JP2011015643A - Culture solution for measuring membrane potential of cell, culture method using the same, and method for measuring membrane potential - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve such a problem that interaction or adsorption frequently occurs among substances such as amino acids and proteins and a test compound for evaluating pharmaceutical effects, and the pharmaceutical effects cannot accurately be evaluated because a conventional culture solution contains large amounts of the substances.SOLUTION: Accurate measurement can be carried out without inhibiting a fluorescent label and without causing the interaction or adsorption among the substances and the test compound for evaluating the pharmaceutical effects during a test for evaluating the pharmaceutical effects by including selenium and/or nitrate ions and iron ions without adding the amino acids and proteins in the culture solution for measuring the membrane potential of a cell. Furthermore, the membrane potential can be measured, particularly in a cardiomyocyte derived from a human induced pluripotent stem (iPS) cell over a long period of time by including the selenium and/or nitrate ions and iron ions in the culture solution.

Description

  The present invention relates to a culture solution for measuring membrane potential of cells, a culture method using the same, and a membrane potential measurement method, and in particular, a culture solution for measuring membrane potential of cardiomyocytes derived from human iPS cells, and The present invention relates to a culture method and a membrane potential measurement method used.

In the development of new drugs, it is required to quickly and appropriately evaluate the efficacy and toxicity of drugs.
And the voltage-gated ion channel (a transmembrane protein existing on the cell surface that allows specific ions to selectively pass) present in the cell membrane has a physiologically important function, It is a target for evaluation of efficacy and toxicity of drug development.
For example, a drug-induced (acquired) QT prolongation syndrome is a disease that causes severe arrhythmia in a patient by acting on an ion channel. Drug-induced long QT syndrome is a prolongation of the electrocardiogram QT interval after drug administration, often due to TdP (Torsades de points, non-persistent polymorphic ventricular tachycardia) Is a serious disease that causes fainting, fainting, and sudden death. The cause of this drug-induced QT prolongation syndrome is known to be that the drug acts on the ion channels of cardiomyocytes to inhibit harmonious heart pulsation and prolong the QT interval. (Non-Patent Document 1).

One example of a method for evaluating whether a drug causes drug-induced long QT syndrome is a method for measuring a membrane potential of a cell. Referring to Non-Patent Document 2, a patch clamp method and a fluorescent membrane potential measurement method are described as an example of a membrane potential measurement method.
Here, the fluorescent membrane potential measurement method uses a fluorescent calcium probe, Bis-oxonol, and the membrane-permeable and anionic fluorescent dye moves in and out of the cell membrane in accordance with the movement of ions. This is a method for measuring the membrane potential of a cell by measuring. The fluorescent membrane potential measurement method is characterized by extremely high throughput.
The fluorescent membrane potential measurement method can be used as a drug efficacy evaluation of drug-induced QT prolongation syndrome by measuring fluorescence when a test compound drug is given to the above-described cells. Specifically, when the hERG channel activity is inhibited by a toxic drug, the fluorescence intensity changes when an electromagnetic wave of a predetermined wavelength is irradiated. Therefore, a change in membrane potential can be detected by detecting this. Thereby, it can test | inspect whether the drug which is a test compound causes the drug-induced QT prolongation syndrome.

Pharmacology Journal 121, 384-392, 2003 Pharmacological Journal 126, 321-327, 2005

  However, in the conventional culture solution used for measuring the membrane potential of cells, the culture solution itself has fluorescence or emits fluorescence when irradiated with electromagnetic waves of a predetermined wavelength. Could not be applied. This is because it contains a lot of fluorescent substances such as basal medium components and serum-derived proteins. In addition, since conventional culture solutions contain many substances such as amino acids and proteins, many interactions and adsorptions occur with test compounds for which the drug efficacy is evaluated, and accurate drug efficacy evaluation cannot be performed.

  This invention is made | formed in view of such a condition, and it aims at providing the culture solution which can solve the said problem.

The culture solution for measuring the membrane potential of cells of the present invention is characterized by containing selenium and / or nitrate ions and iron ions without adding amino acids and proteins that are not derived from cells.
In the culture solution for measuring the membrane potential of cells of the present invention, the selenium is supplied at a sodium selenite concentration of 0.001 to 0.1 μM, and / or the nitrate ions and iron ions are iron nitrate (III). It is characterized by being supplied at a concentration of 0.1 to 9 μM.
The culture solution for measuring the membrane potential of cells of the present invention is characterized in that the cells are human iPS cell-derived cardiomyocytes.
The cell culture method of the present invention is characterized by using the culture solution of the present invention.
The cell membrane potential measurement method of the present invention is characterized by using the culture solution of the present invention.

  Since the culture solution for measuring the membrane potential of cells of the present invention does not contain a fluorescent substance such as protein and is almost non-fluorescent, a fluorescent calcium probe or a fluorescent dye such as a fluorescent film-sensitive dye is used. In particular, a suitable measurement result can be obtained without inhibiting the fluorescent label. In the culture solution for measuring the membrane potential of cells of the present invention, the amino acid and protein concentrations can be measured by the fluorescent membrane potential measurement method and / or the amino acid and protein concentrations can interact with or adsorb the test compound. Since the concentration is less than the concentration at which it does not occur, accurate measurement can be performed without interaction or adsorption with the test compound for evaluating drug efficacy during the test for evaluating drug efficacy. This makes it possible to quickly and appropriately evaluate the efficacy and toxicity of the new drug.

It is the figure which showed the example of a composition of the culture solution which concerns on embodiment of this invention. It is the figure which showed the comparative example of the waveform change between the culture solution which concerns on embodiment of (a) embodiment of this invention, and (b) general buffer solution. (A) It is the figure which showed the comparative example of the noise between the culture solution which concerns on embodiment of this invention, and the (b) conventional culture solution. It is the figure which showed the structural formula of the reagent (astemisol) which extends QT space | interval to the culture solution which concerns on embodiment of this invention. It is the figure which showed the density | concentration dependence curve with respect to the extension of QT interval of the reagent (astemisol) which extends QT interval to the culture solution which concerns on embodiment of this invention. It is the figure which showed in detail the waveform change at the time of adding the reagent (astemisol) which extends QT space | interval to the culture solution which concerns on embodiment of this invention. It is the figure which showed in detail the waveform change at the time of adding the reagent (astemisol) which extends QT space | interval to the culture solution which concerns on embodiment of this invention. It is the figure which showed in detail the waveform change at the time of adding the reagent (astemisol) which extends QT space | interval to the culture solution which concerns on embodiment of this invention. It is the figure which showed the fluctuation | variation of the QT interval of a reagent (aspirin) judged that it does not influence the QT interval (within +/- 5%) in the culture solution which concerns on embodiment of this invention. It is the figure which showed the density | concentration dependence curve with respect to the fluctuation | variation of the QT interval of the reagent (aspirin) judged that it does not influence the QT interval to the culture solution which concerns on embodiment of this invention (within +/- 5%). It is the figure which showed in detail the waveform change at the time of adding the reagent (aspirin) judged to have no influence on QT space | interval (within +/- 5%) to the culture solution which concerns on embodiment of this invention. It is the figure which showed that only the culture solution which concerns on embodiment of this invention does not affect a QT space | interval under the same experiment environment.

As described above, the conventional culture solution used for measuring the membrane potential of cells is not suitable for fluorescence observation under a microscope because the culture solution itself emits fluorescence. Therefore, the inventor of the present invention has been searching for a culture solution for more accurately detecting the membrane potential and detecting the fluorescent label in place of the conventional culture solution.
Here, for those skilled in the art, it is thought that proteins such as various ions, sugars, essential amino acids, and albumin are commonly required as a culture solution for measuring the membrane potential of cells in order for the cells to maintain normal pulsation. The culture solution has been improved by adjusting the optimum concentration of these substances.

However, the inventor of the present invention has found that substances such as amino acids and proteins that are abundantly contained in the conventional culture medium emit fluorescence and interact with or adsorb compounds with which the drug efficacy is examined. However, if these substances were removed, it was difficult to culture the cells while pulsating, and fluorescence observation itself could not be performed in the first place.
On the other hand, the inventor of the present invention adds selenium and / or nitrate ions and iron ions to remove cells even in a state in which substances related to fluorescence, interaction, and adsorption are removed except for amino acids and proteins. It has been found that a remarkable effect can be obtained in which fluorescence can be observed while suppressing the interaction and adsorption while pulsing, and the culture solution according to the present invention exemplified in the present embodiment has been invented.

  That is, the cells can be maintained in a normal state for a long period of time by using the cell membrane potential measuring medium of the present invention. In addition, since the culture solution of the present invention is non-fluorescent, a suitable result can be obtained without disturbing the detection system even when the cell membrane potential is measured by a fluorescence method or observed using a fluorescence method or a luminescence method. Can do. In the culture solution of the present invention, the concentration of amino acids and proteins is less than the concentration that can be measured by the fluorescent membrane potential measurement method and / or the concentration of amino acids and proteins does not cause interaction or adsorption with the test compound. In the test for evaluating the drug efficacy, accurate measurement can be performed without causing interaction or adsorption with the test compound for which the drug efficacy is evaluated. Furthermore, since the culture solution of the present invention optimizes the glucose level and various ionic strengths, normal pulsations such as cardiomyocytes can be observed for a long time.

(Composition of culture solution)
With reference to FIG. 1, the composition of the culture solution according to the embodiment of the present invention will be described.
As shown in FIG. 1, the components of the culture solution according to the embodiment of the present invention are mainly composed of each ion such as sodium, potassium, magnesium, calcium, sugar, buffer solution, and pH adjusting reagent. The concentration of each substance shown in FIG. 1 indicates the final concentration. This will be specifically described below.

The concentration of each ion can be appropriately changed in order to optimize the measurement of the membrane potential of the cell. For example, as shown in FIG. 1, 140 mM sodium chloride (NaCl), 4.8 mM potassium chloride (KCl), 0.8 mM magnesium sulfate (MgSO ₄ ), 2.8 mM calcium chloride (CaCl ₂ ) are used. be able to. Other ions can also be added as appropriate depending on the characteristics of each cell, the purpose of measuring the membrane potential, and the like.

  The sugar concentration can be changed as appropriate in optimizing membrane potential measurement. Preferably, glucose is glucose. For example, 11.2 mM glucose can be used.

The buffer solution can be appropriately changed in order to optimize the membrane potential measurement. Preferably, the buffer is made using Hepes (C ₈ H ₁₈ N ₂ O ₄ S). The buffer also has the purpose of preventing pH changes due to acidosis of the culture medium during long-term cell culture.
In addition, as a buffer solution, in order to obtain optimal pH, arbitrary buffering agents can be used, for example, PBS etc. can also be used.

  As the pH adjusting reagent, any substance can be used as long as the pH can be adjusted. For example, the pH is adjusted using an acid-base substance such as NaOH or HCl. Preferably, the pH is adjusted to an optimum pH for cell culture, and in the case of cardiomyocyte culture, the pH is adjusted in the range of 7.2 to 7.4. It should be noted that the pH can be arbitrarily changed as appropriate depending on the characteristics of each cell, the purpose of measuring the membrane potential, and the like.

In the culture solution according to the embodiment of the present invention, the concentration of substances contained in the conventional culture solution such as amino acids such as essential amino acids and non-essential amino acids, proteins such as albumin, vitamins, yeast extract, etc. is measured by fluorescent membrane potential. It is below the measurable concentration of the method and / or the concentration at which no interaction or adsorption occurs with the test compound. In particular, the amino acid and protein concentrations are measurable concentrations of the fluorescent membrane potential measurement method and / or the amino acid and protein concentrations are below the concentration at which no interaction or adsorption with the test compound occurs. Here, the amino acid and protein concentrations indicate the concentration of amino acids and proteins combined. Preferably, amino acids and proteins that are not derived from cells are not added or contained at all.
In a culture solution containing substances such as amino acids, proteins, vitamins, and yeast extract, the culture solution itself becomes fluorescent, and it is possible to accurately measure the membrane potential of cells using a highly sensitive fluorescence detection device. Have difficulty. Moreover, since the fluorescent substance used for fluorescent membrane potential measurement and the test compound for evaluating the drug effect interact and adsorb to these substances, accurate measurement results cannot be guaranteed.
On the other hand, in the culture solution according to the embodiment of the present invention, it is involved in the fluorescence of the culture solution itself such as amino acids, proteins, vitamins, yeast extract and the like, or interacts with or adsorbs the test compound. Since such a substance is excluded, the disadvantage that the accurate measurement result which the conventional culture solution had is not ensured is overcome.

Furthermore, the culture solution according to the embodiment of the present invention can appropriately measure the fluorescent membrane potential of cells by containing an appropriate concentration of selenium. This has a remarkable effect that normal membrane potential measurement can be performed for a long period of time, particularly in the measurement of cardiomyocytes derived from human iPS cells.
Preferably, the selenium is provided by Na ₂ SeO ₃ (sodium selenite). More preferably, the concentration of Na ₂ SeO ₃ can be varied from 0.001 to 0.1 μM.

Furthermore, the culture solution according to the embodiment of the present invention can appropriately measure the fluorescent membrane potential of cells by containing appropriate concentrations of nitrate ions and iron ions. This has a remarkable effect that normal membrane potential measurement can be performed for a long period of time, particularly in the measurement of cardiomyocytes derived from human iPS cells.
Preferably, nitrate ions and iron ions _are provided by a _{Fe (NO 3) 3 · 9H} 2 O ( iron (III) nitrate hydrate). More _{preferably, Fe (NO 3) 3 ·} 9H 2 O concentration can be varied in 0.1～9MyuM.

The culture solution according to the embodiment of the present invention can be prepared by an ordinary method that can be used by those skilled in the art. For example, there is a method of adding each component to a liquid prepared in advance to obtain a final concentration. The test substance can be used from any concentration, and may be dissolved in a suitable solvent such as DMSO or directly in a measurement solution such as a measurement medium.
Note that the composition and concentration of the culture solution shown in FIG. 1 are merely examples in which the concentration of each substance is optimized, and the concentration of each substance can be changed as appropriate in order to optimize potential measurement. For example, the composition of the culture solution can be adjusted depending on the origin of the cells to be used, the type of the cells, and the like. For example, in the case of cardiomyocytes having different properties, it is necessary to appropriately adjust the composition of the culture solution.

By using the test culture solution according to the embodiment of the present invention, cells can be cultured for a long period of time such as one day or longer. At this time, it is possible to maintain the state in which the cells are beating. Thus, since it is optimized as a culture solution for measuring the membrane potential of cells, it is possible to observe the membrane potential for a long time with more stable cell functions. Therefore, it can be said that it is a culture solution suitable for performing measurement used for evaluating the drug efficacy of pulsatile cells such as cardiomyocytes.
In addition, when the composition of the culture solution is not preferred, cell pulsation is stopped and eventually killed. Paused or dead cells are no longer suitable for observation. For this reason, it has been difficult to realize a pulsating cell culture solution in a state in which amino acids, proteins, and the like are removed as in the culture solution according to the embodiment of the present invention.

(Membrane potential measurement method using the culture solution of the present invention)
The actual membrane potential measurement method using the culture solution according to the embodiment of the present invention will be described with reference to the following protocol examples. For specific membrane potential measurement methods, see the description in Non-Patent Document 2.

(1) First, a cell to be measured or a colony of cells is prepared to an appropriate size (about 10 μm to 1 mm) in an appropriate culture solution such as DMEM.
Arbitrary cells, such as a nerve cell, a hepatocyte, and a cardiac muscle cell, can be used as a measurement object cell. Moreover, these cells can be used singly or in plural. The plurality of cells may be cell clusters (colony). More specific cells to be measured include stem cell-derived or primary cultured cell beating myocardial mass.

(2) Next, the prepared cells are moved using a pipette or the like into a measurement container containing an appropriate culture solution such as DMEM.
The cells that have moved into the culture solution settle down to the bottom of the measurement vessel in the culture solution due to their own weight. Alternatively, centrifugal force may be applied using a centrifuge to settle on the bottom surface of the measurement container.

(3) Next, the cells are arranged at a preferred position such as on the electrode in the measurement container, and left to stand until the cells adhere. This standing is performed for an optimum predetermined period depending on the cells. In the case of cardiomyocytes derived from human iPS cells, it is common to leave them for about 1 hour to 1 day.
Here, the measurement container for measuring the membrane potential may have any shape. For example, a multi-electrode single dish with a plurality of electrodes accumulated in the center of a culture dish or a cell in which the bottom of the container has a mortar shape and naturally settled cells adhere securely on the measurement electrode It can be performed using a measurement container (see PCT / JP2009 / 059359) or the like.

(4) Next, after the cell adheres to the electrode or the like, it is replaced with the culture solution according to the embodiment of the present invention.
The term “adhesion” as used herein means fixing at an appropriate distance for measuring the potential. Such an operation is performed for each operation of cell placement and culture medium replacement.
At this time, the cells can be cultured as they are for about one day until the cells are adapted to the culture solution according to the embodiment of the present invention. Further, at the time of this culture, the culture solution according to the embodiment of the present invention can be replaced like a normal method for culturing adherent cells. As described above, when the culture solution according to the embodiment of the present invention is used, the pulsation state can be maintained even if long-term culture is performed.

(5) Then, it is used for membrane potential measurement.
When there is a test compound or the like, the compound is dissolved and added to the culture medium of the present invention. If necessary, the test compound can be diffused in the culture solution by, for example, vibrating after supplying the test compound. By performing this operation after the cells are adhered, it is possible to prevent the cells from being detached from the electrodes or the like due to the influence of vibration or the like. When the test compound is supplied, the measured value of the membrane potential of the cell changes, so from the state of change in the measured value before and after the supply of the test compound, by evaluating the effect of each compound on the cell, Evaluate drug efficacy. A multi-electrode system can be used for this medicinal evaluation.

  The culture medium which concerns on embodiment of this invention is demonstrated further more concretely with the following example. This embodiment is only an example, and the present invention is not limited to this.

(Measurement example of cardiomyocytes)
Using a culture solution according to an embodiment of the present invention, cardiomyocytes derived from human iPS cells that pulsate as cells to be measured and differentiated by a known method are used to measure the membrane potential using a fluorescent membrane potential measurement method. The Example which measured was shown.

FIG. 2 shows a comparative example of pulsation in the measurement of the membrane potential of cardiomyocytes. FIG. 2 (a) shows the result of membrane potential measurement using the culture solution according to the embodiment of the present invention, and FIG. 2 (b) is one of common buffer solutions (11.2 mM glucose). The result of having measured the membrane potential using (containing PBS) is shown. The time indicated by the arrow indicates 2 minutes. A vertical spike-like signal indicates the occurrence of heartbeat pulsation.
From these experimental results, in the culture solution according to the embodiment of the present invention, pulsation is regularly and continuously observed from the cardiomyocytes, but pulsation from the cardiomyocytes is intermittently observed in a general buffer solution. A regular and unstable beat is observed.
As described above, the culture solution according to the embodiment of the present invention can be used to measure membrane potential at which more accurate pulsation is observed.

Next, FIG. 3 shows a comparative example of noise in the measurement of the membrane potential of cardiomyocytes. FIG. 3 (a) shows the result of measuring the membrane potential using the culture solution according to the embodiment of the present invention, and FIG. 3 (b) shows the result of measuring the membrane potential using the conventional culture solution. Show. Here, the conventional culture solution is obtained by adding bovine serum (10-20%, manufactured by Gibco) to DMEM (manufactured by Gibco, etc.).
From these experimental results, when the culture solution according to the embodiment of the present invention is used, K ⁺ as indicated by an arrow together with a clear sharp Na ⁺ peak (Na ⁺ current peak: QRS wave-like waveform). A waveform considered to be a peak (K ⁺ current peak: T-wave like waveform) can be confirmed. In addition, since the noise is reduced by using the culture solution according to the embodiment of the present invention, it is possible to easily measure the membrane potential waveform by observing the detailed parameters of drug efficacy evaluation. On the other hand, in the conventional culture solution, the waveform was noisy, the interval between the Na ⁺ peak and the Na ⁺ peak was irregular, and the amplitude of the potential waveform was also nonuniform. Further, a K ⁺ peak-like waveform as confirmed in the culture solution according to the embodiment of the present invention could not be confirmed.
As described above, the culture solution according to the embodiment of the present invention can accurately measure the membrane potential of a cell with less noise, and thus can detect a K ⁺ peak-like waveform. It is shown that it is a culture solution that can be used for evaluating the efficacy of the compound.

Next, a measurement example as a positive control using a reagent (astemizole, Wako Pure Chemical Industries) that extends the QT interval is shown using FIGS. FIG. 4A shows the structural formula of astemizole. FIG. 4B shows a concentration-dependent curve for QT interval (norm QT-Interval) when astemizole was added from a concentration of 0.10 nM to a concentration of 0.01 M. FIGS. 4C to E show in detail the potential change (FP [μV]) for each time course (Time [ms]) when astemizole was added, and the figures are enlarged as it becomes FIGS. 4C, 4D, and 4E. ing.
From these experimental results, as shown in FIG. 4B, it can be seen that when the QT interval is measured using astemizole, the QT interval is extended depending on the concentration. Moreover, as shown in FIG. 4C, FIG. 4D, and FIG. 4E, when astemizole is added, Na ⁺ peak and K ⁺ peak are detected in the non-added, added low concentration region, and high concentration region, and measurement is good. It turns out that it is possible.

Next, a measurement example as a negative control using a reagent (aspirin, Cayman chemical company) that does not affect the QT interval (within ± 5%) is shown using FIGS. FIG. 5A shows that the QT interval of aspirin is not affected (within ± 5%). FIG. 5B shows a concentration dependence curve with respect to the QT interval (norm QT-Interval) when aspirin was added at a concentration of 0.10 nM to 0.10 M. FIG. 5C shows in detail the potential change (FP [μV]) over time (Time [ms]) when aspirin was added.
From these experimental results, as shown in FIG. 5A, it can be seen that when the QT interval is measured using aspirin, the QT interval does not change in the test concentration range. In addition, as shown in FIG. 5B, it can be seen that when the QT interval is measured using aspirin, the QT interval does not change in the test concentration range. Further, as shown in FIG. 5C, it can be seen that when aspirin is added, Na ⁺ peak and K ⁺ peak are detected from the added low concentration region to the high concentration region without addition, and good measurement is possible.

Next, FIG. 6 shows an example in which the culture medium according to the embodiment of the present invention that does not affect the QT interval is measured by the same procedure as when astemizole and aspirin were experimented.
From this experimental result, as shown in FIG. 6, it can be seen that when the QT interval is measured using the medium according to the embodiment of the present invention, the QT interval does not change depending on the concentration.

  As described above, when the membrane potential is measured using the culture solution according to the embodiment of the present invention, even if any compound is used regardless of whether or not there is an influence on the QT interval, a membrane with less detailed noise. A potential waveform could be detected. This shows that the culture solution according to the embodiment of the present invention is suitable for evaluating the efficacy of a test compound that affects the ion channel.

  Conventionally, the fluorescent membrane potential measurement method has a very high throughput, but has a problem that the ratio of false positives is also high. However, since the detailed membrane potential waveform can be detected by using the culture solution according to the embodiment of the present invention, the accurate efficacy of the test compound can be reduced by reducing the false positive ratio. Evaluation is expected. As a result, the efficacy evaluation can be accelerated, and the development of new drugs can be speeded up and made efficient.

  According to the present invention, by providing a culture solution for beating cells that does not have fluorescence and does not cause interaction or adsorption with a test compound, it is possible to quickly evaluate the efficacy of a new drug and reduce costs. .

Claims (5)

  A culture solution for measuring the membrane potential of a cell, comprising selenium and / or nitrate ions and iron ions without adding amino acids and proteins not derived from cells.   The selenium is supplied at a sodium selenite concentration of 0.001 to 0.1 μM, and / or the nitrate ions and iron ions are supplied at a concentration of iron (III) nitrate of 0.1 to 9 μM. The culture solution according to claim 1.   The culture medium according to claim 1 or 2, wherein the cells are human iPS cell-derived cardiomyocytes.   A method for culturing cells using the culture solution according to any one of claims 1 to 3.   A method for measuring a membrane potential of a cell using the culture solution according to any one of claims 1 to 3.

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