Frozen stem cell modification method

Abstract

To provide a frozen stem cell modification method, with which a cryopreserved mesenchymal stem cell may be modified to an activated mesenchymal stem cell having sufficient activity.SOLUTION: In a case where mesenchymal stem cells are cryopreserved at a predetermined temperature together with a cryoprotectant and the mesenchymal stem cells that have been cryopreserved are used, a cryopreservation container 43 is immersed in warm water at 35 to 37°C for 2 to 3 minutes so that the mesenchymal stem cells 35 are thawed to a temperature of 2 to 3°C. Washing solution 46 is injected into the cryopreservation container storing the thawed mesenchymal stem cells and the cryoprotectant 44, by which the cryoprotectant is removed from the mesenchymal stem cells with the washing solution. The mesenchymal stem cells from which the cryoprotectant has been removed, the cryoprotectant and the washing solution are subjected to centrifugal separation so as to be separated into layers. The mesenchymal stem cells having been subjected to the centrifugal separation, which are located in the bottom layer of the cryopreservation container, are extracted. Culture solution is injected into a first culture vessel storing the extracted mesenchymal stem cells. The mesenchymal stem cells are cultured in the first culture vessel at 36 to 37°C for 24 hours so that the mesenchymal stem cells are activated and modified to activated mesenchymal stem cells.SELECTED DRAWING: Figure 12

Description

[Technical Field]

[0001] The present invention relates to a method for modifying frozen stem cells, which activates mesenchymal stem cells that have been frozen to a predetermined temperature and modifies them into activated mesenchymal stem cells. [Background technology]

[0002] A stem cell preservation method has been disclosed that includes a detachment step of detaching stem cells using a pronase solution and a freezing step of slowly freezing the detached stem cells in a stem cell preservation medium, where the stem cell preservation medium is a medium containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), or a medium containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and a culture medium or an albumin solution (see Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication WO2013 / 187077 Summary of the Invention [Problem to be solved by the invention]

[0004] Various stem cells are used for the treatment of various diseases (such as cardiovascular disease and central nervous system diseases), regeneration in regenerative medicine, and non-therapeutic purposes such as cosmetic treatment. When cultured stem cells are cryopreserved, the stem cells are placed in a designated storage container and a cryoprotectant such as dimethyl sulfoxide (DMSO), hydroxyethyl starch (HES), or ethylene glycol (EG) is injected into the container to prevent cell destruction during freezing. When cryopreserved frozen stem cells are used, they must be thawed, but the cryoprotectant is not removed during thawing; the thawed stem cells are used as is, along with the cryoprotectant.

[0005] If the cryoprotectant is not removed when thawing frozen stem cells, approximately 50% or more of the frozen stem cells will die and the stem cells will not be fully activated. If the number of surviving stem cells decreases and the stem cells cannot be fully activated, the effectiveness of stem cells in treating various diseases, regenerative effects in regenerative medicine, and non-therapeutic uses such as cosmetic treatments will be reduced, and the desired effects may not be achieved. Furthermore, if stem cells containing a cryoprotectant are administered to the human body, there is a concern that the cryoprotectant may have adverse effects.

[0006] An object of the present invention is to provide a method for modifying frozen stem cells, which, when thawing cryopreserved mesenchymal stem cells, can remove a cryoprotectant while preserving the survival of most mesenchymal stem cells and can modify cryopreserved mesenchymal stem cells into activated mesenchymal stem cells with sufficient activity. Another object of the present invention is to provide a method for modifying frozen stem cells, when thawing cryopreserved mesenchymal stem cells, that can produce mesenchymal stem cells that are sufficiently and highly effective in the treatment of various diseases, regeneration in regenerative medicine, non-therapeutic uses such as cosmetics, and can achieve the desired effects. [Means for solving the problem]

[0007] The premise of the present invention to solve the above problems is a method for modifying frozen stem cells, which activates mesenchymal stem cells frozen at a predetermined temperature and modifies them into activated mesenchymal stem cells.

[0008] The present invention is characterized in that the frozen stem cell modification method includes a cryopreservation step of placing mesenchymal stem cells prepared by culturing bone marrow fluid collected from a donor in a cryopreservation container of a predetermined volume, injecting a cryoprotectant into the cryopreservation container containing the mesenchymal stem cells, and freezing and preserving the mesenchymal stem cells contained in the cryopreservation container together with the cryoprotectant at a predetermined temperature until the mesenchymal stem cells contained in the cryopreservation container are used; a stem cell thawing step of immersing the cryopreserved mesenchymal stem cells and the cryoprotectant in warm water maintained at 35-37°C for 2-3 minutes to thaw the mesenchymal stem cells to a temperature of 2-3°C when the mesenchymal stem cells frozen and preserved in the cryopreservation step are to be used; and a stem cell thawing step of injecting a washing solution into the cryopreservation container containing the mesenchymal stem cells and the cryoprotectant thawed from a frozen state in the stem cell thawing step. the mesenchymal stem cells located in the bottom layer of the cryopreservation container after the cryoprotectant has been removed from the mesenchymal stem cells by the stirring and removal step is placed in a centrifuge, and the mesenchymal stem cells, cryoprotectant, and washing solution in the cryopreservation container are centrifuged in layers to extract the mesenchymal stem cells located in the bottom layer of the cryopreservation container after the centrifugation step is performed; and the mesenchymal stem cells extracted by the mesenchymal stem cell extraction step are placed in a first culture vessel having a predetermined volume and a predetermined bottom area, a predetermined culture medium is poured into the first culture vessel containing the mesenchymal stem cells, and the mesenchymal stem cells are cultured in the first culture vessel at 36 to 37°C for 24 hours to activate the mesenchymal stem cells in the first culture vessel and convert them into activated mesenchymal stem cells.

[0009] In one example of the present invention, the frozen stem cell modification method includes a stem cell transport step in which, after extracting activated mesenchymal stem cells from a first culture container, the extracted activated mesenchymal stem cells are transported to the site where the activated mesenchymal stem cells will be used within 18 to 20 hours while being maintained at 20 to 21°C.

[0010] In another example of the present invention, the stem cell activation step involves injecting a culture product solution produced during the culture process of culturing bone marrow fluid collected from a donor into a first culture vessel, and culturing mesenchymal stem cells in the first culture vessel using the culture solution and culture product solution at a temperature of 35 to 37°C for 24 hours.

[0011] In another example of the present invention, the stem cell activation step comprises leaving the first culture vessel statically at a temperature of 35 to 37°C for 24 hours with the first culture vessel tilted at an angle of 2 to 5 degrees.

[0012] In another example of the present invention, the stem cell activation process comprises statically leaving the first culture vessel tilted at an angle of 2 to 5 degrees in one direction at a temperature of 35 to 37°C for 12 hours, and then tilting the first culture vessel tilted in one direction at an angle of 2 to 5 degrees in the opposite direction at a temperature of 35 to 37°C for 12 hours.

[0013] In another example of the present invention, the cryoprotectant is at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), and the washing solution is Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS).

[0014] Another example of the present invention includes a first stem cell settling step in which bone marrow fluid collected from a donor is separated into layers, intermediate layer bone marrow fluid located in the intermediate layer of the separated layers is extracted, and the intermediate layer bone marrow fluid and a predetermined culture medium are poured into a second culture vessel having a predetermined volume and a predetermined bottom area to settling first mesenchymal stem cells contained in the intermediate layer bone marrow fluid on the bottom surface of the second culture vessel; and after the first mesenchymal stem cells have settled on the bottom surface of the second culture vessel by the first stem cell settling step, the culture medium in the second culture vessel is discharged while the second culture vessel is being settling. a first stem cell culture step of injecting the new culture medium into a second culture vessel to culture the first mesenchymal stem cells and proliferating the first mesenchymal stem cells until the total planar area of ​​the first mesenchymal stem cells relative to the bottom area of ​​the second culture vessel reaches a first target ratio; and a second mesenchymal stem cell culture step of placing the second culture vessel containing the first mesenchymal stem cells proliferated in the first stem cell culture step and the culture medium in a centrifuge, centrifuging the first mesenchymal stem cells and the culture medium in the second culture vessel in layers, and extracting second mesenchymal stem cells located in the bottom layer of the second culture vessel after centrifugation. a first cell extraction step, a second stem cell fixation step of placing the second mesenchymal stem cells extracted in the first second mesenchymal stem cell extraction step in a third culture vessel having a predetermined volume and a predetermined bottom area and a volume larger than that of the second culture vessel, injecting a new culture medium into the third culture vessel, and fixing the second mesenchymal stem cells to the bottom surface of the third culture vessel; and a second stem cell fixation step of fixing the second mesenchymal stem cells to the bottom surface of the third culture vessel by the second stem cell fixation step, injecting a new culture medium into the third culture vessel while discharging the culture medium in the third culture vessel. The mesenchymal stem cells are produced by a mesenchymal stem cell culture method comprising: a second stem cell culture step of culturing cells and proliferating the second stem cells until the total planar area of ​​the second mesenchymal stem cells relative to the bottom area of ​​the third culture vessel reaches a second target ratio; and a second mesenchymal stem cell extraction step of placing the third culture vessel containing the second mesenchymal stem cells cultured by the second stem cell culture step in a centrifuge, centrifuging the second mesenchymal stem cells and culture solution in the third culture vessel in layers, and extracting the second mesenchymal stem cells located in the bottom layer of the third culture vessel after centrifugation.

[0015] In another example of the present invention, the cryopreservation step involves placing the second mesenchymal stem cells immediately after extraction in the second mesenchymal stem cell extraction step in a cryopreservation container, and immediately freezing and preserving the mesenchymal stem cells placed in the cryopreservation container together with a cryoprotectant.

[0016] In another example of the present invention, the culture solution is the culture solution remaining after extracting the cultured mesenchymal stem cells from the second culture vessel or the third culture vessel, and contains specific metabolic substances secreted from the mesenchymal stem cells during the mesenchymal stem cell culture process. [Effects of the Invention]

[0017] According to the frozen stem cell modification method of the present invention, a cleaning solution is poured into a cryopreservation container containing mesenchymal stem cells and a cryoprotectant that have been thawed from a frozen state, the cryopreservation container into which the cleaning solution has been poured is agitated to remove the cryoprotectant from the mesenchymal stem cells in the cryopreservation container using the cleaning solution, the mesenchymal stem cells, cryoprotectant, and cleaning solution in the cryopreservation container are centrifuged in layers, and the mesenchymal stem cells located in the bottom layer of the cryopreservation container after centrifugation are extracted. Therefore, when thawing frozen mesenchymal stem cells, the cleaning solution can be used to remove the cryoprotectant, and the majority (70 to 90%) of the mesenchymal stem cells thawed from a frozen state can be kept alive. The frozen stem cell modification method involves placing mesenchymal stem cells thawed from a frozen state in a first culture vessel having a predetermined volume and a predetermined bottom area, pouring a predetermined culture medium into the first culture vessel containing the mesenchymal stem cells, and culturing the mesenchymal stem cells in the first culture vessel at 35-37°C for 24 hours to activate the mesenchymal stem cells in the first culture vessel. Therefore, when the cryopreserved mesenchymal stem cells are thawed, the thawed mesenchymal stem cells can be modified into activated mesenchymal stem cells with sufficient activity. The frozen stem cell modification method can produce activated mesenchymal stem cells that are sufficiently and highly effective in the treatment of various diseases (such as cardiovascular disease and central nervous system disease), regeneration in regenerative medicine, and non-therapeutic uses such as cosmetics, and can produce activated mesenchymal stem cells that can achieve desired effects. The frozen stem cell modification method can produce activated mesenchymal stem cells that can be used suitably and in a timely manner to treat various diseases, can produce activated mesenchymal stem cells that can be used suitably and in a timely manner to regenerate various tissues and organs, and can produce activated mesenchymal stem cells that can be used suitably and in a timely manner for non-therapeutic purposes such as cosmetics. Because the frozen stem cell modification method does not contain a cryoprotectant in the thawed activated mesenchymal stem cells, administering activated mesenchymal stem cells produced by the frozen stem cell modification method to the human body will not have any adverse effects.

[0018] When the activated mesenchymal stem cells are to be used within 18 to 20 hours after extraction from the first culture vessel, the frozen stem cell modification method includes a stem cell transport step in which the extracted activated mesenchymal stem cells are transported to the site of use within 18 to 20 hours while maintained at 20 to 21°C. By transporting the activated mesenchymal stem cells to the site of use within 18 to 20 hours while maintained at 20 to 21°C, the activity of the activated mesenchymal stem cells is not lost, and the activated mesenchymal stem cells can be used to treat various diseases (such as cardiovascular disease and central nervous system disease).The activated mesenchymal stem cells can be used for regeneration in regenerative medicine, and can also be used for non-therapeutic purposes such as cosmetic treatment.

[0019] In the stem cell activation step, the culture product solution produced during the culture process of culturing bone marrow fluid collected from a donor is poured into a first culture vessel, and mesenchymal stem cells are cultured in the first culture vessel at a temperature of 35-37°C for 24 hours using the culture solution and culture product solution.This frozen stem cell modification method reliably promotes the activation of mesenchymal stem cells in the first culture vessel by utilizing the culture product solution produced during the culture process of mesenchymal stem cells before cryopreservation.Therefore, the culture product solution can be used to activate thawed mesenchymal stem cells within 24 hours, allowing for the efficient production of activated mesenchymal stem cells with sufficient activity, and for the rapid use of mesenchymal stem cells from which the cryoprotectant has been removed.

[0020] In the stem cell activation step, a frozen stem cell modification method involves leaving the first culture vessel tilted at an angle of 2 to 5 degrees and statically leaving it at a temperature of 35 to 37°C for 24 hours. By tilting the first culture vessel at an angle of 2 to 5 degrees, the thawed mesenchymal stem cells and culture medium (and culture product solution) within the first culture vessel are biased to one side of the first culture vessel, increasing the pressure of the mesenchymal stem cells and culture medium (and culture product solution) on one side of the first culture vessel and concentrating the mesenchymal stem cells on one side of the first culture vessel, thereby increasing the activity of the mesenchymal stem cells and modifying the thawed mesenchymal stem cells into activated mesenchymal stem cells with sufficient activity.

[0021] In the stem cell activation step, the first culture vessel is tilted at an angle of 2 to 5 degrees in one direction and left statically at a temperature of 35 to 37°C for 12 hours, and the first culture vessel tilted in one direction is tilted at an angle of 2 to 5 degrees in the opposite direction from the one direction and left statically at a temperature of 35 to 37°C for 12 hours. The frozen stem cell modification method includes tilting the first culture vessel at an angle of 2 to 5 degrees in one direction and further tilting the first culture vessel tilted in one direction at an angle of 2 to 5 degrees in the opposite direction from the one direction. By tilting the container at a certain angle, the thawed mesenchymal stem cells and culture medium (and culture product solution) in the first culture container are biased to one side or the other of the first culture container, and the pressure of the mesenchymal stem cells and culture medium (and culture product solution) on one side or the other of the first culture container increases, and the mesenchymal stem cells are concentrated evenly on one side or the other of the first culture container, thereby increasing the activity of the mesenchymal stem cells and modifying the thawed mesenchymal stem cells into activated mesenchymal stem cells with sufficient activity.

[0022] The method for modifying frozen stem cells, in which the cryoprotectant is at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), and the washing solution is Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS), involves injecting the washing solution (Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS)) into a cryopreservation container containing mesenchymal stem cells thawed from a frozen state and the cryoprotectant, stirring the cryopreservation container into which the washing solution has been injected, and removing the cryoprotectant (at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)) from the mesenchymal stem cells in the cryopreservation container by the washing solution. The cryoprotectant (hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO)), which is a component of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), is removed from the cryopreservation container, and the mesenchymal stem cells, cryoprotectant, and washing solution are centrifuged in layers within the cryopreservation container. After centrifugation, the mesenchymal stem cells located at the bottom layer of the cryopreservation container are extracted. Therefore, when thawing frozen mesenchymal stem cells, a washing solution (Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS)) is used to reliably remove the cryoprotectant (at least dimethyl sulfoxide (DMSO) among hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)). This allows the survival of most (70-90%) of the mesenchymal stem cells upon thawing, and also enables the thawed mesenchymal stem cells to be converted into activated mesenchymal stem cells with sufficient activity.

[0023] a first stem cell settling step in which bone marrow fluid collected from a donor is separated into layers, intermediate layer bone marrow fluid located in the intermediate layer of the separated layers is extracted, and the intermediate layer bone marrow fluid and a predetermined culture medium are poured into a second culture vessel having a predetermined volume and a predetermined bottom area, thereby causing first mesenchymal stem cells contained in the intermediate layer bone marrow fluid to settle on the bottom surface of the second culture vessel; and after the first mesenchymal stem cells have settled on the bottom surface of the second culture vessel by the first stem cell settling step, a new culture medium is poured into the second culture vessel while the culture medium in the second culture vessel is discharged, thereby culturing the first mesenchymal stem cells, and causing the first mesenchymal stem cells to settle on the bottom surface of the second culture vessel. a first stem cell culture step of growing the first mesenchymal stem cells until the total planar area of ​​the first mesenchymal stem cells relative to the surface area reaches a first target ratio; a first second mesenchymal stem cell extraction step of placing a second culture vessel containing the first mesenchymal stem cells grown in the first stem cell culture step and a culture medium in a centrifuge, centrifuging the first mesenchymal stem cells and the culture medium in the second culture vessel in layers, and extracting the second mesenchymal stem cells located in the bottom layer of the second culture vessel after centrifugation; and a first second mesenchymal stem cell extraction step of placing the second mesenchymal stem cells in a third culture vessel having a predetermined volume and a bottom surface with a predetermined area and a larger capacity than the second culture vessel. a second stem cell settling step of injecting a new culture medium into a third culture vessel while accommodating the second mesenchymal stem cells extracted by the method described above, and settling the second mesenchymal stem cells on the bottom surface of the third culture vessel; a second stem cell culture step of culturing the second mesenchymal stem cells by injecting a new culture medium into the third culture vessel while discharging the culture medium from the third culture vessel, and culturing the second mesenchymal stem cells until the total planar area of ​​the second mesenchymal stem cells to the bottom surface area of ​​the third culture vessel reaches a second target ratio; and a second mesenchymal stem cell extraction step of placing a third culture vessel containing second mesenchymal stem cells in a centrifuge, centrifuging the second mesenchymal stem cells and culture solution in the third culture vessel in layers, and extracting the second mesenchymal stem cells located in the bottom layer of the third culture vessel after centrifugation; and a second mesenchymal stem cell extraction step of extracting the second mesenchymal stem cells located in the bottom layer of the third culture vessel after centrifugation. The method for modifying frozen stem cells is implemented by a mesenchymal stem cell culture method including: allowing the first mesenchymal stem cells to settle on the bottom surface of the second culture vessel; discharging the culture solution from the second culture vessel; injecting new culture solution into the second culture vessel; and culturing the first mesenchymal stem cells settled on the bottom surface of the second culture vessel using the new culture solution.The second mesenchymal stem cells proliferated in the second culture vessel are placed in a third culture vessel, and after the second mesenchymal stem cells have settled on the bottom surface of the third culture vessel, the culture medium is discharged from the third culture vessel and new culture medium is poured into the third culture vessel, and the second mesenchymal stem cells that have settled on the bottom surface of the third culture vessel are cultured using the new culture medium, and the second mesenchymal stem cells are proliferated in the third culture vessel. This makes it possible to proliferate mesenchymal stem cells efficiently and reliably, and to efficiently produce mesenchymal stem cells with sufficient activity.

[0024] In the frozen storage step, the second mesenchymal stem cells immediately after extraction in the second mesenchymal stem cell second extraction step are placed in a frozen storage container, and the mesenchymal stem cells placed in the frozen storage container are immediately frozen and stored together with a cryoprotectant.This frozen stem cell modification method immediately freezes and stores mesenchymal stem cells with sufficient activity produced by the mesenchymal stem cell culture method, so that the majority of the mesenchymal stem cells placed in the frozen storage container can be frozen and stored alive, and the mesenchymal stem cells produced by the mesenchymal stem cell culture method can be stored for a long period of time.

[0025] The frozen stem cell modification method, in which the culture solution is the culture solution remaining after extracting the cultured mesenchymal stem cells from the second or third culture vessel and contains the specified metabolic substances secreted by the mesenchymal stem cells during the mesenchymal stem cell culture process, triggers the mesenchymal stem cells to quickly begin to become active, and the culture solution can be used to quickly activate the mesenchymal stem cells, allowing activated mesenchymal stem cells to be produced efficiently in a short period of time. [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a schematic diagram of an example frozen stem cell reforming system. [Figure 2] An explanatory diagram showing an example of a bone marrow fluid separation step in a mesenchymal stem cell culture method. [Figure 3] An explanatory diagram of the bone marrow fluid separation process, continuing from Figure 2. [Figure 4] FIG. 10 is an explanatory diagram showing an example of a first shape deformation observation step. [Figure 5] FIG. 2 is a side view of the first flat culture vessel (second culture vessel). [Figure 6] FIG. 2 is an explanatory diagram showing an example of the second mesenchymal stem cell first extraction step in the mesenchymal stem cell culture method. [Figure 7] FIG. 10 is an explanatory diagram showing an example of the second shape deformation observation step in the mesenchymal stem cell culture method. [Figure 8] FIG. 10 is a side view of the second flat culture vessel (third culture vessel). [Figure 9] FIG. 1 is an explanatory diagram showing an example of the cryopreservation process in the frozen stem cell modification method. [Figure 10] FIG. 1 is an explanatory diagram showing an example of a stem cell thawing process in a frozen stem cell modification method. [Figure 11] FIG. 1 is an explanatory diagram showing an example of the stirring and removal process in the frozen stem cell modification method. [Figure 12] FIG. 1 is an explanatory diagram showing an example of a mesenchymal stem cell extraction process in a frozen stem cell modification method. [Figure 13] FIG. 1 is an explanatory diagram showing an example of a stem cell activation step in a frozen stem cell modification method. [Figure 14] FIG. 10 is a side view of the third flat culture vessel (first culture vessel). [Figure 15] FIG. 2 is a partially enlarged view showing an example of the planar shape of the second mesenchymal stem cells. [Figure 16] FIG. 10 is a partially enlarged view showing another example of the planar shape of the second mesenchymal stem cells. [Figure 17] FIG. 1 is an explanatory diagram showing an example of a stem cell transport process in a frozen stem cell modification method. DETAILED DESCRIPTION OF THE INVENTION

[0027] The frozen stem cell modification method according to the present invention will be described in detail below with reference to the accompanying drawings, such as Figure 1, which shows a schematic diagram of an example of a frozen stem cell modification system 10. Figure 2 is an explanatory diagram showing an example of the bone marrow fluid separation step in the mesenchymal stem cell culture method, and Figure 3 is an explanatory diagram of the bone marrow fluid separation step continuing from Figure 2. Figure 4 is an explanatory diagram showing an example of the first shape deformation observation step, and Figure 5 is a side view of the first flat culture vessel (second culture vessel).

[0028] The frozen stem cell modification system 10 cultures (manufactures) a specific type of single-species mesenchymal stem cells using a stem cell culture method, and modifies cryopreserved single-species mesenchymal stem cells into activated mesenchymal stem cells using the frozen stem cell modification method. The stem cell culture method uses a first bone marrow fluid collected from multiple donors (people) and performs a first stem cell colonization step, a first stem cell culture step, a first second mesenchymal stem cell extraction step, a second stem cell colonization step, a second stem cell culture step, and a second second mesenchymal stem cell extraction step, thereby culturing (manufacturing) a specific type of single-species mesenchymal stem cells from multiple types of mesenchymal stem cells contained in the first bone marrow fluid.

[0029] The frozen stem cell modification system 10 is made up of a management server 11, an IC tag reader / writer 12 connected to the management server 11 via an interface (wireless or wired), an electron microscope 13 connected to the management server 11 via an interface (wireless or wired), and a refrigerator 14 or freezer 14 connected to the management server 11 via an interface (wireless or wired). The electron microscope 13 has an image capturing function that captures an enlarged image of a subject using an imaging element, and an image transmission function that transmits the enlarged image to the management server 11.

[0030] The management server 11 is a physical computer or cloud computing system equipped with a central processing unit (CPU or MPU, virtual CPU or virtual MPU) and memory (main memory and cache memory, virtual main memory and virtual cache memory), and has a built-in large-capacity storage area or a large-capacity virtual storage area. The cloud may be Infrastructure as a Service (IaaS), Platform as a Service (PaaS), or Software as a Service (SaaS). Input devices such as a keyboard 15 and a mouse 16, and output devices such as a display 17 and a printer (not shown) are connected to the management server 11 via interfaces (wireless or wired).

[0031] In the frozen stem cell reforming system 10, various donor data (donor identification information) are managed using an IC tag 18 (IC chip), and stem cell data related to mesenchymal stem cells is also managed using the IC tag 18. Donor data includes the donor's name, address, telephone number, date of birth, sex, blood type, height, weight, email address, etc., while stem cell data includes stem cell identification information, stem cell production date, culture product solution production date, culture product solution identification information, etc. In the first stem cell settlement process, a person in charge (such as a doctor, nurse, researcher, or bioengineer) collects 2-3 cc (2-3 ml) of first bone marrow fluid from the donor. Simultaneously with the collection of the first bone marrow fluid, the person in charge starts the frozen stem cell reforming system 10 on the management server 11 and appropriately inputs donor data and stem cell data into the management server 11 using input devices such as the keyboard 15 and mouse 16.

[0032] Management server 11 generates a unique donor identifier that identifies each donor each time donor data is input (each time first bone marrow fluid is collected from the donor). Management server 11 writes the input donor data and stem cell data to IC tag 18 using IC tag reader / writer 12. Management server 11 stores (memorizes) the donor data and stem cell data in a large-capacity storage area or a large-capacity virtual storage area in a state where they are associated with the donor identifier and the IC tag identification information (IC tag identifier) ​​of IC tag 18 (donor data storage process).

[0033] As shown in FIG. 2, the first bone marrow fluid 19 collected from the donor is poured (contained) into a vertically long glass test tube 20 (separation container). Note that 2 to 3 cc of the first bone marrow fluid 19 contains 0.5 to 1 ml (approximately 5×10 7 The glass test tube 20 contains multiple types of mesenchymal stem cells (cells / ml). An IC tag 18 is affixed to the outer surface of the glass test tube 20. When the donor data and stem cell data written in the IC tag 18 are read by the IC tag reader / writer 12, the management server 11 displays the donor data and stem cell data on the display 17. The glass test tube 20 into which the first bone marrow fluid 19 has been injected is set in a test tube rack (not shown), and the test tube rack is then housed inside a thermostatic bath (not shown).

[0034] The glass test tube 20 into which the first bone marrow fluid 19 has been injected is left statically (left quietly without movement) in an incubator for a predetermined time (approximately 2 hours). The temperature inside the incubator is maintained at approximately 35-37°C, which is approximately the same as body temperature. By leaving the glass test tube 20 statically in the incubator for the predetermined time (approximately 2 hours), the first bone marrow fluid 19 injected into the glass test tube 20 separates into several layers (three layers) vertically within the glass test tube 20, as shown in FIG. 3 (bone marrow fluid separation process). After the first bone marrow fluid 19 has separated into layers, the test tube rack is removed from the incubator, the glass test tube 20 is pulled out of the test tube rack, and the second bone marrow fluid 21 (intermediate layer bone marrow fluid) present in a specific layer (the intermediate layer (second layer) of the three layers (layers)) of the separated first bone marrow fluid 19 is extracted (aspirated) using a syringe (not shown) or a pipette (not shown) (bone marrow fluid extraction process).

[0035] After extracting a specific second bone marrow fluid 21 (intermediate layer bone marrow fluid) located in the intermediate layer from the first bone marrow fluid 19, the second bone marrow fluid 21 and culture medium 26 are poured (contained) into a first flat culture vessel 22 (second culture vessel) (cell culture vessel). The culture vessel 22 is maintained at a temperature approximately equal to body temperature (approximately 35-37°C) and left statically (left unmoved) for 12-24 hours. During the 12-24 hours, the deformation of the first mesenchymal stem cells 21 contained in the second bone marrow fluid 21 in the culture vessel 22 from their initial planar shape is observed using a display 17 (electron microscope) at approximately 1-2 hour intervals to determine whether the first mesenchymal stem cells 21 have settled on the bottom surface 23 of the first flat culture vessel 22 (first shape deformation observation step). An IC tag 18, on which donor data and stem cell data are written, is attached to the bottom surface 23 (outer surface of the bottom wall) of the first flat culture vessel 22.

[0036] The first flat culture vessel 22 (second culture vessel) is made of transparent glass or transparent plastic, and is a flat vessel with a small capacity and a predetermined area on the bottom 23, with a planar shape of an approximately regular square. The injection port 24 of the first flat culture vessel 22 is watertightly closed by a lid 25. The first flat culture vessel 22 has a capacity of about 20 to 30 cc (preferably 25 cc), and the area of ​​the bottom 23 is about 25 to 36 mm 2 The length of one side is 5 to 6 mm. Note that the first flat culture vessel 22 may be a flat vessel having a small capacity, a bottom surface with a predetermined area, and a circular or elliptical planar shape.

[0037] The person in charge removes the lid 25 from the injection port 24, and injects (contains) the second bone marrow fluid 21 drawn into a syringe or pipette into the first flat culture vessel 22 through the injection port 24, and also injects (contains) the culture solution 26 into the culture vessel 22, and then closes the injection port 24 with the lid 25. The culture solution 26 contains a mineral salt solution and amino acids supplemented with penicillin (approximately 100 U / ml), amphotericin (approximately 100 ng / ml), streptomycin (approximately 100 mkg / ml), L-glutamine (approximately 2-4 ml), and 20% fetal bovine serum.

[0038] Note that Dulbecco's Modified Eagle's Medium (DMEM), Grasgow Minimum Essential Medium (GMEM), RPMI 640, etc. may also be used as the culture medium 26. Insulin, transferrin, ethanolamine, selenium, 2-mercaptoethanol, L-alanyl-L-glutamine, sodium pyruvate, L-alanine, L-asparagine, L-aspartic acid, glycine, L-proline, L-serine, etc. may also be added to the culture medium 26. The first mesenchymal stem cells 21 contained in the second bone marrow fluid 21 injected into the first flat culture vessel 22 settle on the bottom surface 23 of the first flat culture vessel 22 over time, while being cultured in the culture medium 26, and gradually proliferate (differentiate) and form colonies on the bottom surface 23 of the culture vessel 22.

[0039] After the second bone marrow fluid 21 and the culture medium 26 are poured into the first flat culture vessel 22, the culture vessel 22 is placed (set) in the specimen holder 27 of the electron microscope 13. A spacer 30 is interposed between the upper surface 28 of the specimen holder 27 of the electron microscope 13 and the bottom 29 of the first flat culture vessel 22, and the bottom 29 of the culture vessel 22 is held in an elevated position by the spacer 30. The culture vessel 22 is held in an inclined position at a predetermined angle so that the bottom 29 of the culture vessel 22 faces up and the top 31 (inlet 24) of the culture vessel 22 faces down. Alternatively, the top 31 of the first flat culture vessel 22 may be held in an elevated position by the spacer 30, and the culture vessel 22 may be held in an inclined position at a predetermined angle so that the top 31 of the culture vessel 22 faces up and the bottom 29 of the culture vessel 22 faces down. The inclination angle α1 of the first flat culture vessel 22 with respect to the upper surface 28 of the sample holder 27 is in the range of 2 to 5°, and preferably in the range of 2 to 3°.

[0040] By tilting the first flat culture vessel 22 at the above-mentioned tilt angle relative to the upper surface 28 of the sample holder 27, the second bone marrow fluid 21 (or the first mesenchymal stem cells 21) and the culture medium 26 within the culture vessel 22 are biased toward the top 31 side (or the bottom 29 side) of the culture vessel 22, and the water pressure between the second bone marrow fluid 21 (or the first mesenchymal stem cells 21) and the culture medium 26 on the top 31 side (or the bottom 29 side) of the culture vessel 22 increases, causing the second bone marrow fluid 21 (or the first mesenchymal stem cells 21) to concentrate on the top 31 side of the culture vessel 22, thereby increasing the activity of the first mesenchymal stem cells 21 and allowing the first mesenchymal stem cells 21 to easily and quickly settle (or proliferate (differentiate)) on the bottom surface 23 of the culture vessel 22.

[0041] The electron microscope 13 captures enlarged images of the planar shape of the first mesenchymal stem cells 21 contained in the second bone marrow fluid 21 injected into the first flat culture vessel 22 at intervals of approximately 1 to 2 hours, and transmits the captured enlarged images of the planar shape of the first mesenchymal stem cells 21 to the management server 11 at intervals of approximately 1 to 2 hours. The management server 11 stores (memorizes) the enlarged images of the planar shape of the first mesenchymal stem cells 21 transmitted from the electron microscope 13 and the time of imaging in a memory area in association with the IC tag identification information (IC tag identifier) ​​and the donor identifier. The management server 11 outputs (displays) the enlarged images of the planar shape of the first mesenchymal stem cells 21 transmitted from the electron microscope 13 and the time of imaging on the display 17. The person in charge (doctor, nurse, researcher, bioengineer, etc.) checks (visually observes) the enlarged image of the planar shape of the first mesenchymal stem cells 21 displayed on the display 17 at intervals of approximately 1 to 2 hours over a period of 12 to 24 hours, and observes changes in the planar shape of the first mesenchymal stem cells 21 contained in the second bone marrow fluid 21.

[0042] Although not shown, the initial planar shape of the first mesenchymal stem cells 21 is approximately circular, and when the planar shape of the first mesenchymal stem cells 21 is approximately circular, the first mesenchymal stem cells 21 have not yet settled on the bottom surface 23 (inner bottom wall surface) of the first flat culture vessel 22, and the first mesenchymal stem cells 21 have not yet begun to proliferate (differentiate). The planar shape of the first mesenchymal stem cells 21 after deformation is a flattened shape in which the first mesenchymal stem cells 21 have stretched (expanded) in an indefinite manner in one direction (predetermined direction) with the approximately circular shape before settling as a nucleus, and the first mesenchymal stem cells 21 have settled on the bottom surface 23 (inner bottom wall surface) of the first flat culture vessel 22, and the first mesenchymal stem cells 21 have begun to proliferate (differentiate).

[0043] If, as a result of the observation in the first shape deformation observation step, the planar shape of the first mesenchymal stem cells 21 output (displayed) on the display 17 is transformed from an approximately circular shape to an amorphous flat shape with an approximately circular nucleus, it is determined that the first mesenchymal stem cells 21 have settled on the bottom surface 23 of the first flat culture vessel 22 (first stem cell settlement step). After it is confirmed as a result of the observation in the first shape deformation observation step that the first mesenchymal stem cells 21 are transformed from an approximately circular shape (initial planar shape) to an amorphous flat shape with an approximately circular nucleus and that the first mesenchymal stem cells 21 have settled on the bottom surface 23 of the first flat culture vessel 22 (first culture vessel), the first total planar area observation step is carried out.

[0044] The person in charge drains the culture solution 26 poured into the first flat culture vessel 22 from the culture vessel 22 and pours (contains) new culture solution 26 into the culture vessel 22. Next, the first flat culture vessel 22 is left statically (left quietly without moving) for 36 to 48 hours at a temperature substantially equal to body temperature (approximately 35 to 37°C) to culture the first mesenchymal stem cells 21, while observing the total planar area of ​​the first mesenchymal stem cells 21 settled on the bottom surface 23 of the culture vessel 22 relative to the bottom surface area of ​​the culture vessel 22 on the display 17 (electron microscope 13) at intervals of approximately 1 to 2 hours during the 36 to 48 hours, and determines whether the total planar area of ​​the first mesenchymal stem cells 21 relative to the bottom surface area of ​​the culture vessel 22 has reached a first target ratio (first total planar area observation step). The first target ratio of the total planar area of ​​the first mesenchymal stem cells 21 to the bottom area of ​​the first flat culture vessel 22 is 70 to 80% (70 to 80% confluence).

[0045] In the first observation step of the total planar area, the culture medium 26 poured into the first flat culture vessel 22 is discharged from the culture vessel 22, new culture medium 26 is poured (contained) into the culture vessel 22, and the first flat culture vessel 22 with the poured new culture medium 26 is placed (set) in the specimen holder 27 of the electron microscope 13. A spacer 30 is interposed between the upper surface 28 of the specimen holder 27 of the electron microscope 13 and the bottom 29 of the first flat culture vessel 22, and the bottom 29 of the culture vessel 22 is held elevated by the spacer 30. The culture vessel 22 is held in a tilted state at a predetermined angle so that the bottom 29 of the culture vessel 22 faces upward and the top 31 (inlet 24) of the culture vessel 22 faces downward (see FIG. 6 ). The tilt angle α1 of the first flat culture vessel 22 with respect to the upper surface 28 of the specimen holder 27 is in the range of 2 to 5°, preferably in the range of 2 to 3°.

[0046] The electron microscope 13 captures enlarged images of the planar shape of the first mesenchymal stem cells 21 in the first flat culture vessel 22 at intervals of approximately 1 to 2 hours, and transmits the captured enlarged images of the planar shape of the first mesenchymal stem cells 21 to the management server 11 at intervals of approximately 1 to 2 hours. The management server 11 stores (memorizes) the enlarged images of the planar shape of the first mesenchymal stem cells 21 transmitted from the electron microscope 13 and the time of imaging in a memory area in association with the IC tag identification information (IC tag identifier) ​​and the donor identifier. The management server 11 outputs (displays) the enlarged images of the planar shape of the first mesenchymal stem cells 21 transmitted from the electron microscope 13 and the time of imaging on the display 17.

[0047] The person in charge (doctor, nurse, researcher, bioengineer, etc.) checks (visually inspects) the enlarged image of the planar shape of the first mesenchymal stem cells 21 displayed on the display 17 at approximately 1 to 2 hour intervals over a period of 36 to 48 hours, and while observing the total planar area of ​​the first mesenchymal stem cells 21 settled on the bottom 23 of the first flat culture vessel 22 relative to the bottom area of ​​the culture vessel 22, determines whether the total planar area of ​​the first mesenchymal stem cells 21 relative to the bottom area of ​​the first flat culture vessel 22 has reached the first target ratio (70 to 80% confluent).

[0048] The first mesenchymal stem cells 21 grow and form colonies on the bottom surface 23 (inner surface of the bottom wall) of the first flat culture vessel 22 (first stem cell culture step). When the first mesenchymal stem cells 21 form colonies and, as a result of observation in the first total planar area observation step, the planar shape of the first mesenchymal stem cells 21 expands, and the total planar area of ​​the first mesenchymal stem cells 21 relative to the bottom surface area of ​​the first flat culture vessel 22 displayed on the display 17 reaches a first target ratio (70 to 80% confluent), the first mesenchymal stem cells 21 are extracted from the culture vessel 22.

[0049] The culture medium 26 (first culture product liquid 32) during the total planar area first observation step poured into the first flat culture vessel 22 is discharged from the first flat culture vessel 22, the culture vessel 22 is washed with phosphate buffered saline (PBS), and then a trypsin solution is poured into the culture vessel 22. When the trypsin solution is poured into the first flat culture vessel 22, the first mesenchymal stem cells 21 that have settled on the bottom surface 23 of the culture vessel 22 are detached from the bottom surface 23 by the trypsin solution and rise to the surface of the trypsin solution. The person in charge uses a pipette to aspirate the first mesenchymal stem cells 21 that have risen to the surface.

[0050] The culture solution 26 discharged from the first flat culture vessel 22 (second culture vessel) is the culture solution 26 remaining after the first mesenchymal stem cells 21 are extracted from the culture vessel 22, and has been transformed into a first culture product solution 32 (culture product solution) containing predetermined metabolic substances secreted from the first mesenchymal stem cells 21 during the culture process of the first mesenchymal stem cells 21. The first culture product solution 32 containing metabolic substances activates mesenchymal stem cells whose activity has decreased. The first culture product solution 32 is poured into a cryopreservation container 43 (described below) to which an IC tag 18 is attached and is quick-frozen using a quick-freezer (not shown). The first culture product solution 32 is then stored in the cryopreservation container 43 at a predetermined temperature (chilled storage at 0 to 5°C or frozen storage at 0°C or below) for a predetermined period of time in the refrigerator 14 or freezer 14.

[0051] FIG. 6 is an explanatory diagram showing an example of the first step of extracting second mesenchymal stem cells in the mesenchymal stem cell culture method, FIG. 7 is an explanatory diagram showing an example of the second step of observing shape deformation in the mesenchymal stem cell culture method, and FIG. 8 is a side view of a second flat culture vessel 33 (third culture vessel). After extracting the first mesenchymal stem cells 21 from the first flat culture vessel 22, a person in charge (such as a doctor, nurse, researcher, or bioengineer) injects (contains) the first mesenchymal stem cells 21 into a glass test tube 34 and places (sets) the glass test tube 34 in a centrifuge (not shown). After centrifuging the first mesenchymal stem cells 21 in the centrifuge for a predetermined time, the glass test tube 34 is removed from the centrifuge. The first mesenchymal stem cells 21 in the glass test tube 34 are centrifuged vertically by the centrifuge to form two layers.

[0052] After separating the first mesenchymal stem cells 21 into layers, second mesenchymal stem cells 35 located in the lower layer (bottom layer) are extracted (aspirated) from the separated layer of first mesenchymal stem cells 21 using a syringe (not shown) or a pipette (not shown) (first second mesenchymal stem cell extraction step), as shown in Figure 6. The first mesenchymal stem cells 21 containing unnecessary stem cells are centrifuged in a centrifuge to separate them into layers in the vertical direction, and the second mesenchymal stem cells 35 located in the lower layer (bottom layer) of the centrifuged layered first mesenchymal stem cells 21 are extracted, thereby ensuring the extraction of specific second mesenchymal stem cells 35 from the first mesenchymal stem cells 21 and removing unnecessary mesenchymal stem cells from the first mesenchymal stem cells 21.

[0053] After specific second mesenchymal stem cells 35 located in the lower layer (bottom layer) are extracted from the first mesenchymal stem cells 21, a second shape deformation observation step is performed. In the second shape deformation observation step, the second mesenchymal stem cells 35 and a culture solution 36 are poured (contained) into a second flat culture vessel 33 (third culture vessel) (cell culture vessel), and the culture vessel 33 is left statically (left quietly without moving) for 36 to 48 hours at a temperature approximately equal to body temperature (approximately 35 to 37°C). During the 36 to 48 hours, the deformation of the second mesenchymal stem cells 35 (mesenchymal stem cells) from their initial flat shape is observed on a display 17 (electron microscope) at approximately 1 to 2 hour intervals, and it is determined whether the second mesenchymal stem cells 35 have settled on the bottom surface 37 of the culture vessel 33 (second shape deformation observation step). An IC tag 18, on which donor data and stem cell data are written, is attached to the bottom surface 37 (outer surface of the bottom wall) of the second flat culture vessel 33.

[0054] The second flat culture vessel 33 (third culture vessel) is made of transparent glass or transparent plastic, and is a flat vessel with a small capacity and a predetermined bottom area, with a planar shape of an approximately regular square, and its volume is larger than that of the first flat culture vessel 22 (second culture vessel). The injection port 38 of the second flat culture vessel 33 is watertightly closed by a lid 39. The second flat culture vessel 33 has a capacity of approximately 40 to 60 cc (preferably 50 cc), and its bottom area is approximately 50 to 72 mm 2 The second flat culture vessel 33 has a length of approximately 7 to 8.5 mm on each side. A flat vessel with a small capacity and a bottom surface of a predetermined area, having a circular or oval planar shape, can also be used as the second flat culture vessel 33. The second mesenchymal stem cells 35 injected into the second flat culture vessel 33 settle on the bottom surface 37 of the culture vessel 33 over time, while being cultured in the culture solution 36, and gradually proliferate (differentiate) on the bottom surface 37 of the culture vessel 33 to form colonies.

[0055] The person in charge removes the lid 39 from the injection port 38, and injects (contains) the second mesenchymal stem cells 35 aspirated into the syringe or pipette into the interior of the second flat culture vessel 33 through the injection port 38 of the second flat culture vessel 33, and also injects (contains) the culture medium 36 into the interior of the culture vessel 33, and then closes the injection port 38 with the lid 39. The culture medium 36 is the same as that injected in the first shape deformation observation step. After injecting the second mesenchymal stem cells 35 and the culture medium 36 into the second flat culture vessel 33, the person in charge places (sets) the culture vessel 33 in the specimen holder 27 of the electron microscope 13.

[0056] A spacer 30 is interposed between the upper surface 28 of the sample holder 27 of the electron microscope 13 and the bottom 40 of the second flat culture vessel 33, and the bottom 40 of the culture vessel 33 is held in a raised position by the spacer 30, and the culture vessel 33 is held in a tilted position at a predetermined angle so that the bottom 40 of the culture vessel 33 is on top and the top 41 (inlet 38) of the culture vessel 33 is on the bottom. Alternatively, a spacer 30 may be interposed between the upper surface 28 of the sample holder 27 of the electron microscope 13 and the top 41 of the second flat culture vessel 33, and the top 41 of the culture vessel 33 is held in a raised position by the spacer 30, and the culture vessel 33 may be held in a tilted position at a predetermined angle so that the top 41 of the culture vessel 33 is on top and the bottom 40 of the culture vessel 33 is on the bottom. The inclination angle α2 of the second flat culture vessel 33 with respect to the upper surface 28 of the sample holder 27 is in the range of 2 to 5°, and preferably in the range of 2 to 3°.

[0057] By tilting the second flat culture vessel 33 at the above-mentioned tilt angle relative to the upper surface 28 of the sample holder 27, the second mesenchymal stem cells 35 and culture medium 36 within the culture vessel 33 are biased toward the top 41 side (or bottom 40 side) of the culture vessel 33, and the water pressure between the second mesenchymal stem cells 35 and the culture medium 36 on the top 41 side (or bottom 40 side) of the culture vessel 33 increases, causing the second mesenchymal stem cells 35 to concentrate on the top 41 side (or bottom 40 side) of the culture vessel 33, thereby increasing the activity of the second mesenchymal stem cells 35 and allowing the second mesenchymal stem cells 35 to easily and quickly settle (or proliferate (differentiate)) on the bottom surface 37 of the culture vessel 33.

[0058] The electron microscope 13 captures enlarged images of the planar shape of the second mesenchymal stem cells 35 injected into the second flat culture vessel 33 at approximately 1-2 hour intervals, and transmits the captured enlarged images of the planar shape of the second mesenchymal stem cells 35 to the management server 11 at approximately 1-2 hour intervals. The management server 11 stores (memorizes) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of the image capture in a memory area, in association with the IC tag identification information (IC tag identifier) ​​and the donor identifier. The management server 11 displays the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of the image capture on the display 17. The person in charge checks (visually confirms) the enlarged images of the planar shape of the second mesenchymal stem cells 35 displayed on the display 17 at approximately 1-2 hour intervals over a period of 36-48 hours, and observes changes in the planar shape of the second mesenchymal stem cells 35.

[0059] The initial planar shape of the second mesenchymal stem cells 35 is approximately circular, and when the planar shape of the second mesenchymal stem cells 35 is approximately circular, the second mesenchymal stem cells 35 have not yet settled on the bottom surface 37 (inner bottom wall surface) of the second flat culture vessel 33, and the second mesenchymal stem cells 35 have not yet begun to proliferate (differentiate). The planar shape of the second mesenchymal stem cells 35 after deformation is a flattened shape in which the second mesenchymal stem cells 35 have stretched indefinitely in one direction, with the approximately circular shape before settling as a nucleus, and the second mesenchymal stem cells 35 have settled on the bottom surface 37 (inner bottom wall surface) of the second flat culture vessel 33, and the second mesenchymal stem cells 35 have begun to proliferate (differentiate).

[0060] If, as a result of the observation in the second shape deformation observation step, the planar shape of the second mesenchymal stem cells 35 displayed on the display 17 is transformed from an approximately circular shape to an amorphous flat shape with an approximately circular nucleus, it is determined that the second mesenchymal stem cells 35 have settled on the bottom surface 37 of the second flat culture vessel 33 (second stem cell settlement step). After the observation in the second shape deformation observation step confirms that the second mesenchymal stem cells 35 have changed from an approximately circular shape (initial planar shape) to an amorphous flat shape with an approximately circular nucleus and that the second mesenchymal stem cells 35 have settled on the bottom surface 37 of the second flat culture vessel 33 (third culture vessel), the second total planar area observation step is carried out.

[0061] The culture medium 36 poured into the second flat culture vessel 33 is discharged from the culture vessel 33, and new culture medium 36 is poured into (contained in) the culture vessel 33. Next, the second flat culture vessel 33 is left statically (left quietly without moving) for 36 to 48 hours at a temperature substantially equal to body temperature (approximately 35 to 37°C) to culture the second mesenchymal stem cells 35, while the total planar area of ​​the second mesenchymal stem cells 35 that have settled on the bottom surface 37 of the culture vessel 33 relative to the bottom surface area of ​​the culture vessel 33 is observed on the display 17 (electron microscope) at intervals of approximately 1 to 2 hours during the 36 to 48 hours, and it is determined whether the total planar area of ​​the second mesenchymal stem cells 35 relative to the bottom area of ​​the second flat culture vessel 33 has reached a second target ratio (second total planar area observation step). A second target ratio of the total planar area of ​​the second mesenchymal stem cells 35 to the bottom area of ​​the second flat culture vessel 33 is 88 to 92% (88 to 92% confluent).

[0062] In the second total planar area observation step, the culture medium 36 poured into the second flat culture vessel 33 is discharged from the culture vessel 33, new culture medium 36 is poured (contained) into the culture vessel 33, and the culture vessel 33 with the poured new culture medium 36 is placed (set) in the sample holder 27 of the electron microscope 13. A spacer 30 is interposed between the upper surface 28 of the sample holder 27 of the electron microscope 13 and the bottom 40 of the second flat culture vessel 33, and the bottom 40 of the culture vessel 33 is held elevated by the spacer 30. The second flat culture vessel 33 is held in a tilted state at a predetermined angle so that the bottom 40 of the culture vessel 33 faces up and the top 41 (inlet 38) of the culture vessel 33 faces down (see FIG. 8 ). The tilt angle α1 of the second flat culture vessel 33 with respect to the upper surface 28 of the sample holder 27 is in the range of 2 to 5°, preferably 2 to 3°.

[0063] The electron microscope 13 captures enlarged images of the planar shape of the second mesenchymal stem cells 35 in the second flat culture vessel 33 at approximately 1-2 hour intervals, and transmits the captured enlarged images of the planar shape of the second mesenchymal stem cells 35 to the management server 11 at approximately 1-2 hour intervals. The management server 11 stores (memorizes) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of photographing in a memory area in association with the IC tag identification information (IC tag identifier) ​​and the donor identifier. The management server 11 outputs (displays) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of photographing on the display 17.

[0064] The person in charge (doctor, nurse, researcher, bioengineer, etc.) checks (visually confirms) the enlarged image of the planar shape of the second mesenchymal stem cells 35 displayed on the display 17 at approximately 1 to 2 hour intervals over a period of 36 to 48 hours, and while observing the total planar area of ​​the second mesenchymal stem cells 35 that have settled on the bottom 37 of the second flat culture vessel 33 relative to the bottom area of ​​the culture vessel 33, determines whether the total planar area of ​​the second mesenchymal stem cells 35 relative to the bottom area of ​​the culture vessel 33 has reached a second target ratio (70 to 80% confluent).

[0065] The second mesenchymal stem cells 35 grow and form colonies on the bottom surface 37 (inner surface of the bottom wall) of the second flat culture vessel 33 (second stem cell culture step). The second mesenchymal stem cells 35 form colonies, the planar shape of the second mesenchymal stem cells 35 expands, and as a result of observation in the second total planar area observation step, when the total planar area of ​​the second mesenchymal stem cells 35 relative to the bottom surface area of ​​the second flat culture vessel 33 displayed on the display 17 reaches a second target ratio (70 to 80% confluent), the second mesenchymal stem cells are extracted from the culture vessel 33.

[0066] The culture medium 36 injected into the second flat culture vessel 33 during the second observation step for the total planar area is discharged from the culture vessel 33, the culture vessel 33 is washed with phosphate-buffered saline (PBS), and then a trypsin solution is injected into the culture vessel 33. When the trypsin solution is injected into the second flat culture vessel 33, the second mesenchymal stem cells 35 that have settled on the bottom surface 37 of the culture vessel 33 are detached from the bottom surface 37 by the trypsin solution and rise to the surface of the trypsin solution. The person in charge uses a pipette to aspirate the floating second mesenchymal stem cells 35 (second mesenchymal stem cell second extraction step).

[0067] The culture solution 36 discharged from the second flat culture vessel 33 (third culture vessel) is the culture solution 36 remaining after the second mesenchymal stem cells 35 are extracted from the culture vessel 33, and has been transformed into a second culture product solution 42 (culture product solution) containing predetermined metabolic substances secreted from the second mesenchymal stem cells 35 during the culture process of the second mesenchymal stem cells 35. The second culture product solution 42 containing metabolic substances activates mesenchymal stem cells whose activity has decreased. The second culture product solution 42 is poured into a cryopreservation container 43 to which an IC tag 18 is attached and flash-frozen using a flash freezer. The second culture product solution 42 is then stored in the cryopreservation container 43 at a predetermined temperature (chilled storage at 0 to 5°C or frozen storage at 0°C or below) for a predetermined period of time in the refrigerator 14 or freezer 14.

[0068] The frozen stem cell modification method involves first mesenchymal stem cells 21 settling on the bottom surface 23 of a first flat culture vessel 22 (second culture vessel), then discharging the culture medium 26 from the culture vessel 22 while injecting new culture medium 26 into the culture vessel 22, culturing the first mesenchymal stem cells 21 settling on the bottom surface 23 of the culture vessel 22 using the new culture medium 26, and placing second mesenchymal stem cells 35 proliferated in the culture vessel 22 and the culture medium 36 in a second flat culture vessel 33 (third culture vessel) to form the second mesenchymal stem cells 35. After the second mesenchymal stem cells 35 have settled on the bottom surface 37 of the culture vessel 33, the culture medium 36 is discharged from the culture vessel 33 and new culture medium 36 is poured into the culture vessel 33, and the second mesenchymal stem cells 35 that have settled on the bottom surface 37 of the culture vessel 33 are cultured using the new culture medium 36, and the second mesenchymal stem cells 35 are proliferated in the culture vessel 33.This makes it possible to proliferate the mesenchymal stem cells 35 efficiently and reliably, and to efficiently produce mesenchymal stem cells 35 with sufficient activity.

[0069] FIG. 9 is an explanatory diagram showing an example of the cryopreservation step in the frozen stem cell modification method, and FIG. 10 is an explanatory diagram showing an example of the stem cell thawing step in the frozen stem cell modification method. FIG. 11 is an explanatory diagram showing an example of the stirring and removal step in the frozen stem cell modification method, and FIG. 12 is an explanatory diagram showing an example of the mesenchymal stem cell extraction step in the frozen stem cell modification method. FIG. 13 is an explanatory diagram showing an example of the stem cell activation step in the frozen stem cell modification method, and FIG. 14 is a side view of a third flat culture vessel (first culture vessel). FIG. 15 is a partially enlarged view showing an example of the planar shape of second mesenchymal stem cells 35, and FIG. 16 is a partially enlarged view showing another example of the planar shape of second mesenchymal stem cells 35. FIG. 17 is an explanatory diagram showing an example of the stem cell transport step in the frozen stem cell modification method. FIGS. 15 and 16 show enlarged images of the planar shape of second mesenchymal stem cells 35 photographed by an electron microscope 13.

[0070] The frozen stem cell modification method involves carrying out a cryopreservation process to freeze and preserve second mesenchymal stem cells 35 (mesenchymal stem cells) cultured by a stem cell culture method, and then modifying the frozen and preserved second mesenchymal stem cells 35 (mesenchymal stem cells) into activated mesenchymal stem cells 55 by carrying out a stem cell thawing process, a stirring and removal process, a mesenchymal stem cell extraction process, a stem cell activation process, and a stem cell transport process.

[0071] A person in charge (such as a doctor, nurse, or researcher) injects (contains) second mesenchymal stem cells 35 (cultured mesenchymal stem cells) from a pipette into a cryopreservation container 43 of a predetermined volume. The second mesenchymal stem cells 35 injected into the cryopreservation container 43 are active pure mesenchymal stem cells of a specific type (almost a single type) to be cultured, from which unnecessary mesenchymal stem cells have been removed. An IC tag 18 containing donor data and stem cell data is attached to the outer surface of the cryopreservation container 43.

[0072] A cryoprotectant 44 (at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)) is poured into the cryopreservation container 43. The cryopreservation container 43 containing the second mesenchymal stem cells 35 and the cryoprotectant 44 is rapidly frozen using a rapid freezer (not shown). The rapidly frozen second mesenchymal stem cells 35 are stored in the cryopreservation container 43 at a predetermined temperature (chilled storage at 0 to 5°C or frozen storage at 0°C or below) in a refrigerator 14 or freezer 14 for a predetermined period of time until they are used, as shown in Figure 9 (freezing storage process).

[0073] When using second mesenchymal stem cells 35 that have been frozen and stored in a refrigerator 14 or freezer 14 (using mesenchymal stem cells 35 that have been frozen and stored through a cryopreservation process), as shown in Figure 10, a temperature-controllable insulated container 45 (constant temperature bath) of a predetermined volume is prepared, and the cryopreservation container 43 is immersed (submerged) in the warm water in the insulated container 45 for 2 to 3 minutes to thaw the second mesenchymal stem cells 35 to a temperature of 2 to 3°C (stem cell thawing process). The temperature of the warm water in the insulated container 45 is maintained at 35 to 37°C. The cryopreservation container 43 containing the first culture product solution 32 and the second culture product solution 42 is immersed (submerged) in the warm water in the insulated container 45 for 2 to 3 minutes to thaw the first and second culture product solutions 32, 42 to a temperature of 2 to 3°C.

[0074] Next, the cryopreservation container 43 is removed from the insulated container 45, and a washing solution 46 (Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS)) is poured into the cryopreservation container 43 containing the thawed second mesenchymal stem cells 35 and the cryoprotectant 44. After pouring the washing solution 46 into the cryopreservation container 43, as shown in FIG. 11 , the cryopreservation container 43 containing the washing solution 46 is shaken side to side to agitate the cryopreservation container 43, and the cryoprotectant 44 is removed (separated) from the second mesenchymal stem cells 35 in the cryopreservation container 43 by the washing solution 46 (Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS)) (agitation and removal process).

[0075] After removing (separating) the cryoprotectant 44 from the second mesenchymal stem cells 35 through the stirring and removal process, the person in charge (doctor, nurse, researcher, etc.) places (sets) the cryopreservation container 43 containing the mixed solution containing the second mesenchymal stem cells 35, the cryoprotectant 44 separated from the mesenchymal stem cells 35, and the washing solution 46 in a centrifuge 47. The mixed solution is centrifuged for a predetermined time in the centrifuge 47, and the second mesenchymal stem cells 35, cryoprotectant 44, and washing solution 46 in the cryopreservation container 43 are centrifuged in layers. The mixed solution in the cryopreservation container 43 is centrifuged in an up-and-down direction to form two layers.

[0076] After separating the mixture into two layers, the person in charge extracts (sucks) the second mesenchymal stem cells 35 located in the lower (bottom) layer from the separated mixture using a syringe (not shown) or pipette (not shown) (mesenchymal stem cell extraction step), as shown in Figure 12. The second mesenchymal stem cells 35 extracted by the mesenchymal stem cell extraction step have had the cryoprotectant 44 (at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)) injected during freezing removed (separated) from them.

[0077] After specific second mesenchymal stem cells 35 located in the lower layer (bottom layer) are extracted from the mixed solution, a stem cell activation step is performed. In the stem cell activation step, the washed second mesenchymal stem cells 35, culture solution 48, and first culture product solution 32 and / or second culture product solution 42 are poured (contained) into a third flat culture vessel 49 (first culture vessel). The first culture product solution 32 poured into the third flat culture vessel 49 is a culture product solution containing specific metabolic substances secreted from the first mesenchymal stem cells 21 during the culture process of the first mesenchymal stem cells 21 that are the source of the thawed second mesenchymal stem cells 35 (mesenchymal stem cells), and the second culture product solution 42 poured into the third flat culture vessel 49 is a culture product solution containing specific metabolic substances secreted from the second mesenchymal stem cells 35 during the culture process of the thawed second mesenchymal stem cells 35 (mesenchymal stem cells). Furthermore, the injection ratio of the culture solution 32, 42 injected into the third flat culture vessel 49 is 5 to 15%, preferably 8 to 12%, and more preferably 10%, when the total injection amount of the culture solution 48 injected into the culture vessel 49 is 100%.

[0078] The third flat culture vessel 49 (first culture vessel) is made of transparent glass or transparent plastic, and is a flat vessel with a small capacity and a predetermined bottom area, with a planar shape of an approximately regular square. An inlet 50 of the third flat culture vessel 49 is watertightly closed by a lid 51. The third flat culture vessel 49 has a capacity of about 20 to 30 cc (preferably 25 cc), and a bottom area of ​​about 25 to 36 mm 2The third flat culture vessel 49 has a diameter of 1 / 4" and a side length of 5 to 6 mm. A flat vessel with a small capacity and a bottom surface of a predetermined area, and a circular or elliptical planar shape, can also be used as the third flat culture vessel 49. An IC tag 18, on which donor data and stem cell data are written, is attached to the bottom surface 52 (outer surface of the bottom wall) of the third flat culture vessel 49.

[0079] After containing the washed second mesenchymal stem cells 35, culture medium 48, first culture product solution 32 and / or second culture product solution 42, the third flat culture vessel 49 is placed (set) on the specimen holder 27 of the electron microscope 13. A spacer 30 is interposed between the upper surface 28 of the specimen holder 27 of the electron microscope 13 and the bottom 53 of the third flat culture vessel 49, and the bottom 53 of the culture vessel 49 is held in a raised position by the spacer 30, and the culture vessel 49 is held in a tilted position at a predetermined angle (tilted in one direction) so that the bottom 53 of the culture vessel 49 is facing up and the top 54 (inlet 50) of the culture vessel 49 is facing down. The top 54 of the third flat culture vessel 49 may be held in a raised position by the spacer 30, and the culture vessel 49 may be held in a tilted position at a predetermined angle (tilted in one direction) so that the top 54 of the culture vessel 49 is on top and the bottom 53 of the culture vessel 49 is on the bottom. The tilt angle α3 of the third flat culture vessel 49 in one direction relative to the upper surface 28 of the sample holder 27 is in the range of 2 to 5°, and preferably in the range of 2 to 3°.

[0080] After the third flat culture vessel 49 is placed (set) in an inclined position on the specimen holder 27 of the electron microscope 13, the third flat culture vessel 49 containing the second mesenchymal stem cells 35, culture medium 48, first culture product liquid 32 and / or second culture product liquid 42 is left statically (left quietly without moving) for 24 hours at a temperature approximately equal to body temperature (approximately 35 to 37°C), and the second mesenchymal stem cells 35 (mesenchymal stem cells) are cultured in the third flat culture vessel 49 (first culture vessel) at 36 to 37°C for 24 hours.

[0081] When 12 hours have passed since the third flat culture vessel 49 was installed (set) on the sample holder 27 of the electron microscope 13, the spacer 30 interposed between the upper surface 28 of the sample holder 27 of the electron microscope 13 and the bottom 53 of the culture vessel 49 is removed, and the spacer 30 is interposed between the upper surface 28 of the sample holder 27 of the electron microscope 13 and the top 54 of the culture vessel 49, the top 54 of the culture vessel 49 is held in a raised position by the spacer 30, and the culture vessel 49 is held in a tilted position at a predetermined angle (tilted in the other direction) so that the top 54 of the culture vessel 49 is on top and the bottom 53 of the culture vessel 49 is on the bottom. Furthermore, when the third flat culture vessel 49 is initially tilted so that the top 54 of the culture vessel 49 is on top and the bottom 53 of the culture vessel 49 is on the bottom, a spacer 30 is interposed between the upper surface 28 of the sample holder 27 of the electron microscope 13 and the bottom 53 of the culture vessel 49, and the bottom 53 of the culture vessel 49 is kept elevated by the spacer 30, and the culture vessel 49 is kept tilted at a predetermined angle (tilted in the other direction) so that the bottom 53 of the culture vessel 49 is on top and the top 54 of the culture vessel 49 is on the bottom.

[0082] The tilt angle α3 of the third flat culture vessel 49 in the other direction relative to the upper surface 28 of the specimen holder 27 is in the range of 2 to 5°, and preferably in the range of 2 to 3°. Note that the third flat culture vessel 49 may be left statically (left quietly without moving) for 24 hours at a temperature substantially the same as body temperature (approximately 35 to 37°C) while continuously tilted in one direction (or the other direction), and the second mesenchymal stem cells 35 (mesenchymal stem cells) may be cultured in the third flat culture vessel 49 (first culture vessel) at 36 to 37°C for 24 hours.

[0083] By tilting the third flat culture vessel 49 in one direction and the other at the tilt angle relative to the upper surface 28 of the sample holder 27, the second mesenchymal stem cells 35 (mesenchymal stem cells), culture medium 48, and culture effluents 32, 42 within the culture vessel 49 are biased toward the top 54 side (or bottom 53 side) of the culture vessel 49, and the water pressure of the second mesenchymal stem cells 35, culture medium 48, and culture effluents 32, 42 on the top 54 side (or bottom 53 side) of the culture vessel 49 increases, causing the second mesenchymal stem cells 35 to concentrate on the top 54 side (or bottom 53 side) of the culture vessel 49, thereby increasing the activity of the second mesenchymal stem cells 35 among themselves and allowing the second mesenchymal stem cells 35 to be easily and quickly activated (settled) on the bottom surface 52 of the culture vessel 49.

[0084] The electron microscope 13 captures enlarged images of the planar shape of the second mesenchymal stem cells 35 injected into the third flat culture vessel 49 at intervals of approximately 1 to 2 hours, and transmits the captured enlarged images of the planar shape of the second mesenchymal stem cells 35 to the management server 11 at intervals of approximately 1 to 2 hours. The management server 11 stores (memorizes) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of photographing in a memory area in association with the IC tag identification information (IC tag identifier) ​​and the donor identifier. The management server 11 outputs (displays) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of photographing on the display 17. The person in charge (doctor, nurse, researcher, bioengineer, etc.) checks (visually observes) the enlarged image of the planar shape of the second mesenchymal stem cells 35 displayed on the display 17 at approximately 1 to 2 hour intervals over a period of 12 to 24 hours, observes changes in the planar shape of the second mesenchymal stem cells 35, and determines whether the second mesenchymal stem cells 35 have been activated (whether they have settled on the bottom surface 52 of the third flat culture vessel 49) (third shape deformation observation process).

[0085] The planar shape of the second mesenchymal stem cells 35 after thawing is approximately circular. When the planar shape of the second mesenchymal stem cells 35 is approximately circular, the second mesenchymal stem cells 35 are not activated (they are not attached to the bottom surface 52 (inner bottom wall surface) of the third flat culture vessel 49) and activation of the second mesenchymal stem cells 35 has not yet begun. The planar shape of the second mesenchymal stem cells 35 after deformation is a flattened shape in which the second mesenchymal stem cells 35 have elongated (expanded) in an indefinite manner in one direction (a predetermined direction) with the approximately circular shape before attachment as a nucleus. The second mesenchymal stem cells 35 have attached to the bottom surface 52 (inner bottom wall surface) of the third flat culture vessel 49, the second mesenchymal stem cells 35 have been activated, and the second mesenchymal stem cells 35 (mesenchymal stem cells) in the culture vessel 49 (in the first culture vessel) have been converted into activated mesenchymal stem cells.

[0086] If, as a result of the observation in the third shape transformation observation step, the planar shape of the second mesenchymal stem cells 35 displayed on the display 17 remains approximately circular as shown in Figure 14, the person in charge determines that the second mesenchymal stem cells 35 have not settled on the bottom surface 52 (inner surface of the bottom wall) of the third flat culture vessel 49, and continues to observe changes in the planar shape of the second mesenchymal stem cells 35 at intervals of approximately 1 to 2 hours. If, as a result of the observation in the third shape transformation observation step, the planar shape of the second mesenchymal stem cells 35 output (displayed) on the display 17 changes from approximately circular to an amorphous flat shape with the approximately circular shape as the nucleus as shown in Figure 15, the person in charge determines that the second mesenchymal stem cells 35 have been activated (settled on the bottom surface of the third flat culture vessel) and have been transformed into activated mesenchymal stem cells 55 (stem cell activation step).

[0087] After determining that the second mesenchymal stem cells 35 have been transformed into activated mesenchymal stem cells 55, the culture medium 48 and culture products 32 and 42 injected into the third flat culture vessel 49 are discharged from the culture vessel 49, the culture vessel 49 is washed with phosphate-buffered saline (PBS), and then trypsin solution is injected into the culture vessel 49. When the trypsin solution is injected into the third flat culture vessel 49, the activated mesenchymal stem cells 55 that have settled on the bottom surface 52 of the culture vessel 49 are detached from the bottom surface 52 by the trypsin solution and rise to the surface of the trypsin solution. The person in charge aspirates the floating activated mesenchymal stem cells 55 using a pipette, extracts the activated mesenchymal stem cells 55 from the third flat culture vessel 49 (first culture vessel), and stores the activated mesenchymal stem cells 55 in a storage container 56.

[0088] When the activated mesenchymal stem cells 55 are to be used within 18 to 20 hours after extraction, the person in charge places the storage container 56 containing the activated mesenchymal stem cells 55 in a portable thermostatic case 57, maintains the temperature inside the thermostatic case 57 at 20 to 21°C, and transports the extracted activated mesenchymal stem cells 55, while maintained at 20 to 21°C, to the site of use within 18 to 20 hours (stem cell transport process), as shown in Figure 17. The activated mesenchymal stem cells 55 transported to the site of use are removed from the thermostatic case 57 and used for the treatment of various diseases (such as cardiovascular disease and central nervous system disease), regeneration in regenerative medicine, and non-therapeutic purposes such as beauty.

[0089] The frozen stem cell modification method (frozen stem cell modification system 10) involves injecting a cleaning solution 46 (Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS)) into a cryopreservation container 43 containing second mesenchymal stem cells 35 (mesenchymal stem cells) thawed from a frozen state and a cryoprotectant 44 (hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), at least dimethyl sulfoxide (DMSO)), and stirring the cryopreservation container 43 containing the cleaning solution 46 to remove the cleaning solution 46. 6 removes (separates) the cryoprotectant 44 from the second mesenchymal stem cells 35 in the cryopreservation container 43, and then the second mesenchymal stem cells 35, cryoprotectant 44, and washing solution 46 in the cryopreservation container 43 are centrifuged in layers, and the second mesenchymal stem cells 35 located in the bottom layer of the cryopreservation container 43 after centrifugation are extracted.Therefore, when thawing the frozen-preserved second mesenchymal stem cells 35, the cryoprotectant 44 can be removed using the washing solution 46, and the majority (70 to 90%) of the second mesenchymal stem cells 35 thawed from a frozen state can survive.

[0090] The frozen stem cell modification method (frozen stem cell modification system 10) involves placing second mesenchymal stem cells 35 (mesenchymal stem cells) that have been thawed from a frozen state in a third flat culture vessel 49 (first culture vessel) with a predetermined volume and a predetermined bottom area, injecting a predetermined culture medium 48 and culture yield solutions 32, 42 into the culture vessel 49 containing the second mesenchymal stem cells 35, and culturing the second mesenchymal stem cells 35 in the culture vessel 49 at 35 to 37°C for 24 hours to activate the second mesenchymal stem cells 35 in the culture vessel 49.Therefore, when the frozen stored second mesenchymal stem cells 35 are thawed, the thawed second mesenchymal stem cells 35 can be modified into activated mesenchymal stem cells 55 with sufficient activity.

[0091] The frozen stem cell modification method (frozen stem cell modification system 10) can produce activated mesenchymal stem cells 55 that are sufficiently and highly effective in the treatment of various diseases (such as cardiovascular disease and central nervous system disease), regeneration in regenerative medicine, and non-therapeutic uses such as cosmetics, and can produce activated mesenchymal stem cells 55 that can achieve the desired effects. The frozen stem cell modification method (frozen stem cell modification system 10) can produce activated mesenchymal stem cells 55 that can be used suitably and in a timely manner for the treatment of various diseases, can produce activated mesenchymal stem cells 55 that can be used suitably and in a timely manner for the regeneration of various tissues and organs, and can produce activated mesenchymal stem cells 55 that can be used suitably and in a timely manner for non-therapeutic uses such as cosmetics. Because the frozen stem cell modification method (frozen stem cell modification system 10) does not contain a cryoprotectant 44 in the activated mesenchymal stem cells 55, administering the activated mesenchymal stem cells 55 produced by the frozen stem cell modification method to the human body will not have any adverse effects.

[0092] In the frozen stem cell modification method (frozen stem cell modification system 10), the culture product solution 32, 42 is the culture solution remaining after extracting the first and second mesenchymal stem cells 21, 35 (mesenchymal stem cells) after culture from the first flat culture vessel 22 (second culture vessel) or the second flat culture vessel 33 (third culture vessel), and contains specific metabolic substances secreted from the mesenchymal stem cells 21, 35 during the culture process of the mesenchymal stem cells 21, 35. Therefore, the metabolic substances of the mesenchymal stem cells 21, 35 themselves act as a trigger, causing the second mesenchymal stem cells 35 to quickly begin to become active, and the culture product solution 32, 42 can be used to quickly culture the second mesenchymal stem cells 35, making it possible to efficiently produce activated mesenchymal stem cells 55 in a short period of time.

[0093] In the frozen stem cell modification method (frozen stem cell modification system 10), when the activated mesenchymal stem cells 55 are to be used within 18 to 20 hours after extraction, the activated mesenchymal stem cells 55 are maintained at 20 to 21°C and transported to the site of use within 18 to 20 hours, so that the activity of the activated mesenchymal stem cells 55 is not lost, and the activated mesenchymal stem cells 55 can be used to treat various diseases (such as cardiovascular diseases and central nervous system diseases), and the activated mesenchymal stem cells 55 can be used for regeneration in regenerative medicine, as well as for non-therapeutic purposes such as cosmetic applications. [Explanation of symbols]

[0094] 10. Frozen Stem Cell Modification System 11 Management Server 12 IC tag reader / writer 13 Electron microscope 14 Refrigerator or freezer 15 keyboards 16 Mouse 17. Display 18 IC tags 19 First bone marrow fluid 20 glass test tubes 21 Second bone marrow aspirate (first mesenchymal stem cells) 22 1st flat culture container (2nd culture container) 23 bottom 24 Inlet 25 Lid 26 Culture solution 27 Sample holder 28 Top 29 Bottom 30 Spacer 31 Top 32 First culture product solution 33 2nd flat culture container (3rd culture container) 34 Glass test tube 35 Secondary mesenchymal stem cells 36 Culture solution 37 bottom 38 Inlet 39 Lid 40 bottom 41 Top 42 Second culture product 43 Freezer storage container 44 Cryoprotectants 45 Heat insulation container 46 Cleaning Agent 47 Centrifuge 48 Culture solution 49 3rd flat culture container (1st culture container) 50 inlet 51 Lid 52 bottom 53 Bottom 54 Top 55 Activated mesenchymal stem cells 56 Storage container 57 Constant Temperature Case

Claims

1. A method for modifying frozen stem cells, which activates mesenchymal stem cells frozen to a predetermined temperature and modifies them into activated mesenchymal stem cells, comprising: The frozen stem cell modification method includes a cryopreservation step of storing mesenchymal stem cells prepared by culturing bone marrow fluid collected from a donor in a cryopreservation container of a predetermined volume, injecting a cryoprotectant into the cryopreservation container containing the mesenchymal stem cells, and freezing and storing the mesenchymal stem cells stored in the cryopreservation container together with the cryoprotectant at a predetermined temperature until the mesenchymal stem cells stored in the cryopreservation container are used; a stem cell thawing step in which, when the mesenchymal stem cells cryopreserved in the cryopreservation step are to be used, the cryopreservation container containing the cryopreserved mesenchymal stem cells and the cryoprotectant is immersed in warm water maintained at 35 to 37°C for 2 to 3 minutes to thaw the mesenchymal stem cells to a temperature of 2 to 3°C; a stirring and removal step of injecting a washing solution into the cryopreservation container containing the mesenchymal stem cells thawed from a frozen state in the stem cell thawing step and the cryoprotectant, and stirring the cryopreservation container into which the washing solution has been injected to remove the cryoprotectant from the mesenchymal stem cells in the cryopreservation container by the washing solution; a mesenchymal stem cell extraction step of placing the cryopreservation container after the cryoprotectant has been removed from the mesenchymal stem cells by the stirring and removal step in a centrifuge, centrifuging the mesenchymal stem cells, the cryoprotectant, and the washing solution in the cryopreservation container in layers, and extracting the mesenchymal stem cells located in the bottom layer of the cryopreservation container after the centrifugation; a stem cell activation step of accommodating the mesenchymal stem cells extracted in the mesenchymal stem cell extraction step in a first culture vessel having a predetermined volume and a predetermined bottom area, pouring a predetermined culture medium into the first culture vessel containing the mesenchymal stem cells, tilting the first culture vessel containing the mesenchymal stem cells in one direction at an angle of 2 to 5 degrees, and leaving the first culture vessel statically at a temperature of 35 to 37°C for 12 hours, and then tilting the first culture vessel tilted in one direction at an angle of 2 to 5 degrees in the opposite direction, and leaving the first culture vessel statically at a temperature of 35 to 37°C for 12 hours to culture the mesenchymal stem cells, thereby activating the mesenchymal stem cells in the first culture vessel and converting them into the activated mesenchymal stem cells; After extracting the activated mesenchymal stem cells from the first culture vessel, if the activated mesenchymal stem cells are to be used within 18 to 20 hours after extraction, the method for modifying frozen stem cells comprises a stem cell transport step of placing the storage container containing the activated mesenchymal stem cells in a portable thermostatic case, maintaining the temperature within the thermostatic case at 20 to 21°C, and transporting the extracted activated mesenchymal stem cells, while maintained at 20 to 21°C, to a site where the activated mesenchymal stem cells will be used within 18 to 20 hours.

2. The method for modifying frozen stem cells according to claim 1, wherein the stem cell activation step comprises injecting a culture product produced during a culture process in which bone marrow fluid collected from the donor is cultured into the first culture vessel, and culturing the mesenchymal stem cells in the first culture vessel at a temperature of 35 to 37°C for 24 hours using the culture product and the culture product.

3. The frozen stem cell modification method of claim 2, wherein the cryoprotectant is at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), and the washing solution is Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS).

4. The mesenchymal stem cells are obtained by a first stem cell settling step of separating bone marrow fluid collected from the donor into layers, extracting intermediate layer bone marrow fluid located in the intermediate layer of the separated layers of bone marrow fluid, and injecting the intermediate layer bone marrow fluid and a predetermined culture medium into a second culture vessel having a predetermined volume and a predetermined bottom area to settling first mesenchymal stem cells contained in the intermediate layer bone marrow fluid on the bottom surface of the second culture vessel; and after the first mesenchymal stem cells have settled on the bottom surface of the second culture vessel by the first stem cell settling step, discharging the culture medium in the second culture vessel and injecting a new culture medium into the second culture vessel. a first stem cell culture step of culturing the first mesenchymal stem cells in a culture medium containing the first mesenchymal stem cells and growing the first mesenchymal stem cells until a ratio of a total planar area of ​​the first mesenchymal stem cells to a bottom area of ​​the second culture vessel reaches a first target ratio; a first second mesenchymal stem cell extraction step of placing the second culture vessel containing the first mesenchymal stem cells grown in the first stem cell culture step and the culture medium in a centrifuge, centrifuging the first mesenchymal stem cells and the culture medium in the second culture vessel in layers, and extracting second mesenchymal stem cells located in the bottom layer of the second culture vessel after the centrifugation; a second stem cell settling step in which the second mesenchymal stem cells extracted in the first second mesenchymal stem cell extraction step are placed in a third culture vessel having a bottom surface of a predetermined area and a volume larger than the second culture vessel, a new culture medium is poured into the third culture vessel, and the second mesenchymal stem cells are allowed to settle on the bottom surface of the third culture vessel; and after the second mesenchymal stem cells have settled on the bottom surface of the third culture vessel in the second stem cell settling step, the culture medium in the third culture vessel is discharged and a new culture medium is poured into the third culture vessel to culture the second mesenchymal stem cells, and the bottom surface of the third culture vessel is settling.

4. The method for modifying frozen stem cells according to claim 3, wherein the frozen stem cells are produced by a mesenchymal stem cell culture method comprising: a second stem cell culture step of proliferating the second mesenchymal stem cells until the total planar area of ​​the second mesenchymal stem cells relative to the volume of the third culture vessel reaches a second target ratio; and a second mesenchymal stem cell extraction step of placing the third culture vessel containing the second mesenchymal stem cells cultured by the second stem cell culture step in a centrifuge, centrifuging the second mesenchymal stem cells and the culture solution in the third culture vessel in layers, and extracting the second mesenchymal stem cells located in the bottom layer of the third culture vessel after the centrifugation.

5. The frozen stem cell modification method described in claim 4, wherein the freezing and preservation process includes placing the second mesenchymal stem cells in the freezing and preservation container immediately after extraction by the second mesenchymal stem cell second extraction process, and immediately freezing and preserving the mesenchymal stem cells placed in the freezing and preservation container together with the cryoprotectant.

6. The frozen stem cell modification method described in claim 5, wherein the culture solution is the culture solution remaining after extracting cultured mesenchymal stem cells from the second culture vessel or the third culture vessel, and contains specific metabolic substances secreted from the mesenchymal stem cells during the culture process of the mesenchymal stem cells.

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