JP7079319B2 - Acid dissociation equilibrium estimation method, acid dissociation equilibrium estimation device, acid dissociation equilibrium estimation system, and user interface device - Google Patents

Description

The present invention relates to an acid dissociation equilibrium estimation method, an acid dissociation equilibrium estimation device, an acid dissociation equilibrium estimation system, and a user interface device.

The medium for cell culture often contains a pH indicator such as phenol red. The pH indicator exhibits a color corresponding to the pH of the medium. The operator can know the pH of the medium from the color of the medium and predict the growth state or contamination of cells based on the pH.

The medium often contains a colored component such as fetal bovine serum (FBS) in addition to the pH indicator, and the color of the medium is a mixture of the color of the pH indicator and the color of FBS. The concentration of FBS contained in the medium varies depending on the type of cells, the purpose of culturing, and the like. Therefore, it is difficult to accurately determine the pH of the medium based on the color of the medium visually. Therefore, a method of calculating the pH of the medium based on the absorbance of the medium has been proposed (see, for example, Patent Document 1).

In Patent Document 1, the pH of the medium is calculated by measuring the absorbance of the medium at four wavelengths and solving a quaternary simultaneous equation using the measured absorbance.
Specifically, the absorbance of the medium is the sum of the absorbance of phenol red, the absorbance of FBS and the offset. That is, the absorbance of the medium is expressed as a ternary linear equation in which the concentration of phenol red, the concentration of FBS and the offset are unknown. Here, at pH 7.0 to 8.0, the relationship between the extinction coefficient of phenol red and pH becomes linear. In Patent Document 1, the linear relationship between the extinction coefficient of phenol red and pH is used to further transform the above-mentioned three-dimensional linear equation into a four-dimensional linear equation having an unknown pH.

Japanese Unexamined Patent Publication No. 2012-215428

However, in the case of the method of Patent Document 1, there is an inconvenience that the pH range in which accurate estimation is possible is limited to pH 7.0 to 8.0 in which the relationship between the extinction coefficient of phenol red and pH is linear. be.
In addition to the above method, Patent Document 1 describes a method of predicting a pH in a wider range by using the absorbance at predetermined four wavelengths. However, this method has the disadvantage that the measurement wavelength of the absorbance is limited.

The present invention has been made in view of the above circumstances, and is an acid dissociation equilibrium estimation method and an acid dissociation equilibrium estimation device capable of accurately estimating the pH of a medium over a wide range without limiting the measurement wavelength. , Acid dissociation equilibrium estimation system, and user interface device.

In order to achieve the above object, the present invention provides the following means.
One aspect of the present invention is an acid dissociation equilibrium estimation method for estimating the acid dissociation equilibrium state of a pH indicator contained in a liquid medium for cell culture, wherein the pH indicator depends on the hydrogen ion concentration in the medium. It changes between the acid type and the base type, and an acid dissociation equilibrium is established between the acid type pH indicator and the base type pH indicator, and the acid type pH indicator and the base type pH indicator are established. Have different optical spectra from each other and obtain an acid dissociation optical index of the medium at the measurement wavelength, and the acid dissociation optical index correlates with the intensity of the optical spectrum of the pH indicator. This is an acid dissociation equilibrium estimation method including a step of calculating the concentration of the acid-type pH indicator and the concentration of the base-type pH indicator in the medium based on the dissociation optical index.

The acid dissociation equilibrium state of the pH indicator in the medium changes depending on the hydrogen ion concentration (ie, pH) in the medium. That is, as the medium becomes more acidic, the concentration of the acid-type pH indicator increases and the concentration of the basic pH indicator decreases. Further, as the medium becomes more basic, the concentration of the acid-type pH indicator decreases and the concentration of the basic pH indicator increases. The optical spectra of acid-type pH indicators and the optical spectra of base-type pH indicators are different from each other. Therefore, the concentration of each of the acid-type pH indicator and the base-type pH indicator in the medium is calculated based on the acid dissociation optical index of the medium, and the acid dissociation of the medium is calculated based on the concentration of the acid-type and base-type pH indicators. The equilibrium state can be estimated.

In the above embodiment, in the step of acquiring the acid dissociation optical index of the medium, in the step of acquiring the acid dissociation optical index at a single measurement wavelength and calculating the concentration, based on the acid dissociation optical index, The acid-type pH indicator and the acid-type pH indicator and the acid-type pH indicator are calculated by calculating the concentration of one of the acid-type pH indicator and the base-type pH indicator and subtracting the one concentration from the total concentration of the pH indicator contained in the medium. The other concentration of the basic pH indicator may be calculated.
With this configuration, the acid dissociation equilibrium state of the medium can be estimated from only one acid dissociation optical index.

In the above embodiment, in the step of acquiring the acid dissociation optical index, the acid dissociation optical index at the first measurement wavelength and the acid dissociation optical index at the second measurement wavelength are acquired, and the first measurement wavelength and the second measurement wavelength are obtained. May be different from each other.
The acid dissociation optical index of the medium can be expressed by an equation using the concentration of the acid type pH indicator and the concentration of the base type pH indicator. Using two acid dissociation optical indices at two measurement wavelengths, a binary system of equations is obtained in which the concentration of the acid-type pH indicator and the concentration of the base-type pH indicator are unknown. By solving this binary simultaneous equation, the concentration of the acid type pH indicator and the concentration of the base type pH indicator can be calculated.

In the above embodiment, in the step of acquiring the acid dissociation optical index, the acid dissociation optical index at the third measurement wavelength is further acquired, and the third measurement wavelength is the first measurement wavelength and the second measurement wavelength. However, the step of calculating the concentration may further include the step of calculating the offset included in the acid dissociation optical index of the medium.
The acid dissociation optical index of the medium can be expressed by an equation using the concentration of the acid-type pH indicator, the concentration of the basic pH indicator, and the offset. Offset is a component of an acid dissociation optical index based on a substance other than a pH indicator. Using three acid dissociation optical indices at three measurement wavelengths, a ternary simultaneous equation is obtained in which the concentration of the acid-type pH indicator and the concentration and offset of the base-type pH indicator are unknown. By solving this ternary simultaneous equation, the offset can be calculated in addition to the concentration of the acid type pH indicator and the concentration of the base type pH indicator.

In the above embodiment, in the step of acquiring the acid dissociation optical index, the acid dissociation optical index at the fourth measurement wavelength is further acquired, and the fourth measurement wavelength is the first measurement wavelength, the second measurement wavelength, and the above. Unlike the third measurement wavelength, the step of calculating the concentration may further include the step of calculating the concentration of the colored substance contained in the medium.
The acid dissociation optical index of the medium can be expressed by an equation using the concentration of the acid type pH indicator, the concentration of the basic pH indicator, the offset and the concentration of the colored substance. The colored substance is, for example, serum. Using the four acid dissociation optical indices at the four measurement wavelengths, a quaternary simultaneous equation is obtained in which the concentration of the acid-type pH indicator, the concentration of the base-type pH indicator, the offset, and the concentration of the colored substance are unknown. By solving this quaternary simultaneous equation, the concentration of the colored substance can be calculated in addition to the concentration of the acid type pH indicator, the concentration of the basic pH indicator and the offset.

In the above embodiment, the acid dissociation optical index may be the absorbance of the medium.
With this configuration, the acid dissociation optical index can be measured by a simple method and configuration.

In the above aspect, the step of calculating the hydrogen ion index (pH) of the medium based on the following formula may be further included.
pH = pKa + log (C _bass / C _aci )
Here, pKa is the acid dissociation index of the pH indicator, C _{bass is the concentration of the basic pH indicator in the medium, and C aci is} the concentration of the acid type pH indicator in the medium.
With this configuration, the pH of the medium can be accurately estimated without being affected by the color of the colored substance contained in the medium.

In the above embodiment, the acid dissociation index is the acid dissociation index at a predetermined standard temperature, and in the step of acquiring the acid dissociation optical index, the acid dissociation optical index of the medium having the predetermined standard temperature is acquired. May be good.
The acid dissociation constant changes with temperature. Therefore, by using the acid dissociation index at the same standard temperature and the acid dissociation optical index of the medium, a more accurate estimate of the pH of the medium can be calculated from the equation (1).

Another aspect of the present invention is an acid dissociation equilibrium estimation device that estimates the acid dissociation equilibrium state of a pH indicator contained in a liquid medium for cell culture, wherein the pH indicator adjusts to the hydrogen ion concentration in the medium. It changes between the acid type and the base type accordingly, and an acid dissociation equilibrium is established between the acid type pH indicator and the base type pH indicator, and the acid type pH indicator and the base type pH are established. Indicators have different optical spectra from each other and are equipped with a processor, which obtains an acid dissociation optical index of the medium at a measurement wavelength, and the acid dissociation optical index correlates with the intensity of the optical spectrum of the pH indicator. , And the step of calculating the concentration of the acid-type pH indicator and the concentration of the basic pH indicator in the medium based on the obtained acid dissociation optical index. It is a device.

In the above aspect, the communication unit is provided with a communication unit capable of communicating with the monitoring device, the monitoring device acquires the acid dissociation optical index of the medium, and the communication unit receives the acid dissociation optical index of the medium from the monitoring device. You may.
With this configuration, the acid dissociation equilibrium estimator can obtain the acid dissociation optical index from the monitoring device at any location. For example, an acid dissociation equilibrium estimator can obtain an acid dissociation optical index from a monitoring device in the incubator outside the incubator.

In the above embodiment, the communication unit may receive the acid dissociation optical index from the monitoring device via at least one relay device.
With this configuration, the acid dissociation equilibrium estimation device can be realized as a server installed remotely from the monitoring device, for example, as a cloud server on the Internet.

Another aspect of the present invention includes the acid dissociation equilibrium estimation device according to any one of the above, a monitoring device for measuring an acid dissociation optical index of the medium, and a user interface device, and the user interface device is used for the monitoring. A signal instructing the measurement of the acid dissociation optical index is transmitted to the apparatus, and the user is notified of the hydrogen ion index of the medium calculated based on the concentration of the acid-type pH indicator and the concentration of the basic pH indicator. It is an acid dissociation equilibrium estimation system.

Another aspect of the present invention is communicable with the acid dissociation equilibrium estimation device according to any one of the above, and the concentration of the acid-type pH indicator and the concentration of the basic pH indicator are added to the acid dissociation equilibrium estimation device. A user interface including a communication unit for requesting transmission of the above and a notification unit for notifying the user of the hydrogen ion index of the medium calculated based on the concentration of the acid-type pH indicator and the concentration of the base-type pH indicator. It is a device.

According to the present invention, the measurement wavelength is not limited, and the pH of the medium can be accurately estimated over a wide range.

It is an external view of the acid dissociation equilibrium estimation apparatus which concerns on one Embodiment of this invention. It is an internal block diagram of the acid dissociation equilibrium estimation apparatus of FIG. It is a chemical structural formula of acid type and base type phenol red. It is a light absorption spectrum of phenol red from pH 4.0 to pH 11. It is a flowchart which shows the acid dissociation equilibrium estimation method which concerns on one Embodiment of this invention. It is a graph which shows the relationship between the estimated value of pH calculated by the acid dissociation equilibrium estimation method of FIG. 5 and the measured value of pH by a pH sensor. It is a light absorption spectrum in the vicinity of pH 6.5 of the sample containing only phenol red and the sample containing phenol red and red ink. It is a light absorption spectrum in the vicinity of pH 7.5 of the sample containing only phenol red and the sample containing phenol red and red ink. It is an overall block diagram of the acid dissociation equilibrium estimation system which concerns on other embodiment of this invention. It is an overall block diagram of the acid dissociation equilibrium estimation system which concerns on other embodiment of this invention. It is an overall block diagram of the acid dissociation equilibrium estimation system which concerns on other embodiment of this invention. It is a block diagram of the absorbance measuring device of the acid dissociation equilibrium estimator which concerns on other embodiment of this invention. It is an overall block diagram of the acid dissociation equilibrium estimation apparatus which concerns on other embodiment of this invention.

Hereinafter, the acid dissociation equilibrium estimation device 1 and the acid dissociation equilibrium estimation method according to the embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, the acid dissociation equilibrium estimation device 1 according to the present embodiment is a monitoring device for monitoring cells in the culture vessel 2 and the liquid medium M. The monitoring device 1 includes a sealed box-shaped housing 3. The top plate 3a of the housing 3 is a transparent and flat stage, and the culture vessel 2 is placed on the stage 3a. Medium M contains serum and a pH indicator. The monitoring device 1 is placed in the incubator together with the culture vessel 2 on the stage 3a to acquire an image of the cells and estimate the pH of the medium M based on the pH indicator.

As shown in FIG. 2, the monitoring device 1 includes an imaging device 4, an absorbance measuring device 5, a memory 6, and a processor 7.
The culture vessel 2 is, for example, a dish for cell culture. The culture vessel 2 is made of an optically transparent material. The image pickup device 4 uses an image pickup device (not shown) to image the cells in the culture vessel 2 through the stage 3a and the bottom plate 2a of the culture vessel 2, and generates an image of the cells. The image pickup device 4 may include a lighting element that irradiates cells with illumination light via the stage 3a and the bottom plate 2a.

The absorbance measuring device 5 includes a light source (not shown) that irradiates the medium M in the culture vessel 2 with the measuring light L, and a detector (not shown) that detects the measuring light L transmitted through the medium M. The measurement light L emitted from the light source passes through the stage 3a, the bottom plate 2a and the medium M upward, is reflected on the inner surface of the top plate 2b of the culture vessel 2, and moves the medium M, the bottom plate 2a and the stage 3a downward. It penetrates toward you. The detector detects the intensity of the measured light L transmitted through the medium M and the stage 3a. The absorbance measuring device 5 calculates the absorbance of the medium M based on the ratio of the intensity of the measured light L emitted by the light source to the intensity of the measured light L detected by the detector.

Here, the relationship between the absorbance of the medium M (acid dissociation optical index), the pH of the medium M, and the pH indicator will be described.
3 and 4 show the molecular structure and light absorption spectrum (optical spectrum) of phenol red (PR), which is a typical pH indicator, respectively.

As shown in FIG. 3, the pH indicator has either an acid type or a base type molecular structure and changes between the acid type and the base depending on the hydrogen ion concentration in the medium M. The acid-type pH indicator and the base-type pH indicator are conjugated acid-base pairs, and an acid dissociation equilibrium is established between the acid-type pH indicator and the base-type pH indicator. The pH of the medium M is represented by the formula (a).
pH = pKa + log (C _bass / C _aci ) ... (a)
C _aci is the concentration of acid-type pH indicator in medium M. C- _bas is the concentration of the basic pH indicator in medium M. pKa is the acid dissociation constant of the pH indicator.

On the other hand, as shown in FIG. 4, the light absorption spectra of the acid-type pH indicator and the light absorption spectra of the base-type pH indicator are different from each other. Therefore, the concentrations C _aci and C _aci in the medium can be calculated from the absorbance of the medium M. Further, the estimated value of the pH of the medium M can be calculated from the formula (a) using the calculated concentrations C _aci and C _aci .

The absorbance measuring device 5 can change the measurement wavelength of the absorbance. The method of changing the measurement wavelength includes a first method of changing the wavelength of the measurement light L irradiated to the medium M and a second method of changing the wavelength of the light detected by the detector.

In the case of the first method, for example, the absorbance measuring device 5 includes a plurality of light sources that output measurement light having different wavelengths from each other. The absorbance measuring device 5 includes a light source that outputs light in a wide band and a wavelength selection element that selects a specific wavelength from the light output from the light source, and the wavelength selected by the wavelength selection element can be changed. You may. Alternatively, the absorptiometry measuring device 5 includes a light source that outputs light in a wide band and a spectroscope such as a diffraction grid, and the light output from the light source is separated by the spectroscope, and only the light of a specific wavelength is separated from the spectroscope. The medium M may be irradiated. Alternatively, the absorbance measuring device 5 may include a wavelength variable device that modulates the wavelength of the light emitted from the light source to the medium M, and may irradiate the medium M with light modulated to a predetermined wavelength from the wavelength variable device.

In the case of the second method, for example, the absorbance measuring device 5 is provided with a spectroscope in front of the detector, the light transmitted through the medium M is separated by the spectroscope, and only the light of a specific wavelength is detected by the spectroscope. Inject into.

The memory 6 stores an imaging program, an absorbance measurement program, and a calculation program.
The processor 7 controls the image pickup device 4 according to the image pickup program, and causes the image pickup device 4 to perform cell imaging and image generation.
The processor 7 controls the absorbance measuring device 5 according to the absorbance measuring program, and causes the absorbance measuring device 5 to perform the measurement of the absorbance of the medium M. Further, the processor 7 receives the absorbance data from the absorbance measuring device 5 and performs a calculation for calculating the pH of the concentrations C _aci , C _bass and the medium M from the absorbance according to the calculation program.

Next, the acid dissociation equilibrium estimation method according to the present embodiment will be described by taking as an example the case where the pH indicator is phenol red (PR) and the serum is FBS.
The acid dissociation equilibrium estimation method can also be applied to a medium containing a pH indicator other than phenol red. The acid dissociation equilibrium estimation method can also be applied to a medium containing serum other than FBS and a medium containing an absorbent substance other than serum.

As shown in FIG. 5, the acid dissociation equilibrium estimation method is based on the measurement step S1 for measuring the absorptivity of the medium M and the absorptivity of the medium M, and the concentration of acid-type phenol red (acid-type PR) and the base type. It includes a concentration calculation step S2 for calculating the concentration of phenol red (basic PR), and a pH calculation step S3 for calculating the pH of the medium M based on the concentrations of the acid PR and the basic PR.

The measurement step S1 includes a step S11 for measuring the absorbance A _λ1 of the medium M at the first measurement wavelength λ1, a step S12 for measuring the absorbance A _λ2 of the medium M at the second measurement wavelength λ2, and a medium at the third measurement wavelength λ3. The step S13 for measuring the absorbance A _λ3 of M and the step S14 for measuring the absorbance A _λ4 of the medium M at the fourth measurement wavelength λ4 are included. The first measurement wavelength λ1, the second measurement wavelength λ2, the third measurement wavelength λ3, and the fourth measurement wavelength λ4 are different from each other.

The measurement step S1 is realized by the processor 7 controlling the absorbance measuring device 5 according to the absorbance measuring program. Specifically, in step S11, the absorbance measuring device 5 irradiates the medium M in the culture vessel 2 with the measurement light L having the first measurement wavelength λ1, detects the measurement light L transmitted through the medium M, and detects the absorbance A. Calculate _λ1 . In steps S12, S13, and S14, the absorbance measuring device 5 calculates the absorbances A _λ2 , A _λ3 , and A _λ4 in order using the measurement light L having the measurement wavelengths λ2, λ3, and λ4 in the same manner as in step S11.
As described above, instead of changing the wavelength of the measurement light L, by changing the wavelength detected by the detector, the absorbances A _λ1 and A _λ2 at the four measurement wavelengths λ1, λ2, λ3, λ4, A _λ3 and A _λ4 may be measured.

The concentration calculation step S2 and the pH calculation step S3 are realized by the processor 7 calculating according to the calculation program.
In the concentration calculation step S2, the processor 7 has four unknowns C _{PR_aci} , C _{PR_bas} , C _FBS , and A _off by substituting the absorbances A _λ1 , A _λ 2, A λ ₃ , and A _λ 4 into the following equation (1). Obtain simultaneous quaternary linear equations. Next, the processor 7 solves simultaneous quaternary linear equations and calculates the values of the unknowns C _{PR_aci} , C _{PR_bas} , C _FBS , and A _off .

In the formula (1), C _{PR_aci} is the concentration of acid-type PR in the medium M. C _{PR_bas} is the concentration of basic PR in medium M. C _FBS is the concentration of FBS in medium M. A _off is an offset.
α _{FBS_λi} (i = 1, 2, 3, 4) is the absorption coefficient of FBS at the measurement wavelength λi. α _{PR_aci} is the extinction coefficient of acid PR. α _{PR_bas} is the extinction coefficient of basic PR. S _λi (i = 1, 2, 3, 4) is an offset coefficient at the measurement wavelength λi. D is the optical path length of the measurement light L transmitted through the medium M. The extinction coefficient α _{FBS_λi} , α _{PR_aci} , α _{PR_bas} , S _λi and the optical path length D are known values.

Equation (1) will be described.
The absorbance of the medium M is the sum of the absorbance of the acid-type PR, the absorbance of the base-type PR, the absorbance of the FBS, and the offset. In the optical path of the measurement light L, in addition to PR and FBS in the medium M, there is an absorbent substance that absorbs the measurement light L. The absorbent substance is, for example, a component of the medium M other than the bottom plate 2a, the stage 3a, PR and FBS of the culture vessel 2. Offset is the absorbance based on an absorbent substance other than PR and FBS.

On the other hand, the absorbance A is represented by the formula (b) using the extinction coefficient α, the concentration C of the sample, and the optical path length D in the sample of the measured light.
A = αDC ... (b)
Using the formula (b), the absorbances A _λ1 , A _λ2 , A _λ3 , and A _λ4 of the medium M at the measurement wavelength λi (i = 1, 2, 3, 4) are represented by the following formula (c). Equation (1) is obtained by modifying equation (c).

Next, in the pH calculation step S3, the processor 7 calculates the pH of the medium M by substituting the concentrations C _{PR_aci} and C _{PR_bas} into the following formula (2).
pH = pKa + log (C _{PR_bas} / C _{PR_aci} )… (2)
pKa is the acid dissociation constant of PR. The pKa varies with temperature and the PR pKa at 37 ° C. is about 7.7. The value of pKa at a given standard temperature is used in the calculation of pH.

Generally, the pH of medium M is estimated based on the apparent color of medium M. However, when a colored substance such as FBS is contained in the medium M, the color of the medium M is a mixed color of the color of PR and the color of the colored substance. Therefore, it is difficult to know the exact pH of the medium M based on the apparent color of the medium M.

On the other hand, the pH of the medium M is determined by the ratio of the concentration of the acid-type pH indicator to the concentration of the base-type pH indicator. According to this embodiment, by solving the simultaneous quaternary linear equation of the equation (1) using four absorbances A _λ1 , A _λ2 , A _λ3 , and A _λ4 , an absorbent substance other than PR is contained in the medium M. Even in the present situation, the accurate concentrations C _{PR_aci} and C _{PR_bas} of the acid-type PR and the base-type PR in the medium M can be calculated, and the acid dissociation equilibrium state of the PR in the medium M can be estimated. can. Further, by using the concentrations C _{PR_aci} and C _{PR_bas} , there is an advantage that the pH of the medium M can be accurately estimated without being affected by the color of a colored substance such as FBS.

Further, there is an advantage that the pH of the medium M can be accurately calculated over a wide range, not limited to the pH range in which the relationship between the pH of the medium M and the absorbance of the medium M becomes linear.
Further, the measurement wavelengths λ1, λ2, λ3, λ4 used for calculating the concentrations C _{PR_aci} and C _{PR_bas} may be any wavelength as long as the PR exhibits absorption. That is, there is an advantage that any measurement wavelength λ1, λ2, λ3, λ4 can be selected from a wide wavelength range.

FIG. 6 shows the result of an evaluation experiment of pH estimation accuracy by the acid dissociation equilibrium estimation method of the present embodiment. In the graph of FIG. 6, the horizontal axis (X-axis) is the measured value of pH measured by the pH sensor, and the vertical axis (Y-axis) is the estimation of pH calculated by the acid dissociation equilibrium estimation method of the present embodiment. It is a value, and the broken line shows the graph of Y = X.

In the evaluation experiment, three types of samples A, B, and C were prepared. Sample A is a solution containing only PR. Sample B is a solution containing PR and low concentration red ink. Sample C is a solution containing PR and a high concentration of red ink. For Sample B and Sample C, three solutions with different pH were prepared.
FIG. 7A is a light absorption spectrum of a solution containing only PR and a solution containing PR and red ink in the vicinity of pH 6.5. FIG. 7B is a light absorption spectrum of a solution containing only PR and a solution containing PR and red ink in the vicinity of pH 7.5.
As can be seen from FIG. 6, estimated values close to the measured values were obtained for all of the samples A, B, and C.

As mentioned above, pKa varies depending on the temperature of the medium M. Therefore, in the measurement step S1, the temperature of the medium M may be adjusted to a predetermined standard temperature of pKa, and the absorbances A _λ1 , A _λ2 , A _λ3 , and A _λ4 of the medium M at the predetermined standard temperature may be measured. In this case, a temperature control device for controlling the temperature of the medium M to a predetermined standard temperature is further provided.
In this way, by matching the temperature of the medium M with the standard temperature of pKa, a more accurate estimate of pH can be calculated from the equation (1).
When the temperature of the medium M in the measurement step S1 is different from the standard temperature, the pKa at the standard temperature may be corrected based on the temperature of the medium M, and the corrected pKa may be used for the calculation of the pH.

When the pH of the same medium M is estimated multiple times during cell culture, the absorbance of the medium M may be measured at only two wavelengths in the second and subsequent pH estimations.
At the time of the first pH estimation, the FBS concentration C _FBS and the offset A _off are calculated. When the medium M and the measurement position of the absorbance are the same, the FBS concentration C _FBS and the offset A _off do not change. Therefore, from the second time onward, the absorbances at the two measurement wavelengths may be measured, and only the concentrations C _{PR_aci} and C _{PR_bas} may be calculated from the equation (1).

In the present embodiment, the absorbance of the medium M at four measurement wavelengths is measured, and four unknowns are calculated from the quaternary simultaneous equations. However, the number of measurement wavelengths and the number of unknowns to be calculated are the numbers of the medium M. It can be changed as appropriate according to the ingredients and purpose.
For example, when the colored substance contained in the medium M is only PR, the absorbance of the medium M at three measurement wavelengths is measured, and the three unknowns C _{PR_aci} , C _{PR_bas} , and A _off are calculated from the ternary simultaneous equations. You may.

On the other hand, when other colored substances are contained in the medium M in addition to PR and FBS, the number of measurement wavelengths and the number of unknowns in the simultaneous equations can be increased to calculate the concentration of other colored substances. good. For example, when there is only one type of other colored substance, the acid type is obtained by measuring the absorbance of the medium M at five measurement wavelengths and solving a quintuple simultaneous equation in which the concentration of the other colored substance is further added as an unknown number. The concentrations and offsets of PR, basic PR, FBS and other colored substances can be calculated.

The values required for estimating the pH of the medium M are the concentration C _aci of the acid type pH indicator and the concentration C _bass of the base type pH indicator. Therefore, the absorbance of the medium M at the two measurement wavelengths may be measured, and only the pH indicator concentrations C _aci and C _bass may be calculated from the binary simultaneous equations. This method is particularly effective when the concentration C _FBS and offset A _off of FBS in the medium M are known.

If the total concentration of the pH indicator contained in the medium M is known, the absorbance may be measured at only one wavelength.
In this case, the absorbance of the medium M is expressed by a linear equation in which only one of the concentrations C _aci and C _bass is unknown. Therefore, one of the concentrations C _aci and C _bas should be calculated based on the absorbance at one measurement wavelength, and the other of the concentrations C _aci and C _bas should be calculated as the difference between the total concentration of the pH indicator and the concentration of one. Can be done.

In the present embodiment, the acid dissociation optical index is the absorbance of the pH indicator, but instead, the acid dissociation optical index may be another type of index that satisfies the following two conditions. good.
Condition 1: An amount that can be measured optically Condition 2: An amount that changes depending on the pH.

A preferred index satisfying conditions 1 and 2 is, for example, the fluorescence intensity of a pH-sensitive fluorescent dye (pH indicator). Similar to phenol red, pH sensitive fluorescent dyes vary between acid and base types depending on the hydrogen ion concentration. The fluorescence spectrum (optical spectrum) of the acid-type fluorescent dye and the fluorescence spectrum (optical spectrum) of the basic fluorescent dye are different from each other. Therefore, the concentration of the acid-type fluorescent dye and the concentration of the basic fluorescent dye are calculated from the fluorescence intensity of the fluorescent dye at a plurality of measurement wavelengths, and the medium M is calculated from the concentration of the acid-type fluorescent dye and the concentration of the basic fluorescent dye. The pH of can be estimated.

In the present embodiment, the acid dissociation equilibrium estimation method is realized by the monitoring device 1, but the device configuration for realizing the acid dissociation equilibrium estimation method is not limited to this. 8 to 12 show other embodiments of the apparatus and system for realizing the acid dissociation equilibrium estimation method.

The acid dissociation equilibrium estimation system 100 shown in FIG. 8 includes a monitoring device 10 and an image processing device 20 installed outside the incubator. The monitoring device 10 and the image processing device 20 are provided with communication devices 8 and 20a (see FIG. 10), respectively, and can communicate with each other via a wireless or wired communication network.
The monitoring device 10 measures the absorbance of the medium M by the absorbance measuring device 5.

The image processing device 20 includes a memory (not shown) and a processor 20b (see FIG. 10). The communication device (communication unit) 20a of the image processing device 20 receives cell image data and absorbance data from the monitoring device 10 by communicating with the communication device 8 of the monitoring device 10.
An image processing program and a calculation program are stored in the memory. The processor 20b performs image processing on the image according to the image processing program. Further, the processor 20b calculates the pH of the concentrations C _aci , C _bas and the medium M according to the calculation program using the absorbance data. That is, the acid dissociation equilibrium estimation device is realized as the image processing device 20.

The acid dissociation equilibrium estimation system 101 shown in FIG. 9 includes a monitoring device 11, an image processing device 21 installed outside the incubator, a server 30, and a user interface (UI) device 40.
The image processing device 21 can communicate with the monitoring device 11, the server 30, and the UI device 40 via a wireless or wired communication network.

The image processing device (relay device) 21 receives the absorbance data from the monitoring device 11 and transmits the absorbance data to the server 30.
The server 30 is, for example, a cloud server on the Internet or a computer installed at an arbitrary location. Calculations of the concentrations C _aci , C _bas and the pH of medium M are performed on the server 30. That is, the acid dissociation equilibrium estimation device is realized as the server 30. The server 30 may be able to communicate with a plurality of image processing devices 21.

The UI device 40 is, for example, a general-purpose tablet computer. The UI device 40 includes a communication device 40a capable of communicating with the monitoring device 11 and the server 30, and a display device (notification unit) 40b. Further, the UI device 40 is installed with dedicated application software for communicating with the monitoring device 11 and the server 30 and displaying the information acquired from the server 30.

The user can send various instructions to the monitoring device 11 and the server 30 by using the dedicated application software. Specifically, the user can send an instruction for measuring the absorbance from the UI device 40 to the monitoring device 11 and remotely control the absorbance measuring device 5. Further, the user requests the server 30 to transmit at least one of the pH of the concentrations C _aci , C _bass and the medium M from the UI device 40, and data on at least one of the concentrations C _aci , C _bass and the pH of the medium M. It can be obtained from the server 30.
Further, the user can display the pH value of the medium M on the display device 40b by using the dedicated application software.

The acid dissociation equilibrium estimation system 102 shown in FIG. 10 includes a medium exchange device 50 mounted on the monitoring devices 1, 10 and 11.
The medium exchange device 50 discharges the used medium M from the inside of the culture container 2 and supplies the unused medium M into the culture container 2. The medium exchange device 50 adjusts the discharge amount of the used medium M and the supply amount of the unused medium M to adjust the height of the medium M in the culture container 2 (the liquid of the medium M from the bottom surface of the culture container 2). The vertical distance to the surface) can be adjusted.
The optical path length D of the measured light L in the formula (1) is determined by the height of the medium M in the culture vessel 2. The optical path length D can be controlled by combining the monitoring devices 1, 10 and 11 with the medium exchange device 50.

As shown in FIG. 11, the absorbance measuring device 5 may be mounted on the culture medium exchange device 50 instead of the monitoring devices 1, 10 and 11. Specifically, the medium exchange device 50 is provided with a light source 5a that emits the measurement light L toward the medium M in the culture medium 2 and a detector 5b that detects the measurement light L that has passed through the medium M. The detector 5b detects, for example, the measurement light L reflected on the bottom surface of the culture vessel 2. The change of the measurement wavelength is performed by wavelength modulation of the light source 5a or wavelength selection of the detector 5b.

The acid dissociation equilibrium estimation device 12 shown in FIG. 12 is an optical microscope device including a microscope main body 13 and a processor 14 connected to the microscope main body 13.
The microscope main body 13 includes a white light source 13a, a wavelength selection element 13b, and a camera 13c. The white light emitted from the white light source 13a passes through the wavelength selection element 13b and irradiates the medium M and cells in the culture vessel 2 as measurement light. The wavelength selection element 13b can change the wavelength of the transmitted light. That is, the wavelength of the measurement light applied to the medium M can be changed by the wavelength selection element 13b.

The camera 13c images the medium M and cells in the culture vessel 2 when the measurement light is irradiated, and transmits the images of the medium M and cells to the processor 14.
The processor 14 calculates the absorbance of the medium M at the measurement wavelength based on the intensity of the measurement light applied to the medium M and the brightness value of the image. Further, the processor 14 calculates the concentration of each of the acid type pH indicator and the base type pH indicator, and calculates the pH of the medium.

In another embodiment of the acid dissociation equilibrium estimator, the light source and detector of the absorbance measuring device 5 may be arranged separately. For example, the light source may be placed next to the culture vessel 2 in the incubator or on a shelf in the incubator. To prevent contamination, the light source may be housed in another culture vessel.
In another embodiment of the acid dissociation equilibrium estimator, the light source and detector of the absorbance measuring device 5 may be provided in another device around the culture vessel 2. For example, both the light source and the detector may be located on shelves in the incubator.

1 Monitoring device, acid dissociation equilibrium estimation device 2 Culture container 3 Housing 3a Stage 4 Imaging device 5 Absorbance measurement device 6 Memory 7, 14, 20b Processor 8, 40a Communication device (communication unit)
10, 11 Monitoring device 12 Microscope device, acid dissociation equilibrium estimation device 20 Image processing device, acid dissociation equilibrium estimation device 21 Image processing device (relay device)
30 Server, acid dissociation equilibrium estimation device 40 User interface device 40b Display device (notification unit)
50 Medium exchange device 100, 101, 102 Acid dissociation equilibrium estimation system

Claims (15)

An acid dissociation equilibrium estimation method for estimating the acid dissociation equilibrium state of a pH indicator contained in a liquid medium for cell culture containing at least one colored component , wherein the pH indicator adjusts to the hydrogen ion concentration in the medium. Correspondingly, it changes between the acid type and the base type , an acid dissociation equilibrium is established between the acid type and the base type of the pH indicator, and the acid type and the base type of the pH indicator become the hydrogen ion concentration. Depending on the different optical spectral characteristics ,
The step of acquiring the acid dissociation optical index of the medium at the measurement wavelength, and
Acid dissociation including the step of calculating the concentration of one of the acid type and the base type of the pH indicator in the medium based on the obtained acid dissociation optical index and the optical spectral characteristics of the pH indicator. Equilibrium estimation method. In the step of calculating the concentration,
Based on the acid dissociation optical index, the concentration of one of the acid type and the base type of the pH indicator was calculated.
The acid dissociation equilibrium estimation method according to claim 1, wherein the concentration of the other of the acid type and the base type of the pH indicator is calculated by subtracting the concentration of one of the known concentrations of the pH indicator contained in the medium. .. In the step of acquiring the acid dissociation optical index, the acid dissociation optical index at the first measurement wavelength and the acid dissociation optical index at the second measurement wavelength are acquired, and the first measurement wavelength and the second measurement wavelength are different from each other. Item 1. The acid dissociation equilibrium estimation method according to Item 1. In the step of acquiring the acid dissociation optical index, the acid dissociation optical index at the third measurement wavelength is further acquired, and the third measurement wavelength is different from the first measurement wavelength and the second measurement wavelength.
The acid dissociation equilibrium estimation method according to claim 3, further comprising a step of calculating the concentration of the other of the acid type and the base type of the pH indicator in the step of calculating the concentration . In the step of acquiring the acid dissociation optical index, the acid dissociation optical index at the third measurement wavelength is further acquired, and the third measurement wavelength is different from the first measurement wavelength and the second measurement wavelength.
In the step of calculating the concentration,
A claim comprising the step of calculating the ratio of the acid type concentration of the pH indicator to the base type concentration of the pH indicator in the medium based on the acid dissociation optical index and the optical spectral characteristics of the pH indicator. Item 3. The acid dissociation equilibrium estimation method according to Item 3. The acid dissociation equilibrium estimation method according to claim 4 or 5, further comprising calculating the concentration of the colored component in the step of calculating the concentration. In the step of acquiring the acid dissociation optical index, the acid dissociation optical index at the fourth measurement wavelength is further acquired, and the fourth measurement wavelength is the first measurement wavelength, the second measurement wavelength, and the third measurement wavelength. Is different
The acid dissociation equilibrium estimation method according to claim 6 , wherein the step of calculating the concentration further includes a step of calculating the offset of the acid dissociation optical index of the medium. The acid dissociation equilibrium estimation method according to any one of claims 1 to 7 , wherein the acid dissociation optical index is the absorbance of the medium. The acid dissociation equilibrium estimation method according to any one of claims 1 to 8 , further comprising a step of calculating the hydrogen ion index of the medium based on the following formula.
Hydrogen ion index = pKa + log (C _bass / C _aci )
here,
pKa is the acid dissociation constant of the pH indicator,
C _bass is the concentration of the basic pH indicator in the medium,
C _aci is the concentration of the acid-type pH indicator in the medium. The acid dissociation index is an acid dissociation index at a predetermined standard temperature.
The acid dissociation equilibrium estimation method according to claim 9 , wherein in the step of acquiring the acid dissociation optical index, the acid dissociation optical index of the medium having the predetermined standard temperature is acquired. An acid dissociation equilibrium estimation device that estimates the acid dissociation equilibrium state of a pH indicator contained in a liquid medium for cell culture containing at least one colored component , wherein the pH indicator adjusts to the hydrogen ion concentration in the medium. Correspondingly, it changes between the acid type and the base type , an acid dissociation equilibrium is established between the acid type and the base type of the pH indicator, and the acid type and the base type of the pH indicator become the hydrogen ion concentration. Depending on the different optical spectral characteristics ,
Equipped with a processor
The processor
The step of acquiring the acid dissociation optical index of the medium at the measurement wavelength, and
A step of calculating the concentration of one of the acid type and the base type of the pH indicator in the medium based on the obtained acid dissociation optical index and the optical spectral characteristics of the pH indicator. Acid dissociation equilibrium estimator to perform. It is equipped with a communication unit capable of communicating with the monitoring device, and the monitoring device measures the acid dissociation optical index of the medium.

The acid dissociation equilibrium estimation device according to claim 11 , wherein the communication unit receives the acid dissociation optical index of the medium from the monitoring device. The acid dissociation equilibrium estimation device according to claim 12 , wherein the communication unit receives the acid dissociation optical index from the monitoring device via at least one relay device. The acid dissociation equilibrium estimator according to claim 12 or 13 .
A monitoring device that measures the acid dissociation optical index of the medium, and
Equipped with a user interface device
The user interface device transmits a signal instructing the monitoring device to measure the acid dissociation optical index, and is calculated based on the acid type concentration and the base type concentration of the pH indicator. An acid dissociation equilibrium estimation system that notifies the user of the hydrogen ion index of the medium. It is possible to communicate with the acid dissociation equilibrium estimator according to any one of claims 11 to 13 .
A communication unit that requests the acid dissociation equilibrium estimator to transmit the concentration of the acid-type pH indicator and the concentration of the base-type pH indicator.
A user interface device including a notification unit for notifying the user of the hydrogen ion index of the medium calculated based on the acid type concentration and the base type concentration of the pH indicator .

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