Culture support device, culture system, culture support method, and program

Abstract

Determine the timing of operations to be performed on the scaffolding material and the culture medium containing cells. [Solution] A culture support device comprising: an image acquisition unit that acquires images of a culture medium containing a scaffold material and cells at multiple timings; a past cell density estimation unit that estimates the density of cells on the scaffold material based on the images of the culture medium; a future cell density estimation unit that estimates the future cell density on the scaffold material based on the multiple timings and the estimated cell density on the scaffold material in the culture medium; and an operation timing determination unit that determines the timing to perform an operation on the culture medium based on the estimated future cell density on the scaffold material.

Description

Technical Field

[0004] , , , ,

[0001] The present invention relates to a culture assistance device, a culture system, a culture assistance method, and a program.

Background Art

[0002] Techniques have been proposed for examining the characteristics of cultured cells by photographing the culture solution in a culture vessel and analyzing the obtained image. Patent Document 1 discloses estimating the quality of cells. Patent Document 2 discloses determining the timing of passage from a two-dimensional image of the culture solution taken. Patent Document 3 discloses evaluating the quality of cells based on the cell shape. Patent Document 4 discloses evaluating cells from an image of cells taken over time. Patent Document 5 discloses predicting the quality of cells.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

[0005] One aspect of the present invention is a culture support device comprising: an image acquisition unit that acquires images of a culture medium containing a scaffold material and cells at multiple timings; a past cell density estimation unit that estimates the density of the cells on the scaffold material based on the images of the culture medium; a future cell density estimation unit that estimates the future density of cells on the scaffold material based on the multiple timings and the estimated cell density on the scaffold material in the culture medium; and an operation timing determination unit that determines the timing for performing an operation on the culture medium based on the estimated future cell density on the scaffold material. [Effects of the Invention]

[0006] According to the present invention, it is possible to determine the timing of operations performed on a culture medium containing scaffolding material and cells. [Brief explanation of the drawing]

[0007] [Figure 1] This figure shows the configuration of the culture system 1 according to this embodiment. [Figure 2] This figure shows the configuration of the culture support device 14 in this embodiment. [Figure 3] This is a bright-field image of culture medium 100. [Figure 4] This is a binary mask image, where the internal region of the scaffolding material and other regions are represented by different values. [Figure 5] This is a binary mask image, where areas of cells attached to the scaffolding material and other areas are shown with different values. [Figure 6] This is an example of an approximation curve between cell density on scaffolding material and time. [Figure 7] This figure shows the relationship between cell density on scaffolding material and time. [Figure 8]This is a flowchart showing the operation of the culture support device 14 according to this embodiment. [Modes for carrying out the invention]

[0008] Embodiments of the present invention will be described in detail below with reference to the drawings. Figure 1 shows the configuration of the culture system 1 according to this embodiment. The culture system 1 comprises a culture vessel 10, an imaging device 12, a culture support device 14, and a display device 16.

[0009] A culture medium 100 is placed inside the culture vessel 10. The culture medium 100 contains a scaffold material and cells. The scaffold material is, for example, a microfiber, nonwoven fabric, or microcarrier. The cells are adherent cells, and these adherent cells are attached to the scaffold material. In the culture medium 100, the cells are attached to the scaffold material and cultured in suspension agitation.

[0010] The imaging device 12 images the culture medium 100. The image of the culture medium 100 includes the scaffold material and cells contained in the culture medium 100. The imaging device 12 may also image a sample of the culture medium 100 taken from the culture vessel 10. The imaging device 12 images the culture medium 100 at multiple timings. The imaging device 12 may image the culture medium 100 periodically, or it may image the culture medium 100 at any timing.

[0011] The culture support device 14 determines the timing of operations to be performed on the culture medium 100 based on images of the culture medium 100. Details of the culture support device 14 will be described later.

[0012] The display device 16 displays an image indicating the timing of operations to be performed on the culture medium 100, as determined by the culture support device 14. Based on the timing of operations shown on the display device 16, the person performing the culture performs the operations on the culture medium 100.

[0013] The operations performed on the culture solution 100 are, for example, subculture, medium replacement, addition of a differentiation inducer, or detachment of cells from the scaffold material. When performing subculture on the culture solution 100, a new scaffold material is added to the culture solution 100.

[0014] FIG. 2 is a diagram showing the configuration of the culture assisting device 14 in the present embodiment. The culture assisting device 14 includes an image acquisition unit 140, a past cell density estimation unit 142, a future cell density estimation unit 144, an operation timing determination unit 146, an output unit 148, and a storage unit 190.

[0015] The image acquisition unit 140 acquires images at a plurality of timings captured by the imaging device 12. The image acquisition unit 140 records the acquired images in the storage unit 190. The acquired images include information on the timing of imaging of the images.

[0016] The past cell density estimation unit 142 estimates the density of cells on the scaffold material in the culture solution 100 based on the acquired image of the culture solution 100. The past cell density estimation unit 142 processes the image of the culture solution 100 and estimates the density of cells on the scaffold material in the culture solution 100.

[0017] Hereinafter, a method for processing an image of the culture solution 100 will be described in detail. FIG. 3 is an example of an image acquired by the image acquisition unit 140. The image shown in FIG. 3 is a bright-field image of the culture solution 100. In the image shown in FIG. 3, a microcarrier is used as the scaffold material. In the bright-field image shown in FIG. 3, the boundary between the scaffold material and the culture solution 100 is shown by a black boundary.

[0018] In the image of the culture solution 100, the area of the region occupied by cells in the region occupied by the scaffold material with respect to the area of the region occupied by the scaffold material is the density of cells on the scaffold material. The area of the region occupied by the scaffold material can be calculated, for example, by applying a mask process to the image of the culture solution 100 to generate an image in which the internal region of the scaffold material and other regions are indicated by different values. FIG. 4 is an image obtained by processing the image of the culture solution 100. The image shown in FIG. 4 is a binary mask image in which the internal region of the scaffold material and other regions are indicated by different values.

[0019] The area of ​​cells within the region occupied by the scaffolding material can be calculated, for example, by applying a masking process to an image of culture medium 100, generating an image in which the cell region within the region occupied by the scaffolding material and other regions are shown with different values. Figure 5 shows an image of culture medium 100 after processing. The image shown in Figure 5 is a binary mask image, which shows the region of cells attached to the scaffolding material and other regions with different values. The images shown in Figure 4 and Figure 5 are images that have undergone binarization processing with different thresholds. By changing the threshold and binarizing the image of culture medium 100, it is possible to generate an image in which the internal region of the scaffolding material and other regions are shown with different values, and an image in which the cell region within the region occupied by the scaffolding material and other regions are shown with different values.

[0020] By performing the image processing described above, the area occupied by the scaffolding material and the area of ​​cells within the area occupied by the scaffolding material are calculated, thereby calculating the cell density on the scaffolding material. The past cell density estimation unit 142 performs the above image processing on the acquired image of the culture medium 100 to estimate the cell density of the culture medium 100 on the scaffolding material.

[0021] In the estimation of cell density on scaffolding material by the past cell density estimation unit 142, if the scaffolding material included in the image does not resemble a predetermined shape, it may be excluded from the calculation of the area occupied by the scaffolding material and the area of ​​cells within the area occupied by the scaffolding material. The predetermined shape of the scaffolding material is stored in the memory unit 190 in advance, for example. Here, the shape of the scaffolding material is obtained from images of scaffolding material to which cells have not been attached or from images of scaffolding material in the early stages of culture. If the scaffolding material forms aggregates as a result of the proliferation of cells, an image of the scaffolding material may be obtained in the early stages of culture. The past cell density estimation unit 142 may exclude shapes that do not resemble images of scaffolding material to which cells have not been attached or from the calculation of the area occupied by the scaffolding material and the area of ​​cells within the area occupied by the scaffolding material.

[0022] In the example shown in Figure 3, since the shape of the scaffolding material is spherical, in the image where the internal region of the scaffolding material and other regions are shown with different values, regions where the internal region of the scaffolding material does not resemble a circle may be excluded from the calculation of the area occupied by the scaffolding material and the area of ​​the cell region within the area occupied by the scaffolding material. Furthermore, in the estimation of the cell density on the scaffolding material by the past cell density estimation unit 142, if the scaffolding material included in the image is out of focus, it may be excluded from the calculation of the area occupied by the scaffolding material and the area of ​​cells within the area occupied by the scaffolding material. The determination of whether or not the scaffolding material is in focus is performed by a well-known method, for example, by determining areas with large changes in brightness as edges, performing a Fourier transform on the brightness changes of the image at the edges to calculate the spectral distribution of the image, and making a determination based on the presence of high-frequency components.

[0023] The future cell density estimation unit 144 estimates the future cell density on the scaffold material in the culture medium 100 based on the cell density on the scaffold material in the culture medium 100 at multiple timings estimated by the past cell density estimation unit 142. The future cell density estimation unit 144 estimates the future cell density on the scaffold material by, for example, calculating a function between the cell density on the scaffold material and time based on the estimated cell density on the scaffold material and the timing corresponding to that cell density (e.g., time or elapsed time from a certain reference point). In calculating the function between the cell density on the scaffold material and time, for example, an approximation curve between the cell density on the scaffold material and time is calculated. Figure 6 is an example of an approximation curve between the cell density on the scaffold material and time.

[0024] The operation timing determination unit 146 determines the timing for performing an operation on the culture medium 100 based on the future cell density on the scaffold material estimated by the future cell density estimation unit 144. For example, the operation timing determination unit 146 determines the time when the estimated future cell density on the scaffold material exceeds a predetermined threshold as the timing for performing an operation on the culture medium 100.

[0025] When an operation is performed on the culture medium 100, the cell density on the scaffold material changes rapidly. For example, when subculturing is performed on the culture medium 100, the cell density on the scaffold material decreases rapidly. The future cell density estimation unit 144 estimates the future cell density on the scaffold material in the culture medium 100 based on the cell density on the scaffold material in the culture medium 100 at the time immediately following the operation.

[0026] The timing of an operation performed on the culture medium 100 is acquired by the future cell density estimation unit 144 by inputting it, for example, into an input device provided in the culture support device 14. After performing a predetermined operation on the culture medium 100, the person performing the operation inputs the timing of the operation into the input device provided in the culture support device 14. The timing at which the rate of change of cell density per unit time on the scaffold material exceeds a predetermined value may be detected as the timing at which an operation was performed on the culture medium 100.

[0027] Figure 7 shows the relationship between cell density on the scaffold material and time. Prior to the current time shown in Figure 7, the culture medium 100 was subjected to two operations (subculturing). At the two times when the culture medium 100 was subculturised, the cell density on the scaffold material decreased sharply. At the current time, the future cell density estimation unit 144 estimates the future cell density on the scaffold material based on data from the time of the second subculturing. In Figure 7, ○ represents the cell density on the scaffold material estimated based on images of the culture medium 100 that were automatically captured periodically, and ◆ represents the cell density on the scaffold material estimated based on images of the culture medium 100 that were manually captured.

[0028] The output unit 148 outputs to the display device 16 information indicating the timing for performing an operation on the culture medium 100, as determined by the operation timing determination unit 146. The output unit 148 may also output to the display device 16 information indicating the image acquired by the image acquisition unit 140, the time corresponding to the cell density on the scaffold material estimated by the past cell density estimation unit 142, and the time corresponding to the cell density on the scaffold material estimated by the future cell density estimation unit 144.

[0029] The memory unit 190 stores image data acquired by the image acquisition unit 140, software used by the past cell density estimation unit 142 for image processing and estimating the density of cells on the scaffolding material, functions used by the future cell density estimation unit 144 for estimating the density of cells on the scaffolding material in the future, and software used by the operation timing determination unit 146 to determine the timing for performing operations on the culture medium 100.

[0030] Figure 8 is a flowchart illustrating the operation of the culture support device 14 according to this embodiment. The image acquisition unit 140 acquires images captured by the imaging device 12 (step S10). The past cell density estimation unit 142 estimates the cell density on the scaffold material in the culture medium 100 based on the acquired images (step S12). The future cell density estimation unit 144 estimates the future cell density on the scaffold material based on the estimated past cell density on the scaffold material (step S14). The operation timing determination unit 146 determines the timing for performing an operation on the culture medium 100 based on the estimated future cell density on the scaffold material (step S16). The output unit 148 outputs information indicating the timing for performing the operation to the display device 16 (step S18).

[0031] As described above, the culture support device 14 can determine the timing of operations to be performed on the culture medium containing the scaffold material and cells. In particular, in suspension culture, which is the culture of adherent cells using scaffold material, cells may adhere to multiple scaffold materials or to cells adhered to other scaffold materials via the extracellular matrix, etc., thus forming aggregates of scaffold material. As a result, images of overlapping scaffold materials may be included in images of the scaffold material in the culture medium. When scaffold materials overlap, the entire image of the scaffold material closest to the camera is captured, but only a portion of the image of the scaffold material farther away from the camera is captured. In the estimation of the cell density on the scaffold material by the past cell density estimation unit 142, if the scaffold material included in the image does not resemble a predetermined shape, it is excluded from the calculation of the area occupied by the scaffold material and the area of ​​the cell area within the area occupied by the scaffold material. This allows for the removal of images of scaffold materials farther away from the camera from which only a portion of the image has been captured. This allows for the calculation of cell density on scaffolding materials using only images of the entire scaffolding material, resulting in a more accurate calculation of cell density on the scaffolding material.

[0032] Furthermore, because cells are highly light-transmitting, it is difficult to detect the contours of cells on scaffolding material when the image is out of focus. In the estimation of cell density on scaffolding material by the past cell density estimation unit 142, if the scaffolding material included in the image is out of focus, it is excluded from the calculation of the area occupied by the scaffolding material and the area of ​​cells within the area occupied by the scaffolding material. This allows the cell density on scaffolding material to be calculated only from scaffolding material where the contours are clearly visible, enabling a more accurate calculation of cell density on scaffolding material.

[0033] <Other Embodiments> Although one embodiment of this invention has been described in detail above with reference to the drawings, the specific configuration is not limited to that described above, and various design changes can be made without departing from the spirit of this invention.

[0034] Some or all of the culture support device 14 in the above-described embodiment may be implemented using a computer. In that case, the program for implementing this function may be recorded on a computer-readable recording medium, and the program recorded on this recording medium may be loaded into a computer system and executed. Here, "computer system" includes the OS and peripheral hardware. Furthermore, "computer-readable recording medium" refers to portable media such as flexible disks, magneto-optical disks, ROMs, CD-ROMs, and recording devices such as hard disks built into a computer system. In addition, "computer-readable recording medium" may include those that dynamically hold programs for a short period of time, such as communication lines used when transmitting programs via networks such as the Internet or communication lines such as telephone lines, and those that hold programs for a certain period of time, such as volatile memory inside a computer system that acts as a server or client in that case. Furthermore, the above-mentioned program may be for implementing some of the functions described above, and may also be able to implement the above-mentioned functions in combination with programs already recorded in the computer system. Furthermore, some or all of the culture support device 14 may be implemented using programmable logic devices such as FPGAs (Field Programmable Gate Arrays). [Explanation of Symbols]

[0035] 1 Culture system, 10 Culture vessels, 100 Culture medium, 12 Imaging device, 14 Culture support device, 140 Image acquisition unit, 142 Past cell density estimation unit, 144 Future cell density estimation unit, 146 Operation timing determination unit, 148 Output unit, 16 Display device, 190 Storage unit

Claims

1. An image acquisition unit that acquires images of the culture medium containing the scaffold material and cells at multiple timings, A past cell density estimation unit estimates the density of the cells on the scaffold material based on the image of the culture medium, A future cell density estimation unit estimates the future cell density on the scaffold material based on the aforementioned multiple timings and the estimated cell density on the scaffold material in the culture medium. An operation timing determination unit that determines the timing of performing an operation on the culture medium based on the estimated future cell density on the scaffold material, A culture support device equipped with the following features.

2. The aforementioned past cell density estimation unit estimates the area of ​​the cell region within the region occupied by the scaffold material relative to the area of ​​the region occupied by the scaffold material included in the image of the culture medium as the density of the cells on the scaffold material. The culture support device according to claim 1.

3. The aforementioned past cell density estimation unit excludes scaffolding materials that do not have a predetermined shape from the scaffolding materials to be calculated. The culture support device according to claim 2.

4. The aforementioned past cell density estimation unit excludes scaffolding materials that are out of focus from the scaffolding materials to be calculated. The culture support device according to claim 2 or 3.

5. The future cell density estimation unit estimates the future cell density on the scaffold material by calculating an approximate curve showing the relationship between the cell density on the scaffold material and time, based on the multiple timings and the estimated cell density on the scaffold material in the culture medium. A culture support device according to any one of claims 1 to 3.

6. The timing for performing the operation on the culture medium as determined above is displayed by the display device. A culture support device according to any one of claims 1 to 3.

7. A culture support device according to any one of claims 1 to 3, An imaging device for imaging the culture medium, A display device that displays an image indicating the timing of operations to be performed on the culture medium as determined by the culture support device, A culture system equipped with the following features.

8. Image acquisition step to acquire images of the culture medium containing the scaffold material and cells at multiple timings, A past cell density estimation step in which the density of the cells on the scaffold material is estimated based on the image of the culture medium, A future cell density estimation step in which the future cell density on the scaffold material is estimated based on the aforementioned multiple timings and the estimated cell density on the scaffold material in the culture medium, An operation timing determination step, which determines the timing of performing an operation on the culture medium based on the estimated future cell density on the scaffold material, A culture support method having the following characteristics.

9. The computer is made to perform the culture support method described in claim 8. program.

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