factors are hydration at or above the temperature at which lipids change from liquid crystal to crystalline form (the temperature at which lipids change from liquid crystal to crystalline form) and the hydration time. In addition, the water phase during hydration must be at least 50% by volume relative to the lipid.

[0032] The ultrasonic method is also called sonication method, in which lipids are suspended in water and irradiated with ultrasound to obtain a lipid dispersion solution that is almost optically transparent. announced that it would be possible to obtain The outline of the method for preparing liposomes using the ultrasonic method is as follows. (1) Prepare multilamellar liposomes by the thin film method. (2) The multilamellar liposomes are irradiated with ultrasound. In this preparation method, after the multilamellar liposomes are prepared or after the phospholipids are uniformly suspended in the aqueous phase, they are subjected to ultrasound treatment to produce fine liposomes.

[0033] The surfactant treatment method is a method in which a mixture of phospholipids and micelles is prepared by utilizing the solubilizing power of the surfactant, and then the surfactant alone is removed by dialysis. The outline of the preparation method is as follows: (1) The lipid dispersion solution and cholic acid (or deoxycholic acid) are stirred in an aqueous solution to form mixed micelles. (2) The mixed micelles are placed in a dialysis tube, and cholic acid (or deoxycholic acid) is removed in distilled water. First, in (1), cholic acid or deoxycholic acid (preferably Na salt) is added to lipid thin films, multilamellar liposomes, or unilamellar liposomes according to the thin film method or ultrasonic method, and the mixture is stirred or ultrasonicated to prepare mixed micelles. The extracted lipids may be directly mixed with the above-mentioned surfactants. Also, suitable surfactants other than cholic acid or deoxycholic acid may be used. Next, in (2), in addition to dialysis, ultracentrifugation, gel filtration, etc. can also be used. For example, in gel filtration, it is preferable to set the molar ratio of lipid:sodium cholate to 1:2 and remove sodium cholate by gel filtration.

[0034] The solvent injection method is outlined as follows. (1) Dissolve lipids in ethanol (or ether). (2) Inject the lipid-ethanol solution from (1) into a moderately warmed aqueous solution (or inject the lipid-ether solution into an aqueous solution heated to 55-65°C). First, in (1), the particle size of liposomes can be appropriately controlled by changing the lipid concentration. In (2), the lipid-ethanol solution is injected into an aqueous solution heated to above the lipid phase transition temperature. Note that the injection speed does not have a significant effect on the physical properties of the final liposomes. The ethanol injection method is extremely simple and has excellent reproducibility in terms of the size and shape of liposomes. On the other hand, in the ether method, in (1), a mixture of diethyl ether and methanol can be used instead of diethyl ether. When the temperature of the system is 55-65°C, diethyl ether (boiling point 39°C) evaporates through the aqueous phase.

[0035] The French press method is a method in which a sample is forced through a narrow gap under high pressure, and is outlined as follows. (1) Prepare a liposome suspension according to the thin film method. (2) The liposome suspension prepared in (1) was placed in a French press and pressurized at 20,000 lbs / in at 4°C. 2 Press out with pressure.

[0036] The reverse phase evaporation method is outlined as follows. (1) The aqueous phase is added to an organic solvent (diethyl ether, isopropyl ether, etc.) in which lipids have been dissolved, and the mixture is sonicated to prepare a W / 0 emulsion. (2) The organic solvent in the W / O emulsion is distilled off under reduced pressure. (3) The mixture is stirred for about 20 minutes using a vortex mixer to induce phase inversion, and liposomes are obtained when the mixture becomes a 0 / W suspension. (4) The remaining organic solvent is completely removed.

[0037] The outline of the microencapsulation method is as follows. (1) High concentration (approximately 135 mM) lipid is dissolved in an organic solvent (e.g., methylene chloride, chloroform). (2) The aqueous phase in which the marker or drug is dissolved is mixed and emulsified with the lipid organic solution of (1) to prepare a W / 0 type emulsion. (3) The obtained W / O type emulsion is quickly added to a large amount of distilled water or a salt solution and dispersed to form a composite W / O / W type emulsion. (4) Stirring is continued until the organic solvent in which the lipids have been dissolved is gradually dissolved in the aqueous phase and then evaporated and removed from the surface of the aqueous mother liquor to obtain a liposome suspension.

[0038] The outline of the coacervation method is as follows. (1) Dissolve lipids in alcohol such as methanol, ethanol, 1-propanol, or 2-propanol. (2) To a certain amount of this lipid-alcohol solution, water or a salt solution, which is a poor solvent, is gradually added to induce coacervation. (3) The alcohol contained in the obtained coacervate or in the system is dialyzed against a large amount of water to remove the alcohol from the system.

[0039] As a method for forming liposomes for producing the simulated cells of this embodiment, the thin film method (Bangham method) is preferred because it is simple, in which an aqueous solution is added to a thin lipid film to hydrate the lipids, causing the lipids to self-organize and form liposomes. Liposomes of the desired particle size can be easily obtained by forming liposomes using this method and adjusting the size of the liposomes using an extruder or ultrasonic waves depending on the purpose.

[0040] [Mock cell dispersion for verifying cell culture equipment] The simulated cell dispersion for verifying the cell culture device of this embodiment contains the above-mentioned simulated cells for verifying the cell culture device and a dispersion medium. The dispersion medium is not particularly limited, and in addition to the external aqueous phase described above, a culture medium for cell culture, a buffer solution, etc. can be used.

[0041] [Cell culture equipment evaluation method] The simulated cells of this embodiment can be used in place of cells to be cultured in the cell culture device verification method. Since there is no need to culture cells to verify the cell culture device, the facilities, equipment, time, skills, etc. required for cell culture are not required.

[0042] By using the simulated cell of this embodiment, the damage that the cell culture device inflicts on the cells can be visualized, making it easier to verify the cell culture device.

[0043] [Effects] The simulated cells for verifying cell culture devices of the present invention have a specific gravity, surface properties, and strength similar to those of cells because they have a basic structure of liposomes. Therefore, when used instead of cells to verify cell culture devices, the facilities, equipment, techniques, and time required for cell culture that were previously required are no longer necessary, and the time and cost required for verifying cell culture devices can be reduced. In addition, because the lipid bilayer membrane and the inner aqueous phase of the liposome contain different dyes, damage to cells can be visualized. In addition, the impact on cell manipulation can be evaluated by absorbance measurement, etc. Therefore, by using the simulated cell for verifying the cell culture device of the present invention, verification of the cell culture device can be performed more easily. EXAMPLES

[0044] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the examples described below, and various modifications are possible without departing from the gist of the present invention.

[0045] [Example 1] <Preparation of liposomes> (1) Preparation of lipid solution Liquid paraffin was added to a chloroform solution (100 mg / mL concentration) of POPC (1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine) to adjust the final concentration of POPC to 5 mg / mL. This POPC solution was treated at 80°C for 20 minutes using a rotary evaporator to remove the chloroform from the POPC solution. A liquid paraffin solution of POPC (hereinafter referred to as "lipid solution A") was obtained. Liquid paraffin was added to a chloroform solution of cholesterol (3β-hydroxy-5-cholestene) (concentration: 100 mg / mL) to adjust the final cholesterol concentration to 4.55 mg / mL. This cholesterol solution was treated at 80°C for 20 minutes using a rotary evaporator to remove the chloroform from the cholesterol solution. A liquid paraffin solution of cholesterol (hereinafter referred to as "lipid solution B") was obtained. Each of lipid solution A and lipid solution B was stirred at 80° C. for 20 minutes. Lipid solution A and lipid solution B were mixed to a mass ratio of POPC:cholesterol = 9:1 and allowed to stand at 37°C for 10 minutes to obtain a liquid paraffin solution containing POPC and cholesterol (hereinafter referred to as "lipid solution C").

[0046] (2) Preparation of giant liposomes To 90 μL of inner aqueous phase solution (composition: 100 mM HEPES-KOH (pH 7.6), 200 mM sucrose) containing FITC-HSA (FITC-human serum albumin) at a final concentration of 10 mg / mL, 900 μL of lipid solution C was added and vigorously stirred, and then allowed to stand on ice for 10 minutes to obtain an inner aqueous phase solution containing lipids (hereinafter referred to as "inner aqueous phase solution B"). 215 μL of the external aqueous phase solution (composition: 100 mM HEPES-KOH (pH 7.6), 200 mM glucose) was mixed with 10 μL of an ethanol solution of Rhodamine PE (rhodamine B-dioleoylphosphatidylethanolamine) (1 mg / mL concentration) to obtain an external aqueous phase solution containing a red pigment (hereinafter referred to as "external aqueous phase solution B"). To the obtained outer aqueous phase solution B, 250 μL of inner aqueous phase solution B was carefully added to obtain a mixed solution. The resulting mixture was centrifuged at 4°C for 45 minutes under three acceleration conditions: 16,000×g, 8,500×g, and 5,900×g. The supernatant was then discarded and the precipitate was suspended in 300 μL of the external aqueous phase solution to obtain suspensions (hereinafter referred to as "Suspension B1" (centrifuged at 16,000×g), "Suspension B2" (centrifuged at 8,500×g), and "Suspension B3" (centrifuged at 5,900×g). Each of Suspension B1, Suspension B2, and Suspension B3 was centrifuged (16,000 × g) at 4 °C for 10 minutes, and the precipitate was suspended in 300 μL of the external aqueous phase solution to obtain suspensions (hereinafter referred to as "Suspension C1", "Suspension C2", and "Suspension C3", respectively). Suspensions C1, C2, and C3 were centrifuged (16,000×g) at 4° C. for 10 minutes, and the precipitate was suspended in 300 μL of the external aqueous phase solution to obtain liposome suspensions (hereinafter referred to as "Sample 1," "Sample 2," and "Sample 3," respectively).

[0047] <Evaluation method and results> The liquid after mechanical manipulation was evaluated by microscopic observation and fluorescence spectrum measurement. Figure 2 shows the microscopic photograph and Figure 3 shows the fluorescence spectrum. For microscopic observation, Sample 1, Sample 2, and Sample 3, which were diluted 2-fold, were observed at a magnification of 400x using white light or fluorescent light. Rhodamine PE and FITC-HSA were observed in the same locations as the circular images observed under white light in Figure 2, confirming that the respective fluorescent substances were embedded in the outer and inner layers of the liposome. If damage occurs to the simulated cells, the fluorescent substance will flow out into the liquid, and the entire microscope image will be stained with the fluorescent dye, causing a change in the image. The degree of damage to the cells can be determined from the stained range of Rhodamine PE and FITC-HSA and the degree of circularity. For the fluorescence spectrum measurement, Sample 1, Sample 2, and Sample 3 were diluted 10-fold with the external aqueous phase solution (100 mM HEPES-KOH (pH 7.6), 200 mM glucose), and then excited (FITC: excitation wavelength 495 nm; Rhodamine: excitation wavelength 544 nm) using a spectrophotometer (RF5300, Shimadzu Corporation) to measure the fluorescence spectrum. It is presumed that the fluorescence intensity detected in Figure 3 was confirmed by the presence of liposomes. If damage occurs to the simulated cells, fluorescent substances will leak into the liquid, so the degree of damage to the cells can be determined by measuring the fluorescence spectrum of the supernatant based on the intensity.

[0048] [Example 2] <Preparation of liposomes> (1) Preparation of lipid solution Lipids (DPPC (dipalmitoylphosphatidylcholine), DPPG (dipalmitoylphosphatidylglycerol)) and red fluorescent dye (Rhodamine-PE) were weighed out and dissolved in a methanol-chloroform mixture (volume ratio of methanol:chloroform = 2.1) together with an equimolar amount of fructose to the lipids to obtain a solution containing lipids and the red fluorescent dye (hereinafter referred to as "lipid solution D"). While gently mixing lipid solution D, the solvent was distilled off under reduced pressure at 50°C, and then vacuum drying was performed to completely remove the solvent, to obtain a lipid containing a red fluorescent dye (hereinafter referred to as "lipid B").

[0049] (2) Preparation of giant liposomes Lipid B was rehydrated with an aqueous solution of FITC-HSA (FITC-human serum albumin) solution dissolved in HEPES buffer (10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 mM NaCl, pH 7.2), and gently mixed at 50°C to obtain a lipid mixture (hereinafter referred to as "lipid mixture C"). Lipid mixture C was centrifuged at 800 rpm for 10 minutes at 4°C, the supernatant was removed, and HEPES buffer (as above) was added. This procedure was repeated twice to obtain liposome dispersions (hereinafter, "Sample 4" (centrifuged at 16,000 x g), "Sample 5" (centrifuged at 8,500 x g), and "Sample 6" (centrifuged at 5,900 x g).

[0050] <Evaluation method and results> The liquid after mechanical manipulation was evaluated by microscopic observation and fluorescence spectrum measurement. Figure 4 shows the microscopic photograph and Figure 5 shows the fluorescence spectrum. For microscopic observation, 2-fold diluted Sample 4 was observed at 400x magnification using white light or fluorescent light. Rhodamine PE and FITC-HSA were observed in the same locations as the circular images observed under white light in Figure 4, confirming that the respective fluorescent substances were embedded in the outer and inner layers of the liposome. If damage occurs to the simulated cells, the fluorescent substance will flow into the liquid and the entire microscope image will be stained with the fluorescent dye, causing a change in the image. The degree of damage to the cells can be determined from the stained range of Rhodamine PE and FITC-HSA and the degree of circularity, etc. For the fluorescence spectrum measurement, Samples 4, 5, and 6 were diluted 10-fold with the external aqueous phase solution (100 mM HEPES-KOH (pH 7.6), 200 mM glucose), and then excited (FITC: excitation wavelength 495 nm; Rhodamine: excitation wavelength 544 nm) using a spectrophotometer (RF5300, Shimadzu Corporation) to measure the fluorescence spectrum. Since the external aqueous phase solution in which the liposomes are suspended does not contain any dye, it is presumed that the fluorescence intensity detected in Figure 5 was confirmed by the presence of liposomes. If damage occurs to the simulated cells, fluorescent substances will flow out into the liquid, so the degree of damage to the cells can be determined by measuring the fluorescence spectrum of the supernatant based on the intensity. [Explanation of symbols]

[0051] 1...lipid bilayer membrane, 1a...outer membrane, 1b...inner membrane, 2...inner aqueous phase, 3...first dye, 4...second dye, 100...liposome

Claims

1. It is composed of a liposome having a lipid bilayer membrane and an internal aqueous phase, the lipid bilayer membrane comprises at least one first dye; the internal aqueous phase comprises at least one second dye different from the first dye; Simulated cells for verifying cell culture equipment.

2. 2. The simulated cell for verifying a cell culture device according to claim 1, wherein the liposome has a particle diameter of 1 to 100 μm.

3. The simulated cell for verifying a cell culture device according to claim 1 or 2, wherein the first dye and the second dye are both fluorescent dyes.

4. The simulated cell for verifying a cell culture device according to claim 3, wherein one of the first dye and the second dye has a fluorescent wavelength of 570 to 820 nm, and the other has a fluorescent wavelength of 420 to 540 nm.

5. The simulated cell for verifying a cell culture device according to claim 3, wherein one of the first dye and the second dye has an excitation wavelength of 550 to 800 nm, and the other has an excitation wavelength of 400 to 520 nm.

6. The simulated cell for verifying a cell culture device according to any one of claims 1 to 5, wherein the first dye is rhodamine or a rhodamine derivative, and the second dye is FITC or a FITC derivative.

7. The simulated cell for verifying a cell culture device according to any one of claims 1 to 6, wherein the lipid bilayer is a phospholipid bilayer.

8. The simulated cell for verifying a cell culture device according to any one of claims 1 to 7, wherein the liposome is a multilamellar vesicle type, a small unilamellar vesicle type, a large unilamellar vesicle type, or a spiral wound type.

9. A simulated cell dispersion for verifying a cell culture device, comprising the simulated cell for verifying the cell culture device according to any one of claims 1 to 8 and a dispersion medium.

10. A method for verifying a cell culture device, which uses a simulated cell for verifying the cell culture device according to any one of claims 1 to 8 in place of a cell to be cultured.

Images