Production method and culture apparatus for culturing cells to produce substances

Abstract

To provide a method for measuring the number of living cells and a device for measuring the number of living cells which have a small risk of contamination, and can measure the number of living cells with high accuracy by means non-invasive to cells.SOLUTION: A method for measuring the number of living cells includes the steps of: measuring the amount of suspensoid included in cell suspension; measuring capacitance of the cell suspension; and calculating one or more of the number of living cells, the number of dead cells, and the survival rate of cells contained in the cell suspension on the basis of the previously obtained capacitance per unit living cell, the previously obtained capacitance per unit dead cell, the amount of suspensoid, and the capacitance of cell suspension. A device 1 for measuring the number of living cells comprises: a turbidity sensor 2; a measuring device 3 which measures the capacitance of cell suspension; a storage unit which stores date on the capacitance per unit living cell and capacitance per unit dead cell; and a calculation unit which calculates one or more of the number of living cells, the number of dead cells, and the survival rate of cells.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to Production method and culture apparatus for culturing cells to produce substances .

Background Art

[0002] Conventionally, methods for culturing cells to produce useful substances have been used in fields such as the brewing industry, food industry, chemical industry, and pharmaceutical industry. For example, as biopharmaceuticals such as antibody drugs, many products mainly composed of substances produced by cells have been developed. Such biopharmaceuticals are produced by culturing cells in a culture medium and separating and purifying the target substance secreted into the culture medium.

[0003] In the production of useful substances using cells, culture control for improving production efficiency and appropriate quality control regarding the product are required. During culturing, it is desired to keep the cell count high while avoiding an increase in impurities due to cell death or the like, so it is important to monitor the cell count and the state of cell life and death in real time.

[0004] Conventionally, as culture methods suitable for the production of useful substances, a fed-batch culture method, a continuous culture method, etc. are known. In these culture methods, culturing is performed while maintaining the concentration of nutrients and the culture environment. Since it is necessary to control the addition amount of glutamine or the like according to the specific growth rate of cells, monitoring of the viable cell count is particularly important.

[0005] Generally, as methods for measuring the cell count, there are an offline method and an in-line method. The offline cell count measurement method targets cells sampled from the culture system. On the other hand, the in-line cell count measurement method targets cells being cultured in the culture system and is suitable for real-time monitoring and reduction of contamination.

[0006] As offline cell counting methods, there are methods such as observing a culture solution intermittently sampled from a culture system under a microscope and counting the cells visually observed within the microscope field of view using a hemocytometer, or automatically counting the cells in a microscope image by image processing. With these methods, it is possible to determine the life and death of cells by staining with trypan blue, and viable cells and dead cells can be counted separately.

[0007] As in-line cell counting methods, there are methods such as estimating the cell count from the oxygen consumption in the culture solution, or estimating the cell count from the capacitance, impedance, or dielectric constant of the culture solution. With the method based on oxygen consumption, viable cells can be quantified on the premise that the decrease in dissolved oxygen concentration is due to the oxygen consumption of viable cells. With the method based on capacitance, etc., the response to an electric field differs between viable cells and dead cells, and viable cells are quantified based on the premise that only viable cells cause polarization inside and outside the cells.

[0008] Patent Document 1 describes a cell inspection device that accurately estimates the number of viable cells based on the impedance of a culture solution. This cell inspection device includes an impedance sensor that measures the impedance of a culture solution, a storage unit that classifies a predetermined period within the culture period from the start to the death of cells into a plurality of periods, and stores a coefficient for estimating the number of viable cells in the culture solution during the predetermined period using the impedance for each of the classified plurality of periods, and a viable cell number estimation unit that acquires the impedance and estimates the number of viable cells using at least one of the coefficients for each period stored in the storage unit for the impedance.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] There is a need for a technique to accurately quantify the number of viable cells contained in a cell suspension, targeting a culture solution or the like when producing a useful substance. However, conventional offline methods that require sampling and conventional inline methods based on capacitance or the like have the following problems.

[0011] In the offline method, the viability of cells can be determined by staining with trypan blue, but it is necessary to sample the culture solution outside the culture tank. In such a method, there is a risk that the inside of the culture tank will be contaminated with miscellaneous bacteria when the culture tank is temporarily opened. Staining with trypan blue is invasive to viable cells, so it is difficult to use in the inline method.

[0012] Also, in the method of sampling, in order to prevent the invasion of miscellaneous bacteria into the culture tank after sampling, the sampling pipe must be sterilized with high-temperature and high-pressure steam or the like. During steam sterilization, time is required to heat the inside of the pipe, sterilization time, and time to cool the inside of the pipe to room temperature. Since the sterilization time usually requires 20 minutes or more, the sampling interval becomes about 1 hour or more, and real-time monitoring cannot be properly performed.

[0013] In the inline method, since the culture solution is not sampled, problems such as a long sterilization time do not occur, but there are several problems depending on the measurement method.

[0014] In the method based on oxygen consumption, when measuring the dissolved oxygen concentration, it is necessary to stop the control of the oxygen amount, so there is a problem that the culture solution temporarily becomes a low oxygen concentration state, and the production efficiency and quality of the useful substance deteriorate. Also, in the conventional method based on capacitance or the like, viable cells and dead cells with cell membranes remaining cannot be distinguished, and there is a problem that the number of viable cells cannot be accurately measured because dead cells with cell membranes remaining may also be quantified.

[0015] Therefore, the present invention aims to provide a method for accurately measuring the number of live cells with a low risk of contamination and a non-invasive means for cells. Production method and culture apparatus to achieve this objective.

Means for Solving the Problems

[0016] To solve the above problems, the present invention Production method includes A production method for culturing cells to produce substances, comprising a step of culturing cells in a cell suspension that transmits light, and the a step of measuring the amount of suspended matter including live and dead cells contained in a cell suspension, a step of measuring the capacitance of the cell suspension that changes depending on the state of life or death of the cells, and based on the capacitance per unit of live cells, the capacitance per unit of dead cells, the amount of the suspended matter, and the capacitance of the cell suspension, The number of viable cells, the number of dead cells, the cell viability rate, the capacitance of all live cells and the capacitance of all dead cells at least one of are calculated.

[0017] Also, the present invention Culture apparatus includes A culture apparatus for culturing cells to produce substances, comprising a culture tank for culturing cells, and provided in the culture tank, a turbidity sensor for measuring the amount of suspended matter including live and dead cells contained in a cell suspension that transmits light, Provided in the culture tank, a measuring instrument for measuring the capacitance of the cell suspension that changes depending on the state of life or death of the cells, and a calculation unit that calculates The number of viable cells, the number of dead cells, the cell viability rate, the capacitance of all live cells and the capacitance of all dead cells at least one of based on the capacitance per unit of live cells, the capacitance per unit of dead cells, the amount of the suspended matter, and the capacitance of the cell suspension.

Advantages of the Invention

[0018] According to the present invention, it is possible to provide a method for accurately measuring the number of live cells with a low risk of contamination and a non-invasive means for cells. Production method and culture apparatus to achieve this.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2A

Figure 2B

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Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Mode for Carrying Out the Invention

[0020] Hereinafter, according to an embodiment of the present invention A production method for culturing cells to produce substances, a culture apparatus for culturing cells to produce substances, and those used for theseA method for measuring the number of viable cells and a viable cell number measuring apparatus will be described with reference to the drawings. In the following drawings, the same reference numerals are given to the common configurations, and duplicate explanations are omitted.

[0021] The method for measuring the number of viable cells according to the present embodiment relates to a measurement method for quantifying the number of viable cells contained in a cell suspension. In this method for measuring the number of viable cells, a cell suspension such as a culture solution is used as a measurement target, and one or more of the number of viable cells, the number of dead cells, and the cell viability contained in the cell suspension are obtained by measurement and calculation.

[0022] In the method for measuring the number of viable cells according to the present embodiment, the amount of suspended matter contained in the cell suspension is measured, and the capacitance of the cell suspension generated by the polarization of the cell membrane is measured. By combining these measurements, the measurement result of the capacitance can be converted into the amount of suspended matter that has a correlation with the cell number. Therefore, the measurement result of the cell number or cell concentration can be obtained from the measurement result of the capacitance.

[0023] In addition, in the method for measuring the number of viable cells according to the present embodiment, the contributions of viable cells and dead cells to the capacitance of the cell suspension are considered. Specifically, the capacitance per unit viable cell obtained in advance and the capacitance per unit dead cell obtained in advance are incorporated into a simultaneous model equation of the total number of cells contained in the cell suspension and the capacitance of the cell suspension, and the number of viable cells, the number of dead cells, and the cell viability contained in the cell suspension are calculated.

[0024] Generally, when an electric field is applied to a cell suspension, cells having cell membranes behave as dielectrics, so polarization occurs across the cell membrane between the inside and outside of the cell. Since individual cells having cell membranes generate capacitance due to polarization, the amount of cells having cell membranes can be obtained from the measured capacitance of the cell suspension based on the correlation between the capacitance and the cell number.

[0025] In conventional general cell counting methods, there are those that estimate the number of cells from capacitance, impedance, or dielectric constant. Such conventional general methods enable the quantification of viable cell numbers, but are based on the premise that only viable cells cause polarization. In an actual cell suspension, cell death leaving the cell membrane may also occur, so there may be cases where dead cells that have caused polarization are included. In conventional general methods, since such dead cells are also counted as viable cells, there is a problem that the viable cell number cannot be measured with high accuracy.

[0026] On the other hand, if the capacitance per viable cell and the capacitance per dead cell are obtained in advance and these data are incorporated into a simultaneous model equation of the total number of cells contained in the cell suspension and the capacitance of the cell suspension for calculation, the capacitance or viable cell number of all viable cells synthesized for all viable cells and the capacitance or dead cell number of all dead cells synthesized for all dead cells can be obtained. Therefore, even if the viability of the cells is unknown, the viable cell number contained in the cell suspension can be obtained with high accuracy.

[0027] In the viable cell number measurement method according to the present embodiment, the type of cells to be measured for the viable cell number is not particularly limited as long as the total cell number can be measured as the amount of suspended matter. The cells to be measured may be any of animal cells, insect cells, plant cells, microalgae, cyanobacteria, bacteria, yeast, fungi, algae, etc. As the cells to be measured, cells that produce various useful substances such as various antibodies such as human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, various physiologically active substances, pharmaceutical raw materials, chemical raw materials, and food raw materials are preferable.

[0028] Hereinafter, a viable cell number measurement apparatus according to an embodiment of the present invention will be described with reference to the drawings together with the details of the viable cell number measurement method.

[0029] FIG. 1 is a diagram showing the configuration of a viable cell number measurement apparatus according to an embodiment of the present invention. As shown in FIG. 1, the viable cell count measuring apparatus 1 according to the present embodiment includes a turbidity sensor 2, a first measuring instrument 3, a cell separation device 4, a second measuring instrument 5, and an arithmetic device 6. The turbidity sensor 2, the first measuring instrument 3, and the second measuring instrument 5 are connected to the arithmetic device 6 via signal lines, either wired or wirelessly.

[0030] In FIG. 1, the viable cell count measuring apparatus 1 is provided in a culture tank 7 for culturing cells and producing useful substances. A culture solution 8, which is a cell suspension, is placed in the culture tank 7. The turbidity sensor 2 and the first measuring instrument 3 are provided in the culture tank 7. The cell separation device 4 is connected to the culture tank 7 via an extraction pipe 7a and a return pipe 7b. A circulation pump 9 for circulating the culture solution to the cell separation device 4 is installed in the return pipe 7b. A discharge pipe 7c is connected to the cell separation device 4.

[0031] The viable cell count measurement method used in the viable cell count measuring apparatus 1 includes a step of measuring the amount of suspended matter contained in the cell suspension, a step of measuring the capacitance of the cell suspension that changes depending on the state of life and death of the cells, and based on the capacitance per unit viable cell obtained in advance, the capacitance per unit dead cell obtained in advance, the amount of suspended matter, and the capacitance of the cell suspension, a step of calculating one or more of the viable cell count, dead cell count, and cell survival rate contained in the cell suspension.

[0032] The turbidity sensor 2 is provided for measuring the amount of suspended matter contained in the cell suspension. The culture solution 8 in the culture tank 7 is a cell suspension containing cells as suspended matter, and in the culture solution 8, floating viable cells, dead cells, etc. are measured as suspended matter. When the amount of suspended matter is measured by the turbidity sensor 2, the total number of viable and dead cells contained in the culture solution 8, which is a cell suspension, can be obtained. In FIG. 1, the turbidity sensor 2 is provided in-line in the culture tank 7, and the measuring portion is inserted into the culture tank 7.

[0033] As a method for measuring the amount of suspended matter, various methods such as a transmitted light measurement method, a scattered light measurement method, a transmitted light-scattered light comparison method, an integrating sphere method, and a particle counting method can be used. As the turbidity sensor 2, an absorptiometer, a laser-diffraction type measuring device, etc. can be used. By using such an optical method, the amount of suspended matter contained in the cell suspension can be accurately measured simultaneously with the measurement of capacitance.

[0034] FIG. 2A is a diagram showing an example of a turbidity sensor by a transmitted light measurement method. FIG. 2B is a diagram showing a partially enlarged view of FIG. 2A. FIG. 2B shows an enlarged view of a portion surrounded by a broken line a in FIG. 2A. As shown in FIG. 2A, as the turbidity sensor 2 by a transmitted light measurement method, for example, a transmitted light type turbidity sensor 10 in which a light source is integrally provided can be used. The transmitted light type turbidity sensor 10 has a probe-shaped main body. The side surface on the tip side of the main body is recessed in a concave shape from the side of the main body toward the central axis side. The concave portion recessed toward the central axis side becomes a detection portion filled with the liquid to be measured.

[0035] When measuring the amount of suspended matter, the tip side provided with the detection portion of the transmitted light type turbidity sensor 10 is immersed in the cell suspension to be measured. As shown in FIG. 2B, a light source 11 that emits light such as visible light and infrared light is provided on the surface of the base end side of the main body facing the detection portion. On the other hand, a light receiving portion 13 for detecting the transmitted light 12 is provided on the surface of the tip side of the main body. When the measurement light is emitted from the light source 11, the transmitted light 12 that has passed through the cell suspension in the measurement portion reaches the light receiving portion 13, and the intensity of the transmitted light 12 is measured.

[0036] The cell suspension contains suspended cells such as live cells and dead cells in a floating state as suspended matter that absorbs, scatters, or refracts light. When the influence of background by substances other than cells, for example, the influence of suspended matter smaller than cells and culture medium components, is sufficiently small, the amount of suspended matter contained in the cell suspension can be regarded as equivalent to the total number of cells contained in the cell suspension.

[0037] Therefore, when measuring the turbidity (E) of the cell suspension, based on the following formula (1), the total number of cells (N) contained in the cell suspensionT ) can be obtained. However, in Equation (1), I 0 is the intensity of the incident light, I is the intensity of the transmitted light, k is a constant depending on the measurement conditions, and N T represents the total number of cells (cell concentration).

[0038]

Equation

[0039] The relationship represented by Equation (1) holds when the suspended matter contained in the cell suspension is at a low concentration. Therefore, when determining the total number of cells (N T ) contained in the cell suspension, it is preferable to prepare a calibration curve using a cell suspension with a known total number of cells. The calibration curve is preferably prepared in a range where the relationship between the amount of suspended matter and the total number of cells is a linear proportional relationship, but it is not limited to such a range.

[0040] For example, the total number of cells (N T ) contained in the cell suspension can also be determined as a function of the amount of suspended matter (turbidity E) as a variable, as represented by the following Equation (2). The function with the amount of suspended matter as a variable is, for example, a linear function, etc. After measuring the amount of suspended matter for various total numbers of cells using a plurality of cell suspensions with known total numbers of cells, the relationship between the amount of suspended matter and the total number of cells contained in the cell suspension can be obtained by fitting a function to the measurement results showing the relationship.

[0041]

Equation

[0042] Figure 3 is a diagram showing an example of the relationship between the turbidity and the number of cells in a cell suspension. In Figure 3, the vertical axis represents the turbidity of the culture solution [a.u.], and the horizontal axis represents the number of cells in the culture solution [×10 8It shows [cells / mL]. Figure 3 shows the turbidity when culturing Chinese Hamster Ovary (CHO) cells and the results by the conventional counting method using a hemocytometer.

[0043] As shown in Figure 3, in the low-concentration region, the turbidity of the culture medium and the total cell number are in a linear proportional relationship. Therefore, when the cell suspension to be measured for viable cell count is at a low concentration, the measured value of the amount of suspended matter can be converted to the total cell number by multiplying the measured value of the amount of suspended matter by a constant. On the other hand, in the high-concentration region, the turbidity of the culture medium and the total cell number are in a non-linear relationship. Therefore, when the cell suspension is at a high concentration, the measured value of the amount of suspended matter is converted to the total cell number using the function obtained by fitting.

[0044] In Figure 3, the relationship between the turbidity of the culture medium and the total cell number is obtained up to 1.4×10 8 cells / mL. This cell concentration is a level generally used in high-density perfusion culture. Therefore, from the results shown in Figure 3, it can be said that even when culturing animal cells, etc. at a high density as in perfusion culture, the total cell number can be obtained from the amount of suspended matter as long as it is in a range where self-shielding by the suspended matter is unlikely to occur.

[0045] The measurement wavelength of the turbidity sensor 2 can be appropriately set according to the type of cells to be measured for viable cell count, etc. Generally, the shorter the measurement wavelength is on the short-wavelength side, the more the measurement sensitivity tends to improve. From the viewpoint of increasing the measurement sensitivity and non-invasively measuring the cells during culture, the measurement wavelength of the turbidity sensor 2 is preferably from the visible light region to the near-infrared light region.

[0046] The measurement wavelength of the turbidity sensor 2 is preferably 600±10 nm in the case of yeast, bacteria, etc., 660±10 nm in the case of animal cells, Escherichia coli, etc., and 730±10 nm in the case of cyanobacteria, microalgae, etc. Also, from the viewpoint of reducing the influence of ambient light and color, the near-infrared light region of 840 to 910 nm is preferable.

[0047] The first measuring device 3 is provided for measuring the capacitance of a cell suspension that changes depending on the state of life and death of cells. As the first measuring device 3, for example, an impedance analyzer that measures the impedance of a cell suspension, a multimeter, or a measuring device using the reflection transmission method, the lumped constant method, the resonance method, etc. can be used. In FIG. 1, the first measuring device 3 is provided in-line in the culture tank 7, and the measuring unit is inserted into the culture tank 7.

[0048] A cell suspension such as the culture solution 8 in the culture tank 7 may contain both living cells and dead cells, and both living cells and dead cells may cause polarization. When the background capacitance other than cells is sufficiently small, the capacitance of the cell suspension measured by the first measuring device 3 can be regarded as equivalent to the capacitance of all cells, which is the synthesis of the capacitance of all living cells and the capacitance of all dead cells.

[0049] Therefore, when the capacitance of the cell suspension is measured by the first measuring device 3, the number of living cells, the number of dead cells, and the cell survival rate contained in the cell suspension can be calculated from the simultaneous model equations of the capacitance per unit living cell, the capacitance per unit dead cell, the total number of all cells contained in the cell suspension, and the capacitance of the cell suspension.

[0050] The cell separation device 4 is a device that separates cells contained in a cell suspension. The culture solution 8 in the culture tank 7 is drawn out into the cell separation device 4 during culture. In the cell separation device 4, the culture solution 8 drawn out from the culture tank 7 is separated into a concentrated culture solution containing cells and a cell-free solution containing suspended substances and medium components smaller than cells. When the cell separation process is performed in the cell separation device 4, the concentrated culture solution containing cells is returned from the cell separation device 4 to the culture tank 7 through the return pipe 7b. The cell-free solution is discharged from the cell separation device 4 to the outside of the living cell number measuring device 1 through the discharge pipe 7c.

[0051] The cell separation process by the cell separation device 4 may be performed continuously or intermittently during cell culture. However, when monitoring the number of viable cells in real time with high precision or when cell separation is required for perfusion culture, it is preferably performed continuously.

[0052] As a method for separating cells, a centrifugation method, a filtration method using a hollow fiber membrane, a filtration method using a flat membrane, a rotary filter method, a gravity sedimentation method, etc. can be used. As the cell separation device 4, a centrifuge, a membrane separation device, a rotary filter type filtration device, a sedimentation separation tank, etc. can be used. By using such a method, since mechanical damage to the cells during culture is small, the cells can be separated relatively non-invasively. Since the generation of dead cells can be suppressed, efficient production and appropriate quality can be maintained when producing useful substances.

[0053] The second measuring device 5 is provided for measuring the capacitance of the cell-free liquid from which the cells have been separated. As the second measuring device 5, the same measuring device as the first measuring device 3 can be used. In FIG. 1, the second measuring device 5 is provided in-line in the discharge pipe 7c connected to the cell separation device 4, and the measuring part is inserted into the discharge pipe 7c.

[0054] A cell suspension such as the culture solution 8 in the culture tank 7 contains suspended substances and medium components smaller than the cells, and these components may also cause polarization. When the capacitance of the cell suspension measured by the first measuring device 3 is such that the background capacitance caused by factors other than these cells is large, it cannot be regarded as equivalent to the capacitance of all the cells, and thus the number of viable cells cannot be calculated with high precision.

[0055] In such a case, when the capacitance of the cell-free fluid separated from the cell suspension is measured by the second measuring device 5, the background capacitance other than the cells can be excluded from the capacitance of the cell suspension. Since the capacitance of the cell suspension excluding the background capacitance other than the cells can be regarded as equivalent to the capacitance of all the cells, even when the cell suspension contains suspended substances and culture medium components smaller than the cells, the number of live cells, the number of dead cells, and the survival rate of the cells contained in the cell suspension can be obtained with high precision.

[0056] FIG. 4 is a diagram showing an example of a measuring device by the reflection transmission method. As shown in FIG. 4, as the first measuring device 3 and the second measuring device 5, for example, a probe-type dielectric sensor 14 by the reflection transmission method provided with an electrode at the tip can be used. The dielectric sensor 14 has a rod-shaped main body. At the tip of the main body, an electrode probe 15 made of platinum or the like for irradiating the measurement object with electromagnetic waves is provided.

[0057] When measuring the capacitance, the tip side provided with the electrode probe 15 of the dielectric sensor 14 is immersed in the cell suspension as the measurement object. As shown in FIG. 4, the cell suspension as the measurement object contains live cells 16 and dead cells 17. When an electric field 18 is generated by the electromagnetic wave from the electrode probe 15, the live cells 16 and the dead cells 17 generate polarizations of different magnitudes.

[0058] Since the live cells 16 have a healthy cell membrane that becomes a dielectric, they are strongly polarized and have a large capacitance. On the other hand, since the dead cells 17 have a damaged cell membrane, they are polarized less strongly than the live cells 16 or do not polarize. Therefore, when detecting the reflected waves from the live cells 16 and the dead cells 17, the capacitance of all the cells, which is the synthesis of the capacitance of all the live cells and the capacitance of all the dead cells, can be obtained from the reflection transmission characteristics of the reflected waves.

[0059] Cells polarized in a cell suspension can each be regarded as a capacitor. Therefore, the capacitance of all the cells contained in the cell suspension is the value obtained by summing the capacitance per cell over the total number of cells. That is, the capacitance of all the cells is the combination of the capacitance of all living cells obtained by summing the capacitance of a single living cell over the number of living cells, and the capacitance of all dead cells obtained by summing the capacitance of a single dead cell over the number of dead cells.

[0060] Generally, the capacitance (C d ) of a single dead cell is much smaller than the capacitance (C l ) of a single living cell. Therefore, in conventional general methods for estimating the number of cells from capacitance, impedance, or dielectric constant, the capacitance of all the cells is usually regarded as corresponding to the capacitance of all living cells. In contrast, in the method for measuring the number of living cells according to the present embodiment, since the capacitance of dead cells is taken into account, as variables, not only the capacitance (C l ) of a single living cell and the number of living cells (N l ), but also the capacitance (C d ) of a single dead cell and the number of dead cells (N d ) are added.

[0061] The capacitance (C T ) of the cell suspension can be expressed by the following formula (3) with respect to the background capacitance (C B ), the capacitance (C L ) of all living cells, and the capacitance (C D ) of all dead cells. Formula (3) is a relational expression in the case of simplification as capacitors arranged in series for each cell. It is desirable to establish the relational expression according to the cell arrangement within the capacitance measurement region. Also, the total number of cells (N T ) contained in the cell suspension can be expressed by the following formula (4) using the number of living cells (N l ) and the number of dead cells (N d ).

[0062]

Equation

[0063]

Number

[0064] Also, the capacitance (C L ) of all living cells can be expressed by the following formula (5) with respect to the capacitance (C l ) of a single living cell and the number of living cells (N l ). Also, the capacitance (C D ) of all dead cells can be expressed by the following formula (6) with respect to the capacitance (C d ) of a single dead cell and the number of dead cells (N d ).

[0065]

Number

[0066]

Number

[0067] Therefore, from formulas (3) to (6), the simultaneous model formulas expressed by the following formulas (7) and (8) are established. When the background capacitance caused by factors other than cells is sufficiently small, the term of C B can be omitted.

[0068]

Number

[0069]

Number

[0070] The survival rate (V) of cells can be expressed by the following formula (9) using formula (2).

[0071]

Number

[0072] The capacitance (C l ) of a single living cell can be determined based on the capacitance of all cells and the known number of living cells by preparing a cell suspension with a known number of living cells and measuring the capacitance of all cells contained in this cell suspension. As the cell suspension with a known number of living cells, a cell suspension not containing dead cells is preferred, and for example, a suspension of cells in the growth phase can be used.

[0073] The capacitance (C d ) of a single dead cell can be determined based on the capacitance of all cells and the known number of dead cells by preparing a cell suspension with a known number of dead cells and measuring the capacitance of all cells contained in this cell suspension. As the cell suspension with a known number of dead cells, a cell suspension not containing living cells is preferred, and for example, a suspension obtained by subjecting cells in the growth phase to a treatment that induces cell death can be used. Examples of the treatment that induces cell death include a treatment that causes mechanical damage and a treatment that deprives nutrients.

[0074] Figure 5 is a diagram showing an example of the relationship between the capacitance of a cell suspension and the number of living cells. In Figure 5, the vertical axis represents the capacitance of the cell suspension [pF / cm], and the horizontal axis represents the number of cells in the culture solution [×10 8 cells / mL]. Figure 5 shows the capacitance when culturing CHO cells and the results by a conventional counting method using a hemocytometer. As the cell suspension to be measured, a sample in which the cell viability has been confirmed to be 95% or more was used.

[0075] In Figure 5, the relationship between the capacitance of the cell suspension and the number of living cells is shown as a substantially linear relationship of 1.2×10 8It has been obtained up to cells / mL. This cell concentration is a level commonly used in high-density perfusion culture. Therefore, from the results shown in FIG. 5, even when culturing animal cells or the like at a high density as in perfusion culture, by subtracting the background capacitance as necessary, it can be seen that the correlation between the capacitance of the cell suspension and the number of live cells can be utilized.

[0076] The arithmetic unit 6 is composed of a computer or the like including an arithmetic unit that calculates the simultaneous model equations represented by equations (7) and (8), a storage unit that stores data on the capacitance per unit live cell obtained in advance and data on the capacitance per unit dead cell obtained in advance, an input unit that receives an input from the operator of the live cell number measuring device 1, a display unit that displays the result of the calculation, and the like.

[0077] The arithmetic unit of the arithmetic device 6 can be composed of a CPU (Central Processing Unit), an MPU (Micro Processing Unit), etc. The storage unit can be composed of a storage device such as a hard disk drive (HDD) or a solid state drive (SSD). The input unit can be composed of an input device such as a keyboard, a mouse, or a touch pad. The display unit can be composed of various display devices such as a liquid crystal display, a plasma display, an organic EL display, or a cathode ray tube.

[0078] Data on the capacitance (C l ) of a unit live cell and data on the capacitance (C d ) of a unit dead cell are obtained in advance using a cell suspension in which the number of live cells and the number of dead cells are known, and then stored in the storage unit of the arithmetic device 6. As data per unit cell, data of one cell may be prepared, or data of a predetermined cell population may be prepared. In addition, data on a calibration curve showing the relationship between the amount of suspended matter contained in the cell suspension and the total number of cells is obtained in advance using a cell suspension in which the number of cells is known, and then stored in the storage unit of the arithmetic device 6.

[0079] A signal of the measurement result of the amount of suspended matter measured for the cell suspension to be measured is input to the arithmetic unit 6 from the turbidity sensor 2 at a predetermined time interval. The data of the amount of suspended matter is converted into data of the total cell count based on the relationship between the previously obtained amount of suspended matter and the total cell count, and is used for arithmetic operations by the simultaneous model equations.

[0080] In addition, a signal of the measurement result measured for the cell suspension to be measured is input to the arithmetic unit 6 from the first measuring instrument 3 at a predetermined time interval. Further, a signal of the measurement result measured for the cell-free liquid from which the cells have been separated is input to the arithmetic unit 6 from the second measuring instrument 5 at a predetermined time interval. Data such as impedance and transmission parameters measured by the first measuring instrument 3 and the second measuring instrument 5 are converted into data of the capacitance of the cell suspension and used for arithmetic operations by the simultaneous model equations.

[0081] The arithmetic unit 6 calculates the simultaneous model equations represented by formulas (7) and (8) based on the input of the measurement results from the turbidity sensor 2, the first measuring instrument 3, and the second measuring instrument 5, and the data per unit cell stored in the storage unit. That is, based on the capacitance per unit live cell obtained in advance, the capacitance per unit dead cell obtained in advance, the amount of suspended matter contained in the cell suspension, and the measured capacitance of the cell suspension, one or more of the number of live cells, the number of dead cells, and the cell survival rate contained in the cell suspension are calculated.

[0082] The capacitance of the cell suspension measured for the cell suspension to be measured may be used for arithmetic operations by the simultaneous model equations without removing the background capacitance caused by factors other than cells, or may be used for arithmetic operations by the simultaneous model equations after removing the background capacitance caused by factors other than cells. When the background capacitance caused by factors other than cells is sufficiently small or when high precision is not required, the background processing can be omitted.

[0083] According to the arithmetic operations by the simultaneous model equations represented by formulas (7) and (8), the total cell count (N T ) is the number of live cells (Nl ), the number of dead cells (N d ), the capacitance of all living cells (C L ), the capacitance of a single living cell (C l ), the capacitance of a single dead cell (C d ), among which, since the number of living cells (N l ) and the number of dead cells (N d ) are unknowns, by solving the two equations simultaneously, the number of living cells (N l ) and the number of dead cells (N d ) can be obtained. The cell viability (V) can be calculated from these results.

[0084] The number of living cells (N l ) obtained by the calculation of the simultaneous model equations, the number of dead cells (N d ), and the results of the cell viability (V) can be displayed on the display unit of the arithmetic device 6. These results may be displayed in the form of the amount of suspended matter contained in the cell suspension, time-series data or graphs along the measurement time of the capacitance of the cell suspension, etc. Also, together with these results, the data of the capacitance of a single living cell (C l ) and the data of the capacitance of a single dead cell (C d ) may be displayed.

[0085] The living cell number measuring device 1 can measure one or more of the number of living cells (N l ), the number of dead cells (N d ) and the cell viability (V) in real time or according to a predetermined schedule while culturing the cells to be measured. The living cell number measuring device 1 may be used for the culture solution of any culture method such as batch culture, fed-batch culture, chemostat culture, perfusion culture, etc. in addition to the cell suspension prepared in advance.

[0086] <Batch culture> Batch culture (batch culture) is a culture method in which a medium is prepared for each culture and the medium is not supplied during the culture. According to batch culture, when producing useful substances by culture, the quality tends to vary for each culture, but there is an advantage that the risk of contamination can be dispersed and reduced.

[0087] FIG. 6 is a diagram showing a batch culture apparatus equipped with a viable cell number measuring device. As shown in FIG. 6, the viable cell number measuring device can be provided in the batch culture apparatus. The batch culture apparatus includes a turbidity sensor 2, a first measuring device 3, a cell separation device 4, a second measuring device 5, and an arithmetic device 6 that constitute the viable cell number measuring device. Further, it includes a circulation pump 19a, a transfer pump 19b, a return pump 19c, and a measurement tank 22.

[0088] In FIG. 6, the viable cell number measuring device is provided in a culture tank 7 for producing a useful substance by batch culture. The turbidity sensor 2 and the first measuring device 3 are inserted into the culture tank 7. The cell separation device 4 is connected to the culture tank 7 via an extraction pipe 7a and a return pipe 7b. A circulation pump 19a for circulating the culture solution is installed in the extraction pipe 7a.

[0089] The measurement tank 22 is connected to the cell separation device 4 via a transfer pipe 7d. The second measuring device 5 is inserted into the measurement tank 22. A transfer pump 19b for sending the cell-free solution from the cell separation device 4 to the measurement tank 22 is installed in the transfer pipe 7d. The measurement tank 22 is connected to the culture tank 7 via a return pipe 7e. A return pump 19c for returning the cell-free solution from the measurement tank 22 to the culture tank 7 is installed in the return pipe 7e.

[0090] The batch-type culture tank 7 can be provided with a pH sensor, a dissolved oxygen sensor, a temperature sensor such as a thermocouple, a stirring device for stirring the culture solution, a ventilation device for ventilating air, oxygen, nitrogen, carbon dioxide, etc. into the culture solution, a heater for adjusting the temperature of the culture solution, a supply device for supplying an alkaline solution to the culture tank 7, etc. to control the culture environment.

[0091] When culturing floating cells, a stirring device equipped with a stirring blade driven by a motor can be used. The culture atmosphere can be controlled by aeration through the liquid surface, aeration in the liquid, or both. The temperature of the culture solution is usually adjusted to the optimum temperature for culturing and substance production by on-off control of a heater. The pH and dissolved oxygen concentration are maintained at set values by feedback control or the like.

[0092] In a batch culture device, cell culture and substance production by cells are carried out in the culture solution placed in the culture tank 7 without supplying the medium during the culture. In the batch culture device, before the culture, the capacitance per unit live cell (C l ) and the capacitance per unit dead cell (C d ) are determined in advance, and these data are stored in the storage unit of the arithmetic device 6.

[0093] During the batch culture of cells, the amount of suspended matter contained in the culture solution is measured over time by the turbidity sensor 2. The data of the measured amount of suspended matter is converted into data of the total cell number (N T ) based on the relationship between the amount of suspended matter (turbidity) contained in the culture solution and the cell number.

[0094] Also, during the batch culture of cells, the capacitance (C T ) of the culture solution, which is a cell suspension, is measured over time by the first measuring device 3. When the background capacitance caused by factors other than cells is not sufficiently small, the capacitance of the cell-free solution separated from the culture solution is measured over time by the second measuring device 5, and the background capacitance caused by factors other than cells is excluded from the capacitance of the culture solution.

[0095] The arithmetic device 6 uses the data of the total cell number (N T ) contained in the culture solution at a predetermined measurement time and the data of the capacitance (C T ) of the culture solution as inputs, and the total cell number (N T ) of the culture solution, the capacitance (C T ) of the culture solution, and the capacitance per unit live cell (C l) and the capacitance per unit dead cell (C d ) from the simultaneous model equations, calculate the number of live cells (N l ), the number of dead cells (N d ), and the cell viability (V).

[0096] According to the batch culture device equipped with a live cell number measuring device, during batch culture where dead cells tend to accumulate, the number of live cells can be measured in real time with high precision. During batch culture, since the consumption of nutrients and the accumulation of metabolites proceed, the background capacitance caused by factors other than cells increases. However, by removing the background capacitance caused by factors other than cells from the capacitance of the culture solution, higher precision can be obtained.

[0097] <Fed-batch culture> Fed-batch culture (fed-batch culture) is a culture method in which the medium itself or specific medium components are added from outside the system during culture, but the culture solution is not discharged until the culture is completed. According to fed-batch culture, since nutrient supplementation and metabolite dilution are performed during culture, higher density culture is possible compared to batch culture, and medium costs and the like can be reduced compared to perfusion culture.

[0098] Figure 7 is a diagram showing a fed-batch culture device equipped with a live cell number measuring device. As shown in Figure 7, the live cell number measuring device can be provided in a fed-batch culture device. The fed-batch culture device includes a turbidity sensor 2, a first measuring device 3, a cell separation device 4, a second measuring device 5, and an arithmetic device 6 that constitute the live cell number measuring device. It also includes a circulation pump 19a, a transfer pump 19b, a discharge pump 19d, a medium supply pump 19e, a medium tank 21, and a measurement tank 22.

[0099] In Fig. 7, the viable cell count measuring device is provided in a culture tank 7 for producing useful substances by fed-batch culture. The turbidity sensor 2 and the first measuring instrument 3 are inserted into the culture tank 7. The cell separation device 4 is connected to the culture tank 7 via an extraction pipe 7a and a return pipe 7b. A circulation pump 19a for circulating the culture solution is installed in the extraction pipe 7a.

[0100] A measuring tank 22 is connected to the cell separation device 4 via a transfer pipe 7d. The second measuring instrument 5 is inserted into the measuring tank 22. A transfer pump 19b for sending the cell-free solution from the cell separation device 4 to the measuring tank 22 is installed in the transfer pipe 7d. A discharge pipe 7f is connected to the measuring tank 22. A discharge pump 19d for discharging the cell-free solution from the measuring tank 22 to the outside of the device is installed in the discharge pipe 7f.

[0101] A medium tank 21 is connected to the culture tank 7 via a medium supply pipe 7g. Fresh medium is prepared in the medium tank 21. A medium supply pump 19e for sending fresh medium from the medium tank 21 to the culture tank 7 is installed in the medium supply pipe 7g.

[0102] Similar to the batch-type culture tank 7, the fed-batch type culture tank 7 can be equipped with a pH sensor, a dissolved oxygen sensor, a temperature sensor such as a thermocouple for controlling the culture environment, a stirring device for stirring the culture solution, an aeration device for aerating air, oxygen, nitrogen, carbon dioxide, etc. into the culture solution, a heater for adjusting the temperature of the culture solution, a supply device for supplying an alkaline solution to the culture tank 7, etc.

[0103] In the fed-batch culture apparatus, cell culture and substance production by cells are carried out while supplying fresh medium during the culture in the culture solution placed in the culture tank 7. In the fed-batch culture apparatus, similar to the batch culture apparatus, before the culture, the capacitance (C l ) per unit viable cell and the capacitance (C d ) per unit dead cell are obtained in advance, and these data are stored in the storage unit of the arithmetic device 6.

[0104] During the fed-batch culture of cells, the turbidity sensor 2 measures the amount of suspended matter contained in the culture solution over time. Based on the relationship between the amount of suspended matter (turbidity) contained in the culture solution and the number of cells, the data on the amount of measured suspended matter is converted into data on the total number of cells (N T ).

[0105] Also, during the fed-batch culture of cells, the first measuring device 3 measures the capacitance (C T ) of the culture solution, which is a cell suspension, over time. When the background capacitance caused by factors other than cells is not small enough, the second measuring device 5 measures the capacitance of the cell-free solution separated from the culture solution over time, and subtracts the background capacitance caused by factors other than cells from the capacitance of the culture solution.

[0106] The arithmetic unit 6 takes as input the data on the total number of cells (N T ) contained in the culture solution at a predetermined measurement time and the data on the capacitance (C T ) of the culture solution, and calculates the total number of cells (N T ) in the culture solution, the capacitance (C T ) of the culture solution, the capacitance (C l ) per living cell, the capacitance (C d ) per dead cell, the number of living cells (N l ), the number of dead cells (N d ), and the cell viability (V) from the simultaneous model equations.

[0107] According to the fed-batch culture apparatus equipped with a living cell number measuring device, the number of living cells can be measured in real time with high accuracy during the fed-batch culture in which dead cells tend to accumulate. During the fed-batch culture, nutrients are supplied and metabolites accumulate, so the background capacitance caused by factors other than cells may increase. However, by subtracting the background capacitance caused by factors other than cells from the capacitance of the culture solution, higher accuracy can be obtained.

[0108] <Chemostat culture> Chemostat culture is a culture method in which the culture medium is continuously supplied during the culture and the same amount of culture solution containing cells is continuously discharged. According to chemostat culture, since the culture environment is kept substantially constant, the productivity of substances can be stabilized.

[0109] Figure 8 is a diagram showing a chemostat culture apparatus equipped with a viable cell count measuring device. As shown in Figure 8, the viable cell count measuring device can be provided in the chemostat culture apparatus. The chemostat culture apparatus includes a turbidity sensor 2, a first measuring device 3, a cell separation device 4, a second measuring device 5, and an arithmetic device 6 that constitute the viable cell count measuring device. Further, it includes a circulation pump 19a, a transfer pump 19b, a discharge pump 19d, a medium supply pump 19e, a culture solution sampling pump 19f, a medium tank 21, and a measurement tank 22.

[0110] In Figure 8, the viable cell count measuring device is provided in a culture tank 7 for producing useful substances by chemostat culture. The turbidity sensor 2 and the first measuring device 3 are inserted into the culture tank 7. The cell separation device 4 is connected to the culture tank 7 via an extraction pipe 7a and a return pipe 7b. A circulation pump 19a for circulating the culture solution is installed in the extraction pipe 7a.

[0111] The measurement tank 22 is connected to the cell separation device 4 via a transfer pipe 7d. The second measuring device 5 is inserted into the measurement tank 22. A transfer pump 19b for sending the cell-free solution from the cell separation device 4 to the measurement tank 22 is installed in the transfer pipe 7d. A discharge pipe 7f is connected to the measurement tank 22. A discharge pump 19d for discharging the cell-free solution from the measurement tank 22 to the outside of the device is installed in the discharge pipe 7f.

[0112] The culture tank 7 is connected to the medium tank 21 via the medium supply pipe 7g. Fresh medium is prepared in the medium tank 21. A medium supply pump 19e for sending fresh medium from the medium tank 21 to the culture tank 7 is installed in the medium supply pipe 7g. Further, a sampling pipe 7h is connected to the culture tank 7. A culture solution sampling pump 19f for sampling a part of the culture solution containing cells from the culture tank 7 is installed in the sampling pipe 7h. The culture solution sampled from the culture tank 7 is sent to the recovery system 25 together with the useful substances produced by the cells.

[0113] Similar to the batch-type culture tank 7, the chemostat-type culture tank 7 can be equipped with a pH sensor, a dissolved oxygen sensor, a temperature sensor such as a thermocouple, a stirring device for stirring the culture solution, an aeration device for aerating the culture solution with air, oxygen, nitrogen, carbon dioxide, etc., a heater for adjusting the temperature of the culture solution, a supply device for supplying an alkaline solution to the culture tank 7, etc. to control the culture environment.

[0114] In the chemostat culture apparatus, in the culture solution placed in the culture tank 7, while supplying fresh medium during culture and extracting a part of the culture solution containing the grown cells, cell culture and substance production by the cells are performed. The supply rate of the fresh medium and the extraction rate of the culture solution are kept constant, and the temperature, pH, and dissolved oxygen concentration in the culture tank 7 are kept constant. In the chemostat culture apparatus, similar to the batch culture apparatus, etc., before culture, the capacitance (C l ) per unit live cell and the capacitance (C d ) per unit dead cell are obtained in advance, and these data are stored in the storage unit of the arithmetic device 6.

[0115] During the chemostat culture of cells, the amount of suspended matter contained in the culture solution is measured over time by the turbidity sensor 2. The data of the measured amount of suspended matter is converted into data of the total cell number (N T ) based on the relationship between the amount of suspended matter (turbidity) contained in the culture solution and the cell number.

[0116] Also, during the chemostat culture of cells, the capacitance (C T) is measured over time. When the capacitance of the background caused by factors other than cells is not small enough, the second measuring device 5 measures the capacitance of the cell-free liquid separated from the culture solution over time, and subtracts the capacitance of the background caused by factors other than cells from the capacitance of the culture solution.

[0117] The arithmetic unit 6 uses the data of the total number of cells (N T ) contained in the culture solution at a predetermined measurement time and the data of the capacitance (C T ) of the culture solution as inputs, and from the simultaneous model equations of the total number of cells (N T ) in the culture solution, the capacitance (C T ) of the culture solution, the capacitance (C l ) per living cell, and the capacitance (C d ) per dead cell, calculates the number of living cells (N l ), the number of dead cells (N d ), and the survival rate (V) of the cells.

[0118] According to the chemostat culture apparatus equipped with the living cell number measuring device, the number of living cells can be measured with high precision in real time during the chemostat culture in which cells are sequentially extracted. During chemostat culture, the number of cells in the culture tank may vary due to the extraction of cells. However, since the number of living cells can be measured with high precision in real time by the living cell number measuring device, the supply amount of fresh medium and the extraction amount of the culture solution can be appropriately adjusted.

[0119] <Perfusion culture> Perfusion culture is a culture method in which medium is continuously supplied during culture and the same amount of cell-free culture solution is continuously discharged. According to perfusion culture, since the culture environment is easily kept constant, the productivity of substances can be stabilized. In addition, since the extraction of grown cells is suppressed, cells can be cultured at a higher density compared to chemostat culture.

[0120] FIG. 9 is a diagram showing a perfusion culture apparatus equipped with a living cell number measuring device. As shown in Fig. 9, the viable cell count measuring device can be provided in a perfusion culture device. The perfusion culture device includes a turbidity sensor 2, a first measuring instrument 3, a cell separation device 4, a second measuring instrument 5, and an arithmetic device 6 that constitute the viable cell count measuring device. Further, it includes a circulation pump 19a, a transfer pump 19b, a discharge pump 19d, a medium supply pump 19e, a culture solution extraction pump 19f, a medium tank 21, and a measurement tank 22.

[0121] In Fig. 9, the viable cell count measuring device is provided in a culture tank 7 for producing useful substances by perfusion culture. The turbidity sensor 2 and the first measuring instrument 3 are inserted into the culture tank 7. The cell separation device 4 is connected to the culture tank 7 via an extraction pipe 7a and a return pipe 7b. A circulation pump 19a for circulating the culture solution is installed in the extraction pipe 7a.

[0122] The measurement tank 22 is connected to the cell separation device 4 via a transfer pipe 7d. The second measuring instrument 5 is inserted into the measurement tank 22. A transfer pump 19b for sending the cell-free solution from the cell separation device 4 to the measurement tank 22 is installed in the transfer pipe 7d. A discharge pipe 7f is connected to the measurement tank 22. A discharge pump 19d for discharging the cell-free solution from the measurement tank 22 to the outside of the device is installed in the discharge pipe 7f. The cell-free solution extracted from the measurement tank 22 is sent to a recovery system 25 together with the useful substances produced by the cells.

[0123] The culture tank 7 is connected to the medium tank 21 via a medium supply pipe 7g. Fresh medium is prepared in the medium tank 21. A medium supply pump 19e for sending fresh medium from the medium tank 21 to the culture tank 7 is installed in the medium supply pipe 7g. Further, an extraction pipe 7h is connected to the culture tank 7. A culture solution extraction pump 19f for extracting a part of the culture solution containing cells from the culture tank 7 to the outside of the device is installed in the extraction pipe 7h. The extraction pipe 7h is used to extract overly cultured cells as bleeding.

[0124] In the perfusion culture tank 7, similar to the batch culture tank 7, in order to control the culture environment, there can be provided a pH sensor, a dissolved oxygen sensor, a temperature sensor such as a thermocouple, a stirring device for stirring the culture solution, a ventilation device for ventilating air, oxygen, nitrogen, carbon dioxide, etc. into the culture solution, a heater for adjusting the temperature of the culture solution, a supply device for supplying an alkaline solution to the culture tank 7, and the like.

[0125] In the perfusion culture apparatus, in the culture solution placed in the culture tank 7, while supplying fresh medium during the culture and extracting the cell-free liquid from which the cells have been separated, cell culture and substance production by the cells are carried out. The supply rate of the fresh medium and the extraction rate of the cell-free liquid are kept constant, and the temperature, pH, and dissolved oxygen concentration in the culture tank 7 are kept constant. In the perfusion culture apparatus, similar to the batch culture apparatus, etc., before the culture, the capacitance (C l ) per living cell and the capacitance (C d ) per dead cell are obtained in advance, and these data are stored in the storage unit of the arithmetic unit 6.

[0126] During the perfusion culture of the cells, the turbidity sensor 2 measures the amount of suspended matter contained in the culture solution over time. The data of the measured amount of suspended matter is converted into the data of the total cell number (N T ) based on the relationship between the amount of suspended matter (turbidity) contained in the culture solution and the cell number.

[0127] Also, during the perfusion culture of the cells, the first measuring instrument 3 measures the capacitance (C T ) of the culture solution, which is a cell suspension, over time. When the background capacitance caused by factors other than the cells is not sufficiently small, the second measuring instrument 5 measures the capacitance of the cell-free liquid separated from the culture solution over time, and excludes the background capacitance caused by factors other than the cells from the capacitance of the culture solution.

[0128] The arithmetic unit 6 takes as input the data of the total cell number (N T ) contained in the culture solution at a predetermined measurement time and the data of the capacitance (C T ) of the culture solution, and the total cell number (N T) and the capacitance of the culture solution (C T ) and the capacitance per living cell (C l ) and the capacitance per dead cell (C d ), from the simultaneous model equations, calculate the number of living cells (N l ), the number of dead cells (N d ), and the cell viability (V).

[0129] According to the perfusion culture device equipped with the living cell number measurement device, during perfusion culture in which cells are continuously cultured, the number of living cells can be measured in real time with high accuracy. Conventionally, in perfusion culture, cell separation treatment has been performed when a part of the culture solution is extracted. In the living cell number measurement device, cell-free fluid necessary for measuring the background capacitance caused by substances other than cells can be recovered by using an existing cell separation device used for such cell separation treatment.

[0130] According to the above living cell number measurement method and living cell number measurement device, the capacitance per living cell and the capacitance per dead cell are incorporated into the simultaneous model equation of the total number of cells contained in the cell suspension and the capacitance of the cell suspension, and the number of living cells, the number of dead cells, and the cell viability are calculated. Therefore, even when dead cells have capacitance, such as in the case of cell death leaving the cell membrane, the number of living cells can be measured with high accuracy. In addition, since the total number of cells contained in the cell suspension is measured optically as the amount of turbidity, a measurement result expressed in units of the number of cells or cell concentration can be obtained.

[0131] Also, the measurement of the amount of turbidity contained in the cell suspension and the capacitance of the cell suspension can be performed in real time by impedance measurement or turbidity measurement in a predetermined frequency range. Different from the case of sampling cells, there is little risk of contamination with miscellaneous bacteria or the like during measurement, and there is no invasive influence on the cells, such as when controlling staining or dissolved oxygen concentration. Therefore, the risk of contamination is low, and the number of living cells can be measured with high accuracy by a non-invasive means for the cells.

[0132] As described above, the present invention has been explained. However, the present invention is not limited to the above-described embodiments, and various modifications are possible without departing from the spirit of the present invention. For example, the present invention is not necessarily limited to having all the configurations provided in the above-described embodiments. A part of the configuration of one embodiment can be replaced with another configuration, a part of the configuration of one embodiment can be added to another configuration, or a part of the configuration of one embodiment can be omitted.

[0133] For example, although the above-described viable cell count measuring apparatus 1 includes a cell separation apparatus 4 and a second measuring instrument 5, when the background processing of the capacitance of the cell suspension is not performed, etc., the installation of the cell separation apparatus 4 and the second measuring instrument 5 can also be omitted. Further, as the turbidity sensor 2, the first measuring instrument 3, and the second measuring instrument 5, in-line sensors such as probe types are provided, but as long as sampling is not performed, other types of sensors may be used.

[0134] Also, in the above-described viable cell count measuring apparatus 1 and viable cell count measuring method, the simultaneous model equations represented by equations (7) and (8) are used in the calculation. However, as long as the total cell count (N T ), viable cell count (N l ), dead cell count (N d ), capacitance of all viable cells (C L ), capacitance per unit viable cell (C l ), and capacitance per unit dead cell (C d ) are variables, other simultaneous model equations may be used in the calculation according to the spatial arrangement of the cells for which capacitance is measured, the range of the background to be considered, etc.

Example

[0135] Hereinafter, the present invention will be specifically described by showing examples, but the technical scope of the present invention is not limited thereto.

[0136] <Example 1> In batch culture, the amount of suspended matter contained in the cell suspension was measured, the capacitance of the cell suspension was measured, and the viable cell count was measured based on the simultaneous model equations represented by formulas (7) and (8).

[0137] (Cell·Medium) As the cells for which the viable cell count was to be measured, CHO cells (ATCC CRL-12445 cells) that are recombinant genes producing an antibody (IgG) and are adapted to floating cells were used. As the medium, a medium obtained by adding insulin (final concentration: 10 μg / mL), transferrin (final concentration: 10 μg / mL), and fetal bovine serum (FBS) (final concentration: 10 μg / mL) to Dulbecco's Modified Eagle Medium (DMEM) was used.

[0138] (Experimental Apparatus) Figure 10 is a diagram showing the viable cell count measuring apparatus used in Example 1 by batch culture. As shown in Figure 10, as the viable cell count measuring apparatus, an apparatus in which a turbidity sensor 2, a first measuring instrument 3 which is a capacitance meter, a cell separation apparatus 4 that performs filtration with a hollow fiber membrane, and a second measuring instrument 5 which is a capacitance meter are attached to a batch-type culture tank 7 was used.

[0139] In the batch-type culture tank 7, CHO cells, which are the cells to be measured, were cultured, and the viable cell count was measured in real time. Also, as a comparison target, a conventional measurement using a hemocytometer was performed in parallel. The conventional measurement was performed by sampling the culture solution once a day, staining dead cells with trypan blue, and counting the cells visually observed in the microscope field with a hemocytometer.

[0140] (Experimental Results) Figure 11 is a diagram showing the measurement results of the viable cell count in Example 1 by batch culture. In Figure 11, the horizontal axis is the culture time [day], and the vertical axis is the measured viable cell count [×10 6It indicates [cells / mL]. The plots of circles show the measurement results by the conventional offline method of sampling. The plots of triangles show the measurement results by the conventional inline method of estimating the cell count from the measured capacitance. The plots of squares show the measurement results according to Example 1 obtained by measuring the amount of suspended matter and the capacitance of the cell suspension and performing calculations based on the model formula.

[0141] Figure 12 is a diagram showing the deviation of the measurement results of Example 1 from the conventional method. In Figure 12, the measurement difference between the measurement results of the viable cell count according to Example 1 and the conventional offline measurement results, and the measurement difference between the measurement results of the conventional inline method of estimating the cell count from the capacitance and the conventional offline measurement results are compared and shown. Each plot in Figure 12 corresponds to the plot in Figure 11. The measurement results by the conventional offline method are considered to be the most accurate measurement because dead cells are stained with trypan blue and counted using a hemocytometer.

[0142] As shown in Figure 12, the measurement results of the viable cell count according to Example 1 calculated based on the simultaneous model formulas represented by formulas (7) and (8) are less likely to vary with respect to the counting by the hemocytometer compared to the measurement results by the conventional inline method of estimating the cell count from the capacitance. In the conventional inline method, variations with respect to the counting by the hemocytometer frequently occur, and in particular, the variations are expanding in the later stage of the culture where the cell viability decreases.

[0143] From these results, it can be seen that the cell count measurement method of Example 1 that calculates based on the simultaneous model formulas represented by formulas (7) and (8) can obtain high accuracy close to the counting using the conventional hemocytometer in batch culture.

[0144] <Example 2> In perfusion culture, the amount of suspended matter contained in the cell suspension was measured, the capacitance of the cell suspension was measured, and the viable cell count was measured based on the simultaneous model formulas represented by formulas (7) and (8).

[0145] (Cell and Medium) As the cells to be measured for viable cell count, as in Example 1, CHO cells (ATCC CRL-12445 cells) that are gene recombinants producing antibody (IgG) and are adapted to suspension cells were used. As the medium, as in Example 1, a medium obtained by adding insulin (final concentration: 10 μg / mL), transferrin (final concentration: 10 μg / mL), and fetal bovine serum (FBS) (final concentration: 10 μg / mL) to Dulbecco's Modified Eagle Medium (DMEM) was used.

[0146] (Experimental Apparatus) Figure 13 is a diagram showing the viable cell count measuring apparatus used in Example 2 by perfusion culture. As shown in Figure 13, as the viable cell count measuring apparatus, a perfusion-type culture tank 7 was equipped with a turbidity sensor 2, a first measuring instrument 3 which is a capacitance meter, a cell separation device 4 that performs filtration with a hollow fiber membrane, a second measuring instrument 5 which is a capacitance meter, an arithmetic unit 6, and a control device 24. The capacity of the culture tank 7 was set to 1 L. The cell separation device 4 was equipped with a hollow fiber membrane having a fractional molecular weight of 300 kDa.

[0147] In the perfusion-type culture tank 7, CHO cells, which are the cells to be measured, were cultured, and the viable cell count was measured in real time. As the seed cells, CHO cells diluted to 1×10 5 cells / mL in DMEM medium were seeded in the culture tank 7. The culture conditions were maintained at a culture temperature of 37°C, a dissolved oxygen concentration of 2.7 mg / L, and pH 7.2. The circulation rate of the culture solution to the cell separation device 4 was set to 10 mL / min, and the perfusion rate was set to 1 vvd (30 mL / h) by supplying fresh medium.

[0148] During perfusion culture, the viable cell count was measured by the arithmetic unit 6, and bleeding through the sampling tube 7h was controlled based on the measurement results on the 21st day from the start of culture. The amount of bleeding was controlled by the output of the culture solution sampling pump 19f by the control device 24 so that the viable cell count in the culture tank 7 was 1×10 7Adjusted to be maintained at cells / mL.

[0149] (Experimental results) Figure 14 is a diagram showing the measurement results of the number of live cells in Example 2 by perfusion culture. In Figure 14, the horizontal axis represents the culture time [day], and the vertical axis represents the measured number of live cells [×10 6 cells / mL] or the cell survival rate [%]. The ○ plots represent the number of live cells. The △ plots represent the cell survival rate. The section sandwiched by the dashed line indicates the period during which the cell concentration was controlled.

[0150] As shown in Figure 14, the measurement results of the number of live cells in Example 2 calculated based on the simultaneous model equations represented by formulas (7) and (8) also achieved high accuracy in perfusion culture. Therefore, even at the stage when the culture time had elapsed, the number of live cells could be kept constant.

[0151] From these results, it can be seen that the method for measuring the number of cells in Example 2, which is calculated based on the simultaneous model equations represented by formulas (7) and (8), can be used for feedback control to keep the number of live cells constant in perfusion culture.

Explanation of symbols

[0152] 1 Live cell counting device 2 Turbidity sensor 3 First measuring instrument 4 Cell separation device 5 Second measuring instrument 6 Computing device 7 Culture tank 8 Culture medium 10 Transmission light type turbidity sensor 11 Light source 12 Transmission light 13 Light receiving part 14 Dielectric constant sensor 15 Electrode probe 16 Live cell 17 Dead cell 19a Circulation pump 19b Transfer pump 19c Return pump 19d Drain pump 19e Medium supply pump 19f Culture solution extraction pump 21 Medium tank 22 Measurement tank 24 Control device 25 Recovery system

Claims

1. A production method for producing a substance by culturing cells, comprising: Cultivating cells in a light-transmitting cell suspension; measuring the amount of suspended solids, including live and dead cells, contained in the cell suspension; Measuring the capacitance of the cell suspension, which varies depending on the viability of the cells; and calculating at least one of the number of live cells, the number of dead cells, the cell viability, the capacitance of all live cells, and the capacitance of all dead cells contained in the cell suspension based on the capacitance per unit of live cells, the capacitance per unit of dead cells, the amount of the suspended solid, and the capacitance of the cell suspension.

2. 2. The method of claim 1 , further comprising: measuring the capacitance of the cell suspension having a known number of viable cells to determine the capacitance per viable cell; measuring the capacitance of said cell suspension having a known number of dead cells to determine the capacitance per dead cell.

3. 2. The method of claim 1 , further comprising: The capacitance of the cell suspension is used for calculation after subtracting the background capacitance due to non-cells, consisting of live and dead cells.

4. 4. The method of claim 3, further comprising the steps of: Separating the cells contained in the cell suspension; and measuring the capacitance of the cell-free fluid from which the cells have been separated.

5. A culture apparatus for culturing cells to produce a substance, comprising: A culture tank for culturing cells; A turbidity sensor provided in the culture tank for measuring the amount of suspended solids including live cells and dead cells contained in the light-transmitting cell suspension; A measuring device provided in the culture tank for measuring the capacitance of the cell suspension, which changes depending on the viability of the cells; a calculation unit that calculates at least one of the number of live cells, the number of dead cells, the cell viability, the capacitance of all live cells, and the capacitance of all dead cells contained in the cell suspension based on the capacitance per specified unit of live cell, the capacitance per specified unit of dead cell, the amount of the suspended solid, and the capacitance of the cell suspension.

6. The culture device according to claim 5, A culture device having a memory unit that stores data on capacitance per unit of live cells and capacitance per unit of dead cells.

7. The culture device according to claim 5, The capacitance of the cell suspension is used for calculation after background capacitance due to non-cells, consisting of live and dead cells, is subtracted.

8. The culture device according to claim 7, a cell separation device for separating cells consisting of live cells and dead cells contained in the cell suspension; A culture device comprising: a measuring device for measuring the capacitance of the cell-free liquid from which the cells have been separated.

9. The culture device according to claim 8, A culture apparatus in which the method for separating the cells is a centrifugation method, a filtration method using a hollow fiber membrane, a filtration method using a flat plate membrane, a rotary filter method, or a gravitational sedimentation method.

10. The culture device according to claim 5, A culture apparatus in which the method for measuring the amount of suspended solids is a transmitted light measurement method, a scattered light measurement method, a transmitted light / scattered light comparison method, an integrating sphere method, or a particle counting method.

11. A step of measuring the amount of suspended matter including live cells and dead cells contained in a cell suspension that transmits light; Measuring the capacitance of the cell suspension, which varies depending on the viability of the cells; and calculating at least one of the number of live cells, the number of dead cells, the cell viability, the capacitance of all live cells, and the capacitance of all dead cells contained in the cell suspension based on the capacitance per unit of live cells, the capacitance per unit of dead cells, the amount of the suspended solid, and the capacitance of the cell suspension.

12. A turbidity sensor for measuring the amount of suspended solids, including live and dead cells, contained in a light-transmitting cell suspension; A measuring device for measuring the capacitance of the cell suspension, which changes depending on the viability of the cells; and a calculation unit that calculates at least one of the number of live cells, the number of dead cells, the cell viability, the capacitance of all live cells, and the capacitance of all dead cells contained in the cell suspension based on the capacitance per specified unit of live cell, the capacitance per specified unit of dead cell, the amount of the suspended solid, and the capacitance of the cell suspension.

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