Cell peeling method, cell peeling system, and information processing device

Abstract

The present invention provides a cell dissecting method for dissecting cells adhering to a culture surface of a cell culture vessel, the cell dissecting method comprising: an adhesive strength reduction step of reducing the adhesive strength of the cells by applying a stimulus to the cells while the cells remain adhering to the cell culture vessel; a measurement step of measuring the cells at at least two times and acquiring cell information; an exfoliation step for exfoliating the cells from the culture surface; and a determination step of determining, on the basis of the cell information, a time t2 at which the dissection step is to start, the time t2 being after a time t1, the time t1 being a later one of the at least two times.

Description

Technical Field

[0001] The present invention relates to a cell detachment method, a cell detachment system, and an information processing device. Background Art

[0002] In recent years, there has been a major paradigm shift in drug development: from conventional small-molecule drugs and antibody drugs to cell therapy that uses cells themselves as drugs and regenerative medicine aimed at regenerating tissues and organs. In this cell therapy and regenerative medicine, a large number of cells are required, and in particular, adherent cells that make up most of biological tissues need to be supplied efficiently and stably.

[0003] In the culture of adherent cells, target cells are obtained through the following steps: culturing cells on a culture substrate such as a polystyrene dish, detaching the cells from the substrate, and recovering and washing the cells. When an increase in the number of cells is desired, a so-called subculture operation is performed, in which some of the obtained cells are transferred to a new substrate and cultured. In this series of steps, research has been conducted on the detachment step of detaching cells from the substrate to achieve efficient and stable cell supply.

[0004] Patent Document 1 discloses that in a device for sorting and detaching cells, the ratio of the projected areas of individual adherent cells before the start of detachment and during the detachment process is calculated and compared with a predetermined threshold to determine whether the cells have been detached from the substrate.

[0005] Patent Document 2 discloses that in a device for determining cell detachment, based on data obtained by photographing cells, brightness information is compared with a predetermined brightness level to determine whether the cells have been detached.

[0006] Patent Document 3 discloses that in a device for evaluating cell proliferation ability, after applying a detachment agent, individual anchorage-dependent cells are repeatedly photographed, and data on cell proliferation ability is displayed based on the time change in the projected area of the cells. Citation List Patent Documents

[0007] Patent Document 1: Japanese Patent Publication No. 2003-235540 Patent Document 2: International Publication No. 2007 / 136073 Patent Document 3: Japanese Patent Publication No. 2004-344049 Summary of the Invention Technical Problem

[0008] The techniques described in Patent Documents 1 to 3 disclose determining the end of a peeling operation and evaluating cell proliferation ability by measuring cell information. However, Patent Documents 1 to 3 do not disclose a method for determining the start timing of the peeling operation. The present inventors have noted the fact that the adhesion strength of cells varies depending on conditions such as cell type and culture conditions. The present inventors have conducted in-depth research and found that when the peeling operation is performed without setting the start timing to an appropriate timing corresponding to the state of the cells to be peeled, the cell survival rate and cell peeling efficiency obtained by the peeling step sometimes decrease.

[0009] An object of the present invention is to provide a cell peeling method that achieves high cell survival rate and peeling efficiency. Solution to the problem

[0010] The cell peeling method disclosed in this specification is a cell peeling method for peeling cells attached to a culture surface of a cell culture container, the method comprising: an adhesion strength reduction step of reducing the adhesion strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; a measurement step of measuring the cells at at least two times and acquiring cell information; a peeling step of peeling the cells from the culture surface; and a determination step of determining a timing t2 at which the peeling step is to start based on the cell information, the timing t2 being after a timing t1 which is the later of the at least two times.

[0011] The cell peeling method disclosed in this specification is a cell peeling method for peeling cells attached to a culture surface of a cell culture container, the method comprising: an adhesion strength reduction step of reducing the adhesion strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; a measurement step of measuring the cells and acquiring time-series data related to the cells; a peeling step of peeling the cells from the culture surface; and a prediction step of predicting a timing at which the peeling step is to start based on the time-series data.

[0012] The cell peeling system disclosed in this specification is a cell peeling system for peeling cells attached to a culture surface of a cell culture container, the system comprising: an adhesion strength reduction unit that reduces the adhesion strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; a measurement unit that measures the cells at at least two times and acquires cell information; a peeling unit that peels the cells from the culture surface; and a determination unit that determines a timing t2 at which the peeling step of peeling the cells from the culture surface is to start based on the cell information, the timing t2 being after a timing t1 which is the later of the at least two times.

[0013] The cell detachment system disclosed in this specification is a cell detachment system for detaching cells attached to the culture surface of a cell culture container. The system includes: an attachment strength reduction unit that reduces the attachment strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; a measurement unit that measures the cells and acquires time-series data related to the cells; a detachment unit that detaches the cells from the culture surface; and a prediction unit that predicts the timing at which the detachment step of detaching the cells from the culture surface is to start based on the time-series data.

[0014] The information processing device disclosed in this specification is an information processing device for controlling a cell detachment device to detach cells attached to the culture surface of a cell culture container. The information processing device includes: a measurement unit that acquires cell information of cells whose attachment strength to the culture surface has been reduced due to the application of a stimulus at at least two times; and a determination unit that determines the timing t2 at which the detachment step of detaching the cells from the culture surface is to start based on the cell information, where the timing t2 is after the timing t1, and the timing t1 is the later of the at least two times.

[0015] The information processing device disclosed in this specification is an information processing device for controlling a cell detachment device to detach cells attached to the culture surface of a cell culture container. The information processing device includes: a measurement unit that acquires time-series data related to cells whose attachment strength to the culture surface has been reduced due to the application of a stimulus at at least two times; and a prediction unit that predicts the timing at which the detachment step of detaching the cells from the culture surface is to start based on the time-series data. Advantageous Effects of the Invention

[0016] According to the present invention, a cell detachment method capable of achieving high cell survival rate and detachment efficiency can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a diagram showing the configuration of an information processing system capable of executing a program according to an embodiment of the present invention. Figure 2 is a schematic diagram showing a cell detachment device according to an embodiment of the present invention. Figure 3 shows an example of measuring the area fraction of the halo region as cell information in an embodiment of the present invention. Figure 4 shows the cell information measurement results and curve fitting results in Example 1 of the present invention. Figure 5 shows the cell information measurement results and curve fitting results in Example 4 of the present invention. Figure 6Shows the cell information measurement results and curve fitting results in Embodiment 5 of the present invention. Figure 7 Shows the cell information measurement results and curve fitting results in Embodiment 6 of the present invention. Figure 8 Shows the shear stress application mechanism according to Embodiment 7 of the present invention. Figure 9 Is a flowchart for explaining the cell detachment method according to an embodiment of the present invention. Figure 10 Is a flowchart for explaining the cell detachment method according to an embodiment of the present invention. Figure 11 Shows the configuration of the cell detachment system according to an embodiment of the present invention. Detailed Description

[0018] The present invention will be described in detail below with reference to the preferred embodiments.

[0019] The cell detachment method disclosed in this specification is a cell detachment method for detaching cells attached to the culture surface of a cell culture container, and the method includes: a measurement step of measuring the cells at at least two times and acquiring cell information; a detachment step of detaching the cells from the culture surface; and a determination step of determining the time t2 at which the detachment step is to start based on the cell information, where the time t2 is after the time t1, and the time t1 is the later of the at least two times when the cell information is measured.

[0020] The cell detachment method disclosed in this specification is a cell detachment method for detaching cells attached to the culture surface of a cell culture container, and the method includes: an attachment strength reduction step of reducing the attachment strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; a measurement step of measuring the cells and acquiring time-series data related to the cells; a detachment step of detaching the cells from the culture surface; and a prediction step of predicting the time at which the detachment step is to start based on the time-series data.

[0021] As a result of in-depth research on the detachment step, the present inventors found that: when the detachment operation starts before the attachment strength of the cells is sufficiently reduced, the cell detachment efficiency and cell viability are adversely reduced. It was also found that when the step of reducing the cell attachment strength is performed for a long time, the cell viability is adversely reduced because the cells are placed in an environment different from the culture environment for a long time. As described above, the present inventors have found that there is an appropriate start time for cell detachment in the detachment step.

[0022] In addition, the inventors have found that by measuring cell information at at least two times and determining the start time of the peeling operation after the time of measuring the cell information based on the cell information, a preparation operation time until the start time of the peeling operation can be provided. The inventors have found that the existence of the preparation setting operation time can provide a cell peeling method with a high degree of freedom in device design. For example, as a cell peeling system, a configuration can be used in which a measuring device for measuring cell information and a peeling device for performing the peeling operation are provided separately, and during the preparation operation time, the culture vessel is moved from the measuring device to the peeling device. In addition, by providing the preparation operation time, a device for performing the peeling operation, such as an ultrasonic irradiation device, can be prepared to start the operation, so that the peeling operation can be performed at an appropriate timing.

[0023] From the results of the above research, the inventors have found that by providing a method for determining the start time of the peeling operation after the time of measuring cell information in the cell peeling step, a cell peeling method with high cell survival rate and high peeling efficiency can be provided.

[0024] (Cell culture vessel) The substrate in the present embodiment refers to a cell culture vessel for cell culture. The cell culture vessel is not particularly limited as long as it is a cell-adhesive culture vessel. Examples of the cell culture vessel include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multiwell plates, porous plates, multiplates, Petri dishes, culture bags, and bottles.

[0025] The material of the cell culture vessel in the present embodiment only needs to be a chemically stable material capable of culturing the desired cells. The material of the cell culture vessel is, for example, at least one selected from the group consisting of polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, poly(meth)acrylic acid, poly(meth)acrylic acid derivatives, polyacrylonitrile, poly(meth)acrylamide, poly(meth)acrylamide derivatives, polysulfone, cellulose, cellulose derivatives, polysiloxane, polymethylpentene, glass, and metal. Among these materials, polystyrene is preferably used as the material of the cell culture vessel.

[0026] (Cell) The cells in the embodiments are not particularly limited as long as they can adhere to a culture substrate and be cultured in vitro on the culture substrate. Examples thereof include: various cultured cell lines such as CHO cells derived from Chinese hamster ovary, mouse connective tissue L929 cells, mouse skeletal muscle myoblasts (C2C12 cells), normal diploid fibroblasts derived from human fetal lung (TIG-3 cells), cells derived from human fetal kidney (HEK293 cells), A549 cells derived from human alveolar basal epithelial adenocarcinoma, and HeLa cells derived from human cervical cancer. In addition, examples thereof include: epithelial cells and endothelial cells that constitute various tissues and organs in an organism, skeletal muscle cells, smooth muscle cells, and cardiomyocytes that exhibit contractility, neuron cells, glial cells, and fibroblasts that constitute the nervous system, hepatocytes, non-parenchymal liver cells, and adipocytes that participate in organism metabolism, and cells having differentiation potential such as various stem cells, for example, induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germ stem cells, or progenitor cells of various tissues, and cells induced by differentiation of these cells. Among these, the cell detachment method according to the present embodiment is suitable for: cells having strong cell-cell binding and cells having a high adhesion strength to the culture surface of a cell culture container. Since a large amount of cell culture is required, the method is particularly suitable for, for example, CHO cells used for protein production and mesenchymal stem cells that can be used for cell therapy. The cells in the present embodiment may be cell sheets cultured in a sheet form.

[0027] (Culture medium) The type of the culture medium is not particularly limited. Examples thereof include: Dulbecco's Modified Eagles's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagles's Minimum Essential Medium (EMEM), alpha Modified Eagles's Minimum Essential Medium (αMEM), Minimum Essential Medium (MEM Medium), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, Williams Medium E, IPL41 medium, Fischer medium, StemSpan H3000 (manufactured by STEMCELL Technologies), StemSpan SFEM (manufactured by STEMCELL Technologies), StemlineII (manufactured by Sigma-Aldrich), Endothelial Cell Growth Medium 2 Kit (manufactured by PromoCell), Mesenchymal Stem Cell Growth Medium 2 (manufactured by PromoCell), MSCGM Bullet Kit (manufactured by Lonza), mTeSR1 or 2 medium (manufactured by STEMCELL Technologies), ReproFF or ReproFF2 (manufactured by ReproCELL), NutriStem medium (manufactured by Biological Industries), and MF-Medium mesenchymal stem cell growth medium (manufactured by Toyobo Co., Ltd.).

[0028] Among these, a culture medium suitable for culturing the target cells is preferably used.

[0029] (Serum) Serum or antibiotics can be added to the above-mentioned culture medium. Examples of serum that can be used include fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, porcine serum, chicken serum, rabbit serum, and human serum. Generally, FBS is often used because it is easily available. The culture medium can be a serum-free medium that does not contain untreated serum or unrefined serum but contains components derived from serum or compounds (such as growth factors) derived from animal tissues.

[0030] (Antibiotics) Examples of antibiotics added to the culture medium include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, and amphotericin B.

[0031] (Cell culture conditions) Cell culture conditions can be appropriately selected according to the cells to be cultured. Typically, an appropriate culture medium is added to a dish. Approximately 1.0×10 1 to 5.0×10 4 cells / cm 2 are seeded therein, and the cells are cultured in an environment of 37°C and 5% CO2 concentration. In this case, it is preferable to culture the cells until the proportion of the area occupied by the cells in the substrate reaches approximately 70% to 80%, that is, the cells are in a so-called sub-confluent state.

[0032] (Cell detachment method) The cell detachment method according to an embodiment of the present invention is a cell detachment method for detaching cells attached to the culture surface of a cell culture container. Figure 9 is a flowchart showing the cell detachment method according to this embodiment. The cell detachment method includes: an attachment strength reduction step S101 of reducing the cell attachment strength to the culture surface, a measurement step S102 of obtaining cell information, a determination step S103 of determining the time t2 to start the detachment step, and a detachment step S104 of detaching the cells from the culture surface.

[0033] Figure 10 is a flowchart showing another example of the cell detachment method according to an embodiment of the present invention. Other examples of the cell detachment method include: an incubation step S201 of bringing the cells into contact with each other and incubating the cells, and a measurement step S202 of measuring the time change of the average brightness value of the cell image. Other examples of the cell detachment method also include: a step S203 of obtaining a fitting curve from the average brightness value and the time, a determination step S204 of determining the start time of the detachment step, and a detachment step S205 of detaching the cells by ultrasonic vibration.

[0034] Each step will be described below.

[0035] (Step of reducing adhesion strength) The step of reducing adhesion strength according to an embodiment of the present invention is a step of reducing the cell adhesion strength by applying a stimulus to cells while the cells are adhered to a substrate. Specific means for reducing the cell adhesion strength include: chemical stimuli such as cell detachment solutions, mechanical stimuli such as tapping and flow, electromagnetic stimuli such as light, electricity, and magnetism, and thermal stimuli such as heating and cooling. Multiple means can be combined.

[0036] Among the above means, from the viewpoint of the quality of detached cells, chemical stimulation using a cell detachment solution is preferred. Among these, chemical stimulation using a cell detachment solution substantially free of proteolytic enzymes is particularly preferred.

[0037] Here, a proteolytic enzyme is an enzyme that decomposes a part of cells to promote the detachment of cells from the substrate. Examples of proteolytic enzymes include trypsin, accutase, collagenase, natural protease, chymotrypsin, elastase, papain, streptomyces protease, or their recombinants. The phrase "substantially free of proteolytic enzymes" means that the amount of proteolytic enzyme based on the total mass of the cell detachment solution is 0.0005% by mass or less.

[0038] Regarding the state of cells adhered to a culture vessel, cells are observed while shaking the culture vessel, and it can be determined whether the cells are adhered to the culture vessel based on the fact that the cells do not move. In addition, in the step of reducing the cell adhesion strength, some cells with weak adhesion strength, such as cells before and after cell division, can be detached.

[0039] (Cell detachment solution) A cell detachment solution is a solution for reducing the cell adhesion strength.

[0040] The pH value of the cell detachment solution is preferably in the neutral range. Here, the neutral range means that the pH value is 6 or more and the pH value is 8 or less. This is because the neutral range is suitable for cell culture and can stably maintain a high cell survival rate. The pH value can be appropriately adjusted with, for example, hydrochloric acid or sodium hydroxide. To stably maintain the pH value, various buffers can be appropriately used.

[0041] The viscosity of the cell detachment solution is preferably 1.80 mPa·s or less. The viscosity of the cell detachment solution can be appropriately adjusted by adding a polymer or sugar.

[0042] As the cell detachment solution, a solution containing a metal ion chelator (hereinafter, sometimes referred to as "chelator") is particularly preferred. This is because using a cell detachment solution containing a chelator can effectively reduce the cell adhesion strength.

[0043] The chelating agent is not particularly limited. Examples of the chelating agent include at least one selected from the group consisting of ethylenediaminetetraacetic acid (hereinafter sometimes referred to as "EDTA"), ethylenediamine, ethylenediaminetetra(methylenephosphonic acid), diethylene glycol ether diamine tetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethylglycine, dicarboxymethylglutamic acid, ethylenediaminedisuccinic acid, hydroxyethylidene diphosphonic acid, citric acid, gluconic acid, and phosphonobutane triacetic acid. Among these chelating agents, a chelating agent that forms a chelate with a divalent cation is preferred. Particularly preferred is a chelating agent that forms a chelate with Ca 2+ and Mg 2+ to form a chelate. Ethylenediaminetetraacetic acid is most preferred.

[0044] When using ethylenediaminetetraacetic acid as the chelating agent, the pH value of the cell detachment solution is preferably 7.0 or more and 8.0 or less. This is because by setting the pH value to a higher value within the neutral range where a high cell survival rate can be maintained, the chelating ability of ethylenediaminetetraacetic acid can be enhanced, and the efficiency of reducing the cell attachment strength can be further improved. The chelating agent can be used alone or in combination of two or more.

[0045] The content of the chelating agent is preferably 0.01 mM or more and 5.0 mM or less. Within this range, the chelating action can be reliably provided, and the deterioration of cell proliferation ability due to the presence of an excessive chelating agent can be reduced.

[0046] The cell detachment solution may contain a hydrophilic polymer containing a polyalkylene glycol structure. Examples of the hydrophilic polymer containing a polyalkylene glycol structure include polyethylene glycol. The hydrophilic polymer preferably has a peak molecular weight Mp of 800 or more and 50000 or less, more preferably 1200 or more and 20000 or less, measured by gel permeation chromatography. This is because the polymer has little effect on cells, and the thickening effect of the polymer can be reduced.

[0047] (Buffer solution) The buffer solution for the cell detachment solution can be used without any limitation as long as it can maintain a neutral range. Examples of the buffer solution include: Tris buffer solutions such as Tris-HCl buffer solution, phosphate buffer solution, HEPES buffer solution, citrate-phosphate buffer solution, glycylglycine-sodium hydroxide buffer solution, Britton-Robinson buffer solution, and GTA buffer solution. Among these buffer solutions, a phosphate buffer solution close to the in-vivo environment is preferred. More preferably, phosphate buffered saline (PBS) prepared to be isotonic with intracellular fluid is used as the phosphate buffer solution.

[0048] In the step of reducing the adhesion strength according to an embodiment of the present invention, when chemical stimulation using a cell detachment solution is employed, in other words, when an incubation step of bringing the cells into contact with the cell detachment solution and incubating the cells is provided, the incubation step preferably includes the following steps. [1-1] A step of bringing the cells into contact with the cell detachment solution while the cells remain attached to the substrate. [1-2] A step of performing incubation.

[0049] (Step [1-1]) Step [1-1] is a step of replacing the growth medium for cell culture with the cell detachment solution. Before or after step [1-1], the cells can be washed with a buffer solution. For example, when detaching cultured cells from a cell culture container, the effect of the cell detachment solution can be enhanced by the following method: first removing the growth medium and washing the cells with a buffer solution, and then adding the cell detachment solution while the cells remain attached to the cell culture container. This is because the growth medium typically contains factors that inhibit the reduction of cell adhesiveness, such as serum.

[0050] (Step [1-2]) The incubation in step [1-2] refers to performing at least one of the following steps: a step of adjusting the temperature, a step of adjusting the humidity, and a step of adjusting the air composition. The step of adjusting the temperature is, for example, a step of controlling a heater so that the temperature around the cell culture container is about 37°C. The step of adjusting the humidity is, for example, a step of adjusting the humidity to 90% or more by installing a humidifying bucket so that the culture medium does not dry out. The step of adjusting the air composition is, for example, a step of supplying CO2 so that the partial pressure of CO2 is about 5% to prevent oxidation of the culture medium.

[0051] The incubation time is not particularly limited and is, for example, in the range of 30 seconds to 20 minutes. The incubation temperature is not particularly limited, and from the viewpoint of maintaining cell viability, it is preferably 10°C to 40°C. In particular, 30°C to 40°C is the optimal temperature for cell growth and is thus preferred.

[0052] (Measurement step) The measurement step in the cell detachment method according to an embodiment of the present invention is a step of obtaining cell information of the cells attached to the cell culture container. The measurement step (acquisition of cell information) can be performed at a time before the step of reducing the adhesion strength of the cells is executed. The acquisition of cell information can be performed at at least two times. The acquisition of cell information can be performed multiple times at regular intervals. Among the two times of acquiring cell information, the later time is defined as time t1. The time t2 at which the detachment step described below is to start is a time after time t1.

[0053] In the measurement step, time-series data related to cells can be obtained. The time-series data represents cell information measured at at least two time points and the time points at which the cell information is measured.

[0054] The number of time points for obtaining cell information is preferably 2 or more and 10,000 or less, more preferably 3 or more and 1,000 or less, and still more preferably 5 or more and 300 or less. When the number of time points for obtaining cell information is two or more, the start time of the peeling step can be accurately determined. In addition to the cell information at the time points for actually obtaining cell information, for example, cell information obtained in advance under the same conditions can be used as the cell information at the start time of the adhesion strength reduction step. When the number of time points for obtaining cell information is 10,000 or less, the processing time for determining the start time of the peeling step can be reduced.

[0055] Examples of the cell information measured in the measurement step include: the area of the cell, the area of the halo region in a phase-contrast microscope image (also referred to as a "phase-contrast image"), the brightness value in the phase-contrast image, and the brightness value when observing the cell culture surface of a cell culture container under oblique incident illumination.

[0056] As an example of the cell area, for example, a phase-contrast image of adherent cells is obtained and the area occupied by a single cell is measured by using image processing, so that the area of the cell can be digitized.

[0057] It is known that adherent cells appear to have a large area in a phase-contrast image, while cells with reduced adhesion strength appear to have a small area in a phase-contrast image because, due to surface tension, cells have a shape close to spherical. By using this phenomenon, the area of the cells on the phase-contrast image is digitized and its change is observed.

[0058] Another type of cell information is the brightness value or the area of the halo region in the phase-contrast image.

[0059] In a phase-contrast image of cells in an adherent state, only regions with a small phase difference as a whole (the cells are in a flat state) are detected. In contrast, it is known that in a halo region where the adhesion of the cells is observed to become weak, the phase difference increases in the periphery where the cells are lifted from the culture surface, and the image brightness value is higher than other regions. That is, when the adhesion of the cells becomes weak, the area of the halo region increases.

[0060] Another example of cell information is the brightness value when observing the cell culture surface of a cell culture vessel under oblique incident illumination. In the case where the cells have a reduced attachment strength, the pseudopodia contract and the attachment to the microscopic surface of the cell culture vessel decreases, so light scattering easily occurs at the interface between the cell culture vessel and the cells. Therefore, when observing under oblique incident illumination where the proportion of scattered light increases, if the attachment strength of the cells decreases, the brightness value of the cell culture surface of the cell culture vessel increases.

[0061] In this embodiment, various parameters can be used as cell information. The cell information can be, for example, at least one selected from the group consisting of: the area of each cell, the size of each cell, the roundness of each cell, the light transmittance of each cell, the occupied area of the cell group, the brightness value of the cell image, the area of the region where there are no cells, the area of the halo region, the brightness value of the image based on the halo, and the brightness value of the image obtained by observing the cell culture surface of the cell culture vessel under oblique incident illumination.

[0062] In this embodiment, cell information can be measured in various ways. For example, by using a phase contrast microscope to obtain a phase contrast image of the cells and analyzing the image, various types of cell information can be measured. Additionally, the brightness value of the culture surface can be measured by an illumination means that irradiates the cell culture surface of the cell culture vessel obliquely and an observation means arranged in a direction perpendicular to the culture surface. Additionally, the illumination means and the observation means can be arranged at positions opposite to each other across the cell culture vessel, or can be arranged in the same direction, as long as they are arranged to easily receive the scattered light on the culture surface.

[0063] The cell information is preferably brightness information related to the cells, more preferably brightness information of the cell image. The brightness information is preferably the brightness information of the culture surface observed under oblique incident illumination. From the viewpoint of reducing brightness non-uniformity, the oblique incident illumination is preferably annular illumination. The brightness information is preferably the average brightness value of the culture surface or the area of the culture surface where the brightness value exceeds a threshold.

[0064] The observation means for measuring cell information preferably has a field of view of 1 cm 2 or more. By using a field of view of 1 cm 2 or more, it is possible to evaluate the reduction in the attachment strength of the cells on a macroscopic scale without being affected by minor deviations in the cell culture state.

[0065] (Example of measuring the cell area as cell information) Immediately after replacing the growth medium with a cell detachment solution, obtain the phase contrast image of the cells over time. Mark a specific cell and quantify the area of the cell using image processing software.

[0066] As the attachment of cells to the cell culture container weakens, the cell area thus determined decreases over time. For example, after 10 minutes of incubation, the cell area of 237 μm 2 decreases to 113 μm 2 .

[0067] (Example of measuring the cell area change rate as cell information) Immediately after replacing the growth medium with the cell detachment solution, obtain the phase-contrast images of the cells over time. Mark specific cells and use image processing software to quantify the area of the cells. The area of the cells immediately after contact with the cell detachment solution (incubation time: zero) is defined as A1. The area of the cells at the incubation time t in the cell detachment solution is defined as A2. Calculate the change rate of the cell area by the following formula (1). (A1 - A2) / A1 (1)

[0068] As the attachment of cells to the substrate weakens, the change rate of the cell area thus determined increases over time. For example, the change rate of the cell area is 0.15 after 1 minute of incubation and increases to 0.5 after 5 minutes of incubation.

[0069] (Example of measuring the area fraction of the halo region as cell information) Immediately after replacing the growth medium with the cell detachment solution, obtain the phase-contrast images of the cells over time. Measure the area fraction of the halo region by performing binarization, threshold setting, and area measurement of the phase-contrast images of these cells using image processing software. Here, a specific brightness value on the image is set as the threshold. The region with a brightness value higher than or equal to the threshold is defined as the halo region. Determine the area of the halo region in the entire image area.

[0070] As the attachment of cells to the substrate weakens, the area fraction of the halo region thus obtained increases over time. Figure 3 Examples of the obtained phase-contrast microscope images and binarized images are shown. In the binarized image, the halo region is shown in black. For example, when the incubation time in the cell detachment solution is 30 seconds, the area fraction of the halo region in the phase-contrast image is 2.8%; while when the incubation time in the cell detachment solution is 5 minutes, the area fraction of the halo region in the phase-contrast image increases to 49.9%.

[0071] (Example of measuring the brightness value of the phase-contrast image as cell information) Immediately after replacing the growth medium with the cell detachment solution, obtain the phase-contrast images of the cells over time. Use image processing software to measure the brightness values of these cells in the phase-contrast image.

[0072] The brightness value of the phase-contrast image obtained in this way increases over time because the halo area increases as the attachment of the cells to the substrate weakens. For example, when the incubation time in the cell detachment solution is 30 seconds, the brightness value of the phase-contrast image is 7.2; when the incubation time in the cell detachment solution is 5 minutes, the brightness value of the phase-contrast image increases to 127.2.

[0073] (As an example of cell information, measure the brightness value of the image observed under oblique incident illumination) Immediately after replacing the growth medium with the cell detachment solution, acquire the time-lapse images of the cell culture surface observed under oblique incident illumination. Use image processing software to measure the brightness value of the entire cell culture surface of the cell culture container.

[0074] The brightness value of the cell culture surface observed under the oblique incident illumination obtained in this way increases over time because the intensity of the scattered light increases as the attachment of the cells to the cell culture container weakens. For example, when the incubation time in the cell detachment solution is 30 seconds, the average value of the brightness of the entire culture surface of the cell culture container is 96.2. When the incubation time in the cell detachment solution is 5 minutes, the average value of the brightness of the entire culture surface of the cell culture container increases to 108.0.

[0075] (Determination step) The determination step in the cell detachment method according to an embodiment of the present invention is a step of determining the time to start the detachment operation based on the cell information measured at at least two times, and this time is after the time when the cell information has been measured. The cell detachment method according to an embodiment of the present invention may further include a prediction step that predicts the time to start the detachment step based on the time-series data related to the cells. The time used here can be an absolute time, such as 13:30. The determined start time can be a relative time, such as 30 seconds after any one of the start time of the incubation step, the start time of acquiring cell information, and the time point when the start time is determined.

[0076] The interval for measuring cell information is preferably more than 1 second and less than 1 minute. That is, the interval between at least two times of acquiring cell information is preferably more than 1 second and less than 1 minute. Measuring cell information at an interval within this range can accurately determine the start time t2 of the detachment operation. The measurement intervals can be equal or unequal.

[0077] The time t1 for measuring cell information is preferably 1 hour or less, more preferably 15 minutes or less, from the start time t0 of incubation (the start time of the attachment strength reduction step). The time t1 for measuring cell information is preferably 10 seconds or more, more preferably 30 seconds or more, from the start time t0 of incubation (the start time of the attachment strength reduction step). When the time t1 is within this range, a decrease in cell viability can be suppressed.

[0078] t0, t1, and t2 preferably satisfy the relationship (t2 - t0) / (t1 - t0) ≥ 1.1. t0 is the start time of the attachment strength reduction step. When this relationship between t1 and t2 is satisfied, the preparation operation time until the start time of peeling can be sufficiently ensured.

[0079] The determination step may include a step of determining the end time t3 of the peeling operation. The method for determining t3 is not particularly limited. An example thereof is a method of determining t3 by a constant multiple of a value obtained by curve fitting based on time-series data of cell information.

[0080] (Curve fitting) In the determination step, it is preferable to obtain a fitting curve based on the cell information and the time at which the cell information is obtained, and to determine the time t2 based on the fitting curve. In the determination method, it is preferable to use curve fitting to obtain the fitting curve. The function for curve fitting and indicating the fitting curve is not particularly limited. Depending on the cell type used, the cell information, and the means and conditions for reducing the cell attachment strength, an appropriate function is used. Examples of the function include the functions represented by the following formulas (2-1) to (2-9).

[0081] [Equation 1]

[0082] [Equation 2]

[0083] [Equation 3]

[0084] [Equation 4]

[0085] [Equation 5]

[0086] [Equation 6]

[0087] [Equation 7]

[0088] [Number 8]

[0089] f(t) = C0 + C1t + C2t 2 + C3t 3 + C4t 4 ··· (2-9)

[0090] Here, t is the time when cell information is measured with the time when the step of reducing the cell attachment strength starts set to 0, and f(t) is the cell information measured at time t.

[0091] The values of the parameters other than t in formulas (2-1) to (2-9) can be determined in advance, or can be determined based on the time-series data of cell information, and at least one fitting parameter determined by curve fitting of the time-series data of cell information is provided. As an example, in formula (2-1), C0, C1, τ, and β can all be used as fitting parameters. Alternatively, C0 can be the cell information at time 0, β can be determined in advance, and C1 and τ can be used as fitting parameters.

[0092] As the cell information for curve fitting, the cell information measured up to time t1 is used. The cell information obtained by deleting appropriate times from the times when cell information has been measured can be used. The cell information at all times when measurements have been performed can be used. Before performing curve fitting, the time-series data of cell information can be smoothed, such as by filtering.

[0093] The method for determining the fitting parameter values by curve fitting is not particularly limited, and examples thereof include the least squares method and the least absolute deviation method.

[0094] (Determination of start time t2) After curve fitting the time-series data of cell information, the time t2 when the peeling operation is to start is determined. The time t2 is later than the time t1 when the measurement step is performed. The method for determining the time t2 is not particularly limited. As an example, a method will be described in which the average value of the brightness of an image observed under oblique incident illumination is used as cell information, and the function for curve fitting is the function represented by formula (2-1) with β = 1.

[0095] (Example 1 of determination of start time t2) According to the type of cell, the culture conditions, and the peeling operation, a brightness threshold Th1 for starting the peeling operation is provided in advance. The average value of the brightness is measured until time t1. The time when the curve of the function obtained by curve fitting using formula (2-1) reaches Th1 is calculated. This time is defined as the start time t2.

[0096] (Example 2 for determining the start time t2) Measure the average value of the luminance until time t1. Calculate the values of the fitting parameters C0, C1, and τ in Equation (2-1) by curve fitting. The time Aτ (where A is multiplied by τ) is defined as the start time t2. Here, A is provided in advance according to the cell type, culture conditions, and peeling operation. A is preferably 0.3 or more and 2 or less, and particularly preferably 0.5 or more and 1 or less.

[0097] (Example 3 for determining the start time t2) Measure the average value of the luminance until time t1. Calculate the time when the curve of the function obtained by curve fitting using Equation (2-1) reaches C0 + C1 × B. This time is defined as the start time t2. Here, B is provided in advance according to the cell type, culture conditions, and peeling operation. B is a positive value less than 1. B is preferably 0.3 or more and 0.9 or less, and particularly preferably 0.4 or more and 0.7 or less.

[0098] (Peeling operation) The peeling operation to start at the time t2 determined in the determination step includes vibrations caused by ultrasonic waves, vibrations caused by tapping, convection of the culture solution or cell detachment solution, etc. Multiple means can be combined. Examples of the method for causing convection of the culture solution or cell detachment solution include pipetting and using a pump or a stirring impeller. From the viewpoint of the quality of the cells after peeling, it is preferable to use the vibrations caused by ultrasonic waves for the peeling operation.

[0099] (Vibrations caused by ultrasonic waves) Examples of the vibrations caused by ultrasonic waves are vibrations with a frequency of about 10 kHz to 1 MHz, as described in International Publication No. 2016-047368. Means for generating vibrations can be used without particular limitation as long as it can subject the cells to vibrations caused by ultrasonic waves. For example, an ultrasonic transducer made of lead zirconate titanate (PZT) or the like can be used, as described in Japanese Patent Laid-Open No. 2008-92857.

[0100] As described in Japanese Patent Laid-Open No. 2006-314204, a vibrator can be brought into direct contact with the outer surface of a sealed culture vessel filled with a culture medium to subject the culture vessel to vibrations. In addition, as described in International Publication No. 2016-047368, instead of bringing the ultrasonic wave emitting means into direct contact with the container, an ultrasonic wave transmitting substance is inserted between the ultrasonic wave emitting means and the area to be treated to make ultrasonic waves incident on the cells to be peeled.

[0101] From the viewpoint of effectively detaching cells and improving cell viability, there is no particular limitation on the time for which the cells are subjected to vibration caused by ultrasonic waves, for example, it is 1 second to 1 hour, preferably 5 seconds to 30 minutes, more preferably 10 seconds to 15 minutes.

[0102] From the viewpoint of maintaining cell viability, there is no particular limitation on the environmental temperature for which the cells are subjected to vibration caused by ultrasonic waves, preferably it is 20°C to 40°C, particularly preferably 30.0°C to 37.5°C. This is because the temperature at the time of cell detachment is close to the temperature during culturing, thereby enabling reduction of the influence of temperature change on the cells and maintaining high viability.

[0103] (Treatment after cell detachment) The cells detached by the detachment operation can be inoculated in a culture container such as a new substrate, dish, or flask, or steps such as cell labeling can be performed. The cells can also be used as samples for measuring the number of cells or cell viability. In addition, when the obtained cells conjugate with each other to form cell clusters, a cell dissociation treatment can be appropriately performed.

[0104] (Configuration of information processing system) Figure 1 It is a block diagram showing an example of the hardware configuration of an information processing system 110 capable of executing a program according to an embodiment of the present invention.

[0105] The information processing system 110 has the functions of a computer. For example, the information processing system 110 can be configured to be integrated with a desktop personal computer (PC), notebook PC, tablet PC, smartphone, etc.

[0106] The information processing system 110 implements the functions of a computer for performing calculations and storage. The information processing system 110 includes a central processing unit (CPU) 1101, a random access memory (RAM) 1102, a read-only memory (ROM) 1103, and a hard disk drive (HDD) 1104. In addition, the information processing system 110 includes a communication interface (I / F) 1105, a display device 1106, and an input device 1107. The CPU 1101, RAM 1102, ROM 1103, HDD 1104, communication I / F 1105, display device 1106, and input device 1107 are connected to each other via a bus 1110. The display device 1106 and the input device 1107 can be connected to the bus 1110 via a driving device (not shown in the figure) to drive these devices.

[0107] In Figure 1In this case, the devices constituting the information processing system 110 are described in terms of integrated devices, but some of these functions may be implemented using external devices. For example, the display device 1106 and the input device 1107 may be external devices different from those constituting the computer functions including the CPU 1101 and the like.

[0108] The CPU 1101 executes predetermined operations according to programs stored in the RAM 1102, HDD 1104, etc., and also has the function of controlling each device of the information processing system 110. The RAM 1102 includes a volatile storage medium and provides a temporary memory area required for the CPU 1101 to operate. The ROM 1103 includes a non-volatile storage medium and stores necessary information such as programs for the operation of the information processing system 110. The HDD 1104 includes a non-volatile storage medium and is a storage device for storing information related to the number and position of each independent partition, fluorescence intensity, etc.

[0109] The communication I / F 1105 is a communication interface compliant with standards such as Wi-Fi (registered trademark) and 4G, and is a module for communicating with other devices. The display device 1106 is a liquid crystal display, an organic light emitting diode (OLED) display, etc., and is used to display moving images, still images, characters, etc. The input device 1107 is a button, a touch screen, a keyboard, a pointing device, etc., and is used by the user to operate the information processing system 110. The display device 1106 and the input device 1107 may be integrally formed as a touch screen.

[0110] Figure 1 The hardware configuration shown is an example, and devices other than these may be added, or some of these devices may not be provided. Some devices may be replaced with other devices having the same function. In addition, some functions may be provided by other devices via a network, and the functions included in the embodiment may be dispersed and implemented by multiple devices. For example, the HDD 1104 may be replaced with a solid state drive (SSD) using a semiconductor device such as a flash memory, or may be replaced with cloud storage.

[0111] (Cell detachment system) The cell detachment system according to an embodiment of the present invention includes: an adhesion strength reduction unit 201 for reducing the adhesion strength of cells; a measurement unit 202 for measuring cells at at least two times and acquiring cell information; a detachment unit 204 for detaching cells from the culture surface of a cell culture container; and a determination unit 203 for determining the timing at which the step of detaching cells is to start based on the cell information. Figure 11It is a schematic diagram of a cell detachment system. The cell detachment system 200 can be the cell detachment device described below. Additionally, the cell detachment system 200 can include multiple devices, and the functions of the cell detachment system 200 can be achieved by these devices.

[0112] (Cell detachment device) An example of a cell detachment device according to an embodiment of the present invention is a cell detachment device for detaching cells cultured on the culture surface of a cell culture container. The cell detachment device includes: a culture medium replacement unit that brings cells into contact with a cell detachment solution while the cells remain attached to the cell culture container; a cell information acquisition unit for acquiring and measuring cell information of the cells; an ultrasonic irradiation determination unit for determining the timing of ultrasonic irradiation; and an ultrasonic irradiation unit for subjecting the cells to vibrations caused by ultrasonic waves.

[0113] Use Figure 2 a conceptual diagram to describe an example of the configuration of a cell detachment device 100 according to an embodiment of the present invention. Figure 2 The components in are as follows: 100: cell detachment device, 1S: ultrasonic irradiation unit, 40: culture container movement controller, 41: amplifier, 42: function generator, 43: cell information acquisition unit (which may include a cell information analysis unit), 44: cell culture container, 45: pipetting unit, 46: pipette movement controller, 47: waste liquid recovery unit, 48: detached cell recovery unit (cell culture container), 49: detached cell recovery unit (tube), 50: washing culture medium, 51: culturing culture medium, 52: cell detachment solution, 53: liquid delivery controller, and 54: ultrasonic irradiation determination unit (which may include a cell information analysis unit). The positions are as follows: P4: the position of the culture container when acquiring cell information, P5: the position of the culture container during cell detachment (set in the ultrasonic irradiation unit), P6: the position of the culture container when adding cell detachment solution, replacing culture medium, or recovering cells, P7: the position of the new culture container when inoculating the detached cells into a new culture container, P8: the position of the pipette when adding cell detachment solution, replacing culture medium, or recovering cells, P9: the position of the pipette when discarding the culture medium or cell suspension, P10: the position of the pipette when inoculating the cell suspension into a dish, and P11: the position of the pipette when recovering the cell suspension into a tube.

[0114] The culture container movement controller 40 is a unit that holds and moves the culture container 44.

[0115] The culture vessel moving controller 40 may include a heater to control the temperature and humidity of the cell detachment environment. Examples thereof include a heating unit and a humidifying unit, which can maintain the temperature at 37°C. To move the culture vessel 44, an electric XYZ stage is used, for example. As an alternative, a robotic arm can be used to hold and move the culture vessel 44. The culture vessel moving controller 40 horizontally moves the culture vessel 44 between position P4, position P5, and position P6. At position P4, the cell information acquisition unit 43 can acquire information on the cells cultured in the culture vessel 44, such as a phase contrast image. At position P5, the ultrasonic irradiation unit 1S irradiates the inside of the culture vessel 44 with ultrasonic waves. At position P6, the pipetting unit 45 recovers the solution in the culture vessel 44 or adds various solutions (sometimes referred to as "culture media") to the culture vessel 44. The pipetting unit 45 may include a mechanism for automatically or manually discharging or aspirating the culture medium for culture. The configuration of the pipetting unit 45 is not limited as long as it can transport liquids, and it can be, for example, a tube pump. Additionally, multiple pipetting units can be provided and multiple pipetting units can be provided independently according to the type or function of the culture medium.

[0116] Examples of using a cell detachment process utilizing ultrasonic waves are further explained.

[0117] Cells are cultured in the culture vessel 44. When it is time for the cells to be detached, the culture vessel 44 is placed at position P6. The culture vessel 44 is appropriately placed at position P4 by the culture vessel moving controller 40, and the cell information acquisition unit 43 can observe the state of the cells. In the culture vessel 44 placed at position P6, the cells are attached to the vessel. First, the culture medium must be removed and replaced with a cell detachment solution. Therefore, at position P6, the culture medium in the culture vessel 44 is recovered with the pipetting unit 45. The recovered culture medium is discarded into the waste liquid recovery unit 47 at position P9. Various culture media are stored in the washing medium 50, the culture medium 51 for culture, and the cell detachment solution 52, and are transported to the pipetting unit 45 by the liquid transport controller 53. The pipetting unit can move vertically to enter the culture vessel 44. A lid opening and closing system for the culture vessel 44 can be provided appropriately. After removing the culture medium, the cells attached to the vessel can be appropriately washed. At position P6, the washing medium 50 is transported to the pipetting unit 45 by the liquid transport controller 53, and the washing medium 50 is added to the culture vessel 44 through the pipetting unit 45 at position P6. After removing the washing medium 50 in the same manner as the above-described culture medium removal method, the cell detachment solution 52 is transported to the pipetting unit 45 by the liquid transport controller 53, and at position P6, the cell detachment solution 52 is added to the culture vessel 44 through the pipetting unit 45.

[0118] Next, the culture vessel movement controller 40 moves the culture vessel 44 to position P4. Here, the cells are incubated in the cell detachment solution while the cell information acquisition unit 43 acquires cell information. During the incubation, the cell information acquisition unit 43 acquires cell information, analyzes the obtained cell information, and determines the start time of the detachment operation by curve fitting based on the time-series data of the cell information. The determination is performed by the ultrasonic irradiation determination unit 54. Subsequently, the culture vessel movement controller 40 is instructed to move the culture vessel 44, and commands are sent to start the ultrasonic irradiation unit 1S, the function generator 42, and the amplifier 41.

[0119] Next, the culture vessel movement controller 40 moves the culture vessel 44 to position P5. Here, the culture vessel 44 is placed in the ultrasonic irradiation unit 1S. An example configuration is one in which the ultrasonic detachment unit is combined with the function generator 42 and the amplifier 41, and the function generator and the amplifier input freely selectable frequencies, voltages, etc. to the ultrasonic detachment unit. At the determined start time, the culture vessel 44 is irradiated with ultrasonic waves from the ultrasonic irradiation unit 1S, thereby detaching the cells attached to the culture vessel 44. The culture vessel 44 can be appropriately moved to position P4, and the cell information acquisition unit 43 can be used to monitor the cell detachment state.

[0120] After the cell detachment is completed, the culture vessel movement controller 40 moves the culture vessel 44 to position P6. At position P6, the cell detachment solution containing the detached cells in the culture vessel 44 is recovered by the pipetting unit 45. At position P10, the cell detachment solution containing the recovered detached cells is inoculated into the detached cell recovery unit 48, for example, into a new cell culture vessel placed at position P7. As an alternative, at position P11, the cells are recovered in the tube 49 serving as the detached cell recovery unit. Subsequently, these recovered cells are used for cell culture and detection, such as cell measurement and cell labeling.

[0121] The cell detachment device according to an embodiment of the present invention may further include a controller to control the cell detachment process. The controller is a means for controlling the operations of the respective parts (units) in the cell detachment device 100. For example, the controller may include a computer that includes an arithmetic processor such as a CPU, a memory such as a RAM, and a memory such as a hard disk drive.

[0122] The controller may further include an operation unit. The operation unit may include a display unit, an input unit, etc. The display unit may display image data output from the controller and various information related to the operation of the cell detachment device 100. For example, a liquid crystal display is used as the display unit. The input unit receives various instruction inputs from the user. For example, a keyboard or a mouse is used as the input unit. The functions of the display unit and the input unit may be in a single unit, such as a touch screen display.

[0123] (Example) The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited thereto.

[0124] [Example 1] (Cell culture on a substrate) Chinese hamster ovary (CHO) cells were inoculated at a density of 10,000 cells / cm 2 in a φ35 polystyrene dish (manufactured by Corning Inc.) and cultured in an environment of 37 °C and 5% CO2 concentration. The medium used was Ham's F12 (manufactured by Thermo Fisher Scientific), to which 10% fetal bovine serum (manufactured by Sigma-Aldrich) and 1% penicillin-streptomycin (10,000 U / ml, manufactured by Thermo Fisher Scientific) were added. The culture was carried out for 48 hours. The state of the cells was observed using a phase-contrast microscope to confirm cell attachment and proliferation. The proportion of the area occupied by the cells on the dish was approximately 80%.

[0125] (Cell detachment) The medium in the dish was removed, and then phosphate-buffered saline (PBS(-), manufactured by Thermo Fisher Scientific) was added to the dish as a cell detachment solution. Observation images of the cell culture surface of the dish were obtained under oblique incident illumination. The average brightness value of the entire cell culture surface of the dish was measured using image processing software. In this case, the field of view area of the image was 50 cm 2 . Measurement was started immediately after adding the cell detachment solution to the dish, and measurement was carried out while incubating the cells in an incubator at 37 °C. Measurement was carried out at 5-second intervals for 2.5 minutes.

[0126] Figure 4 shows the time change of the average brightness value obtained from the measurement. The time-series data was curve-fitted using the function represented by Equation (2-1). Here, the value of β was set to 1, and the values of the fitting parameters C0, C1, and τ were determined by the least squares method. The obtained values of C0, C1, and τ were 62.9, 30.4, and 4.4, respectively. Figure 4 shows the curve obtained by curve fitting. The moment when the average brightness value reaches 77.5 was preset as the start time t2 of the detachment operation. The start time t2 was calculated from the curve obtained by curve fitting and found to be t2 = 2.9 minutes.

[0127] After measuring the cell information, the disk is placed in an ultrasonic peeling device. At an ambient temperature of 37°C, starting from time t2, the cells are peeled off by sweep-frequency vibration (frequency: 29.5 to 34.5 kHz, sweep-frequency period: 1 second, voltage: 100 V) for 3 minutes.

[0128] After collecting the peeled-off cells, the number of cells is measured using a hemocytometer. The survival rate is calculated by trypan blue staining using a viability test. The suspension of the peeled-off cells is mixed with a 0.4% trypan blue solution at a ratio of 1:1 to stain the dead cells contained in the suspension. The suspension is injected into a hemocytometer (DHC-N01, manufactured by NanoEntek). The number of live and dead cells on the grid of the hemocytometer is counted by microscopic observation. Among the cells peeled off by the vibration of the cell peeling device, the ratio of the number of live cells to the total number of counted cells is calculated as the survival rate.

[0129] The plate that has undergone ultrasonic peeling is treated with a cell scraper to peel off any cells that have not been peeled off by ultrasonic waves. The suspension of the peeled-off cells is injected into a hemocytometer (DHC-N01, manufactured by NanoEntek). The number of peeled-off cells is measured by microscopic observation. The total number of cells peeled off by ultrasonic waves and the number of cells subsequently peeled off by the cell scraper are combined to obtain the total number of peeled-off cells. The ratio of the number of cells peeled off by ultrasonic waves to the total number of peeled-off cells is defined as the peeling efficiency and calculated. When both the survival rate of the peeled-off cells and the cell peeling efficiency are 95% or more, it is determined that the effects of the present invention have been achieved.

[0130] [Example 2] The cell survival rate and cell peeling efficiency are evaluated in the same manner as in Example 1, except that the start time t2 of the peeling operation is set to 0.7 times τ. In this case, the start time t2 of the peeling operation is 3.1 minutes.

[0131] [Example 3] The cell survival rate and cell peeling efficiency are evaluated in the same manner as in Example 1, except that the start time t2 of the peeling operation is set to the time when the average brightness value reaches C0 + C1 × 0.5. The start time t2 of the peeling operation in this case is 3.0 minutes.

[0132] [Example 4] The cells are cultured in the same manner as in Example 1.

[0133] Remove the culture medium from the dish, and then add phosphate-buffered saline (PBS(-), manufactured by Thermo Fisher Scientific) to the dish as a cell detachment solution. Place the dish on the stage of a phase-contrast microscope to obtain a phase-contrast image. Measure the average brightness value of the phase-contrast image using image processing software. In this case, the field-of-view area of the image is 6 mm 2 . Start the measurement immediately after adding the cell detachment solution to the dish, and perform the measurement while incubating the cells in an incubator at 37 °C. Perform the measurement at 15-second intervals for 2.5 minutes.

[0134] Figure 5 shows the time variation of the average brightness value obtained from the measurement. Curve fitting is performed on this time-series data using the function represented by Equation (2-1). The values of the fitting parameters C0, C1, τ, and β are determined by the least squares method. The obtained values of C0, C1, τ, and β are 19.5, 90.4, 1.6, and 1.8, respectively. Figure 5 shows the curve obtained by curve fitting. The moment when the average brightness value reaches 105 is preset as the start time t2 of the detachment operation. Calculate the start time t2 from the curve obtained by curve fitting and find that it is t2 = 2.8 minutes.

[0135] After measuring the cell information, place the dish in an ultrasonic detachment device, and perform swept-frequency vibration (frequency: 29.5 to 34.5 kHz, sweep period: 1 second, voltage: 100 V) on it from time t2 at an ambient temperature of 37 °C for 3 minutes. Subsequently, evaluate the cell viability and cell detachment efficiency in the same manner as in Example 1.

[0136] [Example 5] Culture cells in the same manner as in Example 1.

[0137] Remove the culture medium from the dish, and then add phosphate-buffered saline (PBS(-), manufactured by Thermo Fisher Scientific) to the dish as a cell detachment solution. Place the dish on the stage of a phase-contrast microscope to obtain a phase-contrast image. Measure the cell areas of multiple cells from the phase-contrast image using image processing software. In this case, the field-of-view area of the image is 6 mm 2 . Start the measurement immediately after adding the cell detachment solution to the dish, and perform the measurement while incubating the cells in an incubator at 37 °C. Perform the measurement at 15-second intervals for 2 minutes. Take the average of the measured cell areas of multiple cells. Define the cell area immediately after multiple cells come into contact with the cell detachment solution (incubation time: zero) as A1.

[0138] Thereafter, the cell area at incubation time t is defined as A2. The change rate X of the cell area is calculated by the following formula (3). X = (A1 - A2) / A1 ··· (3)

[0139] Figure 6 The change rate of the cell area obtained from the measurement over time is shown. The time-series data is curve-fitted using the function shown in formula (2-1). Here, the value of C0 is 0, the value of β is 1, and the values of the fitting parameters C1 and τ are determined by the least squares method. The obtained values of C1 and τ are 0.70 and 3.5, respectively. Figure 6 The curve obtained by curve fitting is shown. The time when the change rate of the cell area reaches 0.4 is preset as the start time t2 of the peeling operation. The start time t2 is calculated from the curve obtained by curve fitting and found to be t2 = 2.9 minutes.

[0140] After measuring the cell information, the disk is placed in an ultrasonic peeling device, and from time t2 at an ambient temperature of 37°C, it is subjected to swept-frequency vibration (frequency: 29.5 to 34.5 kHz, sweep period: 1 second, voltage: 100 V) for 3 minutes. Subsequently, the cell viability and cell peeling efficiency are evaluated in the same manner as in Example 1.

[0141] [Comparative Examples 1 to 5] The cell viability and cell peeling efficiency are evaluated in the same manner as in Example 1, except that the measurement of cell information is not performed, and the respective start times t2 are changed as shown in Table 1.

[0142] Table 1 presents the results of the peeled cell viability and cell peeling efficiency in Examples 1 to 5 and Comparative Examples 1 to 5.

[0143] [Table 1] Table 1

[0144] [Example 6] (Cell culture) The C2C12 cells (mouse skeletal muscle myoblast cell line) are seeded at 5000 cells / cm 2Cells were inoculated at a density in φ35 polystyrene dishes (manufactured by Corning) and cultured in an environment of 37°C and 5% CO₂ concentration. The medium used was DMEM / F12 (manufactured by Thermo Fisher Scientific), to which 10% fetal bovine serum (manufactured by Sigma-Aldrich) and 1% penicillin-streptomycin (10000 U / ml, manufactured by Thermo Fisher Scientific) were added. Culturing was carried out for 48 hours. The state of the cells was observed using a phase contrast microscope to confirm cell attachment and proliferation. The proportion of the cell-occupied area of the dish was approximately 80%.

[0145] (Cell detachment) The medium in the dish was removed, and then phosphate buffered saline (PBS(-), manufactured by Thermo Fisher Scientific) was added to the dish as a cell detachment solution. Observation images of the cell culture surface of the dish were obtained under oblique incident illumination. The average brightness value of the entire cell culture surface of the dish was measured using image processing software. At this time, the field of view area of the image was 50 cm 2 . Measurement was started immediately after adding the cell detachment solution to the dish, and measurement was carried out while incubating the cells in an incubator at 37°C. Measurement was carried out at 1-second intervals for 5 minutes.

[0146] Figure 7 shows the time variation of the average brightness value obtained from the measurement. The time-series data was curve-fitted using the function represented by Equation (2-5). The values of the fitting parameters C0, C1, C2, and τ were determined by the least squares method. The obtained values of C0, C1, C2, and τ were 78.4, 16.9, 1.34, and 5.15, respectively. Figure 7 shows the curve obtained by curve fitting. The moment when the average brightness value reached 90.0 was preset as the start time t2 of the detachment operation. The start time t2 was calculated from the curve obtained by curve fitting and found to be t2 = 6.2 minutes.

[0147] After measuring the cell information, the dish was placed in an ultrasonic detachment device. At an environmental temperature of 37°C, starting from time t2, the cells were detached by swept-frequency vibration (frequency: 29.5 to 34.5 kHz, sweep period: 1 second, voltage: 100 V) for 3 minutes, and then, in the same manner as in Example 1, the cell viability and cell detachment efficiency were evaluated.

[0148] [Comparative Examples 6 to 9] The cell viability and cell detachment efficiency were evaluated in the same manner as in Example 6, except that the measurement of cell information was not carried out, and the respective start times t2 were changed as shown in Table 2.

[0149] Table 2 presents the results of the survival rate of detached cells and the cell detachment efficiency in Example 6 and Comparative Examples 6 to 9.

[0150] [Table 2] Table 2

[0151] [Example 7] The process up to the measurement of cell information was carried out in the same manner as in Example 6, except that the start time t2 of the detachment operation was set to the time when the average brightness value reached 92.5. In this case, the start time t2 of the detachment operation was 7.3 minutes.

[0152] After the measurement of cell information, the disk 10 was placed on the holding mechanism 11 by Figure 8 the shear stress applying mechanism shown. Using the rotation mechanism 12 and the impact applying mechanism 13, a load of 5 N was applied to the outer periphery of the disk 12 times at intervals of 5 seconds, while changing the load application position every 30° along the outer periphery of the disk. Then, the cell survival rate and the cell detachment efficiency were evaluated in the same manner as in Example 1.

[0153] [Comparative Examples 10 to 13] The cell survival rate and the cell detachment efficiency were evaluated in the same manner as in Example 7, except that the measurement of cell information was not carried out, and the respective start times t2 were changed as shown in Table 3.

[0154] Table 3 presents the results of the survival rate of detached cells and the cell detachment efficiency in Example 7 and Comparative Examples 10 to 13.

[0155] [Table 3] Table 3

[0156] Embodiments of the present invention can also be provided in the following ways: by a computer of a system or device (such as an application-specific integrated circuit (ASIC)) that reads and executes computer-executable instructions (such as one or more programs) recorded on a storage medium to perform one or more functions of the above embodiments, and / or by a computer of a system or device including one or more circuits to perform one or more functions of the above embodiments, and by reading and executing computer-executable instructions from a storage medium to perform, for example, one or more functions of the above embodiments, and / or by using a method executed by a computer of a system or device to control one or more circuits to perform one or more functions of the above embodiments. The computer may include one or more processors (e.g., a central processing unit (CPU) and a microprocessor (MPU)), and may include a network of independent computers or independent processors for reading and executing computer-executable instructions. Computer-executable instructions can be provided to the computer from, for example, a network or a storage medium. The storage medium may include, for example, one or more of the following: a hard disk, a random access memory (RAM), a read-only memory (ROM), a storage device of a distributed computing system, an optical disc (e.g., a high-density compact disc (CD), a digital versatile disc (DVD), or a Blu-ray disc (BD)), a flash device, a memory card, etc.

[0157] The disclosure of the embodiments includes the following methods and configurations.

[0158] (Method 1) A cell detachment method for detaching cells attached to a culture surface of a cell culture container, the method comprising: an attachment strength reduction step of reducing the attachment strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; a measurement step of measuring the cells at at least two times and obtaining cell information; a detachment step of detaching the cells from the culture surface; and a determination step of determining a time t2 at which the detachment step is to start, the time t2 being after a time t1, the time t1 being the later time of the at least two times.

[0159] (Method 2) The cell detachment method as described in Method 1, wherein the cell detachment method includes an incubation step of bringing the cells into contact with a cell detachment solution and incubating the cells.

[0160] (Method 3) The cell detachment method as described in Method 1 or 2, wherein the time t1 is a time of 30 seconds or more and 15 minutes or less from the start time t0 of the attachment strength reduction step.

[0161] (Method 4) The cell detachment method according to any one of methods 1 to 3, wherein in the measuring step, an interval between at least two times of acquiring the cell information is 1 second or more and 1 minute or less.

[0162] (Method 5) The cell detachment method as described in any one of methods 1 to 4, wherein the start time t0 of the adhesion strength reduction step, the time t1, and the time t2 satisfy the relationship (t2-t0) / (t1-t0)≥1.1.

[0163] (Method 6) The cell detachment method as described in any one of methods 1 to 5, wherein the detachment step includes a step of subjecting the cells to vibration caused by ultrasonic waves.

[0164] (Method 7) The cell detachment method as described in any one of Methods 1 to 6, wherein, in the determining step, a fitting curve is obtained based on the cell information and the time when the cell information is acquired, and the time t2 is determined based on the fitting curve.

[0165] (Method 8) In the cell detachment method as described in Method 7, in the determining step, the time when the cell information calculated based on the fitting curve is equal to a predetermined threshold value of the cell information is determined as the time t2.

[0166] (Method 9) The cell detachment method as described in method 7, wherein, in the determining step, the following formula is used as the fitting curve for curve fitting:

[0167] [Number 9]

[0168] Wherein t is the time t at which the cell information is acquired when the start time of the adhesion strength reduction step is set to 0, f(t) is the cell information acquired at the time t, A greater than 0.3 and less than 2 is provided in advance, and the time Aτ at which A is multiplied by τ is determined as the time t2.

[0169] (Method 10) The cell detachment method as described in method 7, wherein, in the determining step, the following formula is used as the fitting curve for curve fitting:

[0170] [Number 10]

[0171] Among them, t is the time t when cell information was acquired with the start time of the adhesion strength reduction step set to 0, f(t) is the cell information acquired at the time t, B greater than or equal to 0.3 and less than or equal to 0.9 is provided in advance, and the time when f(t) is equal to C0 + C1×B is determined as the time t2.

[0172] (Method 11) The cell detachment method according to any one of Methods 1 to 10, wherein, in the determination step, the time t3 when the detachment step is completed is determined.

[0173] (Method 12) The cell detachment method according to any one of Methods 1 to 11, wherein the cell information is measured from an image of the cell, and the cell information is at least one of the following: the area of the cell, the area change rate of the cell, the brightness value of the cell, the area fraction of the region where the brightness value of the image is greater than a threshold value, and the brightness value of the image.

[0174] (Method 13) The cell detachment method according to any one of Methods 1 to 12, wherein the measurement step is performed after the start of the adhesion strength reduction step.

[0175] (Method 14) The cell detachment method according to any one of Methods 1 to 13, wherein the fitting curve is a fitting curve of the time elapsed after the start of the adhesion strength reduction step and the brightness value of the cell.

[0176] (Method 15) The cell detachment method according to any one of Methods 1 to 13, wherein the fitting curve is a fitting curve of the time elapsed after the start of the adhesion strength reduction step and the area of the cell.

[0177] (Method 16) A cell detachment method for detaching cells attached to a culture surface of a cell culture container, the method comprising: an adhesion strength reduction step of reducing the adhesion strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; a measurement step of measuring the cells and acquiring time-series data related to the cells; a detachment step of detaching the cells from the culture surface; and a prediction step of predicting the time when the detachment step is to start based on the time-series data.

[0178] (Method 17) A cell detachment method as described in Method 16, wherein the time-series data is cell information measured at at least two times and the times when the cell information is measured, and the cell information is at least one of the brightness value and the area of the cell.

[0179] (Configuration 1) A program for causing a computer to execute the cell detachment method according to any one of Methods 1 to 17.

[0180] (Configuration 2) A cell detachment system for detaching cells attached to a culture surface of a cell culture container, the system including: an attachment strength reduction unit that reduces the attachment strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; a measurement unit that measures the cells at at least two times and acquires cell information; a detachment unit that detaches the cells from the culture surface; and a determination unit that determines a time t2 at which a detachment step of detaching the cells from the culture surface is to start based on the cell information, the time t2 being after a time t1, the time t1 being the later of the at least two times.

[0181] (Configuration 3) A cell detachment system for detaching cells attached to a culture surface of a cell culture container, the system including: an attachment strength reduction unit that reduces the attachment strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container, a measurement unit that measures the cells and acquires time-series data related to the cells, a detachment unit that detaches the cells from the culture surface, and a prediction unit that predicts a time at which a detachment step of detaching the cells from the culture surface is to start based on the time-series data.

[0182] (Configuration 4) An information processing device for controlling a cell detachment device to detach cells attached to a culture surface of a cell culture container, the information processing device including: a measurement unit that acquires cell information of cells whose attachment strength to the culture surface has been reduced due to the application of a stimulus at at least two times; and a determination unit that determines a time t2 at which a detachment step of detaching the cells from the culture surface is to start based on the cell information, the time t2 being after a time t1, the time t1 being the later of the at least two times.

[0183] (Configuration 5) An information processing device for controlling a cell detachment device to detach cells attached to a culture surface of a cell culture container, the information processing device including: a measurement unit configured to acquire time-series data related to cells whose attachment strength to the culture surface is reduced due to application of a stimulus at at least two times; and a prediction unit configured to predict a time at which a detachment step of detaching the cells from the culture surface is to start based on the time-series data.

[0184] The present invention is not limited to the above-described embodiments, and various changes and modifications can be made within the spirit and scope of the present invention. Accordingly, the following claims are made to inform the public of the scope of the present invention.

[0185] This application claims priority to Japanese Patent Application No. 2022-190040, filed on Nov. 29, 2022, which is incorporated herein by reference in its entirety.

Claims

1. A cell detachment method for detaching cells attached to a culture surface of a cell culture vessel, the method comprising: An attachment strength reduction step of reducing the attachment strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture vessel; A measurement step of measuring the cells at at least two times and obtaining cell information; A detachment step of detaching the cells from the culture surface; And A determination step of determining a time t2 at which the detachment step is to start based on the cell information, the time t2 being after a time t1, the time t1 being the later of the at least two times.

2. The cell detachment method according to claim 1, wherein, The cell detachment method includes an incubation step of bringing the cells into contact with a cell detachment solution and incubating the cells.

3. The cell detachment method according to claim 1, wherein, The time t1 is a time of 30 seconds or more and 15 minutes or less from the start time t0 of the attachment strength reduction step.

4. The cell detachment method according to claim 1, wherein, In the measurement step, the interval between at least two of the times at which the cell information is obtained is 1 second or more and 1 minute or less.

5. The cell detachment method according to claim 1, wherein, The start time t0 of the attachment strength reduction step, the time t1, and the time t2 satisfy the relationship (t2 - t0) / (t1 - t0) ≥ 1.

1.

6. The cell detachment method according to claim 1, wherein, The detachment step includes a step of subjecting the cells to vibrations caused by ultrasonic waves.

7. The cell detachment method according to claim 1, wherein, In the determination step, a fitting curve is obtained based on the cell information and the time at which the cell information is obtained, and the time t2 is determined based on the fitting curve.

8. The cell detachment method according to claim 7, wherein, In the determination step, the time at which the cell information calculated based on the fitting curve is equal to a predetermined threshold value of the cell information is determined as the time t2.

9. The cell detachment method according to claim 7, wherein, In the determination step, the following formula is used as the fitting curve for curve fitting, [Equation 1] where t is the time t at which the cell information is obtained when the start time of the attachment strength reduction step is set to 0, f(t) is the cell information obtained at the time t, A of 0.3 or more and 2 or less is provided in advance, and the time Aτ obtained by multiplying A by τ is determined as the time t2.

10. The cell detachment method according to claim 7, wherein, In the determination step, the following formula is used as the fitting curve for curve fitting, [Equation 2] where t is the time t at which the cell information is obtained when the start time of the attachment strength reduction step is set to 0, f(t) is the cell information obtained at the time t, B of 0.3 or more and 0.9 or less is provided in advance, and the time when f(t) is equal to C0 + C1 × B is determined as the time t2.

11. The cell detachment method according to claim 1, wherein, In the determination step, the time t3 when the detachment step is completed is determined.

12. The cell detachment method according to claim 1, wherein, The cell information is measured from an image of the cells, and the cell information is at least one of the following: the area of the cells, the area change rate of the cells, the brightness value of the cells, the area fraction of the region of the image where the brightness value is greater than a threshold value, and the brightness value of the image.

13. The cell detachment method according to claim 1, wherein, The measurement step is performed after the start of the attachment strength reduction step.

14. The cell detachment method according to claim 1, wherein, The fitting curve is a fitting curve of the time elapsed after the start of the attachment strength reduction step and the brightness value of the cells.

15. The cell detachment method according to claim 1, wherein, The fitting curve is a fitting curve of the time elapsed after the start of the adhesion strength reduction step and the area of the cells.

16. A cell detachment method for detaching cells attached to a culture surface of a cell culture container, the method comprising: An adhesion strength reduction step of reducing the adhesion strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; A measurement step of measuring the cells and acquiring time-series data related to the cells; A detachment step of detaching the cells from the culture surface; and A prediction step of predicting the time when the detachment step is to start based on the time-series data.

17. The cell detachment method according to claim 16, wherein, The time-series data is cell information measured at at least two times and the time when the cell information is measured, and The cell information is at least one of the brightness value of the cells and the area of the cells.

18. A program for causing a computer to execute the cell detachment method according to any one of claims 1 to 17.

19. A cell detachment system for detaching cells attached to a culture surface of a cell culture container, the system comprising: An adhesion strength reduction unit that reduces the adhesion strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container; A measurement unit that measures the cells at at least two times and acquires cell information; A detachment unit that detaches the cells from the culture surface; And A determination unit that determines the time t2 when the detachment step of detaching the cells from the culture surface is to start based on the cell information, the time t2 being after the time t1, and the time t1 being the later time of the at least two times.

20. A cell detachment system for detaching cells attached to a culture surface of a cell culture container, the system comprising: An adhesion strength reduction unit that reduces the adhesion strength of the cells by applying a stimulus to the cells while the cells remain attached to the cell culture container, A measurement unit that measures the cells and acquires time-series data related to the cells, A detachment unit that detaches the cells from the culture surface, and A prediction unit that predicts the time when the detachment step of detaching the cells from the culture surface is to start based on the time-series data.

21. An information processing device for controlling a cell detachment device to detach cells attached to a culture surface of a cell culture container, the information processing device comprising: A measurement unit for acquiring cell information of cells whose adhesion strength to the culture surface has been reduced due to the application of a stimulus at at least two times; And A determination unit that determines the time t2 when the detachment step of detaching the cells from the culture surface is to start based on the cell information, the time t2 being after the time t1, and the time t1 being the later time of the at least two times.

22. An information processing device for controlling a cell detachment device to detach cells attached to a culture surface of a cell culture container, the information processing device comprising: A measurement unit for acquiring time-series data related to cells whose adhesion strength to the culture surface has been reduced due to the application of a stimulus at at least two times; And A prediction unit that predicts, based on the time-series data, the timing at which the peeling step of peeling the cells from the culture surface is to start.

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