WO2017188370A1

WO2017188370A1 - Method for producing activated stem cells

Info

Publication number: WO2017188370A1
Application number: PCT/JP2017/016702
Authority: WO
Inventors: アレクセイグラドコフ
Original assignee: 株式会社ＣｅｌｌｓＰｏｗｅｒ
Priority date: 2016-04-27
Filing date: 2017-04-27
Publication date: 2017-11-02
Also published as: MY186453A

Abstract

[Problem] To provide a method for producing activated stem cells with which it is possible to efficiently grow a single type of dormant stem cells in a dormant state and to produce the necessary amount of a single type of activated stem cells having adequate activity. [Solution] The method for producing activated stem cells has a dormant stem cell fixation step that injects a cultured product solution produced in the course of culturing a single type of dormant stem cells, a predetermined culture solution, and a single type of stem cells prior to storage of the dormant stem cells into a first culture vessel having a predetermined volume and a bottom surface of a predetermined area and fixes the dormant stem cells to the bottom surface of the first culture vessel, and a dormant stem cell culture step that cultures the dormant stem cells fixed to the bottom surface of the first culture vessel by the dormant stem cell fixation step, grows and activates the dormant stem cells until the total planar area of the dormant stem cells relative to the bottom surface area of the first culture vessel reaches a first target ratio, and changes the dormant stem cells into a single type of activated stem cells.

Description

Method for producing activated stem cells

The present invention activates a single type of dormant stem cell in a dormant state after storing a single type of stem cell produced by culturing bone marrow fluid collected from a donor for a predetermined period of time, thereby producing a single type of activated stem cell. The present invention relates to a method for producing activated stem cells.

A medium for culturing stem cells, and the irradiation energy for stem cells exceeds 0 and is less than 10 joules / cm ² , the laser power density is not more than 0.1 W / cm ² , and the irradiation energy is not less than 0.1 and not more than 2.5 joules / cm ² After defocusing the irradiation light of the following low-power carbon dioxide laser and irradiating the whole medium, and equipped with a laser irradiation means to activate the stem cells, after activating the stem cells by irradiating the low-power laser, A stem cell culturing method is disclosed in which a predetermined rest period is provided for a stem cell to proliferate to a target proliferation number. According to this stem cell culture method, tissue collected from humans and non-humans (animals) or stem cells present in the cells can be activated and proliferated dramatically.

Japanese Patent Laying-Open No. 2015-186465

Stem cells grown by the stem cell culture method or other stem cell culture methods are stored in a refrigerator or freezer, stored at a predetermined temperature for a predetermined period, and taken out from the refrigerator or freezer as needed. Dormant dormant stem cells stored in a refrigerator or freezer have low activity and need to activate dormant stem cells, but only part of the dormant stem cells survive during the activation process, and the rest of dormant stem cells die. End up. Since the growth of dormant stem cells that survive is blocked by dead dormant stem cells, dormant stem cells cannot be efficiently propagated, and it is possible to produce the necessary amount of activated stem cells that are fully activated from the dormant stem cells after thawing Can not.

Since various stem cells exist in the tissue or cells collected from the donor, even if the stem cells present in the tissue or cells are cultured, the various stem cells proliferate and culture only a specific type of stem cell. It is impossible to produce a single type of activated stem cell obtained by proliferating and activating a single type of dormant stem cell. Activated stem cells are used for the treatment of various diseases (cardiovascular diseases, central nervous system diseases, etc.), regenerative medicine, and non-therapeutic applications. Compared to the case of only one kind of activated stem cells, the effect of treatment for various diseases and the effect of regeneration in regenerative medicine are small.

An object of the present invention is to provide an activated stem cell production method capable of efficiently proliferating a single type of dormant stem cells in a dormant state and capable of producing a necessary amount of a single type of activated stem cell having sufficient activity. It is to provide. Another object of the present invention is to activate a single kind of activated stem cell that does not contain various kinds of activated stem cells and has a large therapeutic effect on various diseases and a large regenerative effect in regenerative medicine. It is to provide a method for producing stem cells.

The premise of the present invention to solve the above problems is to activate a single type of dormant stem cells in a dormant state after storing a single type of stem cells made from culturing bone marrow fluid collected from a donor for a predetermined period of time An activated stem cell production method for producing a single type of activated stem cell.

The feature of the present invention based on the above premise is that the method for producing activated stem cells predetermines a single type of dormant stem cells, a predetermined culture solution, and a culture product generated in the culture process of the single type of stem cells before storage of the dormant stem cells. A dormant stem cell fixing step for injecting a dormant stem cell onto the bottom surface of the first culture vessel by injecting the first culture vessel having a volume and a bottom surface of a predetermined area, and a dormancy fixed on the bottom surface of the first culture vessel by the dormant stem cell fixing step A dormant stem cell that cultivates a stem cell, proliferates and activates the dormant stem cell until the total planar area of the dormant stem cell with respect to the bottom surface area of the first culture container reaches the first target ratio, and transforms the dormant stem cell into a single type of activated stem cell A culture process.

As an example of the present invention, in the dormant stem cell culture step, the dormant stem cell is fixed on the bottom surface of the first culture vessel by the dormant stem cell fixing step, and then the mixed culture solution of the culture solution and the culture product solution is discharged from the first culture vessel. However, a new culture solution and a new culture product solution are poured into the first culture vessel, and the dormant stem cells fixed on the bottom surface of the first culture vessel are newly added to the new culture solution and the new culture product solution. Incubate using mixed culture.

As another example of the present invention, in the dormant stem cell fixing step, the first culture container is statically left at a temperature substantially the same as the body temperature for 12 to 24 hours with the first culture container inclined at a predetermined angle. When the deformation from the initial planar shape of the dormant stem cells in the first culture vessel is observed at intervals of about 1 to 2 hours between 12 and 24 hours, and the dormant stem cells are deformed from the initial planar shape to a predetermined planar shape, It is determined that the dormant stem cells have settled on the bottom surface of the first culture container.

As another example of the present invention, the initial planar shape of the dormant stem cell is a substantially circular shape, and the planar shape after the deformation of the dormant stem cell is a flat shape in which the dormant stem cell extends in an indefinite shape in one direction with a substantially circular nucleus. In the dormant stem cell fixing step, it is determined that the dormant stem cell has settled on the bottom surface of the first culture container when the dormant stem cell is transformed into an irregular flat shape.

As another example of the present invention, the first target ratio of the total planar area of the dormant stem cells to the bottom surface area of the first culture container is 70 to 80%. In the dormant stem cell culture step, the first culture container is set at a predetermined angle. The first culture vessel is statically left at an approximately same temperature as the body temperature for 36 to 48 hours in a tilted state, and is placed on the bottom surface of the first culture vessel at intervals of about 1 to 2 hours during 36 to 48 hours. The total planar area with respect to the bottom area of the first culture vessel of the settled dormant stem cells is observed.

As another example of the present invention, the method for producing activated stem cells extracts activated stem cells from the first culture container when the total planar area of the dormant stem cells with respect to the bottom area of the first culture container reaches the first target ratio. Injecting the extracted activated stem cells, a new culture solution, and a new culture product solution into a second culture vessel having a predetermined volume and a bottom area of a predetermined area and having a capacity larger than that of the first culture vessel. Activated stem cell fixing step of fixing the stem to the bottom surface of the second culture vessel, and activating stem cells fixed on the bottom surface of the second culture vessel by the activated stem cell fixing step, and activating the bottom area of the second culture vessel And an activated stem cell culturing step for growing until the total planar area of the stem cells reaches the second target ratio.

As another example of the present invention, in the activated stem cell culturing step, the activated stem cells are fixed on the bottom surface of the second culture vessel by the activated stem cell fixing step, and then the mixed culture solution of the culture solution and the culture product solution is added to the first culture cell. (2) While discharging from the culture vessel, a new culture solution and a new culture product solution are injected into the second culture vessel, and the activated stem cells fixed on the bottom surface of the second culture vessel are added to the new culture solution and a new culture. Cultivate using a new mixed culture with the product.

As another example of the present invention, in the activated stem cell fixing step, the second culture container is statically left at a temperature substantially the same as the body temperature for 36 to 48 hours with the second culture container inclined at a predetermined angle. The deformation of the activated stem cells in the second culture vessel from the initial planar shape is observed at intervals of about 1 to 2 hours during 36 to 48 hours, and the activated stem cells are deformed from the initial planar shape to a predetermined planar shape. In this case, it is determined that the activated stem cells have settled on the bottom surface of the second culture container.

As another example of the present invention, the initial planar shape of the activated stem cells is a substantially circular shape, and the deformed planar shape of the activated stem cells is a flat shape in which the activated stem cells extend in an indefinite shape in one direction with a substantially circular shape as a nucleus. In the activated stem cell fixing step, it is determined that the activated stem cells have settled on the bottom surface of the second culture container when the activated stem cells are deformed into an irregular flat shape.

As another example of the present invention, the second target ratio of the total planar area of the activated stem cells to the bottom area of the second culture container is 88 to 92%. In the activated stem cell culture step, the second culture container is While the second culture vessel is statically left for 36 to 48 hours at a temperature substantially the same as the body temperature while being inclined at an angle, the second culture vessel is placed at intervals of about 1 to 2 hours between 36 and 48 hours. The total planar area with respect to the bottom area of the second culture vessel of the activated stem cells settled on the bottom surface is observed.

As another example of the present invention, the culture product solution contains a predetermined metabolite secreted from a single type of stem cell during the culture process of the single type of stem cell.

As another example of the present invention, a single type of stem cell is obtained by separating a bone marrow fluid collected from a donor into layers, extracting a middle layer bone marrow fluid located in an intermediate layer of the separated bone marrow fluid, Stem cell first that injects the intermediate layer bone marrow fluid and the predetermined culture solution into a third culture vessel having a bottom surface with a predetermined volume and a predetermined area to fix the first stem cells contained in the intermediate layer bone marrow fluid to the bottom surface of the third culture container. After fixing the first stem cells on the bottom surface of the third culture vessel by the fixing step and the stem cell first fixing step, a new culture solution is injected into the third culture vessel while discharging the culture solution in the third culture vessel. A first stem cell culturing step for culturing the first stem cells and proliferating the first stem cells until the total planar area of the first stem cells with respect to the bottom surface area of the third culture vessel reaches a third target ratio, and a stem cell first culturing step The cultured first stem cells are separated into layers The second stem cell located in the lowermost layer of the first stem cells separated and separated into layers is extracted, and a fourth culture having a predetermined volume and a bottom surface with a predetermined area and a larger volume than the third culture vessel. Injecting the second stem cell and a new culture solution into the container to fix the second stem cell to the bottom surface of the fourth culture container, and the second stem cell in the fourth culture container by the stem cell second fixing process. After fixing on the bottom surface, a new culture solution is poured into the fourth culture vessel while discharging the culture solution in the fourth culture vessel to culture the second stem cell, and the second stem cell relative to the bottom surface area of the fourth culture vessel And the stem cell second culturing step for proliferating the second stem cells until the total planar area reaches the fourth target ratio.

Another example of the present invention is a culture solution remaining after the culture product solution has extracted a single second stem cell from the fourth culture vessel.

As another example of the present invention, the stem cells are mesenchymal stem cells.

According to the method for producing activated stem cells according to the present invention, a single type of dormant stem cell with respect to the bottom surface of the first culture vessel is injected by injecting a culture product produced in the culture process of the single type of stem cell before storage of the dormant stem cell. And the growth of the dormant stem cells are promoted, so that the dormant stem cells can be quickly fixed on the bottom surface of the first culture vessel using the culture product solution, and the total surface can be obtained using the culture product solution. The dormant stem cells can be rapidly proliferated until the area reaches the first target ratio, and a single type of dormant stem cells in a dormant state can be efficiently and reliably proliferated, and it is necessary to have sufficient activity from the dormant stem cells An amount of a single type of activated stem cell can be efficiently produced. The method for producing activated stem cells can produce pure single types of activated stem cells that do not contain unnecessary stem cells, and does not contain a variety of stem cells. It is possible to produce activated stem cells that have a high regenerative effect in medicine, and to produce activated stem cells that are suitable for the treatment of various diseases and can be used in a timely manner, as well as various tissues and organs. Activated stem cells that are suitable for regeneration and can be used in a timely manner can be produced.

After the dormant stem cells are fixed on the bottom surface of the first culture vessel, a new culture solution and a new culture product solution are first discharged while discharging the mixed culture solution of the culture solution and the culture product solution from the first culture vessel. An activated stem cell production method in which dormant stem cells injected into a culture vessel and cultured on a bottom surface of a first culture vessel are cultured using a new mixed culture solution of a new culture solution and a new culture product solution is a dormancy method. Since the growth of dormant stem cells is surely promoted by the new culture product produced in the culture process of a single type of stem cell before storage of the stem cells, the total target area is the first target using the new culture product. A dormant stem cell can be rapidly proliferated until the ratio is reached, and a dormant single type of dormant stem cell can be efficiently and reliably proliferated, and a necessary amount of a single type of activated stem having sufficient activity can be obtained. It can be produced vesicles efficiently. The method for producing activated stem cells can produce pure single types of activated stem cells that do not contain unnecessary stem cells, and does not contain a variety of stem cells. It is possible to produce activated stem cells that have a high regenerative effect in medicine, and to produce activated stem cells that are suitable for the treatment of various diseases and can be used in a timely manner, as well as various tissues and organs. Activated stem cells that are suitable for regeneration and can be used in a timely manner can be produced.

While the first culture container is inclined at a predetermined angle, the first culture container is statically left at a temperature substantially the same as the body temperature for 12 to 24 hours, and the interval of about 1 to 2 hours is set between 12 and 24 hours. When the deformation of the dormant stem cells in the first culture container from the initial planar shape is observed and the dormant stem cells are deformed from the initial planar shape to the predetermined planar shape, it is determined that the dormant stem cells have settled on the bottom surface of the first culture container. In the method for producing activated stem cells, dormant stem cells can be reliably fixed on the bottom surface of the first culture container by statically leaving the first culture container inclined at a predetermined angle for 12 to 24 hours. The proliferation of dormant stem cells can be surely promoted. In the method for producing activated stem cells, the first culture of dormant stem cells is observed by observing deformation of the dormant stem cells from the initial planar shape in the first culture container at intervals of about 1 to 2 hours in a standing time of 12 to 24 hours. Since the colonization of the bottom of the container is confirmed as appropriate, it is possible to reliably grasp the dormant stem cell colonization, and to reliably proliferate the dormant stem cell and produce a single type of activated stem cell having sufficient activity. it can.

The initial planar shape of the dormant stem cell is a substantially circular shape, and the deformed planar shape of the dormant stem cell is a flat shape in which the dormant stem cell extends in one direction in an irregular shape with the substantially circular shape as the nucleus, and the dormant stem cell has an irregular shape. An activated stem cell manufacturing method for determining that dormant stem cells have settled on the bottom surface of the first culture container when deformed into a first shape by observing the deformation of the dormant stem cells from a substantially circular shape to a flat shape on the bottom surface of the first culture container. Since the establishment of the dormant stem cells on the bottom surface of the first culture container is accurately determined, the dormant stem cells can be firmly established in the first culture container, and the dormant stem cells can be reliably proliferated and have sufficient activity. A single type of activated stem cell can be produced.

The first target ratio of the total planar area of dormant stem cells to the bottom area of the first culture container is 70 to 80%, and the first culture container is substantially the same as the body temperature with the first culture container inclined at a predetermined angle. The total plane with respect to the bottom area of the first culture vessel of the dormant stem cells that have settled on the bottom surface of the first culture vessel at intervals of about 1 to 2 hours during 36 to 48 hours while standing statically at temperature for 36 to 48 hours In the method for producing activated stem cells for observing the area, the first culture vessel is allowed to stand statically for 36 to 48 hours in a state where the first culture vessel is inclined at a predetermined angle, thereby allowing the dormant stem cells that have settled on the bottom surface of the first culture vessel to grow. Can be surely promoted. In the method for producing activated stem cells, the total planar area of dormant stem cells relative to the bottom area of the first culture container is accurately confirmed by observing the total planar area at intervals of about 1 to 2 hours. The proliferation of dormant stem cells can be reliably grasped, and the dormant stem cells can be reliably proliferated to produce a single type of activated stem cell having sufficient activity. In the method for producing activated stem cells, when the total planar area of the dormant stem cells with respect to the bottom area of the first culture container exceeds 80% and the dormant stem cells proliferate, the activity of the dormant stem cells is gradually lost. Since the activated dormant stem cells are extracted from the first culture container when the total planar area of the dormant stem cells grows to 70 to 80% of the area, the activated dormant stem cell activity is retained. A single type of activated stem cell having sufficient activity for dormant stem cells can be produced.

When the total planar area of dormant stem cells with respect to the bottom surface area of the first culture container reaches the first target ratio, the activated stem cells are extracted from the first culture container, and the extracted activated stem cells, a new culture medium, and a new culture are extracted. The product solution is injected into a second culture container having a predetermined volume and a bottom surface with a predetermined area and larger than the first culture container, and the activated stem cells are fixed on the bottom surface of the second culture container. An activated stem cell production method in which activated stem cells fixed on the bottom surface of a container are cultured and proliferated until the total planar area of the activated stem cells with respect to the bottom surface area of the second culture container reaches a second target ratio is an activated stem cell ( The culture product produced in the course of culturing a single type of stem cell before storage of the dormant stem cells promotes the establishment of activated stem cells on the bottom of the second culture vessel and the proliferation of activated stem cells. The activated stem cells can be quickly established on the bottom surface of the second culture vessel using the culture product solution, and the activated stem cells can be rapidly used until the total planar area reaches the second target ratio using the culture product solution. And a single type of activated stem cells can be efficiently and reliably proliferated, and a necessary amount of single type of activated stem cells having sufficient activity can be efficiently produced. The method for producing activated stem cells can produce pure single types of activated stem cells that do not contain unnecessary stem cells, and does not contain a variety of stem cells. It is possible to produce activated stem cells that have a high regenerative effect in medicine, and to produce activated stem cells that are suitable for the treatment of various diseases and can be used in a timely manner, as well as various tissues and organs. Activated stem cells that are suitable for regeneration and can be used in a timely manner can be produced.

After the activated stem cells are fixed on the bottom surface of the second culture vessel, a new culture solution and a new culture product solution are removed while discharging the mixed culture solution of the culture solution and the culture product solution from the second culture vessel. A method for producing activated stem cells in which activated stem cells injected into two culture vessels and cultured on a bottom surface of a second culture vessel are cultured using a new mixed culture solution of a new culture solution and a new culture product solution, Because the growth of activated stem cells is surely promoted by a new culture product generated in the process of culturing a single type of stem cell before preservation of activated stem cells (dormant stem cells), a new culture product solution is used. The activated stem cells can be rapidly proliferated until the total planar area reaches the second target ratio, and a single type of activated stem cells can be efficiently and surely proliferated, and a necessary amount of sufficient activity can be obtained. single Can be efficiently activated stem cells be manufactured. The method for producing activated stem cells can produce pure single types of activated stem cells that do not contain unnecessary stem cells, and does not contain a variety of stem cells. It is possible to produce activated stem cells that have a high regenerative effect in medicine, and to produce activated stem cells that are suitable for the treatment of various diseases and can be used in a timely manner, as well as various tissues and organs. Activated stem cells that are suitable for regeneration and can be used in a timely manner can be produced.

While the second culture container is tilted at a predetermined angle, the second culture container is statically left at a temperature substantially the same as the body temperature for 36 to 48 hours, and the interval of about 1 to 2 hours is set between 36 and 48 hours. When the deformation of the activated stem cells in the second culture container from the initial planar shape is observed and the activated stem cells are deformed from the initial planar shape to a predetermined planar shape, the activated stem cells are fixed on the bottom surface of the second culture container. The method for producing activated stem cells, which is judged to have been established, ensures that the activated stem cells are firmly fixed on the bottom surface of the second culture vessel by statically leaving the second culture vessel inclined at a predetermined angle for 36 to 48 hours. And the proliferation of activated stem cells can be surely promoted. The method for producing activated stem cells comprises observing the deformation of the activated stem cells from the initial planar shape in the second culture container at intervals of about 1 to 2 hours in a standing time of 36 to 48 hours, thereby (2) Since the colonization of the bottom of the culture vessel is confirmed appropriately, it is possible to reliably grasp the colonization of the activated stem cells, and a single type of activated stem cells having sufficient activity by reliably proliferating the activated stem cells. It can be manufactured efficiently.

The initial planar shape of the activated stem cell is a substantially circular shape, and the planar shape after deformation of the activated stem cell is a flat shape in which the activated stem cell extends indefinitely in one direction with the substantially circular shape as the nucleus. An activated stem cell manufacturing method for determining that activated stem cells have settled on the bottom surface of the second culture container when deformed into a regular flat shape is obtained from a substantially circular shape of activated stem cells on the bottom surface of the second culture container to a flat shape. By observing the deformation, the establishment of the activated stem cells on the bottom surface of the second culture container can be accurately determined, so that the establishment of the activated stem cells in the second culture container can be reliably grasped, A single type of activated stem cell that is reliably proliferated and has sufficient activity can be efficiently produced.

The second target ratio of the total planar area of the activated stem cells to the bottom surface area of the second culture container is 88 to 92%, and the second culture container is substantially the same as the body temperature with the second culture container inclined at a predetermined angle. With respect to the area of the bottom surface of the second culture vessel of activated stem cells that have settled on the bottom surface of the second culture vessel at intervals of about 1 to 2 hours during 36 to 48 hours In the method for producing activated stem cells for observing the total planar area, the activated stem cells fixed on the bottom surface of the second culture container are statically left for 36 to 48 hours with the second culture container inclined at a predetermined angle. Can be surely promoted. In the method for producing activated stem cells, the total planar area of the activated stem cells relative to the bottom area of the second culture container is accurately confirmed by observing the total planar area at intervals of about 1 to 2 hours. The proliferation of the activated stem cells in the container can be reliably grasped, and the activated stem cells can be reliably proliferated and a single kind of activated stem cells having sufficient activity can be efficiently produced. In the activated stem cell production method, when the total planar area of the activated stem cells with respect to the bottom surface area of the second culture container exceeds 92% and the activated stem cells proliferate, the activity of the activated stem cells is gradually lost. By extracting the activated stem cells from the second culture container when the total planar area of the activated stem cells has grown to 88-92% with respect to the bottom area of the container, the activity of the activated stem cells is retained. A single type of activated stem cell with sufficient activity can be produced.

An activated stem cell manufacturing method in which a culture product contains a predetermined metabolite secreted from a single type of stem cell in the course of culturing a single type of stem cell is a culture that includes a predetermined metabolite secreted from a single type of stem cell. By using the generated solution, the metabolite of the stem cell itself becomes a trigger, and the dormant stem cell and the activated stem cell start to activate quickly. Therefore, the establishment of dormant stem cells on the bottom surface of the first culture container and the establishment of activated stem cells on the bottom surface of the second culture container are promoted, and the proliferation of dormant stem cells in the first culture container and the activated stem cells in the second culture container are promoted. The growth product is promoted, and dormant stem cells and activated stem cells can be quickly established on the bottom of the culture vessel using the culture product, and the total plane area reaches the target ratio using the culture product. Dormant stem cells and activated stem cells can be rapidly proliferated, and a single type of dormant stem cells and activated stem cells in a dormant state can be efficiently and reliably proliferated. A kind of activated stem cells can be efficiently produced.

The intermediate bone marrow fluid located in the intermediate layer of the bone marrow fluid separated into layers is extracted, the first stem cells contained in the intermediate layer bone marrow fluid are fixed on the bottom surface of the third culture vessel, and the first stem cells are cultured. The first stem cells are expanded until the total planar area of the first stem cells with respect to the bottom surface area of the third culture container reaches the third target ratio, and the second stem cell located in the lowest layer of the first stem cells separated in layers is used. Stem cells are extracted, the second stem cells are fixed on the bottom surface of the fourth culture container having a large capacity, the second stem cells are cultured, and the total planar area of the second stem cells relative to the bottom surface area of the fourth culture container is the fourth target ratio. The activated stem cell production method in which a single type of stem cell is produced by proliferating the second stem cell until it reaches the target stem cell does not contain a variety of stem cells before storage, and as a result includes unnecessary stem cells Not pure (pure Single type of activated stem cells can be produced, and activated stem cells that are highly effective in the treatment of various diseases and regenerative medicine can be produced, and are suitable for the treatment of various diseases and used in a timely manner. It is possible to produce activated stem cells that can be used, and it is possible to produce activated stem cells that can be used for regeneration of various tissues and organs in a timely manner.

The activated stem cell production method in which the culture product solution is a culture solution remaining after extraction of a single second stem cell from the fourth culture vessel is secreted from a single kind of stem cell into the culture solution remaining after extraction of the second stem cell. The metabolite of the stem cell itself is used as a trigger, and the dormant stem cell or the activated stem cell immediately starts to be activated. Therefore, the establishment of dormant stem cells on the bottom surface of the first culture container and the establishment of activated stem cells on the bottom surface of the second culture container are promoted, and the proliferation of dormant stem cells in the first culture container and the activated stem cells in the second culture container are promoted. The growth product is promoted, and dormant stem cells and activated stem cells can be quickly established on the bottom of the culture vessel using the culture product, and the total plane area reaches the target ratio using the culture product. Dormant stem cells and activated stem cells can be rapidly proliferated, and a single type of dormant stem cells and activated stem cells in a dormant state can be efficiently and reliably proliferated. A kind of activated stem cells can be obtained.

The method for producing activated stem cells in which the stem cells are mesenchymal stem cells can efficiently and reliably proliferate a single type of mesenchymal dormant stem cells or mesenchymal activated stem cells that are in a dormant state. A necessary amount of a single species of mesenchymal activated stem cells having various activities can be efficiently produced. The activated stem cell production method can produce pure (pure) activated mesenchymal stem cells that do not contain unnecessary stem cells, and does not contain a variety of mesenchymal stem cells. Mesenchymal activated stem cells that can produce mesenchymal activated stem cells that are highly effective in the treatment of various diseases and regenerative medicine, and are suitable for the treatment of various diseases and can be used in a timely manner It is possible to produce mesenchymal activated stem cells that can produce various stem cells and various tissues and organs that are suitable for regeneration and can be used in a timely manner.

The schematic block diagram of the activated stem cell culture system shown as an example. The figure explaining an example of a dormant stem cell fixing process. The side view of a 1st flat culture container. The elements on larger scale which show an example of the planar shape of a dormant stem cell. The elements on larger scale which show another example of the planar shape of a dormant stem cell. The elements on larger scale which show another example of the planar shape of a dormant stem cell. The figure explaining an example of the activated stem cell fixing process. The side view of a 2nd flat culture container. The elements on larger scale which show an example of the planar shape of an activated stem cell. The elements on larger scale which show another example of the planar shape of an activated stem cell. The elements on larger scale which show another example of the planar shape of an activated stem cell. The perspective view of the glass test tube used in a stem cell 1st fixing process. The figure explaining the stem cell 1st fixing process following FIG. The figure explaining the stem cell 1st fixing process following FIG. The elements on larger scale which show an example of the planar shape of a stem cell. The elements on larger scale which show another example of the planar shape of a stem cell. The elements on larger scale which show another example of the planar shape of a stem cell. The perspective view of the glass test tube and centrifuge used in a stem cell 2nd fixing process. The perspective view of the glass test tube after centrifugation. The elements on larger scale which show an example of the planar shape of a stem cell (2nd stem cell). The elements on larger scale which show another example of the planar shape of a stem cell (2nd stem cell). The elements on larger scale which show another example of the planar shape of a stem cell (2nd stem cell). The figure which shows an example of preservation | save of a stem cell (2nd stem cell) and a culture production liquid.

Referring to the accompanying drawings such as FIG. 1 which is a schematic configuration diagram of an activated stem cell culture system 10 shown as an example, details of the method for producing a single type (specific type) of mesenchymal activated stem cells according to the present invention will be described. Then, it is as follows. 2 is a diagram for explaining an example of a dormant stem cell fixing process, and FIG. 3 is a side view of the first flat culture vessel 21. FIG. 4 is a partially enlarged view showing an example of the planar shape of the dormant stem cell 22, and FIG. 5 is a partially enlarged view showing another example of the planar shape of the dormant stem cell 22. 4 and 5 show enlarged images of the planar shape of the dormant stem cell 22 photographed by the electron microscope 13.

The single species of mesenchymal activated stem cells 27 are in a dormant state after storing a single species (specific type) of mesenchymal stem cells 30 made by culturing bone marrow fluid 29 collected from a donor for a predetermined period. The mesenchymal dormant stem cell 22 is activated. The activated stem cell 27 uses the culture solution 24 generated in the culture process of the stem cell 30 that is the source of the dormant stem cell 22 before the preservation of the dormant stem cell 22, and establishes the dormant stem cell 22 in the dormant stem cell 22 in the dormant state. It is manufactured from performing a process and a dormant stem cell culture process.

In addition, the activated stem cell uses the culture product solution 24 generated in the culture process of the stem cell 30 which is the source of the dormant stem cell 22 before the preservation of the dormant stem cell 22, and the dormant stem cell 22 in the dormant state is used as the dormant stem cell 22. It is made by performing a fixing step and a dormant stem cell culture step, and performing an activated stem cell fixing step and an activated stem cell culture step on activated stem cells. The stem cell 30 before storage and the dormant stem cell 22 after storage are the same stem cell.

The culture product solution 24 contains a predetermined metabolite secreted from the single type of stem cell 30 in the culture process of the single type of stem cell 30. By using the culture product solution 24 containing a predetermined metabolite secreted from a single type of stem cell 30, the metabolite of the stem cell 30 itself becomes a trigger, and the dormant stem cell 22 and the activated stem cell 27 are activated quickly. Start. Therefore, the establishment of the dormant stem cells 22 and the activated stem cells 27 is promoted, and the proliferation of the dormant stem cells 22 and the activated stem cells 27 is promoted. The dormant stem cell 22 and the activated stem cell 27 can be rapidly proliferated using the culture solution 24, and the dormant stem cell 22 and the activated stem cell 27 can be efficiently and reliably proliferated. be able to.

The activated stem cell culture system 10 includes a computer 11, an IC tag reader / writer 12, an electron microscope 13, a refrigerator 14, or a freezer 14. The computer 11 includes a central processing unit (CPU or virtual CPU), a storage device (memory or virtual memory), and a large-capacity storage area (hard disk, virtual hard disk, etc.), and a physical OS (operating system) or virtual OS ( Virtual operating system).

The computer 11 is connected to input devices such as a keyboard 15 and a mouse 16 and output devices such as a display 17 and a printer (not shown) via an interface (wireless or wired). The IC tag reader / writer 12 and the electron microscope 13 are connected to the computer 11 via an interface (wireless or wired). The electron microscope 13 has an image capturing function for capturing an enlarged image of a subject using an image sensor, and also has an image transmission function for transmitting the enlarged image to the computer 11.

In the activated stem cell culture system 10, various types of donor data (donor specific information) are managed using the IC tag 18, and stem cells, dormant stem cells 22, and stem cell data regarding activated stem cells are managed using the IC tag 18. The Donor data includes the donor's name, address, phone number, date of birth, sex, blood type, height, weight, email address, etc., and is stored (stored) in the IC tag in association with the donor identifier. . Stem cell data includes stem cell identification information, stem cell production date, culture production liquid production date, culture production liquid identification information, etc., and is stored (stored) in the IC tag 18 in a state associated with the donor identifier and the stem cell identifier. Has been.

A single type (specific type) of stem cells 30 produced by culturing the bone marrow fluid 29 is stored in the stem cell storage container 19 in the refrigerator 14 or the freezer 14 for a predetermined period (3 to 5 ° C. or frozen). Saved). An IC tag 18 storing donor data and stem cell data is attached to the outer peripheral surface of the stem cell storage container 19 that stores the stem cells 30.

The culture solution 24 generated in the process of culturing the stem cell 30 that is the source of the activated stem cell 27 (the dormant stem cell 22) is stored in the product storage container 20 in the refrigerator 14 or the freezer 14 for a predetermined period of time at a predetermined temperature. Saved in. An IC tag 18 in which donor data and stem cell data are stored is affixed to the outer peripheral surface of the product solution container 20 containing the culture product solution 24.

When the system 10 is started in the computer 11, an initial screen (not shown) is displayed on the display 17. On the initial screen, a stem cell culture button, a dormant stem cell culture button, an activated stem cell culture button, and a logout button are displayed. A person in charge such as a doctor, nurse or researcher clicks a dormant stem cell culture button displayed on the display 17. When the dormant stem cell culture button is clicked, the computer 11 displays a data comparison button on the display 17.

In the dormant stem cell fixing step, the person in charge clicks on the data comparison button, takes out the stem cell storage container 19 and the product liquid storage container 20 from the refrigerator 14 or the freezer 14, and is attached to the stem cell storage container 19 or the product liquid storage container 20. The IC tag reader / writer 12 is caused to read the data of the IC tag 18. The computer 11 compares the donor identifier or stem cell identifier of the IC tag 18 of the stem cell storage container 19 with the donor identifier or stem cell identifier of the IC tag 18 of the product solution storage container 20, and the donor identifier and stem cell identifier of the IC tag 18 are If they match, a matching OK message and a data storage button are displayed on the display 17. When the donor identifier and the stem cell identifier of these IC tags do not match, an error message is displayed on the display 17.

The person in charge confirmed by the matching OK message that the culture product liquid 24 stored in the product liquid storage container 20 was generated in the course of culturing the dormant stem cells 22 (stem cells) stored in the stem cell storage container 19. Thereafter, the stem cell storage container 19 and the product liquid storage container 20 are stored in a thermostat (not shown), and the dormant stem cells 22 stored in the stem cell storage container 19 and the culture product liquid 24 stored in the product liquid storage container 20 are stored. Return to room temperature. Alternatively, the stem cell storage container 19 and the product liquid storage container 20 are left in the room for a predetermined time, and the dormant stem cells 22 stored in the stem cell storage container 19 and the culture product liquid 24 stored in the product liquid storage container 20 are returned to room temperature.

The person in charge prepares the first flat culture vessel 21 (first culture vessel), attaches the IC tag 18 to the outer peripheral surface of the culture vessel 21, clicks the data storage button, and uses the IC tag reader / writer 12 Then, data of the IC tag 18 attached to the stem cell container 19 and the IC tag 18 attached to the product liquid container 20 are stored in the IC tag 18. The computer 11 displays a data storage completion message, a dormant stem cell fixation observation button, and a dormant stem cell fixation completion button on the display 17.

The person in charge returns the dormant stem cells 22 and the culture solution 24 to room temperature, and then injects the dormant stem cells 22 from the stem cell storage container 19 into the first flat culture container 21 (first culture container) using a syringe or pipette ( And injecting (accommodating) the culture solution 23 into the culture container 21 using a syringe or pipette, and injecting (accommodating) the culture solution 24 from the product solution storage container 20 into the culture container 21 using a syringe or pipette ( Contain). The injection rate of the culture product solution 24 injected into the first flat culture vessel 21 is 5 to 15%, preferably 8 to 8 when the total injection amount of the culture solution 23 injected into the culture vessel 21 is 100%. 12%, more preferably 10%.

Next, the person in charge keeps the first flat culture vessel 21 infused with the dormant stem cells 22, the culture solution 23, and the culture product solution 24 at a temperature (about 37 ° C.) substantially the same as the body temperature, And leave the stem cell 22 in the culture vessel 21 for deformation from the initial planar shape at intervals of about 1 to 2 hours during 12 to 24 hours. Then, it is determined whether or not the dormant stem cell 22 has settled on the bottom surface 25 of the culture vessel 21.

The culture solution 23 is a mineral salt to which penicillin (about 100 U / ml), amphotericin (about 100 ng / ml), streptomycin (about 100 mg / ml), L-glutamine (about 2 to 4 ml) and 20% fetal bovine serum are added. Solutions and amino acids are included. The dormant stem cells 22 injected into the first flat culture vessel 21 are cultured with the mixed culture solution 26 of the culture solution 23 and the culture product solution 24 while being fixed to the bottom surface 25 of the culture vessel 21 as time passes. It gradually grows (differentiates) on the bottom surface 25 of 21 to form a colony.

For the culture solution 23, Dulbecco's Modified Eagle's Medium (DMEM), GrasgowｉMinimumｓｓEssential Medium (GMEM), RPMI640 or the like can also be used. The culture solution 23 includes insulin, transferrin, ethanolamine, selenium, 2-mercaptoethanol, L-alanyl-L-glutamine, sodium pyruvate, L-alanine, L-asparagine, L-aspartic acid, glycine, L- Proline, L-serine and the like can also be added.

The first flat culture vessel 21 (first culture vessel) is a flat vessel that is made of transparent glass or transparent plastic, has a small volume and has a bottom surface with a predetermined area, and has a substantially square shape in plan view. As the first flat culture vessel 21, a flat vessel having a small volume and having a bottom surface with a predetermined area and having a circular or elliptical planar shape may be used. The first flat culture vessel 21 used in the dormant stem cell colonization step has a capacity of about 20 to 30 cc (preferably 25 cc) and a bottom area of about 25 to 36 mm ² . The culture vessel 21 has a side length of 5 to 6 mm.

The person in charge clicks the dormant stem cell fixation observation button and installs (sets) the first flat culture vessel 21 in the sample holder 39 of the electron microscope 13. In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 13 and the bottom 41 of the first flat culture vessel 21, and the bottom 41 of the culture vessel 21 is held in a state where it is lifted by the spacer 42. The culture vessel 21 is held at a predetermined angle so that the bottom 41 of the vessel 21 is on the top and the top 43 (injection port 44) of the culture vessel 21 is on the bottom. In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 13 and the top 43 of the first flat culture vessel 21, and the top 43 of the culture vessel 21 is held in a state where it is lifted by the spacer 42. You may hold | maintain the culture container 21 in the state inclined to the predetermined angle so that the top part 43 of the container 21 may become upper and the bottom part 41 of the culture container 21 may become lower. The inclination angle α1 of the first flat culture vessel 21 with respect to the upper surface 40 of the sample holder 39 is in the range of 2 to 5 °, preferably in the range of 2 to 3 °.

In the culture product production method, the dormant stem cells 22, the culture solution 23, and the culture product solution 24 are contained in the culture vessel 21 by inclining the first flat culture vessel 21 at the inclination angle with respect to the upper surface 40 of the sample holder 39. The pressure is biased toward the top 43 side (or the bottom 41 side) of the culture vessel 21, and the water pressure of the dormant stem cells 22, the culture solution 23, and the culture product solution 24 is increased on the top 43 side (or the bottom 41 side) of the culture vessel 21. The dormant stem cells 22 become concentrated and concentrate on the top 43 side (or the bottom 41 side) of the culture vessel 21, thereby increasing the activity of the dormant stem cells 22. It can be fixed quickly.

The display 17 displays a dormant stem cell colonization observation message and a dormant stem cell colonization completion button. The electron microscope 13 takes a magnified image of the planar shape of the dormant stem cell 22 injected into the first flat culture vessel 21 at intervals of about 1 to 2 hours, and the magnified image of the planar shape of the photographed dormant stem cell 22 is approximately 1 to It transmits to the computer 11 at intervals of 2 hours. The image capturing interval and the image transmission interval in the electron microscope 13 can be freely set within 1 to 2 hours by an input device such as a keyboard 14 or a mouse 15.

The computer 11 stores (stores) the enlarged image of the planar shape of the dormant stem cell 22 and the imaging time transmitted from the electron microscope 13 in a storage area in a state associated with the donor identifier and the stem cell identifier. The computer 11 displays the enlarged image of the planar shape of the dormant stem cell 22 transmitted from the electron microscope 13 and the imaging time on the display 17. The person in charge checks (views) the enlarged image of the planar shape of the dormant stem cell 22 displayed on the display 17 at intervals of about 1 to 2 hours during 12 to 24 hours, and changes the planar shape of the dormant stem cell 22. Observe. The person in charge may directly observe the change in the planar shape of the dormant stem cell 22 from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 12 to 24 hours.

The initial planar shape (planar shape before colonization) of the dormant stem cell 22 is substantially circular. When the planar shape of the dormant stem cell 22 is substantially circular, the dormant stem cell 22 is the bottom surface 25 (bottom wall inner surface) of the first flat culture vessel 21. The dormant stem cell 22 has not started to proliferate (differentiate). The planar shape after deformation of the dormant stem cell 22 (planar shape after colonization) is a flat shape in which the dormant stem cell 22 extends (expands) in one direction (predetermined direction) in an irregular shape with the substantially circular shape before colonization as the nucleus. The stem cells 22 have settled on the bottom surface 25 (bottom wall inner surface) of the first flat culture vessel 21, and the dormant stem cells 22 have started to proliferate (activate).

As shown in FIG. 4, when the person in charge observes the enlarged image of the planar shape of the dormant stem cell 22 displayed on the display 17 in a substantially circular shape as shown in FIG. 1 It is judged that the flat culture container 21 has not settled on the bottom surface 25 (bottom wall inner surface), and the change in the planar shape of the dormant stem cells 22 is continuously observed at intervals of about 1 to 2 hours. As shown in FIG. 5, when the planar shape of the dormant stem cell 11 displayed on the display 17 is changed from a substantially circular shape to an irregular flat shape with the substantially circular shape as a nucleus, the person in charge makes the dormant stem cell 11 in the first flat culture. It is determined that the toner is fixed on the bottom surface 25 of the container 21.

When a large culture container having a capacity exceeding 30 cc and a bottom area exceeding 36 mm ² is used when the dormant stem cells 22 are fixed, the dormant stem cells 22 are difficult to settle on the bottom surface of the container and the proliferation of the dormant stem cells 22 is delayed. By using the first flat culture vessel 21 having the capacity and the bottom area, the dormant stem cells 22 can be easily fixed on the bottom surface 25 of the culture vessel 21, and the dormant stem cells 22 are rapidly proliferated in the culture vessel 21. be able to. While the first flat culture vessel 21 is statically left at a temperature substantially the same as the body temperature for 12 to 24 hours, the initial period of the dormant stem cells 22 in the culture vessel 21 is set at intervals of about 1 to 2 hours during 12 to 24 hours. Since the deformation from the planar shape is observed, the deformation of the dormant stem cell 22 is not overlooked, and the dormant stem cell 22 can be accurately confirmed on the bottom surface 25 of the culture vessel 21.

FIG. 6 is a partially enlarged view showing another example of the planar shape of the dormant stem cell 22. FIG. 6 shows an enlarged image of the planar shape of the dormant stem cell 22 photographed by the electron microscope 13. As a result of observation in the dormant stem cell fixing step, as shown in FIG. 5, the mesenchymal dormant stem cell 22 is deformed from a substantially circular shape (initial planar shape) to an indeterminate flat shape with a substantially circular shape as a nucleus. After confirming fixation on the bottom surface 25 of the first flat culture vessel 21, a dormant stem cell culture step is performed.

A person in charge such as a doctor, a nurse, or a researcher confirms that the dormant stem cells 22 are fixed on the bottom surface 25 of the first flat culture vessel 21 and then clicks a dormant stem cell fixing completion button displayed on the display 17. When the dormant stem cell colonization completion button is clicked, the computer 11 displays a dormant stem cell colonization completion message, a dormant stem cell culture observation button, and a dormant stem cell culture completion button on the display 17.

In the dormant stem cell culture step, the mixed culture solution 26 of the culture solution 23 and the culture product solution 24 injected into the first flat culture vessel 21 is discharged from the culture vessel 21, and a new culture solution 23 and a new one are added to the culture vessel 21. A fresh culture product solution 24 (a culture product solution 24 produced before the preservation of the dormant stem cells 22 and in the course of culturing the stem cells 30 that are the source of the dormant stem cells 22) is injected (accommodated). Note that the injection rate of the new culture solution 24 to be injected into the first flat culture vessel 21 is 5 to 15%, preferably 100% when the total injection amount of the new culture solution 23 to be injected into the culture vessel 21 is 100%. Is 8 to 12%, more preferably 10%.

The person in charge removes the first flat culture vessel 21 from the sample holder 39 of the electron microscope 16 and uses a syringe or pipette to mix the mixed culture solution 26 of the culture solution 23 and the culture product solution 24 injected into the culture vessel 21 in the dormant stem cell fixing step. The culture medium is discharged from the culture container 21 using a syringe, and a new culture solution 23 is injected (contained) into the culture container 21 using a syringe or pipette, and a new culture solution 24 is generated using the syringe or pipette. Injection (accommodation) is performed from the liquid storage container 120 to the culture container 21. The new culture solution 23 and the new culture product solution 24 are the same as those injected into the first flat culture vessel 21 in the dormant stem cell fixing step.

The person in charge statically left the first flat culture vessel 21 at a temperature substantially the same as the body temperature (about 37 ° C.) for 36 to 48 hours (to leave it quietly without moving), and for about 36 to 48 hours. The total planar area of the dormant stem cells 22 fixed on the bottom surface 25 of the culture vessel 21 at intervals of 1 to 2 hours with respect to the bottom surface area of the culture vessel 21 is observed with the electron microscope 13. It is determined whether the target ratio (first target ratio) has been reached with respect to the bottom surface area. The target ratio of the total planar area of the dormant stem cells 22 to the bottom area of the first flat culture vessel 21 is 70 to 80% (70 to 80% confluent).

The person in charge injects a new culture solution 23 and a new culture product solution 24 into the first flat culture vessel 21, then clicks a dormant stem cell culture observation button, and uses the culture vessel 21 as a sample holder of the electron microscope 13. Install (set). In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 16 and the bottom 41 of the first flat culture vessel 21, and the bottom 41 of the culture vessel 21 is held in a state where it is lifted by the spacer 42. The culture vessel 21 is held at a predetermined angle so that the bottom 41 of the vessel 21 is on the top and the top 43 (injection port 44) of the culture vessel 21 is on the bottom (see FIG. 3). Further, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 16 and the top 43 of the first flat culture vessel 21, and the top 43 of the culture vessel 21 is held in a state where it is lifted by the spacer 42. You may hold | maintain the culture container 21 in the state inclined to the predetermined angle so that the top part 43 of the container 21 may become upper and the bottom part 41 of the culture container 21 may become lower. The inclination angle α1 of the first flat culture vessel 21 with respect to the upper surface 40 of the sample holder 39 is in the range of 2 to 5 °, preferably in the range of 2 to 3 °.

In the method for producing a culture product solution, after confirming that the dormant stem cells 22 are fixed, a new culture solution 23 and a new culture product solution 24 are injected into the culture vessel 21 while discharging the mixed culture solution 26 in the culture vessel 21. Thus, the proliferation of the dormant stem cells 22 can be surely promoted. In the culture product production method, the dormant stem cells 22, the culture solution 23, and the culture product solution 24 are contained in the culture vessel 21 by inclining the first flat culture vessel 21 at the inclination angle with respect to the upper surface 40 of the sample holder 39. It is biased toward the top 43 side (or the bottom 41 side) of the culture vessel 21, and the water pressure of the dormant stem cells 22, the culture solution 23, and the culture product solution 24 is large on the top 43 side (or the bottom 41 side) of the culture vessel 21. Thus, the dormant stem cells 22 are concentrated on the bottom 41 side (or the top 43 side) of the culture vessel 21, thereby increasing the activity of the dormant stem cells 22, and the dormant stem cells 22 can be easily and quickly formed on the bottom surface 25 of the culture vessel 21. Can be proliferated (differentiated).

The display 17 displays a dormant stem cell culture observation message and a dormant stem cell culture completion button. After confirming the establishment of the dormant stem cells 22, the mixed culture solution 26 of the culture solution 23 and the culture product solution 24 was discharged from the first flat culture vessel 21 and secreted from the new culture solution 23 and a single kind of stem cells. By injecting a new culture product solution 24 containing a predetermined metabolite into the culture vessel 21, the metabolite of the stem cell 30 itself becomes a trigger, and the dormant stem cell 22 quickly starts to be activated. Proliferation can be reliably promoted.

The electron microscope 13 takes a magnified image of the planar shape of the dormant stem cell 22 in the first flat culture vessel 21 at intervals of about 1 to 2 hours, and takes a magnified image of the planar shape of the photographed dormant stem cell 22 at intervals of about 1 to 2 hours. To the computer 11. The image capturing interval and the image transmission interval in the electron microscope 13 can be freely set within 1 to 2 hours by an input device such as a keyboard 14 or a mouse 15. The computer 11 stores (stores) the planar enlarged image of the dormant stem cell 22 transmitted from the electron microscope 13 and the imaging time in a storage area in a state associated with the donor identifier and the stem cell identifier. The computer 11 displays the enlarged image of the planar shape of the dormant stem cell 22 transmitted from the electron microscope 13 and the imaging time on the display 17.

The person in charge confirms (views) the enlarged image of the planar shape of the dormant stem cell 22 displayed on the display 17 at intervals of about 1 to 2 hours during 36 to 48 hours, and then the bottom surface 25 of the first flat culture vessel 21. While observing the total planar area of the dormant stem cells 22 settled on the bottom surface area of the culture vessel 21, the total planar area of the dormant stem cells 22 is the target ratio (first target ratio) (70 to 70) with respect to the bottom surface area of the culture container 21. 80% confluent) is determined. The person in charge directly observes the total planar area of the dormant stem cells 22 with respect to the bottom surface area of the first flat culture vessel 21 from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 36 to 48 hours. It may be determined whether or not the total plane area of 22 has reached the target ratio (70 to 80% confluent) with respect to the bottom area of the culture vessel 21.

As a result of the observation in the dormant stem cell culture process, the person in charge, as shown in FIG. 4, determines that the total planar area of the dormant stem cells 22 displayed on the display 17 with respect to the bottom area of the first flat culture vessel 21 is the target ratio (first target). If the ratio (70-80% confluent) is not reached, the total planar area of the dormant stem cells 22 relative to the bottom area of the culture vessel 21 is continuously observed at intervals of about 1 to 2 hours. When the total planar area of the dormant stem cells 22 reaches the target ratio (first target ratio) with respect to the entire area of the enlarged image displayed on the display 17, the bottom surface of the first flat culture vessel 21 of the dormant stem cells 22 is used. It is assumed that the total plane area with respect to the area has reached the target ratio.

As a result of the observation in the dormant stem cell culture process, the dormant stem cell 22 grows on the bottom surface 25 (bottom wall inner surface) of the first flat culture vessel 21 to form a colony, and the planar shape of the dormant stem cell 22 expands. As shown in FIG. 5, when the total planar area of the dormant stem cells 22 displayed on the display 17 with respect to the bottom surface area of the culture vessel 21 reaches the target ratio (first target ratio) (70 to 80% confluent), The dormant stem cells 22 are sufficiently proliferated and activated, and the dormant stem cells 22 are transformed into activated stem cells 27. When the total planar area of the dormant stem cells 22 with respect to the bottom surface area of the first flat culture container 21 reaches a target ratio, the activated stem cells 27 are extracted from the culture container 21 and the extracted activated stem cells 27 are treated with various diseases. Used for regenerative medicine.

The person in charge discharges the mixed culture solution 26 injected into the first flat culture vessel 21 from the culture vessel 21 using a syringe or pipette, and the culture vessel 21 is washed with PBS, and then the trypsin solution is added to the culture vessel. 21. When a trypsin solution is injected into the first flat culture vessel 21, activated stem cells 27 fixed (proliferated) on the bottom surface 25 of the culture vessel 21 are detached from the bottom surface 25 by the trypsin solution and float on the water surface of the trypsin solution. The person in charge aspirates the activated stem cells 27 using a pipette, and accommodates the activated stem cells 27 in the pipette. The activated stem cells 27 cultured in the dormant stem cell culturing step are stored in the stem cell storage container 19, and the stem cell storage container 19 storing the activated stem cells 27 is placed in the refrigerator 14 or the freezer 14 and activated in the refrigerator 14 or the freezer 14. Stem cells 27 can be stored.

The person in charge confirms that the total planar area of the dormant stem cells 22 with respect to the bottom surface area of the first flat culture vessel 21 has reached the target ratio (first target ratio), and then displays the dormant stem cell culture completion button displayed on the display 17. Click. When the dormant stem cell culture completion button is clicked, the computer 11 displays an initial screen on the display 17. To end the culture, click the logout button on the initial screen. When the logout button is clicked, the computer 11 logs out from the system.

The activated stem cell production method is a culture product solution containing a predetermined metabolite that is generated in the culture process (proliferation process) of the single mesenchymal stem cell 30 before storage of the dormant stem cell 22 and secreted from the stem cell 30. 24, the metabolite of the stem cell itself is used as a trigger, and the dormant stem cell 22 starts to be activated quickly, and the dormant stem cell 22 is fixed to the bottom surface 25 of the first flat culture vessel 21 (first culture vessel). The proliferation of the dormant stem cell 22 can be promoted, and the dormant stem cell 22 can be rapidly proliferated while maintaining the activity of the dormant stem cell 22.

The activated stem cell production method uses the culture product solution 24 generated in the culture process (proliferation process) of the single species of mesenchymal stem cells 30 before the preservation of the dormant stem cells 22 to convert the dormant stem cells 22 into the first flat culture container. The mesenchymal system of a single species is allowed to settle on the bottom surface 25 of the 21 (first culture vessel) and the dormant stem cells 22 are expanded using the culture product solution 24 until the total planar area reaches the first target ratio. The dormant stem cells 22 can be efficiently and reliably established and proliferated, and the necessary amount of single-type mesenchymal activated stem cells 27 having sufficient activity can be efficiently produced.

In the method for producing activated stem cells, the mesenchymal dormant stem cells 22 are subjected to a dormant stem cell fixing step and a dormant stem cell culturing step, whereby pure (pure) single mesenchymal activation without unnecessary mesenchymal stem cells is performed. Since the stem cell 27 can be produced and no miscellaneous mesenchymal stem cells are included, the activated stem cell 27 having a high effect of treatment for various diseases and a regeneration effect in regenerative medicine can be produced. Activated stem cells 27 that can be used for the treatment of various diseases and can be used in a timely manner, and can be used for regeneration of various tissues and organs in a timely manner. 27 can be manufactured.

FIG. 7 is a diagram for explaining an example of the activated stem cell fixing step, and FIG. 8 is a side view of the second flat culture vessel 28. FIG. 9 is a partially enlarged view showing an example of the planar shape of the activated stem cell 27, and FIG. 10 is a partially enlarged view showing another example of the planar shape of the activated stem cell 27. FIG. 11 is a partially enlarged view showing another example of the planar shape of the activated stem cell 27. 9 to 11 show enlarged images of the planar shape of the activated stem cells 27 photographed by the electron microscope 13.

In order to continue culturing the activated stem cells 27 after culturing the dormant stem cells 22 (activated stem cells 27) by the dormant stem cell culture process, the initial screen displayed on the display 17 by clicking the dormant stem cell culture completion button is clicked. Click the Activated Stem Cell Culture button. When the activated stem cell culture button is clicked, the computer 11 displays a data storage button on the display 17.

A person in charge such as a doctor, a nurse, or a researcher prepares the second flat culture container 28 (second culture container), attaches the IC tag 18 to the outer peripheral surface of the culture container 28, and then clicks the data storage button. At the same time, the IC tag reader / writer 12 is used to store the data of the IC tag 18 attached to the first flat culture vessel 21 (first culture vessel) in the IC tag 18. The computer 11 displays a data storage completion message, an activated stem cell fixation observation button, and an activated stem cell fixation completion button on the display 17.

The person in charge injects (accommodates) the activated stem cells 27 from the first flat culture vessel 21 (first culture vessel) into the second flat culture vessel 28 using a syringe or pipette, and cultures using the syringe or pipette. The liquid 23 is injected (accommodated) into the second flat culture container 28, and the culture product liquid 24 is injected (accommodated) from the product liquid storage container 20 into the second flat culture container 28 using a syringe or pipette. The injection ratio of the culture product solution 24 to be injected into the second flat culture vessel 28 is 5 to 15% when the total injection amount of the culture solution 23 to be injected into the second flat culture vessel 28 is 100%, preferably 8-12%, more preferably 10%. The culture solution 23 and the culture product solution 24 are the same as those injected into the first flat culture vessel 21.

Next, the person in charge maintains the second flat culture vessel 28 into which the activated stem cells 27, the culture solution 23, and the culture product solution 24 have been injected at a temperature substantially equal to the body temperature (about 37 ° C.) for 36 to 48 hours. Electrostatic microscope 13 is used to statically leave (slowly leave without moving), and to transform the activated stem cells 27 in the culture vessel 28 from the initial planar shape at intervals of about 1 to 2 hours in 12 to 24 hours. Observe and determine whether the activated stem cells 27 have settled on the bottom surface 29 of the culture vessel 28.

The second flat culture vessel 28 (second culture vessel) is a flat vessel made of transparent glass or transparent plastic, having a small volume and having a bottom surface with a predetermined area and having a substantially quadrangular planar shape. Is about twice as large as the first flat culture vessel 21 (first culture vessel). As the second flat culture vessel 28, a flat vessel having a small volume and a bottom surface having a predetermined area and having a circular or elliptical planar shape can be used. The second flat culture vessel 28 used in the activated stem cell fixing step has a capacity of about 40 to 60 cc (preferably 50 cc) and a bottom area of about 50 to 72 mm ² . The culture container 28 has a side length of about 7 to 8.5 mm.

The person in charge clicks the activated stem cell fixation observation button and installs (sets) the second flat culture vessel 28 in the sample holder 39 of the electron microscope 13. In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 13 and the bottom 45 of the second flat culture vessel 28, and the bottom 45 of the culture vessel 28 is held in a state where it is lifted by the spacer 42. The culture vessel 28 is held at a predetermined angle so that the bottom 45 of the vessel 28 is on top and the top 46 (inlet 47) of the culture vessel 28 is on the bottom. In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 13 and the top 46 of the second flat culture vessel 28, and the top 46 of the culture vessel 28 is held in a state lifted by the spacer 42. You may hold | maintain the culture container 28 in the state inclined by the predetermined angle so that the top part 46 of the container 28 may become the upper side and the bottom part 45 of the culture container 28 may become the lower side. The inclination angle α2 of the second flat culture vessel 28 with respect to the upper surface 40 of the sample holder 39 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

In the culture product production method, the second flat culture vessel 28 is inclined at the inclination angle with respect to the upper surface 40 of the sample holder 39, whereby the activated stem cells 27, the culture solution 23, and the culture product solution 24 are contained in the culture vessel 28. Is biased to the top 46 side (or the bottom 45 side) of the culture vessel 28, and the water pressure of the activated stem cells 27, the culture solution 23, and the culture product solution 24 on the top 46 side (or the bottom 45 side) of the culture vessel 28. And the activated stem cells 27 are concentrated on the top 46 side (or the bottom 45 side) of the culture vessel 28, thereby increasing the activity of the activated stem cells 27, and the activated stem cells on the bottom surface 29 of the culture vessel 28. 27 can be fixed easily and quickly.

The display 17 displays a message indicating that activated stem cell colonization is being observed and an activated stem cell colonization completion button. The electron microscope 13 takes enlarged images of the planar shape of the activated stem cells 27 injected into the second flat culture vessel 28 at intervals of about 1 to 2 hours. It is transmitted to the computer 11 at intervals of 1 to 2 hours. The image capturing interval and the image transmission interval in the electron microscope 13 can be freely set within 1 to 2 hours by an input device such as a keyboard 14 or a mouse 15.

The computer 11 stores (stores) the enlarged image of the planar shape of the activated stem cell 27 transmitted from the electron microscope 13 and the imaging time in a storage area in a state associated with the donor identifier and the stem cell identifier. The computer 11 displays the enlarged image of the planar shape of the activated stem cell 27 transmitted from the electron microscope 13 and the imaging time on the display 17. The person in charge confirms (views) the enlarged image of the planar shape of the activated stem cells 27 displayed on the display 17 at intervals of about 1 to 2 hours during 12 to 24 hours. Observe changes. Note that the person in charge may directly observe the change in the planar shape of the activated stem cell 27 from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 12 to 24 hours.

The initial planar shape (planar shape before colonization) of the activated stem cells 27 is substantially circular. When the planar shape of the activated stem cells 27 is substantially circular, the activated stem cells 27 are the bottom surface 29 (bottom) of the second flat culture vessel 28. The inner surface of the wall is not settled, and the activated stem cells 27 have not started to grow (differentiate). The deformed planar shape of the activated stem cell 27 (planar shape after colonization) is a flat shape in which the activated stem cell 27 is expanded (expanded) in one direction (predetermined direction) with the substantially circular shape before settlement as a nucleus. The activated stem cells 27 have settled on the bottom surface 28 (bottom wall inner surface) of the second flat culture vessel 28, and the activated stem cells 27 have started to proliferate (activate).

As a result of the observation in the activated stem cell fixing step, the person in charge is able to activate the stem cell when the enlarged image of the planar shape of the activated stem cell 27 displayed on the display 17 is observed in a substantially circular shape as shown in FIG. 27 is determined not to have settled on the bottom surface 29 (bottom wall inner surface) of the second flat culture vessel 28, and the change in the planar shape of the activated stem cells 27 is continuously observed at intervals of about 1 to 2 hours. As shown in FIG. 9, when the planar shape of the activated stem cell 27 displayed on the display 17 is deformed from a substantially circular shape to an irregular flat shape with the substantially circular shape as a nucleus, the person in charge displays the activated stem cell 27 as the second shape. It is determined that it has settled on the bottom surface 29 of the flat culture vessel 28.

When a large culture container having a capacity exceeding 60 cc and a bottom area exceeding 72 mm ² is used when the activated stem cells 27 are fixed, the activated stem cells 27 are difficult to settle on the bottom surface of the container and the activated stem cells 27 are proliferated. Although slow, by using the second flat culture vessel 28 having the capacity and the bottom area, the activated stem cells 27 can be easily fixed on the bottom surface 29 of the culture vessel 28, and the activated stem cells are cultured in the culture vessel 28. 27 can be propagated quickly. While the second flat culture vessel 28 is left statically at a temperature substantially the same as the body temperature for 36 to 48 hours, the activated stem cells 27 in the culture vessel 28 are separated at intervals of about 1 to 2 hours during 12 to 24 hours. Since the deformation from the initial planar shape is observed, the deformation of the activated stem cells 27 is not overlooked, and the fixation of the activated stem cells 27 to the bottom surface 29 of the culture vessel 28 can be confirmed accurately.

As a result of the observation in the activated stem cell fixing step, as shown in FIG. 9, the mesenchymal activated stem cell 27 is deformed from a substantially circular shape (initial planar shape) to an indeterminate flat shape with the substantially circular shape as a nucleus, and the activated stem cell After confirming the fixation on the bottom surface 29 of the 27 second flat culture vessel 28, an activated stem cell culture step is performed. A person in charge such as a doctor, a nurse, or a researcher confirms that the activated stem cells 27 are fixed on the bottom surface 29 of the second flat culture vessel 28, and then clicks an activation stem cell fixing completion button displayed on the display 17. When the activated stem cell colonization completion button is clicked, the computer 11 displays an activated stem cell colonization completion message, an activated stem cell culture observation button, and an activated stem cell culture completion button on the display 17.

In the activated stem cell culturing step, the mixed culture solution 26 of the culture solution 23 and the culture product solution 24 injected into the second flat culture vessel 28 is discharged from the culture vessel 28, and the new culture solution 23 and A new culture product solution 24 (a culture product solution 24 produced in the course of culturing the stem cells 30 before storage of the dormant stem cells 22 (which is the source of the dormant stem cells 22)) is injected (accommodated). Note that the injection rate of the new culture solution 24 to be injected into the second flat culture vessel 28 is 5 to 15%, preferably 100% when the total injection amount of the new culture solution 23 to be injected into the culture vessel 28 is 100%. Is 8 to 12%, more preferably 10%.

The person in charge removes the second flat culture vessel 28 from the sample holder of the electron microscope 16 and uses a syringe or pipette to mix the mixed culture solution 26 of the culture solution 23 and the culture product solution 24 injected into the culture vessel 28 in the activated stem cell fixing step. The culture medium is discharged from the culture container 28 using a syringe, and a new culture solution 23 is injected (contained) into the culture container 28 using a syringe or pipette, and a new culture solution 24 is generated using a syringe or pipette. It is injected (accommodated) from the liquid container 20 into the culture container 28. The new culture solution 23 and the new culture product solution 24 are the same as those injected into the second flat culture vessel 28 in the activated stem cell fixing step.

The person in charge left the second flat culture vessel 28 statically at the same temperature (about 37 ° C.) as the body temperature for 36 to 48 hours (silently left without moving), and for about 36 to 48 hours. The total planar area of the activated stem cells 27 fixed on the bottom surface 29 of the culture vessel 28 at intervals of 1 to 2 hours with respect to the bottom surface area of the culture vessel 28 is observed with the electron microscope 13, and the total planar area of the activated stem cells 27 is the culture vessel. It is determined whether or not the target ratio (second target ratio) has been reached with respect to the bottom surface area of 28. The target ratio of the total planar area of the activated stem cells 27 to the bottom area of the second flat culture vessel 28 is 88 to 92% (88 to 92% confluent).

The person in charge injects a new culture solution 23 and a new culture product solution 24 into the second flat culture vessel 28 and then clicks an activated stem cell culture observation button, and the culture vessel 28 is placed in the sample holder of the electron microscope 13. Install (set) on. A spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 16 and the bottom 45 of the second flat culture vessel 28, and the bottom 45 of the culture vessel 28 is held in a state where it is lifted by the spacer 42. The culture vessel 28 is held at a predetermined angle so that the bottom 45 of the vessel 28 is up and the top 46 (injection port 47) of the culture vessel 28 is down (see FIG. 8). In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 16 and the top 46 of the second flat culture vessel 28, and the top 46 of the culture vessel 28 is held in a state lifted by the spacer 42. You may hold | maintain the culture container 28 in the state inclined by the predetermined angle so that the top part 46 of the container 28 may become the upper side and the bottom part 45 of the culture container 28 may become the lower side. The inclination angle α2 of the second flat culture vessel 28 with respect to the upper surface 40 of the sample holder 39 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

In the culture product production method, after confirming that the activated stem cells 27 are fixed, a new culture solution 23 and a new culture product 24 are injected into the culture vessel 28 while discharging the mixed culture solution 26 in the culture vessel 28. Thus, the proliferation of the activated stem cells 27 can be surely promoted. In the culture product production method, the second flat culture vessel 28 is inclined at the inclination angle with respect to the upper surface 40 of the sample holder 39, whereby the activated stem cells 27, the culture solution 23, and the culture product solution 24 are contained in the culture vessel 28. Is biased to the top 46 side (or the bottom 45 side) of the culture vessel 28, and the water pressure of the activated stem cells 27, the culture solution 23, and the culture product solution 24 on the top 46 side (or the bottom 45 side) of the culture vessel 28. And the activated stem cells 27 are concentrated on the bottom 45 side (or the top 46 side) of the culture vessel 28, thereby increasing the activity of the activated stem cells 27, and the activated stem cells on the bottom surface 29 of the culture vessel 27. 27 can be propagated (differentiated) easily and rapidly.

The display 17 displays a message indicating that the activated stem cell culture is being observed and an activated stem cell culture completion button. After confirming the establishment of the activated stem cells 27, the mixed culture solution 26 of the culture solution 23 and the culture product solution 24 is discharged from the second flat culture vessel 28 and secreted from the new culture solution 23 and a single type of stem cell 30. By injecting a new culture product solution 24 containing the prescribed metabolite into the second flat culture vessel 28, the metabolite of the stem cell 30 itself becomes a trigger, and the activated stem cell 27 starts to be activated quickly. Thus, the proliferation of the activated stem cells 27 can be surely promoted.

The electron microscope 13 takes a magnified image of the planar shape of the activated stem cells 27 in the second flat culture vessel 28 at intervals of about 1 to 2 hours, and captures a magnified image of the planar shape of the photographed activated stem cells 27 about 1-2. It transmits to the computer 11 at time intervals. The image capturing interval and the image transmission interval in the electron microscope 13 can be freely set within 1 to 2 hours by an input device such as a keyboard 14 or a mouse 15. The computer 11 stores (stores) the enlarged image of the planar shape of the activated stem cell 27 transmitted from the electron microscope 13 and the imaging time in a storage area in a state associated with the donor identifier and the stem cell identifier. The computer 11 displays the enlarged image of the planar shape of the activated stem cell 27 transmitted from the electron microscope 13 and the imaging time on the display 17.

The person in charge confirms (views) the enlarged image of the planar shape of the activated stem cells 27 displayed on the display 17 at intervals of about 1 to 2 hours during 36 to 48 hours, and the bottom surface of the second flat culture vessel 28 While observing the total planar area of the activated stem cells 27 fixed to the bottom surface area of the culture vessel 28, the total planar area of the activated stem cells 27 is a target ratio (second target ratio) with respect to the bottom surface area of the culture container 28. Judge whether or not (88-92% confluent) has been reached. The person in charge directly observes the total planar area of the activated stem cells 27 with respect to the bottom area of the second flat culture vessel 28 from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 36 to 48 hours. It may be determined whether the total planar area of the stem cell 27 has reached a target ratio (88-92% confluent) with respect to the bottom area of the culture vessel 28.

As a result of the observation in the activated stem cell culture process, the person in charge, as shown in FIG. 9, determines that the total planar area of the activated stem cells 27 displayed on the display 17 with respect to the bottom area of the second flat culture vessel 28 is the target ratio (first (2 target ratio) (88 to 92% confluent) is not reached, the total planar area with respect to the bottom area of the culture vessel 28 of the activated stem cells 27 is continuously observed at intervals of about 1 to 2 hours. In addition, when the total planar area of the activated stem cells 27 reaches the target ratio (second target ratio) with respect to the entire area of the enlarged image displayed on the display 17, the second flat culture vessel 28 of the activated stem cells 27. It is assumed that the total plane area with respect to the bottom area of the target has reached the target ratio.

As a result of observation in the activated stem cell culturing step, the activated stem cells 27 proliferate on the bottom surface 29 (bottom wall inner surface) of the second flat culture vessel 28 to form colonies, and the planar shape of the activated stem cells 27 is obtained. As shown in FIG. 10, the total planar area of the activated stem cells 27 displayed on the display 17 with respect to the bottom surface area of the culture vessel 28 is a target ratio (second target ratio) (88 to 92% confluent). In this case, the activated stem cells 27 are further activated and proliferated. When the total planar area of the activated stem cells 27 with respect to the bottom area of the second flat culture vessel 28 reaches a target ratio, the activated stem cells 27 are extracted from the culture vessel 28, and the extracted activated stem cells 27 are treated for various diseases. And used for regenerative medicine.

The person in charge discharges the mixed culture solution 26 injected into the second flat culture vessel 28 from the culture vessel 28 using a syringe or pipette, and the culture vessel 28 is washed with PBS, and then the trypsin solution is added to the culture vessel. 28. When a trypsin solution is injected into the second flat culture vessel 28, activated stem cells 27 fixed (proliferated) on the bottom surface 29 of the culture vessel 28 are detached from the bottom surface 29 by the trypsin solution and float on the water surface of the trypsin solution. The person in charge aspirates the activated stem cells 27 using a pipette, and accommodates the activated stem cells 27 in the pipette. The activated stem cells 27 cultured in the activated stem cell culturing step are accommodated in the stem cell storage container 19, and the stem cell storage container 19 containing the activated stem cells 27 is placed in the refrigerator 14 or the freezer 14 and activated in the refrigerator 14 or the freezer 14. Stem cells 27 can be stored.

The person in charge confirms that the total planar area of the activated stem cells 27 with respect to the bottom surface area of the second flat culture vessel 28 has reached the target ratio (second target ratio), and then the activated stem cell culture displayed on the display 17. Click the Done button. When the activation stem cell culture completion button is clicked, the computer 11 displays an initial screen on the display 17. To end the culture, click the logout button on the initial screen. When the logout button is clicked, the computer 11 logs out from the system 10.

The method for producing activated stem cells is a predetermined metabolite produced and secreted from the stem cells 30 in the culture process (proliferation process) of a single species of mesenchymal stem cells 30 before storage of the activated stem cells 27 (dormant stem cells 22). Is used as a trigger, and the activated stem cell 27 starts to be activated quickly, and the bottom surface of the second flat culture vessel 28 (second culture vessel) is used. It is possible to promote the establishment of the activated stem cells 27 with respect to 29 and the proliferation of the activated stem cells 27, and it is possible to rapidly proliferate the activated stem cells 27 while maintaining the activity of the activated stem cells 27.

The activated stem cell production method is activated using the culture product solution 24 generated in the culturing process (proliferation process) of the single species of mesenchymal stem cells 30 before the preservation of the activated stem cells 27 (dormant stem cells 22). The stem cells 27 are fixed on the bottom surface 29 of the second flat culture vessel 28, and the activated stem cells 27 are grown using the culture product solution 24 until the total planar area reaches the second target ratio, whereby a single type of stem cell 27 is obtained. The mesenchymal activated stem cells 27 can be efficiently and reliably established and proliferated, and the required amount of single-type mesenchymal activated stem cells 27 having sufficient activity can be efficiently produced.

In the activated stem cell production method, the activated stem cell 27 is subjected to an activated stem cell fixing step and an activated stem cell culturing step to activate a pure single species of mesenchymal system that does not contain unnecessary mesenchymal stem cells. Since the stem cells 27 can be produced and do not contain a variety of mesenchymal stem cells, activated stem cells 27 having a high therapeutic effect on various diseases and a high regenerative effect in regenerative medicine can be produced. Activated stem cells 27 that can be used for treatment of diseases and that can be used in a timely manner can be produced, and activated stem cells 27 that can be used for regeneration of various tissues and organs in a timely manner. Can be manufactured.

FIG. 12 is a perspective view of the glass test tube 32 used in the stem cell first fixing step, and FIG. 13 is a diagram for explaining the stem cell first fixing step continued from FIG. FIG. 14 is a diagram for explaining the stem cell first fixing step continued from FIG. 13. A method for producing a single type of mesenchymal stem cell 30 that is the source of the dormant stem cell 22 before storage of the dormant stem cell 22 will be described with reference to FIGS.

A single type of stem cell 30 uses a raw bone marrow fluid 31 collected from a plurality of donors (people) and is activated by the activated stem cell culture system 10 for the first stem cell first fixing step, the first stem cell first culturing step, 2 It is made by performing the second stem cell second fixing step and the second stem cell second culturing step. The system 10 cultures a single type of single mesenchymal stem cell 38 (first stem cell) from among a plurality of types of mesenchymal stem cells contained in the raw bone marrow fluid 31.

In the first stem cell first fixing step, the raw bone marrow fluid 31 first collected from the donor is separated into layers. In the bone marrow fluid collection, 2 to 3 cc (2 to 3 ml) of the raw bone marrow fluid 31 is collected from the donor's sternum or iliac bone (pelvis). The raw bone marrow fluid 31 is collected by “bone marrow puncture” (Marc) in which the donor is locally anesthetized and then punctured with the bone marrow to aspirate the bone marrow fluid (bone marrow blood). A person in charge such as a doctor, a nurse, or a researcher starts the system 10 in the computer 11 simultaneously with the collection of the raw bone marrow fluid 31, and clicks the stem cell culture button on the initial screen. A data acquisition button and a logout button are displayed on the display 17.

The person in charge clicks the data acquisition button, and then inputs donor data and stem cell data into the computer 11 using an input device such as the keyboard 14 or the mouse 15. The computer 11 generates a unique donor identifier and a stem cell identifier that specify each donor each time donor data and stem cell data are input (every time a raw bone marrow fluid is collected from the donor), and the donor data and stem cell data are generated. Store (store) in the storage area in a state associated with the donor identifier and the stem cell identifier. The display 17 displays a donor data acquisition message, a data storage button, and a logout button.

As shown in FIG. 12, 2-3 cc of raw bone marrow fluid 31 collected from a donor is injected (accommodated) into a glass test tube 32 (separation container) extending in the vertical direction. The 2-3 cc raw bone marrow fluid 31 contains 0.5 to 1 ml (about 5 × 10 ⁷ (cells / ml)) of a plurality of types of mesenchymal stem cells 38 (mesenchymal first stem cells). .

The IC tag 18 is attached to the outer peripheral surface of the glass test tube 32 into which the raw bone marrow fluid 31 is injected. The person in charge writes donor data and stem cell data to the IC tag 18 of the glass test tube 32 using the IC tag reader / writer 12. When donor data or stem cell data is written to the IC tag 18, the computer 11 displays a data storage end message, a bone marrow fluid separation button, and a logout button on the display 17.

As shown in FIG. 13, the glass test tube 32 into which the raw bone marrow fluid 31 has been injected is set in a test tube stand 33 and is housed in a thermostatic chamber 34 together with the test tube stand 33. The person in charge clicks on the bone marrow fluid separation button displayed on the display 17, then injects the raw bone marrow fluid 31 from the syringe into the glass test tube 32, and sets the glass test tube 32 into which the raw material bone marrow fluid 31 is injected as a test tube holder 33. Insert (set) into. The display 17 displays a bone marrow fluid separation in-progress message and a bone marrow fluid separation end button.

The person in charge accommodates the test tube stand 33 in the thermostat 34 and statically leaves the glass test tube 32 into which the raw material bone marrow fluid 31 has been injected in the thermostat 34 for a predetermined time (about 2 hours) without moving. put. The temperature in the thermostat 34 is maintained at approximately 37 ° C., which is substantially the same as the body temperature. By leaving the glass test tube 32 statically in the thermostatic chamber 34 for a predetermined time (about 2 hours), the raw bone marrow fluid 31 injected into the test tube 32 is vertically moved in the test tube 32 as shown in FIG. It is separated into several layers (in FIG. 14, it is separated into three layers).

After the raw bone marrow fluid 31 is separated into layers, the bone marrow fluid is extracted. In the extraction of the bone marrow fluid, the intermediate layer bone marrow fluid 35 is extracted from the raw material bone marrow fluid 31 separated into layers. The person in charge takes out the test tube holder 33 from the thermostat 34, pulls out the glass test tube 32 from the test tube holder 33, confirms that the raw bone marrow fluid 31 has been separated into layers, and then separates the raw bone marrow fluid 31 separated into layers. The intermediate bone marrow fluid 35 existing in a specific layer is extracted.

The person in charge aspirates the intermediate layer bone marrow fluid 35 having a layer thickness of 3 to 4 mm located in the intermediate layer of the raw material bone marrow fluid 31 separated into layers using a syringe or a pipette. After separating the raw bone marrow fluid 31 containing various mesenchymal stem cells into layers in the vertical direction, a specific intermediate layer bone marrow fluid 35 is extracted from the raw material bone marrow fluid 31, so that it is unnecessary in the raw material bone marrow fluid 31 The mesenchymal stem cells can be removed.

After extracting the intermediate layer bone marrow fluid 35, click the bone marrow fluid separation end button. The display 17 displays a bone marrow fluid separation end message and a data storage button. The person in charge prepares a third flat culture vessel (third culture vessel) (not shown), attaches an IC tag to the outer peripheral surface of the culture vessel, clicks a data storage button, and also uses an IC tag reader / writer 12. The donor data and stem cell data of the IC tag 18 attached to the glass test tube 32 are stored in the IC tag. The computer 11 displays a data storage completion message, a stem cell first fixing observation button, and a stem cell first fixing completion button on the display 17.

The person in charge extracts the specific intermediate layer bone marrow fluid 35 located in the intermediate layer from the raw material bone marrow fluid 31, and then uses the syringe or pipette to transfer the intermediate layer bone marrow fluid 35 and the culture solution 23 to the third flat culture container (the first (3 culture vessels), and the culture vessel is left to stand statically (without being moved) for 12 to 24 hours while maintaining the culture vessel at a temperature approximately the same as the body temperature (about 37 ° C.). During an interval of 24 hours, the mesenchymal stem cells 38 (first stem cells) contained in the intermediate layer bone marrow fluid 35 in the culture vessel are observed for deformation from the initial planar shape with an electron microscope at intervals of about 1 to 2 hours. It is determined whether 36 has settled on the bottom surface of the culture vessel.

The third flat culture vessel used in the first stem cell colonization step or stem cell first culture step is the same as the first flat culture vessel 21 (first culture vessel) used in the dormant stem cell colonization step or dormant stem cell culture step ( (Refer to FIG. 2). The third flat culture vessel has a capacity of about 20 to 30 cc (preferably 25 cc) and a bottom area of about 25 to 36 mm ² . The third flat culture vessel has a side length of 5 to 6 mm.

FIG. 15 is a partially enlarged view showing an example of the planar shape of the stem cell 38. FIG. 16 is a partially enlarged view showing another example of the planar shape of the stem cell 38, and FIG. 17 is a partially enlarged view showing another example of the planar shape of the stem cell 38. 15 to 17 show enlarged images of the planar shape of the stem cell 38 taken by the electron microscope 13.

The person in charge clicks the first fixation observation button and installs (sets) the third flat culture vessel in the sample holder 39 of the electron microscope 13. In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 13 and the bottom of the third flat culture vessel, and the bottom of the third flat culture vessel is held in a state lifted by the spacer 42. The third flat culture vessel is held at a predetermined angle so that the bottom of the flat culture vessel is up and the top (inlet) of the third flat culture vessel is down (see FIG. 3). In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 13 and the top of the third flat culture container, and the top of the third flat culture container is held in a state of being lifted by the spacer 42. You may hold | maintain the 3rd flat culture container in the state inclined by the predetermined angle so that the top part of a flat culture container may become an upper side and the bottom part of a 3rd flat culture container may become a lower side. The inclination angle α1 of the third flat culture vessel with respect to the upper surface 40 of the sample holder 39 is in the range of 2 to 5 °, preferably in the range of 2 to 3 °.

In the method for producing a culture solution, the third flat culture vessel is inclined at the inclination angle with respect to the upper surface 40 of the sample holder 39, so that the stem cells 38 and the culture solution 23 are contained in the third flat culture vessel in the third flat culture vessel. Of the third flat culture vessel, the water pressure of the stem cell 38 and the culture solution 23 is increased on the top flat side (or the bottom side) of the third flat culture vessel, and the stem cell 38 is removed from the third flat culture vessel. By concentrating on the top side (or the bottom side), the activity of the stem cells 38 is increased, and the stem cells 38 can be easily and quickly fixed on the bottom surface 25 of the third flat culture vessel.

The display 17 displays a message indicating that the first stem cell colonization observation is being performed and a stem cell first colonization completion button. The electron microscope 13 takes enlarged images of the planar shape of the stem cells 38 contained in the intermediate layer bone marrow fluid 35 injected into the third flat culture vessel at intervals of about 1 to 2 hours, and enlarges the planar shape of the taken stem cells 38. Images are sent to the computer 11 at approximately 1-2 hour intervals.

The image capturing interval and the image transmission interval in the electron microscope 13 can be freely set within 1 to 2 hours by an input device such as a keyboard 14 or a mouse 15. The mesenchymal stem cells 38 (first stem cells) injected into the third flat culture vessel are cultured on the bottom of the culture vessel while being fixed on the bottom of the culture vessel over time, and gradually grow (differentiate) on the bottom of the culture vessel. ) To form colonies.

The computer 11 stores (stores) the enlarged image of the planar shape of the mesenchymal stem cell 38 (first stem cell) transmitted from the electron microscope 13 and the imaging time in a storage area in association with the donor identifier and the stem cell identifier. To do. The computer 11 displays the enlarged image of the planar shape of the stem cell 38 transmitted from the electron microscope 13 and the imaging time on the display 17. The person in charge confirms (views) the enlarged image of the planar shape of the stem cell 38 displayed on the display 17 at intervals of about 1 to 2 hours during 12 to 24 hours, and observes the change in the planar shape of the stem cell 38. . Note that the person in charge may directly observe the change in the planar shape of the stem cell 38 from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 12 to 24 hours.

The initial planar shape of the stem cell 38 (first stem cell) is substantially circular, and when the planar shape of the stem cell 38 is substantially circular, the stem cell 38 is not fixed on the bottom surface (inner wall inner surface) of the third flat culture vessel, The stem cell 38 has not started proliferation (differentiation). The planar shape after deformation of the stem cell 38 (first stem cell) is a flat shape in which the stem cell 38 is expanded (expanded) in one direction (predetermined direction) with a substantially circular shape before fixing as a nucleus, and the stem cell 38 is a culture vessel. The stem cells 38 have started to proliferate.

As a result of observation in the stem cell first fixing step, the person in charge, as shown in FIG. 15, when the enlarged image of the planar shape of the stem cell 38 (first stem cell) displayed on the display 17 is observed in a substantially circular shape, It is determined that the stem cells 38 have not settled on the bottom surface (bottom wall inner surface) of the third flat culture vessel, and changes in the planar shape of the stem cells 38 are continuously observed at intervals of about 1 to 2 hours. As shown in FIG. 16, the person in charge cultivates the stem cell 38 when the planar shape of the stem cell 38 (first stem cell) displayed on the display 17 is changed from a substantially circular shape to an irregular flat shape with the substantially circular shape as a nucleus. Judged to have settled on the bottom of the container.

If a large culture container with a capacity exceeding 30 cc and a bottom area exceeding 36 mm ² is used when the stem cells 38 (first stem cells) are established, the stem cells 38 are difficult to settle on the bottom of the container and the proliferation of the stem cells 38 is slow. However, by using a third flat culture vessel having the same capacity and bottom surface area as the first flat culture vessel 21, the stem cells 38 can be easily fixed on the bottom surface of the culture vessel, and the stem cells 38 in the culture vessel can be obtained. Can be propagated quickly. Stem cells 38 (first stem cells) in the culture vessel at intervals of about 1 to 2 hours between 12 and 24 hours, while the third flat culture vessel is left statically at a temperature substantially the same as the body temperature for 12 to 24 hours. Since the deformation from the initial planar shape is observed, the deformation of the stem cell 38 is not missed, and the establishment of the stem cell 38 on the bottom surface of the third flat culture vessel can be confirmed accurately.

As a result of observation in the first stem cell colonization step, the mesenchymal stem cell 38 (first stem cell) is deformed from a substantially circular shape (initial planar shape) to an irregular flat shape with the substantially circular shape as a nucleus, and the third flat culture of the stem cell 38 is performed. After confirming the fixation on the bottom surface of the container, the stem cell first culture step is performed. A person in charge such as a doctor, a nurse, or a researcher confirms that the stem cell 38 is fixed on the bottom surface of the third flat culture container, and then clicks the first fixing completion button displayed on the display 17. When the first fixing completion button is clicked, the computer 11 displays a stem cell first fixing completion message, a stem cell first culture observation button, and a stem cell first culture completion button on the display 17.

In the stem cell first culture step, the culture solution 23 injected into the third flat culture vessel is discharged from the culture vessel, and a new culture solution 23 is injected (accommodated) into the culture vessel. The person in charge removes the third flat culture container from the sample holder of the electron microscope 16, discharges the culture solution 23 injected into the culture container in the stem cell first fixing step from the culture container using a syringe or pipette, and then injects the syringe or pipette. A new culture solution 23 is injected (accommodated) into the culture vessel by using.

The person in charge statically left the third flat culture vessel at the same temperature (about 37 ° C.) as the body temperature for 36 to 48 hours (leaves it silently without moving), and for about 36 to 48 hours, The total planar area of the stem cells 38 (first stem cells) fixed on the bottom surface of the culture vessel at intervals of ˜2 hours with respect to the bottom surface area of the third flat culture vessel is observed with the electron microscope 13, and the total planar area of the stem cells 38 is It is determined whether or not the target ratio (third target ratio) has been reached with respect to the bottom surface area. The third target ratio of the total planar area of the stem cells 38 to the bottom area of the culture vessel is 70-80% (70-80% confluent).

The person in charge injects a new culture solution 23 into the culture vessel, clicks the first culture observation button, and installs (sets) the culture vessel in the sample holder 39 of the electron microscope 13. In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 16 and the bottom of the third flat culture vessel, and the bottom of the third flat culture vessel is held in a state of being lifted by the spacer 42. The third flat culture vessel is held at a predetermined angle so that the bottom of the flat culture vessel is up and the top (inlet) of the third flat culture vessel is down (see FIG. 3). In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 16 and the top of the third flat culture container, and the top of the third flat culture container is held in a state of being lifted by the spacer 42. You may hold | maintain the 3rd flat culture container in the state inclined by the predetermined angle so that the top part of a flat culture container may become an upper side and the bottom part of a 3rd flat culture container may become a lower side. The inclination angle α1 of the third flat culture vessel with respect to the upper surface 40 of the sample holder 39 is in the range of 2 to 5 °, preferably in the range of 2 to 3 °.

In the culture product production method, after confirming the establishment of the stem cells 38, the culture solution 23 in the third flat culture vessel is discharged, and the new culture solution 23 is injected into the third flat culture vessel, thereby proliferating the stem cells 38. Can be surely promoted. In the method for producing a culture solution, the third flat culture vessel is inclined at the inclination angle with respect to the upper surface 40 of the sample holder 39, so that the stem cells 38 and the culture solution 23 are contained in the third flat culture vessel in the third flat culture vessel. The stem cell 38 and the culture solution 23 are increased in water pressure on the top 43 side (or the bottom 41 side) of the third flat culture vessel, and the stem cell 38 is third. It concentrates on the bottom 41 side (or the top 43 side) of the flat culture vessel, thereby increasing the activity of the stem cells 38 and allowing the stem cells 38 to proliferate (differentiate) easily and rapidly on the bottom surface 25 of the third flat culture vessel. be able to.

On the display 17, a stem cell first culture observation execution message and a stem cell first culture completion button are displayed. After confirming the establishment of the stem cells, the growth of the stem cells 38 can be surely promoted by injecting a new culture solution 23 into the culture vessel while discharging the culture solution 23 in the third flat culture vessel.

The electron microscope 13 takes a magnified image of the planar shape of the stem cell 38 in the third flat culture vessel at intervals of about 1-2 hours, and the computer 11 takes a magnified image of the planar shape of the photographed stem cell 38 at intervals of about 1-2 hours. Send to. The image capturing interval and the image transmission interval in the electron microscope 13 can be freely set within 1 to 2 hours by an input device such as a keyboard 14 or a mouse 15. The computer 11 stores (stores) the planar shape enlarged image of the stem cell 38 (first stem cell) and the imaging time transmitted from the electron microscope 13 in a storage area in a state associated with the donor identifier and the stem cell identifier. The computer 11 displays the enlarged image of the planar shape of the stem cell 38 transmitted from the electron microscope 13 and the imaging time on the display 17.

The person in charge confirmed (viewed) the enlarged image of the planar shape of the stem cell 38 displayed on the display 17 at intervals of about 1 to 2 hours during 36 to 48 hours, and settled on the bottom surface of the third flat culture vessel. While observing the total planar area of the stem cell 38 (first stem cell) relative to the bottom surface area of the culture container, the total planar area of the stem cell 38 is a target ratio (third target ratio) (70 to 80%) with respect to the bottom surface area of the culture container. It is determined whether or not confluence has been reached. The person in charge directly observes the total plane area of the stem cell 38 with respect to the bottom area of the culture vessel from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 36 to 48 hours. It may be determined whether or not a target ratio (70 to 80% confluence) has been reached with respect to the bottom surface area of the culture vessel.

As shown in FIG. 16, the person in charge of the person in charge of the stem cell first culturing step has a target ratio of the total planar area of the stem cell 38 (first stem cell) displayed on the display 17 with respect to the bottom area of the third flat culture vessel. If the third target ratio (70 to 80% confluent) has not been reached, the total planar area of the stem cell 38 relative to the bottom area of the culture vessel is continuously observed at intervals of about 1 to 2 hours. When the total planar area of the stem cells 38 reaches the target ratio (third target ratio) with respect to the entire area of the enlarged image displayed on the display 17, the total of the stem cells 38 relative to the bottom area of the third flat culture vessel Assume that the planar area has reached the target percentage.

As a result of the observation in the stem cell first culture step, as shown in FIG. 17, the stem cells 38 (first stem cells) proliferate on the bottom surface (bottom wall inner surface) of the third flat culture container, and the stem cells form colonies. When the total planar area of the stem cells 38 displayed on the display 17 with respect to the bottom area of the culture vessel reaches the target ratio (third target ratio) (70 to 80% confluent) 3. Extract mesenchymal stem cells 38 grown (differentiated) from the flat culture vessel.

After confirming that the total planar area of the mesenchymal stem cell 38 (the mesenchymal first stem cell) with respect to the bottom area of the third flat culture vessel has reached the target ratio (third target ratio), the stem cell second colonization step is performed. Done. The person in charge confirms that the total planar area of the stem cells 38 with respect to the bottom surface area of the third flat culture vessel has reached the target ratio (third target ratio), and then presses the stem cell first culture completion button displayed on the display 17. click. When the stem cell first culture completion button is clicked, the computer 11 displays a stem cell first culture end button and a stem cell first extraction end button on the display 17.

The person in charge discharges the culture solution 23 injected into the third flat culture vessel from the culture vessel, rinses the culture vessel with PBS, and then injects the trypsin solution into the culture vessel. When the trypsin solution is injected into the third flat culture vessel, the mesenchymal stem cells 38 (first stem cells) fixed on the bottom surface of the culture vessel are detached from the bottom surface by the trypsin solution and float on the water surface of the trypsin solution. The person in charge aspirates the stem cells 38 using a pipette, and accommodates the stem cells 38 in the pipette.

FIG. 18 is a perspective view of the glass test tube 36 and the centrifuge 37 used in the stem cell second fixing step, and FIG. 19 is a perspective view of the glass test tube 36 after centrifugation. After extracting the stem cells 38 (first stem cells) from the third flat culture vessel, the stem cell second fixing step is performed. In the stem cell second fixing step, the extracted stem cells 38 are centrifuged in layers by a centrifuge 37. A person in charge such as a doctor, nurse, researcher or the like clicks the stem cell first extraction end button displayed on the display 17 after sucking the stem cells 38 from the third flat culture container into the pipette. The display 17 displays a stem cell separation message and a stem cell separation end button.

The person in charge injects (accommodates) the stem cells 38 (first stem cells) in the pipette into the glass test tube 36 and installs (sets) the glass test tube 36 in the centrifuge 37. The person in charge centrifuges the stem cells 38 with the centrifuge 37 for a predetermined time, and then removes the glass test tube 36 from the centrifuge 37. Stem cells 38 in the glass test tube 36 are separated into several layers in the vertical direction by a centrifuge 37.

After the stem cells 38 (first stem cells) are separated into layers, the mesenchymal stem cells 30 (second stem cells) located in the lower layer (lowermost layer) are extracted from the stem cells 38 separated in layers. Stem cells 38 (first stem cells) containing unnecessary stem cells are centrifuged in a centrifuge 37 to be separated into layers in the vertical direction, and stem cells 30 (second cells) located in the lowermost layer of the stem cells 38 centrifuged in layers. The specific stem cell 30 can be reliably extracted from the stem cell 38, and unnecessary stem cells can be removed from the stem cell 38.

When the total planar area of the stem cells 38 (first stem cells) exceeds 80% with respect to the bottom area of the third flat culture vessel (third culture vessel) and the stem cells 38 proliferate, the activity of the stem cells 38 is gradually lost. Since the stem cell 38 is extracted from the culture container when the total planar area of the stem cell 38 (first stem cell) grows to 70 to 80% with respect to the bottom surface area of the container, the activity of the stem cell 38 can be retained. The stem cell 38 can be grown while maintaining the activity, and the stem cell 30 (second stem cell) having activity can be extracted from the stem cell 38.

The person in charge uses the centrifuge 37 to separate the stem cells 38 (first stem cells) into layers in the vertical direction, and then removes the glass test tube 36 from the centrifuge 37 and presses the stem cell separation end button displayed on the display 17. click. The display 17 displays a stem cell separation end message and a data storage button. The person in charge prepares a fourth flat culture vessel (fourth culture vessel) (not shown), attaches the IC tag 18 to the outer peripheral surface of the fourth flat culture vessel, clicks the data storage button, and Using the tag reader / writer 12, the IC tag 18 stores donor data and stem cell data of the IC tag attached to the third flat culture vessel. The computer 11 displays a data storage completion message, a stem cell second fixation observation button, and a stem cell second fixation completion button on the display 17.

The fourth flat culture vessel used in the stem cell second colonization step and the stem cell second culture step is the second flat culture vessel 28 (second culture vessel) used in the activated stem cell colonization step and the activated stem cell culture step. They are the same (same shape and size) (see FIG. 6). The fourth flat culture vessel has a capacity of about 40 to 60 cc (preferably 50 cc) and a bottom area of about 50 to 72 mm ² . The culture container 28 has a side length of about 7 to 8.5 mm.

FIG. 20 is a partially enlarged view showing an example of the planar shape of the stem cell 30 (second stem cell), and FIG. 21 is a partially enlarged view showing another example of the planar shape of the stem cell 30 (second stem cell). FIG. 22 is a partially enlarged view showing another example of the planar shape of the stem cell 30 (second stem cell). 20 to 22 show enlarged images of the planar shape of the stem cell 30 (second stem cell) taken by the electron microscope 13.

A doctor, nurse, researcher, or the like is responsible for the mesenchymal stem cell 30 (second stem cell) present in the lower layer (lowermost layer) of the stem cells 38 (first stem cells) separated in layers in the glass test tube 36. ) Is extracted using a syringe or pipette, and the stem cells 30 and the culture solution 23 are injected (contained) into a fourth flat culture container (fourth culture container), and the culture container is heated to a temperature substantially equal to the body temperature (about 37 ° C.) for 36 to 48 hours, and left statically (without moving), and the stem cells 30 (second The deformation of the stem cells from the initial planar shape is observed with the electron microscope 13, and it is determined whether or not the stem cells 30 have settled on the bottom surface of the culture vessel.

The person in charge clicks the stem cell second fixation observation button and installs (sets) the fourth flat culture vessel in the sample holder of the electron microscope 13. A spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 13 and the bottom of the fourth flat culture vessel, and the bottom of the fourth flat culture vessel is held in a state lifted by the spacer 42. The fourth flat culture vessel is held at a predetermined angle so that the bottom of the flat culture vessel is on the top and the top (inlet) of the fourth flat culture vessel is on the bottom. In addition, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 13 and the top of the fourth flat culture vessel, and the top of the fourth flat culture vessel is held in a state lifted by the spacer 42. You may hold | maintain the 4th flat culture container in the state inclined at the predetermined angle so that the top part of a flat culture container may become an upper side and the bottom part of a 4th flat culture container may become a lower side. The inclination angle α2 of the fourth flat culture vessel with respect to the upper surface 40 of the sample holder 39 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

In the culture product production method, the fourth flat culture vessel is inclined at the inclination angle with respect to the upper surface 40 of the sample holder 39 so that the stem cells 30 and the culture solution 23 are contained in the fourth flat culture vessel in the fourth flat culture vessel. Of the fourth flat culture vessel, the water pressure of the stem cell 30 and the culture solution 23 is increased on the top side (or the bottom side) of the fourth flat culture vessel, and the stem cell 30 becomes a portion of the fourth flat culture vessel. By concentrating on the top side (or the bottom side), the activities of the stem cells 30 are increased, and the stem cells 30 can be easily and quickly fixed on the bottom surface of the fourth flat culture vessel.

On the display 17, a message indicating that the stem cell second colonization observation is being performed and a stem cell second colonization completion button are displayed. The electron microscope 13 takes a magnified image of the planar shape of the stem cell 30 (second stem cell) injected into the fourth flat culture container at intervals of about 1 to 2 hours, and the magnified image of the planar shape of the photographed stem cell 30 is approximately It is transmitted to the computer 11 at intervals of 1 to 2 hours. The image capturing interval and the image transmission interval in the electron microscope 13 can be freely set within 1 to 2 hours by an input device such as a keyboard 14 or a mouse 15. The mesenchymal stem cells 30 (second stem cells) injected into the fourth flat culture vessel are cultured on the bottom of the culture vessel while being fixed on the bottom of the culture vessel over time, and gradually grow ( Differentiation) to form colonies.

The computer 11 stores (stores) the enlarged image of the planar shape of the mesenchymal stem cell 30 (second stem cell) transmitted from the electron microscope 13 and the imaging time in a storage area in association with the donor identifier and the stem cell identifier. To do. The computer 11 displays the enlarged image of the planar shape of the stem cell 30 and the imaging time transmitted from the electron microscope 13 on the display 17. The person in charge confirms (views) the enlarged image of the planar shape of the stem cell 30 displayed on the display 17 at intervals of about 1 to 2 hours during 36 to 48 hours, and observes the change in the planar shape of the stem cell 30. . The person in charge may directly observe the change in the planar shape of the stem cell 30 from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 36 to 48 hours.

The initial planar shape of the stem cell 30 (second stem cell) is substantially circular. When the planar shape of the stem cell 30 is substantially circular, the stem cell 30 is not fixed on the bottom surface (bottom wall inner surface) of the fourth flat culture vessel, The stem cell 30 has not started proliferation (differentiation). The planar shape after deformation of the stem cell 30 (second stem cell) is a flat shape in which the stem cell 30 is expanded (expanded) in one direction (predetermined direction) in an irregular shape with the substantially circular shape before fixing as a nucleus. The stem cells 30 have been established on the bottom surface (bottom wall inner surface) of the flat culture container, and have started to proliferate.

As a result of the observation in the stem cell second fixing step, the person in charge, as shown in FIG. 20, when the enlarged image of the planar shape of the stem cell 30 (second stem cell) displayed on the display 17 is observed in a substantially circular shape, It is determined that the stem cell 30 has not settled on the bottom surface (inner wall inner surface) of the fourth flat culture vessel, and the change in the planar shape of the stem cell 30 is continuously observed at intervals of about 1 to 2 hours. As shown in FIG. 21, when the planar shape of the stem cell 30 (second stem cell) displayed on the display 17 is changed from a substantially circular shape to an irregular flat shape with the substantially circular shape as a nucleus, the person in charge cultivates the stem cell 30. Judged to have settled on the bottom of the container.

When a large culture container having a capacity exceeding 60 cc and a bottom area exceeding 72 mm ² is used when the stem cell 30 (second stem cell) is fixed, the stem cell 30 becomes difficult to settle on the bottom of the container and the growth of the stem cell 30 is slow. However, by using the fourth flat culture container having the capacity and the bottom area, the stem cells 30 can be easily fixed on the bottom surface of the culture container, and the stem cells 30 can be rapidly proliferated in the culture container. Stem cells 30 (second stem cells) in the culture vessel at intervals of about 1 to 2 hours between 36 and 48 hours while the fourth flat culture vessel is statically left at a temperature substantially the same as the body temperature for 36 to 48 hours. Since the deformation from the initial planar shape is observed, the deformation of the stem cell 30 is not missed, and the establishment of the stem cell 30 on the bottom surface of the culture container can be confirmed accurately.

As a result of the observation in the stem cell second fixing step, as shown in FIG. 22, the mesenchymal stem cell 30 (second stem cell) is deformed from a substantially circular shape (initial planar shape) to an indeterminate flat shape with a substantially circular shape as a nucleus. After confirming that the stem cells 30 are fixed on the bottom surface of the fourth flat culture vessel, the stem cell second culture step is performed. A person in charge, such as a doctor, nurse, or researcher, confirms that the stem cell 30 has settled on the bottom surface of the fourth flat culture container, and then clicks the stem cell second colonization completion button displayed on the display 17. When the stem cell second fixing completion button is clicked, the computer 11 displays a stem cell second fixing completion message, a stem cell second culture observation button, and a stem cell second culture completion button on the display 17.

In the stem cell second culture step, the culture solution 23 injected into the fourth flat culture vessel is discharged from the culture vessel, and a new culture solution 23 is injected (accommodated) into the culture vessel. The person in charge removes the fourth flat culture container from the sample holder of the electron microscope 16 and discharges the culture solution 23 injected into the culture container in the stem cell first fixing step from the culture container using a syringe or pipette, and the syringe or pipette. A new culture solution 23 is injected (accommodated) into the culture vessel by using.

The person in charge kept the 4th flat culture container statically for 36 to 48 hours at about the same temperature (about 37 ° C.) as the body temperature (silently left without moving), and about 36 to 48 hours. The total planar area of the stem cells 30 (second stem cells) fixed on the bottom surface of the culture container at intervals of 1 to 2 hours with respect to the bottom surface area of the culture container is observed with an electron microscope 13, and the 28 total planar areas of the stem cells are the bottom surface of the culture container. It is determined whether the target ratio (fourth target ratio) has been reached with respect to the area. The fourth target ratio of the total planar area of the stem cells 30 to the bottom area of the culture vessel is 88 to 92% (88 to 92% confluent).

The person in charge injects a new culture solution 23 into the fourth flat culture container, and then clicks the second culture observation button and installs (sets) the culture container in the sample holder of the electron microscope 13. A spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 16 and the bottom of the fourth flat culture vessel, and the bottom of the fourth flat culture vessel is held in a state lifted by the spacer 42. The fourth flat culture vessel is held at a predetermined angle so that the bottom of the flat culture vessel is up and the top (inlet) of the fourth flat culture vessel is down (see FIG. 8). Further, a spacer 42 is interposed between the upper surface 40 of the sample holder 39 of the electron microscope 16 and the top of the fourth flat culture vessel, and the top of the fourth flat culture vessel is held in a state of being lifted by the spacer 42. You may hold | maintain the 4th flat culture container in the state inclined at the predetermined angle so that the top part of a flat culture container may become an upper side and the bottom part of a 4th flat culture container may become a lower side. The inclination angle α2 of the fourth flat culture vessel with respect to the upper surface 40 of the sample holder 39 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

After confirming the establishment of the stem cells 30, the culture product production method injects a new culture solution 23 into the fourth flat culture vessel while discharging the culture solution 23 in the fourth flat culture vessel, thereby proliferating the stem cells 30. Can be surely promoted. In the culture product production method, the fourth flat culture vessel is inclined at the inclination angle with respect to the upper surface 40 of the sample holder 39 so that the stem cells 30 and the culture solution 23 are contained in the fourth flat culture vessel in the fourth flat culture vessel. Of the fourth flat culture vessel, the water pressure of the stem cell 30 and the culture solution 23 is increased on the top side (or the bottom side) of the fourth flat culture vessel, and the stem cell 30 becomes a portion of the fourth flat culture vessel. By concentrating on the bottom side (or the top side), the activity of the stem cells 30 is increased, and the stem cells 30 can be easily and rapidly grown (differentiated) on the bottom surface of the fourth flat culture vessel.

The display 17 displays a message indicating that the stem cell second culture observation is in progress and a stem cell second culture completion button. After confirming the establishment of the stem cells 30, the growth of the stem cells 30 (second stem cells) can be surely promoted by injecting a new culture solution 23 into the culture vessel while discharging the culture solution 23 in the culture vessel. .

The electron microscope 13 takes a magnified image of the planar shape of the stem cell 30 in the fourth flat culture container at intervals of about 1 to 2 hours, and the computer 11 takes a magnified image of the planar shape of the photographed stem cell 30 at intervals of about 1 to 2 hours. Send to. The image capturing interval and the image transmission interval in the electron microscope 13 can be freely set within 1 to 2 hours by an input device such as a keyboard 14 or a mouse 15. The computer 11 stores (stores) the enlarged image of the planar shape of the stem cell 30 (second stem cell) and the imaging time transmitted from the electron microscope 13 in a storage area in a state associated with the donor identifier and the stem cell identifier. The computer 11 displays the enlarged image of the planar shape of the stem cell 30 and the imaging time transmitted from the electron microscope 13 on the display 17.

The person in charge confirmed (viewed) the enlarged image of the planar shape of the stem cell 30 displayed on the display 17 at intervals of about 1 to 2 hours during 36 to 48 hours, and settled on the bottom surface of the fourth flat culture vessel. While observing the total planar area of the stem cell 30 (second stem cell) relative to the bottom surface area of the culture vessel, the total planar area of the stem cell 30 is a target ratio (fourth target ratio) with respect to the bottom area of the fourth flat culture container (88 Judgment is made on whether to reach 92% confluence. The person in charge directly observes the total plane area of the stem cell 30 with respect to the bottom area of the culture vessel from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 36 to 48 hours. It may be determined whether or not a target ratio (88-92% confluence) has been reached with respect to the bottom surface area of the culture vessel.

As a result of the observation in the stem cell second culture step, the person in charge shows the target ratio of the total planar area with respect to the bottom area of the fourth flat culture vessel of the stem cells 30 (second stem cells) displayed on the display 17 as shown in FIG. If the (fourth target ratio) (88-92% confluent) is not reached, the total planar area of the stem cell 30 relative to the bottom area of the culture vessel is continuously observed at intervals of about 1 to 2 hours. In addition, when the total planar area of the stem cells 30 reaches the target ratio (fourth target ratio) with respect to the total area of the enlarged image displayed on the display 17, the total of the stem cells 30 with respect to the bottom area of the fourth flat culture vessel Assume that the planar area has reached the target percentage.

As a result of the observation in the stem cell second culturing step, as shown in FIG. 22, the stem cell 30 (second stem cell) proliferates on the bottom surface (bottom wall inner surface) of the fourth flat culture container, and the stem cell 30 forms a colony. When the total planar area of the stem cell 30 displayed on the display 17 with respect to the bottom surface area of the culture container reaches the target ratio (fourth target ratio) (88 to 92% confluent) by expanding the planar shape of 30, A single type of mesenchymal stem cell 30 grown (differentiated) is extracted from the fourth flat culture vessel.

It should be noted that the culture solution 23 contained (remaining) in the fourth flat culture vessel has a predetermined secreted from the stem cell 30 during the culture process (proliferation process) of the single mesenchymal stem cell 30 (second stem cell). A culture solution 23 containing a metabolite and remaining in the fourth flat culture vessel is a culture product solution 24. The person in charge confirms that the total planar area of the stem cells 30 (second stem cells) with respect to the bottom area of the fourth flat culture vessel has reached the target ratio (fourth target ratio), and then displays the stem cell number displayed on the display 17. 2 Click the culture completion button. When the stem cell second culture completion button is clicked, the computer 11 displays a stem cell second culture end button and a stem cell second extraction end button on the display 17.

FIG. 23 is a diagram showing an example of storage of the stem cells 30 (second stem cells) and the culture product solution 24. The person in charge uses the pipette to aspirate the culture product solution 24 (culture solution 23) injected into the fourth flat culture vessel from the culture vessel, and stores the culture product solution 24 in the pipette. Next, after the inside of the fourth flat culture vessel is washed with PBS, a trypsin solution is injected into the culture vessel. When the trypsin solution is injected into the fourth flat culture vessel, the mesenchymal stem cells 30 (second stem cells) fixed on the bottom surface of the culture vessel are detached from the bottom surface by the trypsin solution and float on the water surface of the trypsin solution. The person in charge uses the pipette to aspirate the stem cell 30 and stores the stem cell 30 in the pipette, and then clicks the stem cell second extraction end button. When the stem cell second extraction end button is clicked, the display 17 displays a stem cell storage container data storage button and a product liquid storage container data storage button.

The person in charge extracts the culture product solution 24 and the stem cells 30 (second stem cells), prepares the stem cell storage container 19 and the product solution storage container 20, and attaches the IC tag 18 to the outer peripheral surface of the stem cell storage container 19. At the same time, the IC tag 18 is attached to the outer peripheral surface of the product liquid storage container 20. The person in charge clicks on the stem cell storage container data storage button and uses the IC tag reader / writer 12 to use the IC tag 18 of the stem cell storage container 19 and the donor data and stem cell data of the IC tag 18 attached to the fourth flat culture container. Is stored. Further, the product solution container data storage button is clicked, and the donor data and stem cell data of the IC tag 18 attached to the fourth flat culture vessel to the IC tag 18 of the product solution container 20 using the IC tag reader / writer 12. Is stored. After these data are stored in the IC tags 18 of the

containers

19 and 20, an initial screen is displayed on the display 17.

The person in charge injects (accommodates) the stem cells 30 (second stem cells) from the pipette into the stem cell storage container 19. A single type of mesenchymal stem cell 30 injected into the stem cell storage container 19 is a specific type of single type of mesenchymal stem cell to be cultured having an activity from which unnecessary mesenchymal stem cells are removed. The person in charge injects the stem cells 30 (single mesenchymal stem cells) from the pipette into the stem cell storage container 19 and then stores the stem cell storage container 19 in the refrigerator 14 or the freezer 14. A single type of mesenchymal stem cell 30 (second stem cell) is stored at a predetermined temperature (3 to 5 ° C. or frozen storage) for a predetermined period in the refrigerator 14 or the freezer 14 while being stored in the stem cell storage container 19.

The person in charge injects (accommodates) the culture product solution 24 from the pipette into the product solution storage container 20. The culture product solution 24 injected into the product solution storage container 20 is a predetermined metabolite secreted from a single kind of mesenchymal stem cell 30 of a specific type to be cultured having an activity from which unnecessary mesenchymal stem cells are removed. Is included. The person in charge injects the culture product solution 24 from the pipette into the product solution storage container 20 and then stores the product solution storage container 20 in the refrigerator 14 or the freezer 14. The culture product solution 24 is stored in the product solution storage container 20 in the refrigerator 14 or the freezer 14 at a predetermined temperature (3 to 5 ° C. or frozen storage) for a predetermined period.

DESCRIPTION OF SYMBOLS 10 Activated stem cell culture system 11 Computer 12 IC tag reader / writer 13 Electron microscope 14 Refrigerator or freezer 15 Keyboard 16 Mouse 17 Display 18 IC tag 19 Stem cell storage container raw material bone marrow fluid 20 Production liquid storage container 21 1st flat culture container (1st Culture vessel)
22 Dormant stem cells (Mesenchymal dormant stem cells)
23 culture solution 24 culture product solution 25 bottom surface 26 mixed culture solution 27 activated stem cells (mesenchymal activated stem cells)
28 Second flat culture vessel (second culture vessel)
29 Bottom 30 Stem cells (second mesenchymal stem cells)
31 Raw material bone marrow fluid 32 Glass test tube 33 Test tube stand 34 Thermostatic chamber 35 Intermediate bone marrow fluid 36 Glass test tube 37 Centrifuge 38 Stem cell (first mesenchymal stem cell)
39 Sample holder 40 Upper surface 41 Bottom 42 Spacer 43 Top 44 Inlet 45 Bottom 46 Top 47 Inlet

Claims

An activated stem cell that activates a single type of dormant stem cell in a dormant state after storing a single type of stem cell made by culturing bone marrow fluid collected from a donor for a predetermined period of time to produce a single type of activated stem cell In the manufacturing method,
In the activated stem cell production method, the single type of dormant stem cells, a predetermined culture solution, and a culture product solution generated in the culture process of the single type of stem cells before storage of the dormant stem cells have a predetermined volume and a predetermined area. A dormant stem cell fixing step of injecting the dormant stem cell onto the bottom surface of the first culture vessel by injecting the dormant stem cell into a first culture vessel having a bottom surface, and the dormancy fixed on the bottom surface of the first culture vessel by the dormant stem cell fixing step. Stem cells are cultured, the dormant stem cells are proliferated and activated until the total planar area of the dormant stem cells with respect to the bottom surface area of the first culture container reaches a first target ratio, and the dormant stem cells are activated by the single type of activated stem cells. A method for producing activated stem cells, comprising the step of culturing dormant stem cells to be transformed into
In the dormant stem cell culturing step, the dormant stem cell fixing step fixes the dormant stem cells on the bottom surface of the first culture vessel, and then the mixed culture solution of the culture solution and the culture product solution is removed from the first culture vessel. While discharging, a new culture solution and a new culture product solution are poured into the first culture vessel, and the dormant stem cells fixed on the bottom surface of the first culture vessel are put into a new culture solution and a new culture product solution. The method for producing activated stem cells according to claim 1, wherein the cells are cultured using a new mixed culture solution.
In the dormant stem cell colonization step, the first culture vessel is left to stand for 12 to 24 hours at a temperature substantially the same as the body temperature while the first culture vessel is inclined at a predetermined angle, and the 12 to 24 hours. When the dormant stem cells in the first culture vessel are deformed from the initial planar shape at intervals of about 1 to 2 hours, and the dormant stem cells are deformed from the initial planar shape to a predetermined planar shape, the dormant stem cells The method for producing activated stem cells according to claim 1 or 2, wherein the stem cells are determined to have settled on the bottom surface of the first culture container.
The initial planar shape of the dormant stem cell is substantially circular, and the deformed planar shape of the dormant stem cell is a flat shape in which the dormant stem cell extends in an indefinite shape in one direction with the substantially circular shape as a nucleus, and the dormant stem cell The method for producing activated stem cells according to claim 3, wherein, in the stem cell fixing step, when the dormant stem cells are deformed into the irregular flat shape, it is determined that the dormant stem cells have settled on the bottom surface of the first culture container.
The first target ratio of the total planar area of the dormant stem cells to the bottom area of the first culture vessel is 70 to 80%, and the dormant stem cell culturing step is a state where the first culture vessel is inclined at a predetermined angle. Then, the first culture vessel is statically left at the same temperature as the body temperature for 36 to 48 hours, and is fixed to the bottom surface of the first culture vessel at intervals of about 1 to 2 hours during the 36 to 48 hours. The method for producing activated stem cells according to any one of claims 1 to 4, wherein the total planar area of the dormant stem cells with respect to the bottom area of the first culture container is observed.
The method for producing activated stem cells extracts and extracts the activated stem cells from the first culture container when the total planar area of the dormant stem cells with respect to the bottom area of the first culture container reaches the first target ratio. The activated stem cell, a new culture solution, and a new culture product solution are injected into a second culture vessel having a predetermined volume and a bottom area of a predetermined area and having a capacity larger than that of the first culture vessel. An activated stem cell fixing step for fixing stem cells on the bottom surface of the second culture vessel; and culturing the activated stem cells fixed on the bottom surface of the second culture vessel by the activated stem cell fixing step; and The activated stem cell production according to any one of claims 1 to 5, further comprising the step of cultivating the activated stem cells until the total planar area of the activated stem cells with respect to the bottom surface area of the cell reaches a second target ratio. Law.
In the activated stem cell culturing step, the activated stem cell is fixed on the bottom surface of the second culture container by the activated stem cell fixing step, and then the mixed culture solution of the culture solution and the culture product solution is added to the second culture vessel. While discharging from the culture vessel, a new culture solution and a new culture product solution are injected into the second culture vessel, and the activated stem cells fixed on the bottom surface of the second culture vessel are renewed as a new culture solution. The method for producing activated stem cells according to claim 6, wherein the cells are cultured using a new mixed culture solution with a fresh culture product solution.
In the activated stem cell fixing step, the second culture vessel is left to stand statically at a temperature substantially the same as the body temperature for 36 to 48 hours while the second culture vessel is inclined at a predetermined angle, and the 36 to 48 are performed. When the deformation of the activated stem cells in the second culture vessel from the initial planar shape is observed at intervals of about 1 to 2 hours during the time, and the activated stem cells are deformed from the initial planar shape to a predetermined planar shape The method for producing activated stem cells according to claim 6 or 7, wherein the activated stem cells are determined to have settled on the bottom surface of the second culture container.
The initial planar shape of the activated stem cell is a substantially circular shape, and the planar shape after deformation of the activated stem cell is a flat shape in which the activated stem cell extends indefinitely in one direction with the substantially circular shape as a nucleus. The activity according to claim 8, wherein, in the activated stem cell fixing step, the activated stem cell is determined to have settled on the bottom surface of the second culture container when the activated stem cell is deformed into the irregular flat shape. Stem cell production method.
The second target ratio of the total planar area of the activated stem cells to the bottom surface area of the second culture vessel is 88 to 92%. In the activated stem cell culture step, the second culture vessel is inclined at a predetermined angle. In this state, the second culture container is left statically at a temperature substantially the same as the body temperature for 36 to 48 hours, and the bottom surface of the second culture container is spaced at intervals of about 1 to 2 hours during the 36 to 48 hours. The method for producing activated stem cells according to any one of claims 6 to 9, wherein the total planar area of the activated stem cells settled on the bottom surface area of the second culture container is observed.
The method for producing activated stem cells according to any one of claims 1 to 10, wherein the culture product solution contains a predetermined metabolite secreted from the single type of stem cells in the culture process of the single type of stem cells.
The single type of stem cells separates the bone marrow fluid collected from the donor into layers, extracts the intermediate layer bone marrow fluid located in the intermediate layer of the bone marrow fluid separated into layers, and has a predetermined volume and a predetermined area. A stem cell first fixing step of injecting the intermediate layer bone marrow fluid and a predetermined culture solution into a third culture vessel having a bottom surface to fix the first stem cells contained in the intermediate layer bone marrow fluid to the bottom surface of the third culture container. And, after the first stem cells are fixed on the bottom surface of the third culture container by the first stem cell fixing step, a new culture solution is discharged while discharging the culture solution in the third culture vessel. A first stem cell culturing step for culturing the first stem cells until the total planar area of the first stem cells with respect to the bottom surface area of the third culture vessel reaches a third target ratio; The stem cell first culture step The first stem cells cultured in this manner are centrifuged in layers to extract the second stem cells located in the lowermost layer of the first stem cells separated in layers, and have a bottom surface with a predetermined volume and a predetermined area. A stem cell second fixing step of injecting the second stem cells and a new culture solution into a fourth culture container having a larger capacity than the third culture container to fix the second stem cells on the bottom surface of the fourth culture container; Then, after the second stem cells are fixed on the bottom surface of the fourth culture vessel in the second stem cell fixing step, a new culture solution is discharged into the fourth culture vessel while discharging the culture solution in the fourth culture vessel. Injecting and culturing the second stem cells, and a second culturing step of the stem cells to proliferate the second stem cells until the total planar area of the second stem cells with respect to the bottom surface area of the fourth culture container reaches a fourth target ratio. Claim 1 made Activated stem cells The production method according to any one of claims 11 to.
The method for producing activated stem cells according to claim 12, wherein the culture product solution is a culture solution remaining after extraction of the single second stem cells from the fourth culture vessel.
The method for producing activated stem cells according to any one of claims 1 to 13, wherein the stem cells are mesenchymal stem cells.