JP7186433B2 - Cell viability determination method and cell viability determination kit - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cell viability determination method and a cell viability determination kit.

Determination of life and death of cells plays an important role in basic research, hygiene control, quality control, etc. in various industrial fields such as medicine, cosmetics, food, and industry, and there is a wide range of demand for it. As a method for determining cell life and death, for example, Non-Patent Document 1 describes a method for easily determining cell life and death using a dye such as trypan blue that selectively stains dead cells. Trypan blue, in particular, is widely used as a dye that stains dead cells in a short period of time and is not taken up by living cells to determine the viability of cells.

A. Bonora and D. Mares, Current Microbiology, 1982, 7, pp. 217-221

However, trypan blue is toxic to cells, including potential teratogenic effects and induction of increased permeability of cell membranes through pore formation. Therefore, there is a demand for staining agents that are non-toxic to cells and can be used safely. In addition, when cells are stained with trypan blue, the number of dead cells increases with the passage of time due to the toxicity of trypan blue. Therefore, for example, when confirming the transition of the action of a drug on cells, it is necessary to prepare a plurality of samples corresponding to desired elapsed times in advance, which is troublesome and costly.

The present invention has been made in view of the above, and can be easily evaluated using a highly safe staining agent, and can continuously evaluate the same specimen for a long period of time. Intended to provide a kit.

Specific means for solving the above problems include the following embodiments.
<1> A step of contacting the cells with a staining agent that stains dead cells, and a step of determining whether the cells are alive or dead based on the presence or absence of the staining of the cells, wherein the staining agent is selected from Monascus dye and anthocyanin dye. A method for determining viability of cells, comprising at least one dye that

<2> The method for determining cell viability according to <1>, wherein the anthocyanin pigment is a purple sweet potato pigment.

<3> A cell life-and-death determination kit used in the method for determining cell life and death according to <1>, comprising a staining agent for staining dead cells, wherein the staining agent is selected from Monascus dye and anthocyanin dye A kit for determining viability of cells, comprising at least one kind of dye.

<4> The cell viability determination kit according to <3>, wherein the anthocyanin pigment is a purple sweet potato pigment.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a cell viability determination method and a cell viability determination kit that can be easily evaluated using a highly safe staining agent and that can continuously evaluate the same specimen for a long period of time. .

FIG. 2 shows absorption spectra and microscopic images of Euglena gracilis cells of Example 1. FIG. 1 is a diagram showing absorption spectra and microscopic images of Euglena gracilis cells in a medium containing monascus pigment in Example 1. FIG. 1 is a diagram showing an absorption spectrum and a microscopic image of Euglena gracilis cells in a medium containing the purple sweet potato pigment of Example 1. FIG. FIG. 2 shows microscopic images of Euglena gracilis cells in a medium containing monascus pigment or purple yam pigment in Example 2. FIG. FIG. 10 is a diagram showing a microscopic image of a mixture of multiple types of plant cells and tissue pieces in a liquid containing monascus pigment or purple yam pigment in Example 3. FIG. FIG. 10 is a diagram showing a microscopic image of Paramecium cells in a liquid containing monascus pigment or purple yam pigment in Example 4. FIG. FIG. 10 is a diagram showing a growth curve of Euglena gracilis in a medium containing monascus pigment or purple yam pigment of Example 5. FIG. FIG. 10 is a diagram showing a growth curve of Euglena gracilis in a medium containing trypan blue or methylene blue in Example 5. FIG. FIG. 10 is a diagram showing changes in percentage of stained cells of Euglena gracilis in a mixed medium of monascus pigment and purple yam pigment in Example 7. FIG.

Embodiments of the present invention will be described below. However, the present invention is not limited to the following embodiments.

<Method for Determining Viability of Cells>
The cell life-and-death determination method according to the present embodiment includes a step of contacting cells with a staining agent that stains dead cells (hereinafter referred to as "contacting step"), and a step of determining whether the cells are alive or dead based on the presence or absence of staining of the cells. (hereinafter referred to as “life-and-death determination step”).

(Contact process)
First, in the contacting step, cells to be determined for life and death are brought into contact with a staining agent for staining dead cells.

There are no particular restrictions on the types of cells to be used for viability determination, and for example, they may be prokaryotic or eukaryotic cells. In the case of eukaryotic cells, they may be animal, plant, protist, or fungal cells.

Staining agents for staining dead cells include monascus dyes and anthocyanin dyes. As a staining agent, only the monascus dye may be used, only the anthocyanin dye may be used, or both dyes may be used.

Monascus pigments are pigments that can be obtained from Aspergillus aspergillus, for example, from ankaflavin, monascorubrin, monascin, rubropunctatin, monascorbramin, rubropunctamine, rubropunctaridin, xatone monacin, etc. It includes one or more selected types. The monascus pigment may be one extracted from Aspergillus oryzae or one obtained by chemical synthesis. The color tone of the monascus pigment is preferably red to purple.

Aspergillus aspergillus is not particularly limited as long as it is an ascomycete aspergillus aspergillus belonging to the genus Monascus, for example Monascus pilosus, Monascus purpureus, Monascus anka ) and the like.

The types of anthocyanin pigments are not particularly limited, and examples include purple potato pigment, red perilla pigment, red cabbage pigment, red radish pigment, cranberry pigment, grape pigment, mulberry pigment, boysenberry pigment, haskap pigment, blueberry pigment, and prune pigment. , bilberry pigment, acai pigment, strawberry pigment, raspberry pigment, eggplant pigment, apple pigment, currant pigment, blackberry pigment, black rice pigment, black sesame pigment, black soybean pigment, daisho pigment, anastasia gray pigment, camellia pigment, adzuki bean pigment, red onion pigments, and the like. Among these, purple sweet potato pigment and red cabbage pigment are particularly preferred.

Purple sweet potato pigments are pigments that can be obtained from purple tuberous roots of the genus Convolvulaceace Ipomoea of the Convolvulaceae family, and include, for example, cyanidin acyl glucosides and peonidin acyl glucosides. The purple sweet potato pigment may be extracted from purple tuberous roots of the genus Ipomoea of the Convolvulaceae family, or may be obtained by chemical synthesis.

The method of contacting the cells with the staining agent is not particularly limited, and examples thereof include a method of adding the staining agent to a medium or liquid containing cells.

The concentration of the dye in the medium or liquid is not particularly limited, but is preferably 2 to 15 mg/mL, for example. If the concentration of the staining agent is 2 mg/mL or more, dead cells can be stained in a short period of time in inverse proportion to the concentration, and the stained state can be maintained for a longer period of time. Particularly in the case of monascus dye, the concentration is more preferably 9 to 15 mg/mL, and even more preferably 15 mg/mL. When the monascus dye has a concentration of 20 to 80 mg/mL, no significant increase in dead cells is observed, and almost no fading of the dye is observed. However, at the above concentration, almost no swimming cells can be seen.

Additives may be added to the medium or liquid in addition to the staining agent. Examples of additives include glucose and the like. Although the concentration of glucose is not particularly limited, it is preferably 5 to 40 g/L, more preferably 10 to 40 g/L.

The contact time between the cells and the staining agent is not particularly limited. For example, in the case of monascus dye, the contact time is preferably 2 minutes or longer, more preferably 3 minutes or longer, and even more preferably 10 minutes or longer. . In the case of purple sweet potato pigment, the time is preferably 2 minutes or longer, more preferably 1 hour or longer, still more preferably 3 hours or longer, and particularly preferably 5 hours or longer.

(Life and death determination process)
In the life-and-death determination step, the presence or absence of staining of the cells is evaluated by the evaluation method described later, and the stained cells are determined as dead cells.

The method for evaluating staining of cells is not particularly limited as long as it is a method that can confirm the staining state of cells. Examples thereof include a method of visually confirming the color tone of cells through a microscope, a method of measuring the absorption spectrum of cells, and the like. . From the viewpoint of easier evaluation, a method of visually confirming the color tone of cells through a microscope is preferred. At this time, the observation magnification is preferably 40 times (×4 objective lens, numerical aperture 0.1) or more, more preferably 100 times (×10 objective lens, numerical aperture 0.25) or more. In addition, it is preferable that the observation magnification for judging life and death of non-stained cells is 400 times (×40 objective lens, numerical aperture 0.65) or more.

Then, based on the cell staining evaluation results, the cells that maintain the cell-specific color tone are discriminated as viable cells, and the cells stained with the staining agent are discriminated as dead cells.

Here, the method for judging life and death of cells of the present embodiment utilizes the difference in membrane permeability between live cells and dead cells to a specific dye, such as the dye exclusion method using trypan blue. Specifically, the staining agent used in this embodiment is not taken up into the cells through the cell membrane of living cells, or is excreted even if it is taken up. On the other hand, since the cell membrane of dead cells is damaged, the dye is taken up into the cell and binds to a specific substance such as protein, thereby accumulating the dye in the cell and staining it strongly.

In addition, unlike trypan blue, the staining agent of the present embodiment is a food coloring agent, and therefore is highly safe and has no cytotoxicity or the like. Therefore, it is possible to more safely determine whether cells are alive or dead. In addition, since dead cells do not increase due to staining agents, the effects of drugs such as anticancer drugs on cells can be monitored over a long period of time using the same sample. This saves the trouble of preparing in advance a plurality of samples corresponding to the desired elapsed time in order to confirm the effect of the drug over time. Moreover, since the same sample is evaluated, there is no variation in measurement data between samples. In other words, it is possible to accurately and simply confirm the transition of the action of the drug on the cells.

<Kit for judging viability of cells>
The cell life-and-death determination kit according to the present embodiment is used for the above-described cell life-and-death determination method, and contains a staining agent for staining dead cells.

Staining agents contained in the kit for judging viability of cells include the above-described monascus dye or anthocyanin dye. Among the anthocyanin pigments, purple sweet potato pigments and red cabbage pigments are particularly preferred.

The life-and-death determination kit may contain an additive in addition to the staining agent. Examples of additives include glucose and the like.

By using the cell viability determination kit according to the present embodiment, cell viability determination can be performed simply and safely.

Examples of the present invention will be described below, but the scope of the present invention is not limited to these examples.

<Example 1: Evaluation of absorbance of Euglena gracilis cells by red yeast rice pigment and anthocyanin pigment>
In Example 1, the presence or absence of staining of Euglena gracilis cells with monascus dyes and anthocyanin dyes was confirmed by measuring the absorption spectrum of the cells.
First, the Z strain of wild-type E. gracilis (Euglena Co., Ltd.) was placed under continuous lighting at 26° C. and 40 μmol/m ² /sec white fluorescent lamp at pH 3.0. The cells were statically cultured in a No. 5 Cramer-Myers medium (hereinafter referred to as "CM medium") for 40 days. Next, 1 mL of the cell suspension of the statically cultured cells was dispensed into microtubes. A sample containing only the cell suspension was designated as sample A. To a microtube containing 1 mL of the above cell suspension, powder of monascus dye (hereinafter referred to as "MP") (My Kitchen Co., Ltd.) was added so that the final concentration was 10 mg/mL, and completely mixed by pipetting. Sample B was obtained by mixing. Anthocyanin dye (hereinafter referred to as "AP") (My Kitchen Co., Ltd.) powder was added to a microtube containing 1 mL of the above cell suspension so that the final concentration was 10 mg / mL, and completely pipetted. Sample C was obtained by mixing MP is a pigment extracted from Aspergillus oryzae, and AP is a pigment extracted from purple sweet potato. Then, samples A to C were allowed to stand at 26° C. under continuous illumination with a white fluorescent lamp of 100 μmol/m ² /sec for two days with the lid of the microtube open.

As control samples for samples B and C, the following samples containing no cell suspension were prepared.
Sample B1: Prepared by adding MP to purified water (industrial purified water, Sanei Chemical Co., Ltd.) to a final concentration of 10 mg/mL.
Sample B2: Prepared by adding MP to CM medium to a final concentration of 10 mg/mL.
Sample C1: Prepared by adding AP to purified water to a final concentration of 10 mg/mL.
Sample C2: Prepared by adding AP to CM medium to a final concentration of 10 mg/mL.

Then, the absorbance of the suspension or solution in each sample and the absorbance of single cells were measured using a previously reported non-scanning single-cell absorption spectroscopic imaging A(x, y, λ) method as follows: (T. Isono et al., PLOS ONE 10:e0128002).
The non-scanning single-cell absorption spectroscopic imaging A(x, y, λ) method uses a white light lamp as the light source to instantaneously image the absorption spectrum distribution A(x, y, λ) in living cells. It is a technology that can Specifically, a bundle fiber array that converts a two-dimensional image (60 μm pitch, 16 × 16 (0.96 × 0.96 mm ² )) into a one-dimensional slit image (60 μm pitch, 1 × 256) enables cell absorption. Spectral images can be mapped one at a time onto a two-dimensional CCD detector. Moreover, the absorption spectrum A(x, y, λ) can be instantaneously measured by a spectroscope capable of forming an image with a long vertical width without aberration and a CCD with a large area. The spectroscope (IsoPlane SCT-Advance, Roper Scientific) used in this example has a wide imaging range of 27×27 mm ² , and the CCD camera (PIXIS2048BUV, Roper Scientific) has a pixel size of 13.5 μm and a pixel The number is 2048×2048. This allows a 16×16×2048 data cube to be acquired instantaneously without spatial scanning or wavelength scanning. By this non-scanning single-cell absorption spectroscopic imaging A(x, y, λ) method, absorbance was measured according to the following measurement procedure.
First, samples A to C were diluted 10-fold with purified water. Next, the 10-fold diluted samples A to C and the control samples B1, B2, C1, and C2 were transferred to a glass bottom dish and examined using an inverted microscope (IX71, Olympus Co., Ltd.) with a numerical aperture of 0.85 and a magnification of 100. Observed with a magnifying lens. A 150 W xenon lamp (Hamamatsu Photonics Co., Ltd.), which is a light source, was used to expose a range of 2 mm in diameter of the sample. The photon flux density for the sample was 2200 μmol/m ² /sec for 0.5 second exposure. The spectrometer automatically switches between three gratings: 1200, 300, and 150 groove/300 nm blaze. In this example, a 150-groove grating with a wavelength resolution of 1.1 nm at 546 nm (50 μm slit width determined by the fiber core size) and a wavelength span of 390-790 nm was used. Measurements were performed at room temperature. In addition, as a method for calculating the absorbance, in the case of samples A to C, the absorbance at the local site of each single cell is extracted from the absorption image, and the absorbance in the region where no cells exist in each solution is subtracted. Cell absorbance was calculated. The local sites referred to here are chloroplast sites in live cells, sites where a characteristic spectrum is obtained in sample A dead cells, and stained sites in sample B and C dead cells. In the case of dye solution samples B1, B2, C1, and C2 that do not contain cell suspension, the absorbance extracted from the entire measurement area of the absorption image in the solution and purified water is averaged, and the purified water is subtracted as a background. was calculated. Absorption spectra of live and dead cells in sample A are shown in FIG. 1, absorption spectra of live and dead cells in sample B, sample B1, and sample B2 are shown in FIG. FIG. 3 shows absorption spectra of C1 and sample C2. The cell images shown in FIGS. 1 to 3 are 1000-fold magnification images of live cells or dead cells for which absorbance was measured.

As shown in FIG. 1, in the absorption spectrum of living cells, the absorption spectrum of chloroplasts having two peaks at 441 nm and 675 nm due to chlorophyll-a (hereinafter referred to as "Chl-a") appears predominantly. It also has a peak attributed to carotenoid at 490 nm. In contrast, the spectra of dead cells 2, in which most of the cellular contents have been lost, did not reveal any characteristic structures derived from pigments such as Chl-a. In the spectra of Dead Cell 1 and Dead Cell 3, a shape characteristic of Chl-a can be confirmed, but the peaks corresponding to 441 nm and 675 nm of live cells are broadened, and the carotenoid peak cannot be confirmed. rice field.

As shown in FIG. 2, the spectra of samples B1 and B2 containing no cell suspension are characterized by having two peaks in the wavelength region of 400 nm to 500 nm. Sample B1, in which the solvent is purified water, has peak wavelengths of 422 nm and 499 nm. Sample B2, in which the solvent is CM medium, has peak wavelengths of 426 nm and 505 nm. It can be confirmed that the peak when the solvent is CM medium shifts to the longer wavelength side compared to when the solvent is purified water. The absorption spectrum of the living cells of Sample B has two peaks at 436 nm and 673 nm and a carotenoid peak at 491 nm, and exhibits substantially the same shape as the absorption spectrum of the living cells of Sample A. From this, it was confirmed that the viable cells of sample B were not stained with MP and maintained the original color tone of the cells. On the other hand, the spectrum of the dead cells of sample B has a feature that the absorbance increases toward the short wavelength side in the wavelength region of 490 to 600 nm so as to cancel out the color of the chloroplast. Thus, the dead cell spectrum of sample B exhibited a characteristic shape of the MP spectrum. From this, it was confirmed that the dead cells were stained with MP.

As shown in FIG. 3, the spectra of samples C1 and C2 without cell suspension are characterized by having an absorption maximum at 529 nm. The absorption spectrum of the viable cells of sample C has two peaks at 433 nm and 674 nm and a carotenoid peak at 489 nm, showing substantially the same shape as the absorption spectrum of the viable cells of sample A. From this, it can be confirmed that the viable cells of sample C were not stained with AP and maintained the original spectrum of the cells. On the other hand, the spectrum of the dead cells of sample C has a feature that the absorbance increases toward the short wavelength side in the wavelength region of 540 to 645 nm so as to cancel out the color of the chloroplast. Thus, the dead cell spectrum of sample C exhibited a characteristic shape of the AP spectrum. This confirmed that the dead cells were stained with AP.

<Example 2: Determination of life and death of Euglena gracilis cells using monascus dye and anthocyanin dye>
In Example 2, cell viability was determined by microscopic observation of Euglena gracilis cells using MP or AP as a staining agent.
The Z strain of Euglena gracilis was statically cultured in a pH 3.5 CM medium for 7 days at 26° C. under continuous illumination with a white fluorescent lamp of 40 μmol/m ² /sec while maintaining aerobic conditions. Using the cell suspension of the statically cultured cells, the following samples were prepared.
Sample D: Prepared by adding MP to a microtube containing 1 mL of cell suspension to a final concentration of 10 mg/mL.
Sample E: Prepared by adding AP to a final concentration of 10 mg/mL and adding glucose to a final concentration of 12 g/L to a microtube containing 1 mL of cell suspension.
Samples D and E were allowed to stand at 26° C. under continuous illumination with a white fluorescent lamp of 100 μmol/m ² /sec for 3 days with the lid of the microtube open. Then, each sample is diluted with purified water as necessary, and is subjected to a bacterial counting board (model A-161, Sunlead Glass Co., Ltd.) using a bright field microscope (digital biological microscope GR-D8T2, Matsuden Co., Ltd.). Observed in the shed). FIG. 4A is a bright-field microscopic image of sample D and (B) of sample E at a magnification of 400 (×40 objective lens, numerical aperture 0.65, Shodensha Co., Ltd.).

As shown in FIG. 4, live cells of Euglena gracilis were green, whereas dead cells were stained red by MP and AP. This indicates that MP and AP stain dead cells so clearly that they cancel out the underlying color of Euglena gracilis, which is a green pigmented cell. Also, MP-stained dead cells showed discernible staining for 6 days with no significant bleaching for 3 days. On the other hand, dead cells stained with AP did not fade over 6 days.

<Example 3: Confirmation of staining of a mixture of dried Euglena gracilis-containing plant cells and tissue fragments with Monascus pigment and anthocyanin pigment>
In Example 3, the presence or absence of staining of MP and AP on a mixed powder (euglena farm green juice, Euglena Co., Ltd.) mainly containing dry powders of Euglena gracilis, barley grass, Angelica keiskei, and chlorella was confirmed.
MP and AP were dissolved in purified water and prepared to a final concentration of 10 mg/mL. The mixed powder was added to and suspended in the MP aqueous solution and the AP aqueous solution. Each sample was observed using a bright field microscope (GR-D8T2, Shodensha Co., Ltd.). FIG. 5A is a bright-field microscope image of sample F and (B) of sample G at a magnification of 400 (×40 objective lens, numerical aperture 0.65, Shodensha Co., Ltd.).

As shown in FIG. 5, in sample F containing MP, dead cells such as Euglena gracilis were stained, but the fiber pieces were not stained. On the other hand, in sample G containing AP, not only the plant cells but also the fiber pieces were strongly stained. This is probably because AP also stains polysaccharides such as cellulose and pectin in addition to intracellular proteins and the like.

<Example 4: Confirmation of staining of dead cells of Paramecium with Monascus dye and anthocyanin dye>
In Example 4, the presence or absence of staining of dead paramecium cells with MP and AP was confirmed.
First, paramecium cells were observed using a bright field microscope (GR-D8T2, Shodensha Co., Ltd.). Next, MP or AP was added to the solution containing Paramecium to prepare a solution having an MP concentration or AP concentration of 10 mg/mL. A 0.05% (W/W) nutrient (strong Wakamoto, Wakamoto Pharmaceutical Co., Ltd.) was used as the paramecium culture solution. A liquid containing MP was designated as sample H, and a liquid containing AP was designated as sample I. Next, 0.5 mL of each of Samples H and I was dropped into a microtube and heat-treated for 20 seconds in a microwave oven (MM820S-N5, Kakuda Musen Denki Co., Ltd.). After 3 minutes, paramecium cells were observed using a bright field microscope. Fig. 6 shows Paramecium cells before dye addition and Paramecium cells in samples H and I after heat treatment observed in bright field at a magnification of 400x (x40 objective lens, NA 0.65, Shodensha Co., Ltd.). It is a microscope image with. 6A is a microscopic image of Paramecium cells before dye addition, (B) is Paramecium cells in sample H, and (C) is a microscopic image of Paramecium cells in Sample I. FIG.

As shown in FIG. 6, live paramecium cells were colorless. On the other hand, the inside of the paramecium cells after heat treatment with a microwave oven was uniformly and clearly stained with MP and AP. From this result, it was found that MP and AP stain the dead cells of Paramecium.

<Example 5: Confirmation of the growth of Euglena gracilis in a medium containing a natural or synthetic pigment>
In Example 5, the natural pigment MP or AP or the synthetic pigment trypan blue (hereinafter referred to as "TB") (Tokyo Chemical Industry Co., Ltd.) or methylene blue (hereinafter referred to as "MB") (Kokusan Chemical Co., Ltd.) The growth of Euglena gracilis in the medium containing was confirmed.
First, the Z strain of Euglena gracilis was statically cultured in a pH 3.5 CM medium for 5 days while maintaining aerobic conditions at 21° C. under continuous illumination with a white fluorescent lamp of 40 μmol/m ² /sec. The cell density at this time was 5.6×10 ⁵ cells/mL. A sample was prepared according to the composition shown in Table 1 below using the cell suspension of the statically cultured cells. A microtube was used as a sample container.

１）メチレンブルーをＣＭ培地で希釈して１％（Ｗ／Ｗ）溶液とした。

1) Methylene blue was diluted with CM medium to give a 1% (W/W) solution.

Then, each sample was allowed to stand at 21° C. under continuous illumination with a white fluorescent lamp of 100 μmol/m ² /sec for 3 days with the lid of the microtube open. At this time, once a day, a bacteria calculator (model A-161, Sunlead Glass Co., Ltd.) and a bright field microscope (GR-D8T2, Shodensha Co., Ltd.) were used with a magnification of 100 times (×10 objective lens). , numerical aperture 0.25, Shodensha Co., Ltd.) or 400 times (×40 objective lens, numerical aperture 0.65, Shodensha Co., Ltd.). Then, the cells in each sample were counted using a plankton counter (MPC-200, Matsunami Glass Industry Co., Ltd.) to obtain the cell density (cell/mL). At this time, each sample was appropriately diluted with a 10% (W/V) solution of benzalkonium chloride (Nihon Pharmaceutical Co., Ltd.) diluted 50 times with purified water, and Euglena gracilis cells were fixed at the same time. The results are shown in FIGS. 7 and 8. FIG.

As shown in Figure 8, the cell density in samples containing MB and TB after 20 hours is lower than in CM medium with or without glucose. These results show that MB and TB inhibit the growth of Euglena gracilis cells.
In contrast, as shown in FIG. 7, the cell density in samples containing MP and AP at 70 hours was found to be higher than in samples CM containing no dye in the absence of glucose. It was also found that in the presence of glucose, the cell density of the sample with MP was higher than the sample CM without dye, and the cell density in the sample with AP was approximately equivalent to the sample CM without dye. confirmed. From this result, it was found that CM to which MP or AP was added did not inhibit the growth of Euglena gracilis cells, unlike MB or TB.

<Example 6: Confirmation of staining time of Euglena gracilis with natural dye or synthetic dye>
In Example 6, the staining time of Euglena gracilis dead cells with the natural dyes MP or AP or the synthetic dyes TB or MB was confirmed.
The samples listed in Table 1 were prepared in the same manner as in Example 5 above. A 10% (W/V) benzalkonium chloride solution was diluted 50-fold with the sample for algicidal treatment, and staining of dead cells was confirmed. At this time, the preparation of the samples shown in Table 1 and the addition of benzalkonium chloride to each sample were performed almost simultaneously, and the elapsed time from the mixing of each sample and benzalkonium chloride was measured.
As a result, within 2 minutes of benzalkonium chloride addition, all dead cells were clearly stained in samples containing MP, AP and MB. However, no significant staining of TB was observed, and aggregates of TB appeared throughout the solution. Since viable cells were very rarely present in the sample containing TB even after 6 minutes, it is considered that benzalkonium chloride reacted with TB and no longer exhibited toxicity.
In addition, when the test of Example 6 was performed on a sample prepared by adding 0.1 mg of TB to 1 mL of cell suspension, all cells were stained within 2 minutes, but Euglena viable cells Since the color is close to that of chloroplasts, the staining was slightly more bluish than before the addition.

<Example 7: Confirmation of staining of Euglena gracilis in mixed medium of monascus pigment and purple sweet potato pigment>
In Example 7, the transition of cell staining was confirmed when Euglena gracilis was cultured in a medium containing a mixed dye of MP and AP.
The Z strain of Euglena gracilis was statically cultured in a pH 3.5 CM medium for 6 days at 21° C. under continuous illumination with a white fluorescent lamp of 40 μmol/m ² /sec while maintaining aerobic conditions. Using the cell suspension of the statically cultured cells, samples were prepared according to the compositions shown in Table 2 below. A microtube was used as a sample container.

Then, each sample was statically cultured for 3 days at 21° C. under continuous illumination with a white fluorescent lamp of 100 μmol/m ² /sec with the lid of the microtube opened. At this time, once a day, a bacteria calculator (model A-161, Sunlead Glass Co., Ltd.) and a bright field microscope (GR-D8T2, Shodensha Co., Ltd.) were used with a magnification of 100 times (×10 objective lens). , numerical aperture 0.25, Shodensha Co., Ltd.) or 400 times (×40 objective lens, numerical aperture 0.65, Shodensha Co., Ltd.).

FIG. 9 is a graph showing the transition of the ratio of stained cells to all cells in each sample from the time of cell culture of each sample. The percentage of stained cells indicates the percentage of dead cells to the total number of cells including live and dead cells.
As shown in FIG. 9, it was confirmed that within 65 hours from the start of culture, the number of stained dead cells was 10% or less in all samples. In addition, clear and stable staining of dead cells was confirmed over the entire measurement period from immediately after dye mixing.
In addition to samples J to L, samples each containing 10 mg/mL of MP or AP alone were prepared and tested in the same manner as in this example. At this time, in the sample containing MP, dead cells were stained in about 10 minutes, and fading of the stained dead cells was confirmed after 3 days. In the sample containing AP, dead cells were stained in about 1 hour, and no fading of dead cells was observed even after 6 days.
On the other hand, in samples J to L containing mixed dyes of MP and AP, the dead cells were stained immediately after the dyes were mixed, and even after 5 days from the start of the culture, the stained state was easily visually distinguishable with a microscope. was maintained. From this result, the property of MP that it can be dyed in a short period of time but is easier to fade than AP, and the property of AP that it takes time to dye but is less likely to fade than MP acted in a complementary manner. It is thought that there are

In addition to the examples described above, MP was used to confirm the presence or absence of staining of human breast cancer cells by microscopic observation. Specifically, a sample obtained by adding cisplatin (final concentration 20 μmol/L) and MP (final concentration 10 mg/mL) to a medium containing MCF7, which is a human breast cancer cell, and MP (final concentration 10 mg/mL) alone. A spiked sample was prepared. After 12 hours, the medium of each sample was replaced with phosphate-buffered saline (PBS) to make a clear medium, and then observed using a bright field microscope. It was previously confirmed by staining with trypan blue that cell death of MCF7 was induced by treatment with 20 μmol/L cisplatin for 12 hours. Some of the MCF7 in the cisplatin-containing sample stained, and almost no MCF7 in the cisplatin-free sample. Based on this result, it is considered that MP does not stain live MCF7 cells, but stains dead cells.

Claims (4)

Contacting the cells with a staining agent that stains dead cells;
A step of determining whether the cell is alive or dead based on the presence or absence of staining of the cell,
A method for determining cell viability, wherein the staining agent contains at least one dye selected from monascus dye and anthocyanin dye. 2. The method for judging life and death of cells according to claim 1, wherein said anthocyanin pigment is purple sweet potato pigment. A cell viability determination kit for use in the cell viability determination method according to claim 1,
containing a staining agent that stains dead cells;
A kit for judging viability of cells, wherein the staining agent contains at least one dye selected from monascus dye and anthocyanin dye. 4. The kit for judging life and death of cells according to claim 3, wherein said anthocyanin pigment is purple sweet potato pigment.

Images