Method for producing cell product and method for controlling quality of cells

Abstract

The present invention comprises: a step for culturing and proliferating cells separated from a cell source; a step for recovering the proliferated cells; and a step for filling a container with the recovered cells. In at least one stage selected from the group consisting of a stage before the culturing and proliferating step, a stage between the culturing and proliferating step and the step for filling the container, and a stage after the step for filling the container, a soft agar colony formation evaluation test is performed on the cells, and whether or not the cells filled in the container can be used is determined on the basis of the result of the soft agar colony formation evaluation test.

Description

Methods for manufacturing cell products and for controlling cell quality.

[0001] This disclosure relates to a method for manufacturing cell products and a method for quality control of cells.

[0002] In recent years, advances in regenerative medicine technology, cell therapy technology, and related basic research have led to advances in cell culture and proliferation technology. Cells used in regenerative medicine technology, cell therapy technology, and related basic research are developed to possess the desired properties, and then cultured and proliferated to produce products. Cells produced as products (hereinafter referred to as cell products) are evaluated for safety, such as tumorigenicity, during the development process. Cells whose safety has been confirmed during the development process are stored in the form of frozen cells and used as raw materials for cell products in the manufacturing process as needed.

[0003] For example, Japanese Patent Publication No. 2012-157263 discloses a process for developing a cell population that is free from tumorigenicity or chromosomal abnormalities in ultra-low-immunity NOG mouse tumorigenicity tests, chromosomal aberration tests, and soft agar colony tests, providing highly safe adipose tissue-derived mesenchymal stem cells effective in treating Alzheimer's disease. Furthermore, Sci Rep. 2015 Dec 8:5:17892. doi:10.1038 / srep17892 discloses a technique for evaluating the contamination of normal cells with cancer cells using a digital soft agar colony formation test.

[0004] [Patent Document 1] Japanese Unexamined Patent Publication No. 2012-157263

[0005] [Non-patent Document 1] Sci Rep. 2015 Dec 8:5:17892. doi:10.1038 / srep17892.

[0006] However, while both Japanese Patent Publication No. 2012-157263 and Sci Rep. 2015 Dec 8:5:17892. doi:10.1038 / srep17892 disclose the evaluation of tumorigenicity during the cell development process, they do not disclose the evaluation of safety, such as tumorigenicity, for cell products to be shipped. Therefore, this disclosure aims to provide a method for manufacturing cell products and a method for quality control of cells that can provide cell products with ensured safety, such as tumorigenicity.

[0007] Having achieved the objectives described above, this disclosure includes the following:

[0008] <1> A method for producing a cell product, comprising the steps of culturing and growing cells isolated from a cell source, collecting the grown cells, and filling the collected cells into a container, wherein a soft agar colony formation evaluation test is performed on the cells at at least one stage selected from the group consisting of before the culturing and growing step, between the culturing and growing step and the filling step into the container, and after the filling step into the container, and the suitability of the cells filled into the container is determined based on the results of the soft agar colony formation evaluation test. <2> The method for producing a cell product according to <1>, wherein the step of filling into a container is to freeze the cells filled into the container. <3> The method for producing a cell product according to <1> or <2>, wherein a soft agar colony formation evaluation test is performed using 0.1% to 10% of the collected cells based on cell number. <4> The method for producing a cell product according to any one of <1> to <3>, wherein the test is deemed successful if the cell viability in the soft agar colony formation evaluation test is 70% or more. <5> A method for producing a cell product according to any one of <1> to <4>, wherein the soft agar colony formation evaluation test is performed multiple times. <6> A method for producing a cell product according to any one of <1> to <5>, wherein the cells are allogeneic cells isolated from a cell source and cryopreserved. <7> A method for producing a cell product according to any one of <1> to <6>, wherein the cells are at least one type of cell selected from the group consisting of somatic stem cells, induced pluripotent stem cells, embryonic stem cells, differentiated cells derived from somatic stem cells, differentiated cells derived from induced pluripotent stem cells, somatic stem cells derived from induced pluripotent stem cells, differentiated cells derived from embryonic stem cells, and somatic stem cells derived from embryonic stem cells. <8> A method for producing a cell product according to any one of <1> to <7>, wherein the cells are adherent cells. <9> A method for producing a cell product according to any one of <1> to <8>, wherein the cells are mesenchymal stem cells. <10> A method for producing a cell product according to any one of <1> to <9>, wherein the cells are cells used as joint treatment drugs. <11> A method for producing a cell product according to any one of <1> to <10>, wherein the cells are cells for drug discovery.<12> A method for producing a cell product according to any one of <1> to <11>, wherein in the step of culturing and growing cells, multiple different lots are prepared, the cells contained in each lot are cultured and grown, and the suitability of each recovered cell is determined based on the results of the soft agar colony formation evaluation test.

[0009] <13> A cell quality control method comprising the steps of culturing and growing cells isolated from a cell source, collecting the grown cells, and filling the collected cells into a container, wherein a soft agar colony formation evaluation test is performed on the cells at at least one stage selected from the group consisting of before the culturing and growing step, between the culturing and growing step and the filling step into the container, and after the filling step into the container, and the suitability of the cells filled into the container is determined based on the results of the soft agar colony formation evaluation test. <14> The cell quality control method according to <13>, wherein the step of filling into a container includes a step of freezing the cells filled into the container. <15> The cell quality control method according to <13> or <14>, wherein a soft agar colony formation evaluation test is performed using 0.1% to 10% of the collected cells based on cell number. <16> The cell quality control method according to any one of <13> to <15>, wherein the test is deemed successful if the cell viability in the soft agar colony formation evaluation test is 70% or more. <17> A cell quality control method according to any one of <13> to <16>, wherein the soft agar colony formation evaluation test is performed multiple times. <18> A cell quality control method according to any one of <13> to <17>, wherein the cells are allogeneic cells isolated from a cell source and cryopreserved. <19> A cell quality control method according to any one of <13> to <18>, wherein the cells are at least one type of cell selected from the group consisting of somatic stem cells, induced pluripotent stem cells, embryonic stem cells, differentiated cells derived from somatic stem cells, differentiated cells derived from induced pluripotent stem cells, somatic stem cells derived from induced pluripotent stem cells, differentiated cells derived from embryonic stem cells, and somatic stem cells derived from embryonic stem cells. <20> A cell quality control method according to any one of <13> to <19>, wherein the cells are adherent cells. <21> A cell quality control method according to any one of <13> to <20>, wherein the cells are mesenchymal stem cells. <22> A method for controlling the quality of cells according to any one of <13> to <21>, wherein the cells are cells used as joint treatment drugs. <23> A method for controlling the quality of cells according to any one of <13> to <22>, wherein the cells are cells for drug discovery.<24> A cell quality control method according to any one of <13> to <23>, wherein in the step of culturing and growing cells, multiple different lots are prepared, the cells contained in each lot are cultured and grown separately, and the suitability of each recovered cell for use is determined based on the results of the soft agar colony formation evaluation test.

[0010] According to the method for manufacturing cell products and the method for quality control of cells described herein, it is possible to provide cell products that ensure safety, such as tumorigenicity.

[0011] The embodiments of this disclosure are described below. The description is illustrative and does not limit the scope of this disclosure.

[0012] In the following embodiments, the components (including elemental steps, etc.) are not essential unless otherwise explicitly stated. The same applies to numerical values ​​and their ranges, and these do not limit the disclosure. For example, the disclosure allows for additions, omissions, substitutions, and changes to numbers, quantities, locations, ratios, materials, compositions, types, and sequences, etc., without departing from the intent of the disclosure.

[0013] In this disclosure, numerical ranges indicated using "~" include the numbers before and after "~" as the minimum and maximum values, respectively. In numerical ranges described in stages in this disclosure, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Also, in numerical ranges described in this disclosure, the upper or lower limit of that numerical range may be replaced with the values ​​shown in the examples. In this disclosure, each component may contain multiple types of the corresponding substance. If multiple types of the substance corresponding to each component exist in the composition, the content or amount of each component means the total content or amount of the multiple types of substances present in the composition, unless otherwise specified. The term "process" in this specification includes not only independent processes but also processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved.

[0014] <Method for Manufacturing Cell Products and Method for Controlling Cell Quality> The method for manufacturing cell products and the method for controlling cell quality described herein (hereinafter collectively referred to as the "method of disclosure") include the steps of culturing and growing cells isolated from a cell source, collecting the grown cells, and filling the collected cells into a container. At least one stage (period) selected from the group consisting of before the culturing and growing step, between the culturing and growing step and the filling into the container step, and after the filling into the container step, a soft agar colony formation evaluation test is performed on the cells, and the suitability of the cells filled into the container is determined based on the results of the soft agar colony formation evaluation test. According to the method of disclosure, the suitability of the cultured and grown cells for use is determined in terms of safety at the latest before use. Therefore, according to the method of disclosure, it is possible to provide cell products with ensured safety, such as tumorigenicity. As a result, by applying the method of disclosure, cell products with ensured safety can be used in regenerative medicine technology, cell therapy technology, or research on these technologies. Ensuring safety means that it has been confirmed that the product does not have tumorigenic properties during the manufacturing process, and does not guarantee, for example, that tumors will not form when used in regenerative medicine or cell therapy technologies. In the method disclosed herein, if a soft agar colony formation evaluation test is conducted at a stage after the process of filling into containers, it may be conducted not only by the cell product manufacturing facility, but also by the pharmaceutical company that received the cell product, healthcare professionals, etc. Furthermore, the cell product manufacturing facility or the pharmaceutical company that received the cell product may encourage healthcare professionals, etc. to conduct a soft agar colony formation evaluation test through product labels, etc. In other words, in the method disclosed herein, the decision on whether or not to use the cell product may be made not only by the cell product manufacturing facility, but also by the pharmaceutical company that received the cell product, healthcare professionals, etc. Furthermore, the method disclosed herein also includes the possibility that the cell product manufacturing facility or the pharmaceutical company that received the cell product may encourage healthcare professionals, etc. to make a decision on whether or not to use the cell product through product labels, etc.

[0015] The method disclosed herein manufactures cell products used in regenerative medicine technologies, cell therapy technologies, or research related to these technologies, as described above. Generally, the manufacture of cell products involves a process of developing the target cells, such as isolating the desired type of cells from a cell source, and a process of culturing and growing the developed cells to produce the product. Conventionally, the safety of cell products was ensured by the results of tumorigenicity tests (ultra-low immunity NOG mouse tumorigenicity test, chromosomal aberration test, soft agar colony formation evaluation test) conducted during the development process. In other words, conventionally, cells that were judged to have low tumorigenicity and be safe during the development process were cultured and grown to become cell products. Unlike conventional methods, the method disclosed herein ensures the safety of cell products by conducting a soft agar colony formation evaluation test on the cells at the manufacturing stage, which is performed after the development stage. As described above, the method disclosed herein makes it possible to manufacture extremely safe cell products, as cells that should be avoided for use as cell products, such as cells that acquire tumorigenicity during the cell culture and growth process, do not become cell products.

[0016] The method of this disclosure allows for the storage of cell products until they are used in regenerative medicine technologies, cell therapy technologies, or research related to these technologies, by filling cells into containers. In this case, the cells stored in the containers can be stored at low temperatures, for example, 0°C to 10°C, preferably 2°C to 8°C. Furthermore, in the method of this disclosure, it is preferable to freeze the cells filled into the containers during the filling step. In this case, the cells stored in the containers can be stored at a freezing temperature, for example, -80°C or lower, preferably -100°C or lower, more preferably -140°C or lower.

[0017] When filling a container with cells, they can be suspended in a liquid medium (e.g., culture medium, dimethyl sulfoxide (DMSO), etc.). The composition containing cells and medium in the container can have any cell concentration. For example, the cell concentration can be 1.0 × 10⁶ cells / mL, 1.0 × 10⁶ cells / mL, etc. ２ pieces / mL, 1.0×10 ３ pieces / mL, 1.0×10 ４ pieces / mL, 1.0×10 ５ pieces / mL, 1.0×10６ cells / mL, 1.0 × 10 ７ cells / mL, 1.0 × 10 ８ cells / mL, 1.0 × 10 ９ cells / mL, 1.0 × 10 １０ cells / mL, or a cell concentration higher than or lower than the above values, but the cell concentration is not limited thereto.

[0018] When freezing cells filled in a container, it is preferable that the cells are in a state of being suspended in a cryopreservation solution. Examples of the cryopreservation solution include CP-1 (manufactured by Kyokuto Pharmaceutical Industrial Co., Ltd.), BAMBANKER (manufactured by Lymphotec Inc.), STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.), ReproCryo RM (manufactured by Reprocell Inc.), CryoNovo (manufactured by Akron Biotechnology), MSC Freezing Solution (manufactured by Biological Industries), CryoStor (manufactured by HemaCare), and the like. The cell concentration in the cryopreservation solution is also the same as the cell concentration in the composition comprising the cells and the liquid medium described above.

[0019] As described above, in the method of the present disclosure, by cryopreserving or preserving cells at low temperature in a container, highly safe cell products can be maintained until they are used in regenerative medicine technology, cell therapy technology, research related to these technologies, or the like. In addition, in the method of the present disclosure, by preserving cells at low temperature or cryopreserving them in a container, the cells can be maintained until a cell product with ensured safety based on the results of a tumorigenicity test is obtained.

[0020] In the method disclosed herein, the soft agar colony formation evaluation test is performed before the cell culture and growth step, between the cell culture and growth step and the container filling step, or after the container filling step, but it is preferable to perform the test using cells recovered in the cell recovery step. In this case, in the method disclosed herein, it is preferable to perform the soft agar colony formation evaluation test using 0.1% to 10% of the recovered cells by cell number, more preferably 0.1% to 5%, and even more preferably 0.1% to 1%. Furthermore, in the method disclosed herein, the soft agar colony formation evaluation test can be performed using less than 0.1% of the recovered cells by cell number, for example, cells of 0.01% or less by cell number may be used, or cells of 0.001% or less may be used. In the method disclosed herein, by using cells within the above range from the recovered cells, a cell product containing a sufficient number of cells can be obtained, and a sufficient number of cells can also be secured for the soft agar colony formation evaluation test.

[0021] In the soft agar colony formation evaluation test described herein, the test is deemed successful if the cell viability is 70% or higher, preferably 75% or higher, more preferably 80% or higher, and even more preferably 85% or higher. Cell viability is measured using a hemocytometer with trypan blue staining. The soft agar colony formation evaluation test involves coating a semi-solid soft agar medium with a cell population that may contain tumorigenic cells, thereby preventing normal cells from adhering to and proliferating. In the soft agar colony formation evaluation test, tumorigenic cells proliferate without adhering to the substrate surface, and as a result, tumorigenic cells can be selectively separated and measured. The soft agar colony formation evaluation test can be performed using a commercially available kit. Commercially available kits for performing the soft agar colony formation evaluation test are not particularly limited, but include the CytoSelect 96-Well Cell Transformation Assay. Passing the soft agar colony test means that the cell product can be used as a safe product. As a criterion for passing, for example, if the measured value for each group on Day 21 is less than or equal to the measured value for that group on Day 0 + 3.3 × SD, then it can be determined that there is no tumorigenicity, i.e., that it is a pass.

[0022] In the method disclosed herein, a soft agar colony formation evaluation test is performed as an indicator for determining tumorigenicity, but it is particularly preferable not to perform genome instability tests such as karyotype analysis as other tumorigenicity tests. Genome instability tests, such as karyotype analysis, test for changes in chromosomes and genes, and determine whether the chromosomes of cells are normal, abnormal, or changed by examining them. For example, cells isolated from animal tissue, including human tissue, have a very high probability of containing cells with karyotype changes or chromosomal changes, and tumorigenicity cannot be accurately determined from genome instability tests. Therefore, in this disclosure, it is preferable not to use the results of genome instability tests such as karyotype analysis as an indicator of tumorigenicity.

[0023] Furthermore, in the method disclosed herein, it is preferable to perform the soft agar colony formation evaluation test multiple times before the culture and growth step, between the culture and growth step and the container filling step, or after the container filling step. In the method disclosed herein, the number of times the soft agar colony formation evaluation test is performed is not particularly limited, but it may be once, two or more times, three or more times, or four or more times. In particular, by performing the soft agar colony formation evaluation test multiple times and passing these multiple soft agar colony formation evaluation tests, it is possible to provide a cell product with superior safety.

[0024] For example, the soft agar colony formation evaluation test can be performed once after the step of collecting the proliferated cells, or after the step of filling the collected cells into a container. Furthermore, if the cells are frozen in the step of filling the container in the method disclosed herein, the soft agar colony formation evaluation test may be performed after thawing the cells stored in the container in a frozen state.

[0025] Furthermore, the method disclosed herein can be used to manufacture cell products based on cell lines isolated from a cell source, cell lines stored under refrigeration or cryopreservation, or cell lines provided from a cell bank (hereinafter referred to as intermediate cell lines). In this case, the soft agar colony formation evaluation test can be performed after the cultured intermediate cell lines are recovered, after the recovered intermediate cell lines are filled into containers, or after the frozen intermediate cell lines are thawed. In this way, by performing a soft agar colony formation evaluation test on intermediate cell lines and manufacturing cell products based on intermediate cell lines that pass the soft agar colony formation evaluation test, it is possible to provide cell products with superior safety.

[0026] Furthermore, in the method of this disclosure, it is preferable to prepare multiple different lots in the step of culturing and growing cells, culture and grow the cells contained in each lot, and determine whether each recovered cell is suitable for use based on the results of the soft agar colony formation evaluation test. A lot refers to a generally uniform population of cells. A uniform population of cells means that the content of different cells is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%. In the method of this disclosure, the cells contained in different lots may be the same cells or different cells. In the method of this disclosure, the same cells contained in different lots can be cultured and grown lot by lot, and the safety of the cells cultured lot by lot can be evaluated. Then, those whose safety has been ensured based on the results of the soft agar colony formation evaluation test can be combined into a single cell product.

[0027] -Cells- In this disclosure, cells may be either the organism's own cells (autologous cells) or cells isolated from an allogeneic or heterogeneous biological sample (allogeneic cells). In particular, the cells cultured and proliferated in this disclosure may be allogeneic cells that have been separated from a cell source and cryopreserved. Allogeneic cells refer to cells isolated from an allogeneic or heterogeneous biological sample. For example, when applying regenerative medicine technology, cell therapy technology, etc., to a patient, human or animal cells other than those of that patient are referred to as allogeneic cells. When manufacturing cell products using allogeneic cells, using cryopreserved allogeneic cells in particular can stabilize the quality of the cell product and allow for low-cost manufacturing.

[0028] Cryopreserved allogeneic cells are a form of the intermediate cell line described above. Therefore, when using cryopreserved allogeneic cells as cells in this disclosure, the soft agar colony formation evaluation test can be performed after the cultured and grown allogeneic cells are collected, after the collected allogeneic cells are filled into a container, or after the frozen allogeneic cells are thawed. In this way, by performing the soft agar colony formation evaluation test on allogeneic cells and manufacturing cell products based on allogeneic cells that pass the soft agar colony formation evaluation test, it is possible to provide cell products with superior safety.

[0029] The cell source is not particularly limited and may include bone marrow, adipose tissue, dental pulp, placenta, umbilical cord, amniotic membrane, synovial membrane, etc. Furthermore, these cell sources may be of human origin or of non-human animal origin. Examples of non-human animals include, but are not limited to, mammals such as dogs, cats, cattle, horses, pigs, goats, sheep, monkeys (crab-eating macaques, rhesus macaques, common marmosets, Japanese macaques), ferrets, rabbits, and rodents (mice, rats, gerbils, guinea pigs, hamsters), as well as birds such as chickens and quail.

[0030] Furthermore, in this disclosure, the cells that form the basis of the cell product are not particularly limited and may be at least one type of cell selected from the group consisting of somatic stem cells, induced pluripotent stem cells, embryonic stem cells, differentiated cells derived from somatic stem cells, differentiated cells derived from induced pluripotent stem cells, somatic stem cells derived from induced pluripotent stem cells, differentiated cells derived from embryonic stem cells, and somatic stem cells derived from embryonic stem cells.

[0031] Somatic stem cells are stem cells that exist in various tissues of adults, have not yet completed terminal differentiation, and possess multipotency. Somatic stem cells are also called adult stem cells or tissue stem cells. Specific examples of somatic stem cells include mesenchymal stem cells, neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, and pigment stem cells. Mesenchymal stem cells (MSCs) are cells that primarily differentiate into mesenchymal cells such as osteoblasts, adipocytes, muscle cells, and chondrocytes. Neural stem cells are cells that primarily differentiate into nerve cells and glial cells. Intestinal epithelial stem cells are cells that primarily differentiate into epithelial cells that make up the inner wall of the digestive tract, such as the small and large intestines. Hematopoietic stem cells are cells that primarily differentiate into blood cells such as red blood cells, white blood cells, and platelets. "Hair follicle stem cells" are cells that have the ability to differentiate into hair follicle epithelial cells such as hair stem cells and hair root sheath cells, as well as sebaceous gland cells and basal cells. "Pigment stem cells" are cells that primarily have the ability to differentiate into pigment cells.

[0032] Induced pluripotent stem cells (iPS cells) are pluripotent cells obtained by reprogramming somatic cells. Induced pluripotent stem cells are produced using, for example, somatic cell nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, and Myc family genes, as well as somatic cell nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, Sox family genes, and Myc family genes. The type of somatic cell used to produce iPS cells is not particularly limited, and any somatic cell can be used. Somatic cells include all cells that make up a living organism except germ cells, and may be differentiated somatic cells or undifferentiated stem cells. When using human somatic cells, fetal, neonatal, or adult somatic cells may be used. Specific examples of somatic cells include, for instance, fibroblasts (e.g., skin fibroblasts), epithelial cells (e.g., gastric epithelial cells, hepatic epithelial cells, alveolar epithelial cells), endothelial cells (e.g., blood vessels, lymphatic vessels), nerve cells (e.g., neurons, glial cells), pancreatic cells, blood cells, bone marrow cells, muscle cells (e.g., skeletal muscle cells, smooth muscle cells, cardiomyocytes), hepatocytes, non-hepatocytes, adipocytes, osteoblasts, cells that make up periodontal tissue (e.g., periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), and cells that make up the synovial membrane, kidneys, eyes, and ears.

[0033] Embryonic stem cells (ES cells) are stem cell lines derived from the inner cell mass of an embryo during the blastocyst stage, an early stage of animal development. They can be proliferated almost indefinitely while maintaining pluripotency, the ability to differentiate into any tissue outside the body. As ES cells, for example, cells into which a reporter gene has been introduced near the Pdx1 gene can be used to facilitate confirmation of the degree of differentiation. For example, 129 / Sv-derived ES cell lines with the LacZ gene incorporated into the Pdx1 locus, or ES cell line SK7 with a GFP reporter transgene under the control of the Pdx1 promoter can be used. Alternatively, ES cell line PH3, which has an mRFP1 reporter transgene under the control of an Hnf3β endoderm-specific enhancer fragment and a GFP reporter transgene under the control of the Pdx1 promoter, can also be used. Additionally, known ES cell lines such as SEES1, SEES2, SEES3, SEES4, SEES5, SEES6, or SEES7, or cell lines obtained by introducing further genes into these ES cell lines, can also be used.

[0034] Differentiated cells derived from somatic stem cells, differentiated cells derived from induced pluripotent stem cells, and differentiated cells derived from embryonic stem cells refer to cells obtained by differentiating somatic stem cells, induced pluripotent stem cells, and embryonic stem cells, respectively. For example, differentiation of stem cells can be induced and promoted by culturing somatic stem cells, induced pluripotent stem cells, and embryonic stem cells in a differentiation induction medium. This differentiation induction medium contains components that induce or promote the differentiation of stem cells, etc. Examples of components that suppress the differentiation of stem cells, etc. include leukemia inhibitory factor (LIF), fibroblast growth factor (FGF), and transforming growth factor (TGF)-β, which are differentiation inhibitors of embryonic stem cells, and bone morphogenetic protein (BMP) and Notch protein, which suppress the differentiation of neural stem cells.

[0035] Further, examples of components that induce or promote differentiation include activin A, which induces embryonic stem cells into endodermal cells, retinoic acid, a bone morphogenetic protein (BMP) inhibitor (such as noggin) that induces differentiation of iPS cells into neuroectoderm, transforming growth factor (TGF)-β, exocrine glycoprotein (WNT) that induces differentiation of iPS cells into mesoderm, activin that induces differentiation of iPS cells into mesoderm and endoderm, and glycogen synthase kinase 3 (GSK3) inhibitors. Furthermore, after initial differentiation into ectodermal cells, mesodermal cells, and endodermal cells, when differentiating into cells of each organ or tissue, necessary growth factors, nutritional factors, and the like can be added depending on the organ or tissue to be subjected to differentiation induction. For example, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), and the like are used.

[0036] In particular, in the method of the present disclosure, it is preferable that the cell is an adherent cell. Adherent cell means a cell that exhibits adhesion to plastic under standard culture conditions using a standard medium. Mesenchymal stromal cells and mesenchymal stem cells are a type of adherent cell. Therefore, in the method of the present disclosure, it is particularly preferable that the cell is a mesenchymal stem cell. When an adherent cell transforms into a tumorigenic cell (transformed cell), anchorage-independent proliferation becomes possible. When a soft agar colony formation test is applied as a tumorigenicity test, the presence or absence of tumorigenic cells can be measured by observing colony formation.

[0037] Examples of adherent stem cells include, among somatic stem cells (tissue stem cells), bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, stem cells in umbilical cord blood, umbilical cord-derived stem cells, amniotic membrane-derived stem cells, amniotic fluid stem cells, placental villus cell-derived mesenchymal stem cells, neural stem cells, adipose tissue-derived stem cells, pancreatic stem cells, synovial mesenchymal stem cells, dental pulp stem cells, exfoliated deciduous tooth-derived dental pulp stem cells, spermatogonial stem cells (GS cells), testicular multipotent stem cells (mGS cells), corneal epithelial stem cells, corneal stromal stem cells, pigment stem cells, tissue stem cells in organs, and the like.

[0038] Among others, in the method of the present disclosure, it is preferable that the cells are cells used as a therapeutic agent for joints. Specifically, examples of cells used as a therapeutic agent for joints include synovial mesenchymal stem cells. However, the method of the present disclosure is not limited to those for producing cell products used as therapeutic agents for joints, and for example, may be those for producing cell products by culturing cells for drug discovery.

[0039] Cells for drug discovery is meant to include both forms used as pharmaceutical compositions that can be used for treatment of various diseases and forms used for developing pharmaceutical compositions for treating various diseases. The various diseases are not particularly limited, and examples include immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defect, meniscus injury, osteochondritis dissecans, avascular necrosis, osteoarthritis of the knee, inflammatory arthritis, rheumatoid arthritis, ocular diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, wound, epithelial injury, fibrosis, pulmonary diseases, and cancer.

[0040] Mesenchymal stem cells can be confirmed by detecting molecules characteristic of mesenchymal stem cells, for example, enzymes, receptors, low-molecular-weight compounds, and the like. Examples of molecules characteristic of mesenchymal stem cells include cell surface markers (positive markers) such as CD73, CD90, CD105, and CD166. Further, examples of negative markers that are not expressed in mesenchymal stem cells include CD19, CD34, CD45, HLA-DR, CD11b, and CD14. CD is an abbreviation for Clusters of differentiation, and HLA-DR is an abbreviation for human leukocyte antigen D-related. These positive markers and negative markers can be used to confirm that the cells are mesenchymal stem cells.

[0041] Detection of these markers may include, but is not limited to, flow cytometry or cell staining. In flow cytometry using a fluorescently labeled antibody, if cells that fluoresce more strongly than the negative control (isotype control) are detected, those cells are judged to be "positive" for the marker. Any antibody known in the art can be used as the fluorescently labeled antibody, and is not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc. In cell staining, if cells that are stained or fluoresce are observed under a microscope, those cells are judged to be "positive" for the marker. Cell staining may be immunohistochemistry using antibodies or non-immunohistochemistry without antibodies. Furthermore, detection of the markers may be performed by quantifying the mRNA amount of each molecule.

[0042] In particular, synovial mesenchymal stem cells are stem cells contained in the synovial membrane. Synovial mesenchymal stem cells can be identified, for example, by detecting CD90 positivity, CD45 negativity, and chondrogenic differentiation potential. Synovial stem cells can also be identified, for example, by the method described in Nakamura, Kentaro et al., International Journal of Molecular Sciences 25.19 (2024):10510.

[0043] -Culture and Growth- The method of this disclosure involves the step of culturing and growing the cells described above. The culture medium used for culture and growth can be prepared by using any liquid culture medium for animal cells as the base medium and adding other components (albumin, serum, serum substitute reagent, growth factor, human platelet lysate, etc.) as appropriate as needed.

[0044] The basal media are not particularly limited, but include BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification of Minimum Essential Medium Eagle) medium, and DMEM medium (Dulbecco's Modified Eagle's). Medium, Ham F10 medium, Ham F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media of these (for example, DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)) can be used.

[0045] Furthermore, commercially available serum-free media may be used as the culture medium for growth in this disclosure. Examples include STK1, STK2 (manufactured by DS Pharma Biomedical), EXPREP MSC Medium (manufactured by Biomimetics Sympathies), and Corning stemgro Human Adherent Stem Cell Medium (manufactured by Corning).

[0046] Other components to be added to the basal medium include, for example, albumin, serum, serum substitute reagents, growth factors, or human platelet lysates. In embodiments where albumin is added to the basal medium, the albumin concentration is preferably more than 0.05% and 5% or less. In embodiments where serum is added to the basal medium, the serum concentration is preferably 5% or more. In embodiments where growth factors are added, reagents for stabilizing the growth factors in the medium (such as proteins like heparin, gels, or polysaccharides) may be added in addition to the growth factors, or pre-stabilized growth factors may be added to the basal medium. Examples of growth factors that can be used include, but are not particularly limited to, fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and their families.

[0047] To culture and grow the cells described above in this disclosure, first, the cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in culture medium. Next, the cells are seeded in a plastic culture vessel and a CO2 solution of 3% to 5% is added. ２ The culture is performed using a culture medium at a concentration and temperature of 37°C, so that the confluence rate is 95% or less. The culture period for one culture can range from 2 to 15 days, and more specifically, it can be 2, 3, 4, 5, 6, 8, 10, 12, 14, or 15 days.

[0048] These cells, cultured once, can be further subcultured as follows: First, the cells that have been cultured once are detached from the plastic culture vessel using a cell detachment device. Next, the resulting cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in culture medium. Finally, the cells are seeded in a plastic culture vessel and cultured in a CO2 solution of 3% to 5%. ２The cells are cultured in a culture medium at a concentration of 95% or less, at a temperature of 37°C, using a culture medium. Cells obtained by this subculturing method are cells that have been subculturished once. By performing similar subculturing and culturing, cells that have been subculturished n times can be obtained (n is an integer of 1 or more). From the viewpoint of mass production of cells, the lower limit of the number of subculturing steps n is, for example, 1 or more, preferably 2 or more, more preferably 4 or more, even more preferably 6 or more, even more preferably 8 or more, even more preferably 10 or more, even more preferably 12 or more, even more preferably 14 or more, even more preferably 16 or more, even more preferably 18 or more, even more preferably 20 or more, and even more preferably 25 or more. Furthermore, from the viewpoint of suppressing cell senescence, the upper limit of the number of subculturing steps n is preferably, for example, 50 or less, 40 or less, or 30 or less. As the cell detachment means described above, for example, a cell detachment agent may be used. As a cell detachment agent, trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA), etc. can be used, but are not particularly limited. Commercially available cell detachment agents may be used as cell detachment agents. Examples include, but are not limited to, Trypsin-EDTA solution (Thermo Fisher Scientific), TrypLE Select (Thermo Fisher Scientific), Accutase (Stemcell Technologies), and Accumax (Stemcell Technologies). Furthermore, physical cell detachment methods may be used as cell detachment means, such as, but are not limited to, a cell scraper (Corning). Cell detachment means may be used individually or in combination.

[0049] The present disclosure will be described in more detail below with reference to examples, but the technical scope of the present disclosure is not limited to the following examples.

[0050] [Example 1] In Example 1, we investigated a quality control method that involves evaluating soft agar colony formation during the shipment inspection of each production lot.

[0051] -In vivo tumorigenicity test in the development process- As a non-clinical trial in the development process of the cell product, an in vivo tumorigenicity evaluation was conducted. Specifically, 0.5 g of synovial tissue derived from a human donor was collected, and synovial mesenchymal stem cells (hereinafter referred to as "development process cells") were obtained by isolation and culture. The obtained "development process cells" were subcutaneously administered to 40 NOG mice, and in vivo tumorigenicity was evaluated. Eight weeks after administration, the presence of the administered substance was not observed, and histopathological observation of the administration site did not reveal any findings suggestive of tumorigenicity, such as mitotic figures. From this, it was considered that the development process cells are safe cells that do not exhibit tumorigenicity.

[0052] -Soft Agar Colony Formation Evaluation Test in the Development Process- As a non-clinical test and characterization in the development process of the cell product, a soft agar colony formation evaluation test was conducted. Specifically, cells for the development process (synovial mesenchymal stem cells) were obtained in the same manner as in the tumorigenicity test described above. The obtained "cells for the development process," negative control (MRC-5 cells), and positive control (mixed cells in which MRC-5 cells were spiked with 0.1% HeLa cells) were seeded in soft agar, and fluorescence intensity was measured using the CytoSelect 96-Well Cell Transformation Assay on the seeding day (Day 0) and 3 weeks after culturing (Day 21). The cell count (concentration) used as the judgment criterion was performed as follows. Specifically, a portion of the cell suspension was mixed with an equal volume of trypan blue solution, and the number of viable and dead cells (in integer form) was counted using a hemocytometer [Burker-Turk hemocytometer, certified by the Japan Hematology Instruments Inspection Association (JHS)]. Subsequently, the cell concentration (viable and dead cell concentration) and viability were calculated according to the following formula: Cell concentration (cells / mL) = Number of cells per compartment [average value of 4 compartments (in integer form)] × 2 (dilution ratio by trypan blue solution) × 10 ４ × Cell suspension dilution ratio Survival rate (%) = Live cell concentration ÷ (Live cell concentration + Dead cell concentration) × 100 (in integer form)

[0053] As a result, the cell viability rate in the development process exceeded the 70% threshold, and the in-process control tests met the criteria for successful testing. Furthermore, the viability rates in both positive and negative controls were above 80%. Microscopic observation of the Day 21 culture plate was performed 7 and 14 days after the start of culture, and no turbidity of the culture medium or contamination by microorganisms was observed.

[0054] Tumor formation was determined as follows: if the measured value for each group on Day 21 exceeded the measured value for that group on Day 0 + 3.3 × SD, it was determined to be tumorigenic; otherwise, it was determined to be non-tumoric. In the negative control group, the measured value on Day 21 was less than or equal to the measured value on Day 0 + 3.3 × SD, so it was determined to be non-tumoric (safe). In the positive control group, the measured value on Day 21 exceeded the measured value on Day 0 + 3.3 × SD, so it was determined to be tumorigenic (not safe). Therefore, the results for the positive and negative controls were as expected. The positive control was prepared by adding 10 HeLa cells / well to 10,000 MRC-5 cells / well, and the detection sensitivity was 0.1%. On the other hand, in the cells used in the development process, the measurement value on Day 21 was less than or equal to the measurement value on Day 0 + 3.3 × SD, so it was determined that there was no tumorigenicity (it was safe).

[0055] - Preparation of intermediate cell lines in the manufacturing process - 0.5 g of donor-derived synovial tissue was collected, cut with scissors, immersed in 5.0 mL of liberase aqueous solution (liberase MNP-S (Roche)) to digest the tissue, centrifuged at 400 g for 5 minutes, and the cell suspension was collected. After washing, the supernatant was removed, the obtained cell suspension was suspended in culture medium, and the entire amount was seeded into a 10-cell stack flask.

[0056] CO ２ Incubator (37°C, 5% CO2) ２ After culturing for two weeks in the following manner, cells were harvested from the flask to obtain synovial-derived mesenchymal stem cells. After adding a cryoprotectant, these were used as intermediate cell lines, with 1 × 10⁶ cells per flask. ６ The solution was dispensed into individual cells and stored frozen at -80°C or below.

[0057] - Preparation of the final product in the manufacturing process - One of the intermediate cell lines obtained in the "Preparation of intermediate cell lines in the manufacturing process" described above was thawed, mixed with microcarriers in culture medium, and then placed in a 37°C bioreactor (2L). Culture was continued at 37°C for 10 days with agitation, and cells that had proliferated more than 1000 times were detached and collected to obtain synovial-derived mesenchymal stem cells, which would become the final product. After adding a cryoprotectant, these were used as the final product, with 2 x 10⁶ cells per cell. ７ The mixture was dispensed into individual cells and stored frozen at -80°C or below. In this example, 100 bottles of the final product were obtained.

[0058] - Final product shipment inspection in the manufacturing process - A soft agar colony formation evaluation was performed on one final product obtained in the "production of the final product in the manufacturing process" described above as a shipment inspection. In other words, in this example, the soft agar colony formation evaluation was performed at a stage after the process of filling into containers.

[0059] The final product, negative control (MRC-5 cells), and positive control (mixed cells of MRC-5 cells spiked with 0.1% HeLa cells) were seeded in soft agar, and fluorescence intensity was measured using the CytoSelect 96-Well Cell Transformation Assay on the seeding day (Day 0) and 3 weeks after culturing (Day 21). The cell count (concentration) used for the judgment criteria was performed as follows: A portion of the cell suspension was mixed with an equal volume of trypan blue solution, and the number of viable and dead cells (in integer form) was counted using a hemocytometer [Burker-Turk hemocytometer, certified by the Japan Society for Health Sciences (JHS)]. Subsequently, cell concentration (viable cell concentration and dead cell concentration) and viability were calculated according to the following. Cell concentration (cells / mL) = Number of cells per compartment [average value of 4 compartments (in integer form)] × 2 (dilution ratio with trypan blue solution) × 10 ４ × Cell suspension dilution ratio Survival rate (%) = Live cell concentration ÷ (Live cell concentration + Dead cell concentration) × 100 (in integer form)

[0060] As a result, the survival rate of the final product exceeded the 70% threshold, and the in-process control inspections met the criteria for successful testing. Furthermore, the survival rates of both the positive and negative controls were over 80%. Microscopic observation of the Day 21 culture plate was performed 7 and 14 days after the start of cultivation, and no turbidity of the culture medium or contamination by microorganisms was observed.

[0061] Tumor formation was determined as follows: if the measured value for each group on Day 21 exceeded the measured value for that group on Day 0 + 3.3 × SD, it was determined to be tumorigenic; otherwise, it was determined to be non-tumoric. In the negative control group, the measured value on Day 21 was less than or equal to the measured value on Day 0 + 3.3 × SD, so it was determined to be non-tumoric (safe). In the positive control group, the measured value on Day 21 exceeded the measured value on Day 0 + 3.3 × SD, so it was determined to be tumorigenic (not safe). Therefore, the results for the positive and negative controls were as expected. The positive control was prepared by adding 10 HeLa cells / well to 10,000 MRC-5 cells / well, and the detection sensitivity was 0.1%. On the other hand, in the final product, the measurement value on Day 21 was less than or equal to the measurement value on Day 0 + 3.3 × SD, so it was determined that there was no tumorigenicity (it was safe).

[0062] As described above, the safety of the final product was ensured by the soft agar colony formation evaluation conducted as part of the shipping inspection. In this example, the proportion of the number of units manufactured that were subjected to the soft agar colony formation evaluation was 1 out of 100 units, which is 1%. If 1,000 units were manufactured, the proportion would be 0.1%.

[0063] [Example 2] In Example 2, we investigated a quality control method that involves evaluating soft agar colony formation as part of the shipment inspection for each production lot and as an in-process control inspection for intermediate cell lines.

[0064] -In vivo tumorigenicity test in the development process- This was performed in the same manner as in Example 1. In this example as well, the cells used in the development process were considered to be safe cells that did not exhibit tumorigenicity.

[0065] - Evaluation Test of Soft Agar Colony Formation in the Development Process - This test was conducted in the same manner as in Example 1. As a result, in this example as well, the viability of cells in the development process exceeded the 70% threshold, and the in-process control tests met the criteria for successful testing. Furthermore, the viability of both the positive and negative controls was 80% or higher. Observation of the Day 21 culture plate under a microscope was performed 7 and 14 days after the start of culture, and no turbidity of the culture medium or contamination by microorganisms was observed.

[0066] Tumor formation was determined as follows: if the measured value for each group on Day 21 exceeded the measured value for that group on Day 0 + 3.3 × SD, it was determined to be tumorigenic; otherwise, it was determined to be non-tumoric. In the negative control group, the measured value on Day 21 was less than or equal to the measured value on Day 0 + 3.3 × SD, so it was determined to be non-tumoric (safe). In the positive control group, the measured value on Day 21 exceeded the measured value on Day 0 + 3.3 × SD, so it was determined to be tumorigenic (not safe). Therefore, the results for the positive and negative controls were as expected. The positive control was prepared by adding 10 HeLa cells / well to 10,000 MRC-5 cells / well, and the detection sensitivity was 0.1%. On the other hand, in the cells used in the development process, the measurement value on Day 21 was less than or equal to the measurement value on Day 0 + 3.3 × SD, so it was determined that there was no tumorigenicity (it was safe).

[0067] - Preparation of intermediate cell lines in the manufacturing process - In the same manner as in Example 1, 1 × 10⁶ cells per tube ６ Intermediate cell lines were prepared by dispensing them into individual cells and then cryopreserved at -80°C or below.

[0068] -In-process control inspection of intermediate cell lines in the manufacturing process- For each intermediate cell line obtained, a soft agar colony formation evaluation was performed as an in-process control inspection. That is, in this example, the soft agar colony formation evaluation was performed at the stage before the culture and growth process. The soft agar colony formation evaluation test was performed in the same manner as in Example 1. As a result, the viability of the intermediate cell line exceeded the judgment criterion of 70%, and the in-process control inspection met the test success criteria. In addition, the viability of both the positive control and the negative control was 80% or higher. Observation of the Day 21 culture plate under a microscope was performed 7 and 14 days after the start of culture, and no turbidity of the culture medium or contamination by microorganisms was observed.

[0069] Tumor formation was determined as follows: if the measured value for each group on Day 21 exceeded the measured value for that group on Day 0 + 3.3 × SD, it was determined to be tumorigenic; otherwise, it was determined to be non-tumoric. In the negative control group, the measured value on Day 21 was less than or equal to the measured value on Day 0 + 3.3 × SD, so it was determined to be non-tumoric (safe). In the positive control group, the measured value on Day 21 exceeded the measured value on Day 0 + 3.3 × SD, so it was determined to be tumorigenic (not safe). Therefore, the results for the positive and negative controls were as expected. The positive control was prepared by adding 10 HeLa cells / well to 10,000 MRC-5 cells / well, and the detection sensitivity was 0.1%. On the other hand, in the intermediate cell line, the measurement value on Day 21 was less than or equal to the measurement value on Day 0 + 3.3 × SD, so it was determined that there was no tumorigenicity (it was safe). Therefore, the safety of the intermediate cell line was ensured in the soft agar colony formation evaluation conducted as an in-process control test.

[0070] - Preparation of the final product in the manufacturing process - One of the intermediate cell lines obtained in the "Preparation of intermediate cell lines in the manufacturing process" described above was thawed, mixed with microcarriers in culture medium, and then placed in a 37°C bioreactor (2L). Culture was continued at 37°C for 10 days with agitation, and cells that had proliferated more than 1000 times were detached and collected to obtain synovial-derived mesenchymal stem cells, which would become the final product. After adding a cryoprotectant, these were used as the final product, with 2 x 10⁶ cells per cell. ７The mixture was dispensed into individual cells and stored frozen at -8°C. In Example 2, 100 bottles of the final product were obtained.

[0071] - Final product shipment inspection in the manufacturing process - For one final product obtained in the "production of the final product in the manufacturing process" described above, a soft agar colony formation evaluation was performed as a shipment inspection, similar to Example 1. That is, in this example, a soft agar colony formation evaluation was also performed at a stage after the process of filling into containers. As a result, the viability rate of the final product exceeded the judgment criterion of 70%, and the in-process control inspection met the test success criteria. In addition, the viability rates of both the positive control and the negative control were 80% or higher. Observation of the Day 21 culture plate under a microscope was performed 7 and 14 days after the start of culture, and no turbidity of the culture medium or contamination by microorganisms was observed.

[0072] Tumor formation was determined as follows: if the measured value for each group on Day 21 exceeded the measured value for that group on Day 0 + 3.3 × SD, it was determined to be tumorigenic; otherwise, it was determined to be non-tumoric. In the negative control group, the measured value on Day 21 was less than or equal to the measured value on Day 0 + 3.3 × SD, so it was determined to be non-tumoric (safe). In the positive control group, the measured value on Day 21 exceeded the measured value on Day 0 + 3.3 × SD, so it was determined to be tumorigenic (not safe). Therefore, the results for the positive and negative controls were as expected. The positive control was prepared by adding 10 HeLa cells / well to 10,000 MRC-5 cells / well, and the detection sensitivity was 0.1%. On the other hand, in the final product, the measurement value on Day 21 was less than or equal to the measurement value on Day 0 + 3.3 × SD, so it was determined that there was no tumorigenicity (it was safe). Therefore, the safety of the final product was ensured in the soft agar colony formation evaluation conducted as a shipping inspection. In this case, the proportion of the number of manufactured units subjected to the soft agar colony formation evaluation is 1 out of 100 units, which is 1%. If 1,000 units were manufactured, it would be 0.1%.

[0073] This embodiment demonstrates that by passing the soft agar colony formation evaluation test as an in-process control inspection for intermediate cell lines in the manufacturing process, safety is ensured, and furthermore, by passing the soft agar colony formation evaluation test as a final product shipment inspection in the manufacturing process, it is possible to manufacture cell products with extremely high safety. This is because, although cells are proliferated more than 1,000 times from the intermediate cell line to the final product, safety can be ensured in the intermediate cell line that forms the basis of the cell product.

[0074] [Evaluation of Comparative Examples and Examples] Table 1 summarizes how the safety differs between Examples 1 and 2 described above and the comparative example to which the conventional quality control method was applied. The quality control method of the comparative example involves conducting in vivo tumorigenicity tests, soft agar colony formation evaluations, and manufacturing method stability evaluations based on genome instability during the development process, but does not involve conducting soft agar colony formation evaluation tests during the manufacturing process.

[0075] For Examples 1, 2, and the Comparative Example, the evaluation criteria for comparing the safety of each method are shown below. When performed every time: 1000 points. When performed as a random inspection: 100 points. When performed only a few times: 10 points. When not performed: 1 point. The basis for setting the evaluation values ​​is as follows. First, for the case where it is performed every time, it is assumed that the number of manufacturing cycles will range from 1000 to tens of thousands of times throughout the product lifecycle, and if the detection power is defined by the number of cycles, it will range from 1000 to tens of thousands of points. In this example, a lower value of 1000 points was set for the case where it is performed every time. When it is performed as a random inspection, it is assumed that it will be performed once every 10 manufacturing cycles, and if the detection power is defined by the number of cycles, it will be 100 points when calculated for 1000 manufacturing cycles. Cases where the test is performed only a few times include, for example, performing the test only three times in process validation or only once in GLP (Good Laboratory Practice). Since the number of times is usually in the single digits, the detection power can be defined as 1 to 10 points. The higher of these was adopted, resulting in 10 points. In the case where the test is not performed, the score would normally be 0 points, but due to the need to calculate the safety result as an accumulated value, the minimum score of 1 point was adopted.

[0076]

[0077] As shown in Table 1, the quality control method in Example 1 can ensure 1,000 times greater tumorigenicity safety than the comparative example method, and in Example 2, 1,000,000 times greater tumorigenicity safety than the comparative example method can be ensured. Furthermore, in the quality control methods in Examples 1 and 2, since the shipment inspection is performed using 1% of the proliferated cells based on cell count, it can be seen that a sufficient quantity of cell product can be secured.

[0078] - Use as a treatment for joint diseases - Cell products that met the quality control methods of Example 1 and Example 2 were administered to a rat model of osteoarthritis of the knee. As a result, an improvement in osteoarthritis of the knee was observed compared to the untreated group, and histopathological evaluation also showed improvement in findings such as inflammation, cartilage erosion, and bone deformation. These results demonstrate that, according to the quality control methods of Example 1 and Example 2, cell products with high safety can be shipped as treatments for joint diseases.

[0079] The disclosure of Japanese Patent Application No. 2025-52146, filed on 26 March 2025, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

1. A method for producing a cell product, comprising the steps of: culturing and growing cells isolated from a cell source; collecting the grown cells; and filling the collected cells into a container, wherein a soft agar colony formation evaluation test is performed on the cells at at least one stage selected from the group consisting of before the culturing and growing step, between the culturing and growing step and the filling step into the container, and after the filling step into the container, and the suitability of the cells filled into the container is determined based on the results of the soft agar colony formation evaluation test.

2. The method for producing a cell product according to claim 1, wherein in the step of filling the container, the cells filled in the container are frozen.

3. The method for producing a cell product according to claim 1, wherein the soft agar colony formation evaluation test is performed using 0.1% to 10% of the recovered cells based on cell number.

4. The method for producing a cell product according to claim 1, wherein the test is deemed successful if the cell viability in the soft agar colony formation evaluation test is 70% or more.

5. A method for producing a cell product according to claim 1, wherein the soft agar colony formation evaluation test is performed multiple times.

6. The method for producing a cell product according to claim 1, wherein the cells are allogeneic cells separated from a cell source and cryopreserved.

7. The method for producing a cell product according to claim 1, wherein the cells are at least one type of cell selected from the group consisting of somatic stem cells, induced pluripotent stem cells, embryonic stem cells, differentiated cells derived from somatic stem cells, differentiated cells derived from induced pluripotent stem cells, somatic stem cells derived from induced pluripotent stem cells, differentiated cells derived from embryonic stem cells, and somatic stem cells derived from embryonic stem cells.

8. The method for producing a cell product according to claim 1, wherein the cells are adherent cells.

9. The method for producing a cell product according to claim 1, wherein the cells are mesenchymal stem cells.

10. The method for producing a cell product according to claim 1, wherein the cells are cells used as a joint treatment drug.

11. The method for producing a cell product according to claim 1, wherein the cells are cells for drug discovery.

12. The method for producing a cell product according to claim 1, wherein in the step of culturing and growing the cells, multiple different lots are prepared, the cells contained in each lot are cultured and grown, and the suitability of each recovered cell for use is determined based on the results of the soft agar colony formation evaluation test.

13. A cell quality control method comprising: a step of culturing and growing cells isolated from a cell source; a step of collecting the grown cells; and a step of filling the collected cells into a container, wherein a soft agar colony formation evaluation test is performed on the cells at at least one step selected from the group consisting of before the culturing and growing step, between the culturing and growing step and the step of filling the container, and after the step of filling the container, and the suitability of the cells filled into the container is determined based on the results of the soft agar colony formation evaluation test.

14. The cell quality control method according to claim 13, wherein the step of filling the container is to freeze the cells filled in the container.

15. The cell quality control method according to claim 13, wherein the soft agar colony formation evaluation test is performed using 0.1% to 10% of the recovered cells based on cell number.

16. The cell quality control method according to claim 13, wherein the test is deemed successful if the cell viability in the soft agar colony formation evaluation test is 70% or more.

17. The cell quality control method according to claim 13, wherein the soft agar colony formation evaluation test is performed multiple times.

18. The cell quality control method according to claim 13, wherein the cells are allogeneic cells separated from a cell source and cryopreserved.

19. The cell quality control method according to claim 13, wherein the cell is at least one cell selected from the group consisting of somatic stem cells, induced pluripotent stem cells, embryonic stem cells, differentiated cells derived from somatic stem cells, differentiated cells derived from induced pluripotent stem cells, somatic stem cells derived from induced pluripotent stem cells, differentiated cells derived from embryonic stem cells, and somatic stem cells derived from embryonic stem cells.

20. The cell quality control method according to claim 13, wherein the cells are adherent cells.

21. The cell quality control method according to claim 13, wherein the cells are mesenchymal stem cells.

22. The cell quality control method according to claim 13, wherein the cells are cells used as joint treatment drugs.

23. The cell quality control method according to claim 13, wherein the cells are cells for drug discovery.

24. The cell quality control method according to claim 13, wherein in the step of culturing and growing the cells, multiple different lots are prepared, the cells contained in each lot are cultured and grown, and the suitability of each recovered cell is determined based on the results of the soft agar colony formation evaluation test.

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