JP2015175681A - Oxygen permeable bead containing phosphorescent dye molecule and method for measuring oxygen concentration using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily measure an oxygen concentration in a cell peripheral environment together with intracellular oxygen concentration.SOLUTION: The method for measuring an oxygen concentration comprises the steps of: placing oxygen permeable beads including phosphorescent dye molecules or oxygen permeable beads claimed in any of [1]-[6] in an oxygen concentration measuring object; and irradiating the oxygen permeable beads with excitation light to measure a phosphorescent lifetime or phosphorescent intensity of the phosphorescent dye molecules. An oxygen concentration in a liquid or a gas can be easily measured.

Description

  The present invention relates to an oxygen-permeable bead containing phosphorescent dye molecules and an oxygen concentration measurement method using the same. According to the present invention, the oxygen concentration in a liquid or gas can be easily measured.

  Oxygen is a very important molecule for aerobic organisms. In these organisms, carbohydrates are decomposed into carbon dioxide and water by a glycolysis system, a citric acid cycle, an electron transport system, and the like. In this process, oxygen acts as the final electron acceptor and plays an important role in the production of energy used for life activities. On the other hand, oxygen is also involved in the generation of highly toxic reactive oxygen species. Reactive oxygen species are generated in the process of breathing and are considered to be the cause of disorders such as tumor formation, nerve defects and premature aging. As described above, oxygen plays an important role in the living body and is deeply related to various diseases.

“Journal of Porphyrins and Phthalocyanines” (Canada) 2007, Vol. 11, p. 160-164 “Journal of Materials Science” (Germany) 2013, Vol. 38, p. 4271-4282

The present inventors can measure the oxygen concentration in a cell by incorporating a phosphorescent dye into the cell and developing a method for imaging oxygen concentration under a microscope using the phosphor lifetime of the dye. (Non-Patent Document 1). However, an appropriate method has not been developed for measuring the oxygen concentration in the cell surrounding environment. For example, as a method for measuring the oxygen concentration in the environment around the cell, there is a method using an oxygen sensor utilizing an electrode current. As an oxygen sensor using electrode current, a semiconductor oxygen sensor that measures changes in electrical resistance due to adsorption of oxygen molecules on the semiconductor surface, a zirconia oxygen sensor using oxygen ion conductivity of zirconia, and electrochemistry at the electrode of oxygen molecules There is a Clark-type oxygen electrode that measures the current value due to mechanical reduction (Non-patent Document 2), but these oxygen sensors can only measure local oxygen concentration. In addition, a sensor plate is commercially available in which a phosphorescent dye is applied to the bottom of a cell culture plate and dissolved oxygen in the medium can be measured with a fluorescent plate reader (PreSens Precision Sensing GmbH). However, in this plate, since the phosphorescence intensity depends not only on the oxygen concentration but also on the phosphorescent dye concentration, accurate concentration measurement cannot be performed.
An object of the present invention is to easily measure the oxygen concentration measurement in the surrounding environment together with the intracellular oxygen concentration.

The present inventor has eagerly studied the measurement of oxygen concentration in the surrounding environment of the cells, and considered the measurement of the oxygen concentration by allowing a phosphorescent dye to be present in the cell culture medium. However, when cells are loaded with a small amount of phosphorescent dye, the cells do not die immediately, but surprisingly, the presence of the phosphorescent dye in the medium indicates that the cells die rapidly. It was.
As a result of further diligent research on the measurement of the oxygen concentration in the environment surrounding the cell, the present inventor surprisingly incorporated a phosphorescent dye molecule into the oxygen-permeable bead, and increased the phosphorescence lifetime from the phosphorescent dye molecule. It has been found that by measuring, the oxygen concentration in the environment surrounding the cell can be measured without killing the cell.
The present invention is based on these findings.
Therefore, the present invention
[1] Oxygen permeable beads containing phosphorescent dye molecules,
[2] The oxygen-permeable bead according to [1], which is used for measuring oxygen concentration,
[3] The oxygen-permeable property according to [1] or [2], wherein the phosphorescent dye molecule is a metal complex selected from the group consisting of a platinum complex, a palladium complex, an iridium complex, a ruthenium complex, and an osmium complex. beads,
[4] The oxygen-permeable bead according to [3], wherein the platinum complex or the ligand of the palladium complex is porphyrin or a porphyrin derivative,
[5] The oxygen permeable beads according to any one of [1] to [4], wherein the oxygen permeable beads are polymer beads or gel beads.
[6] The oxygen permeable beads according to [5], wherein the polymer beads are polystyrene beads, and the phosphorescent dye molecule is octaethylporphyrin platinum complex (Pt (II) -Octaethylporphyrin), and [7] ] A step of placing the oxygen permeable beads according to any one of [1] to [6] on an oxygen concentration measurement target; and irradiating the oxygen permeable beads with excitation light to phosphorescence of phosphorescent dye molecules Measuring the lifetime or phosphorescence intensity; a method for measuring oxygen concentration, comprising:
About.

According to the oxygen permeable bead containing the phosphorescent dye molecule of the present invention, the oxygen concentration in the cell peripheral environment can be measured without killing the cell. For example, the oxygen concentration in the surrounding environment of a fertilized egg with high oxygen consumption can also be measured without affecting the fertilized egg. Further, by dispersing the beads locally, the oxygen concentration in each local environment can be measured.
Furthermore, the oxygen permeable bead containing the phosphorescent dye molecule of the present invention can measure not only the oxygen concentration in the liquid but also the oxygen concentration in the gas.

It is the figure which showed typically the relationship between the photoexcited singlet state (Excited Single state), the photoexcited triplet state (Excited Triplet state), the ground state (State), and the quenching by oxygen in the generation of phosphorescence. It is the figure explaining the measurement of the phosphorescence lifetime by the system of the confocal optical microscope used for the measurement of the phosphorescence lifetime in this invention, and the time correlation single photon coefficient method. Polystyrene beads (a: PS concentration 1.1 mg / mL) and polystyrene beads (b: PS concentration 1.3 mg / mL) obtained using PtOEP as the phosphorescent dye molecule and polystyrene (PS) as the raw material of the beads It is the graph which showed the photograph of this, and particle size distribution. It is the figure which showed the phosphorescence lifetime imaging image by a confocal optical system in the oxygen concentration of 0, 1, 5, 10, 15, and 20% using the oxygen sensing bead of this invention. It is the graph which examined about the temperature dependence of the oxygen concentration measurement of an oxygen sensing bead in the temperature range of 20 degreeC-40 degreeC. It is the graph which measured the change of the oxygen concentration at the time of adding the culture medium of the high oxygen concentration from the outside using the oxygen sensing bead of this invention. It is a photograph which shows the result of having performed intracellular oxygen concentration imaging using a brown fat cell, and imaging of the oxygen concentration of a cell surrounding environment simultaneously.

[1] Oxygen permeable bead containing phosphorescent dye molecule The oxygen permeable bead containing the phosphorescent dye molecule of the present invention is irradiated with excitation light and measured for phosphorescence intensity or lifetime, thereby increasing the oxygen concentration. (Hereinafter, “oxygen-permeable beads containing phosphorescent dye molecules” may be referred to as “oxygen sensing beads” for convenience). In addition, although the oxygen-permeable bead containing the phosphorescent dye molecule of the present invention can be used for the measurement of oxygen concentration as described above, it is not limited to use for other purposes.

Polycyclic aromatic hydrocarbons such as pyrene or pyrenebutanoic acid, metal complexes such as tris (2,2'-bipyridyl) ruthenium (II), heterocyclic compound dye molecules such as porphyrin or phthalocyanine are photoexcited by light absorption It becomes a state. As shown in FIG. 1, the dye molecule is photoexcited by excitation light and enters a photoexcited singlet state. The dye molecule in the photoexcited singlet state returns to the ground state by emitting a non-radiative transition or light. The light at this time is fluorescence, and its lifetime is as short as 10 ⁻¹⁰ to 10 ⁻⁷ s. On the other hand, a certain kind of molecule changes from a photoexcited singlet state to a photoexcited triplet state through intersystem crossing. The light emitted when transitioning from the photoexcited triplet state to the ground state is phosphorescence. Since the transition from the triplet state to the singlet state is a forbidden transition, the probability thereof is low, and the phosphorescence lifetime is longer than the fluorescence lifetime (10 ⁻⁵ to 10 ⁻² s). Therefore, a molecule in a photoexcited triplet state undergoes electron transfer, energy transfer, and the like with another molecule. For example, when energy transfer proceeds between oxygen molecules in a photoexcited triplet state, singlet oxygen is generated. Along with this energy transfer to the oxygen molecule, the excited molecule returns to the ground state (quenching). When such a quenching phenomenon occurs, phosphorescence intensity and phosphorescence lifetime decrease. That is, it is possible to measure the oxygen concentration using the fact that phosphorescence is quenched when oxygen is present.

<Phosphorescent dye molecule>
The phosphorescent dye molecule contained in the oxygen sensing beads of the present invention is not limited as long as it emits phosphorescence by photoexcitation, but is not limited to polycyclic aromatic hydrocarbons such as pyrene or pyrenebutanoic acid, porphyrin or Heterocyclic compound dye molecules such as phthalocyanine, silicone complexes, platinum complexes, palladium complexes, iridium complexes, ruthenium complexes, osmium complexes, aluminum complexes, copper complexes, rhodium complexes, silver complexes, gold complexes, zinc complexes, rhenium complexes, A europium complex or a mixture thereof can be mentioned, but a platinum complex or a palladium complex is particularly preferable.

For example, as a ligand of a platinum complex or a palladium complex, a porphyrin, a porphyrin derivative, or a pyridine derivative can be exemplified, but a porphyrin derivative is preferable. Porphyrin or porphyrin derivative platinum complex (hereinafter collectively referred to as “porphyrin platinum complex”), or porphyrin or porphyrin derivative palladium complex (hereinafter collectively referred to as “porphyrin iridium complex”) emits phosphorescence at room temperature. Can do. Therefore, a porphyrin platinum complex or a porphyrin iridium complex is preferable from the viewpoint of measuring the oxygen concentration in the cell surrounding environment.
The porphyrin derivative is not limited as long as it can coordinate with platinum or palladium to form a complex. Tetrabenzoporphyrin, tetranaphthoporphyrin, tetraanthraporphyrin, tetrafluorophenylporphyrin, mesotetrakis (carboxy) Phenyl) porphyrin or octaethylporphyrin. A metal complex composed of these ligands and platinum or palladium can be used as a phosphorescent dye molecule. For example, from the viewpoint of preparing the oxygen sensing beads of the present invention by the below-mentioned liquid drying method, organic A porphyrin derivative that is easily dissolved in a solvent is preferable, and for example, Pt (II) -octaethylporphyrin (PtOEP) represented by the following formula (1) is preferable.

On the other hand, when measuring intracellular oxygen concentration, a water-soluble porphyrin platinum complex or porphyrin iridium complex is preferable. Examples of the water-soluble porphyrin platinum complex include Pt (II) -meso-tetrakis (carboxyphenyl) porphyrin (PtTCPP) represented by the following formula (2).

  For example, examples of the ligand of the iridium complex include a 2-phenylpyridine derivative and a bipyridine derivative.

  For example, examples of the ligand of the ruthenium complex include a bipyridine derivative and a phenanthroline derivative.

  For example, examples of the ligand of the osmium complex include a bipyridine derivative, a phenanthroline derivative, and a carbonyl derivative.

《Oxygen permeable beads》
The oxygen permeable bead containing the phosphorescent dye molecule is not limited as long as it is oxygen permeable, and may include a polymer bead or a gel bead, but the polymer bead or the gel bead is biocompatible. Since it has high property, it can be preferably used in the present invention. In this specification, gel beads include silica gel or alumina.

  Examples of the polymer beads used as a raw material include polystyrene, polystyrene bromide, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, polycarbonate, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, Polyisoprene, polyurethane, polyvinyl acetate, polyvinyl chloride, polyvinyl pyridine, polyvinyl benzyl chloride, polyvinyl toluene, polyvinylidene chloride, polydivinylbenzene, polymethyl methacrylate, polylactic acid, polyglycolic acid, poly (lactic acid-co-glycol Acid), polyanhydride, polyorthoester, polyphosphazene, polysulfone, polytrimethylsilylpropyne and combinations thereof Align, and can be exemplified a polymer selected from the group consisting of a copolymer, preferably polystyrene, or poly trimethylsilyl propyne.

<< Method for producing oxygen-permeable bead containing phosphorescent dye molecule >>
The oxygen-permeable bead containing the phosphorescent dye molecule of the present invention can be produced by a known method, except that the phosphorescent dye molecule is contained in the bead.

  For example, methods for preparing polymer beads can include in-liquid drying methods, coacervation methods, spray drying methods, in situ polymerization methods, or interfacial polymerization methods. Prepare beads at low cost and in a short time. Therefore, the in-liquid drying method is preferable.

  In the liquid drying method, the polymer and the core substance are dissolved in an organic solvent having a low boiling point and added to a continuous phase containing a hydrophilic colloid or an active agent to form a stable O / W emulsion, followed by reduced pressure or heating. In this method, a film is formed by removing the organic solvent. Specifically, a solution in which the polymer forming the beads is dissolved is added to and mixed with an incompatible solvent. Thereafter, by evaporating and removing the solvent dissolving the polymer, spherical beads can be formed while the dissolved polymer is precipitated.

  The coacervation method uses phase separation and interfacial chemical changes. For example, a substance that forms a bead wall is dissolved in an oil phase, and an aqueous phase serving as a core substance is dispersed therein as fine droplets by vigorous stirring. By injecting a liquid (poor solvent) that cannot dissolve the capsule wall material well into the oil phase of such a W / O dispersion, the solubility of the wall material is lowered and the wall material surrounds the microdroplets. Precipitate and form beads.

  In the spray drying method, a core material (solid or liquid of fine particles) is poured into a liquid in which a substance that becomes a bead wall is dissolved, sprayed, and blown into hot air. As a result, since the liquid dissolving the bead wall material evaporates, the bead wall material remains, and beads can be formed.

  The interfacial polymerization method uses a polymerization reaction at the interface for forming beads. For example, acid beads, sebacoyl chloride, terephthalic acid chloride are used as oil-soluble monomers, polyamines and polyphenols are used as water-soluble monomers, and beads are formed by causing a polymerization reaction using polyamide or polyether as bead wall materials. Can do.

  In the in situ polymerization method, raw material monomers, initiators, and the like of the resin for the outer shell can be supplied from either the inner phase or the outer phase of the dispersed phase, and the outer shell can be formed by polymerization to obtain beads.

  The particle size (diameter) of the beads of the present invention is not particularly limited as long as the oxygen concentration can be measured, and can be determined as appropriate according to the local environment in which the oxygen concentration is measured. For example, when it is desired to measure the oxygen concentration in the cell and the oxygen concentration in the medium at the same time, the cell preferably has a cell size, for example, a diameter of 7 μm or more is preferable. In addition, beads having a size of 6 μm or less are likely to move in the culture medium, which may make it difficult to measure the phosphorescence lifetime. Also in this respect, beads having a diameter of 7 μm or more are preferable. Further, the upper limit of the particle diameter is not particularly limited, but is preferably 100 μm or less, more preferably 50 μm or less from the viewpoint of oxygen permeability.

  The content (concentration) of the phosphorescent dye molecule contained in the beads can be appropriately changed according to the type of the phosphorescent dye molecule and the local environment for measuring the oxygen concentration. When the oxygen concentration in the medium is compared with the oxygen concentration in the culture medium, it is preferable that the same phosphorescence emission intensity can be obtained. In order to obtain such phosphorescence intensity, the content (concentration) of phosphorescent dye molecules can be adjusted without excessive burden.

  The “oxygen permeability” in the present specification is not particularly limited as long as the oxygen concentration can be measured.

[2] Method for Measuring Oxygen Concentration The method for measuring the oxygen concentration according to the present invention comprises a step of placing the oxygen permeable beads on an oxygen concentration measurement target; and irradiating the oxygen permeable beads with excitation light to obtain phosphorescence. Measuring the phosphorescence intensity or phosphorescence lifetime of the dye molecule.

<< Placement process >>
In the method for measuring oxygen concentration according to the present invention, oxygen permeable beads are placed on an oxygen concentration measurement target.
In this specification, “place oxygen permeable beads on an oxygen concentration measurement target” means placing oxygen permeable beads on a measurement target. For example, if the measurement target is a cell culture medium, This means that it is put into a culture medium, and the oxygen concentration of the medium around the oxygen permeable beads can be measured. Further, for example, if the measurement target is a sealed gas, it means that the gas is introduced into the gas, and the oxygen concentration of the gas around the oxygen permeable beads can be measured.

  The oxygen concentration measurement target is not particularly limited. For example, the oxygen concentration in the liquid phase or the gas phase can be measured. An example of the liquid phase is a cell culture medium.

<< Measurement process >>
In the measuring step, the oxygen permeable beads are irradiated with excitation light, and the phosphorescence lifetime or phosphorescence intensity of the phosphorescent dye molecules is measured.
The phosphorescence lifetime can be measured using, for example, a confocal optical microscope having a Z-axis resolution. As shown in FIG. 2, the oxygen permeable beads are irradiated with excitation light by a pulse laser, and the lifetime of phosphorescence is detected by a detector. Specifically, the phosphorescence lifetime is measured using a time correlated single photon coefficient method. The time-correlated single photon coefficient method is a lifetime measurement method based on the concept that the emission probability distribution of one photon by one excitation event becomes an actual intensity distribution on the time axis of all photons emitted by excitation. The amount of light is adjusted so that the pulsed light source has a single photon coefficient level, the sample is excited, and the generated photons are detected by a photomultiplier tube (PMT). At this time, a signal synchronized with the pulse light source is a start signal, and a signal output when a photon is detected by the PMT is a stop signal. The time voltage converter (TAC) detects these two signals and outputs a voltage pulse having a magnitude proportional to the time. The voltage pulse is detected by a pulse height analyzer (PHA) to obtain the arrival time of the photon. By analyzing the photon arrival time probability distribution thus obtained, the phosphorescence lifetime can be derived.

  The phosphorescence intensity can be measured using a general fluorescence microscope. Similar to the measurement of phosphorescence lifetime, the oxygen permeable beads are excited and the intensity of phosphorescence is detected by a detector.

  The measurement temperature in the oxygen concentration measurement method of the present invention is not particularly limited, and can be measured at a desired measurement temperature according to the measurement environment. For example, when measuring the oxygen concentration of a medium in cell culture, generally, a temperature range of about 10 to 40 ° C. is often used as the culture temperature, and measurement is performed at such a measurement temperature. As shown in the examples described later, the method for measuring oxygen concentration of the present invention is not easily affected by temperature changes, and can measure oxygen concentration at a wide range of measurement temperatures.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1
In this example, oxygen sensing beads were prepared by submerged drying using Pt (II) -octaethylporphyrin (PtOEP) as the phosphorescent dye molecule and polystyrene (PS) as the raw material for the beads.
PtOEP was dissolved in dichloromethane so that the final concentration was 6.5 μM and the final concentration of PS was 1.1 mg / mL or 1.3 mg / mL to obtain a PtOEP-PS mixed solution. To a 1 L beaker, 700 mL of a 5 g / L PVA aqueous solution and 130 mL of dichloromethane were added and vigorously stirred. By this stirring, the evaporation rate of dichloromethane from the mixed solution of PtOEP and PS was controlled. To the PVA-dichloromethane solution, 10 mL of the PtOEP-PS mixed solution was quickly added dropwise at a speed at which droplets were generated. Dichloromethane in the suspension was evaporated by stirring for 20 hours in the dark. The precipitate was collected by centrifugation (3500 × g, 5 min) and washed with RO water to obtain polystyrene beads containing PtOEP. FIG. 3 (a) shows polystyrene beads (a) obtained at a PS concentration of 1.1 mg / mL, and FIG. 3 (b) shows polystyrene beads (b) obtained at a PS concentration of 1.3 mg / mL. Show.
The particle size of the polystyrene beads (a) was distributed in 2-10 μm, and the average particle size was 4 μm. The particle size of the polystyrene beads (b) was distributed between 2 and 12 μm, and the average particle size was 7 μm. The particle diameter could be controlled by changing the PS concentration. From observation with a microscope, it was considered that particles of 6 μm or less are easily moved by external stimulation such as addition of a solution and are not suitable for observing changes in intracellular oxygen concentration in response to external stimulation. For this reason, in Example 2 and later, oxygen sensing beads having an average particle diameter of 7 μm were used.

Example 2
In this example, the oxygen concentration of the culture medium for cell culture was measured using the oxygen sensing beads produced in Example 1.
RO water was injected into the cell culture stage, and the oxygen concentration was adjusted to 0, 1, 5, 10, 15, and 20%. The oxygen concentration of 0% was controlled by injecting 100% nitrogen gas into the cell culture stage, and the oxygen concentration of 1 to 20% was controlled using a gas mixing device. The CO ₂ concentration was controlled to 5%.
Oxygen sensing beads were introduced into the cell culture stage, and the phosphorescence lifetime was measured with the following confocal optical system.
(Confocal optical system)
Becker & Hickl 2 channel confocal galvano scanner unit (DCS-120sci), galvano scanner power and signal amplification unit (GDA-120), single photon counting PMT detector (HPM-100-40), picosecond pulse semiconductor laser (BDL) -405-SMC), time-correlated single photon counting unit (SIMPLE-TAU-150-DX), image analysis software (SPC-image), galvano scanner control module (GVD-120), detector control module (DCC-100) ), Cable, connection box set (CSET-DCS-BOX-150), piezo actuator for objective lens (Miops100PLSG-TII)

  The phosphorescence lifetime image in each oxygen concentration of the oxygen sensing bead in RO water was shown in FIG. In the phosphorescence lifetime imaging image by this confocal optical system, it is expressed in a pseudo color corresponding to the phosphorescence lifetime, and the phosphorescence lifetime is expressed in red while the long lifetime is expressed in blue. The image of FIG. 4 displays a portion having a phosphorescence lifetime of 10-70 μs in red to blue, and indicates that the shorter the phosphorescence lifetime, the higher the oxygen concentration. Therefore, in the phosphorescence lifetime imaging image, the high oxygen concentration region is expressed in red, and the low oxygen concentration region is expressed in blue. As shown in FIG. 4, the hypoxic beads are blue and red as the oxygen concentration is increased, indicating that the oxygen concentration in the liquid can be measured by the present invention.

Example 3
In this example, the temperature dependence of oxygen concentration measurement of oxygen sensing beads was examined.
The operation of Example 2 was repeated except that the temperature of the RO water was in the range of 20 ° C to 40 ° C. The oxygen concentration was 20% of atmospheric pressure. The results are shown in FIG.
In the temperature range of 20-40 ° C., the phosphorescence lifetime of the oxygen sensing beads was independent of temperature. That is, the oxygen concentration measurement using the oxygen sensing beads can be performed without depending on the temperature in the temperature range of 20 to 40 ° C. Therefore, the oxygen concentration measurement method using the oxygen sensing beads is effective even when there is a minute temperature change during drug addition.

Example 4
In this example, the change in oxygen concentration when an external medium was added was measured. That is, the change in oxygen concentration in the medium due to the addition of a new medium in the medium that had previously been hypoxic was measured. An 8-well cover glass chamber supplemented with medium containing oxygen sensing beads was incubated for 1 hour in a cell culture stage controlled to 5% CO ₂ , 10% O ₂ . A medium not incubated in the cell culture stage was fed to the medium of the chamber. For the measurement of phosphorescence lifetime, the operation of Example 2 was repeated. The results are shown in FIG.
By sending the solution, the phosphorescence lifetime of the oxygen sensing beads in the medium was temporarily shortened and then increased. From this result, the oxygen concentration in the medium increased once due to the feeding of the air-saturated medium, and then returned to the initial oxygen concentration over time. From these results, it was possible to monitor with the oxygen sensing beads of the present invention that the oxygen concentration increased by newly adding a medium in the atmosphere to a medium that had been hypoxic.

Example 5
In this example, brown adipocytes were used, and intracellular oxygen concentration imaging and oxygen concentration imaging of the cell peripheral environment were simultaneously performed.
The third-generation rat brown fat precursor cells stored in the deep freezer were seeded at a cell density of 2 × 10 ³ cells / cm ² in a collagen-coated 8-well cover glass chamber and cultured at 37 ° C. in a 5% CO ₂ atmosphere. One day after the culture, the medium was replaced with a growth medium, and it was confirmed that the medium was 90% confluent. Then, the medium was replaced with a medium for induction of differentiation. As a differentiation induction medium, a growth medium supplemented with 25 μM dexamethasone and 10 μg / mL insulin was used. After culturing in a differentiation-inducing medium for 2 days, the medium was replaced with a maintenance medium. The maintenance medium used was a growth medium supplemented with 10 μg / mL insulin. After changing to the maintenance medium, the cells were cultured for 5 to 7 days and used for experiments.
Among the brown adipocytes in which differentiation was induced, cells in which fat droplets were sufficiently accumulated were used. After washing twice with PBS (pH 7.4), 350 μL of phenol red-free DMEM medium (high glucose) containing 10 μM Pt (II) -meso-tetrakis (carboxyphenyl) porphyrin (PtTCPP) was added to each well. And incubated for 2 hours at 37 ° C. in a 5% CO ₂ environment. Thereafter, the PtTCPP-containing medium was discarded, washed again with PBS (pH 7.4), and then 200 μL of phenol red-free DMEM medium was added. Furthermore, 50 μL of 1 mg / mL oxygen sensing bead-containing DMEM medium was added, and phosphorescence lifetimes of brown adipocytes and oxygen sensing beads were observed. The phosphorescence lifetime was measured in the same manner as in Example 2. The results are shown in FIG.

In the left photograph of FIG. 7, oxygen sensing beads, well-differentiated brown adipocytes, and undifferentiated brown adipocytes are confirmed. It can be seen that brown adipocytes with advanced differentiation have lipid droplets accumulated inside the cells, and brown adipocytes with poor differentiation do not have lipid droplets. PtTCPP was not taken into the lipid droplets, and no luminescence was seen and blackened, but phosphorescence from the intracellular and oxygen sensing beads was observed. In addition, cells that have differentiated and accumulated large lipid droplets have a longer phosphorescence lifetime than cells that have not differentiated and have no lipid droplets. From this result, it was found that the cell in which differentiation is advanced has a low intracellular oxygen concentration. Moreover, the phosphorescence lifetime of the oxygen sensing beads near the cells was 15-17 μs. This value is equal to 20% O ₂ phosphorescence lifetime of the oxygen sensing beads in the medium below the oxygen concentration in the medium that cultured brown fat cells, and the medium an oxygen concentration of under 20% O ₂ It didn't change. Therefore, the simultaneous measurement method of the ambient oxygen concentration imaging and the intracellular oxygen concentration imaging was possible.

  With the oxygen-permeable bead containing the phosphorescent dye molecule of the present invention and the method for measuring oxygen concentration using the same, the oxygen concentration in a liquid or gas such as a medium for cell culture can be easily measured.

Claims (7)

  Oxygen permeable beads containing phosphorescent dye molecules.   The oxygen-permeable bead according to claim 1, which is used for measuring oxygen concentration.   The oxygen-permeable bead according to claim 1 or 2, wherein the phosphorescent dye molecule is a metal complex selected from the group consisting of a platinum complex, a palladium complex, an iridium complex, a ruthenium complex, and an osmium complex.   The oxygen permeable bead according to claim 3, wherein the ligand of the platinum complex or the palladium complex is porphyrin or a porphyrin derivative.   The oxygen-permeable bead according to any one of claims 1 to 4, wherein the oxygen-permeable bead is a polymer bead or a gel bead.   The oxygen-permeable bead according to claim 5, wherein the polymer bead is a polystyrene bead and the phosphorescent dye molecule is an octaethylporphyrin platinum complex (Pt (II) -Octaethylporphyrin). A step of placing the oxygen-permeable bead according to any one of claims 1 to 6 on an oxygen concentration measurement target; and irradiating the oxygen-permeable bead with excitation light and phosphorescence lifetime of the phosphorescent dye molecule Or measuring the phosphorescence intensity;
Method for measuring oxygen concentration including