JP2020120613A - Method for stably fabricating high-quality pluripotent stem cell stock using image analysis - Google Patents

Abstract

To provide a method for fabricating cryopreserved pluripotent stem cells, and cryopreserved pluripotent stem cells obtained by the method.SOLUTION: A method for fabricating a cryopreserved pluripotent stem cell including the following processes. (1) a process of preparing a pluripotent stem cell; (2) a process of culturing the pluripotent stem cell prepared in process (1) in a culture tool to form a colony of pluripotent stem cells; (3) a process of determining the following feature quantity (A):(A) colony coverage, and at least one selected from the following feature quantity (B) to (D): (B) a colony average area, (C) variation of the colony areas, and (D) a colony average major axis, in the culture tool having the formed colony; (4) a process of comparing the determined feature quantity and a previously set standard value to select a culture tool which fulfills the standard value; and (5) a process of cryopreserving cells in the selected culture tool.SELECTED DRAWING: None

Description

The present invention relates to a method for producing cryopreserved pluripotent stem cells, cryopreserved pluripotent stem cells obtained by the above method, cryopreserved pluripotent stem stock cells having specific characteristics, and the like.

In 2014, autologous iPS cell-derived retinal pigment epithelium sheet was transplanted for age-related macular degeneration, and the regenerative medicine using iPS cells is being put to practical use. At the same time, there is a demand for an efficient and stable quality control method for cultured cells such as iPS cells used in regenerative medicine, and "image analysis technology" is drawing attention as one of the methods. Until now, DNA, RNA, and protein quantification methods using genetic markers have been mainly used for quality control of cultured cells (Non-Patent Document 1), but there are problems from the viewpoint of work efficiency and invasiveness. there were. On the other hand, as a non-invasive evaluation, visual observation is performed by a culture expert, but it is difficult to say that it is a stable management method because there are large variations within and between the inspectors. Therefore, it is considered that cell quality evaluation by image analysis, which has advantages in terms of efficiency, stability, and cost and can be performed non-invasively, will become more and more useful in the future.

The present inventors have examined culturing and qualitatively using a scale bar on a microscope image as a method for establishing a stock containing stable frozen cells (hereinafter, also referred to as “frozen cell stock”). Statistical evaluation of induced pluripotent stem cells after sleep by preparing frozen cell stocks before the inflection point of the logarithmic growth phase (culture days ≤ 7 days, colony major axis ≤ 1250 μm) I have found that it is possible. The above method made it possible to define the optimal cell seeding number and culture days for producing a frozen cell stock.

However, these indicators are still unstable, even with the same cell line, because they are easily influenced by the number of passages and the state of cells. Further, in order to see the state of the cells in more detail, it is possible to confirm that the cells are in the early logarithmic growth phase by peeling the cells from the culture dish, but there remains a problem from the viewpoint of work efficiency and invasiveness. Further, the confirmation of the colony size by visual observation is inefficient and inevitable variation among workers because the sizes of some selected colonies are measured with a scale bar. Further, although cell quality control technology by image evaluation in the culture process of induced pluripotent stem cells has been reported so far (Non-Patent Documents 2 and 3 and Patent Documents 1 and 2), these analysis methods require machine learning. Since it is used, it takes a lot of trial and error and time for it to be ready for use in the analysis method because the index is black-boxed, which is not practical.

International Publication No. 2018/101004 International Publication No. 2011/010449

PLoS ONE. 2010 Mar 8;5(3):e9580 J Biosci Bioeng. 2017 May;123(5):642-650. Sci Rep. 2014 Nov 11;4:6996

An object of the present invention is to provide a method for producing cryopreserved pluripotent stem cells, cryopreserved pluripotent stem cells obtained by the above method (hereinafter, also referred to as “frozen stock cells of the present invention”), specific features It is intended to provide cryopreserved pluripotent stem cells (hereinafter, also referred to as “frozen stock cells having specific characteristics of the present invention”) and the like.

Means to try to solve the problem

The present inventors demand for a method for producing cryopreserved pluripotent stem cells, characterized in that the cryopreserved pluripotent stem cells (and frozen cell stock containing the pluripotent stem cells) are "high quality". That is, that is, the number of viable cells after sleep is large enough for passage and is “stable”, that is, the number of frozen cell stocks containing the cryopreserved pluripotent stem cells is once The important point is to obtain a sufficient amount. In order to establish a method for producing cryopreserved pluripotent stem cells that can satisfy the above characteristics, it is possible to perform a clear index search as morphological information, using an image analysis method that does not use machine learning The amount (analysis parameter) was extracted and evaluated. As a result of repeated diligent studies, four indicators of “colony coverage”, “colony average area”, “variation of colony area (standard deviation (SD))”, and “colony average major axis” are The inventors have found that the index is not easily influenced by the state, and have completed the present invention.

That is, the present invention relates to the following.
[1] A method for producing cryopreserved pluripotent stem cells, which comprises the following steps.
(1) preparing pluripotent stem cells,
(2) culturing the pluripotent stem cells prepared in step (1) in a culture device to form colonies of the pluripotent stem cells,
(3) In the culture device having the formed colony, the following characteristic amount (A):
(A) Colony coverage, and the following feature quantities (B) to (D):
(B) average colony area,
(C) colony area variation, and (D) colony average major axis,
Determining at least one selected from
(4) A step of comparing the determined characteristic amount with a preset standard value to select a culture device satisfying the standard value, and (5) cryopreserving the cells in the selected culture device. Step [2] The method according to [1], wherein the cryopreserved pluripotent stem cells are cells that can be cultured for 7 weeks or more.
[3] The method according to [1] or [2], wherein the pluripotent stem cells are induced pluripotent stem cells.
[4] The method according to any one of [1] to [3], which comprises a step of establishing pluripotent stem cells before the step (1).
[5] The method according to any one of [1] to [4], which comprises a step of thawing the cryopreserved pluripotent stem cells before the step (1).
[6] The preset standard value is as follows:
(A) Colony coverage: more than 7%,
(B) Average colony area: less than 141,933 μm ² (C) Variation in colony area: less than 130,257 (D) Average major axis of colony: less than 483 μm, The method according to any one of [1] to [5].
[7] Cryopreserved pluripotent stem cells obtained by the method according to any one of [1] to [6].
[8] A cryopreserved pluripotent stem cell having at least one selected from the following characteristics (A) and (B) to (D).
(A) Coverage of colonies formed by the cells: more than 7% (B) Average area of colonies formed by the cells: less than 141,933 μm ² (C) Variation in colony area formed by the cells: less than 130,257 (D) Average major axis of colonies formed by cells: less than 483 μm [9] The cell according to [7] or [8], wherein the cryopreserved pluripotent stem cells are cells that can be cultured for 7 weeks or more.
[10] The cell according to any one of [7] to [9], wherein the pluripotent stem cell is an induced pluripotent stem cell.

According to the present invention, there is provided a method for producing cryopreserved pluripotent stem cells, cryopreserved pluripotent stem cells obtained by the method, cryopreserved pluripotent stem cells having specific characteristics, and the like. be able to. Cryopreserved pluripotent stem cells produced by the method of the invention are "high quality" (ie, the number of viable cells after nap is high enough for passage), and by the method of the invention Ensuring "high quality" cryopreserved pluripotent stem cells with "stability" (that is, sufficient number of frozen cell stock containing the cryopreserved pluripotent stem cells is obtained at one time) It becomes possible to manufacture. Further, the cryopreserved pluripotent stem cells obtained by the present invention can be cultured for a long period (7 weeks or more).

FIG. 1 is a diagram in which a bivariate correlation was obtained from the result of each extracted feature amount and the culture evaluation result in the 201B7 strain. FIG. 2 is a diagram in which a bivariate correlation was obtained from the results of each extracted characteristic amount and the culture evaluation result in the 1210B2 strain. Figure 3 shows that in the 201B7 strain, the number of viable cells after nap is 0.3 × 10 ⁶ cells or more, the number of cell stocks produced is 12 or more, which is defined as a passing sample, and the one below is defined as a deviation sample. FIG. 3 is a diagram showing the results of calculating a statistically optimum threshold value by ROC-AUC analysis. FIG. 4 shows that in the 1210B2 strain, the number of viable cells after nap is 0.3×10 ⁶ cells or more, the number of cell stocks produced is 12 or more, which is defined as a passing sample, and the number below is defined as a deviation sample, and each characteristic amount is defined. FIG. 3 is a diagram showing the results of calculating a statistically optimum threshold value by ROC-AUC analysis.

1. Method for Producing Cryopreserved Pluripotent Stem Cells The present invention provides a method for producing cryopreserved pluripotent stem cells (hereinafter abbreviated as “production method of the present invention”). The production method of the present invention comprises: (1) preparing pluripotent stem cells; (2) culturing the pluripotent stem cells prepared in step (1) in a culture device to form colonies of the pluripotent stem cells. The step of (3) determining a specific characteristic amount in the culture device having the formed colonies, (4) comparing the determined characteristic amount with a preset standard value, and the standard The method includes the step of selecting a culture device satisfying the values, and (5) the step of cryopreserving the cells in the selected culture device. According to the production method of the present invention, “high quality” (that is, the number of viable cells after nap is large enough for passage) and high “stability” (that is, the number of frozen cell stocks at one time) A sufficient amount is obtained), and cryopreserved pluripotent stem cells are obtained.

In the present specification, "pluripotent stem cells (pluripotent stem cells)" are embryonic stem cells (ES cells) and pluripotency similar thereto, that is, various tissues of the living body (endoderm, mesoderm, Refers to cells that have the potential to differentiate into all of the ectoderm). The pluripotent stem cells used in the present invention include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonal tumor cells (EC cells), embryos Examples thereof include sex germline stem cells (EG cells), preferably ES cells or iPS cells, and among them, iPS cells are preferable. When the pluripotent stem cell is an ES cell or any cell derived from a human embryo, even if the cell is a cell produced by destroying the embryo, it is a cell produced without destroying the embryo. However, it is preferably a cell produced without destroying the embryo.

In the present invention, the “ES cell” means a pluripotent stem cell established from the inner cell mass of an early embryo (eg, blastocyst) of a mammal such as human or mouse. ES cells were found in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and ES cell lines were subsequently established in primates such as humans and monkeys (JA Thomson et al. al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod. ., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165).

In the present invention, “artificial pluripotent stem cells” are pluripotent stem cells derived from somatic cells, and by initializing somatic cells, they artificially have pluripotency similar to embryonic stem cells. It means a sprung cell. For example, iPS cells (induced pluripotent stem cells) having pluripotency established by initializing differentiated cells such as fibroblasts by expression of genes such as Oct3/4, Sox2, Klf4, and Myc can be exemplified. .. In 2006, Yamanaka et al. established induced pluripotent stem cells from mouse fibroblasts (Cell, 2006, 126(4), p663-676). In 2007, from human fibroblasts, induced pluripotent stem cells having pluripotency similar to embryonic stem cells were established (Cell, 2007, 131(5), p861-872; Science, 2007, 318( 5858), p1917-1920; Nat Biotechnol., 2008, 26(1), p101-106).

Examples of pluripotent stem cells include, but are not limited to, cells derived from rodents such as mouse, rat, hamster, and guinea pig, and cells derived from primates such as human, monkey, orangutan, and chimpanzee. Human-derived cells are preferred.

The pluripotent stem cells prepared in the step (1) may be pre-established and stocked cells, or newly established cells. Therefore, the production method of the present invention may further include the following step before the step (1):
(I) establishing pluripotent stem cells, or (ii) thawing the cryopreserved pluripotent stem cells.

The step of establishing pluripotent stem cells in step (i) is not particularly limited as long as it includes a step of introducing a specific reprogramming factor into somatic cells when the pluripotent stem cells are induced pluripotent stem cells. For example, a step of collecting somatic cells, introducing artificially a reprogramming factor such as Oct3/4, Sox2, Klf4, Myc, and the like, and then expanding and culturing by selecting cells that have acquired pluripotency, A step of introducing reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc) into human peripheral blood lymphocytes using a retrovirus vector or a Sendai virus vector and culturing (Nishimura, T. et al. Cell Stem Cell 2013, 12, 114-126).

Somatic cells from which the induced pluripotent stem cells used in the present invention are derived are not particularly limited. As somatic cells, for example, lymphocytes in peripheral blood, fibroblasts such as skin, skin cells, visual cells, brain cells, hair cells, oral mucosa, lung cells, hepatocytes, gastric mucosal cells, intestinal cells, Mesenchymal stem cells derived from splenocytes, pancreatic cells, kidney cells, neural stem cells, hematopoietic stem cells, wisdom teeth, tissue stem cells, tissue precursor cells, blood cells (eg, peripheral blood mononuclear cells (T cells and non-T cells) , Umbilical cord blood cells, etc.), epithelial cells, endothelial cells (eg, vascular endothelial cells), muscle cells, etc., but are not limited thereto.

The genes contained in the reprogramming factor include, for example, Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1 and the like are exemplified, and these reprogramming factors may be used alone or in combination. Examples of the combination of reprogramming factors include WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009/091659, WO2009/101084, WO2009/. 101407, WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250, WO2009/126251, WO2009/126655, WO2009/157593, WO2010/009015, WO2010/033906, WO2010/033920, WO2010/042800, WO2010/050626, WO 2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO 2010/111409, WO 2010/111422, WO2010/115050, WO2010/124290, WO2010/147395, WO2010/147612, Huangfu D, et al. 2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467. -2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008). ), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, Judson. RL et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912. -8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010). , Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M. , et al. (2011), Nature. 474:225-229.

As a method of introducing the reprogramming factor into somatic cells, when the reprogramming factor is in the form of DNA, for example, virus, plasmid, vector such as artificial chromosome, lipofection, liposome, microinjection, etc. In the case of protein form, for example, lipofection, microinjection, etc., for example, lipofection, fusion with cell membrane-penetrating peptides (eg, HIV-derived TAT and polyarginine), microinjection, etc. be able to. As a method using a viral vector, a method using a retrovirus vector, a method using an episomal vector, a Sendai virus typified by an iD Pharma's initialization kit "CytoTune (registered trademark)-iPS 2.0" Examples of the method include, but are not limited to, a method using a vector, a method using a lentivirus vector, a method using an adenovirus vector, and the like. The vector may contain regulatory sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that the nuclear reprogramming substance can be expressed. Furthermore, if necessary, drug resistance genes (eg, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidine kinase gene, selection marker sequences such as diphtheria toxin gene, green fluorescent protein (GFP), β-glucuronidase (GUS), a reporter gene sequence such as FLAG, and the like can be included. Further, in the expression vector, after introduction into a somatic cell, in order to excise the gene encoding the nuclear reprogramming substance or the promoter and the gene encoding the nuclear reprogramming substance bound thereto, the loxP sequence is placed before and after them. May have. Furthermore, the expression vector may also contain the EBNA-1 gene and the oriP sequence or the Large T antigen gene and the SV40 ori sequence so that the expression vector is present episomally (that is, is replicated without being taken up into the chromosome). it can.

When the pluripotent stem cells are ES cells, the step of establishing the pluripotent stem cells in step (i) above is to take out the inner cell mass from the blastocyst of the fertilized egg of the target animal, and to remove the inner cell mass from the fibroblast. It can be performed by culturing on a cell feeder. For the method for establishing and maintaining human and monkey ES cells, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; Suemori H. et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; Ueno M. et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; Suemori H. et al. (2001), Dev. Dyn., 222:273-279; Kawasaki H. et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I. et al. (2006), Nature. 444:481-485. Alternatively, ES cells can be established using only single blastomeres of embryos in the cleavage stage before the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113- 117), and can also be established using a stunted embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676.).

The step of thawing the cryopreserved pluripotent stem cells of the step (ii) can be carried out by any known cell thawing method, for example, cryopreserved cells, water bath, incubator, incubator, etc. It is achieved by contacting with a solid, liquid or gaseous medium (eg, water, culture solution) having a temperature higher than the freezing temperature, using, but is not limited thereto. The temperature of the medium is typically 4-50°C, preferably 30-40°C, more preferably 36-38°C. The thawing time is typically 2 minutes or less, and particularly 20 seconds or less can significantly suppress the decrease in survival rate. The thawing time can be adjusted, for example, by changing the temperature of the thawing means or the immersion medium, the volume or composition of the culture solution or the cryopreservation solution at the time of freezing.

The production method of the present invention may include a step of washing the cells after the step (ii). The cells can be washed by any known method, for example, the cells are suspended in a cell washing solution (eg, a culture solution or a physiological buffer solution that may contain serum or serum components (serum albumin, etc.), It is achieved by, but not limited to, centrifugation, discarding the supernatant and recovering the precipitated cells. In the step of washing the cells, such a cycle of suspension, centrifugation, and collection may be performed once or multiple times (eg, 2, 3, 4, 5 or more times). In one aspect of the invention, the step of washing the cells is performed immediately after the step of thawing the cryopreserved cells. Examples of commercially available cell washing solutions that can be used in the present invention include Cell Lotion (Nippon Zenyaku Kogyo Co., Ltd.).

In the step of culturing the pluripotent stem cells prepared in step (2) (1) in a culture device to form colonies of the pluripotent stem cells, the culture device is used for extracting the characteristic amount described below. The petri dish, flask, plastic bag, Teflon (registered trademark) bag, dish, petri dish, tissue culture dish, multi-culture, which are generally used for culturing pluripotent stem cells, are not particularly limited as long as necessary images can be taken. Examples include dishes, microplates, microwell plates, multiplates, multiwell plates, chamber slides, cell culture flasks, spinner flasks, tubes, trays, culture bags, roller bottles and the like. The material of these culture equipment is not particularly limited, for example, glass, polyvinyl chloride, cellulosic polymers such as ethyl cellulose and acetyl cellulose, polystyrene, polymethylmethacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene, polypropylene, Polyethylene, polybutadiene, poly(ethylene-vinyl acetate) copolymer, poly(butadiene-styrene) copolymer, poly(butadiene-acrylonitrile) copolymer, poly(ethylene-ethyl acrylate) copolymer, poly(ethylene-methacrylate) copolymer, polychloroprene, Examples thereof include styrene resins, chlorosulfonated polyethylene, ethylene vinyl acetate, plastics such as acrylic block copolymers, and the like.

The culture is not particularly limited as long as it can survive or proliferate pluripotent stem cells in a state of maintaining an undifferentiated state, and examples thereof include culturing the cells in a medium to which various factors are added. it can. Further, a method using feeder cells or a method not using feeder cells (feeder-free culture method) may be used. Examples of the feeder-free culture method include matrigel (BD), type I collagen, type IV collagen, gelatin, laminin (eg, Laminin511 E8 (Nippi Corporation)), heparan sulfate proteoglycan, or entactin, and combinations thereof ( (Also referred to as "Matrigel"), pluripotent stem cells are adhered to a coated culture dish, and the cells are cultured using a medium used for culturing animal cells as a basic medium.

Examples of the basic medium include StemFit (eg: StemFit AK03N, StemFit AK02N) (Ajinomoto Co.), PECM (Primate ES Cell Medium), GMEM (Glasgow Minimum Essential Medium), IMDM (Iscove's modified Dulbecco's medium: Iscove's Modified Dulbecco's Medium), 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fisher medium Fischer's medium), a mixed medium thereof, and the like.

ROCK inhibitors (eg: Y-27632, Fasudil/HA1077, SR3677, GSK269962, H-1152, Wf-536, etc.), serum (eg: fetal bovine serum (FBS), human serum, horse serum, etc.) ) Or serum substitute, insulin, various vitamins, various amino acids such as L-glutamine, non-essential amino acids, 2-mercaptoethanol, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cells Factors (SCF (Stem cell factor), activin, etc.), various hormones, various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.), various extracellular matrices, Various cell adhesion molecules, antibiotics such as penicillin/streptomycin, puromycin and the like, pH indicator such as phenol red and the like can be appropriately added. Serum substitutes include albumin, transferrin, fatty acids, insulin, collagen precursors, trace elements, Knockout Serum Replacement (KSR), ITS-supplements and mixtures thereof.

The culture period in the step (2) is preferably 1 day (eg, 5, 6, 7 days) or more, and 10 days (eg, 7, 8, 9, 10 days) or less. preferable. The culture temperature is not particularly limited, but is 30 to 40° C., preferably 37° C., the culture is performed in the presence of CO ₂ -containing air, and the CO ₂ concentration is preferably 2 to 5%.

In the culture device having the formed colonies in the step (3), the step of determining a specific feature amount is as follows:
(I) acquiring an image of the colony in the culture device having the formed colony, and (ii) the following feature amount from the acquired image:
(A) Colony coverage (%) and the following feature quantities (B) to (D):
(B) Colony average area (μm ² ),
(C) Variation in colony area (standard deviation (hereinafter sometimes abbreviated as "SD")), and (D) colony average major axis (μm)
Can be performed by a method including a step of determining at least one selected from

Regarding the step (i) of obtaining an image of the colony in the culture device having the formed colony, the image to be obtained is particularly limited as long as it is an image of a colony formed by pluripotent stem cells taken non-invasively. Although not included, for example, an image taken by using an optical microscope (eg, phase contrast microscope, differential interference microscope, etc.) can be mentioned. The storage format of the captured image is not particularly limited as long as it is normally used for image analysis, and examples thereof include TIFF, GIF, PNG, JPEG and the like.
Immediately before cryopreservation of pluripotent stem cells formed in the culture device (eg, 1 day before cryopreservation, the same day, 5 hours before cryopreservation, 4 hours before, 3 hours before, 2 hours before 1 hour before, etc.).

The image to be acquired may be a partial image of a dish in which colonies formed by pluripotent stem cells are cultured, or an entire image. When capturing an image of part or all of the dish, for example, part or all of the dish is divided into images for each image in each field of the microscope to be used, and then they are combined into one. It may be an image of part or the whole of the dish. In addition, the captured image was cut out (broken) because various corrections known in normal cell image analysis (eg, removal of scratches and bubbles, positioned at the screen edge, etc.) were performed in order to extract the feature amount described later. Removal of colonies) may be performed.
The features of (A) to (D) are as follows: Phase-contrast image acquisition with an all-in-one fluorescence microscope (BZ-X710 and BZ-X700, KEYENCE) (eg 10 cm dish, 18.842 mm (7 fields of view) in the X-axis direction) ), 14.131 mm (7 fields of view) in the Y-axis direction, a total of 266.3 mm ² ) and image analysis technology using analysis software (BZ-X Analyzer).

The (A) colony coverage (%) means the ratio (area ratio) of colonies formed by pluripotent stem cells per acquired image. The area ratio, before being combined as one of the images, after obtaining the area ratio of colonies formed by pluripotent stem cells in each field (each image) of the microscope photographed in a divided manner, the average value thereof May be used as the area ratio, or the area ratio of colonies formed by pluripotent stem cells may be obtained after combining them as one image.

As shown in the examples below, the colony coverage has a significant positive correlation with the number of cell stocks that can be produced from one culture device in which colonies formed by pluripotent stem cells are cultured. The coverage can be appropriately set according to the number of cell stocks desired to be produced from one culture device used for producing cell stocks. The value of the covering rate for efficiently eliminating those from which a desired number of cell stocks cannot be obtained from one culture device is called a standard value. In the present specification, “effectively eliminate” means eliminating 80% or more of cells for which a desired number of cell stocks cannot be obtained from one culture device, preferably 90% or more. , More preferably 95% or more, and most preferably 100%.

The standard value, from the ROC-AUC analysis and bivariate analysis of the cell stock production number and coverage, as long as it is possible to efficiently eliminate those that can not obtain the desired number of cell stock from one culture device The calculated threshold value may be used as the standard value, and a value that is more severe than the calculated threshold value may be used as the standard value. For example, when it is desired to prepare a cell stock of 0.2 or more per unit area (cm ² ) of culture equipment (eg, dish) (that is, a standard with which a sufficient number of cell stocks can be obtained at one time), the colony coverage standard The value is at least 7% (eg: 8% or more, 16% or more, 17% or more, 31% or more, 32% or more).

The (B) colony average area (μm ² ) means the average area (μm ² ) of all colonies formed by pluripotent stem cells present in the acquired image. There is a significant positive correlation between the reciprocal of the colony average area and the number of viable cells after nap, and in the present specification, "the number of viable cells after nap" is the number of cells after seeding after nap and culturing after 5 days of culturing. It means the number of cells. The colony average area can be appropriately set according to the desired number of viable cells after nap. The value of the average area of colonies for efficiently eliminating the culture equipment for culturing pluripotent stem cells that cannot achieve the desired number of viable cells after nap is called a standard value. In the present specification, the term "effectively eliminate" means eliminating 80% or more of the culture equipment for culturing pluripotent stem cells that cannot achieve the desired post-sleeping viable cell number, and preferably 90% or more, more preferably 95% or more, and most preferably 100%.

The standard value, as long as it is possible to efficiently exclude the culture equipment for culturing pluripotent stem cells that can not achieve the desired number of post-sleeping viable cells, two variables of the post-sleeping viable cell number and the reciprocal of the average colony area The threshold value obtained from the analysis and the ROC-AUC analysis may be used as the standard value, and a value that is more severe than the obtained threshold value may be used as the standard value. For example, when the number of viable cells after nap is desired to be 0.3×10 ⁶ cells or more (that is, the standard in which the number of viable cells after nap is large enough for passage), the standard value of the average colony area is at least 141,933 μm ^2. less (example: 141,900μm ² or less, less than 115,395μm ^2, 115,300μm ² below, less than 109,218μm ^2, 109,200μm ² below, less than 66,599μm ^2, 66,500μm ² below, 45,259Myuemu ² greater, 45,300Myuemu ² or more, 23,190 μm ^2, more than a 23,200μm ² or more).

The (C) colony area variation means a variation between the areas of all colonies formed by pluripotent stem cells present in the acquired image, and the variation index is a standard deviation (SD). There is a significant positive correlation between the reciprocal of colony area variation (SD: standard deviation) and the number of viable cells after nap. The variation of the colony area can be appropriately set according to the desired number of viable cells after nap. The standard value is the variation value of the colony area for efficiently eliminating the culture equipment for culturing pluripotent stem cells that cannot achieve the desired number of viable cells after nap. In the present specification, the term "effectively eliminate" means eliminating 80% or more of the culture equipment for culturing pluripotent stem cells that cannot achieve the desired post-sleeping viable cell number, and preferably 90% or more, more preferably 95% or more, and most preferably 100%.

The standard value is the reciprocal of the variation (SD) of the number of post-sleeping viable cells and the colony area (SD) as long as the culture equipment for culturing pluripotent stem cells that cannot achieve the desired number of post-sleeping viable cells can be efficiently excluded. The threshold value obtained from the bivariate analysis and the ROC-AUC analysis may be used as the standard value, and a value that is more severe than the obtained threshold value may be used as the standard value. For example, when the number of viable cells after nap is desired to be 0.3x10 ⁶ cells or more (that is, the standard in which the number of viable cells after nap is high enough for passaging), the standard value of variation (SD) of the average colony area is , At least less than 130,257 (eg 130,200 or less, less than 57,070, 57,000 or less).

The (D) colony average major axis (μm) means an average value of major axes of all colonies formed by pluripotent stem cells present in the acquired image. There is a significant positive correlation between the reciprocal of the average length of the colony and the number of viable cells after nap. The colony average major axis can be appropriately set in accordance with the desired number of viable cells after nap. The value of the average major axis of colonies for efficiently eliminating the culture equipment for culturing pluripotent stem cells that cannot achieve the desired number of viable cells after nap is called a standard value. In the present specification, the term "effectively eliminate" means eliminating 80% or more of the culture equipment for culturing pluripotent stem cells that cannot achieve the desired post-sleeping viable cell number, and preferably 90% or more, more preferably 95% or more, and most preferably 100%.

The standard value, as long as the culture equipment for culturing pluripotent stem cells that can not achieve the desired post-sleeping viable cell number can be efficiently excluded, a bivariate of the post-sleeping viable cell number and the reciprocal of the colony average major axis The threshold value obtained from the analysis and the ROC-AUC analysis may be used as the standard value, and a value that is more severe than the obtained threshold value may be used as the standard value. For example, when the number of viable cells after nap is desired to be 0.3×10 ⁶ cells or more (that is, the standard in which the number of viable cells after nap is high enough for passage), the standard value of the colony average major axis is at least less than 483 μm ( Examples: 480 μm or less, 418 μm or less, 410 μm or less, 402 μm or less, 400 μm or less, 328 μm or less, 320 μm or less, 261 μm or more, 270 μm or more, 186 μm or more, 190 μm or more).

The above feature amounts (B) to (D) may be used individually as indicators for efficiently eliminating culture equipment for culturing pluripotent stem cells that cannot achieve the desired number of viable cells after nap. It is possible to use them in combination.

In the step (4), comparing the determined characteristic amount with a preset standard value, and selecting a culture device that satisfies the standard value, the determined characteristic amount and preset standard value The value means the above-mentioned characteristic amount and standard value.

The step (5) of cryopreserving the colonies in the selected culture device can be performed by any known method. Such a method can be achieved, for example, by leaving the formed colonies in a freezer or a deep freezer, or by bringing the colonies into contact with a low temperature medium (eg, liquid nitrogen). Not limited. The freezing temperature is typically 0°C or lower, preferably -20°C or lower, more preferably -40°C or lower, still more preferably -80°C or lower. The cooling rate in the freezing operation typically includes a cooling rate of 1 to 5 hours, preferably 2 to 4 hours, and particularly about 3 hours to start cooling at 4°C and reach -80°C. .. Such a cooling rate can be achieved by providing the container containing the colony directly to the freezing means set to a desired temperature, or by accommodating the container in the freezing treatment container. The freezing treatment container may have a function of controlling the rate of decrease of the temperature in the container to a predetermined rate. As such a freezing treatment container, for example, BICELL (registered trademark) (Japan Freezer) or the like can be used. Further, the cooling rate can be achieved by using a freezer or a deep freezer capable of controlling the cooling rate by setting a program or the like. As such a freezer or deep freezer, any known freezer such as a program freezer (for example, PDF-2000G (Strex), KRYO-560-16 (Asahi Life Science)) can be used.

For the freezing operation, a culture solution in which colonies are immersed or a physiological buffer is used as a cryopreservation solution, and a cryoprotective agent is added to this, or the culture solution is replaced with a cryopreservation solution containing a cryoprotective agent. You may go after processing. When substituting the culture solution with the cryopreservation solution, the cryopreservation solution may be added after substantially all of the culture solution is removed, or the cryopreservation solution may be added while leaving a part of the culture solution. As the cryopreservation liquid, a commercially available product may be used, and examples thereof include STEM-CELLBANKER (registered trademark) (ZENOAQ).

The cryoprotective agent used in the present invention is not particularly limited, and examples thereof include dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), 1,2-propanediol (1,2-PD), 1 , 3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol (DPG) and glycerin. Of these, DMSO or 1,2-PD is preferable. The cryoprotective agents may be used alone or in combination of two or more kinds. In addition, the cryoprotective agent may be used in combination with the extracellular cryoprotective agent. Examples of extracellular cryoprotectants include polyethylene glycol, sodium carboxymethyl cellulose, polyvinylpyrrolidone, hydroxyethyl starch (HES), dextran, albumin and the like.

The concentration of the cryoprotective agent added to the culture medium, or the concentration of the cryoprotective agent in the cryopreservation liquid is typically 2 to 20% (v/v) with respect to the entire culture medium or the cryopreservation liquid, preferably Is 5 to 15%, more preferably 8 to 13%. The above concentration can be appropriately adjusted depending on the type of cryoprotective agent.For example, when DMSO is used as the cryoprotective agent, the concentration of DMSO is typically 2 with respect to the whole culture solution or cryopreservation solution. -20% (v/v), preferably 2.5-12.5%, more preferably 5-10%.

2. Cryopreserved pluripotent stem cells The present invention also provides the cryopreserved pluripotent stem cells obtained by the production method of the present invention. As described above, the frozen stock cells and the like of the present invention are "high quality" and have high "stability". In addition, the colonies formed by the frozen stock cells of the present invention have the following 1. Each of the standard values of (A) colony coverage, (B) colony average area, (C) colony area variation, and (D) colony average major axis described in (4) can be satisfied.

The frozen stock cells of the present invention have the above 1. After thawing as described in 1., somatic cells can be differentiated. The method of inducing differentiation of pluripotent stem cells is not limited, and various known differentiation methods can be used. Known differentiation techniques include, for example, T cell differentiation technique, mesenchymal stem cell differentiation technique, hematopoietic stem cell differentiation technique, erythrocyte differentiation technique, platelet differentiation technique, vascular endothelial cell differentiation technique. Method, cardiomyocyte differentiation method, skeletal muscle differentiation method, neural stem cell differentiation method, cerebral cortical nerve cell differentiation method, cerebral limbic nerve cell differentiation method, dopamine nerve cell differentiation method , Hepatocyte differentiation technique, bone differentiation technique, cartilage differentiation technique, primordial germ cell differentiation technique, pancreatic cell differentiation technique, retinal cell differentiation technique, corneal cell differentiation technique, kidney Examples thereof include a cell differentiation method, an alveolar epithelial cell differentiation method, a bronchial epithelial cell differentiation method, and an intestinal differentiation method.

3. Cryopreserved pluripotent stem cells with specific characteristics The present invention also provides colony coverage formed by characteristic (A) cells: more than 7% (eg, 8% or more, 16% or more, 17% or more, 31%). ultra, 32% or more), and wherein (B) colony average area cells form: 141,933Myuemu less than ² (eg: 141,900Myuemu ² or less, less than 115,395μm ^2, 115,300μm ² below, less than 109,218μm ^2, 109,200μm ² hereinafter, less than 66,599μm ^2, 66,500μm ² below, 45,259Myuemu ² greater, 45,300Myuemu ² or more, 23,190Myuemu ² greater, 23,200Myuemu ² or more), (C) variation of colony area cells form: less than 130,257 (e.g. 130,200 or less, less than 57,070, 57,000 or less), and (D) colony average major diameter formed by cells: less than 483 μm (eg: 480 μm or less, 418 μm or less, 410 μm or less, 402 μm or less, 400 μm or less, 328 μm, 320 μm or less, 261 μm or more , 270 μm or more, 186 μm or more, 190 μm or more), which are cryopreserved pluripotent stem cells. Regarding whether or not the cryopreserved pluripotent stem cells have the above-mentioned characteristics, the cryopreserved pluripotent stem cells were selected from the above 1. The cryopreserved pluripotent stem cells are placed under the same conditions as the conditions at the time of cryopreserving the cells in the culture device of step (5) of the method of the present invention described in (i.e., cryopreserved pluripotent cells). After the sex stem cells were put to sleep by the method as described in the above 1, colonies were formed, and the characteristic amount was determined, the cells in the culture device were cryopreserved to the same state as when they were cryopreserved. It can be evaluated by culturing pluripotent stem cells).

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

Example 1: Calculation of feature amount
As the iPS cells, 201B7 strain and 1210B2 strain of iPS cells purchased from Academia Japan Co., Ltd. or iPS Portal Co., Ltd. were used. For culturing, a culture vessel coated with iMatrix-511 (Nippi: 892012) was used as a scaffold, and StemFit(R) AK03N was used as a medium. The culture conditions were 5% CO ₂ /37°C. At the time of cell seeding, a medium supplemented with a final concentration of 10 μM Y-27632 (Wako Pure Chemical Industries, Ltd.: 036-24023) was used, and in the evaluation on the next day or later, it was cultured in a medium not supplemented with Y-27632. A 10-cm dish was used as a culture container for preparing the cell stock, and cells were seeded in the range of 1×10 ^{5 to} 6×10 ⁵ cells/sheet in a single cell state. After seeding, the cells were cultured for 5 to 7 days to prepare a cell stock. STEM-CELLBANKER (registered trademark) (ZENOAQ) was used as a cryopreservation solution, and CoolCell (registered trademark) LX (biosicion) was used as a cryopreservation container, and the cells were cryopreserved (2×10 ⁵ cells/200 μL/vial). went. To extract features from cell images before cell stock preparation, use phase-contrast images with all-in-one fluorescence microscope (KEYENCE: BZ-X710 and BZ-X700) and image analysis with analysis software (BZ-X Analyzer) I was there. "Coverage", "colony area", and "colony major axis" were calculated as the characteristic amounts obtained from the images.

Example 2: Confirmation of quality of cryopreserved pluripotent stem cells On the other hand, a 6-well plate was used as the culture container after cell nap, and immediately after nap, 65,000 cells/well for 201B7 strain and 40,000 cells/well for 1210B2 strain. After seeding and culturing for 5 days, the number of viable cells after nap was automatically counted using a live and dead cell autoanalyzer (model number: ViCELL ^™ XR; hereinafter, ViCELL) manufactured by BECKMAN COULTER. The 201B7 strain was subcultured every week thereafter at a seeding number of 13,000 cells/well, and the culture was continued for up to 7 weeks, and it was confirmed that stable culture was possible. At each passage, the number of viable cells was measured by ViCELL and recorded.

Example 3: Setting of each standard value From the results of the characteristic amount extracted from the image analysis by the KEYENCE analysis software and the culture evaluation result, the bivariate correlations were confirmed, and the values shown in FIG. 1 were obtained. After nap, the number of viable cells was 0.3x10 ⁶ cells or more, and the number of cell stocks produced was 12 or more. Accepted sample (marked with "○" in Fig. 1), and less than deviated sample ("×" in Fig. 1) Symbol)). From this, statistically optimum threshold values were calculated by ROC-AUC analysis, and the results shown in FIG. 2 were obtained. From the results, "coverage ratio vs. cell stock production number", "reciprocal of colony average area vs. number of viable cells after nap", "variation of colony area variation (SD: standard deviation) vs. number of viable cells after nap", and It was suggested that there is a significant (p value of 0.01 or less) correlation in "reciprocal of long diameter vs. number of viable cells after nap".

Furthermore, regarding the “judgment result of the coverage rate versus the number of cell stocks produced”, the AUC (transition in the range of 0 to 1, the highest predictive ability at 1) was 0.9423 for 201B7 strain and 0.8788 for 1210B2 strain It was revealed that the predicting ability of "the number of cell stocks produced" by the "coverage" was sufficiently high in both cell lines. Similarly, in the "colony average area", "variation of colony area", "judgment number of viable cells after nap" for each "colony average major axis", AUC is 0.9546 (colony average area) in 201B7 strain, 0.8939 ( The colony area varied), 0.9546 (colony average major axis), and the 1210B2 strain had high values of 0.8889 (colony average area), 0.8095 (colony area variation), and 0.9206 (colony average major axis). It was revealed that the "average area", "variation in colony area", and "average length of colony" were sufficiently high in predicting the "results of determination of the number of viable cells after sleep" (Fig. 2).

From the above, from the results of ROC-AUC analysis, when calculating the cutoff value for each index by Youden index ("sensitivity + specificity -1" is the maximum), in 201B7 strain "coverage" 7%, " The average colony area was 115,395 μm ² , the “variation in colony area” was 57,070, and the average longest colony was 402 μm (Table 1). Similarly, for the 1210B2 strain, the “coverage” was 16%, the “colony average area” was 141,933 μm ² , the “variation of colony area” was 130,257, and the “colony average major axis” was 483 μm (Table 1). Furthermore, the value for 100% exclusion of cells other than the desired cells, that is, the value when the false positive rate is the lowest when the sensitivity is 1, is 7% for the 201B7 strain, 7% for the average colony area, and 66,599 μm for the colony average area. ² , "variation of colony area" 57,070, "colony average major axis" 328 μm (Table 1). Similarly, in the 1210B2 strain, “coverage” was 31%, “colony average area” was 109,218 μm ² , “colony area variation” was 130,257, and “colony average major axis” was 418 μm (Table 1). With the above value as the threshold value, "coverage" exceeds this, and "colony average area", "variation of colony area" and "colony average major axis" fall below this by using standard values. It was considered that cells other than the desired cells could be efficiently eliminated.

The method of the present invention provides for "stability" (ie, frozen cell stock) of "high quality" (ie, viable cell numbers after nap) that are sufficient for passage to cryopreserved pluripotent stem cells. Is obtained at a sufficient amount at one time), which is useful.

Claims (10)

A method for producing cryopreserved pluripotent stem cells, comprising the steps of:
(1) preparing pluripotent stem cells,
(2) culturing the pluripotent stem cells prepared in step (1) in a culture device to form colonies of the pluripotent stem cells,
(3) In the culture device having the formed colony, the following characteristic amount (A):
(A) Colony coverage, and the following feature quantities (B) to (D):
(B) average colony area,
(C) colony area variation, and (D) colony average major axis,
Determining at least one selected from
(4) A step of comparing the determined characteristic amount with a preset standard value to select a culture device satisfying the standard value, and (5) cryopreserving the cells in the selected culture device. Process The method according to claim 1, wherein the cryopreserved pluripotent stem cells are cells that can be cultured for 7 weeks or more. The method according to claim 1 or 2, wherein the pluripotent stem cells are induced pluripotent stem cells. The method according to any one of claims 1 to 3, comprising a step of establishing pluripotent stem cells before the step (1). The method according to any one of claims 1 to 3, which comprises a step of thawing the cryopreserved pluripotent stem cells before the step (1). The preset standard value is as follows:
(A) Colony coverage: more than 7%,
(B) Average colony area: less than 141,933 μm ² (C) Variation in colony area: less than 130,257 (D) Average length of colony: less than 483 μm, The method according to any one of claims 1 to 5. Cryopreserved pluripotent stem cells obtained by the method according to any one of claims 1 to 6. Cryopreserved pluripotent stem cells having at least one selected from the following characteristics (A) and (B) to (D).
(A) Colony coverage formed by the cells: more than 7% (B) Average area of colonies formed by the cells: less than 141,933 μm ² (C) Variation in colony area formed by the cells: less than 130,257 (D) Average major axis of colonies formed by cells: less than 483 μm 9. The cell according to claim 7 or 8, wherein the cryopreserved pluripotent stem cell is a cell that can be cultured for 7 weeks or more. The cell according to any one of claims 7 to 9, wherein the pluripotent stem cell is an induced pluripotent stem cell.