JP2022038293A - Frozen stem cell modification method - Google Patents

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

The present invention relates to a method for modifying frozen stem cells, which activates mesenchymal stem cells frozen at a predetermined temperature and reforms them into activated mesenchymal stem cells.

It comprises an exfoliation step of exfoliating stem cells using a pronase solution and a freezing step of slowly freezing the exfoliated stem cells in a stem cell storage medium, and the stem cell storage media are hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene. Disclosed is a method for storing stem cells, which is a medium containing glycol (EG) or a medium containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and a culture medium or an albumin solution (Patent Document). 1).

International release WO2013 / 187077

Various stem cells are used for non-therapeutic applications such as treatment of various diseases (cardiovascular diseases, central nervous system diseases, etc.), regeneration in regenerative medicine, and beauty. When the cultured stem cells are stored frozen, the stem cells are stored in a predetermined storage container, and the cells in the storage container are destroyed during freezing such as dimethyl sulfoxide (DMSO), hydroxyethyl starch (HES), and ethylene glycol (EG). A cryoprotectant is injected to prevent. When using frozen stem cells that have been cryopreserved, it is necessary to thaw the frozen stem cells, but the cryoprotectant is not removed at the time of thawing, and the stem cells thawed together with the cryoprotectant are used as they are.

If the cryoprotectant is not removed when the frozen stem cells are thawed, about 50% or more of the frozen stem cells will be killed and the stem cells cannot be sufficiently activated. When the number of surviving stem cells decreases and the stem cells cannot be sufficiently activated, the effect of the stem cells on various diseases, the effect of regeneration in regenerative medicine, and the effect in non-therapeutic applications such as cosmetology are reduced, which is desired. May not be effective. Further, when stem cells containing a cryoprotectant are administered to the human body, there is a concern that the cryoprotectant may have an adverse effect.

An object of the present invention is that when the cryopreserved mesenchymal stem cells are thawed, most of the mesenchymal stem cells can be survived while removing the cryoprotectant, and the cryopreserved mesenchymal stem cells can be obtained. It is an object of the present invention to provide a method for modifying frozen stem cells, which can be modified into activated mesenchymal stem cells having sufficient activity. Another object of the present invention is to provide sufficient and high effect in non-therapeutic applications such as treatment for various diseases, regeneration in regenerative medicine, and beauty when thawing cryopreserved mesenchymal stem cells, which is desired. It is an object of the present invention to provide a method for modifying frozen stem cells capable of producing mesenchymal stem cells capable of obtaining an effect.

The premise of the present invention for solving the above-mentioned problems is a method for modifying mesenchymal stem cells, which activates mesenchymal stem cells frozen at a predetermined temperature and reforms them into activated mesenchymal stem cells.

The feature of the present invention in the above premise is that the mesenchymal stem cell reforming method stores the mesenchymal stem cells prepared by culturing the mesenchymal stem fluid collected from the donor in a predetermined volume of the frozen storage container and also the mesenchymal stem cells. Inject the mesenchymal stem cells into the cryoprotectant containing the cells, and freeze the mesenchymal stem cells contained in the cryoprotector together with the cryoprotectant at a predetermined temperature until the mesenchymal stem cells contained in the cryoprotector are used. When using the mesenchymal stem cells that have been cryopreserved by the cryopreservation step and the mesenchymal stem cells that have been cryopreserved by the cryopreservation step, the mesenchymal stem cells and the mesenchymal stem cells that have been cryopreserved are kept at 35 to 37 ° C. The mesenchymal stem cells and the mesenchymal stem cells thawed from the frozen state by the stem cell thawing step and the mesenchymal stem cells thawed by the stem cell thawing step and the mesenchymal stem cells were housed. Mesenchymal stem cells are removed from the mesenchymal stem cells in the mesenchymal stem cells by injecting the washing solution into the frozen storage container, stirring the frozen storage container into which the washing solution is injected, and using the washing solution to remove the mesenchymal stem cells. The mesenchymal stem cells, the mesenchymal stem cells, and the washing solution in the mesenchymal stem cells, the mesenchymal stem cells, and the washing solution are separated into layers by installing the mesenchymal stem cells, the cryoprotectant, and the washing solution in the centrifuge after removing the cryoprotectant from the stem cells. The mesenchymal stem cell extraction step of extracting the mesenchymal stem cells located at the bottom layer of the container and the mesenchymal stem cells extracted by the mesenchymal stem cell extraction step are put into a first culture container having a predetermined volume and a predetermined area with a bottom surface. At the same time, the predetermined culture solution is injected into the first culture vessel containing the mesenchymal stem cells, the mesenchymal stem cells are cultured in the first culture vessel at 36 to 37 ° C. for 24 hours, and the cells are cultured in the first culture vessel for 24 hours. It is to have a stem cell activation step of activating mesenchymal stem cells and modifying them into activated mesenchymal stem cells.

As an example of the present invention, the frozen stem cell reforming method uses activated mesenchymal stem cells within 18 to 20 hours after extraction from the first culture vessel and then the activated mesenchymal stem cells. This includes a stem cell transport step of transporting the extracted activated mesenchymal stem cells to the site of use of the activated mesenchymal stem cells within 18 to 20 hours while maintaining the temperature at 20 to 21 ° C.

As another example of the present invention, in the stem cell activation step, the culture product solution generated in the culture process of culturing the bone marrow fluid collected from the donor is injected into the first culture vessel, and the culture solution and the culture product solution are used. Then, the mesenchymal stem cells are cultured in the first culture vessel at a temperature of 35 to 37 ° C. for 24 hours.

As another example of the present invention, in the stem cell activation step, the first culture vessel is statically left 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. do.

As another example of the present invention, in the stem cell activation step, the first culture vessel is allowed to stand at a temperature of 35 to 37 ° C. for 12 hours with the first culture vessel tilted at an angle of 2 to 5 degrees in one direction. The first culture vessel was tilted at an angle of 2 to 5 degrees in the opposite direction to the other direction, and the first culture vessel was tilted at a temperature of 35 to 37 ° C. for 12 hours. Leave it statically.

As another example of the present invention, the cryoprotectant is at least the dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG), and the cleaning solution is dalbecco. Phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS).

As another example of the present invention, mesenchymal stem cells separate the bone cell fluid collected from the donor into layers, and extract the intermediate layer bone cell fluid located in the intermediate layer among the layered separated bone cell fluids. The mesenchymal stem cells contained in the intermediate layer mesenchymal stem cells are fixed on the bottom surface of the second culture vessel by injecting the intermediate layer bone cell fluid and the predetermined culture solution into the second culture vessel having a predetermined volume and a predetermined bottom surface. After the first mesenchymal stem cells are colonized on the bottom surface of the second culture vessel by the first stem cell colonization step and the first stem cell colonization step, a new culture solution is discharged while draining the culture solution in the second culture vessel. The first mesenchymal stem cells are cultured by injecting into a second culture vessel, and the first mesenchymal stem cells until the total planar area of the first mesenchymal stem cells with respect to the bottom surface area of the second culture vessel reaches the first target ratio. A second culture container containing the first mesenchymal stem cells and the culture solution grown by the first mesenchymal stem cell culture step and the first mesenchymal stem cell growing step was placed in a centrifuge, and the second in the second culture vessel was placed. 1 Mesenchymal stem cells, a second mesenchymal stem cell first extraction step for extracting a second mesenchymal stem cell located in the bottom layer of a second culture vessel after centrifuging the culture solution in a layered manner. The second mesenchymal stem cells extracted by the first extraction step of the second mesenchymal stem cells are housed in a third culture vessel having a predetermined capacity and a bottom surface of a predetermined area and a larger capacity than the second culture vessel. A new culture solution is injected into the third culture vessel, and the second mesenchymal stem cells are settled on the bottom surface of the third culture vessel by the second mesenchymal stem cell colonization step and the second mesenchymal stem cell. 3 After fixing to the bottom of the culture vessel, the culture solution in the third culture vessel is discharged, and a new culture solution is injected into the third culture vessel to culture the second mesenchymal stem cells, and the third culture vessel is used. A second mesenchymal stem cell culture step in which the second stem cell is proliferated until the total planar area of the second mesenchymal stem cell with respect to the bottom surface area of the cell reaches the second target ratio, and a second mesenchymal stem cell cultured by the second stem cell culture step. The third mesenchymal stem cell and the culture solution in the third mesenchymal stem cell and the culture solution are separated into layers, and the third culture vessel containing the above is placed in the lowest layer of the third culture vessel. It is produced by a mesenchymal stem cell culture method having a second mesenchymal stem cell extraction step of extracting a second mesenchymal stem cell.

As another example of the present invention, in the cryopreservation step, the second mesenchymal stem cells immediately after being extracted by the second extraction step of the second mesenchymal stem cells were housed in a frozen storage container and housed in the frozen storage container. Immediately cryopreserve mesenchymal stem cells with a cryoprotectant.

As another example of the present invention, the culture product solution is a culture solution remaining after extracting the mesenchymal stem cells after culturing from the second culture vessel or the third culture vessel, and is a culture solution remaining after culturing the mesenchymal stem cells in the process of culturing the mesenchymal stem cells. Contains certain metabolites secreted from mesenchymal stem cells.

According to the frozen stem cell reforming method according to the present invention, a cleaning solution is injected into a frozen storage container containing mesenchymal stem cells thawed from a frozen state and a cryoprotectant, and the frozen storage container into which the cleaning solution is injected is stirred. The mesenchymal stem cells in the cryopreservation container are removed from the mesenchymal stem cells with a washing solution, and the mesenchymal stem cells, the cryoprotectant, and the washing solution in the cryopreservation container are centrifuged in layers, and the frozen storage container is separated by centrifugation. Since the mesenchymal stem cells located in the lowest layer are extracted, the cryoprotectant can be removed by using a washing solution when thawing the frozen-stored mesenchymal stem cells, and while the frozen state is thawed. Most (70-90%) of mesenchymal stem cells can survive. In the method for modifying frozen stem cells, mesenchymal stem cells thawed from a frozen state are housed in a first culture vessel having a predetermined volume and a bottom surface of a predetermined area, and a predetermined culture vessel containing the mesenchymal stem cells is housed. Mesenchymal stem cells are cryopreserved because the culture medium is injected and the mesenchymal stem cells are cultured in the first culture vessel at 35 to 37 ° C. for 24 hours to activate the mesenchymal stem cells in the first culture vessel. When thawing, the thawed mesenchymal stem cells can be modified into activated mesenchymal stem cells having sufficient activity. The frozen stem cell modification method produces activated mesenchymal stem cells that are sufficiently and highly effective in the treatment of various diseases (cardiovascular disease, central nervous system disease, etc.), regeneration in regenerative medicine, and non-therapeutic applications such as beauty. It is possible to produce activated mesenchymal stem cells capable of obtaining the desired effect. The frozen stem cell modification method can produce activated mesenchymal stem cells suitable for the treatment of various diseases and can be used in a timely manner, and various tissues and organs are suitable for regeneration and used in a timely manner. It is possible to produce activated mesenchymal stem cells capable of producing activated mesenchymal stem cells, which are suitable for non-therapeutic applications such as beauty and can be used in a timely manner. In the frozen stem cell reforming method, since the thawed activated mesenchymal stem cells do not contain a cryoprotectant, even if the activated mesenchymal stem cells produced by the frozen stem cell reforming method are administered to the human body, It does not adversely affect the human body.

When the activated activated mesenchymal stem cells are extracted from the first culture vessel and then the activated mesenchymal stem cells are used within 18 to 20 hours after the extraction, the extracted activated mesenchymal stem cells are 20 to 20 to 20 hours. A frozen stem cell modification method including a stem cell transfer step of transporting activated mesenchymal stem cells to the site of use within 18 to 20 hours while holding the cells at 21 ° C keeps the activated mesenchymal stem cells at 20 to 21 ° C. By transporting the activated mesenchymal stem cells to the place of use within 18 to 20 hours in the same state, the activity of the activated mesenchymal stem cells is not lost, and the activated mesenchymal stem cells are subjected to various diseases (cardiovascular diseases). Can be used for the treatment of central nervous system diseases, etc.), the activated mesenchymal stem cells can be used for regeneration in regenerative medicine, and the activated mesenchymal stem cells can be used for non-therapeutic purposes such as beauty. It can be used for various purposes.

In the stem cell activation step, the culture product solution generated in the culture process of culturing the bone marrow fluid collected from the donor is injected into the first culture vessel, and the mesenchymal stem cells are first used using the culture solution and the culture product solution. The method for modifying frozen stem cells, which is cultured at a temperature of 35 to 37 ° C. for 24 hours in a culture vessel, is to use the culture product solution produced in the process of culturing mesenchymal stem cells before cryopreservation in the first culture vessel. Since the activation of leaf stem cells is surely promoted, the activated mesenchymal stem cells can be activated in 24 hours after thawing using the culture product solution, and the activated mesenchymal stem cells have sufficient activity. Can be efficiently produced, and mesenchymal stem cells from which the cryoprotectant has been removed can be rapidly utilized.

In the stem cell activation step, the method for modifying frozen stem cells, in which the first culture vessel is tilted at an angle of 2 to 5 degrees and the first culture vessel is statically left at a temperature of 35 to 37 ° C. for 24 hours, is the first method. By tilting 1 culture vessel at an angle of 2 to 5 degrees, the thawed mesenchymal stem cells and culture medium (and culture product) are biased toward one of the first culture vessels in the first culture vessel, and the first The pressure of the mesenchymal stem cells and the culture solution (and the culture product) increases in one of the culture vessels, and the mesenchymal stem cells concentrate in one of the first culture vessels, thereby increasing the activity of the mesenchymal stem cells. The thawed mesenchymal stem cells can be modified into activated mesenchymal stem cells having sufficient activity.

In the stem cell activation step, the first culture vessel is statically left at a temperature of 35 to 37 ° C. for 12 hours with the first culture vessel tilted at an angle of 2 to 5 degrees in one direction, and then tilted in one direction. Frozen stem cell modification in which the first culture vessel is left statically for 12 hours at a temperature of 35 to 37 ° C. with the first culture vessel tilted in the opposite direction to the other direction at an angle of 2 to 5 degrees. The method is to tilt the first culture vessel in one direction at an angle of 2 to 5 degrees, and then tilt the first culture vessel in one direction at an angle of 2 to 5 degrees in the opposite direction. By allowing the cells to concentrate the thawed mesenchymal stem cells and culture medium (and culture product) in one or the other of the first culture medium, and the mesenchymal system in one or the other of the first culture medium. As the pressure of the stem cells and the culture medium (and the culture product) increases, the mesenchymal stem cells are evenly concentrated on one or the other of the first culture vessel, whereby the activity of the mesenchymal stem cells can be enhanced and thawed. The resulting mesenchymal stem cells can be modified into activated mesenchymal stem cells having sufficient activity.

The cryoprotectant is at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG), and the washing solution is dalbecco phosphate buffered saline (D-PBS) or Phosphate-buffered saline (PBS) is a method for modifying frozen stem cells in a washing solution (Dalbeco phosphate-buffered saline (D)) in a frozen storage container containing mesenchymal stem cells thawed from a frozen state and a cryoprotectant. -PBS) or phosphate buffered saline (PBS)) is infused, and the frozen storage container infused with the washing solution is stirred, and the mesenchymal stem cells in the frozen storage container are stirred with the washing solution to protect the mesenchymal stem cells (hydroxyethyl starch (HES)). ), Mesenchymal stem cells, cryoprotectant, and washing solution in a cryopreservation vessel are separated by layering, removing at least dimethylsulfoxide (DMSO) from ), dimethylsulfoxide (DMSO), and ethylene glycol (EG). Since the mesenchymal stem cells located in the bottom layer of the cryopreservation vessel after centrifugation are extracted, a washing solution (Dalbecophosphate buffered saline (D-PBS)) is used when thawing the mesenchymal stem cells that have been cryopreserved. Alternatively, use phosphate buffered saline (PBS) to ensure cryoprotective agents (at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)). Most (70-90%) mesenchymal stem cells can survive in thawing, and thawed mesenchymal stem cells can be transformed into activated mesenchymal stem cells with sufficient activity. Can be modified.

Mesenchymal stem cells separate the mesenchymal stem cells collected from the donor in layers, extract the mesenchymal stem cells located in the middle layer of the layered myeloids, and have a predetermined volume and a predetermined area of the bottom surface. The first mesenchymal stem cell colonization step in which the mesenchymal stem cells contained in the mesenchymal stem cells are fixed on the bottom surface of the second culture vessel by injecting the intermediate layer myelocytes and the predetermined culture cells into the second culture vessel, and the stem cells. After the first mesenchymal stem cells are settled on the bottom surface of the second culture vessel by the first colonization step, the culture solution in the second culture vessel is discharged and a new culture solution is injected into the second culture vessel. With the first mesenchymal stem cell culture step in which 1 mesenchymal stem cell is cultured and the 1st mesenchymal stem cell is proliferated until the total planar area of the 1st mesenchymal stem cell with respect to the bottom surface area of the 2nd culture vessel reaches the 1st target ratio. , The second mesenchymal stem cell and the culture solution containing the first mesenchymal stem cell and the culture solution grown by the first stem cell culture step are placed in the centrifuge, and the first mesenchymal stem cell and the culture solution in the second culture vessel are placed. The first extraction step of the second mesenchymal stem cell, which extracts the second mesenchymal stem cell located at the bottom layer of the second culture vessel after centrifugation in a layered manner, and the bottom surface having a predetermined volume and a predetermined area While accommodating the second mesenchymal stem cells extracted by the first extraction step of the second mesenchymal stem cells in the third culture vessel having a larger capacity than the second culture vessel, a new culture solution is placed in the third culture vessel. Was injected, and the second mesenchymal stem cells were colonized on the bottom surface of the third culture vessel by the stem cell second colonization step in which the second mesenchymal stem cells were colonized on the bottom surface of the third culture vessel and the stem cell second colonization step. After that, while draining the culture solution in the third culture vessel, a new culture solution is injected into the third culture vessel to culture the second mesenchymal stem cells, and the second mesenchymal system relative to the bottom surface area of the third culture vessel. Centrifusate a third culture vessel containing a second mesenchymal stem cell cultured by the second mesenchymal stem cell culture step, in which the second stem cell is proliferated until the total planar area of the stem cell reaches the second target ratio. Placed in a vessel, the second mesenchymal stem cells and the culture solution in the third culture vessel are centrifuged in layers, and the second mesenchymal stem cells located in the lowest layer of the third culture vessel are extracted after the centrifugation. The frozen stem cell modification method produced by the mesenchymal stem cell culture method having the second extraction step of the second mesenchymal stem cell is after the first mesenchymal stem cell is fixed on the bottom surface of the second culture vessel. , While draining the culture solution from the second culture vessel, inject a new culture solution into the second culture vessel, and use the new culture medium for the first mesenchymal stem cells colonized on the bottom surface of the second culture vessel. And grow in the second culture vessel. The second mesenchymal stem cells were housed in the third culture vessel, the second mesenchymal stem cells were fixed on the bottom surface of the third culture vessel, and then the culture solution was discharged from the third culture vessel to make a new one. The culture solution is injected into the third culture vessel, and the second mesenchymal stem cells established on the bottom surface of the third culture vessel are cultured using a new culture medium, and the second mesenchymal stem cells are cultured in the third culture vessel. Therefore, mesenchymal stem cells can be efficiently and reliably proliferated, and mesenchymal stem cells having sufficient activity can be efficiently produced.

In the cryopreservation step, the second mesenchymal stem cells immediately after being extracted by the second extraction step of the second mesenchymal stem cells are housed in a freezing storage container, and the mesenchymal stem cells housed in the freezing storage container are combined with a cryoprotectant. Immediately cryopreserving The frozen stem cell modification method immediately freezes and stores the sufficiently active mesenchymal stem cells produced by the mesenchymal stem cell culturing method. The portion can be stored frozen alive, and the mesenchymal stem cells produced by the mesenchymal stem cell culture method can be stored for a long period of time.

The culture product is the culture solution remaining after extracting the mesenchymal stem cells after culturing from the second culture vessel or the third culture vessel, and is a predetermined one secreted from the mesenchymal stem cells in the process of culturing the mesenchymal stem cells. Since the method for modifying frozen stem cells containing a metabolite contains a predetermined metabolite secreted from the mesenchymal stem cell in the process of culturing the mesenchymal stem cell, the mesenchymal stem cell itself is triggered by the metabolite of the mesenchymal stem cell itself. The stem cells start their activity rapidly, and the culture product can be used to rapidly activate the mesenchymal stem cells, so that the activated mesenchymal stem cells can be efficiently produced in a short time.

The schematic block diagram of the frozen stem cell reforming system shown as an example. Explanatory drawing showing an example of the bone marrow fluid separation step in the mesenchymal stem cell culture method The explanatory view of the bone marrow fluid separation process which continues from FIG. The explanatory view which shows an example of the shape deformation 1st observation process. The side view of the 1st flat culture container (2nd culture container). Explanatory drawing which shows an example of the 2nd mesenchymal stem cell 1st extraction step in the mesenchymal stem cell culture method. Explanatory drawing which shows an example of the shape deformation 2nd observation step in the mesenchymal stem cell culture method. The side view of the 2nd flat culture container (the 3rd culture container). Explanatory drawing which shows an example of the freezing storage process in a freezing stem cell modification method. Explanatory drawing which shows an example of the stem cell thawing process in a frozen stem cell modification method. The explanatory view which shows an example of the stirring removal process in the frozen stem cell modification method. Explanatory drawing which shows an example of the mesenchymal stem cell extraction process in the frozen stem cell modification method. Explanatory drawing which shows an example of the stem cell activation process in the frozen stem cell modification method. The side view of the 3rd flat culture vessel (1st culture vessel). A partially enlarged view showing an example of the planar shape of the second mesenchymal stem cell. A partially enlarged view showing another example of the planar shape of the second mesenchymal stem cell. Explanatory drawing which shows an example of the stem cell transport process in the frozen stem cell modification method.

The details of the frozen stem cell reforming method according to the present invention will be described below with reference to the attached drawings such as FIG. 1, which is a schematic configuration diagram of the frozen stem cell reforming system 10 shown as an example. 2 is an explanatory diagram showing an example of the bone marrow fluid separation step in the mesenchymal stem cell culture method, and FIG. 3 is an explanatory diagram of the bone marrow fluid separation step following from FIG. 2. FIG. 4 is an explanatory view showing an example of the shape deformation first observation step, and FIG. 5 is a side view of the first flat culture container (second culture container).

The frozen stem cell reforming system 10 cultures (manufactures) a specific type of single-species mesenchymal stem cells by a stem cell culture method, and activates the cryopreserved single-species mesenchymal stem cells by the frozen stem cell reforming method. It reforms into lineage stem cells. The stem cell culture method uses the first bone marrow fluid collected from a plurality of donors (humans), and is a stem cell first colonization step, a stem cell first culture step, a second mesenchymal stem cell first extraction step, and a stem cell second colonization step. By carrying out the second stem cell culture step and the second mesenchymal stem cell second extraction step, a specific type of single-species mesenchymal stem cell can be obtained from a plurality of types of mesenchymal stem cells contained in the first bone marrow fluid. Cultivate (manufacture).

The frozen stem cell reforming system 10 is connected to the management server 11, the IC tag reader / writer 12 connected to the management server 11 via an interface (wireless or wired), and the management server 11 via an interface (wireless or wired). It is formed of a refrigerator 14 or a freezer 14 connected to an electron microscope 13 and a management server 11 via an interface (wireless or wired). The electron microscope 13 has an image capturing function of capturing a magnified image of a subject by an image pickup element, and also has an image transmitting function of transmitting the magnified image to the management server 11.

The management server 11 is a physical computer or cloud computing including 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). , Large-capacity storage area or large-capacity virtual storage area is built-in. Infrastructure as a Service (IaaS), Platform as a Service (PaaS), and Software as a Service (SaaS) can be used as the cloud. An input device such as a keyboard 15 and a mouse 16 and an output device such as a display 17 and a printer (not shown) are connected to the management server 11 via an interface (wireless or wired).

In the frozen stem cell reforming system 10, various donor data (donor specific information) are managed using the IC tag 18 (IC chip), and stem cell data related to mesenchymal stem cells are managed using the IC tag 18. .. Donor data includes donor name, address, phone number, date of birth, gender, blood type, height, weight, email address, etc., and stem cell data includes stem cell specific information, stem cell production date, culture generation, etc. There is liquid production date, culture product liquid specific information, etc. In the first stem cell colonization step, a person in charge (doctor, nurse, researcher, biotechnologist, etc.) collects 2 to 3 cc (2 to 3 ml) of the first bone marrow fluid from the donor. At the same time as collecting the first bone marrow fluid, the person in charge activates the frozen stem cell reforming system 10 on the management server 11 and transfers donor data and stem cell data to the management server 11 using an input device such as a keyboard 15 and a mouse 16. Enter as appropriate.

The management server 11 generates a unique donor identifier that identifies each donor each time donor data is entered (every time the first bone marrow fluid is collected from the donor). The management server 11 writes the input donor data and stem cell data to the IC tag 18 using the IC tag reader / writer 12. The management server 11 stores (stores) the donor data and the stem cell data in the large-capacity storage area or the large-capacity virtual storage area in a state of being associated with the donor identifier and the IC tag specific information (IC tag identifier) of the IC tag 18. Data storage process).

As shown in FIG. 2, the first bone marrow fluid 19 collected from the donor is injected (contained) into a vertically long glass test tube 20 (separation container). In addition, 2 to 3 cc of the first cerebrospinal fluid 19 contains 0.5 to 1 ml (about 5 × ¹⁰⁷ (cells / ml)) of a plurality of types of mesenchymal stem cells. An IC tag 18 is attached to the outer peripheral surface of the glass test tube 20. When the donor data or stem cell data written in the IC tag 18 is read by the IC tag reader / writer 12, the management server 11 displays the donor data or stem cell data on the display 17. The glass test tube 20 infused with the first bone marrow fluid 19 is set in a test tube rack (not shown), and is housed inside a constant temperature bath (not shown) together with the test tube rack.

The glass test tube 20 infused with the first bone marrow fluid 19 is statically left (leave quietly without movement) for a predetermined time (about 2 hours) in a constant temperature bath. The temperature in the constant temperature bath is maintained at about 35 to 37 ° C, which is substantially the same as the body temperature. By statically leaving the glass test tube 20 in a constant temperature bath for a predetermined time (about 2 hours), as shown in FIG. 3, the first bone marrow fluid 19 injected into the glass test tube 20 is placed in the glass test tube 20. It is separated into several layers (three layers) in the vertical direction (myeloid fluid separation step). After separating the first bone marrow fluid 19 into layers, the test tube stand is taken out from the inside of the constant temperature bath, the glass test tube 20 is pulled out from the test tube stand, and the specific layer (3) of the first bone marrow fluid 19 separated into layers is taken out. Extract (suction) the second bone marrow fluid 21 (intermediate layer marrow fluid) existing in the intermediate layer (second layer) separated into layers (layered) using a test tube (not shown) or a pipette (not shown). ) (Marrow fluid extraction step).

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 the culture fluid 26 are added to the first flat culture vessel 22 (second culture vessel). ) Inject (contain) into (cell culture vessel) and leave the culture vessel 22 statically for 12 to 24 hours (leave quietly without moving) while keeping the culture vessel 22 at a temperature substantially the same as the body temperature (about 35 to 37 ° C). ), And display 17 (electron microscope) the deformation of the first mesenchymal stem cells 21 contained in the second bone marrow fluid 21 in the culture vessel 22 from the initial planar shape at intervals of about 1 to 2 hours between 12 and 24 hours. ), And it is determined whether or not the first mesenchymal stem cells 21 have settled on the bottom surface 23 of the first flat culture vessel 22 (shape deformation first observation step). An IC tag 18 in 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.

The first flat culture container 22 (second culture container) is a flat container made of transparent glass or transparent plastic and having a small capacity and a bottom surface 23 having a predetermined area and a substantially regular square shape. The injection port 24 of the first flat culture vessel 22 is watertightly closed by the lid 25. The capacity of the first flat culture vessel 22 is about 20 to 30 cc (preferably 25 cc), the area of the bottom surface 23 thereof is about 25 to 36 mm ² , and the length of one side thereof is 5 to 6 mm. .. As the first flat culture container 22, a flat container having a small capacity and a bottom surface having a predetermined area and having a circular or elliptical planar shape can also be used.

The person in charge removes the lid 25 from the injection port 24, injects (contains) the second bone marrow fluid 21 sucked into the syringe or pipette from the injection port 24 of the first flat culture container 22 into the inside of the culture container 22, and at the same time. The culture solution 26 is injected (accommodated) into the inside of the culture vessel 22, and the injection port 24 is closed by the lid 25. Mineral salt containing penicillin (about 100 U / ml), amhotericin (about 100 ng / ml), streptomycin (about 100 mg / ml), L-glutamine (about 2-4 ml), and 20% fetal bovine serum in the culture solution 26. Contains solutions and amino acids.

As the culture medium 26, Dulbecco's Modified Eagle's Medium (DMEM), Grassgo Minimium Essential Medium (GMEM), RPMI640 and the like can also be used. In the culture solution 26, insulin, transferase, ethanolamine, selenium, 2-mercaptoethanol, L-alanyl-L-glutamine, sodium pyruvate, L-alanine, L-asparagine, L-asparagine, glycine, L- Proline, L-serine and the like can also be added. The first mesenchymal stem cells 21 contained in the second bone marrow fluid 21 injected into the first flat culture vessel 22 are cultured by the culture fluid 26 while being established on the bottom surface 23 of the first flat culture vessel 22 with the passage of time. Then, it gradually proliferates (differentiates) on the bottom surface 23 of the culture vessel 22 to form a colony.

After injecting the second bone marrow fluid 21 and the culture fluid 26 into the first flat culture vessel 22, the culture vessel 22 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 29 of the first flat culture vessel 22, and the bottom 29 of the culture vessel 22 is held in a state of being lifted by the spacer 30 to hold the culture vessel 22 in a lifted state. The culture vessel 22 is held in a state of being tilted at a predetermined angle so that the bottom portion 29 of the culture vessel 22 is on the top and the top portion 31 (injection port 24) of the culture vessel 22 is on the bottom. The top 31 of the first flat culture container 22 is held in a lifted state by the spacer 30, and the culture container 22 is set at a predetermined angle so that the top 31 of the culture container 22 is on the top and the bottom 29 of the culture container 22 is on the bottom. It may be held in an inclined state. The tilt 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 °, preferably in the range of 2 to 3 °.

By tilting the first flat culture vessel 22 at the tilt angle with respect to the upper surface 28 of the sample holder 27, the second bone marrow fluid 21 (or the first mesenchymal stem cell 21) and the culture fluid 26 can be placed in the culture vessel 22. Biased to the top 31 side (or bottom 29 side) of the culture vessel 22 and with the second myeloid fluid 21 (or first mesenchymal stem cell 21) on the top 31 side (or bottom 29 side) of the culture vessel 22. The water pressure with the culture solution 26 increases and the second mesenchymal fluid 21 (or the first mesenchymal stem cells 21) concentrates on the side of the top 31 of the culture vessel 22, thereby causing the activity of the first mesenchymal stem cells 21 to each other. The first mesenchymal stem cell 21 can be easily and rapidly established (or proliferated (differentiated)) on the bottom surface 23 of the culture vessel 22.

The electron microscope 13 took magnified 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 about 1 to 2 hours, and photographed the first. 1 An enlarged image of the planar shape of the mesenchymal stem cell 21 is transmitted to the management server 11 at intervals of about 1 to 2 hours. The management server 11 stores the enlarged image of the planar shape of the first mesenchymal stem cell 21 transmitted from the electron microscope 13 and the imaging time in the storage area in a state of being associated with the IC tag specific information (IC tag identifier) and the donor identifier. Store (remember). The management server 11 outputs (displays) the enlarged image of the planar shape of the first mesenchymal stem cell 21 transmitted from the electron microscope 13 and the photographing time on the display 17. The person in charge (doctor, nurse, researcher, biotechnologist, etc.) displays a magnified image of the planar shape of the first mesenchymal stem cell 21 displayed on the display 17 for about 1 to 2 hours in 12 to 24 hours. Check (visually) at intervals and observe changes in the planar shape of the first mesenchymal stem cells 21 contained in the second bone marrow fluid 21.

Although not shown, when the initial planar shape of the first mesenchymal stem cell 21 is substantially circular and the planar shape of the first mesenchymal stem cell 21 is substantially circular, the first mesenchymal stem cell 21 is the first. It has not settled on the bottom surface 23 (inner surface of the bottom wall) of the flat culture vessel 22, and the first mesenchymal stem cells 21 have not started proliferation (differentiation). The deformed planar shape of the first mesenchymal stem cell 21 is a flat shape in which the first mesenchymal stem cell 21 is irregularly extended (expanded) in one direction (predetermined direction) with the substantially circular shape before colonization as the nucleus. The first mesenchymal stem cells 21 have settled on the bottom surface 23 (inner surface of the bottom wall) of the first flat culture vessel 22, and the first mesenchymal stem cells 21 have started proliferation (differentiation).

Shape deformation As a result of observation in the first observation step, when the planar shape of the first mesenchymal stem cell 21 output (displayed) on the display 17 is transformed from a substantially circular shape to an irregular flat shape centered on a substantially circular shape, the first 1 It is determined that the mesenchymal stem cells 21 have settled on the bottom surface 23 of the first flat culture vessel 22 (stem cell first colonization step). Shape deformation As a result of observation in the first observation step, the first mesenchymal stem cell 21 is deformed from a substantially circular shape (initial planar shape) to an amorphous flat shape centered on the substantially circular shape, and the first mesenchymal stem cell 21 is the first. 1 After confirming the fixation of the flat culture container 22 (first culture container) to the bottom surface 23, the first observation step of the total plane area is performed.

The person in charge discharges the culture solution 26 injected into the first flat culture container 22 from the culture container 22, and injects (contains) a new culture solution 26 into the culture container 22. Next, the first flat culture vessel 22 is statically left for 36 to 48 hours at a temperature substantially the same as the body temperature (about 35 to 37 ° C.) (leaving quietly without moving) to obtain the first mesenchymal stem cells 21. Display 17 (electron) shows the total plane area of the first mesenchymal stem cells 21 colonized on the bottom surface 23 of the culture vessel 22 at intervals of about 1 to 2 hours with respect to the bottom surface area of the culture vessel 22 while culturing. Observe with a microscope 13) to determine whether or not the total planar area of the first mesenchymal stem cells 21 has reached the first target ratio with respect to the bottom surface area of the culture vessel 22 (total planar area first observation step). .. The first target ratio of the total planar area of the first mesenchymal stem cells 21 to the bottom surface area of the first flat culture vessel 22 is 70 to 80% (70 to 80% confluent).

Total plane area In the first observation step, the culture solution 26 injected into the first flat culture container 22 is discharged from the culture container 22, the new culture solution 26 is injected (accommodated) into the culture container 22, and the new culture solution 26 is used. The first flat culture vessel 22 in which the mixture was injected 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 29 of the first flat culture vessel 22, and the bottom 29 of the culture vessel 22 is held in a state of being lifted by the spacer 30 to hold the culture vessel 22 in a lifted state. The culture vessel 22 is held in a state of being tilted at a predetermined angle so that the bottom portion 29 of the culture vessel 22 is on the top and the top portion 31 (injection port 24) of the culture vessel 22 is on the bottom (see FIG. 6). The tilt 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 °, preferably in the range of 2 to 3 °.

The electron microscope 13 took an enlarged image of the planar shape of the first mesenchymal stem cell 21 in the first flat culture vessel 22 at intervals of about 1 to 2 hours, and took an image of the planar shape of the first mesenchymal stem cell 21. The enlarged image is transmitted to the management server 11 at intervals of about 1 to 2 hours. The management server 11 stores the enlarged image of the planar shape of the first mesenchymal stem cell 21 transmitted from the electron microscope 13 and the imaging time in the storage area in a state of being associated with the IC tag specific information (IC tag identifier) and the donor identifier. Store (remember). The management server 11 outputs (displays) the enlarged image of the planar shape of the first mesenchymal stem cell 21 transmitted from the electron microscope 13 and the photographing time on the display 17.

The person in charge (doctor, nurse, researcher, biotechnologist, etc.) displays an enlarged image of the planar shape of the first mesenchymal stem cell 21 displayed on the display 17 for about 1 to 2 hours between 36 and 48 hours. The first mesenchymal stem cell 21 was confirmed (visually) at intervals, and while observing the total plane area of the first mesenchymal stem cell 21 established on the bottom surface 23 of the first flat culture vessel 22 with respect to the bottom surface area of the culture vessel 22. It is determined whether or not the total plane area of the first flat culture vessel 22 has reached the first target ratio (70 to 80% confluent) with respect to the bottom surface area of the first flat culture vessel 22.

The first mesenchymal stem cells 21 proliferate on the bottom surface 23 (inner surface of the bottom wall) of the first flat culture vessel 22 to form colonies (stem cell first culture step). The first mesenchymal stem cells 21 formed colonies, and as a result of observation in the first observation step, the planar shape of the first mesenchymal stem cells 21 was expanded, and the first mesenchymal stem cells displayed on the display 17 were displayed. When the total plane area of the stem cells 21 with respect to the bottom surface area of the first flat culture vessel 22 reaches the first target ratio (70 to 80% confluent), the first mesenchymal stem cells 21 are extracted from the culture vessel 22.

Total plane area injected into the first flat culture container 22 The culture solution 26 (first culture product solution 32) in the first observation step is discharged from the first flat culture container 22, and the culture container 22 is subjected to phosphate buffering physiology. After washing with saline (PBS), the trypsin solution is poured into the culture vessel 22. When the trypsin solution is injected into the first flat culture vessel 22, the first mesenchymal stem cells 21 established on the bottom surface 23 of the culture vessel 22 are detached from the bottom surface 23 by the trypsin solution and float on the water surface of the trypsin solution. The person in charge sucks the surfaced first mesenchymal stem cells 21 using a pipette.

The culture solution 26 discharged from the first flat culture container 22 (second culture container) is the culture solution 26 remaining after extracting the first mesenchymal stem cells 21 from the culture container 22, and is the first mesenchymal. In the process of culturing the lineage stem cell 21, it is changed to the first culture product solution 32 (culture product solution) containing a predetermined metabolite secreted from the first mesenchymal stem cell 21. The first culture product 32 containing the metabolite activates mesenchymal stem cells with reduced activity. The first culture product liquid 32 is injected into the freezing storage container 43 described later to which the IC tag 18 is attached, is rapidly frozen by a quick freezer (not shown), and then stored in the freezing storage container 43. It is stored in a refrigerator 14 or a freezer 14 for a predetermined period at a predetermined temperature (chilled storage at 0 to 5 ° C. or freezing storage at 0 ° C. or lower).

FIG. 6 is an explanatory diagram showing an example of a second mesenchymal stem cell first extraction step in the mesenchymal stem cell culturing method, and FIG. 7 is an example of a shape deformation second observation step in the mesenchymal stem cell culturing method. It is explanatory drawing which shows, and FIG. 8 is a side view of the 2nd flat culture vessel 33 (the 3rd culture vessel). The person in charge (doctor, nurse, researcher, biotechnologist, etc.) extracts the first mesenchymal stem cell 21 from the first flat culture vessel 22, and then puts the first mesenchymal stem cell 21 into a glass test tube 34. Inject (accommodate) and install (set) the glass test tube 34 in a centrifuge (not shown). After centrifuging the first mesenchymal stem cells 21 with a centrifuge for a predetermined time, the glass test tube 34 is taken out from the centrifuge. The first mesenchymal stem cell 21 in the glass test tube 34 is centrifuged in two layers in the vertical direction by a centrifuge.

After the first mesenchymal stem cells 21 were separated in layers, as shown in FIG. 6, the second mesenchymal stem cells 35 located in the lower layer (bottom layer) from the first mesenchymal stem cells 21 separated in layers were separated. Extraction (suction) using a syringe (not shown) or a pipette (not shown) Extraction (second mesenchymal stem cell first extraction step). The first mesenchymal stem cells 21 containing unnecessary stem cells are centrifuged in a centrifuge to separate them in layers in the vertical direction, and then to the lower layer (bottom layer) of the first mesenchymal stem cells 21 that have been centrifuged in layers. By extracting the located second mesenchymal stem cell 35, a specific second mesenchymal stem cell 35 can be reliably extracted from the first mesenchymal stem cell 21, and it is unnecessary from the first mesenchymal stem cell 21. Mesenchymal stem cells can be removed.

After extracting a specific second mesenchymal stem cell 35 located in the lower layer (bottom layer) from the first mesenchymal stem cell 21, the second observation step of shape deformation is performed. In the second observation step of shape deformation, the second mesenchymal stem cells 35 and the culture solution 36 are injected (contained) into the second flat culture vessel 33 (third culture vessel) (cell culture vessel), and the culture vessel 33 is set to body temperature. In the culture vessel 33 at intervals of about 1 to 2 hours for 36 to 48 hours while statically leaving (leaving quietly without moving) for 36 to 48 hours at substantially the same temperature (about 35 to 37 ° C.). The deformation of the second mesenchymal stem cell 35 (mesenchymal stem cell) from the initial planar shape was observed with a display 17 (electron microscope), and whether or not the second mesenchymal stem cell 35 had settled on the bottom surface 37 of the culture vessel 33. (The second observation step of shape deformation). An IC tag 18 in 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.

The second flat culture container 33 (third culture container) is a flat container made of transparent glass or transparent plastic, having a small capacity and a bottom surface having a predetermined area, and having a substantially regular square shape, and having a volume thereof. It is larger than that of the first flat culture container 22 (second culture container). The injection port 38 of the second flat culture vessel 33 is watertightly closed by the lid 39. The second flat culture vessel 33 has a capacity of about 40 to 60 cc (preferably 50 cc), a bottom surface area of about 50 to 72 mm ² , and a side length of about 7 to 8.5 mm. be. As the second flat culture container 33, a flat container having a small capacity and a bottom surface having a predetermined area and having a circular or elliptical planar shape can also be used. The second mesenchymal stem cells 35 injected into the second flat culture vessel 33 are cultured in the culture medium 36 while being established on the bottom surface 37 of the culture vessel 33 over time, and gradually proliferate on the bottom surface 37 of the culture vessel 33. (Differentiate) to form colonies.

The person in charge removes the lid 39 from the injection port 38, and injects (contains) the second mesenchymal stem cells 35 sucked into the syringe or pipette from the injection port 38 of the second flat culture container 33 into the inside of the culture container 33. At the same time, the culture solution 36 is injected (accommodated) into the inside of the culture container 33, and the injection port 38 is closed by the lid 39. The culture solution 36 is the same as that injected in the first observation step of shape deformation. After injecting the second mesenchymal stem cell 35 and the culture solution 36 into the second flat culture vessel 33, the person in charge installs (sets) the culture vessel 33 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 in a state of being lifted by the spacer 30 to hold the culture vessel 33. The culture vessel 33 is held in a state of being tilted at a predetermined angle so that the bottom portion 40 of the culture vessel 33 is on the top and the top 41 (injection port 38) of the culture vessel 33 is on the bottom. Further, a spacer 30 is 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 lifted state by the spacer 30 for culturing. The culture container 33 may be held in a state of being tilted at a predetermined angle so that the top 41 of the container 33 is on the top and the bottom 40 of the culture container 33 is on the bottom. The tilt 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 °, preferably in the range of 2 to 3 °.

By tilting the second flat culture vessel 33 with respect to the upper surface 28 of the sample holder 27 at the inclination angle, the second mesenchymal stem cells 35 and the culture solution 36 are placed on the side of the top 41 of the culture vessel 33 in the culture vessel 33. (Or the side of the bottom 40), the water pressure between the second mesenchymal stem cell 35 and the culture solution 36 increases on the side of the top 41 (or the side of the bottom 40) of the culture vessel 33, and the second mesenchymal stem cell 35 concentrates on the side of the top 41 (or the side of the bottom 40) of the culture vessel 33, thereby increasing the activity of the second mesenchymal stem cells 35 and the second mesenchymal stem cells 35 on the bottom surface 37 of the culture vessel 33. Can be easily and quickly established (or proliferated (differentiated)).

The electron microscope 13 took a magnified image of the planar shape of the second mesenchymal stem cell 35 injected into the second flat culture vessel 33 at intervals of about 1 to 2 hours, and photographed the planar surface of the second mesenchymal stem cell 35. An enlarged image of the shape is transmitted to the management server 11 at intervals of about 1 to 2 hours. The management server 11 stores the enlarged image of the planar shape of the second mesenchymal stem cell 35 transmitted from the electron microscope 13 and the imaging time in the storage area in a state of being associated with the IC tag specific information (IC tag identifier) and the donor identifier. Store (remember). The management server 11 displays the enlarged image of the planar shape of the second mesenchymal stem cell 35 transmitted from the electron microscope 13 and the photographing time on the display 17. The person in charge confirms (visually recognizes) the enlarged image of the planar shape of the second mesenchymal stem cell 35 displayed on the display 17 at intervals of about 1 to 2 hours between 36 and 48 hours, and the second mesenchymal stem cell. Observe the change in the planar shape of 35.

When the initial planar shape of the second mesenchymal stem cell 35 is substantially circular, and the planar shape of the second mesenchymal stem cell 35 is substantially circular, the second mesenchymal stem cell 35 is the bottom surface 37 of the second flat culture vessel 33. It has not settled on (the inner surface of the bottom wall), and the second mesenchymal stem cell 35 has not started proliferation (differentiation). The deformed planar shape of the second mesenchymal stem cell 35 is a flat shape in which the second mesenchymal stem cell 35 extends irregularly in one direction with the substantially circular shape before colonization as the nucleus, and the second mesenchymal stem cell 35. Has settled on the bottom surface 37 (inner surface of the bottom wall) of the second flat culture vessel 33, and the second mesenchymal stem cell 35 has started proliferation (differentiation).

Shape deformation As a result of observation in the second observation step, when the planar shape of the second mesenchymal stem cell 35 displayed on the display 17 is transformed from a substantially circular shape to an amorphous flat shape centered on a substantially circular shape, the second mesenchyme It is determined that the mesenchymal stem cells 35 have settled on the bottom surface 37 of the second flat culture vessel 33 (stem cell second colonization step). As a result of observation in the second observation step of shape deformation, the second mesenchymal stem cell 35 is deformed from a substantially circular shape (initial planar shape) to an amorphous flat shape centered on the substantially circular shape, and the second mesenchymal stem cell 35 is the second. 2 After confirming the fixation of the flat culture container 33 (third culture container) to the bottom surface 37, the second observation step of the total plane area is performed.

The culture solution 36 injected into the second flat culture container 33 is discharged from the culture container 33, and a new culture solution 36 is injected (accommodated) into the culture container 33. Next, the second flat culture vessel 33 is statically left for 36 to 48 hours at a temperature substantially the same as the body temperature (about 35 to 37 ° C.) (leaving quietly without moving) to obtain the second mesenchymal stem cells 35. Display 17 (electron) shows the total plane area of the second mesenchymal stem cells 35 settled on the bottom surface 37 of the culture vessel 33 with respect to the bottom surface area of the culture vessel 33 at intervals of about 1 to 2 hours while culturing. Observe with a microscope) to determine whether the total planar area of the second mesenchymal stem cells 35 has reached the second target ratio with respect to the bottom area of the second flat culture vessel 33 (total planar area second observation). Process). The second target ratio of the total planar area of the second mesenchymal stem cell 35 to the bottom surface area of the second flat culture vessel 33 is 88 to 92% (88 to 92% confluent).

In the second observation step of the total plane area, the culture solution 36 injected into the second flat culture container 33 is discharged from the culture container 33, the new culture solution 36 is injected (accommodated) into the culture container 33, and the new culture solution 36 is used. The culture container 33 in which the mixture was injected 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 in a state of being lifted by the spacer 30 to hold the culture vessel 33. The second flat culture vessel 33 is held in a state of being tilted at a predetermined angle so that the bottom portion 40 of the culture vessel 33 is on the top and the top 41 (injection port 38) of the culture vessel 33 is on the bottom. The inclination 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 in the range of 2 to 3 °.

The electron microscope 13 took an enlarged image of the planar shape of the second mesenchymal stem cell 35 in the second flat culture vessel 33 at intervals of about 1 to 2 hours, and took an image of the planar shape of the second mesenchymal stem cell 35. The enlarged image is transmitted to the management server 11 at intervals of about 1 to 2 hours. The management server 11 stores the enlarged image of the planar shape of the second mesenchymal stem cell 35 transmitted from the electron microscope 13 and the imaging time in the storage area in a state of being associated with the IC tag specific information (IC tag identifier) and the donor identifier. Store (remember). The management server 11 outputs (displays) the enlarged image of the planar shape of the second mesenchymal stem cell 35 transmitted from the electron microscope 13 and the photographing time on the display 17.

The person in charge (doctor, nurse, researcher, biotechnologist, etc.) displays a magnified image of the planar shape of the second mesenchymal stem cell 35 displayed on the display 17 for about 1 to 2 hours between 36 and 48 hours. The second mesenchymal stem cell 35 while observing the total plane area of the second mesenchymal stem cell 35 with respect to the bottom surface area of the culture vessel 33, which was confirmed (visually) at intervals and settled on the bottom surface 37 of the second flat culture vessel 33. It is determined whether or not the total plane area of the culture vessel 33 has reached the second target ratio (70 to 80% confluent) with respect to the bottom surface area of the culture vessel 33.

The second mesenchymal stem cell 35 proliferates on the bottom surface 37 (inner surface of the bottom wall) of the second flat culture vessel 33 to form a colony (stem cell second culture step). The second mesenchymal stem cell 35 formed a colony, the planar shape of the second mesenchymal stem cell 35 expanded, and the total planar area was observed in the second observation step. As a result, the second mesenchymal stem cell displayed on the display 17 was displayed. When the total plane area of the stem cells 35 with respect to the bottom surface area of the second flat culture vessel 33 reaches the second target ratio (70 to 80% confluent), the second mesenchymal stem cells are extracted from the culture vessel 33.

Total plane area injected into the second flat culture vessel 33 The culture solution 36 in the second observation step is discharged from the culture vessel 33, the culture vessel 33 is washed with phosphate buffered physiological saline (PBS), and then trypsin is used. The liquid is poured into the culture vessel 33. When the trypsin solution is injected into the second flat culture vessel 33, the second mesenchymal stem cells 35 established on the bottom surface 37 of the culture vessel 33 are detached from the bottom surface 37 by the trypsin solution and float on the water surface of the trypsin solution. The person in charge sucks the surfaced second mesenchymal stem cell 35 using a pipette (second mesenchymal stem cell second extraction step).

The culture medium 36 discharged from the second flat culture vessel 33 (third culture vessel) is the culture medium 36 remaining after extracting the second mesenchymal stem cells 35 from the culture vessel 33, and is the second mesenchymal. In the process of culturing the lineage stem cell 35, it is changed to the second culture product solution 42 (culture product solution) containing a predetermined metabolite secreted from the second mesenchymal stem cell 35. The second culture product 42 containing the metabolite activates mesenchymal stem cells with reduced activity. The second culture product 42 is injected into a freezing storage container 43 to which an IC tag 18 is attached, quickly frozen by a quick freezer, and then stored in a freezing storage container 43 in a refrigerator 14 or a freezer 14. It is stored at a predetermined temperature (chilled storage at 0 to 5 ° C or frozen storage at 0 ° C or lower) for a period of time.

In the method for modifying frozen stem cells, the first mesenchymal stem cells 21 are fixed on the bottom surface 23 of the first flat culture vessel 22 (second culture vessel), and then the culture solution 26 is discharged from the culture vessel 22 while being newly discharged. The culture solution 26 was injected into the culture vessel 22, and the first mesenchymal stem cells 21 established on the bottom surface 23 of the culture vessel 22 were cultured using the new culture solution 26, and the second mesenchymal stem cells 21 were grown in the culture vessel 22. The mesenchymal stem cells 35 and the culture medium 36 are housed in the second flat culture vessel 33 (third culture vessel), the second mesenchymal stem cells 35 are fixed on the bottom surface 37 of the culture vessel 33, and then the culture medium 36 is added. While draining from the culture vessel 33, the new culture medium 36 is injected into the culture vessel 33, and the second mesenchymal stem cells 35 colonized on the bottom surface 37 of the culture vessel 33 are cultured using the new culture medium 36. Since the second mesenchymal stem cell 35 is proliferated in the culture vessel 33, the mesenchymal stem cell 35 can be proliferated efficiently and surely, and the mesenchymal stem cell 35 having sufficient activity can be efficiently produced. ..

FIG. 9 is an explanatory diagram showing an example of a freezing storage step in the frozen stem cell modifying method, and FIG. 10 is an explanatory diagram showing an example of a stem cell thawing step in the frozen stem cell modifying method. FIG. 11 is an explanatory diagram showing an example of the stirring and removing step in the frozen stem cell reforming method, and FIG. 12 is an explanatory diagram showing an example of the mesenchymal stem cell extraction step in the frozen stem cell reforming method. FIG. 13 is an explanatory diagram showing an example of a stem cell activation step in the frozen stem cell reforming 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 the second mesenchymal stem cell 35, and FIG. 16 is a partially enlarged view showing another example of the planar shape of the second mesenchymal stem cell 35. FIG. 17 is an explanatory diagram showing an example of a stem cell transport step in the frozen stem cell reforming method. FIGS. 15 and 16 show enlarged images of the planar shape of the second mesenchymal stem cell 35 taken by the electron microscope 13.

In the frozen stem cell modification method, the second mesenchymal stem cells 35 (mesenchymal stem cells) cultured by the stem cell culture method are cryopreserved by carrying out a cryopreservation step, and the second mesenchymal stem cells after the cryopreservation. 35 (mesenchymal stem cells) is modified into activated mesenchymal stem cells 55 by carrying out a stem cell thawing step, a stirring removal step, a mesenchymal stem cell extraction step, a stem cell activation step, and a stem cell transport step.

The person in charge (doctor, nurse, researcher, etc.) injects (contains) the second mesenchymal stem cell 35 (cultured mesenchymal stem cell) from a pipette into a cryopreservation container 43 having a predetermined volume. The second mesenchymal stem cell 35 injected into the cryopreservation vessel 43 is a pure mesenchymal stem cell of a specific type (substantially a single species) to be cultured, which has an activity in which unnecessary mesenchymal stem cells are removed. An IC tag 18 in which donor data and stem cell data are stored is attached to the outer peripheral surface of the freezing storage container 43.

The cryoprotectant 44 (at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG)) is injected into the freezing storage container 43. The freezing storage container 43 containing the second mesenchymal stem cells 35 and the cryoprotectant 44 is rapidly frozen by a quick freezer (not shown). As shown in FIG. 9, the quick-frozen second mesenchymal stem cell 35 is kept in the freezing storage container 43 and kept in the refrigerator 14 or the freezer 14 for a predetermined period of time until it is used. It is stored in (chilled storage at 0 to 5 ° C. or frozen storage at 0 ° C. or lower) (freezing storage step).

When using the second mesenchymal stem cell 35 frozen and stored in the refrigerator 14 or the freezer 14 (when using the mesenchymal stem cell 35 frozen and stored by the freezing storage step), the temperature control is as shown in FIG. Prepare a heat insulating container 45 (constant temperature bath) having a predetermined capacity, soak (immerse) the frozen storage container 43 in the warm water of the heat insulating container 45 for 2 to 3 minutes, and soak the second mesenchymal stem cells 35 in 2 to 3 Thaw to a temperature of ° C (stem cell thaw step). In the heat insulating container 45, the temperature of the hot water is maintained at 35 to 37 ° C. The frozen storage container 43 containing the first culture product liquid 32 and the second culture product liquid 42 is immersed (immersed) in the warm water of the heat insulating container 45 for 2 to 3 minutes, and the first and second culture product liquid 32, Thaw 42 to a temperature of 2-3 ° C.

Next, the freezing storage container 43 was taken out from the heat insulating container 45, and the washing liquid 46 (Dalbecco phosphate buffered saline) was placed in the freezing storage container 43 containing the second mesenchymal stem cells 35 thawed from the frozen state and the cryoprotectant 44. (D-PBS) or phosphate buffered saline (PBS)) is infused. After injecting the cleaning liquid 46 into the frozen storage container 43, as shown in FIG. 11, the frozen storage container 43 in which the cleaning liquid 46 is injected is shaken left and right to stir the frozen storage container 43, and the cleaning liquid 46 (Dalbecco phosphate buffered saline) is stirred. The cryoprotectant 44 is removed (separated) from the second mesenchymal stem cells 35 in the cryopreservation vessel 43 with water (D-PBS) or phosphate buffered saline (PBS) (stirring removal step).

The person in charge (doctor, nurse, researcher, etc.) removes (separates) the cryoprotectant 44 from the second mesenchymal stem cell 35 by a stirring removal step, and then removes (separates) the second mesenchymal stem cell 35 and the mesenchymal stem cell. A cryopreservation container 43 containing a mixed solution containing the freeze-protecting agent 44 and the cleaning solution 46 separated from 35 is installed (set) in the centrifuge 47. The mixture is centrifuged by a centrifuge 47 for a predetermined time, and the second mesenchymal stem cells 35, the freeze protectant 44, and the washing solution 46 in the cryopreservation container 43 are centrifuged in layers. The mixed solution in the freezing storage container 43 is centrifuged in two layers in the vertical direction.

After separating the mixed solution into two layers, the person in charge pipettes the second mesenchymal stem cells 35 located in the lower layer (bottom layer) from the layered mixed solution as shown in FIG. 12 (Fig. 12). Extraction (not shown) or extraction (suction) using a pipette (not shown) Extraction (mesenchymal stem cell extraction step). The second mesenchymal stem cell 35 extracted by the mesenchymal stem cell extraction step is a cryoprotectant 44 (hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG)) injected at the time of freezing. At least dimethyl sulfoxide (DMSO)) has been removed (separated).

After extracting a specific second mesenchymal stem cell 35 located in the lower layer (bottom layer) from the mixture, a stem cell activation step is performed. In the stem cell activation step, the washed second mesenchymal stem cells 35, the culture solution 48, the first culture product 32 and / or the second culture product 42 are placed in a third flat culture vessel 49 (first culture vessel). Inject (contain) into. The first culture product 32 injected into the third flat culture vessel 49 is the first in the process of culturing the first mesenchymal stem cell 21 which is the source of the thawed second mesenchymal stem cell 35 (mesenchymal stem cell). The second culture product 42, which is a culture product containing a predetermined metabolite secreted from the first mesenchymal stem cell 21 and is injected into the third flat culture vessel 49, is the thawed second mesenchymal stem cell 35. It is a culture product solution containing a predetermined metabolite secreted from the second mesenchymal stem cell 35 in the culture process of (mesenchymal stem cell). The injection ratio of the culture product liquids 32 and 42 to be injected into the third flat culture vessel 49 is 5 to 15%, preferably 5 to 15% when the total injection amount of the culture solution 48 to be injected into the culture vessel 49 is 100%. It is 8 to 12%, more preferably 10%.

The third flat culture container 49 (first culture container) is a flat container made of transparent glass or transparent plastic, having a small capacity and a bottom surface having a predetermined area, and having a substantially regular square shape. The injection port 50 of the third flat culture vessel 49 is watertightly closed by the lid 51. The volume of the third flat culture vessel 49 is about 20 to 30 cc (preferably 25 cc), the bottom surface area thereof is about 25 to 36 mm ² , and the length of one side thereof is 5 to 6 mm. As the third flat culture container 49, a flat container having a small capacity and a bottom surface having a predetermined area and having a circular or elliptical planar shape can also be used. An IC tag 18 in 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.

After accommodating the washed second mesenchymal stem cells 35, culture solution 48, first culture product solution 32 and / or second culture product solution 42, the third flat culture vessel 49 is placed in the sample holder 27 of the electron microscope 13. Install (set). 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 third flat culture vessel 49, and the bottom 53 of the culture vessel 49 is held in a state of being lifted by the spacer 30 to hold the culture vessel 49. The culture vessel 49 is held in a state of being tilted at a predetermined angle (tilted in one direction) so that the bottom portion 53 of the culture vessel 49 is on the top and the top 54 (injection port 50) of the culture vessel 49 is on the bottom. The top 54 of the third flat culture vessel 49 is held in a lifted state by the spacer 30, and the culture vessel 49 is set at a predetermined angle so that the top 54 of the culture vessel 49 is on the top and the bottom 53 of the culture vessel 49 is on the bottom. It may be held in a state of being tilted (tilted in one direction). The unidirectional tilt angle α3 of the third flat culture vessel 49 with respect to the upper surface 28 of the sample holder 27 is in the range of 2 to 5 °, preferably in the range of 2 to 3 °.

After the third flat culture vessel 49 is placed (set) on the sample holder 27 of the electron microscope 13 in an inclined state, the second mesenchymal stem cell 35, the culture medium 48, the first culture product 32 and / or the second. The third flat culture vessel 49 containing the culture product 42 was statically left for 24 hours at substantially the same temperature as the body temperature (about 35 to 37 ° C.) (leave quietly without moving), and the second mesenchymal stem cells. 35 (mesenchymal stem cells) are cultured in a third flat culture vessel 49 (first culture vessel) at 36 to 37 ° C. for 24 hours.

When 12 hours have passed after the third flat culture vessel 49 was placed (set) in the sample holder 27 of the electron microscope 13, it was 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. The spacer 30 was removed, the spacer 30 was 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, and the top 54 of the culture vessel 49 was held in a lifted state by the spacer 30. Then, the culture vessel 49 is held in a state of being tilted at a predetermined angle (tilted in the other direction) so that the top 54 of the culture vessel 49 is on the top and the bottom 53 of the culture vessel 49 is on the bottom. When the top 54 of the third flat culture vessel 49 is at the top and the bottom 53 of the culture vessel 49 is at the bottom, the spacer 30 is placed on the upper surface 28 of the sample holder 27 of the electron microscope 13 and the culture vessel 49. The bottom 53 of the culture vessel 49 is held in a state of being lifted by the spacer 30 so that the bottom 53 of the culture vessel 49 is on the top and the top 54 of the culture vessel 49 is on the bottom. The container 49 is held in a state of being tilted at a predetermined angle (tilted in the other direction).

The inclination angle α3 of the third flat culture vessel 49 with respect to the upper surface 28 of the sample holder 27 in the other direction is in the range of 2 to 5 °, preferably in the range of 2 to 3 °. In addition, in a state where the third flat culture vessel 49 is continuously tilted in one direction (or the other direction), it is statically left for 24 hours at a temperature (about 35 to 37 ° C) substantially equal to the body temperature (quiet without moving). In some cases, the second mesenchymal stem cells 35 (mesenchymal stem cells) are cultured in a third flat culture vessel 49 (first culture vessel) at 36 to 37 ° C. for 24 hours.

By tilting the third flat culture vessel 49 in one direction and the other direction at the tilt angle with respect to the upper surface 28 of the sample holder 27, the second mesenchymal stem cells 35 (mesenchymal stem cells) in the culture vessel 49. The culture solution 48 and the culture product solutions 32 and 42 are biased toward the top 54 side (or bottom 53 side) of the culture vessel 49, and the second mesenchymal system is located on the top 54 side (or bottom 53 side) of the culture vessel 49. The water pressure of the stem cells 35, the culture medium 48, and the culture product 32, 42 increases, and the second mesenchymal stem cells 35 concentrate on the side of the top 54 (or the side of the bottom 53) of the culture vessel 49, thereby the second. The activity of the mesenchymal stem cells 35 is increased, and the second mesenchymal stem cell 35 can be easily and quickly activated (fixed) on the bottom surface 52 of the culture vessel 49.

The electron microscope 13 took a magnified image of the planar shape of the second mesenchymal stem cell 35 injected into the third flat culture vessel 49 at intervals of about 1 to 2 hours, and photographed the planar surface of the second mesenchymal stem cell 35. An enlarged image of the shape is transmitted to the management server 11 at intervals of about 1 to 2 hours. The management server 11 stores the enlarged image of the planar shape of the second mesenchymal stem cell 35 transmitted from the electron microscope 13 and the imaging time in the storage area in a state of being associated with the IC tag specific information (IC tag identifier) and the donor identifier. Store (remember). The management server 11 outputs (displays) the enlarged image of the planar shape of the second mesenchymal stem cell 35 transmitted from the electron microscope 13 and the photographing time on the display 17. The person in charge (doctor, nurse, researcher, biotechnologist, etc.) displays an enlarged image of the planar shape of the second mesenchymal stem cell 35 displayed on the display 17 for about 1 to 2 hours in 12 to 24 hours. Check (visually) at intervals, observe changes in the planar shape of the second mesenchymal stem cells 35, and check whether the second mesenchymal stem cells 35 were activated (whether they settled on the bottom surface 52 of the third flat culture vessel 49). ) Whether or not (shape deformation third observation step).

When the planar shape of the second mesenchymal stem cell 35 after thawing is substantially circular, and the planar shape of the second mesenchymal stem cell 35 is substantially circular, the second mesenchymal stem cell 35 is not activated (second mesenchymal stem cell 35). 3 Not established on the bottom surface 52 (inner surface of the bottom wall) of the flat culture vessel 49), and the second mesenchymal stem cell 35 has not started activation. The deformed planar shape of the second mesenchymal stem cell 35 is a flat shape in which the second mesenchymal stem cell 35 is irregularly extended (expanded) in one direction (predetermined direction) with the substantially circular shape before colonization as the nucleus. The second mesenchymal stem cell 35 settles on the bottom surface 52 (inner surface of the bottom wall) of the third flat culture vessel 49, the second mesenchymal stem cell 35 is activated, and the second in the culture vessel 49 (in the first culture vessel). 2 Mesenchymal stem cells 35 (mesenchymal stem cells) are modified into activated mesenchymal stem cells.

As a result of the observation in the shape deformation third observation step, the person in charge is in the second case where the planar shape of the second mesenchymal stem cell 35 displayed on the display 17 is observed in a substantially circular shape as shown in FIG. It was judged that the mesenchymal stem cells 35 had not settled on the bottom surface 52 (inner surface of the bottom wall) of the third flat culture vessel 49, and the change in the planar shape of the second mesenchymal stem cells 35 was continued at intervals of about 1 to 2 hours. And observe. As a result of observation in the third observation step of shape deformation, the person in charge said that the planar shape of the second mesenchymal stem cell 35 output (displayed) on the display 17 is substantially circular to substantially circular as a nucleus, as shown in FIG. When transformed into an amorphous flat shape, the second mesenchymal stem cell 35 is activated (established on the bottom surface of the third flat culture vessel), and the second mesenchymal stem cell 35 is changed to the activated mesenchymal stem cell 55. Judged as quality (stem cell activation step).

After determining that the second mesenchymal stem cell 35 was modified to the activated mesenchymal stem cell 55, the culture medium 48 and the culture product 32, 42 injected into the third flat culture vessel 49 were transferred from the culture vessel 49. After draining and washing the culture vessel 49 with phosphate buffered physiological saline (PBS), the 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 established on the bottom surface 52 of the culture vessel 49 are detached from the bottom surface 52 by the trypsin solution and float on the water surface of the trypsin solution. The person in charge sucks the surfaced 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 activates the activated mesenchymal stem cells 55. The mesenchymal stem cells 55 are housed in the storage container 56.

When the activated mesenchymal stem cells 55 are used within 18 to 20 hours after extraction, the person in charge can carry the storage container 56 containing the activated mesenchymal stem cells 55 as shown in FIG. 17 in a constant temperature case. Activated mesenchymal within 18 to 20 hours while housed in 57, the temperature inside the constant temperature case 57 is maintained at 20 to 21 ° C, and the extracted activated mesenchymal stem cells 55 are maintained at 20 to 21 ° C. Transport the line stem cells 55 to the place of use (stem cell transport step). The activated mesenchymal stem cells 55 transported to the place of use are taken out from the constant temperature case 57 and are non-therapeutic such as treatment of various diseases (cardiovascular disease, central nervous system disease, etc.), regeneration in regenerative medicine, beauty, etc. Used for applications.

Frozen stem cell reforming method (frozen stem cell reforming system 10) includes a second mesenchymal stem cell 35 (medium-lobed stem cell) thawed from a frozen state, a cryoprotectant 44 (hydroxyethyl starch (HES), and dimethyl sulfoxide (". Washing solution 46 (Dalbeco phosphate buffered physiological saline (D-PBS) or phosphate buffered physiological saline (D-PBS) or phosphate buffered physiological saline (D-PBS) in a cryopreservation container 43 containing at least dimethyl sulfoxide (DMSO) of DMSO) and ethylene glycol (EG). PBS))) is injected, the frozen storage container 43 in which the washing liquid 46 is injected is stirred, and the cryoprotectant 44 is removed (separated) from the second mesenchymal stem cells 35 in the frozen storage container 43 by the washing liquid 46, and frozen storage is performed. The second mesenchymal stem cells 35, the cryoprotectant 44, and the washing solution 46 in the container 43 are centrifuged in a layered manner, and after the centrifugation, the second mesenchymal stem cells 35 located in the lowest layer of the cryopreservation container 43 are extracted. Therefore, when the frozen-stored second mesenchymal stem cell 35 is thawed, the cryoprotectant 44 can be removed by using the washing solution 46, and the second mesenchymal stem cell 35 thawed from the frozen state can be removed. Most (70-90%) of the disease can survive.

In the frozen stem cell reforming method (frozen stem cell reforming system 10), a third mesenchymal stem cell 35 (mesenchymal stem cell) thawed from a frozen state has a predetermined volume and a bottom surface of a predetermined area, and is a third flat culture vessel 49. The second mesenchymal stem cell 35 is cultured by injecting the predetermined culture solution 48 and the culture product 32, 42 into the culture vessel 49 containing the second mesenchymal stem cell 35 while being housed in (the first culture container). Since the second mesenchymal stem cells 35 in the culture vessel 49 are activated by culturing in the vessel 49 at 35 to 37 ° C. for 24 hours, the second mesenchymal stem cells 35 that have been cryopreserved are thawed when they are thawed. The second mesenchymal stem cell 35 can be modified into an activated mesenchymal stem cell 55 having sufficient activity.

The frozen stem cell reforming method (frozen stem cell reforming system 10) has sufficient and high effects in the treatment of various diseases (cardiovascular disease, central nervous system disease, etc.), regeneration in regenerative medicine, and non-therapeutic applications such as beauty. Activated mesenchymal stem cells 55 can be produced, and activated mesenchymal stem cells 55 capable of obtaining a desired effect can be produced. The frozen stem cell reforming method (frozen stem cell reforming system 10) can produce activated mesenchymal stem cells 55 suitable for the treatment of various diseases and can be used in a timely manner, and can produce various tissues and organs. Activated mesenchymal stem cells 55 suitable for regeneration and can be used in a timely manner can be produced, and activated mesenchymal cells suitable for non-therapeutic applications such as beauty and can be used in a timely manner. Mesenchymal stem cells 55 can be made. In the frozen stem cell reforming method (frozen stem cell reforming system 10), since the activated activated mesenchymal stem cells 55 do not contain the cryoprotectant 44, the activated interval produced by the frozen stem cell reforming method Even if the mesenchymal stem cells 55 are administered to the human body, there is no adverse effect on the human body.

In the frozen stem cell reforming method (frozen stem cell reforming system 10), after the culture products 32 and 42 are cultured from the first flat culture vessel 22 (second culture vessel) or the second flat culture vessel 33 (third culture vessel). It is a culture solution remaining after extracting the first and second mesenchymal stem cells 21, 35 (mesenchymal stem cells), and is secreted from the mesenchymal stem cells 21, 35 in the process of culturing the mesenchymal stem cells 21, 35. Since it contains the predetermined metabolites, the metabolites of the mesenchymal stem cells 21 and 35 themselves are the triggers, the second mesenchymal stem cells 35 rapidly start their activity, and the culture products 32 and 42 are used. Therefore, the second mesenchymal stem cell 35 can be rapidly cultured, and the activated mesenchymal stem cell 55 can be efficiently produced in a short time.

The frozen stem cell reforming method (frozen stem cell reforming system 10) maintained the activated mesenchymal stem cells 55 at 20 to 21 ° C. when the activated mesenchymal stem cells 55 were used within 18 to 20 hours after extraction. By transporting the activated mesenchymal stem cells 55 to the place of use within 18 to 20 hours in the state, the activity of the activated mesenchymal stem cells 55 is not lost, and the activated mesenchymal stem cells 55 are caused by various diseases (heart). It can be used for the treatment of vascular diseases, central nervous system diseases, etc.), its activated mesenchymal stem cells 55 can be used for regeneration in regenerative medicine, and its activated mesenchymal stem cells 55 can be used for beauty, etc. Can be used for non-therapeutic applications.

10 Frozen stem cell reforming system 11 Management server 12 IC tag reader / writer 13 Electron microscope 14 Refrigerator or freezer 15 Keyboard 16 Mouse 17 Display 18 IC tag 19 1st bone marrow fluid 20 Glass test tube 21 2nd mesenchymal fluid (1st mesenchymal system) Stem cells)
22 1st flat culture vessel (2nd culture vessel)
23 Bottom 24 Injection 25 Lid 26 Culture solution 27 Sample holder 28 Top surface 29 Bottom 30 Spacer 31 Top 32 1st culture product 33 2nd flat culture container (3rd culture container)
34 Glass test tube 35 Second mesenchymal stem cell 36 Culture solution 37 Bottom 38 Injection port 39 Lid 40 Bottom 41 Top 42 Second culture product 43 Freezing storage container 44 Freezing protection agent 45 Insulation container 46 Cleaning agent 47 Centrifugal separator 48 Culture solution 49 3rd flat culture container (1st culture container)
50 Injection 51 Lid 52 Bottom 53 Bottom 54 Top 55 Activated mesenchymal stem cells 56 Storage container 57 Constant temperature case

Claims (9)

In a frozen stem cell reforming method in which mesenchymal stem cells frozen to a predetermined temperature are activated and reformed into activated mesenchymal stem cells.
In the frozen stem cell reforming method, the mesenchymal stem cells prepared by culturing the bone marrow fluid collected from the donor are housed in a cryopreservation container having a predetermined volume, and the mesenchymal stem cells are housed in the frozen storage container. Is injected with a cryoprotectant, and the mesenchymal stem cells housed in the cryopreservation vessel are cryopreserved at a predetermined temperature together with the cryoprotectant until the mesenchymal stem cells housed in the cryopreservation container are used. Freezing storage process and
When the mesenchymal stem cells cryopreserved by the cryopreservation step are used, the mesenchymal stem cells cryopreserved and the cryopreservation container containing the cryopreservative are kept in warm water at 35 to 37 ° C. Stem cell thawing step of thawing the mesenchymal stem cells to a temperature of 2 to 3 ° C.
The cleaning solution is injected into the freezing storage container containing the mesenchymal stem cells thawed from the frozen state by the stem cell thawing step and the freezing protective agent, and the frozen storage container in which the cleaning solution is injected is stirred by the cleaning solution. A stirring removal step of removing the cryoprotectant from the mesenchymal stem cells in the freezing storage container, and
After removing the cryoprotectant from the mesenchymal stem cells by the stirring removal step, the cryoprotector is placed in a centrifuge, and the mesenchymal stem cells, the cryoprotectant, and the washing solution in the cryopreservation vessel are placed. A step of extracting mesenchymal stem cells by centrifuging in layers and extracting mesenchymal stem cells located at the bottom layer of the cryopreservation vessel after the centrifugation.
The mesenchymal stem cells extracted by the mesenchymal stem cell extraction step are housed in a first culture vessel having a predetermined volume and a bottom surface having a predetermined area, and a predetermined culture is performed in the first culture vessel containing the mesenchymal stem cells. The solution is injected, the mesenchymal stem cells are cultured in the first culture vessel at 36 to 37 ° C. for 24 hours, and the mesenchymal stem cells in the first culture vessel are activated to activate the mesenchymal stem cells. A method for modifying frozen stem cells, which comprises a stem cell activation step of modifying stem cells. When the frozen stem cell reforming method is used within 18 to 20 hours after the activated activated mesenchymal stem cells are extracted from the first culture vessel and then the activated mesenchymal stem cells are extracted. The freezing according to claim 1, further comprising a stem cell transport step of transporting the extracted activated mesenchymal stem cells to the site where the activated mesenchymal stem cells are used within 18 to 20 hours while holding the activated mesenchymal stem cells at 20 to 21 ° C. Stem cell modification method. In the stem cell activation step, the culture product solution generated in the culture process of culturing the bone marrow fluid collected from the donor is injected into the first culture vessel, and the culture solution and the culture product solution are used during the above period. The method for modifying frozen stem cells according to claim 1 or 2, wherein the leaf stem cells are cultured in the first culture vessel at a temperature of 35 to 37 ° C. for 24 hours. Claims 1 to claim that the stem cell activation step statically leaves the first culture vessel 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. Item 3. The method for modifying frozen stem cells according to any one of. In the stem cell activation step, the first culture vessel was statically left at a temperature of 35 to 37 ° C. for 12 hours with the first culture vessel tilted at an angle of 2 to 5 degrees in one direction. The first culture vessel is tilted in one direction at an angle of 2 to 5 degrees in the opposite direction to the other direction, and the first culture vessel is statically tilted at a temperature of 35 to 37 ° C. for 12 hours. The method for modifying frozen stem cells according to claim 4, which is left in the container. The cryoprotectant is at least the dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG), and the cleaning solution is Dulbecco phosphate buffered saline (Dulbecco phosphate buffered saline (DMSO). The method for modifying frozen stem cells according to any one of claims 1 to 5, which is D-PBS) or phosphate buffered saline (PBS). The mesenchymal stem cells separate the mesenchymal stem cells collected from the donor into layers, and extract the mesenchymal stem cells located in the middle layer of the layered separated bone cells, and have a predetermined volume and a predetermined area. A stem cell number that injects the mesenchymal stem cell and a predetermined culture cell into a second culture vessel having a bottom surface and establishes the first mesenchymal stem cells contained in the middle layer mesenchymal stem cells on the bottom surface of the second culture vessel. After the first mesenchymal stem cells are settled on the bottom surface of the second culture vessel by one colonization step and the first stem cell colonization step, a new culture medium is discharged while discharging the culture medium in the second culture vessel. Is injected into the second culture vessel to culture the first mesenchymal stem cells, and the total plane area of the first mesenchymal stem cells with respect to the bottom surface area of the second culture vessel reaches the first target ratio. A second culture vessel containing the first mesenchymal stem cells and the culture solution grown by the first mesenchymal stem cell culture step and the first mesenchymal stem cell culture step is placed in a centrifuge. Then, the first mesenchymal stem cells in the second culture vessel and the culture solution are centrifuged in layers, and the second mesenchymal stem cells located in the lowest layer of the second culture vessel after the centrifugation. The second mesenchymal stem cell first extraction step, and the second mesenchymal stem cell first in a third culture vessel having a predetermined volume and a bottom surface of a predetermined area and a capacity larger than that of the second culture vessel. While accommodating the second mesenchymal stem cells extracted by the extraction step, a new culture solution is injected into the third culture vessel, and the second mesenchymal stem cells are fixed on the bottom surface of the third culture vessel. After the second mesenchymal stem cells are settled on the bottom surface of the third culture vessel by the two colonization step and the second stem cell colonization step, a new culture medium is discharged while draining the culture medium in the third culture vessel. Is injected into the third culture vessel to culture the second mesenchymal stem cells, and the total plane area of the second mesenchymal stem cells with respect to the bottom surface area of the third culture vessel reaches the second target ratio. The third culture vessel containing the second mesenchymal stem cells cultured by the second stem cell culture step and the second mesenchymal stem cell culture step for proliferating the second stem cells is placed in a centrifuge, and the third culture vessel is provided. The second mesenchymal stem cells and the culture solution are centrifuged in a layered manner, and the second mesenchymal stem cells located in the lowermost layer of the third culture vessel after the centrifugation are extracted. The frozen stem cell modification method according to any one of claims 1 to 6, which is produced by a mesenchymal stem cell culture method having a mesenchymal stem cell second extraction step. The mesenchymal system in which the second mesenchymal stem cells immediately after being extracted by the second extraction step of the second mesenchymal stem cells in the cryopreservation step are housed in the frozen storage container and housed in the frozen storage container. The method for modifying frozen stem cells according to claim 7, wherein the stem cells are immediately cryopreserved together with the cryoprotectant. The culture product is a culture solution remaining after extracting the mesenchymal stem cells after culturing from the second culture vessel or the third culture vessel, and the mesenchymal stem cells are in the process of culturing the mesenchymal stem cells. The method for modifying frozen stem cells according to any one of claims 3 to 8, which comprises a predetermined metabolite secreted from.

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