JP-7856634-B2 - Method for determining cell viability, device for determining cell viability, and system for determining cell viability

JP7856634B2JP 7856634 B2JP7856634 B2JP 7856634B2JP-7856634-B2

Inventors

亀井翔太
守本隆史
下田知之

Assignees

富士フイルム株式会社

Dates

Publication Date: 20260511
Application Date: 20220413
Priority Date: 20210428

Claims (15)

A step of acquiring an image of a cell, which is illuminated with light, from a direction opposite to the side of the cell that was illuminated with light, and which is captured at multiple focal planes including the focal plane of the cell. A step of obtaining an image fragment from each of the aforementioned images, including the central part and the outer periphery of the cell. A step of creating a stitched image for analysis by stitching together image fragments in the order of the imaging direction of the focal plane, A step of extracting features from the aforementioned linked image for analysis, and a step of determining the viability of the cells based on the features of the linked image for analysis and a predetermined range of features. A method for determining cell viability, including the method for determining cell viability.
The cell viability determination method according to claim 1, wherein determining the viability of the cell includes determining whether or not the cell is a target cell.
The cell viability determination method according to claim 1 , wherein a machine learning model is used to perform the viability determination based on the features of a known reference concatenated image of the cell and the determination result of whether or not it is a living cell.
The cell viability determination method according to claim 1, wherein the feature quantities of the analytical concatenated image include one or more selected from the group consisting of feature quantities relating to the lens effect of the cell, feature quantities relating to the average refractive index of the cell, feature quantities relating to the diameter of the cell, and feature quantities relating to the specific gravity of the cell.
A method for determining cell viability according to any one of claims 1 to 4, comprising performing the viability determination on a plurality of cells in a cell suspension.
The cell viability determination method according to claim 5, comprising the step of determining the concentration of viable cells in the cell suspension based on the results of the viability determination.
The cell viability determination method according to claim 5 , further comprising the step of determining the cell viability of the cells in the cell suspension based on the results of the viability determination.
An image acquisition unit acquires images of a cell from a direction opposite to the side of the cell that has been irradiated with light, capturing images of the cell at multiple focal planes including the in-focus plane of the cell. An image fragment acquisition unit that acquires an image fragment from each of the aforementioned images, including the central and outer regions of the cell. An analytical concatenation image creation unit creates an analytical concatenation image by concatenating image fragments in the order of the imaging direction of the focal plane. A feature extraction unit that extracts feature quantities from the aforementioned linked image for analysis, and a cell viability determination unit that determines the viability of the cells based on the feature quantities of the linked image for analysis and a predetermined range of feature quantities. A cell viability determination device equipped with the following features.
The cell viability determination device according to claim 8, wherein the viability determination unit is composed of a machine learning model, and the viability determination is performed based on the known reference concatenated image features of the cells and the determination result of whether or not the cells are alive.
The cell viability determination device according to claim 8 , further comprising a viability concentration determination unit that determines the viability concentration of the cells in the cell suspension based on the results of viability determination of a plurality of the cells in the cell suspension.
The cell viability determination device according to claim 10, further comprising a cell viability determination unit that determines the cell viability of the cells in the cell suspension based on the results of the viability determination.
A cell viability determination system comprising: a light source for irradiating the aforementioned light; an imaging device for imaging the aforementioned cells; means for changing the focal plane; and a cell viability determination device according to any one of claims 8 to 11.
The cell viability determination system according to claim 12, wherein the means for changing the focal plane is a stage movement mechanism that moves a stage on which a holding container for holding the cells is mounted, thereby changing the distance between the cells and the imaging device.
The cell viability determination system according to claim 12, wherein the means for changing the focal plane is an imaging device movement mechanism that moves the imaging device to change the distance between the cell and the imaging device.
The cell viability determination system according to claim 12, wherein the imaging device is equipped with a liquid lens as the means for changing the focal plane.

Description

This disclosure relates to a method for determining cell viability, a device for determining cell viability, and a system for determining cell viability. In cell culture technology, it is necessary to use living cells, and therefore, the viability of the cells used for seeding is determined. Such cell viability determination techniques are disclosed, for example, in Japanese Patent Publication No. 2013-517460. Figure 1 is a schematic diagram showing an example of a cell viability determination system.Figure 2 is a schematic diagram showing an example of a focal plane.Figure 3 is a schematic diagram showing an example of an image obtained by imaging living cells while changing the focal plane.Figure 4 is a schematic diagram showing an example of an image obtained by imaging dead cells while changing the focal plane.Figure 5 is a schematic diagram showing an example of a linked image obtained from living cells.Figure 6 is a schematic diagram showing an example of a linked image obtained from dead cells.Figure 7 is a schematic diagram showing an example of processing in the training and operation phases of a machine learning model.Figure 8 is a flowchart showing an example of the life/death determination process.Figure 9 is a block diagram showing an example of a control unit that constitutes a cell viability determination device.Figure 10 is a block diagram showing an example of the processing performed by a cell viability determination device. The following describes the details of the cell viability determination method and cell viability determination device related to this disclosure. In this disclosure, the numerical range indicated using "~" means the range that includes the numbers written before and after "~" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this disclosure, the upper or lower limit stated in one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in the numerical ranges described in this disclosure, the upper or lower limit stated in one numerical range may be replaced with the values shown in the examples. In this disclosure, a combination of two or more preferred embodiments is a more preferred embodiment. In this disclosure, unless otherwise specified, the amount of each component refers to the total amount of multiple substances if there are multiple substances corresponding to each component. In this disclosure, the term "process" includes not only independent processes but also processes that cannot be clearly distinguished from other processes, as long as their intended purpose is achieved. The drawings referenced in the following description are illustrative and schematic, and this disclosure is not limited to these drawings. The same reference numerals indicate the same components. Reference numerals in the drawings may also be omitted. <Method for determining cell viability> The cell viability determination method relating to this disclosure is: The process of acquiring images of a cell, which are captured from the opposite side of the cell to the light-emitting side, at multiple focal planes including the cell's focal plane (hereinafter sometimes referred to as the "image acquisition process"), The process of obtaining image fragments from each image, including the central and peripheral parts of the cell (hereinafter sometimes referred to as the "image fragment acquisition process"), The process of creating a stitched image for analysis by connecting image fragments in the order of the imaging direction of the focal plane (hereinafter sometimes referred to as the "stitched image creation process for analysis"), The process includes a step of extracting features from a stitched image for analysis (hereinafter sometimes referred to as the "feature extraction step"), and a step of determining whether a cell is alive or dead based on the features of the stitched image for analysis and a predetermined range of features (hereinafter sometimes referred to as the "vitality determination step"). As a method for determining cell viability, for example, Japanese Patent Publication No. 2013-517460 describes a method of imaging cells at different focal planes and then determining cell viability based on the brightness of the images. However, the technique described in Japanese Patent Publication No. 2013-517460 may be insufficient for determining cell viability. In contrast, the cell viability determination method described herein utilizes the optical properties of cells to create a stitched image from images of cells captured at different focal planes, thereby determining whether a cell is viable or not. Living cells, being covered by a cell membrane, have a tendency to form a spherical shape in cell suspension (though not necessarily a perfect sphere), and they are also translucent. Therefore, living cells possess the properties of a spherical lens, which is called th