Patent Application
20250002836
This application is based upon and claims the benefit of priority from Japanese Patent Application No. 2023-104917, filed on Jun. 27, 2023, the entire contents of which are incorporated herein by reference.
The present disclosure relates to a cell sample evaluation method and a cell sample evaluation apparatus for evaluating a culture state of a cell sample containing a plurality of cells cultured on a culture container, and a culture state analysis apparatus for analyzing the culture state of the cell sample.
A technique for culturing cells is important not only in a basic research on cells, but also in a practical field such as, for example, regenerative medicine. In the culture of the cells, in order to further proliferate a cell system and a cell line, an operation called a passage is performed in which a culture medium is removed from the cell culture system, and the cultured cells are transferred to a new culture medium. Further, the number of times of performing the passage operation in the cell sample in which a plurality of cells are cultured is referred to as a passage number.
In the cell sample obtained by culturing the plurality of cells as described above, the passage number, a condition of each passage operation, and the like affect a quality, a culture state, and the like of the cells grown in the cell sample (see, for example, Patent Document 1 and Non Patent Document 1). That is, in the passage operation of the cell sample, in many cases, the cultured cells are subjected to damage, stress, and the like by being transferred to a new environment, and thus, the quality and the state of the cells deteriorate.
In general, it is known that, as the passage number increases, the quality of the cultured cells deteriorates due to a change in differentiation and proliferation capacity, a change in form, a change in gene, and the like. Therefore, the culture state such as the passage number, the passage condition and the like of the cell sample is very important information in performing an experiment, a measurement, and the like using the cell sample. However, a technique for measuring and evaluating the culture state of the cells in the cell sample described above has not been established until now.
An object of the present invention is to provide a cell sample evaluation method and a cell sample evaluation apparatus capable of suitably evaluating a culture state of a cell sample containing a plurality of cells, and a culture state analysis apparatus capable of analyzing the culture state of the cell sample.
The present inventors have studied effects of activation of the cells and the like by light irradiation performed using laser light supplied from a quantum cascade laser (QCL) and the like on the cell sample in which the plurality of cells are cultured. In the above research on the effects of the light irradiation on the cell sample, the present inventors have found that dead cells are generated when the light irradiation is performed at an irradiation amount of a certain level or more. In addition, when cell death due to the light irradiation described above was examined in detail, it was found that the generation of dead cells depends on the culture state such as the passage number of the cell sample, and the present invention has been achieved.
An embodiment of the present invention is a cell sample evaluation method. The cell sample evaluation method is an evaluation method for evaluating a culture state of an object cell sample of an evaluation object containing a plurality of cells cultured on a culture container, and includes (1) an evaluation light irradiation step of irradiating an irradiation region set in a culture region of the plurality of cells in the object cell sample with evaluation light having a wavelength in a mid-infrared region under a predetermined irradiation condition; (2) a dead cell number measurement step of measuring a dead cell number generated according to an irradiation amount of the evaluation light in the irradiation region of the object cell sample; and (3) a culture state analysis step of evaluating a culture state of the object cell sample by obtaining an object correlation between the irradiation amount of the evaluation light and the dead cell number for the object cell sample, and comparing a reference correlation between the irradiation amount of the evaluation light and the dead cell number for a reference cell sample with a known culture state and the object correlation.
An embodiment of the present invention is a cell sample evaluation apparatus. The cell sample evaluation apparatus is an evaluation apparatus for evaluating a culture state of an object cell sample of an evaluation object containing a plurality of cells cultured on a culture container, and includes (1) an evaluation light irradiation unit for irradiating an irradiation region set in a culture region of the plurality of cells in the object cell sample with evaluation light having a wavelength in a mid-infrared region under a predetermined irradiation condition; (2) a dead cell number measurement unit for measuring a dead cell number generated according to an irradiation amount of the evaluation light in the irradiation region of the object cell sample; and (3) a culture state analysis unit for analyzing a culture state of the object cell sample, and (4) the culture state analysis unit includes (a) an object correlation generation unit for obtaining an object correlation between the irradiation amount of the evaluation light and the dead cell number for the object cell sample; (b) a reference correlation database for storing a reference correlation between the irradiation amount of the evaluation light and the dead cell number for a reference cell sample with a known culture state; and (c) a culture state evaluation unit for evaluating the culture state of the object cell sample by comparing the reference correlation and the object correlation.
An embodiment of the present invention is a culture state analysis apparatus. The culture state analysis apparatus is an analysis apparatus for analyzing a culture state of an object cell sample of an evaluation object containing a plurality of cells cultured on a culture container, and includes (a) an object correlation generation unit for obtaining, based on a measurement result obtained by irradiating an irradiation region set in a culture region of the plurality of cells in the object cell sample with evaluation light having a wavelength in a mid-infrared region under a predetermined irradiation condition, and measuring a dead cell number generated according to an irradiation amount of the evaluation light in the irradiation region of the object cell sample, an object correlation between the irradiation amount of the evaluation light and the dead cell number for the object cell sample; (b) a reference correlation database for storing a reference correlation between the irradiation amount of the evaluation light and the dead cell number for a reference cell sample with a known culture state; and (c) a culture state evaluation unit for evaluating a culture state of the object cell sample by comparing the reference correlation and the object correlation.
In the cell sample evaluation method, the cell sample evaluation apparatus, and the culture state analysis apparatus described above, the evaluation light having the wavelength in the mid-infrared region is used, and the irradiation region set in the object cell sample is irradiated with the evaluation light, and further, the dead cell number (the number of dead cells) generated according to the irradiation amount of the evaluation light is measured. In addition, based on the above measurement result, the object correlation between the irradiation amount of the evaluation light and the dead cell number is obtained for the object cell sample, and the reference correlation prepared for the reference cell sample is compared with the object correlation to evaluate the culture state of the object cell sample.
According to the above configuration, for the object cell sample in which the plurality of cells are cultured in the culture container, it is possible to suitably evaluate the culture state of the cells in the object cell sample using the object correlation between the irradiation amount of the evaluation light and the dead cell number as an index. In addition, an irradiation condition of the evaluation light for the irradiation region of the object cell sample is preferably appropriately set such that the object correlation between the irradiation amount of the evaluation light and the dead cell number which is used for the evaluation of the object cell sample can be acquired.
According to the cell sample evaluation method, the cell sample evaluation apparatus, and the culture state analysis apparatus of the present invention, it is possible to suitably evaluate the culture state of the cell sample containing the plurality of cells.
The present invention will be more fully understood from the detailed description given hereinbelow and the accompanying drawings, which are given by way of illustration only and are not to be considered as limiting the present invention.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description.
Hereinafter, embodiments of a cell sample evaluation method, a cell sample evaluation apparatus, and a culture state analysis apparatus will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference signs, and redundant description will be omitted. Further, the dimensional ratios in the drawings are not always coincident with those in the description.
In the object cell sample S illustrated in
The object cell sample S is placed on the sample stage 18. In the present embodiment, when an irradiation optical axis of evaluation light by the evaluation light irradiation unit 20 is set to a z axis, the sample stage 18 is arranged in parallel to an xy plane perpendicular to the z axis. Further, the sample stage 18 is configured to be movable in the xy plane perpendicular to the irradiation optical axis of the evaluation light.
Further, a stage drive unit 19 is provided for the sample stage 18 described above. The stage drive unit 19 drives the sample stage 18 on which the object cell sample S is placed in the xy plane, thereby setting and controlling an irradiation position of the evaluation light in the culture region of the object cell sample S. In addition, as a drive mechanism of the sample stage 18, for example, a stepping motor, piezo actuator, or the like can be used.
The evaluation light irradiation unit (evaluation light irradiation apparatus) 20 is arranged below the sample stage 18 on which the object cell sample S is placed, and is optically coupled to a bottom surface 14 of the culture dish 13 in the object cell sample S via an opening provided at a predetermined position of the sample stage 18. In the present embodiment, the evaluation light irradiation unit 20 includes an irradiation light source 21, a reflection mirror 22, and an objective lens 23.
The evaluation light irradiation unit 20 irradiates an irradiation region set in the culture region of the plurality of cells 10 in the object cell sample S with the evaluation light having a predetermined wavelength in a mid-infrared region under a predetermined irradiation condition. The evaluation light supplied from the irradiation light source 21 is reflected by the reflection mirror 22, and applied to the plurality of cells 10 contained in the object cell sample S via the objective lens 23 and the bottom surface 14 of the culture dish 13 along the predetermined irradiation optical axis.
Specifically, for example, in the object cell sample S, the evaluation light irradiation unit 20 sets N irradiation regions (a plurality of irradiation regions, N is an integer of 2 or more) by dividing the culture region of the plurality of cells 10 on the culture dish 13. Further, the N irradiation regions described above are irradiated with the evaluation light supplied from the irradiation light source 21 with irradiation amounts different from each other.
The dead cell number measurement unit (dead cell number measurement apparatus) 30 is arranged above the sample stage 18 on which the object cell sample S is placed. The dead cell number measurement unit 30 measures the number of dead cells (the dead cell number) generated according to the irradiation amount of the evaluation light by the evaluation light irradiation unit 20 in the irradiation region of the object cell sample S. When the N irradiation regions are set in the culture region of the object cell sample S as described above, the dead cell number measurement unit 30 measures the dead cell number generated according to the irradiation amount of the evaluation light in each of the N irradiation regions of the object cell sample S. The specific configuration of the dead cell number measurement unit 30 is appropriately set according to a measurement method of the number of dead cells in the dead cell number measurement unit 30.
In addition, in the configuration of
The control unit 40 controls the operations of respective parts of the sample stage 18, the evaluation light irradiation unit 20, and the dead cell number measurement unit 30 in the evaluation apparatus 1A, and further, performs necessary analysis on the measurement result by the evaluation light irradiation unit 20 and the dead cell number measurement unit 30. In the present embodiment, the control unit 40 includes a measurement control unit 41 and a culture state analysis unit 50.
The measurement control unit (measurement control apparatus) 41 controls the drive operation of the sample stage 18 by the stage drive unit 19, and the irradiation operation of the evaluation light on the object cell sample S by the evaluation light irradiation unit 20 (for example, the supply operation of the evaluation light by the irradiation light source 21). Further, in the case in which the dead cell number measurement unit 30 is provided integrally with the evaluation light irradiation unit 20, the measurement control unit 41 may be configured to further control the measurement operation of the dead cell number in the object cell sample S by the dead cell number measurement unit 30, as illustrated by a dashed arrow in
The culture state analysis unit 50 is a culture state analysis apparatus for analyzing the culture state of the object cell sample S containing the plurality of cells 10. Further, in the configuration illustrated in
In the control unit 40, each of the measurement control unit 41 and the culture state analysis unit 50 may be constituted by, for example, a computer including a CPU as a processing unit and a ROM, a RAM, an external storage device, and the like as a storage unit. The measurement control unit 41 and the culture state analysis unit 50 may be constituted by separate computers, or may be constituted by a single computer. Further, in the case in which the measurement control unit 41 is provided separately from the culture state analysis unit 50, the measurement control unit 41 may be provided with an input unit and a display unit as in the case of the culture state analysis unit 50.
The dead cell number calculation unit 51 calculates the dead cell number generated in the irradiation region of the object cell sample S based on the measurement data of the dead cell number input from the dead cell number measurement unit 30. In the case in which the N irradiation regions are set for the object cell sample S, the dead cell number calculation unit 51 calculates the dead cell number generated in each of the N irradiation regions. In addition, in the case in which the dead cell number is directly acquired in the measurement data by the dead cell number measurement unit 30, it is not necessary to provide the dead cell number calculation unit 51.
The object correlation generation unit 52 obtains an object correlation between the irradiation amount of the evaluation light and the dead cell number for the object cell sample S based on the measurement result including the irradiation data of the evaluation light for the irradiation region input from the evaluation light irradiation unit 20 or the measurement control unit 41 and the measurement data of the dead cell number input from the dead cell number measurement unit 30 or the dead cell number calculation unit 51. In the case in which the N irradiation regions are set for the object cell sample S, the object correlation generation unit 52 obtains the object correlation between the irradiation amount of the evaluation light and the dead cell number for the N irradiation regions. The object correlation described above serves as an index of the evaluation of the culture state of the cells in the object cell sample S.
The reference correlation database 53 is a reference correlation storage unit for storing a reference correlation between the irradiation amount of the evaluation light and the dead cell number for a reference cell sample with a known culture state of the cells. In the case in which a single reference cell sample is assumed, the reference correlation database 53 stores a single reference correlation. Further, in the case in which a plurality of reference cell samples with culture states different from each other are assumed, the reference correlation database 53 stores a plurality of reference correlations for the reference cell samples.
The culture state evaluation unit 54 compares the reference correlation stored in the reference correlation database 53 and the object correlation obtained by the object correlation generation unit 52, and evaluates the culture state of the cells in the object cell sample S based on the comparison result. In the case in which the plurality of reference correlations are stored in the reference correlation database 53, the culture state evaluation unit 54 evaluates the culture state of the cells in the object cell sample S by comparing each of the plurality of reference correlations and the object correlation.
In the configuration example illustrated in
A cell sample evaluation method performed by the cell sample evaluation apparatus 1A illustrated in
In the dead cell number measurement step, the dead cell number measurement unit 30 measures the dead cell number generated according to the irradiation amount of the evaluation light in the irradiation region of the object cell sample S irradiated with the evaluation light by the evaluation light irradiation unit 20. In the culture state analysis step, the culture state analysis unit 50 evaluates the culture state of the cells in the object cell sample S by obtaining the object correlation between the irradiation amount of the evaluation light and the dead cell number for the object cell sample S, and comparing the reference correlation for the reference cell sample and the object correlation.
The effects of the cell sample evaluation apparatus 1A, the cell sample evaluation method, and the culture state analysis apparatus 50 according to the above embodiment will be described.
In the evaluation apparatus 1A and the evaluation method illustrated in
According to the configuration described above, for the object cell sample S in which the plurality of cells 10 are cultured in the culture container 12 such as the culture dish 13 or the like, it is possible to suitably evaluate the culture state of the cells in the object cell sample S using the object correlation between the irradiation amount of the evaluation light and the dead cell number as the index. In addition, as described later, it is preferable that the irradiation condition of the evaluation light for the irradiation region of the object cell sample S is appropriately set such that the object correlation between the irradiation amount of the evaluation light and the dead cell number used for the evaluation of the object cell sample S can be acquired.
In addition, in Non Patent Document 2 (E. Larson et al., J. Biomed. Opt. Vol. 26 (2021), pp. 043012-1-043012-10), it is described that the cell sample is irradiated with the laser light in the mid-infrared region to examine a generation threshold value of dead cells. However, Non Patent Document 2 does not describe or suggest that there is a correlation between the generation of dead cells in the cell sample and the culture state such as the passage number of the cell sample.
Further, in the evaluation apparatus 1A and the evaluation method illustrated in
According to the configuration described above, for the object cell sample S in which the plurality of cells 10 are cultured on the culture dish 13, the plurality of irradiation regions are set in the culture region, and the irradiation regions are irradiated with the evaluation light with the irradiation amounts different from each other, and thus, it is possible to suitably measure the object correlation between the irradiation amount of the evaluation light and the dead cell number, which can be used as the index of the evaluation of the culture state of the cells in the object cell sample S.
In the evaluation apparatus 1A and the evaluation method of the above embodiment, in the evaluation light irradiation unit 20, a plurality of irradiation points may be set for irradiating with the evaluation light in each of the N irradiation regions, and the dead cell number measurement unit 30 may measure the dead cell number at each of the plurality of irradiation points in each of the N irradiation regions.
As described above, the plurality of irradiation points are set in each of the N irradiation regions set in the culture region, and the irradiation of the evaluation light and the measurement of the dead cell number are performed, and thus, the object correlation between the irradiation amount of the evaluation light and the dead cell number can be suitably measured.
Further, in the case in which the plurality of irradiation points are set in each of the irradiation regions as described above, in the evaluation light irradiation unit 20, all the irradiation points set in the N irradiation regions may be arranged in a grid pattern in the culture region, and the irradiation points may be sequentially irradiated with the evaluation light. Further, the culture state analysis unit 50 may obtain the object correlation between the irradiation amount of the evaluation light in each irradiation region and an average value of the dead cell numbers at the plurality of irradiation points in each irradiation region for the N irradiation regions. The details of the setting and the like of the irradiation region and the irradiation point described above will be described later.
In the evaluation apparatus 1A and the evaluation method of the above embodiment, the culture state evaluation unit 54 of the culture state analysis unit 50 preferably evaluates, specifically, the culture state including at least one of the passage number, the passage condition (the condition of the passage operation), and the culture condition between the passages of the object cell sample S as the culture state of the cells in the object cell sample S.
As described above, for the object cell sample S, the object correlation between the irradiation amount of the evaluation light and the dead cell number described above is used as the index of the evaluation, and thus, the culture state such as the passage number and the like of the object cell sample S can be suitably evaluated. Further, in the case in which the passage condition and the culture condition between the passages are considered to be relatively constant, the case in which the information of the passage number is considered to be important, or the like, the passage number may be evaluated as the culture state of the cells in the object cell sample S.
In the evaluation apparatus 1A and the evaluation method of the above embodiment, as described above, the reference correlation database 53 of the culture state analysis unit 50 may store the plurality of reference correlations for the plurality of reference cell samples with the culture states different from each other, and the culture state evaluation unit 54 may evaluate the culture state of the object cell sample S by comparing each of the plurality of reference correlations and the object correlation.
As described above, by preparing the plurality of reference correlations for the plurality of reference cell samples with the known culture states and the culture states different from each other in the reference correlation database 53, and comparing each of the reference correlations and the object correlation, it is possible to accurately evaluate the culture state of the object cell sample S in more detail.
In the evaluation apparatus 1A and the evaluation method of the above embodiment, the culture state evaluation unit 54 of the culture state analysis unit 50 may compare, in the comparison between the reference correlation and the object correlation, at least one of a parameter representing an approximate straight line or an approximate curve of the correlation, a dead cell number at a predetermined irradiation amount, and a threshold irradiation amount for generating dead cells.
As described above, for the correlation between the irradiation amount of the evaluation light and the dead cell number, specifically, by comparing the reference correlation and the object correlation by using at least one of the parameter representing the approximate straight line or the approximate curve of the correlation, the dead cell number at the predetermined irradiation amount, and the threshold irradiation amount for the dead cell generation, it is possible to suitably evaluate the culture state of the object cell sample S.
In the evaluation apparatus 1A and the evaluation method of the above embodiment, as described above, the culture state analysis unit 50 may include the reference correlation generation unit 55 for obtaining, for the reference cell sample, the reference correlation between the irradiation amount of the evaluation light and the dead cell number based on the measurement result obtained by using the evaluation light irradiation unit 20 and the dead cell number measurement unit 30.
As described above, for the reference cell sample, by using the configuration in which the reference correlation between the irradiation amount of the evaluation light and the dead cell number is acquired using the same apparatus and method as those for the object cell sample S of the evaluation object, it is possible to suitably realize the evaluation of the culture state of the object cell sample S by the comparison between the reference correlation and the object correlation. In addition, for the reference cell sample, the reference correlation may be acquired using an apparatus different from that for the object cell sample S. In this case, it is necessary to match the measurement conditions such as the irradiation conditions of the evaluation light between the reference cell sample and the object cell sample. Further, for the reference correlation, it may be acquired in advance and stored in the reference correlation database 53.
The configuration of the evaluation apparatus 1A and the evaluation method according to the above embodiment will be further described. First, the object cell sample S, which is the evaluation object of the culture state, the type of the cells contained in the cell sample, and the like will be described.
The cells used in the research, experiment, and the like related to the cells are roughly classified into established cell lines and primary cultured cells. The established cell line is a cell which has become able to proliferate indefinitely without being restricted by a cell cycle. Further, the primary cultured cell is a cell directly collected from a tissue and cultured. In general, in the primary cultured cell, when a cell division is repeated, the cell division stops at a finite number of times due to the Hayflick limit. However, in the primary culture, a chromosomal abnormality is rarely caused, and an infinitely proliferating cell may appear in some cases. This is a so-called immortalization of the cells, and such cells are called the established cell lines.
Further, the cells used in the research and the like are roughly classified into adherent cells and floating cells from the viewpoint of the culture method. The adherent cells grow while adhering to the culture container, and on the other hand, the floating cells grow in a culture medium in a floating state. The cell sample evaluation by the evaluation apparatus 1A described above is considered to be difficult to apply to the cell sample containing the floating cells in consideration of the optical system, and it is preferable to use the cell sample containing the adherent cells as the object cell sample S of the evaluation object.
In general, mammalian cell lines such as CHO, HeLa, MCF-7, 293, 3T3, COS, and the like are known as examples of the established adherent cell lines, and are widely used in molecular biological research, biochemical research, and the like. Further, in an example of the cell sample evaluation described later, the C6 cells which are the established adherent cell line derived from rat glioma are used. In addition, the well-known cell line as described above has been established for a long time, and thus, the passage number of the cell line is often unknown.
Further, stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic carcinoma cells (EC cells), mesenchymal stem cells, and the like are more difficult to culture than general cell lines, and specific techniques are required to perform stable culture. In the use of the above stem cells, it is important to accurately evaluate the culture state such as the passage number.
On the other hand, the primary cultured cells accurately reproduce a physiological state of the cells in a body, and thus, it is possible to obtain appropriate data indicating a state of a living body, and the primary cultured cells are used in preclinical research, biological research, and the like. However, the primary cultured cell itself is very fragile, and thus, sufficient care should be taken when applying the cell sample evaluation by the evaluation apparatus 1A described above.
Further, in the object cell sample S, it is preferable that the plurality of cells 10 cultured on the culture container 12 are uniformly distributed in the culture region. In the actual case, it is preferable to perform necessary measurement and evaluation for the object cell sample S by confirming that the cultured cells are in the state of being 100% confluent on the culture container 12, that is, in the state in which the adherent cells cover an adhesion surface of the culture container 12.
The culture container 12 and the culture dish 13 used for the culture of the cells in the object cell sample S will be described. In the configuration illustrated in
Specifically, for example, for each of various commercially available culture dishes, an infrared absorption spectrum of the bottom surface is measured, and a culture dish having a high transmittance for the evaluation light may be selected and used. In the example described later, a film bottom dish (product name: lumox dish) manufactured by Sarstedt is used as the culture dish 13. In the above culture dish, a polymer film is provided on the bottom surface.
Further, the culture container 12 used in the object cell sample S is not limited to the culture dish, and further, for example, a flask, a multiwell plate, or the like may be used as the culture container 12. In addition, in each of the above culture containers, a transmittance for the mid-infrared light on the bottom surface needs to be high as in the culture dish described above.
As the material of the bottom surface of the culture container, for example, silicon, germanium, calcium fluoride, barium fluoride, zinc selenide, zinc sulfide, chalcogenide glass, and the like, which are known in general to have a high infrared transmittance, can be used in addition to the polymer film described above. Further, for the culture container, it is preferable to select a culture container having low water solubility and low cytotoxicity.
The evaluation light used for the irradiation on the object cell sample S in the evaluation light irradiation unit 20, and the irradiation light source 21 used for supplying the evaluation light will be described. In the above evaluation apparatus 1A, light having a predetermined wavelength in the mid-infrared region is used as the evaluation light as described above. The energy of the mid-infrared light matches the energy of fundamental vibrations of many molecules. Therefore, the mid-infrared light has a large absorption coefficient for the molecules. The absorption coefficient of the mid-infrared light for the molecules described above is, for example, several orders of magnitude higher than that of near-infrared light or visible light.
The light energy required to induce a change of the cell state by light absorption depends on the absorption coefficient of the cells of the object. Therefore, by using the mid-infrared light as the evaluation light for the object cell sample S, it is possible to induce the change of the cell state with small irradiation energy compared with the case in which the near-infrared light or the visible light is used.
For example, when the near-infrared light or the visible light is used as the evaluation light, large light energy is required to induce the change of the cell state, and thus, heat generation caused by the light irradiation may damage cells or a culture environment around the irradiation point of the evaluation light. Further, in the case of the near-infrared light or the visible light, a large size high intensity laser light source or the like may be necessary as the irradiation light source 21, and thus, there is a problem that the size of the apparatus becomes large.
On the other hand, in the configuration in which the mid-infrared light is used as the evaluation light, it is possible to realize light absorption by directly providing the energy to the cell molecules of the object, or the fundamental vibration of water contained in the cells, and thus, it is possible to selectively induce the change of the cell state only at the object position with small light energy. In this case, for example, a longitudinal single mode laser light source having a wavelength corresponding to a specific absorption line, that is, a laser light source having a narrow oscillation spectrum band can be suitably used. Further, the evaluation light using the mid-infrared light requires small light energy, and thus, for example, the change of the cell state can be induced even in the case of using a compact semiconductor laser. In this case, it is possible to reduce the size of the apparatus.
Further, as to the wavelength of the mid-infrared light used as the evaluation light, specifically, for example, in the evaluation light irradiation unit 20, the wavelength of the evaluation light is preferably set in the wavelength region of 3 μm or more and 20 μm or less. In this case, the object correlation between the irradiation amount of the evaluation light and the dead cell number serving as the index of the evaluation of the culture state of the object cell sample S can be suitably acquired.
In the above wavelength region, for example, there is a lot of absorption of water in a 3 μm band, and effective heating through water is possible. Further, in a wavelength range from 10 μm to 20 μm, there are absorption lines specific to relatively large molecules, for example, such as a molecule containing a benzene ring. In addition, as to a wavelength region of a long wavelength exceeding 20 μm, it is considered that it is difficult to incorporate an appropriate light source into the evaluation apparatus 1A described above.
As the irradiation light source 21 used for supplying the evaluation light in the evaluation light irradiation unit 20, for example, a quantum cascade laser (QCL) can be used. The quantum cascade laser is a monopolar type laser element for generating light by transitions of electrons between subbands by using a level structure including the subbands formed in a semiconductor quantum well structure, and can realize a high efficiency and high output operation by cascade-coupling emission layers of the quantum well structure in multiple stages via injection layers.
In the case in which the QCL is used in the irradiation light source 21, the irradiation light source 21 may be constituted by, for example, the QCL element, a cooling mechanism, and a collimating lens. As a specific example of the QCL element, a DFB-QCL (model number: L12004-2190H-C) having a wavelength of 4.6 μm manufactured by Hamamatsu Photonics K.K. can be used. The irradiation light source described above is preferably a transverse single mode light source, that is, a light source having high spatial coherence in order to realize a small focusing diameter. Further, in the irradiation light source, it is preferable to perform temperature control, for example, by a TEC in order to stabilize the wavelength and output of the evaluation light. Further, a secondary cooling mechanism using a fan or water cooling may be provided.
As the collimating lens, it is necessary to use a lens made of a material which transmits the evaluation light having the wavelength from 3 μm to 20 μm described above. As the material of the lens described above, for example, ZnSe, Ge, CaF2, and the like can be used. Further, it is preferable to provide anti-reflection coating on both surfaces of the collimating lens such that the transmittance for the wavelength of the evaluation light is, for example, 95% or more. Further, a plurality of lenses may be used in combination as the collimating lens, and further, it is preferable to use a single aspheric lens capable of correcting the spherical aberration because loss of the evaluation light increases in the case of using the plurality of lenses.
Further, as the light source element in the irradiation light source 21, in addition to the QCL described above, for example, an interband cascade laser (ICL), a CO2 laser, a CO laser, a free electron laser, and the like can be used. In addition, a wavelength conversion technique using an optical parametric oscillator (OPO) may be used. Further, in the case in which the QCL or the ICL is used, from the viewpoint that the longitudinal single mode oscillation is possible, the configuration is not limited to the DFB type configuration described above, and further, an external cavity type configuration may be used. In addition, as to the wavelength region of the evaluation light, for example, the QCL can supply light having a wavelength of about 4 μm to 20 μm, and the ICL can supply light having a wavelength of about 3 μm to 7 μm.
Further, as the evaluation light supplied from the irradiation light source 21, continuous light may be use, or pulsed light may be used. Further, as the evaluation light, a burst pulse including a plurality of light pulses and having a range which is defined by an envelope of a predetermined shape may be used.
The configuration of the irradiation optical system used for the irradiation of the evaluation light on the object cell sample S in the evaluation light irradiation unit 20 will be described. In the evaluation apparatus 1A described above, the evaluation light from the irradiation light source 21 is reflected by 90° by the reflection mirror 22, and the object cell sample S is irradiated via the objective lens 23. In the above configuration, a height of an irradiation plane (operation plane) can be kept low by bending an optical path of the evaluation light. As the reflection mirror 22, for example, a gold coated flat mirror, a high reflection flat mirror by using a dielectric multilayer film, and the like can be used.
The configuration of the irradiation optical system in the evaluation light irradiation unit 20 is not limited to the configuration described above, and for example, the irradiation light source 21 and the objective lens 23 may be linearly arranged without using the reflection mirror 22. In the above configuration, an emission position of the light source element in the irradiation light source 21, a center position of the collimating lens, and a center position of the objective lens 23 are coaxially arranged on the same straight line. Further, in the dead cell number measurement unit 30, in the case in which the objective lens is used for the observation of the optical image of the object cell sample S, a center position of the observation objective lens is preferably arranged on the same straight line.
As described above, in the configuration in which the components of the irradiation optical system are linearly arranged, the number of components used in the evaluation light irradiation unit 20 is reduced, and thus, it is possible to reduce loss of the light and reduce a cost of the apparatus. In addition, in the above configuration, the optical path of the evaluation light is linearly formed, and thus, it is necessary to pay attention to the point that the height of the irradiation plane becomes high.
In addition, in the configuration of the evaluation light irradiation unit 20 illustrated in
Further, in the case in which the lens is provided in the irradiation optical system, similarly to the collimating lens in the irradiation light source 21, it is preferable to select an appropriate material, and further, provide anti-reflection coating on both surfaces of the lens. Further, as the objective lens 23, a reflection type objective lens may be used. Further, as the objective lens 23, for example, a single lens such as an aspheric lens, a plano convex lens, or the like may be used instead of a configuration in which a plurality of lenses are used in combination. In addition, in order to achieve both a narrow irradiation spot of the evaluation light and a certain depth of focus, it is preferable to use the objective lens in which the plurality of lenses are used in combination.
The irradiation condition of the evaluation light on the object cell sample S in the evaluation light irradiation unit 20 will be described. As described above, the irradiation condition of the evaluation light for the irradiation region of the object cell sample S needs to be set such that the object correlation between the irradiation amount of the evaluation light and the dead cell number used for the evaluation of the object cell sample
S can be acquired. In this case, it is preferable that the evaluation light irradiation unit 20 irradiates the irradiation region of the object cell sample S with the evaluation light while changing the irradiation amount, or with the plurality of irradiation amounts different from each other.
Further, in the evaluation light irradiation unit 20, it is preferable that the irradiation amount of the evaluation light is set by the irradiation condition including at least one of the irradiation time, the irradiation power, and the focusing condition of the evaluation light for the irradiation region. In this case, the irradiation condition capable of acquiring the object correlation between the irradiation amount of the evaluation light and the dead cell number used for the evaluation of the object cell sample S can be suitably realized by controlling the irradiation amount.
In the above configuration, in consideration of the fact that adjustment of the irradiation optical system is necessary to control and change the focusing condition, it is preferable to set the irradiation amount of the evaluation light by controlling one or both of the irradiation time and the irradiation power on the irradiation region of the evaluation light with the focusing condition being constant. In addition, in the case in which the focusing condition is changed, the irradiation power density of the evaluation light on the object cell sample S is changed, and the generation condition of the dead cells is changed. Hereinafter, as a specific example, setting of the irradiation amount in the case in which the burst pulse including the plurality of light pulses is used as the evaluation light will be described.
As illustrated in
Further, in the plurality of light pulses P included in the burst pulse, as illustrated in
In the burst pulse described above, as a method for changing the average irradiation power to be the irradiation power of the evaluation light, there are the following three methods. In the first method, an injection current applied to the light source element in the irradiation light source 21 and the repetition period t2 are fixed, and the pulse width t1 is changed. In the second method, the injection current applied to the light source element and the pulse width t1 are fixed, and the repetition period t2 is changed. In the third method, the pulse width t1 and the repetition period t2 are fixed, and the injection current applied to the light source element is changed. In the case of the third method, a pulse height value of the light pulse P constituting the burst pulse is changed by changing the injection current applied to the light source element.
In this case, the change of the average irradiation power when the pulse width t1 of the light pulse P is changed is shown under the conditions in which the temperature of the QCL in the irradiation light source 21 is set to 20° C., the injection current applied to the QCL is fixed, and the repetition frequency of the light pulses P in the burst pulse is fixed at 30 kHz. As described above, in the configuration in which the burst pulse is used as the evaluation light, the average irradiation power of the evaluation light can be controlled by changing any one of the injection current applied to the light source element, the pulse width of the light pulse P, and the repetition period (repetition frequency).
The irradiation condition of the evaluation light on the object cell sample S in the evaluation light irradiation unit 20 will be further described. In the irradiation of the evaluation light onto the object cell sample S, in order to suitably acquire the object correlation between the irradiation amount of the evaluation light and the dead cell number for the object cell sample S, it is preferable to set the N irradiation regions (plurality of irradiation regions) by dividing the culture region in the object cell sample S, and the N irradiation regions are irradiated with the evaluation light supplied from the irradiation light source 21 with the irradiation amounts different from each other for the respective irradiation regions.
Further, in the configuration example illustrated in
Further, in the present configuration example, all the 160 irradiation points of the irradiation points P1 to P8 set for the eight irradiation regions R1 to R8 are arranged in the grid pattern (lattice pattern, two-dimensional array pattern) in the culture region R0, and as illustrated by an arrowed line in the configuration of
Further, in the configuration in which the plurality of irradiation points set in the object cell sample S are arranged in the grid pattern and irradiated with the evaluation light sequentially, it is possible to easily perform the change of the irradiation position of the evaluation light by moving the sample stage 18 in the xy plane, and the scanning of the respective irradiation points by the evaluation light according thereto. In the configuration example illustrated in
According to the above configuration, it is possible to reduce the movement distances of the sample stage 18 in the scanning of the plurality of irradiation points by the evaluation light. In general, it is preferable that, for the plurality of scanning lines arranged in the predetermined direction set for scanning the plurality of irradiation points arranged in the grid pattern, the scanning of the irradiation points from left to right and the scanning of the irradiation points from right to left are alternately performed in the odd-numbered scanning lines and the even-numbered scanning lines so that the scanning directions are set to be alternately different.
In the case in which the plurality of irradiation points are set in each of the irradiation regions in the object cell sample S as described above, it is preferable that the dead cell number measurement unit 30 measures the dead cell number at each of the plurality of irradiation points in each of the N irradiation regions. Further, it is preferable that the object correlation generation unit 52 of the culture state analysis unit 50 obtains the object correlation between the irradiation amount of the evaluation light in each irradiation region and an average value of the dead cell numbers at the plurality of irradiation points in each irradiation region for the N irradiation regions. In this case, it is possible to appropriately and accurately acquire the object correlation between the irradiation amount of the evaluation light and the dead cell number for the N irradiation regions.
In addition, in the configuration example described above, the single culture dish 13 is assumed as the culture container 12 of the object cell sample S, and the N irradiation regions are set by dividing the culture region on the culture dish 13, and further, a plurality of culture dishes may be used as the culture container 12. For example, in the case in which N culture dishes (N is an integer of 2 or more) are used as the culture container 12, the evaluation light irradiation unit 20 can set the N irradiation regions as a whole of the object cell sample S by setting the irradiation region in the culture region on each of the N culture dishes.
In addition, in the case in which the cell samples on the N culture dishes are used as the object cell sample S, it is necessary to pay attention to the point that the culture states such as the passage numbers serving as the evaluation object need to be substantially the same in the N cell samples. Further, when the plurality of culture dishes are used as described above, the irradiation of the evaluation light and the measurement of the dead cell number are performed by placing the plurality of culture dishes on the sample stage 18 in the configuration illustrated in
In the above configuration, as illustrated by arrows A1, A2, and A3 in the diagram, by moving the sample stage 18 in the x axis direction, the y axis direction, and the rotation direction in the xy plane perpendicular to the irradiation optical axis of the evaluation light, it is possible to select the culture dish 13 to be set as the measurement object, and set the measurement position on the culture dish 13.
In the above configuration, as illustrated by arrows A6 and A7 in the diagram, by moving the sample stage 18 in the x axis direction and the y axis direction in the xy plane perpendicular to the irradiation optical axis of the evaluation light, it is possible to select the culture dish 13 to be set as the measurement object, and set the measurement position on the culture dish 13.
Further, as to the setting and change of the irradiation position of the evaluation light in the evaluation light irradiation unit 20, in the configuration illustrated in
The measurement method of the dead cell number generated in the irradiation region of the object cell sample S in the dead cell number measurement unit 30 will be described. For the measurement of the dead cell number by the dead cell number measurement unit 30, a configuration can be used in which the dead cell number is measured by capturing an optical image of the object cell sample S stained with a staining agent for selectively staining the dead cells. According to the above configuration, it is possible to suitably measure the dead cell number generated according to the irradiation amount of the evaluation light for the irradiation region of the object cell sample S.
Further, as the staining agent for selectively staining the dead cells described above, for example, a fluorescent dye can be used. In this case, the dead cell number measurement unit 30 measures the dead cell number by capturing a fluorescent image generated by irradiating the object cell sample S stained with the fluorescent dye with excitation light having a predetermined wavelength. As the fluorescent dye described above, for example, a chemical material called propidium iodide, a fluorescent dye called CellEvent manufactured by Invitrogen, and the like can be used.
In the counting of the dead cell number in the fluorescent image, for example, for the acquired fluorescent image, a binarized image in which a pixel with a fluorescence intensity exceeding a threshold value is set to 1, and a pixel with the fluorescence intensity equal to or less than the threshold value is set to 0 may be used. Further, as a method for more easily counting the dead cell number, it is also possible to use a method of referring to an area of a region of the dead cells. In this case, for example, the binarized image of the fluorescent image is generated as described above, and the number of pixels having the intensity of 1 is obtained in the region for counting the dead cell number. The above number of pixels corresponds to the area of the cell nuclei belonging to the dead cells.
Further, in addition to the calculation of the number of pixels described above, an average area of the cell nucleus is obtained. In the calculation of the area of the cell nucleus in this case, for example, a method can be used in which an operator manually measures the number of cell nuclei and the area for an image including a plurality of dead cells, and obtains the area of the cell nucleus by taking the average thereof. An estimated value of the dead cell number can be easily calculated by dividing the area (number of pixels) of the cell nuclei belonging to the dead cells by the average area (number of pixels) of the cell nucleus described above. According to the above method, the estimated value of the dead cell number included in each of the images can be easily obtained in a short time by the simple analysis algorithm, for example, even in the case in which the number of images is 100 or more.
As the staining agent for selectively staining the dead cells used in the measurement of the dead cell number in the object cell sample S, in addition to the fluorescent dye described above, for example, a staining agent for staining the cells in an absorptive manner, such as trypan blue or the like, can be used. Further, as to the measurement of the dead cell number, in addition to the method using the staining agent, for example, a method can be used in which images of the object cell sample S are acquired in time series, and cells with motion are counted as live cells and cells without motion are counted as dead cells.
The excitation light source 31 supplies the excitation light having the wavelength corresponding to the fluorescent dye used for staining of the object cell sample S. The excitation filter 32 selectively transmits, as the excitation light, light in a wavelength region including the wavelength used for the excitation of the fluorescent dye in the light supplied from the excitation light source 31, and for example, a bandpass filter may be used. The dichroic mirror 33 reflects the excitation light supplied from the excitation light source 31 through the excitation filter 32 to the objective lens 34.
The plurality of cells 10 stained with the fluorescent dye in the object cell sample S are irradiated with the excitation light reflected by the dichroic mirror 33 through the objective lens 34. As the objective lens 34, for example, a water immersion objective lens may be used.
The fluorescence generated by the fluorescent dye contained in the dead cells in the object cell sample S due to the irradiation of the excitation light passes through the objective lens 34, the dichroic mirror 33, the imaging lens 35, and the fluorescence filter 36 to reach the imaging device 37, and the fluorescent image of the object cell sample S is captured by the imaging device 37. The fluorescence filter 36 is a filter (excitation light cut filter) for cutting light in the wavelength region of the excitation light and selectively transmitting light in a wavelength region of the fluorescence in the light output from the object cell sample S, and for example, a bandpass filter or a notch filter may be used.
The operation, effect, and the like of the cell sample evaluation apparatus 1A and the cell sample evaluation method of the configuration described above will be described together with a specific example and measurement data. In the example, as the cells 10 in the object cell sample S, the C6 cells which are the rat glioma cell line (obtained from JCRB cell bank) were used. As the culture medium, the Dulbecco's modified Eagle's medium (DMEM culture medium) containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/mL penicillin, and 400 μg/mL streptomycin was used, and flask static culture was performed in an incubator (37° C., 5% CO2). The DMEM culture medium was replaced every three days.
The C6 cells reaching 100% confluence were detached with 0.25% trypsin EDTA, resuspended in the DMEM culture medium, and seeded in the lumox dish (manufactured by Sarstedt) which is the culture dish. Static culture was carried out in the incubator for three days, and the cells reaching 100% confluence again were subjected to the irradiation experiment of the evaluation light. Further, three types of cell samples having the passage numbers different from each other were prepared as the cell sample S. The passage numbers in the three types of the cell samples are 45, 53, and 55, respectively.
As to the irradiation condition and the irradiation method of the evaluation light for the cell sample S in the evaluation light irradiation unit 20, as illustrated in
As to the irradiation of the evaluation light on the cell sample S, the DFB-QCL described above having the wavelength of 4.6 μm was used as the irradiation light source 21, and the focusing condition of the evaluation light was set to be fixed. Further, the burst pulse illustrated in
Further, as to the irradiation time of the evaluation light, as illustrated in
Further, three cell samples were prepared for each of the three types of the cell samples with the passage numbers of 45, 53, and 55, and the measurements were performed. The graph of
As shown in the respective graphs of
As shown in the respective graphs of
In the above example, the correlation in the case in which the irradiation power and the focusing condition of the evaluation light are set to be fixed and the irradiation time is changed is shown, and in addition, for example, in the case in which the irradiation time and the focusing condition are set to be fixed and the irradiation power is changed, or in the case in which the focusing condition is set to be fixed and both the irradiation time and the irradiation power are changed, the correlation between the irradiation amount of the evaluation light and the dead cell number serving as the index of the evaluation of the culture state of the cell sample can be similarly acquired.
As shown in the respective graphs of
In the graph of the dead cell number f(P, τ) in which the irradiation time τ and the irradiation power P are variables as described above, in the case in which a cross section is taken with the irradiation power P being fixed, a graph representing the correlation between the irradiation time τ of the evaluation light and the dead cell number is obtained. Further, in the case in which a cross section is taken with the irradiation time τ being fixed, a graph representing the correlation between the irradiation power P of the evaluation light and the dead cell number is obtained. As described above, in the evaluation of the culture state of the cell sample S, it is also possible to use the correlation between both the irradiation time and the irradiation power of the evaluation light and the dead cell number. In addition, when the evaluation of the culture state of the object
cell sample S is performed, for the comparison between the reference correlation and the object correlation, various methods may be specifically used. For example, in the case in which the single reference correlation is prepared, the evaluation can be performed by using a magnitude relationship between the reference correlation and the object correlation. Further, in the case in which the plurality of reference correlations are prepared, the evaluation can be performed by referring to the reference correlation closest to the object correlation out of the plurality of reference correlations. Further, for example, for the plurality of reference correlations, a relational formula or a lookup table representing a relationship between a parameter serving as the index such as the slope of the approximate straight line of the correlation or an intercept on the horizontal axis or the vertical axis of the approximate straight line, and the passage number may be prepared, and the evaluation may be performed by using the relational formula or the lookup table described above.
The cell sample evaluation method, the cell sample evaluation apparatus, and the culture state analysis apparatus are not limited to the embodiments and configuration examples described above, and may be modified in various ways. For example, the configuration of each of the evaluation light irradiation unit 20 and the dead cell number measurement unit 30 is not limited to the configuration described above, and various configurations may be specifically used.
Further, the irradiation condition of the evaluation light in the evaluation light irradiation unit 20 is not limited to the configuration in which the N irradiation regions are irradiated with the evaluation light with the irradiation amounts different from each other as described above, and for example, a configuration in which the irradiation region is scanned by using the evaluation light while continuously changing the irradiation amount may be used. In this case, the correlation between the irradiation amount of the evaluation light and the dead cell number can be acquired by comparing a distribution of the irradiation amount of the evaluation light in the irradiation region and a distribution of the measured dead cell number.
The cell sample evaluation method of a first aspect according to the above embodiment is an evaluation method for evaluating a culture state of an object cell sample of an evaluation object containing a plurality of cells cultured on a culture container, and includes (1) an evaluation light irradiation step of irradiating an irradiation region set in a culture region of the plurality of cells in the object cell sample with evaluation light having a wavelength in a mid-infrared region under a predetermined irradiation condition; (2) a dead cell number measurement step of measuring a dead cell number generated according to an irradiation amount of the evaluation light in the irradiation region of the object cell sample; and (3) a culture state analysis step of evaluating a culture state of the object cell sample by obtaining an object correlation between the irradiation amount of the evaluation light and the dead cell number for the object cell sample, and comparing a reference correlation between the irradiation amount of the evaluation light and the dead cell number for a reference cell sample with a known culture state and the object correlation.
The cell sample evaluation apparatus of a first aspect according to the above embodiment is an evaluation apparatus for evaluating a culture state of an object cell sample of an evaluation object containing a plurality of cells cultured on a culture container, and includes (1) an evaluation light irradiation unit for irradiating an irradiation region set in a culture region of the plurality of cells in the object cell sample with evaluation light having a wavelength in a mid-infrared region under a predetermined irradiation condition; (2) a dead cell number measurement unit for measuring a dead cell number generated according to an irradiation amount of the evaluation light in the irradiation region of the object cell sample; and (3) a culture state analysis unit for analyzing a culture state of the object cell sample, and (4) the culture state analysis unit includes (a) an object correlation generation unit for obtaining an object correlation between the irradiation amount of the evaluation light and the dead cell number for the object cell sample; (b) a reference correlation database for storing a reference correlation between the irradiation amount of the evaluation light and the dead cell number for a reference cell sample with a known culture state; and (c) a culture state evaluation unit for evaluating the culture state of the object cell sample by comparing the reference correlation and the object correlation.
The culture state analysis apparatus of a first aspect according to the above embodiment is an analysis apparatus for analyzing a culture state of an object cell sample of an evaluation object containing a plurality of cells cultured on a culture container, and includes (a) an object correlation generation unit for obtaining, based on a measurement result obtained by irradiating an irradiation region set in a culture region of the plurality of cells in the object cell sample with evaluation light having a wavelength in a mid-infrared region under a predetermined irradiation condition, and measuring a dead cell number generated according to an irradiation amount of the evaluation light in the irradiation region of the object cell sample, an object correlation between the irradiation amount of the evaluation light and the dead cell number for the object cell sample; (b) a reference correlation database for storing a reference correlation between the irradiation amount of the evaluation light and the dead cell number for a reference cell sample with a known culture state; and (c) a culture state evaluation unit for evaluating a culture state of the object cell sample by comparing the reference correlation and the object correlation.
In the cell sample evaluation method of a second aspect, in the configuration of the first aspect, the culture container may be a single culture dish, and in the evaluation light irradiation step, the irradiation region may be set in the culture region on the single culture dish.
In the cell sample evaluation apparatus of a second aspect, in the configuration of the first aspect, the culture container may be a single culture dish, and the evaluation light irradiation unit may set the irradiation region in the culture region on the single culture dish.
In the cell sample evaluation method of a third aspect, in the configuration of the first aspect, the culture container may be N culture dishes (N is an integer of 2 or more), and in the evaluation light irradiation step, N irradiation regions may be set by setting the irradiation region in the culture region on each of the N culture dishes.
In the cell sample evaluation apparatus of a third aspect, in the configuration of the first aspect, the culture container may be N culture dishes (N is an integer of 2 or more), and the evaluation light irradiation unit may set N irradiation regions by setting the irradiation region in the culture region on each of the N culture dishes.
As described above, for the culture container used for the culture of the cells in the object cell sample, specifically, for example, the single culture dish or the plurality of culture dishes can be used. Further, for the irradiation region of the evaluation light set in the object cell sample, it is preferably appropriately set according to the configuration of the culture container in the object cell sample.
In the cell sample evaluation method of a fourth aspect, in the configuration of any one of the first to third aspects, in the culture state analysis step, a culture state including at least one of a passage number, a passage condition, and a culture condition between passages of the object cell sample may be evaluated as the culture state of the object cell sample.
In the cell sample evaluation apparatus of a fourth aspect, in the configuration of any one of the first to third aspects, the culture state evaluation unit may evaluate a culture state including at least one of a passage number, a passage condition, and a culture condition between passages of the object cell sample as the culture state of the object cell sample.
In the culture state analysis apparatus of a second aspect, in the configuration of the first aspect, the culture state evaluation unit may evaluate a culture state including at least one of a passage number, a passage condition, and a culture condition between passages of the object cell sample as the culture state of the object cell sample.
As described above, for the object cell sample, the object correlation between the irradiation amount of the evaluation light and the dead cell number described above is used as the index of the evaluation, and thus, the culture state such as the passage number and the like of the object cell sample can be suitably evaluated.
In the cell sample evaluation method of a fifth aspect, in the configuration of any one of the first to fourth aspects, in the dead cell number measurement step, the dead cell number may be measured by capturing an optical image of the object cell sample stained with a staining agent for selectively staining dead cells.
In the cell sample evaluation apparatus of a fifth aspect, in the configuration of any one of the first to fourth aspects, the dead cell number measurement unit may measure the dead cell number by capturing an optical image of the object cell sample stained with a staining agent for selectively staining dead cells.
As described above, by using the staining agent for selectively staining the dead cells, it is possible to suitably measure the dead cell number generated according to the irradiation amount of the evaluation light for the irradiation region of the object cell sample. Further, as the above staining agent, for example, a fluorescent dye can be used.
In the cell sample evaluation method of a sixth aspect, in the configuration of any one of the first to fifth aspects, in the evaluation light irradiation step, the irradiation amount of the evaluation light may be set by an irradiation condition including at least one of an irradiation time, an irradiation power, and a focusing condition of the evaluation light for the irradiation region.
In the cell sample evaluation apparatus of a sixth aspect, in the configuration of any one of the first to fifth aspects, in the evaluation light irradiation unit, the irradiation amount of the evaluation light may be set by an irradiation condition including at least one of an irradiation time, an irradiation power, and a focusing condition of the evaluation light for the irradiation region.
As described above, for the irradiation of the evaluation light on the irradiation region of the object cell sample, by setting and controlling at least one of the irradiation time, the irradiation power, and the focusing condition of the evaluation light, the irradiation condition capable of acquiring the object correlation between the irradiation amount of the evaluation light and the dead cell number used for the evaluation of the object cell sample can be suitably realized.
In the cell sample evaluation method of a seventh aspect, in the configuration of any one of the first to sixth aspects, in the evaluation light irradiation step, the wavelength of the evaluation light may be set in a wavelength region of 3 μm or more and 20 μm or less.
In the cell sample evaluation apparatus of a seventh aspect, in the configuration of any one of the first to sixth aspects, in the evaluation light irradiation unit, the wavelength of the evaluation light may be set in a wavelength region of 3 μm or more and 20 μm or less.
As described above, by setting the wavelength of the evaluation light in the mid-infrared region used for the evaluation of the culture state of the object cell sample, for example, within the wavelength region described above, the object correlation between the irradiation amount of the evaluation light and the dead cell number serving as the index of the evaluation can be suitably acquired.
In the cell sample evaluation method of an eighth aspect, in the configuration of any one of the first to seventh aspects, in the culture state analysis step, the culture state of the object cell sample may be evaluated by comparing each of a plurality of reference correlations for a plurality of reference cell samples with culture states different from each other and the object correlation.
In the cell sample evaluation apparatus of an eighth aspect, in the configuration of any one of the first to seventh aspects, the reference correlation database may store a plurality of reference correlations for a plurality of reference cell samples with culture states different from each other, and the culture state evaluation unit may evaluate the culture state of the object cell sample by comparing each of the plurality of reference correlations and the object correlation.
In the culture state analysis apparatus of a third aspect, in the configuration of the first or second aspect, the reference correlation database may store a plurality of reference correlations for a plurality of reference cell samples with culture states different from each other, and the culture state evaluation unit may evaluate the culture state of the object cell sample by comparing each of the plurality of reference correlations and the object correlation.
As described above, by preparing the plurality of reference correlations for the plurality of reference cell samples with known culture states and the culture states different from each other, and comparing each of the reference correlations and the object correlation, it is possible to accurately evaluate the culture state of the object cell sample in detail.
In the cell sample evaluation method of a ninth aspect, in the configuration of any one of the first to eighth aspects, in the culture state analysis step, in the comparison between the reference correlation and the object correlation, at least one of a parameter representing an approximate straight line or an approximate curve of the correlation, a dead cell number at a predetermined irradiation amount, and a threshold irradiation amount for generating dead cells may be compared.
In the cell sample evaluation apparatus of a ninth aspect, in the configuration of any one of the first to eighth aspects, the culture state evaluation unit may compare, in the comparison between the reference correlation and the object correlation, at least one of a parameter representing an approximate straight line or an approximate curve of the correlation, a dead cell number at a predetermined irradiation amount, and a threshold irradiation amount for generating dead cells.
In the culture state analysis apparatus of a fourth aspect, in the configuration of any one of the first to third aspects, the culture state evaluation unit may compare, in the comparison between the reference correlation and the object correlation, at least one of a parameter representing an approximate straight line or an approximate curve of the correlation, a dead cell number at a predetermined irradiation amount, and a threshold irradiation amount for generating dead cells.
As described above, by comparing the reference correlation and the object correlation by using, for example, at least one of the parameter representing the approximate straight line or the approximate curve such as a slope of the approximate straight line of the correlation, the dead cell number at the predetermined irradiation amount, and the threshold irradiation amount for the dead cell generation, it is possible to suitably evaluate the culture state of the object cell sample.
In the cell sample evaluation method of a tenth aspect, in the configuration of any one of the first to ninth aspects, in the culture state analysis step, for the reference cell sample, the reference correlation between the irradiation amount of the evaluation light and the dead cell number may be obtained based on a measurement result obtained by using the evaluation light irradiation step and the dead cell number measurement step.
In the cell sample evaluation apparatus of a tenth aspect, in the configuration of any one of the first to ninth aspects, the culture state analysis unit may include a reference correlation generation unit for obtaining, for the reference cell sample, the reference correlation between the irradiation amount of the evaluation light and the dead cell number based on a measurement result obtained by using the evaluation light irradiation unit and the dead cell number measurement unit.
The culture state analysis apparatus of a fifth aspect, in the configuration of any one of the first to fourth aspects, may include a reference correlation generation unit for obtaining, for the reference cell sample, the reference correlation between the irradiation amount of the evaluation light and the dead cell number based on a measurement result.
As described above, for the reference cell sample, by using the configuration in which the reference correlation between the irradiation amount of the evaluation light and the dead cell number is acquired using the same apparatus and method as those for the object cell sample of the evaluation object, it is possible to suitably realize the evaluation of the culture state of the object cell sample by the comparison between the reference correlation and the object correlation. In addition, for the reference cell sample, the reference correlation may be acquired using an apparatus different from that for the object cell sample. Further, for the reference correlation, it may be acquired in advance and stored in the database.
The present invention can be used as a cell sample evaluation method and a cell sample evaluation apparatus capable of suitably evaluating a culture state of a cell sample containing a plurality of cells, and a culture state analysis apparatus capable of analyzing the culture state of the cell sample.
From the invention thus described, it will be obvious that the invention may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended for inclusion within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2023-104917 | Jun 2023 | JP | national |
