Cell culture apparatus and method for adjusting the concentration of viable cells

Abstract

A cell culture system comprising: a first container that holds a cell suspension; a second container that holds a diluent for diluting the cell suspension; a viable cell concentration measurement device that measures the viable cell concentration of target cells in the cell suspension by individually determining the life-or-death of the multiple target cells in the cell suspension; a controller that maintains the viable cell concentration and liquid volume data of the cell suspension and controls the same; a mixer unit that mixes the cell suspension with the diluent; and a culture container in which the diluted cell suspension is seeded and the cells are cultured. The controller determines the liquid volume of the cell suspension to be seeded and the liquid volume of the diluent required for adjusting the cell suspension to be seeded to a preset viable cell concentration, on the basis of the viable cell concentration and the liquid volume data of the cell suspension, supplies the aforesaid liquid volume of the cell suspension and the aforesaid liquid volume of the diluent to the mixer unit, and seeds the diluted cell suspension. A method for adjusting the viable cell concentration.

Description

[Technical Field]

[0001] This disclosure relates to a cell culture apparatus and a method for adjusting the concentration of viable cells. [Background technology]

[0002] In cell culture technology, it is necessary to appropriately adjust the viable cell concentration of the target cells in the cell suspension used for seeding. Techniques for such viable cell concentration adjustment are disclosed, for example, in International Publication No. 2016 / 013394. [Overview of the project] [Problems that the invention aims to solve]

[0003] Although techniques for adjusting the concentration of living cells have been studied for some time, including in International Publication No. 2016 / 013394, the current situation is that they are not sufficient.

[0004] This disclosure has been made in view of the circumstances described herein, and one embodiment of this disclosure aims to solve the problem of providing a cell culture device that can rapidly adjust the viable cell concentration of target cells. Another problem that other embodiments of this disclosure aim to solve is to provide a method for adjusting the viable cell concentration of target cells that can be rapidly adjusted. [Means for solving the problem]

[0005] This disclosure includes the following aspects: <1> A first container for holding the cell suspension, A second container for holding the diluent used to dilute the cell suspension, A cell viability measuring device that measures the viability of target cells in a cell suspension by individually determining the viability of multiple target cells in the cell suspension. A control device that maintains and controls data on the concentration of living cells and the volume of cell suspension. A mixing section for mixing the cell suspension and the diluent, and A culture vessel in which a diluted cell suspension is seeded and cultured. It has, A cell culture apparatus comprising a control device that determines the volume of cell suspension to be seeded and the volume of diluent needed to adjust the cell suspension to a predetermined viable cell concentration based on live cell concentration and volume data of the cell suspension, and then delivers the above volumes of cell suspension and diluent to a mixing unit to seed the diluted cell suspension. <2> The live cell concentration measuring device is located in a channel branched from the channel between the first container and the mixing section, or in the channel between the first container and the mixing section. <1> The cell culture apparatus described above. <3> The viable cell concentration is measured during at least a portion of the time it takes for the cell suspension to reach the mixing section from the first container. <2> The cell culture apparatus described above. <4> The mixing section also serves as the culture vessel. <1> ~ <3> A cell culture apparatus as described in any one of the following. <5> The mixing unit also serves as the culture vessel for the suspension culture system. <1> ~ <4> A cell culture apparatus as described in any one of the following. <6> The mixing section also serves as the second container. <1> ~ <3> A cell culture apparatus as described in any one of the following. <7> The culture vessel is a multi-layer culture vessel. <1> ~ <6> A cell culture apparatus as described in any one of the following. <8> A live cell concentration measuring device, A light source that irradiates target cells with light. An imaging device for imaging target cells, Means for changing the focal plane, An image acquisition unit acquires images of the target cell, taken from the opposite direction to the side of the target cell that was illuminated, and from multiple focal planes including the focal plane of the target cell. Image fragment acquisition unit that acquires image fragments from each image, including the central and peripheral regions of the target cell. An analytical image creation unit that creates an analytical image by connecting image fragments in the order of the imaging direction of the focal plane. A feature extraction unit that extracts features from a concatenated image for analysis, and A viability determination unit that determines the viability of a plurality of target cells in a cell suspension based on the feature amount of the connection image for analysis and a predetermined range of feature amounts, and A viable cell concentration determination unit that determines the viable cell concentration of the target cells in the cell suspension based on the result of the viability determination, The cell culture apparatus according to any one of <1> to <7>, comprising: <9> The cell culture apparatus according to <8>, wherein the means for changing the focal plane is a stage moving mechanism that moves a stage on which a holding container holding the target cells is placed to change the distance between the target cells and the imaging device. <10> The cell culture apparatus according to <8>, wherein the means for changing the focal plane is an imaging device moving mechanism that moves the imaging device to change the distance between the target cells and the imaging device. <11> The cell culture apparatus according to <8>, wherein the imaging device includes a liquid lens as the means for changing the focal plane. <12> The viable cell concentration measuring device is A digital holographic microscope that images the target cells, A phase image acquisition unit that acquires the phase image of the target cells, A viability determination unit that determines the viability of a plurality of target cells in a cell suspension based on the phase amount distribution of the phase image and a predetermined phase amount distribution, and A viable cell concentration determination unit that determines the viable cell concentration of the target cells in the cell suspension based on the result of the viability determination, The cell culture apparatus according to any one of <1> to <7>, comprising: <13> The cell culture apparatus according to any one of <1> to <12>, wherein the viable cell concentration measuring device includes a cell viability determination unit that determines the cell viability of the target cells in the cell suspension based on the result of the viability determination. <14> A step of acquiring an image of the target cells, which is imaged at a plurality of focal planes including the in-focus plane of the target cells, from a direction opposite to the side irradiated with light of the target cells irradiated with light, A step of measuring the viable cell concentration of the target cells in the cell suspension by individually determining the viability of a plurality of target cells in the cell suspension, Based on the viable cell concentration and the volume data of the cell suspension, determining the volume of the cell suspension to be seeded and the volume of the diluent required to prepare the cell suspension to be seeded to a predetermined viable cell concentration, and Mixing the cell suspension of the above volume with the diluent of the above volume, A viable cell concentration adjustment method including. <15> The step of measuring the viable cell concentration is Obtaining an image of the target cells, which is captured at a plurality of focal planes including the in-focus plane of the target cells, from the direction opposite to the side irradiated with light of the target cells irradiated with light, Obtaining an image piece including the central part and the outer peripheral part of the target cells from each image, Connecting the image pieces in the order of the imaging direction of the focal planes to create a connected image for analysis, Extracting feature amounts from the connected image for analysis, Based on the feature amounts of the connected image for analysis and a predetermined range of feature amounts, performing a determination of the life and death of a plurality of target cells in the cell suspension, and Based on the result of the life and death determination, determining the viable cell concentration of the target cells in the cell suspension, The viable cell concentration adjustment method according to <14>, including. <16> The step of measuring the viable cell concentration is Imaging the target cells with a digital holographic microscope to obtain the phase image of the target cells, Based on the phase amount distribution of the phase image and a predetermined phase amount distribution, performing a determination of the life and death of a plurality of target cells in the cell suspension, and Based on the result of the life and death determination, determining the viable cell concentration of the target cells in the cell suspension, The viable cell concentration adjustment method according to <l4>, including. <17> The viable cell concentration adjustment method according to any one of <14> to <16>, including a step of determining the cell viability of the target cells in the cell suspension based on the result of the life and death determination. <18> The cell culture device according to any one of <1> to <13>, wherein the target cells are human synovium-derived mesenchymal stem cells. <19> The target cells are human synovial membrane-derived mesenchymal stem cells. <14> ~ <17> A method for producing an arthropathy treatment agent using a method for adjusting the concentration of living cells as described in any one of the following. <20> <19> An arthritis treatment agent manufactured by the method for manufacturing arthritis treatment agents described above. [Effects of the Invention]

[0006] According to one embodiment of the present disclosure, a cell culture apparatus is provided that can rapidly adjust the viable cell concentration of target cells. According to other embodiments of this disclosure, a method for adjusting the viable cell concentration of target cells is provided, which can rapidly adjust the viable cell concentration of target cells. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 is a schematic diagram showing an example of a cell culture apparatus. [Figure 2] Figure 2 is a block diagram showing an example of a control device that makes up a cell culture system. [Figure 3] Figure 3 is a schematic diagram showing an example of a cell culture apparatus. [Figure 4] Figure 4 is a schematic diagram showing an example of a cell culture apparatus. [Figure 5] Figure 5 is a schematic diagram showing an example of a cell culture apparatus. [Figure 6] Figure 6 is a schematic diagram showing an example of a cell culture apparatus. [Figure 7] Figure 7 is a schematic diagram showing an example of a cell culture apparatus. [Figure 8] Figure 8 is a schematic diagram showing an example of a cell culture apparatus. [Figure 9] Figure 9 is a schematic diagram showing an example of a cell culture apparatus. [Figure 10] Figure 10 is a schematic diagram showing an example of a live cell concentration measuring device. [Figure 11] Figure 11 is a schematic diagram showing an example of a focal plane. [Figure 12]Figure 12 is a schematic diagram showing an example of an image obtained by imaging living cells while changing the focal plane. [Figure 13] Figure 13 is a schematic diagram showing an example of an image obtained by imaging dead cells while changing the focal plane. [Figure 14] Figure 14 is a schematic diagram showing an example of a linked image obtained from living cells. [Figure 15] Figure 15 is a schematic diagram showing an example of a ligature image obtained from dead cells. [Figure 16] Figure 16 is a schematic diagram showing an example of processing during the training and operation phases of a machine learning model. [Figure 17] Figure 17 is a flowchart showing an example of the life / death determination process. [Figure 18] Figure 18 is a block diagram showing an example of a control unit that constitutes a cell viability determination device. [Figure 19] Figure 19 is a block diagram showing an example of the processing performed by a cell viability determination device. [Modes for carrying out the invention]

[0008] The details of the cell culture apparatus and live cell concentration adjustment method related to this disclosure are described below.

[0009] In this disclosure, a numerical range indicated using "~" means a range that includes the numbers before and after "~" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this disclosure, the upper or lower limit stated in one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in the numerical ranges described in this disclosure, the upper or lower limit stated in one numerical range may be replaced with the values ​​shown in the examples. In this disclosure, a combination of two or more preferred embodiments is a more preferred embodiment. In this disclosure, unless otherwise specified, the amount of each component refers to the total amount of multiple substances if there are multiple substances corresponding to each component. In this disclosure, the term "process" includes not only independent processes but also processes that cannot be clearly distinguished from other processes, as long as their intended purpose is achieved.

[0010] The drawings referenced in the following description are illustrative and schematic, and this disclosure is not limited to these drawings. The same reference numerals indicate the same components. Reference numerals in the drawings may also be omitted.

[0011] <Cell culture equipment> The cell culture apparatus relating to this disclosure is A first container for holding the cell suspension, A second container for holding the diluent used to dilute the cell suspension, A cell viability measuring device that measures the viability of target cells in a cell suspension by individually determining the viability of multiple target cells in the cell suspension. A control device that maintains and controls data on the concentration of living cells and the volume of cell suspension. A mixing section for mixing the cell suspension and the diluent, and A culture vessel in which a diluted cell suspension is seeded and cultured. It has, The control device determines the volume of cell suspension to be seeded and the volume of diluent needed to adjust the cell suspension to a predetermined live cell concentration, based on the live cell concentration and volume data of the cell suspension. It then sends the above volumes of cell suspension and diluent to the mixing unit and seeds the diluted cell suspension.

[0012] As a technique for adjusting the concentration of live cells, for example, the technique described in International Publication No. 2016 / 013394, Patent Document 1 can be cited. However, in the technique of International Publication No. 2016 / 013394, Patent Document 1, when measuring the concentration of live cells, a pre-prepared calibration curve is used, so it is necessary to prepare many calibration curves, taking into account the type of target cell and the presence of other cell types in the cell suspension. Furthermore, in the technique of International Publication No. 2016 / 013394, Patent Document 1, when mixing the cell suspension and the diluent, it is necessary to mix them using a circulation path. Therefore, the technique of International Publication No. 2016 / 013394, Patent Document 1 requires a lot of time to adjust the concentration of live cells. In contrast, the cell culture apparatus described herein allows for individual viability testing of multiple target cells in a cell suspension, enabling rapid measurement of the viable cell concentration of the target cells (hereinafter sometimes simply referred to as "viable cell concentration") and rapid adjustment of the viable cell concentration.

[0013] When using cells derived from living tissue, such as human tissue cells, or when manufacturing cell preparations from human tissue cells, there is a limit to the amount of cells that can be collected, and rapid cell preparation is necessary to avoid reducing the performance of the preparation. Specifically, the cell culture apparatus described in this disclosure can also be used in the manufacture of cell preparations for the treatment of arthritis from human synovial membrane-derived mesenchymal stem cells.

[0014] "Target cells" refers to a specific type of cell that is the subject of viability determination, and may be, for example, cells intended for cell culture.

[0015] "Predetermined viable cell concentration" may refer, for example, to the target viable cell concentration determined for the cell suspension of the target cells used for seeding.

[0016] The type of target cell is not particularly limited, and the viability of various living cells can be determined. Specifically, it may be mesenchymal stem cells collected and isolated from living tissue, for example, human synovial membrane-derived mesenchymal stem cells as described above. Synovial membrane-derived mesenchymal stem cells are also called synovial membrane-derived stem cells.

[0017] The following provides a detailed explanation of each component.

[0018] [1st container] The first container holds the cell suspension. A volume of cell suspension, determined based on the viable cell concentration and the volume data of the cell suspension, is delivered to the mixing unit.

[0019] The material, shape, etc., of the first container are not particularly limited and may be appropriately selected considering the configuration of the cell culture apparatus. The type of target cell is not particularly limited, and various target cells can be seeded after measuring and diluting their viable cell concentration. From the viewpoint of facilitating the delivery of a uniform concentration, the first container may be equipped with, for example, a mixing mechanism such as a stirrer, or a rocking mechanism that can rock the first container.

[0020] [Second container] The second container holds the diluent for diluting the cell suspension. The amount of diluent determined based on the live cell concentration and the volume data of the cell suspension is delivered to the mixing unit.

[0021] The material, shape, etc., of the second container are not particularly limited and may be appropriately selected considering the configuration of the cell culture apparatus. The type of diluent is not particularly limited and may be appropriately selected considering the type of target cells. Furthermore, as described later, the mixing section may also serve as the second container.

[0022] [Live cell concentration measuring device] The viable cell concentration measuring device measures the viability of target cells in a cell suspension by individually determining the viability of multiple target cells in the cell suspension.

[0023] The live cell concentration measuring device may include a cell viability determination unit that determines the cell viability of target cells in a cell suspension based on the results of viability determination of multiple target cells in the cell suspension. The cell viability determination unit of the live cell concentration measuring device corresponds to the cell viability determination step of the live cell concentration adjustment method, which will be described in detail later.

[0024] The viable cell concentration measuring device may be placed at any position in the cell culture apparatus. From the viewpoint of measuring the viable cell concentration without exposing the cell suspension to the external environment, it is preferable that the viable cell concentration measuring device is placed in a channel branched from the channel between the first container and the mixing unit, or in the channel between the first container and the mixing unit (hereinafter sometimes referred to as "the viable cell concentration measuring device being inline"). This allows the cell suspension to be kept in a cleaner state.

[0025] If the live cell concentration measuring device is inline, the live cell concentration can be measured during at least a portion of the time it takes for the cell suspension to reach the mixing section from the first container. By measuring the live cell concentration while simultaneously distributing the cell dispersion toward the mixing section, the live cell concentration can be adjusted more quickly.

[0026] The method for measuring the concentration of living cells is not particularly limited.

[0027] For example, as will be described in detail later, there is a viable cell concentration measuring device (hereinafter sometimes referred to as "Viable Cell Concentration Measuring Device of Embodiment 1") that determines whether a target cell is alive or dead by analyzing a concatenated image obtained from multiple images of the target cell, and measures the concentration of viable cells. Another example is a viable cell concentration measuring device (hereinafter sometimes referred to as "viable cell concentration measuring device of embodiment 2") that determines the viability of target cells and measures the viability of viable cells by analyzing the phase image of target cells captured with a digital holographic microscope.

[0028] The following describes in detail the live cell concentration measuring devices of Embodiment 1 and Embodiment 2.

[0029] (Live cell concentration measuring device according to Embodiment 1) The living cell color measuring device of embodiment 1 is A light source that irradiates target cells with light. An imaging device for imaging target cells, Means for changing the focal plane, An image acquisition unit acquires images of the target cell, taken from the opposite direction to the side of the target cell that was illuminated, and from multiple focal planes including the focal plane of the target cell. Image fragment acquisition unit that acquires image fragments from each image, including the central and peripheral regions of the target cell. An analytical image creation unit that creates an analytical image by connecting image fragments in the order of the imaging direction of the focal plane. A feature extraction unit that extracts features from concatenated images for analysis. A cell survival determination unit that determines the viability of multiple target cells in a cell suspension based on the features of the concatenated image for analysis, the features of a known reference concatenated image of the target cell, and the determination result of whether or not the cell is alive, and Based on the results of the viability determination, a viability concentration determination unit determines the viability concentration of target cells in the cell suspension. It is equipped with.

[0030] Living cells, being covered by a cell membrane, have a tendency to form a spherical shape in cell suspension (though not necessarily a perfect sphere), and they are also translucent. Therefore, living cells possess the properties of a spherical lens, which is called the "lens effect." Therefore, when light is shone on a living cell, a focal point (hereinafter sometimes referred to as the "focal point of the lens effect") is formed in the direction opposite to the side of the living cell from which the light is being shone, due to the lens effect. When the living cell is imaged from the direction opposite to the side of the living cell from which the light is being shone, an image including the focal point of the lens effect can be obtained at the focal plane of the living cell. Therefore, for living cells, a stitched image created from images acquired at multiple focal planes, including the plane of focus, contains information specific to living cells.

[0031] On the other hand, dead cells do not form a spherical shape because their cell membranes are ruptured, and the liquid medium of the cell suspension flows into the dead cells. Therefore, dead cells do not exhibit a lens effect. Therefore, even if light is shone on dead cells, no focal point of the lens effect is formed, and even if dead cells are imaged at the focal plane of the dead cells, it is not possible to obtain an image that includes the focal point of the lens effect.

[0032] As described above, concatenated images created from living cells differ from concatenated images created from dead cells, and therefore possess features unique to living cells. For this reason, the viability of target cells can be determined based on the features of the concatenated images used for analysis of target cells whose viability is unknown, and a predetermined range of features.

[0033] Furthermore, since the cell culture apparatus of Embodiment 1 utilizes the optical properties of cells, the concentration of viable cells can be adjusted more easily without using staining reagents.

[0034] The linked images used for analysis and the linked images used for reference are sometimes simply referred to as "linked images." Similarly, "target cells" are sometimes simply referred to as "cells."

[0035] The life / death determination unit may be composed of a machine learning model.

[0036] In the live cell concentration measuring device of Embodiment 1, the image acquisition unit, image fragment acquisition unit, linked image creation unit for analysis, feature extraction unit, viability determination unit, and live cell concentration determination unit correspond to the image acquisition step, image fragment acquisition step, linked image creation step for analysis, feature extraction step, viability determination step, and live cell concentration determination step of the live cell concentration measuring process of Embodiment 1, which will be described in detail later.

[0037] The imaging device consists of a combination of an imaging lens and an area sensor. The imaging lens may be a telecentric lens, a microscope objective lens, or the like.

[0038] The light source is not particularly limited, but examples include LEDs (Light Emitting Diodes).

[0039] The means for changing the focal plane are not particularly limited, and may be, for example, a stage movement mechanism that changes the distance between the cells and the imaging device by moving a stage on which a holding container for holding cells is placed. The living cell concentration measuring device 200 may, for example, as shown in Figure 10, include a light source 10 for irradiating target cells C with light, an imaging device 20 for imaging the target cells C, a holding container 40 for holding the target cells (cell suspension) C, a stage 30 (stage movement mechanism) for moving the holding container 40 to change the distance between the target cells C and the imaging device 20, and a control unit 106.

[0040] The means for changing the focal plane may be an imaging device movement mechanism that moves the imaging device to change the distance between the cell and the imaging device. Furthermore, the imaging device may be equipped with a liquid lens as a means of changing the focal plane.

[0041] The viable cell concentration measuring device may include an input device for inputting data and a display device for displaying the measurement results of viable cell concentration, etc. The live cell concentration measuring device may be configured to receive data via an input device provided in the cell culture apparatus. Furthermore, the live cell concentration measuring device may be configured to display the live cell concentration measurement results on a display device provided in the cell culture apparatus.

[0042] The following will provide a more detailed explanation of the live cell concentration measuring device, using the live cell concentration measuring device 200 shown in Figure 10 as an example.

[0043] Figure 18 is a block diagram showing an example of a control unit 106 that constitutes the live cell concentration measuring device 200 shown in Figure 10. The CPU (Central Processing Unit) 101 in the control unit 106 is a processor that controls the live cell concentration measuring device 200 as a whole. The CPU 101 reads the system program stored in the ROM (Read Only Memory) 102 via the bus 105 and controls the entire live cell concentration measuring device 200 according to the system program. The RAM (Random Access Memory) 103 temporarily stores calculation data, display data for the display device 500 of the control device 350 of the cell culture device, and various data input via the input device 400 of the control device 350 of the cell culture device.

[0044] The non-volatile memory 104 stores data acquired from the light source 10, data acquired from the imaging device 20, data acquired from the stage 30, and data input from the input device 50, for example, using an SRAM (Static Random Access Memory) or SSD (Solid State Drive) backed up by a battery (not shown). The data and programs stored in the non-volatile memory 104 may be loaded into the RAM 103 when needed. The ROM 102 is pre-programmed with various algorithms required for image analysis of image data acquired from the imaging device 20, as well as system programs for executing other necessary processing.

[0045] Figure 19 is a block diagram showing an example of the processing of the live cell concentration measuring device shown in Figure 10. Each functional block shown in Figure 19 is realized by the CPU 101, provided in the control unit 106 shown in Figure 10, executing a system program and controlling the operation of each part of the live cell concentration measuring device 200.

[0046] The control unit 106 controls the light source 10, the imaging device 20, and the stage 30 to image the cell C based on the imaging program stored in the non-volatile memory 104. The control unit 106 turns on the light source 10 to irradiate the cell C with light and drives the stage 30 to move the holding container 40 that holds the cell (cell suspension) C to change the focal plane. After moving the stage 30 to a predetermined focal plane, the control unit 106 commands the imaging device 20 to perform an imaging operation. According to the imaging program, the control unit 106 images the cell from a direction opposite to the side of the irradiated cell, including the focal plane of the cell.

[0047] The image acquisition unit 107 acquires images of target cells captured by the imaging device 20. The images of target cells acquired by the image acquisition unit 107 may be managed together as a set of image data, with multiple images obtained by imaging a single target cell.

[0048] The image fragment acquisition unit 108 performs image processing on the image of the target cell acquired by the image acquisition unit 107 to acquire image fragments from the image of the target cell that include the central and peripheral parts of the target cell. The image fragments acquired by the image fragment acquisition unit 108 may be managed together as a set of image fragment data, with multiple image fragments obtained by imaging a single target cell being collected.

[0049] The analysis-oriented image creation unit 109 performs image processing on the image fragments obtained by the image fragment acquisition unit 108, and concatenates the image fragments in the order of the imaging direction of the focal plane to create an analysis-oriented concatenated image.

[0050] The feature extraction unit 110 performs image processing on the concatenated image for analysis obtained by the concatenated image creation unit 109 and extracts features from the concatenated image for analysis.

[0051] The viability determination unit 111 determines the viability of cells based on the features of the concatenated images for analysis extracted by the feature extraction unit 110 and a predetermined range of features. In another embodiment, the viability determination unit 111 determines the viability of target cells based on the features of the concatenated images for analysis extracted by the feature extraction unit 110, the features of a known reference concatenated image of the target cell, and the determination result of whether or not it is a living cell.

[0052] The viable cell concentration determination unit 112 determines the viable cell concentration of the target cells based on the viability determination result, i.e., the number of analysis linkage images determined to be viable cells. The viable cell concentration determination unit 112 outputs the viable cell concentration of the target cells to the display device 500 via the control device 350 of the cell culture apparatus.

[0053] The cell viability determination unit 113 determines the cell viability rate based on the viability determination results, i.e., the number of analysis linkage images determined to be living cells and the total number of analysis linkage images. The cell viability determination unit 113 outputs the cell viability rate to the display device 500 via the control device 350 of the cell culture apparatus.

[0054] (Live cell concentration measuring device according to embodiment 2) The living cell color measuring device of embodiment 2 is A digital holographic microscope for imaging target cells. Phase image acquisition unit for acquiring phase images of target cells. A viability determination unit that determines the viability of multiple target cells in a cell suspension based on the phase quantity distribution of the above phase image and a predetermined phase quantity distribution, and Based on the results of the viability determination, a viability concentration determination unit determines the viability concentration of target cells in the cell suspension. It is equipped with.

[0055] Living cells have a refractive index difference between the inside and outside of the cell, which is the source of the lens effect, and they also have a refractive index distribution. On the other hand, in dead cells, because the cell membrane is ruptured, the liquid medium of the cell suspension flows into the inside of the cell, resulting in a smaller refractive index difference with the outside of the cell and a smaller refractive index distribution. Since the internal refractive index can be detected as a phase quantity, the phase image of the target cell obtained by digital holographic microscopy has a phase quantity distribution that is unique to living cells.

[0056] Therefore, the viability of target cells can be determined based on the phase quantity distribution of the phase image of target cells whose viability is unknown, and a predetermined range of phase quantity distribution.

[0057] Furthermore, since the cell culture apparatus of embodiment 2 utilizes the optical properties of cells, the concentration of viable cells can be adjusted more easily without using staining reagents.

[0058] The control unit of the live cell concentration measuring device in Embodiment 2 may also be designed appropriately, similar to the example provided for Embodiment 1.

[0059] In the live cell concentration measuring device of Embodiment 2, the phase image acquisition unit, the viability determination unit, and the live cell concentration determination unit correspond to the phase image acquisition step, the viability determination step, and the live cell concentration determination step of the live cell concentration measuring step of Embodiment 2, which will be described in detail later.

[0060] A digital holographic microscope is used to image the target cells. The phase image acquisition unit acquires the phase image of the imaged target cells.

[0061] The living cell concentration measuring devices of Embodiments 1 and 2 have been described above. Further details regarding the method for adjusting the living cell concentration will be described later.

[0062] [Control device] The control device maintains and controls data on the concentration of living cells and the volume of the cell suspension.

[0063] The control unit determines the volume of cell suspension to be seeded and the volume of diluent needed to adjust the cell suspension to a predetermined viable cell concentration, based on data on the viable cell concentration and the volume of the cell suspension. The control unit then delivers the volume of cell suspension and the volume of diluent to the mixing unit and seeds the diluted cell suspension.

[0064] [Mixing section] The mixing section mixes the cell suspension with the diluent.

[0065] In one embodiment, the mixing unit may be a container for mixing a volume of cell suspension and diluent determined based on the live cell concentration and volume data of the cell suspension. The container that is the mixing unit may be equipped with a mixing mechanism such as a stirrer, a rocking mechanism that can rock the mixing unit, and so on. The diluted cell dispersion is then delivered to a culture vessel.

[0066] Examples of the motion include rotation, vertical shaking, and horizontal shaking. The rocking mechanism is not particularly limited, but examples include cam mechanisms, linkage mechanisms, and linear motion mechanisms. More specifically, examples include articulated robots, single-axis robots, combinations of linear cylinders, and shakers. The same applies to the manner of oscillation and the oscillation mechanism in other descriptions of this disclosure.

[0067] In other embodiments, the mixing unit may also serve as the culture vessel. That is, the culture vessel may have a function for mixing the cell suspension and the diluent. When the mixing unit also serves as the culture vessel, "sending the cell culture medium and diluent to the mixing unit" means sending the cell culture medium and diluent to the culture vessel that also serves as the mixing unit.

[0068] If the mixing unit also serves as the culture vessel, the mixing unit may be, for example, a rocking mechanism capable of rocking the culture vessel. This allows for mixing the cell suspension and the diluent while simultaneously seeding the diluted cell suspension. Examples of rocking motions include rotation, vertical shaking, and horizontal shaking.

[0069] The mixing unit may also serve as a culture vessel for a suspension culture system. When the mixing unit also serves as a culture vessel for a suspension culture system, the mixing unit may, for example, be a circulation mechanism capable of circulating a diluted cell suspension. A suspension culture system is a culture system that allows cells to be cultured while suspended by circulating the cells. This allows for seeding of the diluted cell suspension while mixing the cell suspension with the diluent. Examples of circulation mechanisms include a stirring mechanism.

[0070] In other embodiments, the mixing unit may also serve as a second container. That is, the second container may have the function of mixing the cell suspension and the diluent. When the mixing unit also serves as the culture vessel, "sending the cell culture medium and diluent to the mixing unit" means sending the cell culture medium to the culture vessel that also serves as the mixing unit and holds the diluent.

[0071] The material and shape of the mixing section are not particularly limited and may be selected as appropriate considering the configuration of the cell culture apparatus.

[0072] [Culture container] The culture vessel is used to culture cells by seeding a diluted cell suspension.

[0073] The culture vessel may be equipped with a rocking mechanism that allows it to be rocked to facilitate sowing.

[0074] The culture vessel may be a multilayer culture vessel. A multilayer culture vessel is a culture vessel that has multiple layers on which cells can be seeded and cultured. Furthermore, as mentioned above, the culture vessel may be a suspension culture device that also serves as a mixing unit.

[0075] The cell culture apparatus comprises a first container, a second container, a viable cell concentration measuring device, a control device, a mixing unit 330, and a channel connecting the culture containers. Note that, in order to simplify the drawings, the channel is not denoted by reference numerals in this disclosure.

[0076] The cell culture apparatus may be equipped with valves, pumps, flow meters, etc., in the flow path. By adjusting the degree of valve opening and closing, adjusting the output of the pump, etc., it becomes easier to control the flow rate in the flow path. In addition, by using a flow meter, the flow rate can be easily determined at a desired position in the flow path. The valve is not particularly limited and may be a pinch valve, for example. Similarly, the pump is not particularly limited and may be a veristar pump, for example.

[0077] The cell culture apparatus may be equipped with a waste liquid container for disposing of the cell suspension used to measure the concentration of viable cells. The waste liquid container is not particularly limited and may include, for example, a bag, bottle, syringe, etc.

[0078] The cell culture apparatus may include an input device for inputting data, and a display device for displaying the viable cell concentration obtained from a viable cell concentration measuring device, the viable cell concentration of a diluted cell suspension, and the like.

[0079] (Embodiment 1) The cell culture apparatus according to this disclosure will be described in more detail below, using the cell culture apparatus 300 shown in Figure 1 as an example.

[0080] The cell culture apparatus 300 shown in Figure 1 comprises a first container 310, a second container 320, a live cell concentration measuring device 200, a control device 350, a mixing unit 330, and a culture vessel 340. Furthermore, the cell culture apparatus 300 includes an input device 400 and a display device 500.

[0081] The first container 310 and the mixing unit 330 are connected by a flow path via a valve 362 and a pump 372. The second container 320 and the mixing unit 330 are also connected by a flow path via a valve 363 and a pump 373. Furthermore, the mixing unit 330 and the culture container 340 are connected by a flow path via a valve 360 ​​and a pump 370.

[0082] The first container 310 and the live cell concentration measuring device 200 are connected by a flow path via a valve 361 and a pump 371. The live cell concentration measuring device 200 is located in a flow path branched off from the flow path between the first container 310 and the mixing unit 330. A waste liquid container 380 is also connected to the live cell concentration measuring device 200. The cell culture apparatus may include an input device for inputting data and a display device for displaying the results of measurements of viable cell concentration, etc. For example, a keyboard can be used as the input device, and a monitor can be used as the display device.

[0083] The control device 350 is connected to the first container 310, the second container 320, the mixing unit 330, the culture vessel 340, valves 360 to 363, pumps 371 to 373, the live cell concentration measuring device 200, the input device 400, and the display device 500 so as to be able to communicate with them.

[0084] Figure 2 is a block diagram showing an example of a control device 350 that constitutes the cell culture apparatus 300 shown in Figure 1. The CPU (Central Processing Unit) 351 of the control device 350 is a processor that controls the cell culture apparatus 300 as a whole. The CPU 351 reads the system program stored in the ROM (Read Only Memory) 352 via the bus 355 and controls the entire cell culture apparatus 300 according to the system program. The RAM (Random Access Memory) 353 temporarily stores temporary calculation data, display data for the display device 500, and various data input via the input device 400.

[0085] The non-volatile memory 354 stores data such as the volume data of the cell suspension obtained from the first container 310, the volume data of the diluent obtained from the second container 320, and the live cell concentration obtained from the live cell concentration measuring device 200, using, for example, an SRAM (Static Random Access Memory) or SSD (Solid State Drive) backed up by a battery (not shown). The non-volatile memory 354 also stores data obtained from the mixing unit 330, data obtained from the culture vessel 340, data obtained from valves 360 to 363, data obtained from pumps 371 to 373, and data input from the input device 400. The data and programs stored in the non-volatile memory 354 may be loaded into RAM 353 when needed. Furthermore, ROM352 is pre-programmed with various algorithms necessary for determining the volume of cell suspension to be seeded and the volume of diluent required to adjust the cell suspension to a predetermined live cell concentration, based on live cell concentration and cell suspension volume data, as well as system programs for executing other necessary processes.

[0086] Using cell culture apparatus 300 as an example, cell culture examples 1 and 2 below will be explained.

[0087] -Cell culture example 1- The control device 350 opens valve 361 to drive pump 371 and delivers the cell suspension from the first container 310 to the live cell concentration measuring device 200, and then closes valve 361 to stop pump 371.

[0088] The viable cell concentration of the cell suspension is measured using the viable cell concentration measuring device 200, and the cell suspension used for the measurement is discharged into the waste liquid container 380.

[0089] The control device 350 obtains the live cell concentration of the cell suspension from the live cell concentration measuring device 200, and also obtains liquid volume data of the cell suspension from the first container 310.

[0090] The control device 350 determines the volume of the cell suspension to be seeded and the volume of the diluent needed to adjust the cell suspension to a predetermined live cell concentration, based on the live cell concentration and volume data of the cell suspension.

[0091] The control device 350 opens valve 362 to drive pump 372 and delivers the determined amount of cell suspension from the first container 310 to the mixing unit 330, and then closes valve 362 to stop pump 372. Furthermore, the control device 350 opens the valve 363 to drive the pump 373 and delivers the determined amount of diluted liquid from the second container 320 to the mixing unit 330, and then closes the valve 363 to stop the pump 373.

[0092] The control device 350 drives a mixing mechanism (not shown) provided in the mixing unit 330 to mix the cell suspension and the diluent, thereby diluting the fine suspension.

[0093] The control device 350 opens the valve 360 ​​and drives the pump 370, which delivers the diluted cell suspension from the mixing unit 330 to the culture vessel 340, and seeds are seeded into the culture vessel 340.

[0094] -Cell culture example 2- The control device 350 opens valve 361 to drive pump 371 and simultaneously opens valve 362 to drive pump 372. As a result, the control device 350 starts pumping the cell suspension from the first container 310 to the live cell concentration measuring device 200, while simultaneously pumping the cell suspension from the first container 310 towards the mixing unit 330. After pumping the cell suspension to the live cell concentration measuring device 200, the control device 350 closes valve 361 to stop pump 371.

[0095] During at least a portion of the time it takes for the cell suspension to reach the mixing unit 330 from the first container 310, the viable cell concentration of the cell suspension is measured by the viable cell concentration measuring device 200. The cell suspension used for measurement is discharged into the waste liquid container 380.

[0096] The control device 350 obtains the live cell concentration of the cell suspension from the live cell concentration measuring device 200, and also obtains liquid volume data of the cell suspension from the first container 310.

[0097] The control device 350 determines the volume of the cell suspension to be seeded and the volume of the diluent needed to adjust the cell suspension to a predetermined live cell concentration, based on the live cell concentration and volume data of the cell suspension.

[0098] After the control device 350 delivers the determined amount of cell suspension from the first container 310 to the mixing unit 330, it closes the valve 362 and stops the pump 372. Furthermore, the control device 350 opens the valve 363 to drive the pump 373 and delivers the determined amount of diluted liquid from the second container 320 to the mixing unit 330, and then closes the valve 363 to stop the pump 373. As described above, by measuring the viable cell concentration while simultaneously sending the cell dispersion towards the mixing unit 330, the viable cell concentration can be adjusted more quickly.

[0099] In the same manner as in Cell Culture Example 1, the fine suspension is diluted in the mixing unit 330, and then the diluted cell suspension is seeded into the culture vessel 340.

[0100] (Embodiment 2) In another embodiment, the cell culture apparatus 300A differs from the cell culture apparatus 300 in that, as shown in Figure 3, the live cell concentration measuring device 200 is located in the flow path between the first container 310 and the mixing unit 330.

[0101] Using cell culture apparatus 300A as an example, cell culture examples 3 and 4 below will be explained.

[0102] -Cell culture example 3- The control device 350 opens valve 362 to drive pump 372 and delivers the cell suspension from the first container 310 to the live cell concentration measuring device 200, and then closes valve 362 to stop pump 372.

[0103] After the viable cell concentration of the cell suspension is measured by the viable cell concentration measuring device 200, the control device 350 obtains the viable cell concentration of the cell suspension from the viable cell concentration measuring device 200 and also obtains liquid volume data of the cell suspension from the first container 310.

[0104] The control device 350 determines the volume of the cell suspension to be seeded and the volume of the diluent needed to adjust the cell suspension to a predetermined live cell concentration, based on the live cell concentration and volume data of the cell suspension.

[0105] The control device 350 opens valve 362 to drive pump 372 and delivers the determined amount of cell suspension from the first container 310 to the mixing unit 330, and then closes valve 362 to stop pump 372. Furthermore, the control device 350 opens the valve 363 to drive the pump 373 and delivers the determined amount of diluted liquid from the second container 320 to the mixing unit 330, and then closes the valve 363 to stop the pump 373.

[0106] In the same manner as in Cell Culture Example 1, the fine suspension is diluted in the mixing unit 330, and then the diluted cell suspension is seeded into the culture vessel 340.

[0107] -Cell culture example 4- The control device 350 opens the valve 362 and drives the pump 372 to deliver the cell suspension from the first container 310 to the live cell concentration measuring device 200. Then, without closing the valve 362 or stopping the pump 372, it starts delivering the cell suspension from the first container 310 to the mixing unit 330.

[0108] During at least a portion of the time it takes for the cell suspension to reach the mixing unit 330 from the first container 310, the viable cell concentration of the cell suspension is measured by the viable cell concentration measuring device 200.

[0109] The control device 350 obtains the live cell concentration of the cell suspension from the live cell concentration measuring device 200, and also obtains liquid volume data of the cell suspension from the first container 310.

[0110] The control device 350 determines the volume of the cell suspension to be seeded and the volume of the diluent needed to adjust the cell suspension to a predetermined live cell concentration, based on the live cell concentration and volume data of the cell suspension.

[0111] After the control device 350 delivers the determined amount of cell suspension from the first container 310 to the mixing unit 330, it closes the valve 362 and stops the pump 372. Furthermore, the control device 350 opens the valve 363 to drive the pump 373 and delivers the determined amount of diluted liquid from the second container 320 to the mixing unit 330, and then closes the valve 363 to stop the pump 373. As described above, by measuring the viable cell concentration while simultaneously sending the cell dispersion towards the mixing unit 330, the viable cell concentration can be adjusted more quickly.

[0112] In the same manner as in Cell Culture Example 1, the fine suspension is diluted in the mixing unit 330, and then the diluted cell suspension is seeded into the culture vessel 340.

[0113] (Embodiment 3) In another embodiment, the cell culture apparatus 300B differs from the cell culture apparatus 300 in that, as shown in Figure 4, a single pump 372 is positioned between the first container 310 and the mixing unit 330, and between the second container 320 and the mixing unit 330.

[0114] Using cell culture apparatus 300B as an example, the following cell culture example 5 will be explained.

[0115] -Cell culture example 5 The control device 350 opens valve 361 to drive pump 371 and delivers the cell suspension from the first container 310 to the live cell concentration measuring device 200, and then closes valve 361 to stop pump 371.

[0116] The viable cell concentration of the cell suspension is measured using the viable cell concentration measuring device 200, and the cell suspension used for the measurement is discharged into the waste liquid container 380.

[0117] The control device 350 obtains the live cell concentration of the cell suspension from the live cell concentration measuring device 200, and also obtains liquid volume data of the cell suspension from the first container 310.

[0118] The control device 350 determines the volume of the cell suspension to be seeded and the volume of the diluent needed to adjust the cell suspension to a predetermined live cell concentration, based on the live cell concentration and volume data of the cell suspension.

[0119] The control device 350 opens valves 362 and 364 to drive pump 372, delivering the determined amount of cell suspension from the first container 310 to the mixing unit 330, while simultaneously delivering the determined amount of diluent from the second container 320 to the mixing unit 330. After that, the control device 350 closes valves 362 and 364 to stop pump 372.

[0120] In the same manner as in Cell Culture Example 1, the fine suspension is diluted in the mixing unit 330, and then the diluted cell suspension is seeded into the culture vessel 340.

[0121] -Cell culture example 6- The control device 350 opens valve 361 to drive pump 371, and simultaneously opens valves 362 and 364 to drive pump 372. As a result, the control device 350 starts supplying the cell suspension from the first container 310 to the live cell concentration measuring device 200, while simultaneously starting to supply the cell suspension from the first container 310 to the mixing unit 330, and further starts supplying the diluent from the second container 320 to the mixing unit 330. After supplying the cell suspension to the live cell concentration measuring device 200, the control device 350 closes valve 361 to stop pump 371.

[0122] During at least a portion of the time it takes for the cell suspension to reach the mixing unit 330 from the first container 310, the viable cell concentration of the cell suspension is measured by the viable cell concentration measuring device 200. The cell suspension used for measurement is discharged into the waste liquid container 380.

[0123] The control device 350 obtains the live cell concentration of the cell suspension from the live cell concentration measuring device 200, and also obtains liquid volume data of the cell suspension from the first container 310.

[0124] The control device 350 determines the volume of the cell suspension to be seeded and the volume of the diluent needed to adjust the cell suspension to a predetermined live cell concentration, based on the live cell concentration and volume data of the cell suspension.

[0125] The control device 350 delivers the determined amount of cell suspension from the first container 310 to the mixing unit 330, and delivers the determined amount of diluent from the second container 320 to the mixing unit 330, after which it closes valves 362 and 340 to stop pump 372. As described above, by measuring the viable cell concentration while simultaneously sending the cell dispersion towards the mixing unit 330, the viable cell concentration can be adjusted more quickly.

[0126] In the same manner as in Cell Culture Example 1, the fine suspension is diluted in the mixing unit 330, and then the diluted cell suspension is seeded into the culture vessel 340.

[0127] (Embodiment 4) In another embodiment, the cell culture apparatus 300C differs from the cell culture apparatus 300 in that, as shown in Figure 5, the live cell concentration measuring device 200 is located in the flow path between the first container 310 and the mixing unit 330. Additionally, a single pump 372 is located between the first container 310 and the mixing unit 330, and between the second container 320 and the mixing unit 330.

[0128] Using the cell culture apparatus 300C as an example, cell culture examples 7 and 8 below will be explained.

[0129] -Cell culture example 7- The control device 350 opens valve 362 to drive pump 372 and delivers the cell suspension from the first container 310 to the live cell concentration measuring device 200, and then closes valve 362 to stop pump 372.

[0130] After the viable cell concentration of the cell suspension is measured by the viable cell concentration measuring device 200, the control device 350 obtains the viable cell concentration of the cell suspension from the viable cell concentration measuring device 200 and also obtains liquid volume data of the cell suspension from the first container 310.

[0131] The control device 350 determines the volume of the cell suspension to be seeded and the volume of the diluent needed to adjust the cell suspension to a predetermined live cell concentration, based on the live cell concentration and volume data of the cell suspension.

[0132] The control device 350 opens valves 362 and 364 to drive pump 372, delivering the determined amount of cell suspension from the first container 310 to the mixing unit 330, while simultaneously delivering the determined amount of diluent from the second container 320 to the mixing unit 330. After that, the control device 350 closes valves 362 and 364 to stop pump 372.

[0133] In the same manner as in Cell Culture Example 1, the fine suspension is diluted in the mixing unit 330, and then the diluted cell suspension is seeded into the culture vessel 340.

[0134] -Cell culture example 8- The control device 350 opens the valve 362 and drives the pump 372 to deliver the cell suspension from the first container 310 to the live cell concentration measuring device 200. Then, without closing the valve 362 or stopping the pump 372, it starts delivering the cell suspension from the first container 310 to the mixing unit 330.

[0135] During at least a portion of the time it takes for the cell suspension to reach the mixing unit 330 from the first container 310, the viable cell concentration of the cell suspension is measured by the viable cell concentration measuring device 200.

[0136] The control device 350 obtains the live cell concentration of the cell suspension from the live cell concentration measuring device 200, and also obtains liquid volume data of the cell suspension from the first container 310.

[0137] The control device 350 determines the volume of the cell suspension to be seeded and the volume of the diluent needed to adjust the cell suspension to a predetermined live cell concentration, based on the live cell concentration and volume data of the cell suspension.

[0138] The control device 350 opens valve 364 and delivers the determined amount of cell suspension from the first container 310 to the mixing unit 330, while simultaneously delivering the determined amount of diluent from the second container 320 to the mixing unit 330. After that, the control device 350 closes valves 362 and 364 to stop pump 372. As described above, by measuring the viable cell concentration while simultaneously sending the cell dispersion towards the mixing unit 330, the viable cell concentration can be adjusted more quickly.

[0139] In the same manner as in Cell Culture Example 1, the fine suspension is diluted in the mixing unit 330, and then the diluted cell suspension is seeded into the culture vessel 340.

[0140] (Embodiment 5) In another embodiment, the cell culture apparatus 300D differs from the cell culture apparatus 300 in that, as shown in Figure 6, the culture vessel 340 also serves as the mixing unit 330 in a suspension culture apparatus. The culture vessel 340 is equipped with a circulation mechanism (not shown) that allows for the circulation of diluted cell suspension.

[0141] Using the cell culture apparatus 300D as an example, cell culture examples 9 and 10 below will be explained.

[0142] -Cell culture example 9- In the same manner as in Cell Culture Example 1, the viable cell concentration is measured using the viable cell concentration measuring device 200, and the volumes of the cell suspension and diluent are determined. Then, the determined volumes of the cell suspension and diluent are delivered to the culture vessel 340 (mixing section 330).

[0143] The control device 350 drives the circulation mechanism provided in the culture vessel 340 to mix the cell suspension and the diluent, and then seeds the diluted cell suspension.

[0144] -Cell culture example 10- In the same manner as in cell culture example 2, the viable cell concentration is measured using the viable cell concentration measuring device 200, and the volumes of the cell suspension and diluent are determined. Then, the determined volumes of the cell suspension and diluent are delivered to the culture vessel 340 (mixing section 330).

[0145] The control device 350 drives the circulation mechanism provided in the culture vessel 340 to mix the cell suspension and the diluent, and then seeds the diluted cell suspension.

[0146] (Embodiment 6) In another embodiment, the cell culture apparatus 300E differs from the cell culture apparatus 300A in that, as shown in Figure 7, the culture vessel 340 also serves as the mixing unit 330, and the culture vessel 340 is equipped with a rocking mechanism (not shown) that allows the culture vessel 340 to be rocked.

[0147] Using the cell culture apparatus 300E as an example, cell culture examples 11 and 12 below will be explained.

[0148] -Cell culture example 11- In the same manner as in cell culture example 3, the viable cell concentration is measured using the viable cell concentration measuring device 200, and the volumes of the cell suspension and diluent are determined. Then, the determined volumes of the cell suspension and diluent are delivered to the culture vessel 340 (mixing section 330).

[0149] The control device 350 drives the rocking mechanism provided in the culture vessel 340 to mix the cell suspension and the diluent, and then seeds the diluted cell suspension.

[0150] -Cell culture example 12- In the same manner as in cell culture example 4, the viable cell concentration is measured using the viable cell concentration measuring device 200, and the volume of the cell suspension and diluent is determined. Then, the determined volume of the cell suspension and diluent is delivered to the culture vessel 340 (mixing section 330).

[0151] The control device 350 drives the rocking mechanism provided in the culture vessel 340 to mix the cell suspension and the diluent, and then seeds the diluted cell suspension.

[0152] (Embodiment 7) In another embodiment, the cell culture apparatus 300F differs from the cell culture apparatus 300 in that, as shown in Figure 8, the second container 320 also serves as the mixing unit 330. The second container 320 is equipped with a mixing mechanism (not shown).

[0153] Using the cell culture device 300F as an example, the following cell culture example 13 will be explained.

[0154] -Cell culture example 13- In the same manner as in Cell Culture Example 1, measure the viable cell concentration using the viable cell concentration measuring device 200 and determine the volume of the cell suspension and diluent.

[0155] If the amount of diluent held in the second container 320 is greater than the amount determined above, the control device 350 opens the valve 365 to drive the pump 370, sends the excess diluent to the waste liquid container 381, and then closes the valve 365 to stop the pump 370. As a result, the amount of diluent determined above remains in the second container 320.

[0156] The control device 350 delivers the determined volume of cell suspension to the second container 320 (mixing unit 330). It drives a mixing mechanism (not shown) provided in the second container 320 to mix the cell suspension with the diluent and dilute the cell suspension.

[0157] The control device 350 opens the valve 360 ​​and drives the pump 370, which delivers the diluted cell suspension from the second container 320 to the culture container 340, and seeds the cells into the culture container 340.

[0158] (Embodiment 8) In another embodiment, the cell culture apparatus 300G differs from the cell culture apparatus 300A in that, as shown in Figure 9, the second container 320 also serves as the mixing unit 330. The second container 320 is equipped with a mixing mechanism (not shown).

[0159] Using the 300G cell culture system as an example, the following cell culture example 14 will be explained.

[0160] -Cell culture example 14- In the same manner as in cell culture example 3, measure the viable cell concentration using the viable cell concentration measuring device 200 and determine the volume of the cell suspension and diluent.

[0161] If the amount of diluent held in the second container 320 is greater than the amount determined above, the control device 350 opens the valve 365 to drive the pump 370, sends the excess diluent to the waste liquid container 381, and then closes the valve 365 to stop the pump 370. As a result, the amount of diluent determined above remains in the second container 320.

[0162] The control device 350 delivers the determined volume of cell suspension to the second container 320 (mixing unit 330). It drives a mixing mechanism (not shown) provided in the second container 320 to mix the cell suspension with the diluent and dilute the cell suspension.

[0163] The control device 350 opens the valve 360 ​​and drives the pump 370, which delivers the diluted cell suspension from the second container 320 to the culture container 340, and seeds the cells into the culture container 340.

[0164] In Embodiments 1, 3, 5, and 7 (Figures 1, 4, 6, and 8), the cell suspension used for measuring the viable cell concentration is delivered from the first container 310 to the viable cell concentration measuring device 200 by the pump 371, and the cell suspension used for measurement is discharged into the waste liquid container 380. In another embodiment, a syringe is used as the waste liquid container 380 and driven by a syringe pump (not shown), thereby allowing the cell suspension to be delivered to the live cell concentration measuring device 200 and the cell suspension used for measurement to be recovered.

[0165] As described above, the cell culture apparatus relating to this disclosure allows for rapid adjustment of the viable cell concentration of the target cells.

[0166] <Method for adjusting live cell concentration> The method for adjusting the concentration of living cells relating to this disclosure is: A step to measure the viability of target cells in a cell suspension by individually determining the viability of multiple target cells in the cell suspension (hereinafter sometimes referred to as the "viability of target cells measurement step"), A step of determining the volume of the cell suspension to be seeded and the volume of the diluent necessary to prepare the cell suspension to be seeded to a predetermined viable cell concentration, based on the viable cell concentration and volume data of the cell suspension (hereinafter sometimes referred to as the "volume determination step"), and The process of mixing the above-mentioned volume of cell suspension with the above-mentioned volume of diluent (hereinafter sometimes referred to as the "dilution process"), Includes.

[0167] The following explains the details of each step.

[0168] [Live cell concentration measurement process] In the viable cell concentration measurement process, the viability of multiple target cells in the cell suspension is determined individually, thereby measuring the viability of the target cells in the cell suspension.

[0169] The method for measuring the concentration of living cells is not particularly limited.

[0170] For example, as will be described in detail later, one step is to determine whether the target cells are alive or dead by analyzing a concatenated image obtained from multiple images of the target cells, and to measure the concentration of living cells (hereinafter sometimes referred to as the "living cell concentration measurement step of Embodiment 1"). Another example is a step in which the viability of target cells is determined and the concentration of viable cells is measured by analyzing the phase image of target cells captured with a digital holographic microscope (hereinafter sometimes referred to as the "viable cell concentration measurement step of Embodiment 2").

[0171] The following describes in detail the live cell concentration measurement process in Embodiment 1 and Embodiment 2.

[0172] (Method for measuring the concentration of living cells in embodiment 1) The live cell concentration measurement step in embodiment 1 is: The process of acquiring images of the target cell, which is illuminated with light, from the opposite side of the target cell to the light-illuminated side, and captured at multiple focal planes including the focal plane of the target cell (hereinafter sometimes referred to as the "image acquisition process"), The process of obtaining an image fragment from each image that includes the central and peripheral regions of the target cell (hereinafter sometimes referred to as the "image fragment acquisition process"), The process of creating a stitched image for analysis by connecting image fragments in the order of the imaging direction of the focal plane (hereinafter sometimes referred to as the "stitched image creation process for analysis"), The process of extracting features from the concatenated images for analysis (hereinafter sometimes referred to as the "feature extraction process"), A step of determining the viability of multiple target cells in a cell suspension based on the features of the concatenated images for analysis and a predetermined range of features (hereinafter sometimes referred to as the "viability determination step"), and Based on the results of the viability determination, a step is taken to determine the viability of target cells in the cell suspension (hereinafter sometimes referred to as the "viability cell concentration determination step"). including

[0173] Unless otherwise specified, the conditions for creating the linked images for analysis (imaging conditions, etc.) shall be the same as those for creating the reference linked images, to the extent that it is possible to determine the viability of the target cells and identify the target cells. Furthermore, when comparing different linked images for analysis, the conditions for creating these linked images shall be the same, to the extent that it is possible to compare them in terms of determining the viability of the target cells and identifying the target cells.

[0174] -Image acquisition process- In the image acquisition process, images of the target cell are acquired from a direction opposite to the side of the target cell that was illuminated, and are captured at multiple focal planes, including the focal plane of focus of the target cell or a focal plane near it.

[0175] Images of target cells can be obtained by imaging the cell suspension with an imaging device.

[0176] The means for changing the focal plane are not particularly limited; for example, one method is to change the distance between the target cell and the imaging device. Another method for imaging while keeping the distance between the target cell and the imaging device constant is to use an imaging device equipped with a liquid lens and change the focus of the liquid lens. The focal plane moves in accordance with the change in focal length.

[0177] For example, as shown in Figure 10, a holding container 40 containing target cells (cell suspension) C may be placed between the light source 10 and the imaging device 20, and the target cells may be imaged while changing the focal plane by moving the stage 30 on which the holding container 40 is placed.

[0178] Alternatively, a holding container containing cells (cell suspension) may be placed between the light source and the imaging device, and the cells may be imaged while changing the focal plane by moving the imaging device. Alternatively, a holding container containing cells (cell suspension) may be placed between the light source and the imaging device, and the cells may be imaged by changing the focal plane by changing the focus of the liquid lens provided by the imaging device, without moving the imaging device or the stage.

[0179] The target cells may be imaged while moving the focal plane from the light source 10 toward the imaging device 20, or the target cells may be imaged while changing the focal plane toward the light source 10 from the imaging device 20.

[0180] The imaging device consists of a combination of an imaging lens and an area sensor. The imaging lens may be a telecentric lens, a microscope objective lens, or the like.

[0181] The type of lens, aperture angle, magnification, etc. of the imaging device are not particularly limited, but since these can affect the focusing and divergence of light in the stitched image, they may be selected appropriately to ensure proper imaging. As the magnification of the lens increases, the field of view narrows, and the amount of cell suspension that can be imaged per field of view decreases. Therefore, from the viewpoint of measuring a larger amount of cell suspension, it is preferable to set the magnification to, for example, 2x to 4x. In addition, a narrower aperture angle of the lens tends to result in higher sensitivity in identifying the focal point.

[0182] For the area sensor of the imaging device, for example, a CCD (Charge-Coupled Device) or CMOS (Complementary Metal Oxide Semiconductor) can be used. The resolution of the area sensor should preferably be set so that, taking into account the lens magnification, one pixel is approximately 1 μm to 3 μm.

[0183] The light source is not particularly limited, but examples include LEDs (Light Emitting Diodes).

[0184] From the viewpoint of lens effects, it is preferable that the light source emits parallel light.

[0185] The positions and number of multiple focal planes used to image the target cells are not particularly limited as long as they include the focal plane of the target cells, but it is preferable to set them so that light collection and light divergence can be observed in the stitched images.

[0186] For example, the target cells may be imaged while moving the focal plane over a wide area from below to above the holding container, including from the bottom surface to the top surface of the holding container.

[0187] It is preferable that the distance between adjacent focal planes be constant. For example, when using a stage 30 as shown in Figure 10, the position of the stage 30 can be determined by an encoder, and target cells may be imaged while moving it at equal intervals.

[0188] The focal plane movement interval is preferably, for example, about 0.01 mm. Furthermore, since the distance the focal plane moves in a cell suspension is shorter than the distance the stage moves due to the refractive index of the cell suspension, it is preferable to take this into consideration when determining the distance the stage moves.

[0189] For convenience when changing the focal plane, for example, an image may be acquired by focusing on the bottom surface (or its vicinity) of the container holding the target cells and used as the reference plane. Furthermore, by defining a reference plane and obtaining stitched images by moving the focal plane within a predetermined range from the reference plane, stitched images can be created under the same conditions, which is beneficial for the analysis of stitched images.

[0190] Commercially available counting cells and hemocytometers, or measurement cells with similar structures, can be used as holding containers. When the cell suspension is contained in the holding container, for example, if the liquid thickness is about 0.1 mm and the cell concentration is 1 × 10⁶ 6 The cell concentration can be around cells / ml, but if the cell concentration is even lower, a container with a larger liquid thickness may be used.

[0191] For example, if the cell suspension contains other substances (e.g., other cells, red blood cells, oil droplets, etc.), image processing software can be used to pre-select substances whose diameter and roundness are within a predetermined range. By excluding substances that are clearly different from the target cells, the overall time required for cell viability determination can be shortened.

[0192] For example, as shown in Figure 11, light is irradiated onto the target cell C, and multiple focal planes P including the focal plane P0 of the target cell C are used while changing the focal plane. n ~focal plane P -m Then, target cell C may be imaged from the opposite direction to the side from which the light is irradiated, and multiple images may be acquired. Focal plane P n This is the nth focal plane in the direction of the imaging device 20 from the focal plane P0 of the target cell C. -mThis is the m-th focal plane in the direction of the light source 10 from the focal plane P0 of the target cell C. At least one of m and n is an integer of 1 or more, and m+n is an integer of 1 or more. Figure 11 shows an example where m is 2 or more and n is 4 or more.

[0193] The following describes an example where the target cell C is a living cell.

[0194] If the target cell C is a living cell, for example, while changing the focal plane, multiple focal planes P7 to P7, including the focal plane P0 of the living cell, are used. -2 By imaging living cells, images I7 to I7 of living cells can be obtained as shown in Figure 12. -2 You can obtain this.

[0195] Image I0 was captured at the focal plane P0 of a living cell, and the outline of the living cell is clear. In addition, there is a focal point of lens effect with an indistinct outline in the center of the living cell.

[0196] Image I1 was captured from the focal plane P0 of a living cell toward the imaging device 20 at the first focal plane P1, and the outline of the living cell is unclear. However, there is a focal point of the lens effect in the center of the living cell, and the outline of the focal point is clear. In other words, the focal plane P1 is an example of the focal plane of the focal point of the lens effect.

[0197] Images I2 and I3 were captured from the focal plane P0 of a living cell toward the imaging device 20 at the second and third focal planes P2 and P3, respectively, and the outline of the living cell is unclear. In addition, there is a focal point of lens effect with an unclear outline in the center of the living cell.

[0198] Images I4 to I7 were taken from the focal plane P0 of a living cell toward the imaging device 20, using the 4th focal plane P4 to the 7th focal plane P7, respectively. The outlines of the living cells are unclear. Furthermore, no focal point of lens effect is observed.

[0199] Image I -1 The first focal plane P is located in the direction from the focal plane P0 of the living cell toward the light source 10.-1 It is an image captured at -1 , and the outline of the living cell is unclear. Also, at the center of the living cell, there is a converging point of the lens effect with an unclear outline.

[0200] Image I -2 is an image captured at the second focal plane P in the direction of the light source 10 from the focal plane P0 of the living cell. -2 It is an image captured at -2 , and the outline of the living cell is unclear. Also, the converging point of the lens effect is not visible.

[0201] The outline of the living cell, for example, in Images I7 to Image I -2 tends to become larger as the focal plane is farther from the focal plane P0 of the living cell, as shown in. The size of the converging point of the lens effect, on the side where the converging point of the lens effect is formed (i.e., the side of the imaging device 20), for example, as shown in Images I1 and I2, tends to become larger as the focal plane is farther from the focal plane P1 of the converging point of the lens effect. However, at the focal plane P3 that is far from the focal plane P1 of the converging point of the lens effect, as shown in Image I3, the size of the converging point of the lens effect becomes smaller, and at the even farther focal plane P4, the converging point is not visible. Also, the size of the converging point of the lens effect, on the side opposite to the side where the converging point of the lens effect is formed (i.e., the side of the light source 10), for example, as shown in Image I -1 tends to become smaller as it is farther from the focal plane P1 of the converging point of the lens effect. And at the even farther focal plane P -2 the converging point of the lens effect is not visible.

[0202] Next, an example when the target cell is a dead cell will be described.

[0203] When the cell is a dead cell, for example, while changing the focal plane, by imaging the dead cell at a plurality of focal planes P7 to focal plane P -2 including the focal plane P0 of the dead cell, as shown in FIG. 13, images I'7 to Image I' -2 of the dead cell are obtained.

[0204] Image I'0 was captured at the focal plane P0 of dead cells, and the outlines of the dead cells are clearly visible.

[0205] Image I'1 was captured at the first focal plane P1, in the direction from the focal plane P0 of the dead cell toward the imaging device 20, and the outline of the dead cell is unclear.

[0206] Image I'2 was imaged from the focal plane P0 of a dead cell towards the imaging device 20 at the second focal plane P2, and the outline of the dead cell is unclear. Also, dead cells are opaque compared to living cells because their cell membranes are ruptured and light scattering occurs on the outer surface of the dead cell. As a result, when light is shone on a dead cell, a phenomenon similar to diffraction caused by an obstruction placed on a plane wave of light occurs, and a bright area is formed near the center of the dead cell in the direction opposite to the side of the dead cell from which the light is shone. This is the result of the overlapping of diffracted light (hereinafter, the above area may be called the "focus point of diffracted light"). In the center of the dead cell in Image I'2, there is a focus point of diffracted light, and the outline of the focus point is clear. That is, the focal plane P2 is an example of the focal plane of the focus point of diffracted light.

[0207] Images I'3 to I'7 were captured from the focal plane P0 of the dead cell toward the imaging device 20 at the third focal plane P3 to the seventh focal plane P7, respectively, and the outline of the dead cell is unclear. In addition, there is a point of convergence of diffracted light with an unclear outline in the center of the dead cell.

[0208] Image I' -1 and image I' -2 Each of these is the first focal plane P in the direction from the focal plane P0 of the dead cell toward the light source 10. -1 and the second focal plane P -2 The image was taken using [a specific imaging method], and the outlines of dead cells are unclear.

[0209] The outlines of dead cells are similar to those of living cells, for example, in images I7 to I7. -2 As shown, the focal plane tends to become larger the further it is from the focal plane P0 of dead cells. The size of the focal point of the diffracted light tends to increase as it is further away from the focal plane P2 of the focal point, as shown in images I'7 to I'2.

[0210] The above is just one example explained using Figures 12 and 13, and the appearance of the target cell's outline and light-gathering point may differ depending on the sphericity and size of the target cell.

[0211] The focal point of lens effects and the focal point of diffracted light are sometimes simply called "focal points."

[0212] -Image piece acquisition process- In the image fragment acquisition step, image fragments containing the central and peripheral regions of the target cell are obtained from each image acquired in the image acquisition step.

[0213] The image fragment acquisition process may be performed, for example, by image processing using image processing software.

[0214] The position from which an image fragment is acquired from the image of the target cell is not particularly limited, as long as it includes the central and peripheral parts of the target cell. The image fragment preferably includes the portion where the length of the target cell is maximized (i.e., the portion where the length of the straight line connecting any two points on the contour of the target cell is maximized).

[0215] For example, as shown in Figure 12, image fragments S7 to S include the portion where the length of the living cell is maximum. -2 It is possible to obtain the image fragment S'7 to image fragment S' which include the portion where the length of the dead cell is maximum. -2 You may obtain it.

[0216] Before obtaining image patches from each image of the target cells, each image may be preprocessed. As the preprocessing, for example, concentric circles with different radii centered on the center of the target cell are set in pixel units, and for each concentric circle, the average value of the brightness on the circumference is obtained, and a concentric circle (that is, the brightness on the circumference is averaged and has the same radius as the original concentric circle) with the brightness on the circumference being the above average value is drawn. This includes a process of reconstructing the image of the target cell. As a result, a symmetric image centered on the center of the target cell can be obtained. Therefore, when obtaining image patches, the influence of the direction in which the image patches are cut out can be removed. By using the image patches obtained from the image subjected to such preprocessing, a symmetric connected image (for example, a horizontally symmetric connected image as shown in FIGS. 5 and 6) can be obtained, and in the subsequent feature extraction step, it becomes easier to extract features from the connected image.

[0217] - Analytical connected image creation step - In the analytical connected image creation step, each image patch obtained in the image patch acquisition step is connected in the order of the imaging direction of the focal plane to create an analytical connected image.

[0218] The analytical connected image creation step may be performed by image processing using, for example, image processing software.

[0219] The method of connecting the image patches is not particularly limited as long as they are connected in the order of the imaging direction of the focal plane. It is preferable to connect the long sides of the image patches so that they are in contact with each other along a straight line connecting the centers of the target cells of each image patch.

[0220] For example, the image patches S7 to S of the living cells obtained from FIG. 12 -2 are connected as shown in FIG. 14 in the order of the imaging direction of the focal plane along a line connecting the centers of the living cells of each image patch, whereby an analytical connected image L of the living cells is obtained. Also, for example, the image patches S'7 to S' of the dead cells obtained from FIG. 13 -2As shown in Figure 15, by connecting the images along a line connecting the centers of the dead cells in each image piece in the order of the imaging direction of the focal plane, a concatenated image L' for analyzing dead cells is obtained.

[0221] -Feature extraction process- In the feature extraction process, features are extracted from the concatenated images obtained in the concatenated image creation process for analysis.

[0222] The feature extraction process may be performed, for example, by image processing using image processing software.

[0223] (Features extracted from linked images) The concatenated images contain various information about the target cells, and for example, the following features 1 to 11 can be obtained.

[0224] -Feature 1- (Determination of cell viability based on the presence or absence of a focal point in the lens effect) The linked images contain information about cell viability based on the presence or absence of a focal point due to the lens effect.

[0225] The linked images include image fragments of the cell's focal plane. In the focal plane of a cell, living cells have a focal point due to the lens effect, while dead cells do not.

[0226] For example, the linked image L of living cells shown in Figure 14 has a focal point of the lens effect on the image piece S0 of the focal plane P0 of the living cells. On the other hand, for example, the linked image L' of dead cells shown in Figure 15 does not have a focal point of the lens effect on the image piece S'0 of the focal plane P0 of the dead cells. Therefore, it is possible to determine whether a cell is alive or dead based on the presence or absence of a focal point in the lens effect.

[0227] For example, the brightness of the central part of the image fragment of the cell's in-focus plane may be defined as feature quantity 1, and the viability of the cell may be determined based on a predetermined range of feature quantity 1. This allows the cell to be determined to be alive if feature quantity 1 falls within the above range for the concatenated image used for analysis. In another embodiment, the determination of whether a cell is alive or dead may be made based on feature quantity 1 of the analytical concatenated image, feature quantity 1 of a known reference concatenated image of the target cell, and the result of determining whether or not the cell is alive.

[0228] -Feature 2- (Determination of cell viability based on the starting position of the light focus point) The linked images contain information about the viability of cells based on the starting position of the light focus point. "Starting position of the focal point" refers to the focal plane of the focal point of the lens effect or diffracted light. In a stitched image, the "starting position of the focal point" refers to the image piece in the focal plane of the focal point of the lens effect or diffracted light. In living cells, the starting position of the focal point of the lens effect reflects the average refractive index of the cell.

[0229] In the case of living cells, for example, as shown in Figure 12, the focal plane P1 of the focal point of the lens effect is the starting position of the focal point of the lens effect. On the other hand, in the case of dead cells, for example, as shown in Figure 13, the focal plane P2 of the diffracted light collection point is the starting position of the diffracted light collection point.

[0230] For example, as shown in Figures 12 and 13, the starting position of the focal point of the lens effect is closer to the cell than the starting position of the focal point of the diffracted light. This is because the focal point of the lens effect is formed when light that has passed through a living cell is focused near the living cell, whereas the focal point of the diffracted light is formed when light that has passed near the outer surface of a dead cell is focused relatively far from the dead cell. In other words, the focal point of the lens effect formed in a living cell is formed closer to the cell than the focal point of the diffracted light formed in a dead cell.

[0231] For example, in the linked image L of living cells shown in Figure 14, the image piece S1 at the starting position of the focal point of the lens effect (focus plane P1) is adjacent to the image piece S0 at the focus plane P0 of the living cells, and the starting position of the focal point of the lens effect is close to the focus plane P0 of the living cells. On one hand, for example, in the connected image L’ of dead cells shown in FIG. 15, the image piece S’2 of the starting position of the focusing point of the diffracted light (focus plane P2) is not adjacent to the image piece S’0 of the focus plane P0 of the dead cells, and the starting position of the focusing point of the diffracted light is far from the focus plane P0 of the dead cells. Therefore, based on the starting position of the focusing point, it is possible to determine the life or death of cells.

[0232] For example, the starting position of the focusing point may be specified by the distance based on the focus plane of the cells and used as feature quantity 2. For example, the length along the direction in which the image pieces are connected between the image piece of the focus plane of the cells and the image piece of the starting position of the focusing point (i.e., the focus plane of the focusing point) may be used as feature quantity 2, and based on the predetermined range of feature quantity 2, the life or death of the target cells may be determined. Thereby, for the connected image for analysis, when feature quantity 2 is within the above range, it can be determined that the cells are living cells. In another embodiment, the life or death determination may be performed based on feature quantity 2 of the connected image for analysis, feature quantity 2 of the known reference connected image of the target cells, and the determination result of whether the cells are living cells.

[0233] - Feature quantity 3 - (Determination of cell life or death based on the continuous length of the focusing point) The connected image includes information regarding the life or death of cells based on the continuous length of the focusing point. The "continuous length of the focusing point" means the distance from the starting position of the focusing point of the lens effect or diffracted light to the focal plane where the focusing point disappears. In the connected image, the "continuous length of the focusing point" means the shortest length from the image piece of the focus plane of the focusing point of the lens effect or diffracted light to the image piece of the focal plane where the focusing point disappears. In living cells, the continuous length of the focusing point of the lens effect reflects the average refractive index of the cells.

[0234] In the case of living cells, for example, as shown in FIG. 12, the distance from the starting position of the focusing point of the lens effect (focus plane P1) to the focal plane P4 where the focusing point of the lens effect disappears is the continuous length of the focusing point of the lens effect. On the other hand, in the case of dead cells, for example, as shown in Figure 13, the distance from the starting position of the diffracted light's focal point (focus plane P2) to the focal plane where the diffracted light's focal point disappears is the "duration of the focal point" of the diffracted light. Although not shown in Figure 13, the focal point continues beyond the focal plane P4 to a focal plane further away from the focus plane P0.

[0235] At the focal point of the lens effect, light that passes through a part of the living cell closer to its center is focused at a position further away from the living cell, and light that passes through a part of the living cell further away from its center is focused at a position closer to the living cell. As a result, the focal points are connected according to the part of the living cell that is passed through, so the focal point of the lens effect has a duration. Furthermore, at the focal point of the lens effect, the intensity of the light decreases over a short distance due to divergence. In addition, when white light is irradiated onto a living cell, the blue component (short wavelength component) is focused closer to the cell than the red component (long wavelength component), so the difference in wavelength also contributes to the duration of the focal point of the lens effect. On the other hand, the focal point of diffracted light is formed by the focusing of light that has passed near the outer surface of a dead cell, and the change in the interference pattern with respect to the distance from the dead cell extends over a relatively long distance. Therefore, the focal point of diffracted light has a longer duration of focal point than that of a lens effect. In other words, the focal point of a living cell has a shorter duration of focal point compared to that of a dead cell.

[0236] For example, in the linked image L of living cells shown in Figure 14, the focal point of the lens effect is image segment S. -1 The image is continuous from image segment S3. On the other hand, for example, in the linked image L' of dead cells shown in Figure 15, the focal point of the diffracted light is continuous from image segment S'2 to image segment S7. As shown in Figures 14 and 15, the duration of the focal point in the linked image L of living cells is shorter than the duration of the focal point in the linked image L' of dead cells. Therefore, the viability of cells can be determined based on the duration of the light focus point.

[0237] For example, for a region where the focal points are continuous, the length along the direction in which the image fragments are joined may be defined as feature quantity 3, and cell viability may be determined based on a predetermined range of feature quantity 3. This allows for the determination that cells are living cells if feature quantity 3 falls within the above range for the joined image used for analysis. In other embodiments, the determination of whether a cell is alive or dead may be made based on the feature quantity 3 of the analytical concatenated image, the feature quantity 3 of a known reference concatenated image of the target cell, and the determination result of whether or not the cell is alive.

[0238] -Feature 4- (Cell identification based on the starting position of the light focus point) The linked images contain information about cell identification based on the starting position of the light focus point.

[0239] In living cells, the starting position of the focal point of the lens effect reflects the cell's average refractive index. For other substances with a different average refractive index than the target cell (e.g., other cells, red blood cells, oil droplets, etc.), the starting position of the focal point will differ from that of the target cell. Therefore, based on the starting position of the light collection point, it is possible to determine whether or not the cells being tested for viability are target cells.

[0240] For example, the starting position of the focal point may be determined by the distance relative to the cell's focal plane and defined as feature quantity 4. For instance, feature quantity 4 may be defined as the length along the direction in which the image pieces are connected, between the image piece representing the cell's focal plane and the image piece representing the starting position of the focal point (i.e., the focal plane of the focal point). Based on a predetermined range of feature quantity 4, the viability of the cell may be determined. This allows the cell being analyzed to be identified as the target cell if feature quantity 4 falls within the above range. In other embodiments, the determination of whether a cell is alive or dead may be made based on the feature quantities 4 of the analytical concatenated image, the feature quantities 4 of a known reference concatenated image of the target cell, and the determination result of whether or not the cell is alive.

[0241] Feature 4 may be the same as Feature 2. Alternatively, Feature 4 may be different from Feature 2; for example, Feature 4 may be set to be more suitable for cell identification, and Feature 2 may be set to be more suitable for determining whether a cell is alive or dead.

[0242] -Feature 5- (Cell identification based on the duration of the focal point) The linked images contain information about cell identification based on the duration of the focal point.

[0243] In living cells, the duration of the focal point of the lens effect reflects the cell's average refractive index. Therefore, other substances with a different average refractive index than the target cell (e.g., other cells, red blood cells, oil droplets, etc.) will have a different focal point duration than the target cell. Therefore, based on the duration of the light focus point, it is possible to determine whether or not the cells being tested for viability are target cells.

[0244] For example, for a region with consecutive focal points, the length along the direction in which the image fragments are joined may be defined as feature 5, and cell viability can be determined based on a predetermined range of feature 5. This allows the analysis to determine that a cell subject to viability determination is a target cell if feature 5 falls within the above range for the joined image. In other embodiments, the determination of whether a cell is alive or dead may be made based on the feature quantities 5 of the analytical concatenated image, the feature quantities 5 of a known reference concatenated image of the target cell, and the determination result of whether or not the cell is alive.

[0245] Feature 5 may be the same as feature 3. Alternatively, feature 5 may be different from feature 3; for example, feature 5 may be set to be more suitable for cell identification, and feature 3 may be set to be more suitable for determining whether a cell is alive or dead.

[0246] Regarding features 4 and 5, for example, unlike living cells, red blood cells have a concave lens-like shape. Therefore, when light is shone on a red blood cell, a focal point is formed on the side of the red blood cell opposite the side being illuminated, and a focal point is also formed on the side being illuminated. In addition to these differences in how focal points appear, the starting position and duration of the focal point may also differ between red blood cells and living cells.

[0247] -Feature 6- (Cell identification based on cell size in the focal plane) The linked images may contain information regarding cell identification based on the size of cells in the focal plane.

[0248] The linked images include an image fragment of the cell's focal plane. Therefore, if the image fragment of the cell's focal plane includes the portion where the cell's length is maximum (i.e., the portion where the length of the straight line connecting any two points on the cell's contour is maximum), the length of this maximum portion can be considered the cell's diameter. Then, for example, by examining whether the diameter of the cell being tested for viability is similar to the known diameter of the target cell, if it is not, it can be determined that the cell being tested for viability is not the target cell. Therefore, based on the size of the cells in the focal plane, it is possible to determine whether or not the cells being tested for viability are target cells.

[0249] For example, for an image of the in-focus area of ​​a cell to be determined to be viable or viable, the length of the portion where the cell length is maximum (i.e., the portion where the length of the straight line connecting any two points on the cell's contour is maximum) may be defined as feature quantity 6, and the viability of the cell may be determined based on a predetermined range of feature quantity 6. In this way, for the concatenated image for analysis, if feature quantity 6 falls within the above range, it can be determined that the cell to be determined to be viable or viable is the target cell. In other embodiments, the determination of whether a cell is alive or dead may be made based on the feature quantities 6 of the analytical concatenated image, the feature quantities 6 of a known reference concatenated image of the target cell, and the determination result of whether or not the cell is alive.

[0250] -Feature 7- (Cell identification based on the position of the focal plane) The linked images contain information about cell identification based on the position of the focal plane.

[0251] For example, in a cell suspension containing target cells and other substances (e.g., other cells, red blood cells, oil droplets, etc.), if the specific gravity of the cells differs from that of the other substances, there will be a difference in how the cells and the other substances sink in the cell suspension. As a result, the position of the cells will differ from that of the other substances in the depth direction of the cell suspension. Therefore, when cells and other substances are imaged at the same focal plane and a stitched image is created, the position of the image fragment at the focal plane of the cells will differ from the position of the image fragment at the focal plane of the other substances (e.g., the height of the image fragments in the stitched direction). Then, for example, by examining whether the position of the image fragment at the focal plane of the cells to be determined to be viable is similar to the position of the image fragment at the known focal plane of the target cells, if it is not, it can be determined that the cells to be determined to be viable are not the target cells. Therefore, based on the position of the focal plane, it is possible to determine whether or not the cells being tested for viability are target cells.

[0252] For example, feature 7 may be defined as the length along the direction in which the image fragments are connected, between the image fragment at the focal plane of the cell to be determined to be viable or viable and the image fragment at the lowest focal plane. Based on a predetermined range of feature 7, the viability of the cell may be determined. This allows the analysis to determine that the cell to be determined to be viable or viable is the target cell if feature 7 falls within the above range. In another embodiment, the determination of whether a cell is alive or dead may be made based on the feature quantities 7 of the analytical concatenated image, the feature quantities 7 of a known reference concatenated image of the target cell, and the determination result of whether or not the cell is alive.

[0253] -Feature 8- (Cell identification based on the connected shape of light-gathering points) The linked images contain information about cell identification based on the linked shape of the light-gathering points. "Connected shape of condensing points" refers to the shape of the area formed by the connection of condensing points in a stitched image.

[0254] For example, for a concatenated image for analysis, the image of the part where the focal points are connected may be used as feature 8, and a machine learning model may be used to determine whether the cell being subjected to viability determination is the target cell based on feature 8 of the concatenated image for analysis, feature 9 of a known reference concatenated image of the target cell, and the result of the determination of whether or not it is a living cell.

[0255] The shape of the clustering points is related not only to the starting position and duration of the clustering points, but also to the size of the clustering points, and furthermore, in living cells, it reflects the average refractive index of the cell. Therefore, other substances with a different average refractive index than the target cell (e.g., other cells, red blood cells, oil droplets, etc.) will have a different clustering shape than the target cell. Therefore, based on the connected shape of the light-gathering points, it is possible to determine whether or not the cells being tested for viability are target cells.

[0256] -Feature 9- (Cell identification based on the color distribution of the focal point) The linked images may contain information about cell identification based on the color distribution of the focal points.

[0257] When white light is shone on living cells, the blue component (short wavelength component) is concentrated closer to the cell than the red component (long wavelength component), resulting in a color distribution at the focal point of the lens effect. The color distribution at the focal point of the lens effect reflects the average refractive index of the cell. Therefore, other substances with a different average refractive index than the target cell (e.g., other cells, red blood cells, oil droplets, etc.) will have a different color distribution at the focal point than the target cell. Therefore, based on the color distribution of the light focus point, it is possible to determine whether or not the cells being tested for viability are target cells.

[0258] For example, in living cells, the color separation at the focal point of the lens effect described above is observed, showing a color distribution where the area near the cell is colored blue and the area relatively far away is colored red. On the other hand, in red blood cells, for example, due to their concave lens-like shape, the color separation at the focal point is weak, and the focal point tends to appear relatively white.

[0259] For example, for a concatenated image for analysis, the image of the part where the focal points are connected may be used as feature 9, and a machine learning model may be used to determine whether the cell being subjected to viability determination is the target cell, based on feature 9 of the concatenated image for analysis, feature 8 of a known reference concatenated image of the target cell, and the result of the determination of whether or not it is a living cell.

[0260] -Feature 10- (Determination and identification of cell viability based on the degree of matching of linked images) As described above, the linked images contain information regarding cell viability and cell identification. Therefore, based on the degree of agreement between the linked images for analysis and the linked images for reference, it is possible to determine whether the cells being analyzed are target cells or not.

[0261] Therefore, the concatenated image for analysis itself may be used as feature quantity 10, and a machine learning model may be used to determine whether the cell subject to viability determination is a viable cell or a target cell, based on the feature quantity 10 of the concatenated image for analysis, the feature quantity 10 of a known reference concatenated image of the target cell, and the determination result of whether or not it is a viable cell.

[0262] -Feature 11- (Determination of cell viability based on the amount of transmission due to the lens effect of cells) The linked images contain information about the survival of cells based on the amount of light transmitted through the lens effect. The "lens effect transmission rate" of a cell refers to the amount of light transmitted through a cell when the cell exhibits a lens effect. In a stitched image, the "transmittance due to the lens effect" of a cell refers to the weighted sum of the average brightness of multiple image fragments in the imaging direction, starting from the image fragment of the cell's focal plane, and the average brightness of multiple image fragments in the light source direction, starting from the image fragment of the cell's focal plane, when the cell exhibits a lens effect. The weighting coefficients are not particularly limited and may be set as appropriate.

[0263] In dead cells immediately after membrane rupture, an intermediate state may exist where, like living cells, there is a focal point due to the lens effect. Because light scattering occurs at the outer surface due to membrane rupture, the amount of light transmitted through the lens effect is lower compared to living cells. Therefore, cell viability can be determined based on the amount of light transmitted through the lens effect. The output of the light source is assumed to be kept constant.

[0264] The average brightness of an image fragment in the imaging direction relative to the focal plane of a cell is, for example, the range from the focal plane P0 to the focal plane P, as shown in Figure 2. n Image piece S n This is the average brightness. The average brightness of an image fragment in the direction of the light source relative to the focal plane of a cell is, for example, the range from the focal plane P0 to the focal plane P, as shown in Figure 2. -m Image piece S -m This is the average brightness (for example, n=m is preferable).

[0265] A weighted sum of the average brightness of "multiple image fragments in the imaging direction" starting from the image fragment of the cell's focal plane and the average brightness of "multiple image fragments in the direction of the light source" starting from the cell's focal plane may be used as feature quantity 11, and cell viability may be determined based on a predetermined range of feature quantity 11.

[0266] In the case of a multi-wavelength light source (e.g., a white LED) and a spectroscopic imaging device (e.g., an RGB color camera), a concatenated image may be created for each color, and the viability of cells may be determined by combining the average brightness of "multiple image fragments in the imaging direction" and the average brightness of "multiple image fragments in the light source direction" from different colors.

[0267] The features 1 to 11 described above are just examples of features obtained from concatenated images; cell viability and cell identification can also be performed based on other features.

[0268] Feature quantities 1 and 11 relate to the lens effect of cells. Features quantities 2 through 5 relate to the average refractive index of cells. Feature quantity 6 relates to the diameter of cells. Feature quantity 7 relates to the specific gravity of cells. Features quantities 8 and 9 relate to the morphology of the stitched image itself. Furthermore, feature quantity 10 relates to the average refractive index, diameter, and specific gravity of cells. Thus, because many features can be obtained from concatenated images, concatenated images are effective not only for determining whether a cell is alive or dead, but also for identifying cells.

[0269] The features used to determine the viability of target cells preferably include one or more selected from the group consisting of features 1 to 11, namely, one or more selected from the group consisting of features related to the lens effect of cells, features related to the average refractive index of cells, features related to the diameter of cells, and features related to the specific gravity of cells.

[0270] -Life and death determination process- In the viability determination step, the viability of multiple target cells in the cell suspension is determined based on the feature quantities of the concatenated image for analysis and a predetermined range of feature quantities. In other embodiments, the viability of target cells may be determined based on the feature quantities of a known reference concatenated image of the cell and the determination result of whether or not it is a living cell. Determining whether a target cell is alive or dead involves determining whether the cell being evaluated is a target cell, that is, cell identification.

[0271] The "predetermined range of features" refers to a threshold for distinguishing whether a cell is alive or not. If the features fall within this range, the cell can be determined to be alive. The method for determining the "predetermined range of features" is not particularly limited and may be determined appropriately so as to enable the determination of whether a cell is alive or dead. In other embodiments, cell viability may be determined based on the features of a known reference concatenated image of the cell and the determination of whether or not it is a living cell. Such an embodiment is preferred when cell viability is determined by a machine learning model.

[0272] The determination of whether a target cell is viable or not may be performed using one or more of the features 1 to 3 and feature 11 from the perspective of viability determination, and one or more of the features 4 to 9 from the perspective of cell identification. To further improve the accuracy of cell viability determination, cell viability may be determined by combining two or more features from Feature 1 to Feature 3 and Feature 11. You may use any one of the features 4 through 9. From the perspective of further improving the accuracy of cell identification, you may use a combination of two or more features 4 through 9 for cell identification.

[0273] In other embodiments, the determination of whether the target cells are alive or dead may be performed using a feature quantity 10 based on the degree of matching of the linked images. In addition to determining whether the cells are alive or dead, it is also simultaneously determined whether the cells being determined are target cells or not.

[0274] In one embodiment, when determining the viability of cells using feature 2 (or feature 4) based on the starting position of the light collection point, feature 2 functions as feature 4 (or feature 2) also based on the starting position of the light collection point, thus enabling the determination of whether the cells being determined to be viable are target cells or not. In another embodiment, when determining the viability of cells using feature 3 (or feature 4) based on the duration of the focal point, feature 3 can also function as feature 4 (or feature 3) based on the duration of the focal point, thereby enabling the determination of whether the cells being determined to be viable are target cells or not.

[0275] (Machine learning machine) The determination of whether target cells are viable or not may be performed using a machine learning model. The machine learning model may be constructed using one of the following methods: neural networks, support vector machines, or boosting. Preferably, the machine learning model is constructed using a neural network and has undergone deep learning.

[0276] As shown in Figure 16, during the learning phase, the machine learning model learns by being given training data. The training data consists of a pair of known reference concatenated image features of the target cell (hereinafter sometimes referred to as "training features") and the result of determining whether or not the cell is alive corresponding to the training features (hereinafter sometimes referred to as "correct survival / death determination result").

[0277] During the learning phase, the machine learning model receives training features as input. The machine learning model outputs a training survival / death judgment result based on these training survival / death judgment results and the ground truth survival / death judgment result. Based on the results of the loss calculation, the machine learning model's various coefficients are updated, and the machine learning model is updated according to these update settings.

[0278] In the machine learning model's training phase, the above series of processes—inputting training features into the machine learning model, outputting training survival / death judgment results from the machine learning model, calculating loss, setting update parameters, and updating the machine learning model—are repeated while the training data is exchanged. This repetition of the above series of processes ends when the prediction accuracy of the training survival / death judgment results relative to the ground truth survival / death judgment results reaches a predetermined set level. The machine learning model, having thus reached the set level of prediction accuracy, is used in the operational phase and outputs survival / death judgment results in response to the input of features from concatenated images for analysis.

[0279] In the viability determination process, to determine the viability of multiple cells in a cell suspension, a concatenated image for analysis is created for multiple cells in the cell suspension. Then, based on the features of these concatenated images for analysis, the features of a known reference concatenated image of the target cell, and the determination result of whether or not it is a living cell, the viability of the target cell may be determined.

[0280] Furthermore, for example, a cell suspension containing target cells and other substances (e.g., other cells, red blood cells, oil droplets, etc.) may be used to create a concatenated image for analysis of the target cells and other substances. Based on the feature quantities of these concatenated images for analysis, the feature quantities of a known reference concatenated image of the target cells, and the determination of whether or not the cells are alive, the target cells may be identified and their viability may be determined simultaneously.

[0281] An example of the viability determination process flow will be explained using the flowchart shown in Figure 17. The viability determination flow shown in Figure 17 is an example in which target cells are identified using feature quantities 4 to 7, and viability is determined using feature quantities 4 and 5.

[0282] The viability determination flow shown in Figure 17 is an example in which target cells are identified using features 4 to 6, and viability is determined using features 4 and 5.

[0283] -Step S10- Select one image for analysis from among multiple image stitches (S10).

[0284] -Step S12- It is determined whether the feature quantity 6 (diameter) of the concatenated image for analysis falls within a predetermined range of feature quantity 6 (S12). If the feature quantity 6 of the concatenated image for analysis falls within the above range, it is considered a potential target cell and the process proceeds to the next step (S14). If feature quantity 6 of the concatenated image for analysis is outside the above range, it is determined that it is not the target cell (S22).

[0285] -Step S14- It is determined whether the feature quantity 7 (position of the focal plane) of the concatenated image for analysis falls within a predetermined range of feature quantity 7 (S14). If the feature quantity 7 of the concatenated image for analysis falls within the above range, it is assumed to be a target cell and the process proceeds to the next step (S16). If feature quantity 7 of the concatenated image for analysis is outside the above range, it is determined that it is not the target cell (S22).

[0286] -Step S16- It is determined whether the feature quantity 4 (starting position of the focal point) of the concatenated image for analysis falls within a predetermined range of feature quantity 4 (S16). If the feature quantity 4 of the concatenated image for analysis falls within the above range, it is assumed that it may be a living target cell (hereinafter sometimes referred to as "target living cell"), and the process proceeds to the next step (S18). If feature quantity 4 of the concatenated image for analysis is outside the above range, it is determined that the target cell is not a living cell (S22).

[0287] -Step S18- It is determined whether the feature quantity 5 (duration of the focal point) of the concatenated image for analysis falls within a predetermined range of feature quantity 5 (S18). If the feature quantity 5 of the concatenated image for analysis falls within the above range, it is determined that the target cell is a living cell (S20). If feature quantity 5 of the concatenated image for analysis is outside the above range, it is determined that the target cell is not a living cell (S22).

[0288] -Step S20, Step S22- If the target cell is determined to be a living cell (S20), proceed to the next step (S24); if it is determined not to be a target cell (S22), proceed to the next step (S26).

[0289] -Step S24- The number of linked images identified as containing living target cells is counted, and the process proceeds to the next step (S28).

[0290] -Step S26- The number of linked images that are determined not to be living target cells is counted, and the process proceeds to the next step (S28).

[0291] -Step S28- If there are any linked images for analysis whose survival status has not yet been determined, the process returns to step S10. Once survival status has been determined for all linked images for analysis, the survival status determination flow is terminated.

[0292] -Live cell concentration determination process- In the viable cell concentration determination step, the viable cell concentration of the target cells in the cell suspension is determined based on the results of the viability determination.

[0293] In this disclosure, the viable cell concentration of the target cells means the number of viable cells per unit volume [cells / ml].

[0294] The concentration of viable target cells can be determined, for example, as follows: The cell suspension is placed in a holding container, and for a randomly selected field of view, a stitched image for analysis is created of all target cells (which may also include other substances other than target cells, such as other cells, red blood cells, oil droplets, etc.) present between the bottom of the container and the liquid surface of the cell suspension. The viability of these cells is then determined, and the number of stitched images determined to be of viable cells is defined as the number of viable target cells. The concentration of viable target cells in the cell suspension can be determined by dividing the number of viable target cells by the volume of the cell suspension used for measurement (i.e., the product of the area of ​​the field of view and the height of the cell suspension (the height from the bottom of the container to the liquid surface)).

[0295] (Method for measuring the concentration of living cells in embodiment 2) The live cell concentration measurement step in embodiment 2 is: The process of imaging target cells with a digital holographic microscope and obtaining a phase image of the target cells (hereinafter sometimes referred to as the "phase image acquisition process"), A step of determining the viability of multiple target cells in a cell suspension based on the phase distribution of the above phase image and a predetermined phase distribution (hereinafter sometimes referred to as the "viability determination step"), and Based on the results of the viability determination, a step is taken to determine the viability of target cells in the cell suspension (hereinafter sometimes referred to as the "viability cell concentration determination step"). Includes.

[0296] -Phase image acquisition process- In the phase image acquisition process, target cells are imaged using a digital holographic microscope, and phase images of the target cells are acquired.

[0297] The interference pattern generated by the light passing through the target cell and the reference light allows us to obtain a phase image of the target cell.

[0298] The digital holographic microscope is not particularly limited and any known type may be used.

[0299] -Life and death determination process- In the phase image acquisition process, the viability of multiple target cells in the cell suspension is determined based on the phase quantity distribution of the phase image of the target cell and a predetermined phase quantity distribution.

[0300] Living cells have a refractive index distribution within their cells, while dead cells have a smaller refractive index distribution compared to living cells. Therefore, the phase image of a target cell obtained by digital holographic microscopy has a phase distribution characteristic of living cells.

[0301] The "phase distribution" contains information about the refractive index distribution inside the cell, as well as information about the size of the cell.

[0302] The "predetermined range of phase quantity distribution" refers to a threshold for distinguishing whether a cell is a living target cell or not. If the phase quantity distribution falls within this range, the cell can be determined to be a living target cell. The method for determining the "predetermined range of phase quantity distribution" is not particularly limited and may be determined appropriately so as to enable the determination of whether the target cell is alive or dead.

[0303] Furthermore, for example, the viability of the target cells may be determined based on the phase quantity distribution of the acquired phase image, the known phase quantity distribution of the target cells, and the determination result of whether or not they are living cells.

[0304] -Live cell concentration determination process- In the viable cell concentration determination step, the viable cell concentration of the target cells in the cell suspension is determined based on the results of the viability determination. The details are the same as those described above in the viable cell concentration measurement step of Embodiment 1.

[0305] [Liquid volume determination process] In the volume determination step, the volume of the cell suspension to be seeded and the volume of the diluent required to prepare the cell suspension to be seeded to a predetermined viable cell concentration are determined based on the viable cell concentration and volume data of the cell suspension. The volumes of the cell suspension and the diluent may be appropriately determined so that the concentration of viable cells in the diluted cell suspension reaches the desired value.

[0306] [Dilution process] In the dilution step, the determined volume of cell suspension and the determined volume of diluent are mixed. The means of mixing are not particularly limited and may be selected as appropriate.

[0307] As described above, the live cell concentration adjustment method relating to this disclosure allows for the rapid adjustment of the live cell concentration of the target cells.

[0308] [Cell viability determination process] The method for adjusting the concentration of viable cells may include a step of determining the cell viability of target cells in a cell suspension based on the results of viability determination of multiple target cells in the cell suspension (hereinafter sometimes referred to as the "cell viability determination step").

[0309] Cell viability is calculated by dividing the number of living cells by the total number of cells (the sum of living and dead cells).

[0310] When the viable cell concentration measurement step of Embodiment 1 is performed, the cell viability can be calculated by using the number of analytical linked images determined to be viable cells (for example, step S24 in Figure 17) as the number of viable cells, and the total number of analytical linked images (for example, steps S24 and S26 in Figure 17) as the total number of cells. When the viable cell concentration measurement process of embodiment 2 is performed, the cell viability can be calculated from the number of cells determined to be viable and the total number of cells (total number of cells) for which viability was determined. In subculturing and expansion culture, the cell suspension contains only one type of target cell; therefore, the cell viability obtained in this case represents the proportion of target cells that are alive among all target cells.

[0311] The disclosure of Japanese Patent Application No. 2021-076027, filed on 28 April 2021, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually described as being incorporated by reference.

Claims

1. A first container for holding a cell suspension, A second container for holding a diluent for diluting the cell suspension, A cell viability concentration measuring device that measures the viability concentration of target cells in a cell suspension by individually determining the viability of multiple target cells in the cell suspension. A control device that maintains and controls the data of the live cell concentration and the volume of the cell suspension. A mixing unit for mixing the cell suspension and the diluent, A culture vessel in which a diluted cell suspension is seeded and cultured. It has, The control device determines, based on the live cell concentration and the volume data of the cell suspension, the volume of the cell suspension to be seeded and the volume of the diluent necessary to adjust the cell suspension to be seeded to a predetermined live cell concentration, sends the volume of the cell suspension and the volume of the diluent to the mixing unit, and seeds the diluted cell suspension. The aforementioned live cell concentration measuring device, A light source that irradiates the target cells with light, An imaging device for imaging the aforementioned target cells, Means for changing the focal plane, An image acquisition unit acquires images of the target cell, taken from a direction opposite to the side of the target cell that has been irradiated with the light, and from multiple focal planes including the focal plane of the target cell. An image fragment acquisition unit that acquires an image fragment from each of the aforementioned images, including the central and peripheral portions of the target cell. An analytical concatenation image creation unit creates an analytical concatenation image by concatenating the image pieces in the order of the imaging direction of the focal plane. A feature extraction unit that extracts features from the aforementioned concatenated images for analysis, and A life determination unit that determines the life or death of the plurality of target cells in the cell suspension based on the feature quantities of the linked images for analysis and a predetermined range of feature quantities, and Based on the results of the viability determination, a viability concentration determination unit determines the viability concentration of the target cells in the cell suspension. A cell culture device equipped with the following features.

2. The cell culture apparatus according to claim 1, wherein the live cell concentration measuring device is located in a channel branched from the channel between the first container and the mixing section, or in the channel between the first container and the mixing section.

3. The cell culture apparatus according to claim 2, wherein the measurement of the viable cell concentration is performed during at least a portion of the time it takes for the cell suspension to reach the mixing section from the first container.

4. The cell culture apparatus according to claim 1, wherein the mixing section also serves as the culture vessel.

5. The cell culture apparatus according to claim 1, wherein the mixing unit also serves as the culture vessel, which is a suspension culture apparatus.

6. The cell culture apparatus according to claim 1, wherein the mixing section also serves as the second container.

7. The cell culture apparatus according to claim 1, wherein the culture vessel is a multilayer culture vessel.

8. The cell culture apparatus according to claim 1, wherein the means for changing the focal plane is a stage moving mechanism that moves a stage on which a holding container for holding the target cells is placed, thereby changing the distance between the target cells and the imaging device.

9. The cell culture apparatus according to claim 1, wherein the means for changing the focal plane is an imaging device movement mechanism that moves the imaging device to change the distance between the target cell and the imaging device.

10. The cell culture apparatus according to claim 1, wherein the imaging device is equipped with a liquid lens as the means for changing the focal plane.

11. The aforementioned live cell concentration measuring device, A digital holographic microscope for imaging the aforementioned target cells, A phase image acquisition unit that acquires a phase image of the target cell. A viability determination unit that determines the viability of a plurality of target cells in the cell suspension based on the phase quantity distribution of the phase image and a predetermined phase quantity distribution, and Based on the results of the viability determination, a viability concentration determination unit determines the viability concentration of the target cells in the cell suspension. A cell culture apparatus according to any one of claims 1 to 7, comprising:

12. The cell culture apparatus according to claim 1, wherein the viable cell concentration measuring device comprises a cell viability determination unit that determines the cell viability of the target cells in the cell suspension based on the results of the viability determination.

13. A step of measuring the concentration of viable target cells in a cell suspension by individually determining the viability of multiple target cells in the cell suspension. A step of determining the volume of the cell suspension to be seeded and the volume of the diluent necessary to prepare the cell suspension to be seeded to a predetermined live cell concentration, based on the live cell concentration and the volume data of the cell suspension, and A step of mixing the aforementioned liquid volume of the cell suspension with the aforementioned liquid volume of the diluent, Includes, The step of measuring the concentration of living cells is, A step of acquiring an image of the target cell, which is imaged from a direction opposite to the side of the target cell that is irradiated with light, and from multiple focal planes including the focal plane of the target cell. A step of obtaining an image fragment from each of the aforementioned images, including the central and peripheral regions of the target cell. A step of creating a concatenated image for analysis by connecting the image pieces in the order of the imaging direction of the focal plane, A step of extracting features from the aforementioned concatenated images for analysis, A step of determining the viability of a plurality of target cells in the cell suspension based on the feature quantities of the linked images for analysis and a predetermined range of feature quantities, and A step of determining the concentration of viable target cells in the cell suspension based on the results of the viability determination, A method for adjusting the concentration of living cells, including the method described above.

14. The step of measuring the concentration of living cells is, A step of imaging the target cells with a digital holographic microscope and obtaining a phase image of the target cells. A step of determining the viability of a plurality of target cells in the cell suspension based on the phase quantity distribution of the phase image and a predetermined phase quantity distribution, and A step of determining the concentration of viable target cells in the cell suspension based on the results of the viability determination, A method for adjusting the concentration of living cells according to claim 13, including the method described in claim 13.

15. A method for adjusting the concentration of viable cells according to claim 13 or claim 14, comprising the step of determining the cell viability of the target cells in the cell suspension based on the results of the viability determination.

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