JP2019004794A - Proliferation prediction method, proliferation prediction apparatus and program - Google Patents

Abstract

To provide techniques that can predict the future proliferation degree of a cell while suppressing the influence on cells.SOLUTION: The proliferation prediction method comprises calculating an index for predicting the future proliferation state of mesenchymal stem cells based on the culture state of the mesenchymal stem cells at the first time point, and predicting the proliferation state of the mesenchymal stem cells at the second time point after the first time point based on the index and the discriminant.SELECTED DRAWING: Figure 8

Description

  The present disclosure relates to a growth prediction method, a growth prediction apparatus, and a program for predicting the degree of cell proliferation.

  In recent years, cells are collected from a living body and cultured, and the cultured cells are used for medical research or pharmacological tests. When cells are grown for the purpose as described above, there is often a demand to grow cells to a desired amount by a predetermined date. Therefore, various management methods for cultured cells have been devised so far.

  In Patent Document 1, the occupied area ratio of the cultured cells in the culture vessel is measured, and the measured occupied area ratio of the cultured cells and the number and occupation of the cells classified into two regions according to the proliferation state of the cultured cells are disclosed. Based on the relationship with the area ratio, there is disclosed a passage determination method for cultured cells that determines whether passage is possible or not according to whether or not it has an occupied area ratio located at the boundary between the two regions.

JP 2007-124914 A

  However, the passage determination method described in Patent Document 1 is an invention for determining the passage timing so that the growth speed of cultured cells does not decrease, and can a desired amount of cells be obtained on a desired date? Give no information about. Therefore, in the above passage determination method, for example, when using cultured cells for therapeutic purposes, it is difficult to predict whether a desired amount of cells can be obtained on the day of transplantation to a patient.

  In addition, in the invention described in Patent Document 1, it is necessary to acquire images of cells in culture at any time in order to calculate the occupied area ratio of cultured cells. May adversely affect the growth.

  The present disclosure has been made in view of the above points, and provides a technique capable of predicting the degree of future proliferation of cells while suppressing the influence on the cells.

  In order to solve the above-mentioned problem, a calculation step for calculating an index for predicting a future proliferative state of the mesenchymal stem cell based on a culture state of the mesenchymal stem cell at a first time point; And a prediction step of predicting the proliferation state of the mesenchymal stem cells at a second time point after the first time point based on the formula.

  Further, a future proliferative state of the mesenchymal stem cell is predicted based on an acquisition unit that acquires an image of the cultured cell and the cultured state of the mesenchymal stem cell in the first time point that is reflected in the image. A proliferation unit comprising: a calculation unit that calculates an index for predicting; and a prediction unit that predicts a proliferation state of the mesenchymal stem cells at a second time point after the first time point based on the index and the discriminant. A prediction device is provided.

  Further, based on the acquisition step of acquiring an image of the cultured cells and the cultured state of the mesenchymal stem cells at the first time point shown in the image, the future proliferation state of the mesenchymal stem cells is predicted. A calculation step for calculating an index for predicting, and a prediction step for predicting a proliferation state of the mesenchymal stem cells at a second time point after the first time point based on the index and the discriminant. Provide a program to be executed.

  According to the present disclosure, it is possible to predict the degree of future proliferation of cells while suppressing the effect on the cells.

It is a figure explaining the concept of a colony. It is a figure which shows a mode that the collection of colonies is grouped. It is a figure which shows multiple colonies from which the average length of a cell differs. It is a figure which shows the calculation method of a length parameter. It is a figure which shows multiple colonies from which the average density of a cell differs. It is a figure for demonstrating how to count a surrounding cell. It is a figure for demonstrating the method of calculating a density parameter. 1 is a diagram schematically showing a configuration of a growth prediction apparatus of Example 1. FIG. It is a flowchart which shows the flow of the process which the proliferation prediction apparatus of Example 1 performs.

<Overview>
When culturing mesenchymal stem cells by repeating cell culturing experiments, the inventors of the present application, the morphological information of the cells on the fifth day after the start of the culture, and the 15th day after the start of the culture It was confirmed that there was a strong correlation between the degree of cell proliferation. Therefore, the inventors apply the above knowledge and determine whether or not a desired amount of cells can be obtained at the second time point after the first time point based on the cell shape information at the first time point. A prediction method was developed.

  In the specification of the present application, the term “day n after the start of culture” refers to any point in time from 24 × (n−1) hours to 24 × n hours before the start of culture. Further, the morphological information is, for example, a length parameter indicating the length of a cell, a density parameter indicating how densely a plurality of cells are proliferating, and the number of cells. A more detailed definition of the form information will be described later. The prediction method will be described below with reference to the drawings.

[Discriminant]
First, a method for determining a discriminant for predicting the degree of cell proliferation will be described. The discriminant predicts whether or not the cells have grown to a desired amount at the second time point after the first time point when the morphology information of the cell group (colony) at the first time point is specified. It is a standard for judgment. The discriminant is, for example, morphological information of mesenchymal stem cells on the fifth day (within 24 hours after 96 hours have elapsed) and the 15th day (after 336 hours have elapsed since the start of culturing). (Within time) and the number information of mesenchymal stem cells.

  FIG. 1 is a diagram for explaining the concept of a colony. A colony is a term indicating a group of cells in which a plurality of cells are gathered. For example, a set of mesenchymal stem cells originating from the same mesenchymal stem cell is defined as one colony. Defining a colony in this way is preferable for calculating an index for predicting the degree of future proliferation because the growth of cells contained in the same colony is similar. All cells are given a colony ID, and it is possible to determine which colony they belong to.

  As a method for assigning a colony ID to a cell, a culture dish in which cells are cultured at a predetermined time interval using a known cell tracking technique is continuously photographed, and each of a plurality of cells divided from the same cell is photographed. There is a method of assigning the same colony ID. Alternatively, there is a method of assigning the same colony ID to cells existing within a certain range in an image at a certain time.

  Later, in order to know what characteristics the colonies have grown sufficiently, the morphological information of each colony is recorded on the fifth day after the start of the culture. The colony morphological information noted in the present disclosure is a length parameter calculated using the long axis of the cell, a density parameter calculated based on the density of cells in the colony, and the number of cells in the colony.

  Subsequently, it is confirmed whether or not the number of cells of each colony is 500 or more on the 15th day after the start of the culture. Here, a colony in which the number of cells was 500 or more on the 15th day after the start of culture was labeled as 1, and a colony in which the number of cells was less than 500 on the 15th day after the start of culture was labeled as 0. That is, there are four parameters that characterize each colony: a length parameter, a density parameter, the number of cells, and an identifier for identifying the number of cells included in the colony (in the above example, a binary numerical value).

  In the present embodiment, the number of cells contained in the colony on the 15th day after the start of culture is determined using a linear discriminant method based on any combination of the length parameter, the density parameter, and the number of cells on the 5th day after the start of the culture. Binary numbers that are identifiers to identify are grouped so that they are well separated. The number of groups to be divided may be any number. For example, the groups are divided into two groups. This group classification criterion is used as a discriminant. That is, there are at least as many discriminants as combinations of parameters that characterize a colony.

  FIG. 2 is a diagram illustrating how colony sets are grouped. Here, each colony is plotted on the (density, number) plane. FIG. 2A is a distribution diagram showing colonies on the fifth day after the start of culture. FIG.2 (b) is a distribution map which shows each colony of the 15th day after culture | cultivation start. In FIG. 2 (b), colonies with 500 or more cells are plotted with triangles, and colonies with less than 500 cells are plotted with circles. FIG. 2C is a diagram illustrating a state in which a set of colonies is classified into two groups by the linear discrimination method. The straight line shown in FIG. 2C is a straight line indicating the discriminant.

  After obtaining the discriminant, the growth state of the cells on the 15th day after the start of the culture can be predicted based on the cell morphology information on the 5th day after the start of the culture. In the example described above, the first time point for calculating the index is the fifth day after the start of the culture, and the second time point for predicting the cell growth state is the 15th day after the start of the culture. The second time point is not limited to them. For example, the first time point may be the fourth day after the start of the culture, and the second time point may be the 16th day after the start of the culture. Further, although the binary variable is set to 1 when the number of cells at the second time point is 500 or more, it can be arbitrarily determined whether the binary variable is set to 1 when the degree of cell proliferation is high. The identifier is not limited to a binary numerical value. The identifier is 1 when the number of cells at the second time point is 600 or more, the identifier is 0 when the number of cells at the second time point is 400 or more and less than 600, and the number of cells at the second time point If the number is less than 400, the identifier may be -1.

[Indicator calculation method]
As described above, at the first time point, for each colony that is a cell group of mesenchymal stem cells, an index is calculated based on the morphological information of the mesenchymal stem cells. As the cell shape information to be measured, there were a length parameter representing the length of the mesenchymal stem cells and a density parameter representing the density of the mesenchymal stem cells. Below, the calculation method of a length parameter and a density parameter is demonstrated in detail.

(1) Length parameter FIG. 3 is a diagram showing a plurality of colonies having different average lengths of cells. As shown in FIG. 3, the length of the cells in the middle of the culture is not uniform but varies. The inventors of the present application have found that when the proportion of cells whose length is shorter than a certain level is high, the degree of subsequent cell proliferation is high. Therefore, the length parameter, which is a measure of the length of the cells contained in the colony, is used as an index for predicting the degree of future cell growth, and is calculated as follows.

FIG. 4 is a diagram illustrating a method for calculating the length parameter. FIG. 4A is a diagram conceptually showing a colony. As shown in FIG. 4A, in the present specification, the length parameter is calculated using the length of the long axis of the cell. As a method for measuring the length of the long axis of the cell (unit: μm), there is a method of calculating by the following equation using the moment of the cell region.
Length of major axis = 2 × √2 × √ {M _0,2 + M _2,0 + √ [(M _0,2 −M _2,0 ) ² + 4 × M _1,1 ² ]} × A
Here, M _2,0 , M _0,2 , M _1,1 , and A are the second moment in the image X direction, the second moment in the image Y direction, the synergistic moment, and the numerical values obtained from the image resolution. The following formula is used.
M _2,0 = Σ (xx ′) ² ÷ N + (1 ÷ 12)
M _0,2 = Σ (y−y ′) ² ÷ N + (1 ÷ 12)
M _1,1 = Σ {(x−x ′) × (y−y ′)} ÷ N + (1 ÷ 12)
The cell region refers to each closed region represented by white pixels in a binarized image obtained by filtering a phase difference image in which cells are reflected and then binarizing the image. Yes (black pixels are background in the binarized image).
x and y indicate the coordinates of pixels included in the cell region.
x ′ and y ′ indicate the coordinates of the barycentric position of the pixel included in the cell region.
N indicates the number of pixels included in the cell region.
A is the actual length corresponding to one pixel of the image. For example, when the target image is 1 pixel = 4 μm, A = 4.

  The length parameter is calculated as the number of mesenchymal stem cells whose major axis is equal to or less than a predetermined length with respect to the number of mesenchymal stem cells contained in the colony. The predetermined length is, for example, 60 micrometers. As described above, if the proportion of cells having a short cell length is high, the degree of subsequent cell proliferation is high. The inventors of the present application have found that when the ratio of the length of the long axis is 60 micrometers or less is high, it is highly likely that cells will grow to a desired amount in the future. In the example shown in FIG. 4 (a), 11 mesenchymal stem cells are included in the colony A, of which 8 cells have a long axis length of 60 micrometers or less, so the length parameter is It is calculated as 8/11 = 0.727. In addition, the length of the long axis of a cell can be measured using a known long axis measurement technique of a cell other than the above method.

  FIG. 4B is a diagram showing an actual image of the colony. In FIG. 4B, each of the plurality of images indicates a phase difference image, an image obtained by filtering the phase difference image, and a binarized image in order from the left. A region surrounded by a white line in each image indicates a colony A. In the example shown in FIG. 4 (b), since 24 mesenchymal stem cells are included in the colony A, of which 9 cells have a long axis length of 60 micrometers or less, the length parameter Is calculated as 9/24 = 0.375. Therefore, it can be seen that the colony shown in FIG. 4 (b) is a colony having a smaller length parameter and a low degree of future cell growth than the example shown in FIG. 4 (a).

(2) Density parameter FIG. 5 is a diagram showing a plurality of colonies having different average densities of cells. As shown in FIG. 5, the density of the cells in the middle of the culture is not uniform but varies. The inventors of the present application have found that the growing cell group has a correlation between the cell density and the proliferation rate. Therefore, the inventors thought that the growth rate can be accurately predicted by obtaining the cell density as an evaluation index of the culture state. In the present embodiment, how many cells (peripheral cells) are located around one cell is used as a reference for density. In this way, since the value of the density parameter is determined regardless of the size of the entire colony, it is a good index for predicting the degree of cell proliferation by appropriately reflecting the degree of cell density.

  FIG. 6 is a diagram for explaining how to count neighboring cells. To determine the number of surrounding cells, first select one cell and then draw a circle centered on that cell. The radius of the circle is, for example, a value that is larger than half of the representative value (maximum value, average value, intermediate value, etc.) of the length of the long axis of the cell and smaller than the size of the entire colony. Then, the number of cells that are partly included in the circle is counted and determined as the number of surrounding cells.

  FIG. 7 is a diagram for explaining a method of calculating the density parameter. The density parameter is calculated by the following procedure. First, one cell in the colony is selected. The number of cells (number of surrounding cells) in a predetermined range centering on the cell is counted. If the above count is repeated for all cells, the number of surrounding cells is determined for each cell contained in the colony. A value obtained by averaging the determined number of surrounding cells over the entire colony is defined as a colony density parameter.

  Once the discriminant is determined as described above, the degree of cell proliferation at the second time point can be predicted as long as the cell shape information at the first time point is obtained. Below, the proliferation prediction apparatus which can estimate the proliferation degree of a cell is demonstrated.

<Example 1>
[Configuration of growth prediction device]
FIG. 8 is a diagram schematically illustrating a configuration of the growth prediction apparatus 1 according to the first embodiment. The growth prediction device 1 includes a recording unit 2, a control unit 3, a display unit 4, and a microscope 5. The microscope 5 is, for example, an inverted microscope having the photographing unit 6, and can photograph cells in culture and transmit an image to the control unit 3. In addition, the stage of the microscope 5 can be controlled by an instruction signal from the growth prediction device 1, and the imaging unit 6 can image cells in culture from a plurality of positions.

  The recording unit 2 includes, for example, a RAM (Random Access Memory), a ROM (Read Only Memory), and an SSD (Solid State Drive). For example, numerical values in the middle of calculation and cell shape information are temporarily recorded in the RAM. For example, a program executed by the control unit 3 is recorded in the ROM. In the SSD, for example, a captured image of cells in culture and a prediction result regarding the degree of future cell growth are recorded. In addition, a discriminant determined in advance is recorded in the SSD.

  The control unit 3 functions as an acquisition unit 31, a calculation unit 32, and a prediction unit 33 by executing a program recorded in the recording unit 2.

  The acquisition unit 31 acquires an image of the cultured cell from the microscope 5. For example, the acquisition unit 31 continues to acquire cell images from the start of culture until the first time point. The acquisition unit 31 records the acquired image related to the cell in the recording unit 2 and notifies the calculation unit 32 of the image. The acquisition unit 31 can also display the acquired image on the display unit 4. That is, the user can confirm on the display unit 4 an image of cells in culture placed on the microscope.

  The calculation unit 32 gives a colony ID to each cell using the image acquired by the acquisition unit 31. Moreover, the calculation part 32 calculates the parameter | index for predicting the future proliferation state of a mesenchymal stem cell for every colony based on the culture state of the mesenchymal stem cell of a 1st time point. The first time point is, for example, any time point within 24 hours after 96 hours from the start of culture. In the growth prediction device 1 according to the first embodiment, the calculation unit 32 determines the length parameter of each colony as an index. The calculation unit 32 records the calculated index in the recording unit 2 and notifies the prediction unit 33 of it.

  The prediction unit 33 determines the proliferation state of the mesenchymal stem cells at the second time point after the first time point based on the index calculated by the calculation unit 32, the number of cells, and the predetermined discriminant. Predict for each colony. The second time point is, for example, any time point within 24 hours after 336 hours have elapsed since the start of culture. In the growth prediction device 1 according to the first embodiment, the prediction unit 33 predicts the degree of future cell growth for each colony based on the number of cells of each colony and the length parameter. The prediction unit 33 can display the predicted result on the display unit 4.

[Flow of processing executed by the growth prediction device]
FIG. 9 is a flowchart illustrating a flow of processing executed by the proliferation prediction device 1 according to the first embodiment. The operation of the growth prediction apparatus 1 in each step of the processing flow is as follows.

(S901)
The acquisition unit 31 continues to acquire images of cells being cultured from the microscope 5, and the calculation unit 32 assigns a colony ID to each cell.

(S902)
An index for the calculation unit 32 to predict the future proliferation state of the mesenchymal stem cells based on the culture state of the mesenchymal stem cells at the first time point using the image acquired by the acquisition unit 31 at the first time point. For each colony. In addition, the calculation unit 32 counts the number of cells included in each colony at the first time point.

(S903)
Based on the index calculated by the calculation unit 32, the number of cells, and a predetermined discriminant, the prediction unit 33 determines the proliferation state of the mesenchymal stem cells at the second time point after the first time point. Predict for each colony.

  The proliferation predicting apparatus 1 according to the first embodiment having the above-described configuration can predict the degree of cell proliferation at the second time point after the first time point based on the cell shape information at the first time point. Therefore, it is possible to manage the process more efficiently than before, such as discarding a culture dish in which cell growth is not expected to a desired amount and starting cell culture again. In addition, since the degree of future cell proliferation is predicted based on the cell shape information at one time point, unlike the case where the cell culture state is continuously observed, the cell growth is unlikely to be adversely affected.

<Example 2>
In the growth prediction device 1 according to the first embodiment, the calculation unit 32 determines the length parameter of each colony, and the prediction unit 33 determines the future cell growth based on the number of cells and the length parameter of each colony. The degree was predicted. On the other hand, in the growth prediction device 1 according to Example 2, the calculation unit 32 determines the density parameter of each colony, and the prediction unit 33 determines the future based on the number of cells and the density parameter of each colony. Predict the degree of cell growth.

  The growth prediction device 1 of the second embodiment having the above-described configuration is similar to the growth prediction device 1 of the first embodiment, based on the cell shape information at the first time point, at the second time point after the first time point. The degree of cell proliferation can be predicted.

<Example 3>
In the description of Examples 1 and 2, the proliferation predicting apparatus 1 determines the degree of future cell proliferation using a predetermined discriminant. The growth prediction device 1 may further include a discriminant determination unit that determines a discriminant. The discriminant determination unit determines the discriminant by, for example, the same procedure as described in the “discriminant” column.

<Experimental example>
Experimental examples supporting the prediction method of the present disclosure are described below.
In the experiment, the synovial tissue collected at the time of artificial knee joint replacement was cultured. First, the synovial tissue was treated with collagenase, and 1000 nucleated cells obtained were seeded on a 6-well plate. The synovial tissue was adhered to a 6-well plate over 3 hours, the growth medium (αMEM, 10% FBS, 1% Antibiotic) was replaced, and the progress was observed using a time-lapse microscope. In the time lapse microscope, the culture state of the synovial tissue was continuously photographed every 6 hours for 14 days, and a total of 500,000 images were obtained.

  Based on the above image, after seeding synovial stem cells, a phase contrast image on day 5 was analyzed, and a technique for predicting cell proliferation on day 15 after seeding was examined. A group of cells having 4 or more cells was defined as a colony using an image on the fifth day after the start of culture. The colony determination method used here will be described later as a modified example. The number of cells and the density parameter were analyzed for 583 colonies that could be identified as colonies.

  The number of cells on the 15th day after the start of the culture is reflected in a graph in which the number of cells and the density parameter on the 5th day after the start of the culture are plotted two-dimensionally. Group and low density group). Among the colonies classified into the high density group, the number of the colonies having 500 or more cells on the 15th day after the start of the culture was 71% (= 60/84) of the whole. In addition, among the colonies classified into the low density group, 75% (= 376/499) of the colonies had less than 500 cells on the 15th day after the start of the culture.

  As described above, it can be understood that the number of cells and the density parameter on the fifth day after the start of the culture function as an index for predicting the degree of cell proliferation on the 15th day after the start of the culture.

<Modification 1>
In the above description, a set of mesenchymal stem cells originating from the same mesenchymal stem cell is defined as one colony. In the case of this colony definition, it is necessary to keep track of cell division after the start of culture. Below, the method to define a colony based on the image in a single time is demonstrated.

  First, one cell shown in the image is selected, and the same colony ID is assigned to a cell included in a predetermined range from the cell. When four or more cells do not exist within the predetermined range, they are treated as isolated cells that do not form colonies. The above operation is repeated for all the remaining cells except for cells that have already been given a colony ID or have been confirmed not to give a colony ID. In this way, colonies can be defined based on images at a single point in time.

<Modification 2>
In the above description, a set of combinations of the number of cells at the first time point, the length parameter, and the number of cells at the second time point, or the number of cells at the first time point, the density parameter, and the number of cells at the second time point. The discriminant was determined using a set of combinations with.

  The discriminant may be determined based on another combination of parameters. For example, the discriminant may be determined based on the number of cells at the first time point, the length parameter, the density parameter, and the number of cells at the second time point. In that case, the growth prediction device 1 predicts the degree of cell growth based on the number of cells at the first time point of the first time point, the length parameter, the density parameter, and the discriminant.

  DESCRIPTION OF SYMBOLS 1 ... Growth prediction apparatus, 2 ... Recording part, 3 ... Control part, 31 ... Acquisition part, 32 ... Calculation part, 33 ... Prediction part, 4 ... Display part, 5 ... Microscope, 6 ... Imaging | photography part

Claims (11)

Based on the culture state of the mesenchymal stem cells at the first time point, a calculation step for calculating an index for predicting the future proliferation state of the mesenchymal stem cells;
A predicting step of predicting a proliferation state of the mesenchymal stem cells at a second time point after the first time point based on the index and the discriminant;
A method for predicting proliferation, comprising: The index is calculated based on morphological information of the mesenchymal stem cells for each colony that is a cell group of the mesenchymal stem cells,
The growth prediction method according to claim 1. The morphological information is a length parameter that represents the length of the mesenchymal stem cells.
The growth prediction method according to claim 2. The length parameter is calculated as the number of mesenchymal stem cells whose major axis is a predetermined length or less with respect to the number of mesenchymal stem cells contained in the colony.
The growth prediction method according to claim 3. The predetermined length is 60 micrometers;
The growth prediction method according to claim 4. The colony is a collection of mesenchymal stem cells originating from the same mesenchymal stem cell.
The growth prediction method according to claim 2. The index is calculated based on a density parameter representing the density of the mesenchymal stem cells.
The growth prediction method according to claim 2. The discriminant includes the morphological information of the mesenchymal stem cells within 24 hours after 96 hours from the start of culturing, and the mesenchymal stem cells within 24 hours after the 336 hours from the start of culturing. Calculated based on the number information of
The growth prediction method according to claim 1. The first time point is any time point within 24 hours after 96 hours from the start of culture,
The second time point is any time point within 24 hours from the start of culturing for 336 hours.
The growth prediction method according to claim 1. An acquisition unit for acquiring an image of cells in culture;
Based on the culture state of the mesenchymal stem cells at the first time point shown in the image, a calculation unit that calculates an index for predicting the future proliferation state of the mesenchymal stem cells;
Based on the index and the discriminant, a prediction unit that predicts the proliferation state of the mesenchymal stem cells at a second time point after the first time point;
A growth prediction apparatus comprising: An acquisition step of acquiring an image of the cultured cells;
A calculation step for calculating an index for predicting a future proliferation state of the mesenchymal stem cells based on the cultured state of the mesenchymal stem cells at the first time point shown in the image;
A predicting step of predicting a proliferation state of the mesenchymal stem cells at a second time point after the first time point based on the index and the discriminant;
A program that causes a computer to execute.