Method for producing arthropathy treatment agent and arthropathy treatment agent

Abstract

A method for producing a therapeutic agent for arthropathy, said method comprising imaging a population containing a plurality of mesenchymal stem cells under culture to acquire a cell image, analyzing the cell image to derive the density information of the mesenchymal stem cells in the cell image, detecting colonies formed by the mesenchymal stem cells in the population on the basis of the density information, and performing a population sorting depending on the average colony size or colony formation unit derived from the detected colonies; and a therapeutic agent for arthropathy produced by the aforesaid method for producing a therapeutic agent for arthropathy.

Description

[Technical field]

[0001] The present disclosure relates to a method for producing a therapeutic agent for arthropathy, and the therapeutic agent for arthropathy. [Background technology]

[0002] In the field of orthopedic surgery, arthropathy such as articular cartilage injury and meniscus injury is frequently observed in routine medical practice and is widely recognized as a disease affecting a large number of patients. One example of a surgical treatment for cartilage or meniscal injuries is a surgical procedure to remove debris from the injury. Although the above-mentioned treatment inhibited the progression of the disease and promoted the self-regeneration of tissues, the self-regeneration of tissues was not sufficient.

[0003] Meanwhile, cell therapy has become popular due to recent advances in regenerative medicine technology. In particular, mesenchymal stem cells (MSCs) are expected to be a useful cell source for cell therapy. Mesenchymal stem cells can be collected from various biological tissues, and it has been reported that they can be isolated from bone marrow tissue (Prockop, DJ, 1997, Science. 276:71-4), adipose tissue (Zuk, PA et al., 2002, Mol Biol Cell. 13:4279-95), muscle tissue (Cao et al., 2003, Nat Cell Biol. 5:640-6), synovial tissue (De Bari, C. et al., 2001, Arthritis Rheum. 44:1928-42), and periosteal tissue (Fukumoto, T. et al., 2003, OsteoarthritisCartilage. 11:55-64). In addition, it has been reported that synovial-derived mesenchymal stem cells have superior proliferation and chondrogenic capabilities compared to mesenchymal stem cells derived from various mesenchymal tissues such as bone marrow (Sakaguchi, et al., 2005, Arthritis Rhum. 52:2521-9).

[0004] Mesenchymal stem cells are cultured to grow to a desired number and then transplanted into the injured area as a treatment for arthritis. It is important to know whether the mesenchymal stem cells will reach the desired number by the day of transplantation, and it is highly desirable to obtain this information midway through the culture period.

[0005] In response to the above demands, for example, JP 2019-4794 A proposes a proliferation prediction method including a calculation step of calculating an index for predicting the future proliferation state of mesenchymal stem cells based on the culture state of mesenchymal stem cells at a first time point, and a prediction step of predicting the proliferation state of mesenchymal stem cells at a second time point after the first time point based on the index and a discriminant. Summary of the Invention [Problem to be solved by the invention]

[0006] Colonies formed by mesenchymal stem cells have unclear boundaries, unlike colonies of other cells such as epithelial-like cells and lymphoblastoid cells. In JP 2019-4794 A, the number of cells present at a certain distance from a certain cell is counted, and when four or more cells are confirmed, it is determined to be a colony. The present inventors have discovered a new finding that in the colony determination method employed in the proliferation prediction method of JP 2019-4794 A, when proliferated mesenchymal stem cells are present in a densely packed state, these cells are determined to belong to a single colony, and the number of cells belonging to a single colony may become too large, making accurate proliferation prediction difficult.

[0007] The present disclosure has been made in consideration of the above circumstances, and the problem to be solved is to provide a method for producing an arthropathy therapeutic agent, which enables prediction of mesenchymal stem cell proliferation during culture with excellent accuracy and enables the production of an arthropathy therapeutic agent suitable for treating arthropathy, and a therapeutic agent for arthropathy produced by the above-mentioned method for producing an arthropathy therapeutic agent. [Means for solving the problem]

[0008] <1> A cell image is obtained by photographing a group containing multiple cultured mesenchymal stem cells. By analyzing the cell image, density information of mesenchymal stem cells in the cell image is derived, Detecting colonies formed by mesenchymal stem cells in the population based on the density information; sorting the population based on at least one of the average colony size and the colony forming units derived from the detected colonies; A method for producing a therapeutic agent for arthropathy. <2> The density information is an estimated density distribution of mesenchymal stem cells in the cell image derived by kernel density estimation; <1> A method for producing the therapeutic agent for arthropathy described above. <3> Colonies are detected by performing a mean-shift with reference to the estimated density distribution and detecting, as colonies, collections of mesenchymal stem cells that converge to the same maximum point in the estimated density distribution. <2> A method for producing the therapeutic agent for arthropathy described above. <4> Colony detection is performed on the 4th to 6th day from the start of culture, The selection of the population is performed by selecting a population that satisfies at least one of the following conditions a1) and b1): <1> ~ <3> 13. A method for producing the therapeutic agent for arthropathy according to claim 12. a1) The average colony size of mesenchymal stem cells contained in the population at any time point between 4 and 6 days after the start of culture is 0.740 mm or more. b1) The colony-forming units of mesenchymal stem cells contained in the population at any time point between 4 and 6 days after the start of culture are 0.250 / mm 2 That's all. <5> Mesenchymal stem cells are obtained by treating biological tissue with an enzyme. <1> ~ <4> 13. A method for producing the therapeutic agent for arthropathy according to claim 12. <6> In the enzymatic treatment of biological tissue, an enzyme mixture is used which contains at least one collagenase and one neutral protease. <5> A method for producing the therapeutic agent for arthropathy described above. <7> In the enzymatic treatment of the biological tissue, the ratio of the amount of the biological tissue to the amount of the enzyme is 10 to 1000 by mass. <5> or <6> A method for producing the therapeutic agent for arthropathy described above. <8> The mesenchymal stem cells are obtained by washing a tissue digestion fluid obtained by treating a biological tissue with an enzyme until the residual enzyme concentration in the supernatant is 0.5 ng / mL or less. <5> ~ <7> 13. A method for producing the therapeutic agent for arthropathy according to claim 12. <9> The mesenchymal stem cells are cells derived from the synovium. <1> ~ <8> 13. A method for producing the therapeutic agent for arthropathy according to claim 12. <10> The mesenchymal stem cells are cells of human origin. <1> ~ <9> 13. A method for producing the therapeutic agent for arthropathy according to claim 12. <11> <1> ~ <10> 1. A therapeutic agent for arthropathy, produced by the method for producing a therapeutic agent for arthropathy described in any one of the above items. Effect of the Invention

[0009] According to the present disclosure, it is possible to provide a method for producing an arthropathy therapeutic agent that can predict the proliferation of mesenchymal stem cells with excellent accuracy during culture and can produce an arthropathy therapeutic agent suitable for treating arthropathy, and a therapeutic agent for arthropathy produced by the above-mentioned method for producing an arthropathy therapeutic agent. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram showing a cell image analyzer and the like. [Diagram 2] FIG. 2 is a block diagram showing a computer that constitutes the cell image analyzer. [Diagram 3] FIG. 3 is a block diagram showing a processing unit of a CPU of the cell-image analyzing apparatus. [Figure 4] FIG. 4 is a diagram illustrating the density information derivation unit in detail. [Diagram 5] FIG. 5 is a graph showing an outline of kernel density estimation. [Figure 6] FIG. 6 is a graph showing an outline of mean-shift. [Figure 7]FIG. 7 is a graph showing an outline of mean-shift. [Figure 8] FIG. 8 is a diagram showing the details of the detection result. [Figure 9] FIG. 9 is a diagram showing details of the evaluation information. [Figure 10] FIG. 10 shows colony sizes. [Figure 11] FIG. 11 is a diagram showing a cell image display screen. [Figure 12] FIG. 12 is a diagram showing the analysis result display screen. [Figure 13] FIG. 13 is a diagram showing how three types of estimated density distributions are derived by performing kernel density estimation for a specific region using three types of bandwidth. [Figure 14] FIG. 14 is a diagram showing how detection results of three types of colonies corresponding to the three estimated density distributions are output by performing mean-shift on a specific region with reference to each of the three estimated density distributions. [Figure 15] FIG. 15 is a diagram showing the designation screen. [Figure 16] FIG. 16 is a diagram showing how a bandwidth specification is received by the instruction receiving unit. [Figure 17] FIG. 17 is a flowchart showing the processing procedure of the cell-image analyzer. [Figure 18] FIG. 18 is a flowchart showing the processing procedure of the cell-image analyzer. [Figure 19] FIG. 19 is a diagram showing another example of the evaluation information. [Figure 20] FIG. 20 is a diagram showing still another example of the evaluation information. [Figure 21] FIG. 21 shows a mode in which a warning is displayed when the amount of change in the average colony size per culture day is less than a threshold value. [Figure 22] FIG. 22 is a flowchart showing another example of a method for specifying a specified bandwidth. [Figure 23]FIG. 23 is a diagram showing a second embodiment in which the derivation of density information and the detection of colonies are performed only when the area ratio of fibroblast-like cells in a cell image is within a set range. [Figure 24] FIG. 24 is a diagram showing another example of the second embodiment. [Diagram 25] FIG. 25 is a diagram showing yet another example of the second embodiment. [Figure 26] FIG. 26 is a graph summarizing the aspects shown in FIGS. 23 to 25, with the vertical axis representing the area ratio and the horizontal axis representing the number of days of culture. [Figure 27] FIG. 27 is a diagram showing a cell image display screen when the area ratio is outside the set range. [Figure 28] FIG. 28 is a diagram showing a third embodiment in which the number of fibroblast-like cells within a preset search frame is derived as density information. [Figure 29] FIG. 29 is a diagram showing how a search frame is sequentially scanned over a plurality of regions within a cell image. [Diagram 30] FIG. 30 shows a case where the number of fibroblast-like cells in the search frame satisfies the colony condition. [Diagram 31] FIG. 31 is a diagram showing a fourth embodiment in which image analysis is performed using a machine learning model. [Diagram 32] FIG. 32 is a diagram illustrating an overview of the processing in the learning phase of a machine learning model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, the embodiments for carrying out the present disclosure will be described in detail. However, the present disclosure is not limited to the following embodiments. In the following embodiments, the components are not essential unless specifically stated otherwise. The same applies to the numerical values ​​and their ranges, and they do not limit the present disclosure.

[0012] In the present disclosure, a "mesenchymal stem cell" is a somatic stem cell derived from mesodermal tissue (mesenchyme). Mesenchymal stem cells are known to exist in bone marrow, synovium, periosteum, adipose tissue, and muscle tissue, and are known to have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, and muscle cells. In relation to the differentiation of mesenchymal stem cells into chondrocytes, it is known that the addition of BMP or TGF-β to the culture medium promotes the differentiation of undifferentiated mesenchymal stem cells into chondrocytes, and cartilage tissue can be regenerated under in vitro conditions.

[0013] Mesenchymal stem cells can be identified by detecting molecules characteristic of mesenchymal stem cells (enzymes, receptors, low molecular weight compounds, etc.). Molecules characteristic of mesenchymal stem cells include cell surface markers (positive markers), such as CD73, CD90, CD105, and CD166, but are not limited to these. Furthermore, examples of cell surface markers (negative markers) that are not expressed in mesenchymal stem cells include, but are not limited to, CD (Clusters of Differentiation) 19, CD34, CD45, HLA-DR (Human Leukocyte Antigen-D-Related), CD11b, and CD14. Using the above positive and negative markers, it is possible to confirm that the cells are mesenchymal stem cells. Immunological methods are available for the detection of these markers, but detection may also be performed by quantitating the amount of mRNA for each molecule.

[0014] In this disclosure, a "population" refers to a system in which the culture of mesenchymal stem cells is carried out, within the population, the mesenchymal stem cells form colonies.

[0015] In the present disclosure, "average colony size" refers to the average size of colonies formed by mesenchymal stem cells. When the colony size is below a certain level, there is a high possibility that isolated cells or debris in the medium will be detected. Therefore, in the present disclosure, the average colony size is measured for colonies with a colony size of more than 0.54 mm. In this disclosure, a "colony forming unit" is also called a CFU (Colony Forming Unit) and is a unit that is measured within 1 mm of a cell image. 2 The number of colonies per one sample is referred to as the number of colonies per one sample. The cell images will be described later.

[0016] In the present disclosure, "arthropathy" is a disease involving injury, damage or inflammation of a joint. More specifically, examples of arthropathy include, but are not limited to, meniscus injury, traumatic cartilage injury, osteochondritis dissecans, avascular necrosis, osteoarthritis (e.g., knee osteoarthritis, elbow osteoarthritis, shoulder osteoarthritis, etc.), rheumatoid arthritis (e.g., rheumatoid arthritis), gout, reactive arthritis, psoriatic arthritis, juvenile arthritis, inflammatory arthritis, and articular cartilage defect.

[0017] In the present disclosure, a numerical range indicated using "~" includes the numerical values ​​before and after "~" as the minimum and maximum values, respectively. In the numerical ranges described in the present disclosure in stages, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. In addition, in the numerical ranges described in the present disclosure, the upper or lower limit value of the numerical range may be replaced with a value shown in the examples.

[0018] (Method of manufacturing arthropathy treatment agent) The method for producing a therapeutic agent for arthropathy according to the present disclosure includes the steps of: A cell image is obtained by photographing a group containing multiple cultured mesenchymal stem cells. By analyzing the cell image, density information of mesenchymal stem cells in the cell image is derived, Detecting colonies of mesenchymal stem cells in the population based on the density information; The population is selected based on at least one of the average colony size and the colony forming units derived from the detected colonies.

[0019] According to the method for producing an arthropathy treatment agent disclosed herein, it is possible to provide a method for producing an arthropathy treatment agent that can predict the proliferation of mesenchymal stem cells with excellent accuracy during culture and can produce an arthropathy treatment agent suitable for treating arthropathy.

[0020] The reason why the above effect is achieved is presumed to be as follows, but is not limited to this. As described above, in the conventional mesenchymal stem cell proliferation prediction proposed in JP 2019-4794 A and the like, due to the unclear boundaries of the colonies formed by mesenchymal stem cells, it was sometimes difficult to detect colonies with high accuracy, and there was room for improvement in the accuracy of proliferation prediction. In the manufacturing method of an arthropathy treatment agent disclosed herein, density information of mesenchymal stem cells is derived and mesenchymal stem cell colonies are detected based on the density information, making it possible to detect mesenchymal stem cell colonies with excellent accuracy. It is speculated that by deriving the average colony size and colony forming units from the information on colonies detected with high accuracy, and using this as a basis to select populations of mesenchymal stem cells with excellent levels of proliferation and proliferation rates, etc., it is possible to produce an arthropathy therapeutic agent suitable for treating arthropathy.

[0021] (Mesenchymal stem cell culture) For culturing mesenchymal stem cells, a medium generally used for culturing animal cells can be used. Examples of culture media include, but are not limited to, α-MEM, Dulbecco Modified Eagle Medium (DMEM), a mixed medium of DMEM and F12 (DMEM:F12=1:1), RPMI (Roswell Park Memorial Institute) medium (GIBCO (registered trademark) RPMI1640 medium, etc.), a mixed medium of DMEM and F12 and RPMI medium (mixed medium of DMEM and F12:RPMI medium=1:1), and the like. The ratios in parentheses are based on mass.

[0022] The medium may be a serum-containing medium or a serum-free medium. When mesenchymal stem cells are produced from autologous tissue for transplantation into a living body, the medium may contain allogeneic serum. That is, when producing mesenchymal stem cells from human tissue for transplantation into humans, a medium containing human serum may be used. When serum is used, it may be autologous serum or allogeneic serum, but is preferably autologous serum. When serum is used, the amount of serum added to the medium is preferably 20% by volume or less, more preferably 10% by volume or less.

[0023] The cell culture conditions are not particularly limited, and ordinary cell culture conditions can be adopted. For example, the temperature for cell culture can be 30°C to 40°C. In addition, CO in cell culture 2 The concentration can be between 3% and 7%. For example, a temperature of 37°C and CO 2 The concentration may be 5%, but is not limited to this.

[0024] It is preferable not to change the medium during the culture period. Furthermore, when the culture period is 10 days or longer, it is preferable to culture the mesenchymal stem cells without co-culture with cells other than mesenchymal stem cells.

[0025] The differentiation of mesenchymal stem cells into chondrocytes progresses more as the culture period becomes longer. Therefore, it is known that the in situ chondrogenic ability of mesenchymal stem cells decreases when the culture period exceeds a certain length. In order to proliferate mesenchymal stem cells in an undifferentiated state and with good in situ chondrogenic potential, it is preferable to adjust the culture period. Furthermore, in order for the transplanted mesenchymal stem cells to regenerate the affected area, the population must contain a certain amount of undifferentiated mesenchymal stem cells. Considering these, the culture period is preferably 5 days or more, more preferably 7 days or more, and even more preferably 10 days or more. In addition, the culture period is preferably 28 days or less, more preferably 21 days or less, and even more preferably 17 days or less.

[0026] It is known that mesenchymal stem cells can be differentiated into chondrocytes and cartilage tissue can be produced in vitro by culturing them in a medium containing transforming growth factor β3 (TGF-β3), dexamethasone, or bone morphogenetic protein 2 (BMP-2). Therefore, from the viewpoint of inhibiting differentiation of mesenchymal stem cells into chondrocytes, it is preferable that the above-mentioned medium does not contain TGF-β3, dexamethasone, and BMP-2.

[0027] It is also known that the in situ chondrogenic ability of mesenchymal stem cells decreases inversely proportional to the number of in vitro passages of the mesenchymal stem cells. Therefore, the mesenchymal stem cells produced are preferably primary or first passage mesenchymal stem cells.

[0028] When the mesenchymal stem cells after enzyme treatment were seeded in the medium, the cell density was 100 cells / cm 2 ~5000 pieces / cm 2 It is preferable to sow the seeds so that the number of seeds is 200 / cm. 2 ~4500 pieces / cm 2 It is more preferable to sow the seeds so that the density is 300 cells / cm. 2 ~2500 pieces / cm 2 It is more preferable to sow the seeds so that the number of seeds is 400 / cm. 2 ~2000 pieces / cm 2 It is particularly preferable to sow the seeds so that

[0029] From the viewpoint of suitability as a therapeutic agent for osteoarthritis, the number of cells to be collected at the end of the culture should be 0.5 × 10 7 1.0×10 is preferred 7 More preferably, 2.0×10 7More than one is even more preferable. The number of cells harvested at the end of the culture was 3.0 × 10 7 May be 4.0 x 10 cells or more 7 May be 5.0 x 10 cells or more 7 May be more than 6.0 x 10 cells 7 It may be a cell or more.

[0030] Hereinafter, mesenchymal stem cells used in the method for producing a therapeutic agent for arthropathy according to the present disclosure will be described, but the present invention is not limited thereto. The organism from which the mesenchymal stem cells are derived is not particularly limited, but is preferably a cell derived from a mammal, more preferably a cell derived from a primate, and particularly preferably a cell derived from a human.

[0031] Mesenchymal stem cells can be obtained by enzymatically treating biological tissue. Examples of biological tissues include synovial tissue, bone marrow tissue, periosteal tissue, adipose tissue, and muscle tissue. Among these, mesenchymal stem cells obtained by enzymatically treating synovial tissue (synovial-derived mesenchymal stem cells) are preferred, from the viewpoint that the produced therapeutic agent for arthropathy is particularly useful for arthropathy such as a therapeutic agent for meniscus and osteoarthritis. For example, synovial tissue can be harvested from a non-weight-bearing portion of a joint under anesthesia.

[0032] Whether or not the cells are synovial-derived mesenchymal stem cells can be determined, for example, by confirming the presence or absence of cell surface marker expression and the ability to differentiate into cartilage. Synovium-derived mesenchymal stem cells are CD90 positive, CD45 negative, and have the ability to differentiate into cartilage.

[0033] The biological tissue may be biological tissue derived from a single donor or biological tissue derived from multiple donors, but is preferably biological tissue derived from a single donor.

[0034] When synovium-derived mesenchymal stem cells are produced for transplantation into humans, it is preferable to use biological tissue collected from a donor whose histocompatibility antigen type matches or is similar to that of the patient. More preferably, the subject from which the synovium is harvested and the subject to which the synovium-derived mesenchymal stem cells are transplanted are the same subject. That is, it is preferable to use synovial tissue harvested from the patient himself (autologous transplantation).

[0035] The amount of biological tissue to be collected is preferably changed appropriately taking into consideration the type of donor and the amount of mesenchymal stem cells required. For example, the amount collected is preferably 0.10 g to 10.00 g, more preferably 0.20 g to 5.00 g, and even more preferably 0.30 g to 4.50 g. The collected biological tissue is cut into small pieces using scissors or the like as necessary, and then subjected to the enzyme treatment described below.

[0036] The enzyme is not particularly limited as long as it contains a protease, but from the viewpoint of the digestion rate in biological tissue, it is preferably an enzyme mixture containing at least one type each of collagenase and neutral protease. A particularly preferred enzyme is Liberase. For example, Liberase MNP-S (F. Hoffmann-La Roche) can be used as the Liberase, which is an enzyme containing collagenase class I, collagenase class II, and neutral protease (thermocillin).

[0037] The enzyme reaction can be carried out in an aqueous solution containing the enzyme, and even in an aqueous solution containing human serum. The human serum may be autologous serum or allogeneic serum, but is preferably Preferably it is autologous serum. The enzyme concentration in the aqueous solution is preferably 0.01 mg / ml to 10 mg / ml, more preferably 0.1 mg / ml to 10 mg / ml, and even more preferably 0.5 mg / ml to 10 mg / ml. g / ml to 10 mg / ml, and even more preferably 0.5 mg / ml to 5.0 mg / ml, particularly preferably 0.5 mg / ml to 2.0 mg / ml, and most preferably The range is usually 0.7mg / ml to 2.0mg / ml.

[0038] In treating biological tissue with an enzyme, the ratio of the amount of biological tissue used to the amount of enzyme used (amount of biological tissue used / amount of enzyme used) is preferably 10 to 1,000, more preferably 20 to 500, and even more preferably 40 to 200, on a mass basis.

[0039] The enzyme reaction temperature is preferably 15°C to 45°C, more preferably 20°C to 43°C, and even more preferably 25°C to 40°C. The enzyme reaction time is preferably 45 to 180 minutes, more preferably 60 to 180 minutes, further preferably 90 to 180 minutes, and particularly preferably 120 to 180 minutes. The mixture obtained by enzymatically treating biological tissue (hereinafter also referred to as tissue digestion fluid) contains mesenchymal stem cells, which can be recovered by transferring the mixture to a centrifuge tube through a cell strainer and centrifuging. The centrifugation conditions are not particularly limited, but may be, for example, a centrifugal force of 200 g to 600 g and a centrifugation time of 1 minute to 10 minutes.

[0040] The mixture obtained by the enzyme treatment may be washed one or more times, preferably multiple times, and more preferably two times. The washing can be carried out by suspending the mesenchymal stem cells recovered by the above-mentioned centrifugation in a medium and centrifuging again. The centrifugation conditions are not particularly limited, but may be, for example, a centrifugal force of 200 g to 600 g and a centrifugation time of 1 minute to 10 minutes.

[0041] The medium is not particularly limited, and for example, α-MEM (Minimum Essential Medium Eagle Alpha Modification) can be used.

[0042] The mixture is preferably washed until the residual enzyme concentration in the supernatant is 0.5 ng / mL or less, more preferably 0.3 ng / mL or less, even more preferably 0.2 ng / mL or less, and particularly preferably 0.1 ng / mL or less.

[0043] (Acquisition of cell images, extraction of density information and colony detection) In the method for producing an arthropathy treatment agent disclosed herein, cell images of multiple mesenchymal stem cells being cultured are obtained, and density information of the mesenchymal stem cells in the cell images is derived by analyzing the cell images. Based on the derived density information, colonies of mesenchymal stem cells in the population are detected, and the population is selected based on at least one of the average colony size and colony forming units derived from the detected colonies.

[0044] The method for acquiring the cell images is not particularly limited, and the images can be acquired using a conventionally known imaging device. The derived density information is preferably an estimated density distribution of mesenchymal stem cells in the cell image, derived by kernel density estimation. Furthermore, colonies are preferably detected by performing a mean-shift with reference to the estimated density distribution and detecting, as colonies, collections of mesenchymal stem cells that converge to the same maximum point in the estimated density distribution.

[0045] The acquisition of cell images, the derivation of density information and the detection of colonies can be performed using a cell image analysis device comprising at least one processor.

[0046] Hereinafter, one embodiment (also referred to as a first embodiment) of a cell-image analyzer will be described with reference to FIGS. 1 to 22. FIG. According to the method for producing a therapeutic agent for arthropathy using the following cell image analyzer, it is not necessary to use time-lapse images. Therefore, it is not necessary to prepare a dedicated device, and costs can be reduced. In addition, since it is not necessary to use time-lapse images, it is possible to shorten the time required for image acquisition. Furthermore, since it is not necessary to use time-lapse images, it is possible to reduce the analysis load and the cost of data storage.

[0047] 1, the cell-image analysis device 10 is, for example, a desktop personal computer, and is connected to an imaging device 11. The imaging device 11 is, for example, a phase-contrast microscope or a bright-field microscope, and captures images of mesenchymal stem cells 13 being cultured in a culture vessel 12. The imaging device 11 then transmits the cell image 14 thus obtained to the cell-image analysis device 10.

[0048] A population containing an appropriate number of mesenchymal stem cells is seeded in a medium. The mesenchymal stem cells in the population then grow over time to form colonies. A cell image analyzer 10 detects the colonies from a cell image 14. The method for culturing mesenchymal stem cells is described below.

[0049] 2, the computer constituting the cell-image analyzer 10 includes a storage device 30, a memory 31, a CPU (Central Processing Unit) 32, a communication unit 33, a display 34, and an input device 35. These are interconnected via a bus line 36.

[0050] The storage device 30 is a hard disk drive built into the computer constituting the cell image analyzer 10 or connected via a cable or network. Alternatively, the storage device 30 is a disk array in which a plurality of hard disk drives are connected in series. The storage device 30 stores control programs such as an operating system, various application programs, and various data associated with these programs. Note that a solid state drive may be used instead of the hard disk drive.

[0051] The memory 31 is a work memory for executing processes by the CPU 32. The CPU 32 loads programs stored in the storage device 30 into the memory 31 and executes processes according to the programs, thereby providing overall control over each part of the computer.

[0052] The communication unit 33 is a network interface that controls the transmission of various information via a network such as a LAN (Local Area Network). The display 34 displays various screens. The computer constituting the cell-image analyzer 10 accepts input of operation instructions from the input device 35 via the various screens. The input device 35 is a keyboard, a mouse, a touch panel, or the like.

[0053] 3, an operating program 40 is stored in the storage device 30 of the cell-image analyzer 10. The operating program 40 is an application program for causing a computer to function as the cell-image analyzer 10. The storage device 30 also stores the cell image 14 , the specified bandwidth 41 , the colony detection results 42 , and evaluation information 43 indicating the proliferation ability of the mesenchymal stem cells 13 .

[0054] When the operating program 40 is started, the CPU 32 of the computer constituting the cell-image analyzer 10 cooperates with the memory 31 and the like to function as a read / write (hereinafter abbreviated as RW (Read Write)) control unit 50, an instruction receiving unit 51, a density information derivation unit 52, a detection unit 53, an evaluation information derivation unit 54, and a display control unit 55. The CPU 32 is an example of a "processor."

[0055] The RW control unit 50 controls the storage of various data in the storage device 30 and the reading of various data in the storage device 30. For example, the RW control unit 50 receives a cell image 14 from the imaging device 11 and stores it in the storage device 30. The RW control unit 50 also reads the cell image 14 from the storage device 30 and outputs it to the density information derivation unit 52. In other words, the RW control unit 50 is an example of an "acquisition unit."

[0056] The instruction receiving unit 51 receives a user's specification of a bandwidth BW for kernel density estimation performed by the density information derivation unit 52 via the input device 35. The instruction receiving unit 51 outputs the received bandwidth BW to the RW control unit 50 as a specified bandwidth 41. The RW control unit 50 stores the specified bandwidth 41 in the storage device 30. The RW control unit 50 also reads out the specified bandwidth 41 from the storage device 30, and outputs it to the density information derivation unit 52 together with the cell image 14.

[0057] The designated bandwidth 41 is designated for each culture project of the mesenchymal stem cells 13 generated from cells collected from one patient. Therefore, in one culture project, the designated bandwidth 41 once designated is used repeatedly throughout the culture project.

[0058] The density information derivation unit 52 performs image analysis on the cell image 14 to derive density information of the mesenchymal stem cells 13 in the cell image 14 . More specifically, the density information derivation unit 52 performs kernel density estimation in the specified bandwidth 41 to derive an estimated density distribution 60 of the mesenchymal stem cells 13 in the cell image 14 . The density information derivation unit 52 outputs the derived estimated density distribution 60 as density information to the detection unit 53. The density information derivation unit 52 is an example of a "derivation unit".

[0059] The detection unit 53 detects colonies of mesenchymal stem cells 13 based on the estimated density distribution 60. The detection unit 53 outputs colony detection results 42 to the RW control unit 50 and the evaluation information derivation unit 54. The RW control unit 50 stores the detection results 42 in the storage device 30. The detection results 42 are stored in association with the cell image 14.

[0060] The evaluation information derivation unit derives evaluation information based on the detection result. The evaluation information derivation unit outputs the evaluation information to the RW control unit. The RW control unit stores the evaluation information in a storage device. The evaluation information 43 is stored in association with the cell image 14, similar to the detection result 42.

[0061] The RW control unit 50 reads out a set of the cell image 14, the detection result 42, and the evaluation information 43 that are stored in association with each other from the storage device 30, and outputs the set to the display control unit 55. The display control unit 55 controls the display of various screens on the display 34. The various screens include a cell image display screen 80 (see FIG. 11) that displays the cell image 14, an analysis result display screen 85 (see FIG. 12) that displays the cell image 14, the detection result 42, and the evaluation information 43, and the like.

[0062] 4, the density information derivation unit 52 includes a cell centroid position extraction unit 65 and a kernel density estimation unit 66. The cell centroid position extraction unit 65 performs binarization processing on the cell image 14. In the binarization process, first, a brightness threshold value is set to separate mesenchymal stem cells 13 from other areas in the cell image 14. Then, for each pixel of the cell image 14, if the brightness value is less than the threshold value, it is determined that the pixel is a mesenchymal stem cell 13 and the pixel value is set to 0, and if the brightness value is equal to or greater than the threshold value, it is determined that the pixel is a region other than the mesenchymal stem cell 13 and the pixel value is set to 1.

[0063] After the binarization process, the cell center of gravity position extraction unit 65 performs a well-known image recognition process to recognize each individual mesenchymal stem cell 13 for the region in which the pixel values ​​have been replaced with 0 as a mesenchymal stem cell 13. Then, the cell center of gravity position extraction unit 65 extracts the center of gravity 68 (indicated by a cross mark) of each individual mesenchymal stem cell 13 thus recognized. The cell center of gravity position extraction unit 65 outputs the extraction result of the center of gravity 68 to the kernel density estimation unit 66. Specifically, the extraction result of the center of gravity 68 is the XY coordinate of the center of gravity 68 when the axis along the long side of the cell image 14 is the X axis, the axis along the short side is the Y axis, and the upper left corner of the cell image 14 is the origin.

[0064] The kernel density estimation unit 66 performs kernel density estimation using the centroids 68 as sample points, and outputs an estimated density distribution 60 as a result. More specifically, as shown in FIG. 5, the kernel density estimation unit 66 uses a Gaussian function indicated by a dashed line as a kernel function. The Gaussian function has a specified bandwidth 41. The kernel density estimation unit 66 assigns a Gaussian function to each of the multiple centroids 68, and calculates the estimated density distribution 60 by adding (convolving) the assigned multiple Gaussian functions. The estimated density distribution 60 also includes information on the centroids 68. Note that, instead of the Gaussian function, a rectangular function or a triangular function may be used as the kernel function.

[0065] As shown in FIGS. 6 and 7, the detection unit 53 performs a mean-shift with reference to the estimated density distribution. The mean-shift uses a search circle 70 of radius R centered on each center of gravity 68. In the mean-shift, starting from a certain center of gravity 68, the search circle 70 is sequentially moved in the direction of higher density of mesenchymal stem cells 13 using the estimated density distribution 60 as a clue, and finally, a local maximum point 71 of the density of mesenchymal stem cells 13 to which the center of gravity 68 converges is searched for. The detection unit 53 performs a mean-shift on each center of gravity 68. The detection unit 53 detects, as a colony, a collection of mesenchymal stem cells 13 whose centers of gravity 68 converge to the same maximum point 71 in the estimated density distribution 60. 6 and 7 show examples in which a collection of mesenchymal stem cells 13 whose center of gravity 68 converges to a maximum point 71_1 and a collection of mesenchymal stem cells 13 whose center of gravity 68 converges to a maximum point 71_2 are detected as colonies.

[0066] As shown in Fig. 8, the detection unit 53 assigns a number such as No. 1 or No. 2 to each detected colony. The detection unit 53 also generates a rectangular frame 75 that circumscribes the center of gravity 68 of the mesenchymal stem cell 13 for each detected colony. Then, together with the number, the detection unit 53 outputs the XY coordinates of the diagonal points PA and PB at the upper left and lower right corners of the rectangular frame 75 as the detection result 42. Fig. 8 illustrates an example of the detection result 42 including the diagonal points PA_1 and PB_1 of the rectangular frame 75_1 of colony No. 1, and the diagonal points PA_2 and PB_2 of the rectangular frame 75_2 of colony No. 2.

[0067] As shown in FIG. 9, the evaluation information derivation section 54 derives the number of colonies, colony forming units (CFUs), and average colony size detected by the detection section 53 as the evaluation information 43. Thus, the evaluation information 43 is information regarding at least one of the number of colonies, the CFU, and the average colony size at one time point.

[0068] Furthermore, the dilution ratio of the culture medium and the amount of the seeded mesenchymal stem cells 13 are input by the user via the input device 35 .

[0069] 10, the colony size is the sum of the width WX in the X-axis direction and the width WY in the Y-axis direction of the rectangular frame 75, and divided by 2. The average colony size is the sum of the colony sizes calculated in this way and divided by the number of colonies.

[0070] 11 shows a cell image display screen 80 displayed on the display 34 under the control of the display control unit 55. On the cell image display screen 80, a cell image 14 is displayed in response to a user request.

[0071] An analysis button 81 is provided at the bottom of the cell image display screen 80. When the user wishes to perform image analysis of the cell image 14, he or she selects the analysis button 81. When the analysis button 81 is selected, an image analysis instruction is received by the instruction receiving unit 51. As a result, the density information derivation unit 52 derives the estimated density distribution 60, the detection unit 53 detects colonies, and the evaluation information derivation unit 54 derives the evaluation information 43.

[0072] 12 shows an analysis result display screen 85 displayed on the display 34 under the control of the display control unit 55. On the analysis result display screen 85, a cell image 14 with a number and a rectangular frame 75 superimposed thereon is displayed for each colony detected by the detection unit 53. That is, the colony detection results 42 are presented to the user. Evaluation information 43 is displayed below the cell image 14. That is, the evaluation information 43 is presented to the user.

[0073] A confirmation button 86 is provided at the bottom of the analysis result display screen 85. When the confirmation button 86 is selected, a display deletion instruction is received by the instruction receiving unit 51. In this case, the display control unit 55 erases the display of the analysis result display screen 85.

[0074] 13 to 16 show a series of processes for specifying the specified bandwidth 41. First, as shown in Fig. 13, the density information derivation unit 52 extracts the center of gravity 68 of the mesenchymal stem cells 13 in a specific region 90 that is a part of the cell image 14, using the cell center of gravity position extraction unit 65. The specific region 90 is an area within a rectangular frame that is half the size of the cell image 14 and whose center coincides with that of the cell image 14.

[0075] The kernel density estimation unit 66 performs kernel density estimation using a Gaussian function of a bandwidth BW_L, kernel density estimation using a Gaussian function of a bandwidth BW_M, and kernel density estimation using a Gaussian function of a bandwidth BW_S, for the specific region 90. That is, the kernel density estimation unit 66 performs kernel density estimation using a plurality of types of bandwidths BW.

[0076] The bandwidths BW_L, BW_M, and BW_S have a magnitude relationship as shown in the following formula (1). BW_L>BW_M>BW_S···(1) That is, the bandwidth BW_L is the largest and the bandwidth BW_S is the smallest. The bandwidth BW_M is between the bandwidths BW_L and BW_S. The number of types of bandwidth BW is not limited to three. There may be two types, or four or more types.

[0077] The kernel density estimation unit 66 derives an estimated density distribution 60_L corresponding to the bandwidth BW_L, an estimated density distribution 60_M corresponding to the bandwidth BW_M, and an estimated density distribution 60_S corresponding to the bandwidth BW_S. That is, the kernel density estimation unit 66 derives a plurality of types of estimated density distributions 60. The estimated density distribution 60_L has the smoothest shape, and the estimated density distribution 60_S has the most pointed shape. The estimated density distribution 60_M has a shape intermediate between the estimated density distributions 60_L and 60_S.

[0078] As shown in FIG. 14, the detection unit 53 performs a mean-shift on a specific region 90 with reference to each of the estimated density distributions 60_L, 60_M, and 60_S, and outputs a colony detection result 42 corresponding to the estimated density distributions 60_L, 60_M, and 60_S. More specifically, the detection unit 53 outputs as the detection result 42 the XY coordinates of the diagonal points PA_L and PB_L of the rectangular frame 75_L (shown by a dotted line) of the colony detected by performing a mean-shift with reference to the estimated density distribution 60_L. In addition, the detection unit 53 outputs as the detection result 42 the XY coordinates of diagonal points PA_M and PB_M of a rectangular frame 75_M (shown by a solid line) of the colony detected by performing a mean-shift with reference to the estimated density distribution 60_M. Furthermore, the detection unit 53 outputs as the detection result 42 the XY coordinates of the diagonal points PA_S and PB_S of the rectangular frame 75_S (shown by the dashed line) of the colony detected by performing a mean-shift with reference to the estimated density distribution 60_S. The rectangular frame 75_L becomes relatively large, and the rectangular frame 75_S becomes relatively small. The rectangular frame 75_M has a size intermediate between those of the rectangular frames 75_L and 75_S. These detection results 42 are output to the display control unit 55.

[0079] The display control unit 55 displays the specified screen 95 shown in FIG. 15 on the display 34. On the specified screen 95, a specific area 90 in which the rectangular frames 75_L, 75_M, and 75_S as the detection results 42 are superimposed is displayed. The rectangular frames 75_L, 75_M, and 75_S are displayed in different colors (for example, the rectangular frame 75_L is blue, the rectangular frame 75_M is yellow, the rectangular frame 75_S is green, etc.).

[0080] Below the specific area 90, frame designation buttons 96L, 96M, and 96S are provided together with a message indicating that the rectangular frame 75 considered appropriate as a colony is to be designated. The frame designation button 96L corresponds to the rectangular frame 75_L, the frame designation button 96M corresponds to the rectangular frame 75_M, and the frame designation button 96S corresponds to the rectangular frame 75_S. The frame designation buttons 96L, 96M, and 96S are mutually exclusive buttons such that if one is selected, the other two cannot be selected. That is, the display control unit 55 presents a plurality of types of detection results 42 to the user and allows the user to select one of the plurality of types of detection results 42. Further below the frame designation buttons 96L, 96M, and 96S, a designation button 97 is provided.

[0081] As shown in FIG. 16, when the designation button 97 is selected while one of the frame designation buttons 96L, 96M, and 96S is selected, the instruction reception unit 51 receives the designation of the bandwidth BW. The instruction reception unit 51 sets the bandwidth BW corresponding to the one frame designation button 96 selected by the user as the specified bandwidth 41. That is, the instruction reception unit 51 sets the bandwidth BW corresponding to the one detection result 42 selected by the user as the specified bandwidth 41. FIG. 16 illustrates the case where the frame designation button 96M is selected by the user and the bandwidth BW_M is set as the specified bandwidth 41.

[0082] Next, the operation of the above configuration will be described with reference to the flowcharts of FIGS. 17 and 18. First, when the operating program 40 is started in the cell image analysis device 10, as shown in FIG. 3, the CPU 32 of the cell image analysis device 10 functions as a RW control unit 50, an instruction receiving unit 51, a density information derivation unit 52, a detection unit 53, an evaluation information derivation unit 54, and a display control unit 55.

[0083] The RW control unit 50 reads out the cell image 14 in response to the user's request from the storage device 30 (step ST100). The cell image 14 is output from the RW control unit 50 to the display control unit 55.

[0084] Under the control of the display control unit 55, the cell image display screen 80 shown in Fig. 11 is displayed on the display 34 (step ST110), thereby presenting the cell image 14 to the user.

[0085] When the user selects the analysis button 81 and an image analysis instruction is received by the instruction receiving unit 51 (YES in step ST120), and the specified bandwidth 41 has already been stored in the storage device 30 (YES in step ST130), the RW control unit 50 reads out the cell image 14 and the specified bandwidth 41 from the storage device 30 (step ST140). The cell image 14 and the specified bandwidth 41 are output from the RW control unit 50 to the density information derivation unit 52. Step ST140 is an example of an "acquisition step".

[0086] 4 and 5, in the density information derivation unit 52, kernel density estimation is performed in the specified bandwidth 41. As a result, an estimated density distribution 60 is derived as density information (step ST150). The estimated density distribution 60 is output from the density information derivation unit 52 to the detection unit 53. Step ST150 is an example of a "deriving step".

[0087] 6 and 7, the detection unit 53 performs a mean-shift with reference to the estimated density distribution 60. This detects colonies of mesenchymal stem cells 13 in the cell image 14 (step ST160). The colony detection results 42 are output from the detection unit 53 to the RW control unit 50 and stored in the storage device 30. The colony detection results 42 are also output from the detection unit 53 to the evaluation information derivation unit 54. Step ST160 is an example of a "detection step".

[0088] 9, in the evaluation information derivation section 54, the number of colonies, the colony forming unit, and the average colony size are derived as evaluation information 43 based on the detection result 42 (step ST170). The evaluation information 43 is output from the evaluation information derivation section 54 to the RW control section 50 and stored in the storage device 30.

[0089] The detection results 42 and the evaluation information 43 are read out from the storage device 30 by the RW control unit 50 and output to the display control unit 55. Then, under the control of the display control unit 55, the analysis result display screen 85 shown in FIG. 12 is displayed on the display 34 (step ST180). This presents the detection results 42 and the evaluation information 43 to the user. When the confirmation button 86 is selected and an instruction to erase the display is received by the instruction receiving unit 51 (YES in step ST190), the display on the analysis result display screen 85 is erased. Then, the process ends.

[0090] If the instruction receiving unit 51 does not receive an image analysis instruction (NO in step ST120) and does not receive an end instruction (NO in step ST200), the display of the cell image display screen 80 continues. If the instruction receiving unit 51 receives an end instruction (YES in step ST200), the display of the cell image display screen 80 is turned off and the process ends.

[0091] If the specified bandwidth 41 is not stored in the storage device 30 (NO in step ST130), the process shown in FIG. 18 is carried out. Specifically, first, as shown in FIG. 13, in the density information derivation unit 52, kernel density estimation is performed for the specific region 90 using three bandwidths BW_L, BW_M and BW_S, thereby deriving three estimated density distributions 60_L, 60_M and 60_S (step ST1301).

[0092] Next, as shown in FIG. 14, in the detection unit 53, a mean-shift is performed on the specific region 90 with reference to each of the three estimated density distributions 60_L, 60_M and 60_S, thereby outputting detection results 42 of three types of colonies corresponding to the three estimated density distributions 60_L, 60_M and 60_S (step ST1302). The detection results 42 of the three types of colonies are specifically the XY coordinates of diagonal points PA_L and PB_L of rectangular frame 75_L, diagonal points PA_M and PB_M of rectangular frame 75_M, and diagonal points PA_S and PB_S of rectangular frame 75_S.

[0093] 15, under the control of the display control unit 55, a designation screen 95 is displayed on the display 34 (step ST1303). On the designation screen 95, frame designation buttons 96L, 96M, and 96S corresponding to the three rectangular frames 75_L, 75_M, and 75_S, respectively, are provided so as to be alternatively selectable.

[0094] 16, the user selects the frame designation button 96 corresponding to the rectangular frame 75 considered appropriate for a colony, and then selects the designation button 97. This causes the instruction acceptance unit 51 to accept the designation of the bandwidth BW (step ST1304). The instruction acceptance unit 51 sets the bandwidth BW corresponding to the frame designation button 96 selected by the user as the designated bandwidth 41. The designated bandwidth 41 is output from the instruction acceptance unit 51 to the RW control unit 50, and is stored in the storage device 30 by the RW control unit 50 (step ST1305).

[0095] As described above, the cell image analysis device 10 includes the RW control unit 50 as an acquisition unit, the density information derivation unit 52 as a derivation unit, and the detection unit 53. The RW control unit 50 acquires the cell image 14 obtained by photographing the mesenchymal stem cells 13 with the imaging device 11 by reading it from the storage device 30. The density information derivation unit 52 performs image analysis on the cell image 14 to derive an estimated density distribution 60 as density information of the mesenchymal stem cells 13 in the cell image 14. The detection unit 53 detects colonies of mesenchymal stem cells 13 based on the estimated density distribution 60.

[0096] The density information derivation unit 52 derives an estimated density distribution 60 of the mesenchymal stem cells 13 in the cell image 14 by performing kernel density estimation, and outputs the estimated density distribution 60 as density information. Since the estimated density distribution 60 is derived using a commonly used method called kernel density estimation and used as density information, density information that relatively accurately represents the density of the mesenchymal stem cells 13 can be easily obtained.

[0097] The detection unit 53 performs a mean-shift with reference to the estimated density distribution 60, and detects, as a colony, a collection of mesenchymal stem cells 13 that converge to the same maximum point 71 in the estimated density distribution 60. The mean-shift is also a method that is often used along with kernel density estimation. This allows colonies to be detected easily with a relatively high degree of accuracy. Furthermore, compared to detection of colonies by visual inspection by a user, colonies can be detected based on consistent criteria without hesitation.

[0098] The instruction receiving unit 51 receives a user's specification of a bandwidth BW for kernel density estimation. The density information derivation unit 52 performs kernel density estimation using the specified bandwidth 41, which is the bandwidth BW received by the instruction receiving unit 51. This can improve the validity of the estimated density distribution 60. As a result, it is possible to detect colonies that match the user's sense.

[0099] The density information derivation unit 52 derives a plurality of types of estimated density distributions 60 by performing kernel density estimation with a plurality of types of bandwidths BW for a specific region 90 of the cell image 14. The detection unit 53 performs mean-shift with reference to each of the plurality of types of estimated density distributions 60, thereby outputting a plurality of types of colony detection results 42 corresponding to the plurality of types of estimated density distributions 60. The display control unit 55 presents the plurality of types of detection results 42 to the user, and allows the user to select one of the plurality of types of detection results 42. The instruction reception unit 51 sets the bandwidth BW corresponding to the one detection result 42 selected by the user as the specified bandwidth 41. The user can select a detection result 42 that the user thinks is appropriate as a colony while comparing the plurality of types of detection results 42. This makes it easy to select the detection result 42.

[0100] The display control unit 55 presents the colony detection result 42 to the user. This allows the user to check the colony detection result 42.

[0101] The evaluation information derivation unit 54 derives evaluation information 43 representing the proliferation ability of the mesenchymal stem cells 13 based on the colony detection results 42. The display control unit 55 presents the evaluation information 43 to the user. This allows the user to check the evaluation information 43 and evaluate the proliferation ability of the mesenchymal stem cells 13 by referring to the evaluation information 43.

[0102] The evaluation information 43 is information on at least one of the number of colonies, CFU, and average colony size at a single time point, which allows the user to evaluate the proliferation ability of the mesenchymal stem cells 13 at a single time point. Furthermore, it is possible to set the detection unit 53 so that the derived evaluation information 43 is limited to information on mesenchymal stem cells 13 having a colony size exceeding a certain size. Specifically, the detector 53 can be configured to derive an average colony size for colonies with a colony size of greater than 0.54 mm.

[0103] The evaluation information may include evaluation information other than the number of colonies, CFU, and average colony size exemplified in FIG. For example, the evaluation information may be the average number of mesenchymal stem cells 13 constituting a colony, or the average ratio (WX / WY) of the width WX to the width WY of the rectangular frame 75, or the like. The closer the ratio of width WX to width WY of rectangular frame 75 is to 1, that is, the closer rectangular frame 75 is to a square, the more uniformly mesenchymal stem cells 13 that compose the colony can be said to proliferate, which is preferable. 19, statistical information such as a histogram 101 in which the frequency on the vertical axis represents the number of colonies and the class on the horizontal axis represents the size of the colonies may be included. Note that reference numeral 102 denotes a line segment indicating the average colony size.

[0104] Furthermore, the evaluation information is not limited to information at one time point, and may be information at multiple time points. For example, like the evaluation information 105 shown in FIG. 20, it may include a graph 106 showing the time series change in average colony size, with the average colony size on the vertical axis and the number of days the mesenchymal stem cells 13 were cultured on the horizontal axis. Graph 106 displays plots 107 of average colony size against each number of days of culture (indicated by crosses), an approximation line 108 of each plot 107, and a balloon 109. Within balloon 109, the amount of change in average colony size per number of days of culture is shown.

[0105] Also, a threshold value may be set for the average colony size change amount. If the average colony size change amount is less than the threshold value, a warning 110 may be displayed as shown in FIG. 21. The threshold value is a value set from an empirical point of view, and is a value at which it is clear that the quality of the mesenchymal stem cells 13 after the end of the culture will not reach a shipping level. This makes it possible to prompt the user to take various measures, such as stopping the culture of the mesenchymal stem cells 13 or changing the components of the medium to raise the quality of the mesenchymal stem cells 13 to a shipping level.

[0106] As a method for specifying the specified bandwidth 41, the mode shown in FIG. 22 may be adopted. First, the bandwidth BW is set to a first bandwidth BW_I (not shown) which is an initial value (step ST1310). The first bandwidth BW_I is, for example, the upper bandwidth BW_L. Next, in density information derivation unit 52, kernel density estimation is performed on specific region 90 with first bandwidth BW_I, thereby deriving estimated density distribution 60_I (not shown) (step ST1311).

[0107] Next, in the detection unit 53, a mean-shift is performed on the specific region 90 with reference to the estimated density distribution 60_I, and a colony detection result 42 corresponding to the estimated density distribution 60_I is output (step ST1312). Specifically, the colony detection result 42 is the XY coordinates of diagonal points PA_I and PB_I (not shown) of the rectangular frame 75_I (not shown).

[0108] Next, under the control of the display control unit 55, a display screen of the cell image 14 on which the rectangular frame 75_I, which is the colony detection result 42, is superimposed is displayed on the display 34 (step ST1313). The display screen is provided with buttons for allowing the user to select whether or not the area indicated by the rectangular frame 75_I is appropriate as a colony.

[0109] When the user selects a button indicating that the area indicated by the rectangular frame 75_I is not suitable as a colony (NO in step ST1314), the bandwidth BW is changed from the first bandwidth BW_I to a second bandwidth BW_SE (not shown) (step ST1315). The second bandwidth BW_SE is, for example, the bandwidth BW_M in the first embodiment. Then, the process of step ST1311 is performed with the second bandwidth BW_SE, and further the processes of steps ST1312 and ST1313 are also performed. The series of processes from step ST1315 to step ST1313 are repeated until the user selects a button indicating that the area indicated by the rectangular frame 75 is suitable as a colony.

[0110] When the user selects a button indicating that the area indicated by the rectangular frame 75 is suitable as a colony (YES in step ST1314), the instruction receiving unit 51 sets the bandwidth BW set at that time as the designated bandwidth 41. The designated bandwidth 41 is output from the instruction receiving unit 51 to the RW control unit 50, and is stored in the storage device 30 by the RW control unit 50 (step ST1316).

[0111] In this way, the bandwidth BW may be changed stepwise, and each time a rectangular frame 75 is presented to the user as the colony detection result 42, allowing the user to select whether the area indicated by the rectangular frame 75 is appropriate as a colony. Alternatively, the user may be prompted to input a rectangular frame 75 that is considered appropriate as a colony, and the bandwidth BW corresponding to the input rectangular frame 75 may be set as the specified bandwidth 41.

[0112] Another embodiment of the cell-image analyzing apparatus (hereinafter, also referred to as the second embodiment) will be described with reference to FIGS. In the second embodiment, only when the index indicating the degree of proliferation of the mesenchymal stem cells 13 in the cell image 14 is within the set range 123, the density information is derived and the colony is detected.

[0113] 23 to 25, the CPU of the cell-image analyzing apparatus functions as an area ratio calculation unit 120 and a determination unit 121 in addition to the respective processing units 50 to 55 of the first embodiment. The area ratio calculation section 120 calculates the area ratio 122 of the mesenchymal stem cells 13 in the cell image 14. Specifically, the area ratio calculation section 120, like the cell centroid position extraction section 65, performs a binarization process on the cell image 14. Next, the number of pixels whose pixel values ​​have been replaced with 0 as being mesenchymal stem cells 13 is counted. The counted number of pixels is then divided by the total number of pixels in the cell image 14 to calculate an area ratio 122 of the mesenchymal stem cells 13. The area ratio calculation unit 120 outputs the calculated area ratio 122 to the determination unit 121. The area ratio 122 is an example of an "index".

[0114] The determination unit 121 determines whether the area ratio 122 from the area ratio calculation unit 120 is within a set range 123. The set range 123 is stored in the storage device 30, and is read out from the storage device 30 by the RW control unit 50 and passed to the determination unit 121.

[0115] As shown in FIG. 23, when area ratio 122 is within set range 123, determination unit 121 outputs determination result 124A indicating that "derivation of density information and detection of colonies are to be performed." In this case, the density information derivation unit 52 derives an estimated density distribution 60, which is density information, and the detection unit 53 also detects colonies.

[0116] As shown in FIGS. 24 and 25, when area ratio 122 is outside set range 123, determination unit 121 outputs determination result 124B indicating that "density information is not derived and colony detection is not performed." In this case, the density information derivation unit 52 does not derive the estimated density distribution 60, which is density information, and the detection unit 53 does not detect colonies.

[0117] 23 to 25 show an example in which the setting range 123 of the area ratio is set to 25% to 75%. Fig. 23 shows an example in which area ratio 122 is 41%, which is within set range 123, and judgment unit 121 outputs judgment result 124A. Fig. 24 shows an example in which area ratio 122 is 14%, which is outside set range 123, and judgment unit 121 outputs judgment result 124B. Fig. 25 shows an example in which area ratio 122 is 79%, which is outside set range 123, and judgment unit 121 outputs judgment result 124B.

[0118] FIG. 26 is a graph showing the aspects shown in FIGS. 23 to 25, with the vertical axis representing the area ratio and the horizontal axis representing the number of days of culture. Mesenchymal stem cells 13 tend to have a very high proliferation rate, with the average colony size reaching a plateau relatively quickly. However, in the early stages of culture, the cells are not dense enough to form colonies. Therefore, when area ratio 122 is less than 25%, which can be said to be the early stage of culture, determination unit 121 outputs determination result 124B indicating that "density information is not derived and colony detection is not performed." Furthermore, in the final stage of culture where the average colony size reaches a plateau, the colonies cover almost the entire culture vessel 12, making it meaningless to distinguish between colonies. Therefore, even when the area ratio 122 is greater than 75%, which can be said to be the final stage of culture, the determination unit 121 outputs a determination result 124B indicating that "density information is not derived and colony detection is not performed."

[0119] 27 shows the cell image display screen 80 in the case where the area ratio 122 is outside the set range 123 and the judgment unit 121 outputs the judgment result 124B. In this case, when the analysis button 81 is selected, the display control unit 55 pops up a warning window 130. The warning window 130 displays a message that analysis is not possible because the area ratio 122 is outside the set range 123. The warning window 130 disappears when the OK button 131 is selected.

[0120] Thus, in the second embodiment, density information is derived and colonies are detected only when the area ratio 122, which is an index indicating the degree of proliferation of mesenchymal stem cells 13 in the cell image 14, is within the set range 123. This makes it possible to avoid unnecessary image analysis when performing image analysis is meaningless, such as in the early stage of culture (the stage where the mesenchymal stem cells 13 have not yet formed colonies) and the final stage of culture (the stage where the mesenchymal stem cells 13 have formed large colonies covering almost the entire culture vessel 12). In other words, image analysis can be performed only during a period appropriate for evaluating the proliferation ability of the mesenchymal stem cells 13.

[0121] The index showing the degree of proliferation of the mesenchymal stem cells 13 in the cell image 14 is not limited to the exemplified area ratio 122. It may be the number of mesenchymal stem cells 13 in the cell image 14.

[0122] Still another embodiment of the cell-image analyzing apparatus (hereinafter, also referred to as a third embodiment) will be described with reference to FIGS.

[0123] 28, the density information derivation unit 140 derives the number of mesenchymal stem cells 13 within a preset search frame 141 (hereinafter referred to as the number of cells within the frame) 142 as density information. The search frame 141 has a size equivalent to that of the rectangular frame 75_M in the first embodiment, for example. Alternatively, the search frame 141 is set by a user. The number of cells within the frame 142 is obtained, for example, by counting the number of centroids 68 of the mesenchymal stem cells 13 present within the search frame 141. The density information derivation unit 140 outputs the number of cells within the frame 142 to the detection unit 143.

[0124] The detection unit 143 detects colonies based on the number of cells in the frame 142 and the colony condition 144, and outputs the detection result 145. The colony condition 144 is stored in the storage device 30, and is read out from the storage device 30 by the RW control unit 50 and transferred to the detection unit 143.

[0125] As shown in FIG. 29, the density information derivation unit 140 sequentially scans a search frame 141 over a plurality of regions RE1_1, RE1_2, . . . , RE2_1, . . . , REM_N in the cell image 14, and derives the number of cells within the frame 142 in each region. The detection unit 143 detects whether each region RE is a colony region based on whether the number of cells in frame 142 in each region RE satisfies the colony condition 144. The region RE1_2 is a region obtained by shifting the search frame 141 by half in the X-axis direction from the region RE1_1. The region RE2_1 is a region obtained by shifting the search frame 141 by half in the Y-axis direction from the region RE1_1.

[0126] For example, as shown in FIG. 30, when the number of cells within frame 142 in region RE2_1 is 52 and the colony condition 144 is the number of cells within frame ≧50, the detection unit 143 detects region RE2_1 as a colony region.

[0127] In this way, in the third embodiment, the number of cells within the frame 142 is derived as density information instead of the estimated density distribution 60. Therefore, in comparison with the estimated density distribution 60, the density information can be derived more easily.

[0128] The search frame 141 is not limited to one type. A plurality of types of search frames 141 with different sizes or shapes may be set, and colonies may be detected for each search frame 141.

[0129] Still another embodiment of the cell-image analyzing apparatus (hereinafter, also referred to as a fourth embodiment) will be described with reference to FIGS. In the fourth embodiment, the machine learning model 150 is used to perform image analysis.

[0130] 31, the machine learning model 150 is a model that uses a cell image 14 as an input image and uses a cell image 14 superimposed with a rectangular frame 75 as a colony detection result 42 as an output image. The machine learning model 150 is, for example, a convolutional neural network such as U-Net (U-Shaped Neural Network), SegNet, ResNet (Residual Network), or DenseNet (Densely Connected Convolusional Network).

[0131] 32 shows an overview of the process in the learning phase of the machine learning model 150. In the learning phase, the machine learning model 150 is given learning data 155 and learns. The learning data 155 is a set of a learning cell image 14L and a correct answer image 14CA corresponding to the learning cell image 14L. The correct answer image 14CA is an image in which a user manually draws a rectangular frame 75 in an area of ​​the learning cell image 14L that is thought to be a colony.

[0132] In the learning phase, the learning cell image 14L is input to the machine learning model 150. The machine learning model 150 outputs a learning output image 14OL for the learning cell image 14L. The learning output image 14OL is the learning cell image 14L in which a rectangular frame 75 is superimposed on an area estimated to be a colony in the machine learning model 150. A loss calculation is performed on the machine learning model 150 based on the learning output image 14OL and the correct answer image 14CA. Then, update settings of various coefficients of the machine learning model 150 are performed according to the result of the loss calculation, and the machine learning model 150 is updated according to the update settings. If there is a large gap between the rectangular frame 75 of the learning output image 14OL and the rectangular frame 75 of the correct answer image 14CA, the loss becomes large and the degree of update of the machine learning model 150 also becomes large. Conversely, if there is almost no gap between the rectangular frame 75 of the learning output image 14OL and the rectangular frame 75 of the correct answer image 14CA, the loss becomes small and the degree of update of the machine learning model 150 also becomes small.

[0133] In the learning phase, the above series of processes, including input of the learning cell image 14L to the machine learning model 150, output of the learning output image 14OL from the machine learning model 150, loss calculation, update setting, and updating of the machine learning model 150, are repeated while the learning data 155 is exchanged. The repetition of the above series of processes is terminated when the estimation accuracy of the rectangular frame 75 of the learning output image 14OL reaches a predetermined set level. The machine learning model 150 in which the estimation accuracy of the rectangular frame 75 has thus reached the set level is stored in the storage device 30 and used.

[0134] In this way, in the fourth embodiment, colonies are detected using the machine learning model 150. Therefore, colonies can be detected more efficiently.

[0135] In addition, instead of the correct image 14CA, the XY coordinates of diagonal points PA, PB of a rectangular area 75 that is thought to be a colony in the learning cell image 14L may be used as the learning data 155.

[0136] The designated bandwidth 41 may be changed for each cell image 14, not for each culture project. The designated bandwidth 41 may also be changed depending on the type of mesenchymal stem cell 13 (for example, the designated bandwidth 41 for A cells is set to bandwidth BW_M, and the designated bandwidth 41 for B cells is set to bandwidth BW_S, etc.).

[0137] The colony detection result 42 is not limited to the exemplified rectangular frame 75, but may be, for example, a curved frame (such as a circular or elliptical frame) surrounding the area of ​​the detected colony.

[0138] Similar to the bandwidth BW, the radius R of the search circle 70 used in the mean-shift may be specified by the user.

[0139] The manner in which the colony detection result 42 is presented to the user is not limited to the manner in which the cell image 14 with the rectangular frame 75 superimposed thereon is displayed on the display 34. For example, a mode in which the cell image 14 on which the rectangular frame 75 is superimposed is printed out, or a mode in which the cell image 14 on which the rectangular frame 75 is superimposed is delivered to a terminal owned by the user via e-mail or the like may be adopted. Similarly, the manner in which the evaluation information 43 is presented to the user is not limited to the manner in which the evaluation information 43 is displayed on the display 34 . For example, the evaluation information 43 may be printed out or distributed to a terminal owned by the user by e-mail or the like.

[0140] The hardware configuration of the computer constituting the cell-image analyzing device 10 can be modified in various ways. For the purpose of improving processing capacity and reliability, the cell-image analyzing device 10 can be composed of multiple computers separated as hardware. For example, the functions of the density information derivation unit 52 and the detection unit 53 and the function of the evaluation information derivation unit 54 are distributed and assigned to two computers. In this case, the cell-image analyzing device 10 is composed of two computers.

[0141] In this way, the hardware configuration of the computer of the cell-image analyzer 10 can be changed as appropriate depending on the required performance, such as processing power, safety, and reliability. Furthermore, not only the hardware but also application programs such as the operating program 40 can be duplicated and stored in a distributed manner in multiple storage devices in order to ensure safety and reliability.

[0142] In each of the above embodiments, the hardware structure of a processing unit that executes various processes, such as the RW control unit 50, instruction receiving unit 51, density information derivation unit 52, 140, detection unit 53, 143, evaluation information derivation unit 54, display control unit 55, cell center of gravity position extraction unit 65, kernel density estimation unit 66, area ratio calculation unit 120 and judgment unit 121, can use the various processors shown below. As described above, the various processors include the CPU 32, which is a general-purpose processor that executes software (operating program 40) and functions as various processing units, as well as dedicated electrical circuits such as programmable logic devices (PLDs) that are processors whose circuit configuration can be changed after manufacture, such as FPGAs (Field Programmable Gate Arrays), and application specific integrated circuits (ASICs), which are processors having a circuit configuration designed specifically to execute specific processes.

[0143] A single processing unit may be configured with one of these various processors, or may be configured with a combination of two or more processors of the same or different types (for example, a combination of multiple FPGAs, a combination of a CPU and an FPGA, etc.). Also, multiple processing units may be configured with a single processor.

[0144] As an example of configuring multiple processing units in a single processor, first, there is a form in which a single processor is configured by combining one or more CPUs and software, as typified by computers such as client and server computers, and this processor functions as multiple processing units. Secondly, there is a form using a processor that realizes the functions of an entire system including a plurality of processing units on a single IC (Integrated Circuit) chip, as typified by a system on chip (SoC). In this way, the various processing units are configured as a hardware structure using one or more of the various processors described above.

[0145] Furthermore, more specifically, the hardware structure of these various processors can be an electric circuit that combines circuit elements such as semiconductor elements.

[0146] The above description and illustrations are merely an example of the technology of the present disclosure. For example, the above description of the configuration, function, action, and effect is an example of the configuration, function, action, and effect of the parts related to the technology of the present disclosure. Therefore, it goes without saying that unnecessary parts may be deleted, new elements may be added, or replacements may be made to the above description and illustrations, within the scope of the gist of the technology of the present disclosure. In addition, in order to avoid confusion and to facilitate understanding of the parts relating to the technology of the present disclosure, the descriptions and illustrations provided above omit explanations of common technical knowledge and the like that do not require particular explanation in order to enable the implementation of the technology of the present disclosure.

[0147] (Group Selection) In the method for producing a therapeutic agent for arthropathy of the present disclosure, a population is selected based on at least one of the average colony size and the colony forming units derived from the detected colonies. As a specific example, a population can be selected based on evaluation information of the average colony size and colony forming units derived from the evaluation information derivation section.

[0148] More specifically, colony detection is carried out on the 4th to 6th day after the start of culture, and population selection is preferably carried out by selecting a population that satisfies at least one of the following conditions a1) and b1), more preferably by selecting a population that satisfies at least one of the following conditions a1') and b1'), and even more preferably by selecting a population that satisfies at least one of the following conditions a1'') and b1''). a1) The average colony size of mesenchymal stem cells contained in the population at any time point between 4 and 6 days after the start of culture is 0.740 mm or more. b1) The colony-forming units of mesenchymal stem cells contained in the population at any time point between 4 and 6 days after the start of culture are 0.250 / mm 2 That's all. a1') The average colony size of mesenchymal stem cells contained in the population at any time point between 4 and 6 days after the start of culture is 0.750 mm or more. b1') The colony-forming units of mesenchymal stem cells in the population at any time between 4 and 6 days after the start of culture were 0.270 / mm 2 That's all. a1'') The average colony size of mesenchymal stem cells contained in the population at any time point between 4 and 6 days after the start of culture is 0.800 mm or more. b1'') The colony-forming units of mesenchymal stem cells in the population at any time between 4 and 6 days after the start of culture were 0.300 / mm 2 That's all. A population that satisfies at least one of the following conditions a1) and b1) is predicted to have a desired number of mesenchymal stem cells by the day of transplantation, and an arthropathy treatment agent produced by such selection is suitable for treating arthropathy.

[0149] (others) After the culture is completed, the synovium-derived mesenchymal stem cells may be mixed with a pharma- ceutical acceptable carrier to form a suspension or gel-like substance, which may then be used as a therapeutic agent for arthropathy. Furthermore, the synovium-derived mesenchymal stem cells after the culture may be used in a therapeutic agent for arthropathy in the form of a cell sheet or the like without being mixed with a carrier.

[0150] Pharmaceutically acceptable carriers include aqueous carriers such as isotonic solutions containing, for example, saline, glucose, D-sorbitol, D-mannitol, sodium chloride, and the like. Additionally, bioabsorbable gels such as gelatin and collagen may be used as pharma- ceutically acceptable carriers.

[0151] The arthropathy therapeutic agent may contain pharma- ceutically acceptable additives, such as buffers (e.g., phosphate buffer, sodium acetate buffer, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), preservatives, and antioxidants.

[0152] (Arthropathy treatment) The therapeutic agent for arthropathy according to the present disclosure is a therapeutic agent for arthropathy produced by the above-mentioned production method for a therapeutic agent for arthropathy. The therapeutic agent for arthropathy has been described above, so a detailed description thereof will be omitted here.

[0153] An example of a method for using the therapeutic agent for arthropathy according to the present disclosure (a method for treating arthropathy) will be described below, but the present invention is not limited thereto.

[0154] The arthropathy treatment agent can be used to treat arthropathy by transplanting the arthropathy treatment agent (a suspension containing mesenchymal stem cells, a cell sheet of mesenchymal stem cells, or mesenchymal stem cells made into a gel-like substance using a carrier such as gelatin or collagen, etc.) so that the damaged area of ​​cartilage or damaged area of ​​the meniscus is covered with mesenchymal stem cells, differentiating the mesenchymal stem cells into chondrocytes at the damaged area, and regenerating cartilage tissue in situ.

[0155] During the process of in situ chondrogenesis by mesenchymal stem cells, cartilage tissue is regenerated according to the local microenvironment (nutrient supply and cytokine environment, etc.), so no external manipulation is required. As a result of in situ chondrogenesis by mesenchymal stem cells, cartilage tissue is regenerated at the cartilage injury site or meniscus injury site, and the injury is repaired. Specifically, in the case of cartilage injury, the bone region, the boundary between cartilage and bone, the cartilage center, the surface region, and the region adjacent to the original cartilage are formed as original cartilage tissue, and in the case of meniscus injury, meniscus cartilage is formed.

[0156] Transplantation of mesenchymal stem cells can be carried out by exposing the damaged cartilage or meniscus area by surgical treatment, and then covering the damaged area with the therapeutic agent for arthropathy. The method for transplanting mesenchymal stem cells is not limited to the above, and may be, for example, by injecting a suspension containing mesenchymal stem cells into a joint.

[0157] Surgical treatment can be performed by open surgery or arthroscopic surgery, with arthroscopic surgery being preferred since it is less invasive.

[0158] From the viewpoint of adhesiveness of mesenchymal stem cells to the injured site, the injured site is preferably covered with the arthropathy therapeutic agent for 10 minutes or more, and more preferably 15 minutes or more.

[0159] In order to further improve the adhesiveness of mesenchymal stem cells to the damaged area, the area covered with the arthropathy therapeutic agent can be further covered with periosteum.

[0160] When transplanting mesenchymal stem cells into a patient, the number of mesenchymal stem cells contained in the arthritis treatment agent to be transplanted into the damaged area should be 0.50 × 10 7 Preferably, it is 1.00×10 or more. 7 More preferably, 2.00×10 7 It is more preferable that the number of mesenchymal stem cells contained in the arthropathy treatment agent to be transplanted to the injured area is 2.5010 or more. 7It may be 3.0010 or more 7 It may be 4.0010 or more 7 It may be 5 or more. In addition, the upper limit value of the number of mesenchymal stem cells contained in the arthrosis therapeutic agent transplanted into the damaged part is not particularly limited. For example, it may be 1.0×10 11 or less, and it may be 1.0×10 10 or less, and it may be 1.0×10 9 or less, and it may be 1.0×10 8 or less.

[0161] The disclosure of Japanese Patent Application No. 2021-040648 filed on March 12, 2021 is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described in this specification are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually indicated to be incorporated by reference.

Examples

[0162] Hereinafter, the above-described embodiments will be specifically described by way of examples, but the above-described embodiments are not limited to these examples.

[0163] <Example 1> Using human specimens of patients with meniscus injury, after disinfecting the synovial tissue, synovium-derived mesenchymal stem cells were prepared. Specifically, the tissue of M1 specimen (0.85 g of synovial tissue) was minced with scissors and immersed in 5.0 mL of a collagenase aqueous solution, and an enzyme reaction was carried out. The collagenase aqueous solution used was a solution prepared by dissolving 5.0 mg of collagenase MNP-S (manufactured by F. Hoffmann-La Roche) containing an enzyme mixture containing collagenase and neutral protease in 5.0 mL of water for injection containing 20% human autologous serum as the final concentration. The enzyme reaction was carried out at 37°C for 1 hour and 30 minutes. In addition, in the enzyme reaction, the ratio of the usage amount of the M1 specimen to the usage amount of the enzyme was 170 on a mass basis.

[0164] The tissue digestion fluid containing synovium-derived mesenchymal stem cells obtained by enzymatically reacting the M1 sample was transferred to a 50 mL centrifuge tube through a cell strainer and centrifuged at a centrifugal force of 400 g for 5 minutes.

[0165] The supernatant was removed, and the resulting suspension containing synovium-derived mesenchymal stem cells was suspended in medium, and the entire amount was seeded into a flask (1.44 × 10 7 pieces, 1880 pieces / cm 2 ). The medium used was α-modified Eagle's minimum essential medium (α-MEM) containing human autologous serum at a final concentration of 10% by volume. In addition, the culture was carried out in a CO 2 Incubator (37 °C, 5% CO 2 ) and synovium-derived mesenchymal stem cells were collected from the flasks two weeks later. The medium was not exchanged during the culture period.

[0166] The amount of liberase remaining in the medium was quantified to be 59.8 ng / mL.

[0167] The number of cells collected from synovium-derived mesenchymal stem cells was 0.80 × 10 7 The therapeutic applicability of the composition was evaluated as B in Table 1. The ratio of the number of cells recovered two weeks later to the number of cells at the time of seeding (proliferation fold) was 0.6. The number of collected cells was measured by visual cell counting and by using a dual fluorescent optical cell counter LUNA-FL (registered trademark) (manufactured by logos biosystems).

[0168] On the fourth day after the start of culture (seeding), cell images of the population containing synovium-derived mesenchymal stem cells were obtained, and density information (estimated density distribution) of the synovium-derived mesenchymal stem cells was derived from the cell images by kernel density estimation. Next, a mean-shift was performed with reference to the estimated density distribution, and clusters of mesenchymal stem cells that converged to the same maximum point within the estimated density distribution were detected as colonies. The average colony size and colony forming units (CFU) were calculated from the detected colonies. The average colony size was 0.743 mm and the CFU was 0.256 / mm 2 It was. The method of acquiring cell images, the method of deriving an estimated density distribution, the method of detecting colonies, and the method of deriving the average colony size and CFU were performed using the cell-image analyzer according to the first embodiment described above. Additionally, the average colony size was measured for colonies with a colony size of more than 0.54 mm. In the following Example 2, the average colony size and the like were measured in the same manner.

[0169] The expression of cell surface markers specific to the collected synovium-derived mesenchymal stem cells was examined by antigen-antibody reaction, and the cells were found to be CD90 positive and CD45 negative. Furthermore, when the collected synovium-derived mesenchymal stem cells were stained with Alcian blue, their ability to differentiate into chondrocytes was confirmed. From the above, it was confirmed that the collected cells were synovium-derived mesenchymal stem cells.

[0170] <Example 2> Using human specimens from patients with meniscus injuries, synovium-derived mesenchymal stem cells were produced without disinfecting the synovial tissue. Specifically, each tissue of the G0 sample (0.80 g synovial tissue), G1 sample (0.42 g synovial tissue), G2 sample (0.60 g synovial tissue), G3 sample (0.66 g synovial tissue), G4 sample (0.86 g synovial tissue), and G5 sample (0.33 g synovial tissue) was cut into small pieces with scissors and each was immersed in 5.0 mL of the Liberase aqueous solution also used in Example 1 to carry out the enzyme reaction. The enzyme reaction was carried out at 37°C for 3 hours. In the enzyme reaction, the ratios of the amounts of G0, G1, G2, G3, G4 and G5 samples used to the amount of enzyme used were 160, 84, 120, 132, 172 and 66, respectively, on a mass basis.

[0171] The tissue digestion fluid containing synovium-derived mesenchymal stem cells obtained by enzymatic reaction of each of the G0, G1, G2, G3, G4 and G5 samples was passed through a cell strainer and transferred to a 50 mL centrifuge tube, and centrifuged at a centrifugal force of 400 g for 5 minutes.

[0172] After that, as a washing step, the supernatant was discarded, the mixture was resuspended in 20 mL of α-MEM, and centrifuged at a centrifugal force of 400 g for 5 minutes. This washing step was repeated a total of two times.

[0173] The supernatant was removed, and each of the resulting suspensions containing synovium-derived mesenchymal stem cells was suspended in a medium, and the entire amount was seeded into a flask (G0 sample was 1.43 × 10 7 pieces, 1874 pieces / cm 2 , G1 sample was 0.53 × 10 7 pieces, 690 pieces / cm 2 , and G2 sample was 0.81 × 10 7 pieces, 1059 pieces / cm 2 , and G3 specimen was 1.52 × 10 7 pieces, 1987 pieces / cm 2 , and G4 sample was 0.79 × 10 7 pieces, 1028 pieces / cm 2 , G5 sample is 0.60 × 10 7 pieces, 787 pieces / cm 2 ). As in Example 1, α-MEM was used as the medium. In addition, the culture was carried out in a CO 2 Incubator (37 °C, 5% CO 2 ) and synovium-derived mesenchymal stem cells were collected from the flasks two weeks later. The medium was not exchanged during the culture period.

[0174] The amount of liberase remaining in the medium was quantified, and all of the G0 to G5 samples were found to be below 0.1 ng / mL (below the detection limit of the measurement kit).

[0175] The number of cells collected from synovium-derived mesenchymal stem cells was 4.9 × 10 for the G0 sample. 7 , and G1 sample was 4.9 × 10 7 , and G2 sample was 7.6 × 10 7 , and G3 sample was 5.8 × 10 7 , and G4 sample was 7.0 × 10 7 , and G5 specimens were 6.5 × 10 7 There were 100 pieces. The total number of cells of synovium-derived mesenchymal stem cells collected was 1.0 × 10 7 This was significantly higher than the number of cells required for treatment, and was at a level that could provide a sufficient and stable amount of cells required for treatment. The proliferation fold was 3.5 for the G0 specimen, 9.5 for the G1 specimen, 9.6 for the G2 specimen, 3.9 for the G3 specimen, 9.1 for the G4 specimen, and 11.0 for the G5 specimen, demonstrating a significant improvement in proliferation fold. In Table 1, the therapeutic applicability was rated as A.

[0176] On days 4 to 6 after the start of culture (seeding), cell images of the population containing synovium-derived mesenchymal stem cells were obtained, and density information (estimated density distribution) of the synovium-derived mesenchymal stem cells was derived from the cell images using kernel density estimation. Next, a mean-shift was performed with reference to the estimated density distribution, and clusters of mesenchymal stem cells that converged to the same maximum point within the estimated density distribution were detected as colonies. The average colony size and colony forming units (CFU) were calculated from the detected colonies, and the results are as follows: G0 sample: Colonies were detected 6 days after the start of culture, with an average colony size of 1.150 mm and CFU of 0.947 / mm. 2 G1 sample: colonies were detected 5 days after the start of culture, with an average colony size of 0.830 mm and CFU of 0.344 / mm 2 G2 sample: Colonies were detected 4 days after the start of culture, with an average colony size of 0.782 mm and CFU of 0.368 / mm. 2 G3 sample: Colonies were detected 4 days after the start of culture, with an average colony size of 0.816 mm and CFU of 0.311 / mm. 2 G4 sample: colonies were detected 6 days after the start of culture, with an average colony size of 1.030mm and CFU 0.575 / mm 2 G5 sample: Colonies were detected 4 days after the start of culture, with an average colony size of 0.777 mm and CFU of 0.340 / mm 2

[0177] The expression of cell surface markers specific to the collected synovium-derived mesenchymal stem cells was examined by antigen-antibody reaction, and the cells were found to be CD90 positive and CD45 negative. Furthermore, when the collected synovium-derived mesenchymal stem cells were stained with Alcian blue, their ability to differentiate into chondrocytes was confirmed. From the above, it was confirmed that the collected cells were synovium-derived mesenchymal stem cells.

[0178] [Table 1]

[0179] The results shown in Table 1 show that the arthropathy treatment agent produced by the arthropathy treatment method disclosed herein can predict the proliferation of mesenchymal stem cells during culture with excellent accuracy, making it possible to produce an arthropathy treatment agent suitable for treating arthropathy. It is also evident that the average colony size and CFU of the mesenchymal stem cells during culture contribute to the number of recovered cells and the proliferation rate.

[0180] <<Application as a meniscus treatment agent>> The synovium-derived mesenchymal stem cells produced from the G0 to G5 specimens in Example 2 were transplanted as autologous cell therapy into the site of meniscus injury in a patient with meniscus injury. In patients with meniscus injuries who received synovium-derived mesenchymal stem cells, good symptom relief was observed up to 52 weeks after transplantation. The Lysholm score, known as a symptom score (see Table 2 below, performed by an evaluator), showed an improvement of more than 22 points 52 weeks after transplantation compared to before treatment, and MRI (magnetic resonance imaging) also confirmed that the meniscus had been reduced, indicating good treatment outcomes.

[0181] [Table 2]

Claims

1. A cell image is obtained by photographing a group containing multiple cultured mesenchymal stem cells. deriving density information of the mesenchymal stem cells in the cell image by analyzing the cell image; detecting colonies formed by the mesenchymal stem cells in the population based on the density information; sorting the population based on at least one of average colony size and colony forming units derived from the detected colonies; A method for producing a therapeutic agent for arthropathy.

2. The method for producing a therapeutic agent for arthropathy according to claim 1 , wherein the density information is an estimated density distribution of the mesenchymal stem cells in the cell image, derived by kernel density estimation.

3. 3. The method for producing a therapeutic agent for arthropathy according to claim 2, wherein the detection of the colony is carried out by performing a mean-shift with reference to the estimated density distribution and detecting, as a colony, a collection of the mesenchymal stem cells that converge to the same maximum point in the estimated density distribution.

4. The detection of the colonies is carried out on the 4th to 6th days after the start of the culture, The method for producing a therapeutic agent for arthropathy according to any one of claims 1 to 3, wherein the selection of the population is carried out by selecting a population that satisfies at least one of the following conditions a1) and b1): a1) The average colony size of the mesenchymal stem cells contained in the population at any time point between 4th and 6th days from the start of culture is 0.740 mm or more. b1) The colony-forming units of the mesenchymal stem cells contained in the population at any time point between 4 and 6 days after the start of culture are 0.250 / mm ２ That's all.

5. The method for producing a therapeutic agent for arthropathy according to any one of claims 1 to 4, wherein the mesenchymal stem cells are obtained by treating a biological tissue with an enzyme.

6. 6. The method for producing a therapeutic agent for arthropathy according to claim 5, wherein an enzyme mixture containing at least one type each of collagenase and neutral protease is used in the enzymatic treatment of the biological tissue.

7. 7. The method for producing a therapeutic agent for arthropathy according to claim 5, wherein in the enzymatic treatment of the biological tissue, a ratio of an amount of the biological tissue to an amount of the enzyme is 10 to 1000 by mass.

8. The method for producing an arthropathy therapeutic agent according to any one of claims 5 to 7, wherein the mesenchymal stem cells are obtained by washing a tissue digestive fluid obtained by treating the biological tissue with an enzyme until the residual enzyme concentration in the supernatant is 0.5 ng / mL or less.

9. The method for producing a therapeutic agent for arthropathy according to any one of claims 1 to 8, wherein the mesenchymal stem cells are cells derived from synovium.

10. The method for producing a therapeutic agent for arthropathy according to any one of claims 1 to 9, wherein the mesenchymal stem cells are cells derived from a human.

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