Quantitative detection device, quantitative detection method, and cell culturing system

Abstract

The present invention is a quantitative detection device and a quantitative detection method utilizing the device, said device comprising: a laser oscillator that oscillates a pulsed laser beam oscillating at an appropriate wavelength corresponding to lactic acid or lactate, which is a substance to be measured; an irradiation mechanism that irradiates, with the pulsed laser beam, a cell culture into which the substance to be measured has been dissolved; a photosensor that receives detection laser light output from the cell culture; and a control unit that controls operation of each constituent element. The control unit outputs an oscillation command to the laser oscillator so as to oscillate the pulsed laser beam at constant periodicity, extracts the detection signal from the photosensor as interval data for time periods that correspond to the constant periodicity, and on the basis of the interval data, computes the quantity of the substance to be measured within the cell culture. Further, the quantitative detection device may be combined with a culturing device and incorporated into a cell-culturing system so as to be configured to perform measurements on a cell culture under cultivation.

Description

Amount detection device, amount detection method, and cell culture system

[0001] The present invention relates to an amount detection device and an amount detection method, and more particularly to an amount detection technique for obtaining the amount of lactic acid or lactate dissolved in a cell culture.

[0002] In recent years, in the field of regenerative medicine, such as human tissue regeneration, research and development has been progressing on techniques for easily and efficiently culturing human-derived cells. Such cell culture is generally carried out using a culture device called a "bioreactor."

[0003] As one example, Patent Document 1 discloses a bioreactor system including a container defining an internal space for accommodating a cell culture, an impeller for stirring the inside of the container, a monitoring system for measuring parameter concentrations of the cell culture, and a controller, wherein the monitoring system further includes a light source that irradiates coherent light such as a laser, and a Raman spectrometer that receives scattered light reflected from the cell culture, and the controller performs spectral analysis of received light data from the Raman spectrometer to measure the concentrations of one or more parameters in the cell culture and controls the concentrations of the parameters to be maintained within a preset range.

[0004] Special Publication No. 2020-537126

[0005] When culturing the above-mentioned human-derived cells, etc., the concentration of lactic acid or lactate in the cell culture (culture solution) may be measured to determine whether the cell culture has progressed sufficiently, and the culture operation may be terminated when the lactic acid concentration reaches or exceeds a certain reference value. In this regard, for example, in the prior art bioreactor system disclosed in Patent Document 1, one or more parameters including lactic acid are measured in real time and the operation of the system is controlled to maintain the parameters within a predetermined range, but no consideration is given to terminating the culture operation using these parameters (especially the lactic acid concentration).

[0006] In particular, there has been no established method for independently measuring or determining the amount (concentration) of lactic acid or lactate in a cell culture solution. This means that it has not been possible to determine the appropriate timing for ending the culture, resulting in excessive culture times and unnecessary culture costs.

[0007] In light of the above, an object of the present application is to provide an amount detection device and amount detection method that can independently obtain only the amount of lactic acid or lactate contained in a cell culture.

[0008] According to one aspect of the present invention, a quantity detection device includes a laser oscillator that emits pulsed laser light at an appropriate wavelength corresponding to the substance to be measured, lactic acid or lactate, an irradiation mechanism that irradiates the pulsed laser light onto a cell culture in which the substance to be measured has dissolved, a light-receiving sensor that receives detection laser light output from the cell culture, and a control unit that controls the operation of each component.The control unit is configured to output an oscillation command to the laser oscillator to oscillate the pulsed laser light at a constant cycle, extract detection signals from the light-receiving sensor as section data at a time corresponding to the constant cycle, and calculate the quantity of the substance to be measured in the cell culture based on the section data.

[0009] In another aspect of the present invention, a quantity detection method for obtaining the quantity of the substance to be measured in a cell culture in which the substance to be measured has dissolved is irradiated with pulsed laser light oscillated at an appropriate wavelength corresponding to lactic acid or lactate, which is the substance to be measured, and for receiving detection laser light output from the cell culture with a light-receiving sensor, is configured to oscillate the pulsed laser light at a constant period, extract the detection signal from the light-receiving sensor as section data at a time corresponding to the constant period, and calculate the quantity of the substance to be measured based on the section data.

[0010] Furthermore, according to another aspect of the present invention, there is provided a cell culture system including a culture device that contains a cell culture and a quantity detection device that detects the amount of lactic acid or lactate, a measurement target substance, dissolved in the cell culture cultured in the culture device, wherein the quantity detection device includes a laser oscillator that emits pulsed laser light at an appropriate wavelength corresponding to the measurement target substance, an irradiation mechanism that irradiates the cell culture with the pulsed laser light, a light-receiving sensor that receives the detection laser light output from the cell culture, and a control unit that controls the operation of each component, and the control unit is configured to output an oscillation command to the laser oscillator to emit pulsed laser light at a constant cycle, extract the detection signal from the light-receiving sensor as section data at a time corresponding to the constant cycle, and calculate the amount of the measurement target substance in the cell culture based on the section data.

[0011] According to one aspect of the present invention, an oscillation command is output to a laser oscillator to oscillate pulsed laser light at a constant cycle, a detection signal from a light-receiving sensor is extracted as section data at a time corresponding to the constant cycle, and the amount of the substance to be measured in the cell culture is calculated based on the section data.Therefore, it is possible to independently obtain only the amount of lactic acid or lactate, which is the substance to be measured contained in the cell culture.

[0012] FIG. 1 is a schematic diagram showing the configuration of a quantity detection device according to a first embodiment, which is a representative example of the present invention. FIG. 2 is a block diagram showing an example of the configuration of a laser oscillator included in the quantity detection device shown in FIG. 1. FIG. 3 is a block diagram showing an example of the configuration of a control unit included in the quantity detection device shown in FIG. 1. FIG. 4 is a flowchart showing an overview of a quantity detection method according to the first embodiment. FIG. 5 is a plan view showing an overview of an operational procedure in which a cell culture is irradiated with pulsed laser light in the quantity detection device according to the first embodiment. FIG. 6 is a partial front view showing an overview of an operational procedure in which a cell culture is irradiated with pulsed laser light in the quantity detection device according to the first embodiment. FIG. 7 is a time series graph showing the relationship between various command signals and measurement data. FIG. 8 is a plan view showing an overview of an operational procedure in which a cell culture is irradiated with pulsed laser light in the quantity detection device according to a second embodiment. FIG. 9 is a partial front view showing an overview of an operational procedure in which a cell culture is irradiated with pulsed laser light in the quantity detection device according to the second embodiment. 10 is a time series graph showing the relationship between various command signals and measurement data. It is a schematic diagram showing the configuration of a cell culture system according to a third embodiment of the present invention. It is a schematic diagram showing an example of a quantity detection device according to the third embodiment shown in FIG. 9. It is a flowchart showing an overview of the quantity detection operation executed by the quantity detection device of the cell culture system according to the third embodiment. It is a schematic diagram showing a modified example of the quantity detection device according to the third embodiment shown in FIG. 9. It is a schematic diagram showing the configuration of a quantity detection device according to a fourth embodiment. It is a time series graph showing the relationship between various command signals and measurement data. It is a block diagram showing the configuration of a laser oscillator according to a first modified example of the quantity detection device shown in FIG. 13. It is a schematic diagram showing the main parts of a cell culture system to which a quantity detection device according to a second modified example is applied.

[0013] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, an embodiment of a quantity detection device, a quantity detection method, and a cell culture system according to a representative example of the present invention will be described with reference to the drawings.

[0014] As used herein, the term "cell culture" refers to cells of a living organism dispersed in a culture medium. An example of such a cell culture is one in which cells isolated from tissues or organs of a living organism, such as a human, are dispersed in a culture medium containing, for example, serum.

[0015] <First embodiment> Fig. 1 is a schematic diagram showing the configuration of an amount detection device according to a first embodiment, which is a representative example of the present invention. Fig. 2 is a block diagram showing an example of the configuration of a laser oscillator included in the amount detection device shown in Fig. 1. Fig. 3 is a block diagram showing an example of the configuration of a control unit included in the amount detection device shown in Fig. 1.

[0016] As shown in FIG. 1, the quantity detection device 100 according to the first embodiment includes, as an example, a laser oscillator 110 that emits pulsed laser light LB at an appropriate wavelength corresponding to the substance to be measured, which is lactic acid or lactate; an irradiation mechanism 120 that irradiates the pulsed laser light LB onto a cell culture CM containing the substance to be measured; a transport mechanism 130 that moves the position of the irradiation mechanism 120 in the X, Y, and Z directions; a sample holding mechanism 140 that moves the position of a container 142 containing the cell culture CM in the X, Y, and Z directions; a light receiving sensor 150 that receives detection laser light output from the cell culture CM; and a control unit 160 that controls the operation of each component.

[0017] The laser oscillator 110 is a laser light source that outputs a wavelength (e.g., a wavelength with high absorption efficiency) appropriate for detecting lactic acid or lactate (see reference symbol MM in FIG. 5C , which will be described later) that is a measurement target substance contained in the cell culture CM. As an example, as shown in FIG. 2 , the laser oscillator 110 includes an oscillation control unit 112 that controls the oscillation of the pulsed laser light LB at a constant period T based on an oscillation signal from the control unit 160, a drive power supply 113 that supplies drive power to the multiple laser light sources 115 a, 115 b in response to an on / off signal from the oscillation control unit 112, a support unit 114 to which the laser light sources 115 a, 115 b are attached, a focusing lens 116 that focuses the pulsed laser light LB emitted from the multiple laser light sources 115 a, 115 b, a wavelength adjustment unit 117 that adjusts the wavelength of the emitted pulsed laser light LB, and a transmission path 118 (e.g., an optical fiber) that transmits the focused pulsed laser light LB to the irradiation mechanism 120.

[0018] Here, the wavelength of the pulsed laser light LB appropriate for detecting the measurement target substance, lactic acid or lactate, is, for example, 1480 nm. Therefore, in order to selectively irradiate the cell culture CM with the pulsed laser light LB of the appropriate wavelength (1480 nm), the multiple laser light sources 115a, 115b can be an array of light-emitting diodes (LEDs) or semiconductor lasers (LDs), a laser oscillating at millimeter waves, submillimeter waves, or microwaves, or a free electron laser. As a result, when the pulsed laser light LB of the above wavelength is irradiated onto the cell culture CM, it is selectively absorbed or reflected by the measurement target substance, lactic acid or lactate, while being transmitted through other substances such as the cells themselves and the culture medium.

[0019] Then, the plurality of pulsed laser beams emitted from the plurality of laser light sources are coaxially focused, and the high-power pulsed laser beam LB is transmitted through a wavelength tuning unit 117, whereby the pulsed laser beam LB selected to fall within a predetermined appropriate wavelength range is emitted to a transmission path 118. Here, an example of the wavelength tuning unit 117 is a band-pass filter that selectively removes light outside a range between predetermined upper and lower limits.

[0020] 2 illustrates an example in which two light sources are arranged as the plurality of laser light sources 115a, 115b, but a configuration in which a larger number (three or more) of laser light sources are arranged in an array or on a circumference is also possible. Also, while the configuration in which drive power is directly supplied to the plurality of laser light sources 115a, 115b from the drive power source 113 is illustrated, a configuration in which, for example, an amplifier circuit (not shown) is provided in the support portion 114, and the drive power from the drive power source 113 is amplified and supplied to the laser light sources 115a, 115b is also possible.

[0021] As an example, the irradiation mechanism 120 introduces pulsed laser light LB from one end (upper end) via the transmission line 118, shapes the light into a beam profile with a predetermined beam diameter and beam cross-sectional shape by a focusing lens (not shown) disposed inside, and then irradiates the light from the other end (lower end) toward the cell culture CM contained in the container 142. Note that, although the first embodiment illustrates a case in which the pulsed laser light LB is shaped so that the beam spot has a circular cross section, it is also possible to shape the beam profile into any shape, such as a polygon or a line, by appropriately selecting the focusing lens.

[0022] As an example, the transport mechanism 130 is configured as a linear drive body that moves relatively in three mutually orthogonal axis directions of X, Y, and Z, and has the irradiation mechanism 120 attached to one end of the linear drive body. Note that the transport mechanism 130 may also be configured as a six-axis or seven-axis industrial robot equipped with a robot arm having the irradiation mechanism 120 attached to one end thereof.

[0023] As an example, the sample holding mechanism 140 is configured as a table that can move freely in three axes directions (X, Y, and Z) as shown in the figure, with a container 142 containing a cell culture CM obtained in advance from, for example, a culture device, placed on its upper surface. In addition, a light receiving sensor 150 that detects transmitted light from the cell culture CM is disposed between the upper surface of the sample holding mechanism 140 and the lower surface of the container 142.

[0024] The container 142 containing the cell culture CM is made of a material that is transparent to the wavelength of the pulsed laser light LB (i.e., the irradiated pulsed laser light LB passes through). To prevent contamination of the cell culture CM, it is preferable to provide a sealed structure with a lid on the top side where the pulsed laser light LB is incident. In this way, the pulsed laser light irradiated onto the cell culture CM contained in the container 142 passes through the bottom of the container 142 and reaches the light-receiving sensor 150 in areas where the substance to be measured is not present, and the light-receiving sensor 150 detects the irradiation.

[0025] 3, the control unit 160 includes, for example, a main control unit 162 that outputs operation commands to each component of the amount detection device 100, an amount calculation unit 164 that calculates the amount of the target substance contained in the cell culture CM using the detection value from the light-receiving sensor, a display unit 166 that displays the calculated amount of the target substance and other various parameters, and an input interface 168 that allows manual input of information for correcting various parameters such as measurement conditions. The main control unit 162 of the control unit 160 is connected to the laser oscillator 110, the transport mechanism 130, and the sample holding mechanism 140 by wire or wirelessly, and controls the operation of the entire amount detection device 100 by exchanging signals with these peripheral devices.

[0026] For example, when a start signal Ss corresponding to the start of measurement is input by the user via the input interface 168, the main control unit 162 has the function of extracting, from a predetermined measurement program, operation information such as oscillation of the laser oscillator 110 and relative movement of the transport mechanism 130 and the sample holding mechanism 140, generating an oscillation signal So and a relative movement signal Sm for executing these operations and outputting them to each component. The main control unit 162 also has the function of outputting an oscillation signal So to a quantity calculation unit 164 (described later) in synchronization with the output to the laser oscillator 110, and receiving the calculation results from the quantity calculation unit 164, sending the calculation results of the quantity and various current parameters of the quantity detection device 100 to a display unit 166 to display them.

[0027] The quantity calculation unit 164 has a function of receiving a calculation signal Se corresponding to the start of calculation from the main control unit 162 that has received the above-mentioned start signal, and then continuously receiving and accumulating a detection signal Sd corresponding to the detection value at time t from the light-receiving sensor 150. The quantity calculation unit 164 also has a function of receiving an oscillation signal So from the main control unit 162, extracting section data D synchronized with the oscillation timing of the laser oscillator 110 from the time-series data of the detection signal Sd detected by the light-receiving sensor 150, and calculating the quantity of the target substance to be measured contained in the cell culture CM based on the section data D. The calculated quantity of the target substance to be measured is then sent to the main control unit 162.

[0028] Next, specific operational aspects of the amount detection method executed in the amount detection device according to the first embodiment will be described with reference to FIGS.

[0029] Fig. 4 is a flowchart showing an outline of the quantity detection method according to the first embodiment. Fig. 5A is a plan view showing an outline of the operational procedure for irradiating a cell culture with pulsed laser light in the quantity detection device according to the first embodiment. Fig. 5B and Fig. 5C are partial front views showing an outline of the operational procedure for irradiating a cell culture with pulsed laser light in the quantity detection device according to the first embodiment. Fig. 6 is a time series graph showing the relationship between various command signals and measurement data.

[0030] 4, in the amount detection method executed by the amount detection device 100 according to the first embodiment, when a start signal Ss corresponding to the start of measurement is input by the user via the input interface 168, the main control section 162 of the control unit 160 first commands the amount calculation section 164 to start receiving the detection signal Sd from the light receiving sensor 150 (step S101). As a result, the amount calculation section 164 continuously receives and temporarily stores the detection signal Sd from the light receiving sensor 150 as time-series data until the operation shown in the flowchart is completed.

[0031] Next, the main control unit 162 outputs a relative movement signal Sm to the transport mechanism 130 and the sample holding mechanism 140 based on a predetermined measurement program (step S102), thereby determining the position and focal length at which the pulsed laser beam LB is irradiated onto the cell culture CM.

[0032] Next, the main control unit 162 outputs an oscillation signal So to the laser oscillator 110 to cause the laser oscillator 110 to emit a pulsed laser beam LB for a predetermined irradiation time Ton (step S103). In the laser oscillator 110 that has received the oscillation signal So, the oscillation control unit 112 outputs an ON command signal Son to the drive power supply 113 for the irradiation time Ton, and the pulsed laser beam LB adjusted to a predetermined wavelength is emitted.

[0033] Next, the main control unit 162 determines, based on the measurement program, whether or not irradiation of the pulsed laser beam LB has been completed in all measurement ranges defined for the cell culture CM in the container 142 (step S104). That is, if it is determined in step S104 that irradiation has been completed in all measurement ranges, the main control unit 162 outputs a calculation signal Se to the quantity calculation unit 164, indicating that measurement of the cell culture CM has been completed, and proceeds to the subsequent step S105.

[0034] On the other hand, if it is determined in step S104 that irradiation has not been completed in all measurement ranges, the process returns to step S102, and the positioning and irradiation of the pulsed laser beam LB for the unfinished measurement ranges according to the measurement program are repeatedly performed, thereby detecting the measurement target substance in all ranges (regions) to be measured of the cell culture CM contained in the container 142.

[0035] 5A to 5C, a specific example of the operational procedure from step S102 to step S104 will be described. First, as shown in Fig. 5A, a cell culture CM is contained in a container 142 placed on a light-receiving sensor 150 having a sensing surface on its upper surface, and a plurality of rectangular regions C are defined in the cell culture CM, each having a length and width corresponding to the focal diameter (spot diameter) of the focal point FP of the pulsed laser beam LB.

[0036] Then, for the plurality of rectangular regions defined as above, the measurement program further defines an irradiation start position Ps and an irradiation end position Pe of the pulsed laser beam LB, and specifies a scanning path for scanning in the X and Y directions between the irradiation start position Ps and the irradiation end position Pe. Note that the determination in step S104 of the flowchart shown in FIG. 4 is made based on whether the current irradiation position (focus point FP) coincides with the irradiation end position Pe on the scanning path.

[0037] Next, a description will be given below of the relationship between the presence or absence of the measurement target substance MM at the irradiation position (focus point FP) of the pulsed laser beam LB and the detection signal from the light-receiving sensor 150. For example, as shown in Fig. 5B , if the measurement target substance MM is not present at the focus point FP of the irradiated pulsed laser beam LB or on its extension, the pulsed laser beam LB passes through the cell culture CM and the container 142, and transmitted light TB corresponding to the output of the irradiated pulsed laser beam LB is detected at the light-receiving point DP of the light-receiving sensor 150.

[0038] On the other hand, as shown in Fig. 5C , when the measurement target substance MM is present at the focal point FP of the irradiated pulsed laser beam LB or on an extension thereof, the pulsed laser beam LB is absorbed or reflected by the measurement target substance MM, and transmitted light TB having a lower output than the output of the irradiated pulsed laser beam LB is detected at the light-receiving point DP of the light-receiving sensor 150. Note that Fig. 5C illustrates an example in which the measurement target substance MM is larger than the focal diameter of the focal point FP of the pulsed laser beam LB, but a similar tendency is observed even when the measurement target substance MM is smaller than the focal diameter.

[0039] 6, the quantity calculation unit 164, which has received the calculation signal from the main control unit 162, extracts section data D by extracting a section of the detection signal Sd from the light receiving sensor 150 corresponding to the section during which the oscillation signal So from the main control unit 162 was received (step S105). This makes it possible to narrow down the data to only the section of the detection signal Sd corresponding to the irradiation time Ton during which the pulsed laser beam LB was irradiated (i.e., noise during detection can be reduced). Here, the irradiation time Ton and non-irradiation time Toff of the oscillation signal So are defined together as one period T.

[0040] Next, the amount calculation unit 164 calculates the amount of the analyte substance MM relative to the cell culture CM based on the extracted section data D. Specifically, the section data D extracted in step S105 includes, as an example, output values ​​at two levels: reference data Ds (the state shown in FIG. 5B ) when the analyte substance MM is not detected (non-detection section Tn), and detection data Dd (the state shown in FIG. 5C ) when the analyte substance MM is detected (detection section Td).

[0041] At this time, it can be determined that the greater the absolute value of the difference ΔD between the reference data Ds and the detection data Dd, the greater the amount of the target substance MM detected. After measuring all of the target measurement ranges, the amount calculation unit 164 adds up the number of detection data Dd in the entire section data D, outputs this as the "amount" of the target substance MM to the main control unit 162, and then terminates its operation (step S106).

[0042] The amount of the measurement target substance MM may be calculated as a ratio to the total amount rather than as an integrated number. Also, a threshold may be set for the difference ΔD between the reference data Ds and the detection data Dd, and anything exceeding the predetermined threshold may be determined to have been "detected."

[0043] By being configured as described above, the quantity detection device and quantity detection method according to the first embodiment are configured to output an oscillation command to a laser oscillator to oscillate pulsed laser light at a constant cycle, extract the detection signal from the light receiving sensor as interval data at a time corresponding to the constant cycle, and calculate the quantity of the substance to be measured in the cell culture based on the interval data, so that only the quantity of lactic acid or lactate, which is the substance to be measured contained in the cell culture, can be obtained independently.

[0044] Furthermore, according to the amount detection method described in the first embodiment, for example, the amount of lactic acid or lactate dissolved in a cell culture cultured in a culture device can be independently obtained, and therefore, when the amount or concentration of lactic acid or lactate obtained exceeds a predetermined threshold or is within a predetermined range, it is possible to determine at an appropriate time to stop the culture operation in the culture device. This makes it possible to appropriately control the time required for cell culture, thereby improving culture efficiency.

[0045] Second Embodiment Next, an embodiment of an amount detection device and an amount detection method according to a second embodiment, which is another example of the present invention, will be described using Figures 7A to 8. In the second embodiment, in the schematic diagrams shown in Figures 1 to 6, components that can adopt the same or common configurations as those in the first embodiment are assigned the same reference numerals, and repeated description of these components will be omitted.

[0046] Fig. 7A is a plan view showing an outline of an operation procedure for irradiating a cell culture with pulsed laser light in the quantity detection device according to the second embodiment. Fig. 7B and Fig. 7C are a plan view and a partial front view showing an outline of an operation procedure for irradiating a cell culture with pulsed laser light in the quantity detection device according to the second embodiment. Fig. 8 is a time series graph showing the relationship between various command signals and measurement data.

[0047] The quantity detection device 100 according to the second embodiment uses a measurement method of detecting reflected light from a measurement target substance MM contained in the cell culture CM, whereas the device of the first embodiment uses a measurement method of detecting transmitted light from the cell culture CM with a light-receiving sensor 150. That is, as shown in Fig. 7A, the cell culture CM is contained in a container 142 placed on a sample holding mechanism 140, and a pulsed laser beam LB is irradiated onto a predetermined measurement range of the cell culture CM, as in the first embodiment.

[0048] At this time, in the quantity detection device 100 according to the second embodiment, the reflected light RB reflected by the measurement target substance MM contained in the cell culture CM is detected by the light-receiving sensor 250 attached to the irradiation mechanism 120. That is, for example, as shown in Fig. 7B, if the measurement target substance MM is not present at the focal point FP of the irradiated pulsed laser beam LB or on its extension, the pulsed laser beam LB passes through the cell culture CM and the container 142, and therefore the light-receiving sensor 250 detects only a detection value based on the amount of light around the device.

[0049] On the other hand, as shown in Fig. 7C , when the measurement target substance MM is present at the focal point FP of the irradiated pulsed laser beam LB or on an extension thereof, the pulsed laser beam LB is absorbed or reflected by the measurement target substance MM, and reflected light RB, which is a portion of the output of the irradiated pulsed laser beam LB, is detected at the light-receiving point DP of the light-receiving sensor 250. Note that Fig. 7C shows the same tendency even when the measurement target substance MM is smaller than the focal diameter of the focal point FP of the pulsed laser beam LB, as in the first embodiment.

[0050] 8, in the amount detection device 100 according to the second embodiment, a detection signal Sd having a large detection value is received in a section in which the light receiving sensor 250 detects the reflected light RB. The amount calculation unit 164 then extracts the section data D by cutting out the section of the detection signal Sd from the light receiving sensor 250 that corresponds to the section in which the oscillation signal So from the main control unit 162 was received. This makes it possible to narrow down the data to only the section in the detection signal Sd that corresponds to the irradiation time Ton during which the pulsed laser beam LB was irradiated, as in the first embodiment.

[0051] Next, the amount calculation unit 164 calculates the amount of the analyte substance MM relative to the cell culture CM based on the extracted section data D. Specifically, the extracted section data D includes, as an example, output values ​​at two levels: reference data Ds when the analyte substance MM is not detected (non-detection section Tn), and detection data Dd when the analyte substance MM is detected (detection section Td).

[0052] In this case, as in the first embodiment, it can be determined that the greater the absolute value of the difference ΔD between the reference data Ds and the detection data Dd, the greater the detected amount of the measurement target substance MM. Therefore, after measuring all of the target measurement ranges, the amount calculation unit 164 adds up the number of detection data Dd in the entire section data D and outputs the calculation result to the main control unit 162 as the "amount" of the measurement target substance MM.

[0053] By being provided with the above-described configuration, the quantity detection device and quantity detection method according to the second embodiment can, in addition to the effects described in the first embodiment, employ a method of measuring reflected light from the substance to be measured contained in the cell culture, thereby adopting a structure in which the light receiving sensor is arranged alongside the irradiation mechanism, thereby making it possible to make the entire device compact.

[0054] Third Embodiment Next, a cell culture system according to a third embodiment, which is another example of the present invention, will be described with reference to Figures 9 to 12. In the third embodiment, in the schematic diagrams shown in Figures 1 to 8, components that can adopt the same or common configurations as those in the first and second embodiments are designated by the same reference numerals, and repeated description of these components will be omitted.

[0055] Fig. 9 is a schematic diagram showing the configuration of a cell culture system according to a third embodiment as a representative example of the present invention. Fig. 10 is a schematic diagram showing an example of a quantity detection device according to the third embodiment shown in Fig. 9. Fig. 11 is a flowchart showing an outline of a quantity detection operation executed by the quantity detection device of the cell culture system according to the third embodiment. Fig. 12 is a schematic diagram showing a modified example of the quantity detection device according to the third embodiment shown in Fig. 9.

[0056] As shown in Figure 9, the cell culture system 1 according to the third embodiment includes a culture device 10 that contains a cell culture CM, and a quantity detection device 300 that detects the amount of lactic acid or lactate, which is the substance to be measured, that has dissolved into the cell culture CM cultured in the culture device 10.

[0057] As an example, the culture device 10 is composed of a tank 20 supported by multiple legs 22 and containing a cell culture CM therein, a motor 30 attached to the upper end of the tank 20, an upper port 40 connected to a supply unit 42 that supplies additional substances, etc. to the cell culture CM, a lower port 44 provided at the lower end of the tank and connected to an extraction unit 46 that extracts the cell culture CM after cultivation, and a circulation path 50 branching off from the tank 20 and connected to a quantity detection device 300.

[0058] The tank 20 is made of, for example, a resin that is impermeable to liquids and has hydrophilicity, such as a hard polymer or a flexible polymer. Examples of such resins include polymers such as polyolefin, polypropylene, polyethylene, and polyamide. Although not shown, the tank 20 may be provided with a baffle, a gas injection pipe (sparger), a heat exchange unit, and the like, as needed.

[0059] The motor 30 is configured to rotate a rotating shaft 32 to which a plurality of blades (impellers) 34a, 34b are attached to agitate the cell culture CM contained in the tank 20. While Fig. 9 illustrates an example in which two blades 34a, 34b are attached to the rotating shaft 32, the number of blades may be three or more to increase the fluidity of the cell culture CM.

[0060] 10 , the amount detecting device 300, like the first embodiment, includes a laser oscillator 110, an irradiation mechanism 320, a holding mechanism 330 that holds the irradiation mechanism 320, a light receiving sensor 350, and a control unit 160. The amount detecting device 300 in the third embodiment is configured so that pulsed laser light LB emitted from the irradiation mechanism 320 is irradiated toward the culture solution flow MF that flows in the circulation path 50 branching from the tank 20 of the culture device 10.

[0061] 10 , the circulation path 50 has a conduit 52 in the region irradiated with the pulsed laser beam LB, which is made of a material that is transparent to the wavelength of the pulsed laser beam LB (i.e., the irradiated pulsed laser beam LB passes through). As a result, the pulsed laser beam LB irradiated toward the culture solution flow MF of the cell culture CM, which flows continuously through the region of the conduit 52, passes through the conduit 52 and the culture solution flow MF, and is then detected by a light-receiving sensor 350 provided at a position opposite the irradiation mechanism 320 across the conduit 52.

[0062] In this state of arrangement, pulsed laser light LB is irradiated for a predetermined time by on / off control at a constant cycle T, while receiving a detection signal Sd from the light-receiving sensor 350. As a result, instead of using the method of scanning and measuring the measurement area of ​​the cell culture CM in the container 142 in the first embodiment, in the third embodiment, it is possible to acquire time-series data of the cell culture CM continuously flowing in the circulation path 50.

[0063] 11 , in the cell culture system 1 according to the third embodiment, when a start signal Ss corresponding to the start of measurement is input by the user via the input interface 168, the main control section 162 of the control unit 160 first instructs the quantity calculation section 164 to start receiving the detection signal Sd from the light-receiving sensor 150 (step S301). As a result, the quantity calculation section 164 continuously receives and temporarily stores the detection signal Sd from the light-receiving sensor 350 as time-series data until the operation shown in the flowchart is completed.

[0064] Next, the main control unit 162 outputs an oscillation signal So to the laser oscillator 110 to cause the laser oscillator 110 to emit a pulsed laser beam LB for a predetermined irradiation time Ton based on a predetermined measurement program (step S302). In the laser oscillator 110 that has received the oscillation signal So, the oscillation control unit 112 outputs an on-command signal Son to the drive power supply 113 for the irradiation time Ton, as in the first embodiment, and the pulsed laser beam LB adjusted to a predetermined wavelength is emitted.

[0065] Next, the main control unit 162 determines whether the irradiation of the pulsed laser beam LB at the constant period T has been completed for a predetermined number of cycles based on the measurement program (step S303). That is, if it is determined in step S303 that the irradiation has been completed for the predetermined number of cycles, the main control unit 162 outputs a calculation signal Se to the quantity calculation unit 164, indicating that the measurement of the cell culture CM has been completed, and the process proceeds to the subsequent step S304.

[0066] On the other hand, if it is determined in step S303 that the irradiation has not been completed for the predetermined number of cycles, the process returns to step S302 and the irradiation of the pulsed laser beam LB for one cycle is repeated, thereby executing the detection operation for the measurement target substance in the cell culture CM continuously flowing through the circulation path 50 for a predetermined time.

[0067] Next, similarly to the first embodiment, the quantity calculation unit 164 that has received the calculation signal from the main control unit 162 extracts section data D by cutting out a section of the detection signal Sd from the light receiving sensor 350 that corresponds to the section during which the oscillation signal So from the main control unit 162 was received (step S304). This makes it possible to narrow down only the data of the section in the detection signal Sd that corresponds to the irradiation time Ton during which the pulsed laser beam LB was irradiated.

[0068] Next, as in the first embodiment, the quantity calculation unit 164 calculates the quantity of the analyte substance MM in the cell culture CM based on the extracted section data D. That is, after measuring the time-series data for a predetermined number of periods, the quantity calculation unit 164 adds up the number of detection data Dd in all of the section data D, outputs this as the "quantity" of the analyte substance MM to the main control unit 162, and ends the operation (step S305).

[0069] In the cell culture system 1 according to the third embodiment, the quantity detection device 300 may be configured to detect reflected light RB from the cell culture CM in combination with the technology described in the second embodiment. That is, as shown in FIG. 11 , in the quantity detection device 300 according to a modified example of the third embodiment, the reflected light RB reflected by the measurement target substance MM contained in the cell culture CM is detected by a light-receiving sensor 350 attached to the irradiation mechanism 320. At this time, since a portion of the pulsed laser beam LB emitted from the irradiation mechanism 320 is transmitted through the conduit 52 of the circulation path 50 to the rear side, a damper 352 that absorbs the transmitted pulsed laser beam LB is preferably provided at a position facing the irradiation mechanism 320 across the conduit 52.

[0070] By being provided with the above-described configuration, the cell culture system according to the third embodiment has the advantages described in the first and second embodiments, in addition to the advantages described in the first and second embodiments. For example, since the cell culture is measured in-line without removing it from the culture device, contamination during measurement can be reliably prevented and an additional configuration for sample acquisition can be eliminated. Furthermore, since the step of removing the cell culture CM to be measured into a container is not required, the overall measurement time can be shortened.

[0071] Furthermore, according to the cell culture system shown in the third embodiment, as in the first embodiment, for example, only the amount of lactic acid or lactate dissolved in a cell culture cultured in a culture device can be obtained inline and independently during the culture operation, so that when the amount or concentration of lactic acid or lactate obtained exceeds a predetermined threshold or is within a predetermined range, it is possible to make a decision at an appropriate time to stop the culture operation in the culture device. This makes it possible to appropriately control the time required for cell culture, thereby improving culture efficiency.

[0072] 9 to 12, a culture device having a structure using a tank equipped with internal stirring blades (impellers) is exemplified, but a general culture device may also be applied, such as a perfusion culture device that has a function of stirring by vibrating the container while injecting the cell culture medium with a pump, etc. Also, while a case where a circulation path is provided in the tank as a mechanism for irradiating the cell culture medium with pulsed laser light is exemplified, any structure that can irradiate the cell culture medium with pulsed laser light inline (during the culturing operation) may be adopted.

[0073] <Fourth embodiment> Next, an embodiment of a quantity detection device, a quantity detection method, and a cell culture system according to a fourth embodiment, which is yet another example of the present invention, will be described using Figures 13 to 16. In the fourth embodiment, in the schematic diagrams shown in Figures 1 to 12, components that can adopt the same or common configurations as those in the first to third embodiments are assigned the same reference numerals, and repeated description of these components will be omitted.

[0074] Fig. 13 is a schematic diagram showing the configuration of an amount detection device according to a fourth embodiment. Fig. 14 is a time series graph showing the relationship between various command signals and measurement data. Fig. 15 is a block diagram showing the configuration of a laser oscillator according to a first modified example of the amount detection device shown in Fig. 13. Fig. 16 is a schematic diagram showing the main parts of a cell culture system to which a amount detection device according to a second modified example of the fourth embodiment is applied.

[0075] It is generally known that lactic acid and pyruvic acid have similar chemical structures and absorbance characteristics, so when pyruvic acid is present in addition to lactic acid in a cell culture CM, the detection signal may contain detection information for not only lactic acid or lactate but also pyruvic acid.

[0076] Therefore, the quantity detecting device 400 according to the fourth embodiment differs from the first embodiment in that it includes a plurality of laser oscillators 410a, 410b that output a first pulsed laser beam LBa and a second pulsed laser beam LBb that are set to different appropriate wavelengths corresponding to different measurement target substances. This makes it possible for the quantity detecting device 400 according to the fourth embodiment to obtain the quantities of a plurality of measurement target substances MM1, MM2 from a single cell culture CM.

[0077] Specifically, as shown in FIG. 13 , the quantity detection device 400 according to the fourth embodiment includes: a laser oscillator 410 a that oscillates a first pulsed laser beam LBa at an appropriate wavelength corresponding to lactic acid or lactate, which is a first substance to be measured; a laser oscillator 410 b that oscillates a second pulsed laser beam LBb at an appropriate wavelength corresponding to pyruvic acid, which is a second substance to be measured; an irradiation mechanism 120 that irradiates a cell culture CM containing the first substance to be measured and the second substance to be measured with the first pulsed laser beam LBa and the second pulsed laser beam LBb; a transport mechanism 130 that moves the position of the irradiation mechanism 120 in the X, Y, and Z directions; a sample holding mechanism 140 that moves the position of a container 142 containing the cell culture CM in the X, Y, and Z directions; a light-receiving sensor 150 that receives the detection laser beam output from the cell culture CM; and a control unit 160 that controls the operation of each component.

[0078] In the fourth embodiment, the appropriate wavelength of the first pulsed laser beam LBa for detecting lactic acid or lactate as the first analyte is, for example, 1480 nm, as in the first embodiment, whereas the appropriate wavelength of the second pulsed laser beam LBb for detecting pyruvic acid as the second analyte is, for example, 1462 nm.

[0079] The first pulsed laser beam LBa and the second pulsed laser beam LBb output from the laser oscillators 410a and 410b are introduced into one end (upper end) of the irradiation mechanism 120 via transmission lines 418a and 418b, respectively. The pulsed laser beams LBa and LBb are then shaped into coaxial beam profiles by a focusing optical system (not shown) arranged inside the irradiation mechanism 120, and are then irradiated from the other end (lower end) toward the cell culture CM accommodated in the container 142. Note that in the fourth embodiment as well, the beam spots of the first pulsed laser beam LBa and the second pulsed laser beam LBb can be shaped into beam profiles of any shape, such as a polygon or a line.

[0080] 14, the relationship between the detection signal Sd and various signals obtained in the light receiving sensor 150 according to this configuration includes a first detection interval T1d and a second detection interval T2d corresponding to the two oscillation signals S1o and S2o. Similarly to the first embodiment, the quantity calculation unit 164, upon receiving a calculation signal from the main control unit 162, extracts the first interval data D1 and the second interval data D2 by extracting the interval of the detection signal Sd corresponding to the interval during which the oscillation signal S1o was received and the interval of the detection signal Sd corresponding to the interval during which the oscillation signal S2o was received. This allows narrowing down the data to only those intervals corresponding to the irradiation time T1on during which the first pulsed laser beam LBa was irradiated and the irradiation time T2on during which the second pulsed laser beam LBb was irradiated.

[0081] Next, the quantity calculation unit 164 calculates the quantities of the first analyte substance MM1 and the second analyte substance MM2 relative to the cell culture CM based on the extracted first section data D1 and second section data D2. Specifically, the extracted first section data D1 and second section data D2 include output values ​​at two levels, for example, reference data Ds when the first analyte substance MM1 and the second analyte substance MM2 are not detected (non-detection section Tn), and detection data D1d and D2d when the first analyte substance MM1 and the second analyte substance MM2 are detected.

[0082] In this case, it can be determined that the larger the absolute values ​​of the difference ΔD1 between the reference data Ds and the detection data D1d and the difference ΔD2 between the reference data Ds and the detection data D2d, the greater the detected amount of the first analyte MM1 or the second analyte MM2. After measuring all of the target measurement ranges, the amount calculation unit 164 adds up the numbers of detection data D1d and D2d in the entire first section data D1 and the entire second section data D2, and outputs the results to the main control unit 162 as the "amounts" of the first analyte MM1 and the second analyte MM2, respectively.

[0083] The amounts of the first analyte MM1 and the second analyte MM2 thus obtained can be used to determine a more precise amount of lactic acid or lactate. For example, if the first analyte MM1 is likely to contain not only "lactic acid or lactate" but also "pyruvic acid," which has a similar chemical structure, and the amount of "pyruvic acid" is obtained as the second analyte MM2, it is possible to obtain the amount of only "lactic acid or lactate" without including "pyruvic acid" by subtracting the amount of the second analyte MM2 from the amount of the first analyte MM1.

[0084] As in the first embodiment, the amounts of the first and second target substances MM1 and MM2 may be calculated as a ratio to the total rather than as an integrated number. Also, a threshold may be set for the difference ΔD1 or ΔD2, and anything exceeding the predetermined threshold may be determined to have been "detected."

[0085] Next, a first modified example of the fourth embodiment will be described with reference to Fig. 15. The amount detection device 400 according to the first modified example of the fourth embodiment includes, as an example, a single laser oscillator 410 configured instead of the two laser oscillators 410a and 410b shown in Fig. 13.

[0086] That is, as shown in FIG. 15 , the laser oscillator 410 includes an oscillation control unit 112, a drive power supply 113, a support unit 114 to which a plurality of laser light sources 415 a, 415 b are attached, a focusing lens 416 a that focuses the first pulsed laser beam LBa emitted from the laser light source 415 a, a focusing lens 416 b that focuses the second pulsed laser beam LBb emitted from the laser light source 415 b, wavelength adjustment units 417 a, 417 b that adjust the wavelengths of the emitted first pulsed laser beam LBa and second pulsed laser beam LBb, and transmission paths 418 a, 418 b (e.g., optical fibers) that transmit the focused first pulsed laser beam LBa and second pulsed laser beam LBb to the irradiation mechanism 120, respectively.

[0087] As in the first embodiment, the multiple laser light sources 415a, 415b may be configured to have a high output by arranging multiple light emitting diodes (LEDs) or semiconductor lasers (LDs) in an array. Also, a separate amplifier circuit (not shown) may be provided to amplify and supply drive power from the drive power supply 113 to the multiple laser light sources 415a, 415b. This configuration allows the amount detection device 400 to occupy a reduced overall space.

[0088] Furthermore, in the quantity detection device 400 according to the first modified example shown in Figure 15, an example is given in which two different first pulsed laser beams LBa and LBb are used to detect two measurement target substances MM1 and MM2, but it is also possible to apply a combination of pulsed laser beams with three or more different wavelengths to detect three or more measurement target substances.

[0089] Next, a second modified example of the fourth embodiment will be described with reference to Fig. 16. As shown in Fig. 16, a quantity detection device 400 according to the second modified example of the fourth embodiment includes, as an example, an irradiation mechanism 420 that coaxially focuses and emits a first pulsed laser beam LBa emitted from a laser oscillator 410a and a second pulsed laser beam LBb emitted from a laser oscillator 410b onto a cell culture CM flowing in a circulation path 50, and a holding mechanism 430 that holds the irradiation mechanism 420.

[0090] The quantity detection device 400 according to the second modification is configured to detect transmitted light TB that has passed through the conduit 52 of the circulation path 50 and the culture solution flow MF by a light-receiving sensor 450 provided at a position opposite the irradiation mechanism 420 across the conduit 52. Note that, as shown in Fig. 12 in the third embodiment, the light-receiving sensor 450 attached to the irradiation mechanism 420 may be configured to detect reflected light RB that has been reflected by the cell culture CM flowing through the circulation path 50.

[0091] By being provided with the above-described configuration, the quantity detection device and quantity detection method according to the fourth embodiment, and the cell culture system to which they are applied, have the advantages described in the first to third embodiments, in addition to the above-described advantages. Since the detection is performed using multiple laser oscillators that output pulsed laser light set to different appropriate wavelengths corresponding to multiple target substances, it is possible to simultaneously obtain the quantities of multiple target substances from a single cell culture. This makes it possible to obtain a more precise quantity of lactic acid or lactate by excluding the influence of the detection of pyruvic acid, which has a chemical structure and absorbance characteristics similar to those of lactic acid or lactate.

[0092] The present invention is not limited to the above-described embodiments, and can be modified as appropriate without departing from the spirit and scope of the invention. Any of the components of the embodiments can be modified or omitted within the scope of the invention. For example, the specific examples shown in the first to fourth embodiments can be applied by combining their respective features.

[0093] 1 Cell culture system 10 Culture device 20 Tank 22 Leg 30 Motor 32 Rotating shaft 34a, 34b Blade 40 Upper port 42 Supply section 44 Lower port 46 Extraction section 50 Circulation path 52 Pipe 100, 300, 400 Amount detection device 110, 410, 410a, 410b Laser oscillator 112 Oscillation control section 113 Driving power supply 114 Support section 115a, 115b, 415a, 415b Laser light source 116, 416a, 416b Condenser lens 117, 417a, 417b Wavelength adjustment section 118, 418a, 418b Transmission path 120, 320, 420 Irradiation mechanism 130 Transport mechanism 140 Sample holding mechanism 142 Container 150, 250, 350, 450 Light receiving sensor 160 Control unit 162 Main control unit 164 Amount calculation unit 166 Display unit 168 Input interface 330, 430 Holding mechanism 352 Damper

Claims

1. A quantity detection device including a laser oscillator that emits a pulsed laser beam at an appropriate wavelength corresponding to the substance to be measured, which is lactic acid or a lactate salt, an irradiation mechanism that irradiates a cell culture in which the substance to be measured has been dissolved with the pulsed laser beam, a light receiving sensor that receives detection laser beam output from the cell culture, and a control unit that controls the operation of each component, wherein the control unit outputs an oscillation command to the laser oscillator to oscillate the pulsed laser beam at a constant cycle, extracts detection signals from the light receiving sensor as section data at a time corresponding to the constant cycle, and calculates the quantity of the substance to be measured in the cell culture based on the section data.

2. The quantity detection device according to claim 1, wherein the appropriate wavelength is 1480 nm.

3. The amount detection device according to claim 1 or 2, wherein the light receiving sensor is configured to receive reflected light of the pulsed laser light.

4. The amount detection device according to any one of claims 1 to 3, further comprising a second laser oscillator that oscillates a second pulsed laser light oscillated at a second appropriate wavelength corresponding to pyruvic acid, wherein the second pulsed laser light is irradiated onto the cell culture so as to be coaxial with the pulsed laser light, and the control unit further executes the operations of outputting an oscillation command to the second laser oscillator to oscillate the second pulsed laser light at a second constant period, extracting a detection signal from the light receiving sensor as second section data at a time corresponding to the second constant period, and calculating the amount of pyruvic acid in the cell culture based on the second section data.

5. The amount detection device according to claim 4, wherein the second optimum wavelength is 1462 nm.

6. A method for detecting an amount of the substance to be measured, comprising irradiating a cell culture in which the substance to be measured has been dissolved with a pulsed laser beam oscillated at an appropriate wavelength corresponding to lactic acid or a lactate salt, receiving detection laser beam output from the cell culture with a light receiving sensor, and acquiring the amount of the substance to be measured in the cell culture, wherein the pulsed laser beam is oscillated at a constant period, a detection signal from the light receiving sensor is extracted as section data at a time corresponding to the constant period, and the amount is calculated based on the section data.

7. The quantity detection method according to claim 6, wherein the appropriate wavelength is 1480 nm.

8. The quantity detection method according to claim 6 or 7, wherein reflected light of the pulsed laser light is received as the detection laser light.

9. The amount detection method according to any one of claims 6 to 8, further comprising the steps of irradiating the cell culture with a second pulsed laser light oscillated at a second appropriate wavelength corresponding to pyruvic acid so as to be coaxial with the pulsed laser light, the second pulsed laser light being oscillated at a second constant period, extracting a detection signal from the light receiving sensor as second interval data at a time corresponding to the second constant period, and calculating the amount of pyruvic acid in the cell culture based on the second interval data.

10. The quantity detection method according to claim 9, wherein the second optimum wavelength is 1462 nm.

11. A cell culture system including a culture device that contains a cell culture, and an amount detection device that detects the amount of lactic acid or lactate, a measurement target substance, that has dissolved in the cell culture cultured in the culture device, wherein the amount detection device includes a laser oscillator that oscillates a pulsed laser beam at an appropriate wavelength corresponding to the measurement target substance, an irradiation mechanism that irradiates the cell culture with the pulsed laser beam, a light receiving sensor that receives the detection laser beam output from the cell culture, and a control unit that controls the operation of each component, wherein the control unit outputs an oscillation command to the laser oscillator to oscillate the pulsed laser beam at a constant cycle, extracts a detection signal from the light receiving sensor as section data at a time corresponding to the constant cycle, and calculates the amount of the measurement target substance in the cell culture based on the section data.

12. The cell culture system according to claim 11, wherein the suitable wavelength is 1480 nm.

13. The cell culture system according to claim 11 or 12, wherein the light receiving sensor is configured to receive reflected light of the pulsed laser light.

14. The cell culture system according to any one of claims 11 to 13, wherein the amount detection device further includes a second laser oscillator that oscillates a second pulsed laser light oscillated at a second appropriate wavelength corresponding to pyruvic acid, the second pulsed laser light is irradiated onto the cell culture so as to be coaxial with the pulsed laser light, and the control unit further executes an operation of outputting an oscillation command to the second laser oscillator to oscillate the second pulsed laser light at a second constant period, extracting a detection signal from the light receiving sensor as second section data at a time corresponding to the second constant period, and calculating the amount of the pyruvic acid in the cell culture based on the second section data.

15. The cell culture system according to claim 14, wherein the second suitable wavelength is 1462 nm.

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