Cell proliferation control method in automatic culture device

Abstract

To provide a method for causing stem cells to proliferate while suppressing proliferation.SOLUTION: In one embodiment, the present disclosure provides a method for causing stem cells to proliferate while suppressing proliferation, wherein the method includes a step for culturing the stem cells under hibernation conditions of the stem cells. In another embodiment, the present disclosure provides a method for producing a predetermined amount of stem cells in a timely manner, wherein the method includes (a) a step for culturing the stem cells under hibernation conditions of the stem cells and (b) a step for culturing the stem cells at approximately 36-38°C until reaching the predetermined amount of the stem cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to techniques for controlling the proliferation rate of stem cells. [Background technology]

[0002] Regenerative medicine has attracted attention as a new treatment for central nervous system diseases (cerebral infarction, cerebral hemorrhage, head trauma, Parkinson's disease, etc.), and the development of several cell medicines is underway. The present inventors previously developed a treatment method using direct transplantation of cells into the brain, which achieved excellent therapeutic results (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Shichinohe, Kawabori et al. Research on advanced intervention using novel bone marrow stem cell (RAINBOW): a study protocol for a phase I, open-label, uncontrolled, dose-response trial of autologous bone marrow stromal cell transplantation in patients with acute ischemic stroke BMC Neurol. 2017 Sep 8;17(1):179 Summary of the Invention [Means for solving the problem]

[0004] The present inventors have discovered hibernation conditions that suppress stem cell proliferation, thereby making it possible to control stem cell proliferation.

[0005] The present invention provides, for example, the following items. (Item 1) A method for expanding stem cells while inhibiting proliferation, the method comprising culturing the stem cells in hibernation conditions for the stem cells. (Item 2) A method for growing stem cells while inhibiting proliferation, the method comprising the step of culturing the stem cells at a temperature between about 20°C and about 34°C. (Item 3) Item 2. The method according to Item 1, wherein the hibernation conditions are a temperature between about 25°C and about 34°C. (Item 4) Item 2. The method according to Item 1, wherein the hibernation conditions are a temperature between about 28°C and about 32°C. (Item 5) Item 2. The method according to Item 1, wherein the hibernation conditions are a temperature between about 30°C and about 32°C. (Item 6) 6. The method according to any one of items 1 to 5, wherein the method is carried out without passaging. (Item 7) 7. The method according to any one of items 1 to 6, wherein the method is carried out in an automated culture device. (Item 8) 8. The method according to any one of items 1 to 7, wherein the stem cells are mesenchymal stem cells. (Item 9) Item 9. The method according to Item 8, wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, placental tissue, synovial tissue, umbilical cord tissue (e.g., umbilical cord blood), dental pulp, or amniotic membrane, or are mesenchymal stem cells differentiated from ES cells or iPS cells. (Item 10) Item 10. The method according to item 9, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. (Item 11) 11. The method according to any one of items 1 to 10, wherein the cells are cells used in either cell injection therapy or drug discovery research. (Item 12) A method for producing a predetermined amount of stem cells in a timely manner, the method comprising the steps of: (a) culturing stem cells under hibernation conditions for the stem cells; and (b) culturing the stem cells at approximately 36°C to 38°C until the predetermined amount of stem cells is reached. (Item 13) Item 13. The method according to item 12, wherein the hibernation conditions are a temperature between about 20°C and about 34°C. (Item 14) Item 13. The method according to item 12, wherein the hibernation conditions are about 28°C to about 32°C. (Item 15) Item 13. The method according to item 12, wherein the hibernation conditions are about 30°C to about 32°C. (Item 16) A method for producing a predetermined amount of stem cells in a timely manner, the method comprising the steps of (a) culturing the stem cells at a temperature of between about 20°C and about 34°C, and (b) culturing the stem cells at about 36°C to 38°C until the predetermined amount of stem cells is obtained. (Item 17) 17. The method according to any one of items 12 to 16, wherein the method is carried out without passaging. (Item 18) 18. The method according to any one of items 12 to 17, wherein the method is carried out in an automated culture device. (Item 19) 19. The method according to any one of items 12 to 18, wherein the stem cells are mesenchymal stem cells. (Item 20) 20. The method according to item 19, wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, placental tissue, synovial tissue, umbilical cord tissue (e.g., umbilical cord blood), dental pulp, or amniotic membrane, or are mesenchymal stem cells differentiated from ES cells or iPS cells. (Item 21) 20. The method of claim 19, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. (Item 22) 22. The method according to any one of items 12 to 21, wherein the cells are cells used in cell injection therapy and drug discovery research. (Item 23) A cell obtained by the method according to any one of items 1 to 22. (Item 24) 23. A pharmaceutical composition comprising cells obtained by the method according to any one of items 1 to 22. (Item 25) 25. The pharmaceutical composition according to item 24, for treating or preventing severe burns, spinal cord injury, head trauma, cerebral infarction, cerebral hemorrhage, Parkinson's disease, or dementia. (Item 26) A system for growing stem cells while inhibiting proliferation, the system comprising: a culture vessel for culturing stem cells; a temperature controller for adjusting the temperature of the culture vessel; wherein the temperature regulator can be adjusted to culture the stem cells in a hibernation condition for the stem cells. (Item 27) 1. A system for producing a predetermined amount of stem cells in a timely manner, the system comprising: a culture vessel for culturing stem cells; a temperature controller for adjusting the temperature of the culture vessel; a cell amount measuring means for detecting whether the cell amount is a predetermined amount; wherein the temperature regulator can adjust the temperature so as to culture the stem cells under hibernation conditions and normal culture conditions. (Item 28) A system for growing stem cells while inhibiting proliferation, the system comprising: a culture vessel for culturing stem cells; a temperature controller for adjusting the temperature of the culture vessel; wherein the temperature regulator can be adjusted to culture the stem cells at approximately 20°C to 34°C. (Item 29) 1. A system for producing a predetermined amount of stem cells in a timely manner, the system comprising: a culture vessel for culturing stem cells; a temperature controller for adjusting the temperature of the culture vessel; a cell amount measuring means for detecting whether the cell amount is a predetermined amount; wherein the temperature regulator can adjust the temperature so that the stem cells are cultured at about 20°C to 34°C and about 36°C to 38°C.

[0006] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0007] According to the present disclosure, it is possible to control the proliferation of stem cells and provide an appropriate amount of stem cells at an appropriate time. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows a graph of the change in viable cell count when cells were cultured at 37° C. for 2 days, at 30° C. for 3 days, and then at 37° C. for 3 days. [Figure 2] FIG. 2 shows a graph of the change in viable cell count when cells were cultured at 37° C. for 2 days, at 30° C., 32° C. or 34° C. for 3 days, and then at 37° C. for 3 days. [Figure 3] Figure 3 shows a graph that combines the average results from Figure 2 into one graph. [Figure 4] FIG. 4 shows a graph of the change in viable cell count when cells were cultured at 37° C. for 2 days, at 32° C. for 3 days, and then at 37° C. for 8 days. [Figure 5] Figure 5 shows a graph that combines the average results from Figure 4 into one graph. [Figure 6] 6 shows a graph of the HGF ratio per cell after the temperature shift, where the group without the temperature shift was set to 1. [Figure 7] Figure 7 shows a comparison between the final cell number when cultured using an automated culture device and the cell number predicted from the lactate value. [Figure 8] FIG. 8 shows a graph of the change in cell number during culture in the automated culture device. [Figure 9] FIG. 9 shows a graph of the change in cell number when cells were cultured at 37° C. for 2 days, at 20° C. for 3 days, and then at 37° C. for 3 days. [Figure 10] Figure 10 shows a graph averaging the results from Figure 9 into one graph. [Figure 11] FIG. 11 shows a graph of the change in cell number when cells were cultured at 37° C. for 2 days, at 25° C. for 3 days, and then at 37° C. for 3 days. [Figure 12] FIG. 12 shows a graph averaging the results of FIGS. 9 and 11 in one graph. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure will be described below. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the relevant field unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will prevail.

[0010] (definition) As used herein, "about" means ±10% of the value that follows.

[0011] As used herein, the term "stem cell" refers to a cell that has the ability to self-renew, i.e., divide to produce identical cells, and the ability to differentiate into specific cells. Stem cells are not particularly limited as long as they have the ability to self-renew and differentiate, and may be pluripotent stem cells or somatic stem cells. Pluripotent stem cells are cells that have the ability to self-renew and the pluripotency to differentiate into any of the ectoderm, mesoderm, and endoderm. Examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ cells (EG cells), embryonic carcinoma cells (EC cells), pluripotent adult progenitor cells (MAP cells), adult pluripotent stem cells (APS cells), and Muse cells. Examples of somatic stem cells include mesenchymal stem cells, hematopoietic stem cells, and neural stem cells.

[0012] As used herein, "mesenchymal stem cells," also known as mesenchymal stromal cells, refer to stem cells capable of differentiating into mesenchymal cells such as osteoblasts, adipocytes, muscle cells, and chondrocytes. Mesenchymal stem cells may include mesenchymal stem cells derived from bone marrow, adipose tissue, placental tissue, synovial tissue, umbilical cord tissue (e.g., umbilical cord blood), dental pulp, or amniotic membrane, or mesenchymal stem cells differentiated from ES cells or iPS cells.

[0013] As used herein, "proliferation" (of stem cells) refers to the process of dividing to produce cells identical to the stem cell, thereby increasing the number of cells.

[0014] As used herein, "inhibition" of "growth" refers to stopping cell growth or, even if not completely stopping it, slowing down the growth rate compared to culture under normal culture conditions.

[0015] As used herein, "hibernation conditions" refers to conditions under which the cell cycle is arrested and proliferation is suppressed. When hibernation conditions are released, cell proliferation can resume. Such conditions include, but are not limited to, temperature, medium components, pH, osmotic pressure, oxygen or carbon dioxide concentration, etc.

[0016] As used herein, "normal culture conditions" refers to conditions suitable for the proliferation of specific cells. Such conditions include, but are not limited to, temperature, medium components, pH, osmotic pressure, oxygen or carbon dioxide concentration, etc. Normal culture conditions for stem cell culture are typically about 36 to about 38°C (preferably about 37°C), CO 2 The culture medium may be a 5% CO atmosphere, pH 6-8, containing any growth factors.

[0017] As used herein, "without passage" refers to continuing to culture cells in the same vessel without transferring the cells grown in the culture vessel to a new vessel and continuing to maintain the culture.

[0018] In this specification, the term "automated culture device" refers to a device that automatically performs the steps from cell introduction to cell recovery without manual intervention. Specific examples of automated culture devices include, but are not limited to, Quantum (TERUMO BCT), CliniMACS Prodigy (Miltenyi Biotec), and CELLAFORTE (Nipro). Other automated culture devices are disclosed in Shimizu Miyuki et al., Journal of Medical Devices, Vol. 88, No. 4 (2018) (17).

[0019] As used herein, the term "cell infusion therapy" refers to a therapeutic method in which cells are infused to treat a disease.

[0020] As used herein, "drug discovery research" refers to exploratory research, development research, and clinical research in the creation of pharmaceuticals.

[0021] As used herein, "cells used in cell injection therapy" refers to cells injected in cell injection therapy, including mesenchymal stem cells (regardless of origin, such as bone marrow-derived, amniotic membrane-derived, or dental pulp-derived), embryonic stem cells (ES cells), iPS cells, etc.

[0022] As used herein, the term "cells used in drug discovery research" refers to cells used in exploratory research, development research, and clinical research, or cells that are candidates for actual use as pharmaceuticals. Specifically, such cells include ES cells and iPS cells.

[0023] As used herein, "producing a predetermined amount of stem cells at an appropriate time" refers to providing stem cells through culture so as to achieve a desired number or cell density of stem cells at the end of the culture that is determined according to the purpose.

[0024] The predetermined amount can be determined appropriately depending on the purpose, and can be, for example, about 50 to 100 million cells, which is sufficient for injecting 40 million cells in cell injection therapy.

[0025] The desired cell density is preferably a cell density at which the cells reach confluence or a cell density lower than that, and can be set appropriately.

[0026] The end point of culture set according to the purpose refers to the end point of culture set so that a predetermined amount of cells can be provided before the purpose (for example, cell injection therapy) is performed.

[0027] As used herein, the term "medicine" or "pharmaceutical composition" refers to a drug or a composition containing the same used in the prevention or treatment of a disease.

[0028] As used herein, the term "cell preparation" refers to a preparation containing cells in a form suitable for use or in a form prepared just before use. "Prepared just before use" refers to preparing a preparation suitable for use by adding a drug or diluting with a solvent just before administration.

[0029] As used herein, "prevention" (of severe burns, spinal cord injury, head trauma, cerebral infarction, cerebral hemorrhage, Parkinson's disease, or dementia) refers to delaying the onset of these diseases, preventing the onset of these diseases, or alleviating symptoms when these diseases do occur.

[0030] As used herein, "treatment" (of severe burns, spinal cord injury, head trauma, cerebral infarction, cerebral hemorrhage, Parkinson's disease, or dementia) refers to alleviating or eliminating these diseases or the symptoms of these diseases.

[0031] (Preferred embodiment) Although the following description of preferred embodiments is given, it should be understood that these embodiments are merely examples of the present disclosure and that the scope of the present invention is not limited to such preferred embodiments. It should also be understood that those skilled in the art can easily make modifications, changes, etc. within the scope of the present disclosure by referring to the following preferred examples. Those skilled in the art can combine any of these embodiments as appropriate.

[0032] (Application of hibernation conditions) While regenerative medicine products using stem cells are beginning to be approved, the high cost of culturing (manual labor costs for human labor and maintaining the cleanliness of the culture room) remains a problem. The solution is the automation of manufacturing using automated culture equipment. Automated culture equipment, particularly those that do not require cell passaging, promises significant cost reductions due to the fact that they can perform the entire process from the introduction of stem cell sources to the collection of the final product without manual labor and can ensure cleanliness using disposable kits. However, a challenge with automated culture equipment that does not require cell passaging is the difficulty of controlling stem cell proliferation. While conventional flask culture allows for cell number control by adjusting the cell number during cell passaging, automated culture equipment that does not require passaging does not allow for this. This means that controlling proliferation after seeding becomes difficult. The rate of stem cell proliferation varies depending on the cell type, donor characteristics (e.g., age), and the type of culture medium and additives used. Excessive proliferation requires more culture medium than expected to provide nutrients, which increases costs. Furthermore, the prepared volume of culture medium becomes insufficient, necessitating the interruption or early termination of the culture. In this case, stem cells scheduled for shipping without freezing will not be administered on the scheduled date, and in the case of autologous cell products, even if the stem cells proliferate far beyond the amount required for administration, that amount cannot be administered, resulting in waste. The technology to keep the required number of cells within a predetermined range is extremely important for automated culture devices that do not require passaging, but no established method has been established. This disclosure provides a solution to these problems.

[0033] In the present disclosure, the medium that can be used for cell culture is not particularly limited, and any medium can be used. Specifically, it may be a liquid medium such as a basal medium for mammalian cells (e.g., Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F-12)), Eagle's minimal essential medium (EME), Basal Medium Eagle (BME), RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium), E8 base medium, Skeletal Muscle Cell Basal Medium (SkBM), MCDB104, MCDB153, 199, or L15), a commercially available stem cell maintenance medium, a basal medium for insect cells, a yeast medium, or a bacterial medium. Media suitable for cell culture may contain various commonly available additives, such as antibiotics (e.g., penicillin, streptomycin, etc.); vitamins or vitamin derivatives (e.g., ascorbic acid, retinoic acid, etc.); sugar sources (e.g., glucose, etc.); amino acids; inorganic salts; serum, serum substitutes; proteins (e.g., transferrin, etc.); hormones (e.g., insulin, etc.); growth factors; differentiation inhibitors; antioxidants (e.g., 2-mercaptoethanol, dithiothreitol, etc.); and metal ions (e.g., calcium ions, magnesium ions, zinc ions, iron ions, copper ions, etc.). During cell culture, the medium is altered by metabolites secreted by the cells. Therefore, a medium exchange process may be performed at an appropriate time during the culture period, in which the used medium in the cell culture vessel is replaced with fresh medium.

[0034] A cell culture device that can be used in the present disclosure includes a cell supply unit, a medium supply unit, and, if necessary, other component supply units. The cell culture device may also optionally include a cell culture vessel, a storage container, a division processing unit, a waste liquid collection container, and the like. The cell culture device accommodates cells supplied from the cell supply unit in a cell culture vessel together with a medium (culture solution) supplied from a medium supply unit, and cultures the cells in the cell culture vessel, for example, suspended in the medium. The device of the present disclosure may include a temperature detection unit such as a thermometer that detects temperature and a temperature control unit (heating means and / or cooling means, etc.) that controls the temperature. The heating means may be any heater, and the cooling means may be any refrigerator or refrigerant. The temperature control unit may achieve both heating and cooling in a single device. The cell culture device of the present disclosure may include a control unit that controls these components. The control unit may be controlled by a program, etc., described in the present disclosure.

[0035] The cell supply unit may have a cell storage unit that stores cells to be cultured by the cell culture device, and a pump that sends the cells stored in the cell storage unit to a flow path that includes a pipe. The cell supply unit has an on-off valve provided downstream of the pump in the pipe that connects the cell storage unit to the pipe. The cells stored in the cell storage unit may be sent to the flow path by driving the pump and opening the on-off valve.

[0036] The inner wall surface of the cell culture vessel may be made of a plastic material, or may be made of, for example, metal, glass, or ceramic. In this case, a non-flat structure can be formed by, for example, thermal spraying, sandblasting, or etching. The entire cell culture vessel may also be made of metal, glass, or ceramic. From the viewpoint of enhancing the effect of inhibiting cell adhesion, the inner wall surface of the cell culture vessel may also be made of a plastic material. Alternatively, by using a plastic film having a non-flat surface as the base material of the cell culture vessel, it can be used as an inexpensive single-use cell culture bag.

[0037] In one aspect, the present disclosure provides a method for expanding stem cells while inhibiting proliferation, the method comprising culturing the stem cells in conditions for hibernation of the stem cells.

[0038] In another aspect, the present disclosure provides a method for producing a predetermined amount of stem cells in a timely manner, the method comprising the steps of: (a) culturing stem cells under hibernation conditions for the stem cells; and (b) culturing the stem cells at about 36°C to 38°C until the predetermined amount of stem cells is reached.

[0039] The method of the present disclosure is particularly advantageous in culture without subculture, for example, culture in an automated culture device without subculture, and is also advantageous in cell infusion therapy and drug discovery research, which require a specific amount of stem cells at a specific time.

[0040] In some embodiments, the period for which cells are subjected to hibernation conditions can be determined as appropriate, and may be 1 to 10 days, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. Because cells need to grow normally when warmed back to normal temperature (e.g., 37°C), if the period for hibernation conditions is too long, cell growth may become uncontrollable again, and the period is preferably up to 10 days, more preferably up to 5 days, and most preferably up to 3 days.

[0041] In some embodiments, the hibernation conditions can be temperature, which suppresses cell proliferation and allows normal cell proliferation when the hibernation conditions are released, for example, about 20°C to about 34°C, preferably about 25°C to about 34°C, more preferably about 28°C to about 32°C, and most preferably about 30°C to about 32°C.

[0042] The duration and temperature of hibernation conditions, as well as the total culture period, can be appropriately determined with reference to Examples 1 to 7 so as to achieve the number of stem cells or cell density set according to the purpose at the end of the culture.

[0043] In some embodiments, the stem cells can be mesenchymal stem cells. In some embodiments, the mesenchymal stem cells can be mesenchymal stem cells derived from bone marrow, adipose tissue, placenta tissue, synovial tissue, umbilical cord tissue (e.g., umbilical cord blood), dental pulp, or amniotic membrane, or mesenchymal stem cells differentiated from ES cells or iPS cells. In a preferred embodiment, the mesenchymal stem cells can be bone marrow-derived mesenchymal stem cells.

[0044] In a further aspect, the present disclosure provides cells obtained by the above method and pharmaceutical compositions comprising such cells, which can be used to treat or prevent severe burns, spinal cord injuries, head trauma, cerebral infarction, cerebral hemorrhage, Parkinson's disease, or dementia.

[0045] (Culture at a specific temperature) In another aspect, the present disclosure provides a method for expanding stem cells while inhibiting proliferation, the method comprising culturing the stem cells at a temperature of about 20°C to about 34°C.

[0046] In another aspect, the present disclosure provides a method for producing a predetermined amount of stem cells in a timely manner, the method comprising the steps of (a) culturing the stem cells at about 20°C to about 34°C, and (b) culturing the stem cells at about 36°C to 38°C until the predetermined amount of stem cells is obtained.

[0047] The method of the present disclosure is particularly advantageous in culture without subculture, for example, culture in an automated culture device without subculture, and is also advantageous in cell infusion therapy and drug discovery research, which require a specific amount of stem cells at a specific time.

[0048] In some embodiments, the period for which cells are subjected to hibernation conditions can be determined as appropriate, but may be 1 to 10 days, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. Because cells need to grow normally when warmed back to normal temperature (e.g., 37°C), the period for hibernation conditions is preferably up to 10 days, more preferably up to 5 days, and most preferably up to 3 days.

[0049] In some embodiments, the hibernation conditions can be temperature, which suppresses cell proliferation and allows normal cell proliferation when the hibernation conditions are released, for example, about 20°C to about 34°C, preferably about 25°C to about 34°C, more preferably about 28°C to about 32°C, and most preferably about 30°C to about 32°C.

[0050] The duration and temperature of hibernation conditions, as well as the total culture period, can be appropriately determined with reference to Examples 1 to 7 so as to achieve the number of stem cells or cell density set according to the purpose at the end of the culture.

[0051] In some embodiments, the stem cells can be mesenchymal stem cells. In some embodiments, the mesenchymal stem cells can be mesenchymal stem cells derived from bone marrow, adipose tissue, placenta tissue, synovial tissue, umbilical cord tissue (e.g., umbilical cord blood), dental pulp, or amniotic membrane, or mesenchymal stem cells differentiated from ES cells or iPS cells. In a preferred embodiment, the mesenchymal stem cells can be bone marrow-derived mesenchymal stem cells.

[0052] In a further aspect, the present disclosure provides cells obtained by the above method and pharmaceutical compositions comprising such cells, which can be used to treat or prevent severe burns, spinal cord injuries, head trauma, cerebral infarction, cerebral hemorrhage, Parkinson's disease, or dementia.

[0053] (Cultivation systems and programs, etc.) In one aspect, the present disclosure provides a system for expanding stem cells while inhibiting proliferation, the system comprising a culture vessel for culturing stem cells and a temperature regulator for regulating the temperature of the culture vessel, the temperature regulator being capable of adjusting the temperature so as to culture the stem cells in a hibernation condition for the stem cells.

[0054] In one aspect, the present disclosure provides a system for producing a predetermined amount of stem cells at an appropriate time, the system comprising a culture vessel for culturing stem cells, a temperature regulator for regulating the temperature of the culture vessel, and a cell quantity measuring means for detecting whether the cell quantity is a predetermined amount, wherein the temperature regulator can be adjusted to culture the stem cells under hibernation conditions and normal culture conditions.

[0055] In one aspect, the present disclosure provides a system for growing stem cells while inhibiting proliferation, the system comprising a culture vessel for culturing stem cells and a temperature regulator for regulating the temperature of the culture vessel, the temperature regulator being capable of adjusting the temperature so that the stem cells are cultured at approximately 20°C to 34°C.

[0056] In one aspect, the present disclosure provides a system for producing a predetermined amount of stem cells in a timely manner, the system comprising a culture vessel for culturing stem cells, a temperature regulator for adjusting the temperature of the culture vessel, and a cell quantity measuring means for detecting whether the cell quantity is a predetermined quantity, wherein the temperature regulator can adjust the temperature so that the stem cells are cultured at approximately 20°C to 34°C and approximately 36°C to 38°C.

[0057] In some embodiments, the system of the present disclosure may be an automated culture device further including a control unit that automatically controls the temperature adjustment unit. The control unit may be controlled by a program or the like described in the present disclosure. Examples of automated culture devices that can be used include, but are not limited to, Quantum (TERUMO BCT), CliniMACS Prodigy (Miltenyi Biotec), and CELLAFORTE (Nipro). Other automated culture devices are disclosed in Shimizu Miyuki et al., Journal of Medical Devices, Vol. 88, No. 4 (2018) (17).

[0058] In the present disclosure, the culture vessel may be any vessel as long as it can culture stem cells.

[0059] In the present disclosure, the temperature regulator may be any device capable of regulating the temperature of the culture vessel. In addition to a mechanism for raising or lowering the temperature, the device may be equipped with a thermometer for measuring the temperature, a CPU having a program for regulating the temperature, or the like, but is not limited to these.

[0060] The present disclosure also provides a program for controlling a culture device so that stem cells can be cultured according to the method of the present disclosure.

[0061] In one aspect, the present disclosure provides a program that instructs a system for expanding stem cells while inhibiting proliferation to perform a method for expanding stem cells while inhibiting proliferation, the method comprising culturing the stem cells in hibernation conditions for the stem cells.

[0062] In one aspect, the present disclosure provides a program that instructs a system for expanding stem cells while inhibiting proliferation to execute a method for expanding stem cells while inhibiting proliferation, the method comprising culturing the stem cells under stem cell hibernation conditions and normal culture conditions.

[0063] In one aspect, the present disclosure provides a program that instructs a system for producing a predetermined amount of stem cells in a timely manner to execute a method for producing a predetermined amount of stem cells in a timely manner, the method including culturing the stem cells under stem cell hibernation conditions and normal culture conditions.

[0064] In one aspect, the present disclosure provides a program that instructs a system for producing a predetermined amount of stem cells in a timely manner to execute a method for producing a predetermined amount of stem cells in a timely manner, the method including the steps of culturing the stem cells at about 20°C to 34°C and about 36°C to 38°C, and, if necessary, confirming whether the cell amount is the predetermined amount.

[0065] The present disclosure also provides a computer program for implementing the methods described elsewhere in the present disclosure or a storage medium for storing the same, as well as a system or user equipment constituting part of the system.

[0066] Machine learning, neural networks, and the like can be used to implement the method of the present disclosure. The weight coefficients of each node in the hidden layer of a neural network can be calculated based on previously acquired data. The process of calculating these weight coefficients is a learning process. The learning process can be supervised learning or unsupervised learning. In the case of supervised learning, for example, the weight coefficients of each node can be calculated so that the value of the output layer when a certain parameter value is input to the input layer is an appropriate amount or level. This can be performed, for example, by backpropagation (error backpropagation). Although a large amount of training data set used for learning may be preferable, if it is too large, overfitting can easily occur. A trained model can be used to implement the present disclosure. The trained model is not limited to the neural network model described above, and any other machine learning model can be used. For example, a random forest can be used. For example, a simple model constructed by simply setting weight coefficients obtained through prior learning can also be used.

[0067] The program of the present disclosure may be operated on any terminal. The terminal may be, for example, any terminal device such as a smartphone, tablet computer, smartwatch, laptop computer, or desktop computer. The user device may communicate with the server device via a network. Here, the type of network does not matter. For example, the user device may communicate with the server device via the Internet or via a LAN. The number of user devices is not limited to this and may be any number greater than or equal to one. In addition, a database unit connected to the server device may store eye information of multiple targets acquired in advance. The stored information may be used, for example, to construct a trained model. For example, the database unit may store the constructed trained model.

[0068] The user device includes a communication interface unit, an input unit, a display unit, a memory unit, and a processor unit.

[0069] The communication interface unit controls communication via the network. The processor unit of the user device can receive information from outside the user device and transmit information to outside the user device via the communication interface unit. For example, the processor unit of the user device can receive information from a server device and transmit information to the server device via the communication interface unit. The communication interface unit can control communication in any manner.

[0070] The input unit allows a user to input information into the user device. It does not matter in what manner the input unit allows a user to input information into the user device. For example, if the input unit is a touch panel, the user may input information by touching the touch panel. Alternatively, if the input unit is a mouse, the user may input information by operating the mouse. Alternatively, if the input unit is a keyboard, the user may input information by pressing keys on the keyboard. Alternatively, if the input unit is a microphone, the user may input information by voice.

[0071] The calculation results or measurements of the present disclosure can be displayed on a display unit included in the system of the present disclosure. The display unit can be any display for displaying information.

[0072] The memory unit that may be included in the system of the present disclosure stores programs for executing processes in the user device and data required for executing the programs. The memory unit stores, for example, a program that realizes the system of the present disclosure and / or part or all of a program for analyzing or evaluating information related to a subject. The memory unit may also store an application that implements any function.

[0073] The processor unit controls the overall operation of the user device. The processor unit reads and executes programs stored in the memory unit. This allows the user device to function as a device that executes desired steps. The processor unit may be implemented by a single processor or by multiple processors.

[0074] The server device includes a communication interface unit, a memory unit, and a processor unit.

[0075] The communication interface unit controls communication via the network. The communication interface unit also controls communication with the database unit. The processor unit of the server device can receive information from outside the server device and can transmit information to outside the server device via the communication interface unit. The processor unit of the server device can receive information from a user device and can transmit information to a user device via the communication interface unit. The communication interface unit can control communication in any manner.

[0076] The memory unit stores programs required for executing processes of the server device, data required for executing the programs, and the like.

[0077] The processor unit controls the operation of the entire server device. The processor unit reads and executes programs stored in the memory unit. This allows the server device to function as a device that executes desired steps. The processor unit may be implemented by a single processor or by multiple processors.

[0078] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0079] The cells of the present disclosure may be cells used in cell infusion therapy and drug discovery research. In cell infusion therapy, the cells may be cells obtained from a subject or from a different subject.

[0080] In some embodiments, any culture medium may be used, for example, platelet lysate is preferred. The platelets may be platelets obtained from the subject or from a different subject. The cells and platelets to be cultured may be of the same origin (i.e., autologous) or of different origins (i.e., allogeneic). In a preferred embodiment, the platelets may be autologous.

[0081] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0082] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0083] The present disclosure will be described in more detail below with reference to examples. The following examples were conducted in accordance with the Declaration of Helsinki and other ethical standards.

[0084] (Example 1: Temperature change experiment 1 in cell culture) In this example, it was confirmed whether cell proliferation could be suppressed by lowering the culture temperature from 37°C to 30°C during cell culture.

[0085] (material) Cells: Human bone marrow-derived mesenchymal stem cells (BMSCs) (3 samples) Culture medium: MEM-α + 5% (platelet lysate) PL + (penicillin-streptomycin solution) PS ·Container…FALCON EasyGrip TissueCultureDish35mm (bottom area 9cm 2 ) Incubator: 37℃ and 30℃ settings (method) -Day 0- 1. Cultured human BMSCs were detached and collected, and the cell number was counted. Thirteen 2.35 mm dishes were prepared, six for group A and six for group B. 3. 2 ml of medium was added to each well. 4. 1.5 x 10 each 4 The cells were seeded one by one. 5. The cells were cultured in a 37°C incubator. -Day 2- 6. One dish was taken out, the medium was removed, and the dish was washed with PBS. 7. 1 ml of TrypLE was added and reacted at 37°C for 5 minutes. TrypLE was inactivated by adding 8.1 ml of medium, and the mixture was collected into a Falcon tube by pipetting. 9. Centrifuge at 1500 rpm for 5 minutes. 10. Cells were suspended in 1 ml of medium and counted with LUNA-FL™ (three independent experiments). 11. The six dishes in group A were moved to a 30°C incubator. -Days 3 to 5- 12. One dish from group A and one dish from group B were taken out and steps 6 to 10 on the second day were carried out. On day 13.5, the remaining dishes of group A were returned to the 37°C incubator. -Days 6 to 8 -14. Procedure 12 was performed on days 3 to 5.

[0086] (result) The results are shown in Figure 1. As shown, the proliferation rate of stem cells decreased only during the low temperature (30°C) period, and proliferation returned to normal for 4 days after rewarming (Figure 1).

[0087] (Example 2: Temperature change experiment 2 in cell culture) In this example, we confirmed how cell proliferation would change when the culture temperature was lowered from 37°C to 30°C, 32°C, and 34°C during cell culture. We also confirmed how cell proliferation would be affected by returning the temperature from 30°C, 32°C, and 34°C to 37°C.

[0088] (material) Cells: Human BMSC (3 samples) Culture medium: MEM-α + 5% PL + PS ·Container…Nunc EasY Flask (bottom area 25cm) 2 ) Incubator: Set to 30°C, 32°C, 34°C, or 37°C (method) -Day 0- 1. BMSCs stored frozen in liquid nitrogen were extracted, thawed, and counted. The cells were diluted appropriately according to the number of cells. 2.25cm 2 Fifteen flasks were prepared for each temperature group. 3. 5 ml of medium was added to each well. 4. 1.0 x 10 for each 4 The cells were seeded one by one. 5. The cells were cultured in a 37°C incubator. -Day 2- 6. One flask was taken out, the medium was removed, and the flask was washed with PBS. 7. 1 ml of TrypLE was added and reacted at 37°C for 5 minutes. TrypLE was inactivated by adding 8.1 ml of medium, and the mixture was collected into a Falcon tube by pipetting. 9. Centrifuge at 1500 rpm for 5 minutes. 10. Cells were suspended in 1 ml of medium and counted using LUNA-FL™. 11. The remaining four flasks in the temperature change group were moved to a 30°C (32°C, 34°C) incubator. -Days 3 to 5- 12. One flask was removed and steps 6 to 10 on the second day were carried out. On day 13.5, the remaining dishes of group A were returned to the 37°C incubator. -Days 6 to 8- 14. Procedure 12 was performed on days 3 to 5.

[0089] (result) The results are shown in Figures 2 and 3. As shown, cell growth was inhibited at 30°C and 32°C. Cell growth was also slightly inhibited at 34°C. Furthermore, after rewarming to 37°C, normal cell growth was observed regardless of the temperature change.

[0090] (Example 3: Temperature change experiment 3 in long-term cultured cells) In this example, the effect of temperature change was confirmed by extending the incubation period to 14 days in total so that confluence could be observed. However, if confluence was confirmed during the incubation period, the incubation period was stopped at that point. The 3-day period for applying the temperature change was not changed.

[0091] (method) Human BMSCs (3 samples) Culture medium (MEM-α + 5% PL + P / S) Nunc Easy Flask 25cm 2 Incubators (37°C and 32°C) (method) -Day 0- 1. BMSCs stored frozen in liquid nitrogen were extracted, thawed, and counted. The cells were diluted appropriately according to the number of cells. 2.25cm 2 Twelve flasks were prepared in duplicate for each temperature condition. 3. 5 ml of medium was added to each well. 4. 1.0 x 10 for each 4 The cells were seeded one by one. 5. The cells were cultured in a 37°C incubator. -Day 2- 6. One flask was taken out, the medium was removed, and the flask was washed with PBS. 7. 1 ml of TrypLE was added and reacted at 37°C for 5 minutes. TrypLE was inactivated by adding 8.1 ml of medium, and the mixture was collected into a Falcon tube by pipetting. 9. Centrifuge at 1500 rpm for 5 minutes. 10. Cells were suspended in 1 ml of medium and counted using LUNA-FL™. 11. The remaining flasks in the temperature shift group were moved to a 32°C incubator. -Day 5- 12. One flask was removed from 32°C and one from 37°C, and steps 6 to 10 on day 2 were carried out. 13. The flasks that had been moved to the 32°C incubator were returned to the 37°C incubator. -Day 6 to Day 14- One sheet was counted each day from the group that had the temperature changed to 14.32°C and the group that remained at 37°C. 15. If confluence was confirmed by microscopic observation, no further counting was performed the following day.

[0092] (result) As in Examples 1 and 2, the proliferation rate of stem cells decreased only during the low temperature period, and proliferation returned to normal after rewarming (FIGS. 4 and 5).

[0093] Example 4: Changes in nutritional factors due to temperature changes We confirmed whether the application of temperature change and the duration of the temperature change would affect the secretion of cellular trophic factors.

[0094] (material) Cells: Human BMSC (2 samples) Culture medium: MEM-α + 5% PL + PS ·Container…Nunc EasY Flask25cm 2 Incubator: 37℃ and 32℃ settings ·ELISA kit…HGF (a nutrient factor not contained in PL) (method) Human BMSCs were cultured at 37°C for 2 days and then transferred to lower temperatures (days 0, 1, 3, 5, and 7). They were then reincubated at 37°C for 3 days, after which trophic factors were measured. 1000 μL of the supernatant was collected, and the trophic factor concentration was determined by ELISA. The cells were counted, and the amount of trophic factor per cell was calculated.

[0095] Experiments were conducted on two samples for each of the groups: no temperature change, 1-day temperature change, 3-day temperature change, 5-day temperature change, and 7-day temperature change (10 samples in total).

[0096] (result) The results are shown in Figure 6. There appears to be no significant difference in trophic factor secretion between the temperature shift groups. HGF levels appear to be low in the 3-day temperature shift group, but this is thought to be due to the unexpectedly rapid cell proliferation, large cell numbers, and a large denominator.

[0097] (Example 5: Study using an automatic culture device) In this example, the influence of temperature changes on cell culture using an automatic culture device was confirmed.

[0098] (material) Cells: Human PBMC ·Culture device…Quantum automatic culture device Culture medium: MEM-α + 5% PL + PS (method) The cells were cultured at 37°C until the cell count reached 10-50 million (4-11 days), then transferred to a lower temperature of 28-30°C for culture (4-10 days). The cells were then returned to 37°C and cultured for 4-6 days. Since it was impossible to measure anything other than the final cell count, the lactate value was converted to cell count (4.34e-8 mmol / day / cell).

[0099] (result) When the final cell count was compared with the cell count calculated from the lactate value, the cell count calculated from the lactate value matched the actual measured value with an accuracy of 97±19% (Figure 7). Even in the automated culture device, cell proliferation was suppressed during the low temperature period, but proliferation occurred without any problems after rewarming (Figure 8).

[0100] (Example 6: Temperature change experiment 1 in cell culture (20°C)) In this example, it was confirmed what effect a temperature change to an even lower temperature (20° C.) than in Examples 1 to 5 would have on cell proliferation.

[0101] (material) Cells: Human BMSCs Culture medium: MEM-α + 5% PL + PS ·Container…FALCON EasyGrip TissueCultureDish 35mm (bottom area 9cm 2 ) Incubator: A regular cell culture incubator was used for 37°C. 20°C was maintained in 5% CO using Anaeropack in the AS ONE ICI100. 2 I created the environment.

[0102] (method) -Day 0- 1. Human BMSCs were detached and collected during culture, and the cell number was counted. Thirteen 2.35 mm dishes were prepared: six for group A and six for group B. 3. 2 ml of medium was added to each well. 4. 3.6 x 10 each 3 Individual cells were seeded. 5. The cells were cultured in a 37°C incubator. -Day 2- 6. One dish was taken out, the medium was removed, and the dish was washed with PBS. 7. 1 ml of TrypLE was added and reacted at 37°C for 5 minutes. TrypLE was inactivated by adding 8.1 ml of medium, and the mixture was collected into a Falcon tube by pipetting. 9. Centrifuge at 1500 rpm for 5 minutes. 10. Cells were suspended in 1 ml of medium and counted using LUNA-FL™. 11. Place the six dishes from Group A into the Anaeropack jar, add the Anaeropack, and immediately close the lid. 12. Place 11 in the AS ONE ICI100 set to 20°C. -Days 3 to 5- 13. Open the Anaeropack jar, remove one sheet from Group A and one sheet from Group B from the 37°C incubator, and perform steps 6 to 10 on the second day. 14. A new Anaeropack was opened, transferred into the Anaeropack jar, and returned to 20°C. On day 15.5, all remaining dishes in group A were transferred to a 37°C incubator. -Days 6 to 8- 16. One dish from group A and one dish from group B were taken out and steps 6 to 10 on the second day were carried out.

[0103] (result) When cultured at 20°C, cell growth was suppressed, and after warming to 37°C, cell growth recovered, although the growth rate was relatively slow (Figs. 9 and 10).

[0104] (Example 7: Temperature change experiment 1 in cell culture (25°C)) In this example, it was confirmed what effect a temperature change to an even lower temperature (25° C.) than in Examples 1 to 5 would have on cell proliferation.

[0105] (material) Cells: Human BMSCs Culture medium: MEM-α + 5% PL + PS ·Container…FALCON EasyGrip TissueCultureDish 35mm (bottom area 9cm 2 ) Incubator: set to 37°C 25℃, 5% CO using Anaeropack in AS ONE ICI100 2 I created the environment. (method) -Day 0- 1. Human BMSCs were detached and collected during culture, and the cell number was counted. Thirteen 2.35 mm dishes were prepared: six for group A and six for group B. 3. 2 ml of medium was added to each well. 4. 7.2 x 10 each 3 Individual cells were seeded. 5. The cells were cultured in a 37°C incubator. -Day 2- 6. One dish was taken out, the medium was removed, and the dish was washed with PBS. 7. 1 ml of TrypLE was added and reacted at 37°C for 5 minutes. TrypLE was inactivated by adding 8.1 ml of medium, and the mixture was collected into a Falcon tube by pipetting. 9. Centrifuge at 1500 rpm for 5 minutes. The cells were suspended in 10.1 ml of medium and counted using LUNA-FL™. 11. Place the six dishes from Group A into the Anaeropack jar, add the Anaeropack, and immediately close the lid. 11. was placed in the AS ONE ICI100 set to 12.25°C. -Days 3 to 5- 13. Open the Anaeropack jar, remove one sheet from Group A and one sheet from Group B from the 37°C incubator, and perform steps 6 to 10 on the second day. 14. A new Anaeropack was opened, transferred into the Anaeropack jar, and returned to 25°C. On day 15.5, all remaining dishes in group A were transferred to a 37°C incubator. -Days 6 to 8- 16. One dish from group A and one dish from group B were taken out and steps 6 to 10 on the second day were carried out.

[0106] (result) When the temperature was raised to 25°C, cell proliferation was suppressed, and after rewarming, cells proliferated normally. The proliferation rate after rewarming was faster than that in the culture at 20°C in Example 6 (Figs. 11 and 12).

[0107] (Consideration) From the above, we have clarified the hibernation conditions for stem cells (e.g., bone marrow-derived mesenchymal stem cells) by suppressing cell proliferation at 20°C to 34°C. Furthermore, normal cell proliferation is important after rewarming, and cell proliferation recovered under all hibernation conditions, although the proliferation rate varied. The temperature of the hibernation conditions can be appropriately set depending on the target cell number. However, at 20°C, the rate of cell proliferation after rewarming was slow, and at 34°C, there was little difference in the final cell number compared to when cultured constantly at 37°C. Therefore, hibernation conditions at 25°C to 32°C are preferred, and hibernation conditions at 30°C to 32°C are even more preferred. The proliferation rate of bone marrow-derived mesenchymal stem cells varies greatly depending on the donor, the nationality of the subject, and the facility where the cells are cultured (Stroncek1 et al., Front Cell Dev Biol. 2020 Jun 16;8:458. doi: 10.3389 / fcell.2020.00458. eCollection 2020.), but the method disclosed herein makes it possible to achieve the target final cell number by appropriately adjusting the temperature and duration of culture.

[0108] (Example 8) Example of a protocol for actually producing an appropriate amount 50 ml of bone marrow fluid, the number of stem cells contained in which is unknown, is placed into an automated culture device, and the attached stem cells are cultured over a period of four weeks until they reach 100 million cells. The degree of proliferation during this process is estimated from the known daily lactate secretion level of each stem cell.

[0109] (Example 9) Treatment example after producing an appropriate amount of cell preparation For localized brain damage (such as cerebral infarction, intracerebral hemorrhage, and traumatic brain injury), functional recovery can be achieved by administering 40 million cells around the damaged area.

[0110] As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the scope of the claims. It is understood that the patents, patent applications, and literature cited in this specification should be incorporated by reference in their entirety as if the contents themselves were specifically set forth in this specification. This application claims priority to Japanese Patent Application No. 2023-011235 filed with the Japan Patent Office on January 27, 2023, the contents of which are incorporated by reference in their entirety. [Industrial Applicability]

[0111] According to the present disclosure, it is possible to control the proliferation rate of stem cells, and produce an appropriate amount of stem cells at an appropriate time. The cultured cells can be used for cell injection therapy, and can be used in fields such as pharmaceuticals.

Claims

[Claim 1] The invention described in the specification.

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