WO2017155055A1

WO2017155055A1 - Method for manufacturing culture product liquid

Info

Publication number: WO2017155055A1
Application number: PCT/JP2017/009546
Authority: WO
Inventors: アレクセイグラドコフ
Original assignee: 株式会社ＣｅｌｌｓＰｏｗｅｒ
Priority date: 2016-03-10
Filing date: 2017-03-09
Publication date: 2017-09-14

Abstract

[Problem] To provide a method for manufacturing a culture product liquid, whereby a culture product liquid is manufactured which includes a predetermined metabolite secreted from a single type of stem cell. [Solution] The method for manufacturing a culture product liquid according to the present invention comprises: extracting an intermediate-layer bone marrow liquid positioned in an intermediate layer from bone marrow liquid separated in layered fashion and culturing the intermediate-layer bone marrow liquid together with a culture liquid; fixing first stem cells to a bottom surface of a first culture container and collecting the first stem cells from the first culture container when the total area of the first stem cells reaches a first target ratio of the bottom-surface area of the first culture container; culturing second stem cells together with the culture liquid, the second stem cells being positioned in a bottom layer from among the first stem cells separated in layered fashion, while collecting the second stem cells; and fixing the second stem cells to the bottom surface of a second culture container, and extracting a culture product liquid including a predetermined metabolite secreted from a single type of second stem cells grown from the second culture container when the total area of the second stem cells reaches a second target ratio of the bottom-surface area of the second culture container.

Description

Production method of culture product

The present invention relates to a culture product production method for producing a culture product solution using stem cells made from bone marrow fluid collected from a donor.

A stem cell culture comprising a culture tub, a culture solution filling the culture tub, and a carrier having a height portion on its surface and coated with a stem cell adhesion substance, wherein there are a plurality of low portions that form a height on the carrier surface. An apparatus is disclosed (see Patent Document 1). In this stem cell culture apparatus, the carrier is suspended in the culture solution, and the stem cells attached in a dispersed state to the carrier surface via the stem cell adhesion substance are proliferated without being detached from the carrier surface.

JP 2014-183770 A

In the stem cell culture apparatus disclosed in Patent Document 1, the culture solution after culturing the stem cells is discarded. The culture solution after culturing the stem cells contains a metabolite secreted from the stem cells, and the culture solution can be used for treatment of various diseases and beauty. However, since various stem cells are present in the tissue or cells collected from the donor, the various stem cells proliferate even if the stem cells present in the tissue or cells collected from the donor are cultured. Since various kinds of metabolites secreted from the culture medium are contained in the culture solution, it is difficult to produce a culture product solution having only a specific effect on various diseases and various cosmetics.

An object of the present invention is to provide a culture product production method capable of producing a culture product solution containing a predetermined metabolite secreted from a single type of stem cell and having a single specific effect on various diseases and various cosmetics. It is to provide.

The premise of the present invention for solving the above problems is a culture product production method for producing a culture product using stem cells made from bone marrow fluid collected from a donor.

The feature of the present invention based on the above premise is that the method for producing a culture solution includes a bone marrow fluid separation step in which bone marrow fluid collected from a donor is separated into layers, and an intermediate layer of the bone marrow fluid separated in layers by the bone marrow fluid separation step. A bone marrow fluid extraction step for extracting the located intermediate layer bone marrow fluid, and the intermediate layer bone marrow fluid extracted by the bone marrow fluid extraction step and a predetermined culture solution are injected into a first culture vessel having a predetermined volume and a predetermined area bottom surface, The first culture cell is left statically for a predetermined time, and the first stem cell is fixed by the stem cell first fixing step for fixing the first stem cell contained in the intermediate layer bone marrow fluid to the bottom surface of the first culture container, and the stem cell first fixing step. After fixing on the bottom surface of the first culture vessel, a new culture solution is poured into the first culture vessel while discharging the culture solution in the first culture vessel, and the first culture vessel is left statically for a predetermined time. Against the bottom area of the first culture vessel In the first stem cell culturing step of culturing the first stem cells until the total planar area of the first stem cells reaches the first target ratio, and the culturing process of the first stem cells in the first stem cell culturing step, the total planar area of the first stem cells is A first stem cell collection step for collecting the first stem cells from the first culture container when the first target ratio is reached, a stem cell separation step for centrifuging the first stem cells collected in the first stem cell collection step, and a stem cell The second stem cell collecting step for collecting the second stem cell located in the lowermost layer of the first stem cells separated in a layered manner by the separating step, the second stem cell collected by the second stem cell collecting step, and a predetermined culture solution Pour into a second culture container having a larger volume and a larger bottom area than the one culture container, and leave the second culture container statically for a predetermined time to fix the second stem cells to the bottom surface of the second culture container. The stem cell second fixing step and the stem cell second fixing step fix the second stem cell on the bottom surface of the second culture vessel, and then discharge the culture solution in the second culture vessel to the second culture. A second stem cell that is poured into the container and statically left in the second culture container for a predetermined time, and the second stem cell is cultured until the total planar area of the second stem cell with respect to the bottom surface area of the second culture container reaches a second target ratio. In the culturing process and the culturing process in the stem cell second culturing process, when the total planar area of the second stem cells reaches the second target ratio, culturing includes a predetermined metabolite secreted from the single stem cell that has proliferated. And having a culture product solution first extraction step of extracting the product solution from the second culture vessel.

As an example of the method for producing a culture product of the present invention, in the stem cell first fixing step and the stem cell first culturing step, the first culture vessel is allowed to stand statically for a predetermined time in a state where the first culture vessel is inclined at a predetermined angle, thereby stem cell second fixing. In the step and the stem cell second culture step, the second culture vessel is statically left for a predetermined time in a state where the second culture vessel is inclined at a predetermined angle.

As another example of the culture product production method of the present invention, the culture product production method extracts the culture product solution from the second culture vessel in the culture product solution first extraction step, and then extracts the culture product solution from the second culture vessel. A stem cell second collecting step for collecting two stem cells, and a stem cell second collecting step in a third culture vessel having a bottom surface with a predetermined volume and a predetermined area and a larger volume and a larger bottom area than the second culture vessel. Injecting the collected second stem cells and a new culture solution, and statically leaving the third culture vessel for a predetermined time to establish the second stem cells on the bottom surface of the third culture vessel; After fixing the second stem cells on the bottom surface of the third culture vessel by the 3 fixing step, a new culture solution is poured into the third culture vessel while discharging the culture solution in the third culture vessel. Leave it statically for a certain period of time In the third stem cell culturing step for culturing the second stem cells until the total planar area of the second stem cells with respect to the bottom surface area of the container reaches the third target ratio, and in the culturing process in the third stem cell culturing step, the total planar area of the second stem cells When the third target ratio is reached, a culture product solution second extraction step of extracting a culture product solution containing a predetermined metabolite secreted from the proliferated single species of second stem cells from the third culture vessel is included.

As another example of the method for producing a culture product solution of the present invention, in the third stem cell fixing step and the third stem cell culture step, the third culture vessel is statically left for a predetermined time in a state where the third culture vessel is inclined at a predetermined angle.

As another example of the culture product production method of the present invention, the bone marrow fluid separation step collects 2 to 3 cc of bone marrow from a donor, and injects 2 to 3 cc of bone marrow into a separation container extending vertically. The separation container is statically left at a temperature substantially the same as the body temperature for a predetermined time to separate the bone marrow fluid into layers in the vertical direction in the separation container, and the bone marrow fluid extraction step is performed to remove the bone marrow fluid separated into layers in the separation container. The middle layer bone marrow fluid located in the middle layer is extracted.

As another example of the culture product production method of the present invention, the capacity of the first culture vessel is about 20 to 30 cc, the initial planar shape of the first stem cells is substantially circular, and the first stem cells are deformed. The planar shape is a flat shape in which the first stem cells are indefinitely elongated in one direction with a substantially circular nucleus, and in the first stem cell fixing step, the first culture vessel is kept at a temperature substantially the same as the body temperature for 12 to 24 hours. While standing statically, the deformation of the first stem cells in the first culture vessel from the initial planar shape is observed at intervals of about 1 to 2 hours during 12 to 24 hours. When deformed into a regular flat shape, it is determined that the first stem cells have settled on the bottom surface of the first culture vessel.

As another example of the method for producing a culture product of the present invention, in the first stem cell collection step, the first culture container is statically allowed to stand for 36 to 48 hours at a temperature substantially the same as the body temperature for 36 to 48 hours. The total planar area of the first stem cells that have settled on the bottom surface of the first culture vessel at about 1 to 2 hours is observed with respect to the bottom surface area of the first culture vessel.

As another example of the method for producing a culture product solution of the present invention, the first target ratio of the total planar area of the first stem cells to the bottom area of the first culture vessel is 70 to 80%.

As another example of the method for producing a culture product solution of the present invention, the stem cell separation step injects the first stem cells into a separation container, sets the separation container in a centrifuge, and centrifuges the first stem cells. 1 collection process collect | recovers the 2nd stem cell located in the lowest layer among the 1st stem cells centrifuged in the layer form in the isolation | separation container.

As another example of the method for producing a culture product of the present invention, the capacity of the second culture container is about 40-60 cc, the capacity of the third culture container is about 70-80 cc, and the initial planar shape of the second stem cell Is substantially flat, and the planar shape of the second stem cell after deformation is a flat shape in which the second stem cell is indefinitely elongated in one direction with the substantially circular shape as a nucleus. In the stem cell second fixing step and the stem cell third fixing step, The second and third culture containers are allowed to stand for 36 to 48 hours statically at the same temperature as the body temperature for 36 to 48 hours while the second and third culture containers are spaced at about 1 to 2 hours between 36 and 48 hours. When the deformation of the second stem cell from the initial planar shape was observed and the second stem cell was deformed from the initial planar shape to an indeterminate flat shape, the second stem cell was fixed on the bottom surfaces of the second and third culture vessels. Judge.

As another example of the method for producing a culture product of the present invention, in the second stem cell collection step and the third stem cell collection step, the second culture vessel and the third culture vessel are statically kept at a temperature substantially the same as the body temperature for 36 to 48 hours. The total planar area of the second stem cells established on the bottom surfaces of the second and third culture vessels at intervals of about 1 to 2 hours during 36 to 48 hours with respect to the bottom surface areas of the second and third culture vessels Observe.

As another example of the culture product production method of the present invention, the second target ratio of the total planar area of the second stem cells to the bottom area of the second culture container is 88 to 92%, and the bottom area of the third culture container The third target ratio of the total planar area of the second stem cells is 88-92%.

As another example of the method for producing a culture product solution of the present invention, the first and second stem cells are mesenchymal stem cells.

According to the culture product production method of the present invention, the intermediate layer bone marrow fluid located in the intermediate layer of the bone marrow fluid separated into layers is extracted, and the intermediate layer bone marrow fluid is cultured with the culture solution to obtain the first stem cells. When the total planar area of the first stem cells reaches the first target ratio with respect to the bottom area of the first culture container while culturing the first stem cells while being fixed on the bottom surface of the first culture container, Collecting one stem cell and collecting the second stem cell located in the lowermost layer of the first stem cells separated into layers, culturing the second stem cell together with a culture solution, and cultivating the second stem cell to the bottom surface of the second culture vessel The total planar area of the second stem cells reached the second target ratio with respect to the bottom area of the second culture vessel while injecting a new culture solution into the second culture vessel and culturing the second stem cells. A single species grown in the course of culturing second stem cells Since the culture product solution containing the predetermined metabolite secreted from the second stem cell is extracted from the second culture vessel, various kinds secreted from various stem cells can be obtained by using the cultured single type of second stem cell. It does not contain a variety of metabolites, contains only certain metabolites secreted from a single type of second stem cell, and is only specific for a given disease, skin care, hair care, body care, etc. A culture product that can produce a culture product solution that can be used effectively for the treatment of a given disease, and that can be used effectively for a given beauty. The product liquid can be made. In the culture product production method, a culture product solution is produced using pure second stem cells from which unnecessary stem cells have been removed, and a predetermined kind of secreted from a single kind of second stem cells is produced in the culture product solution. Since it can contain only metabolites, it has a high therapeutic effect on a given disease, can make a culture product that has a high probability of completely curing the disease, and has a high effect on a given beauty. A culture product solution can be made.

In the stem cell first fixing step and the stem cell first culturing step, the first culture vessel is statically left for a predetermined time in a state inclined at a predetermined angle, and in the stem cell second fixing step and the stem cell second culturing step, the second culture is performed. The method of producing a culture product solution in which the container is statically left for a predetermined time in a state where the container is inclined at a predetermined angle is obtained in a state where the first culture container is inclined at a predetermined angle in the stem cell first fixing step and the stem cell first culture step. By standing statically for a predetermined time, the first stem cells can be reliably fixed on the bottom surface of the first culture vessel, and the proliferation of the first stem cells can be surely promoted. The method for producing a culture product includes the step of statically leaving the second culture vessel tilted at a predetermined angle for a predetermined time in the second stem cell fixing step and the second stem cell culture step, so that the bottom of the second culture vessel The second stem cell can be firmly established, and the proliferation of the second stem cell can be surely promoted.

After extracting the culture product solution from the second culture vessel, the second stem cells are collected from the second culture vessel, the second stem cells are cultured with the culture solution, and the second stem cells have a larger capacity and a larger bottom surface than the second culture vessel. The bottom surface of the third culture vessel is fixed to the bottom surface of the third culture vessel having a bottom surface area, a new culture solution is injected into the third culture vessel, and the second stem cells are cultured while the total planar area of the second stem cells is the bottom surface of the third culture vessel. When the third target ratio is reached with respect to the area, a culture product solution containing a predetermined metabolite secreted from a single type of second stem cell grown in the culture process of the second stem cell is extracted from the third culture vessel. In the production method, the second stem cells are further cultured in the third culture container, and when the total planar area of the second stem cells reaches the third target ratio with respect to the bottom area of the third culture container, Single species grown during the culture process Since the culture product solution containing a predetermined metabolite secreted from the second stem cell is extracted from the third culture vessel, the various stem cells are not cultured in the stem cell culturing process and are secreted from the various stem cells. A variety of metabolites that contain only a specific metabolite secreted from a single type of second stem cell, and that produce a specific effect on a specific disease or a specific beauty A culture product that can be used for the treatment of a given disease and can be used effectively for a given beauty. it can. The culture product production method can produce a large amount of culture product at a time by using a third culture vessel having a larger capacity than the second culture vessel.

In the stem cell third fixing step and the stem cell third culturing step, the method for producing a culture product solution in which the third culture vessel is statically left for a predetermined time in a state where the third culture vessel is inclined at a predetermined angle includes the stem cell third fixing step and the stem cell third culture. In the process, the second stem cells can be reliably fixed on the bottom surface of the second culture container by allowing the third culture container to stand statically for a predetermined time in a state where the third culture container is inclined at a predetermined angle. Can be surely promoted.

In the bone marrow fluid separation step, 2 to 3 cc of bone marrow fluid is collected from the donor, and 2 to 3 cc of bone marrow fluid is injected into a separation container extending in the vertical direction, and the separation container is statically fixed at a temperature substantially equal to the body temperature for a predetermined time. Culturing the bone marrow fluid by separating it into layers in the vertical direction in the separation container and extracting the intermediate layer bone marrow fluid located in the middle layer of the bone marrow fluid separated into layers in the separation container in the bone marrow fluid extraction step In the production method, bone marrow fluid containing various stem cells is statically left for a predetermined time at approximately the same temperature as the body temperature and separated into layers in the vertical direction. Since it is reliably extracted and a single type of second stem cell is cultured from the intermediate layer bone marrow fluid, it contains only a predetermined metabolite secreted from the single type of stem cell from which unnecessary stem cells have been removed, For a given beauty It is possible to make a culture product solution that exhibits only a specific effect, to make a culture product solution that can be used effectively for treatment of a given disease, and to be used effectively for a given beauty. Possible culture products can be made.

The capacity of the first culture vessel is about 20-30 cc, the initial planar shape of the first stem cells is substantially circular, and the first stem cells are deformed in one direction with the deformed planar shape of the first stem cells as a nucleus. In the stem cell first colonization step, the first culture vessel is statically left at a temperature substantially the same as the body temperature for 12 to 24 hours, and is about 1-2 for 12 to 24 hours. When the deformation from the initial planar shape of the first stem cells in the first culture container is observed at time intervals, and the first stem cells are deformed from the initial planar shape to an indeterminate flat shape, the first stem cells are in the first culture container. In the method for producing a culture product solution that is determined to have settled on the bottom surface, when the capacity of the first culture container exceeds 30 cc, the first stem cells are difficult to settle on the bottom surface of the first culture container and the growth of the first stem cells is slowed. Use the first culture vessel of the above capacity. As a result, the first stem cells quickly and easily settle on the bottom surface of the first culture container, and the first stem cells proliferate quickly in the first culture container, so that a single type of stem cells can be made in a short period of time and unnecessary. A large amount of a culture product solution containing only a predetermined metabolite secreted from a single kind of stem cell from which various stem cells have been removed can be produced in a short period of time. The method for producing a culture product solution comprises the first culture container being left to stand for 12 to 24 hours at a temperature substantially the same as the body temperature, and the first culture container in the first culture container at intervals of about 1 to 2 hours during 12 to 24 hours. When the deformation of the stem cell from the initial planar shape was observed and the first stem cell was deformed into a flat shape extending in an irregular shape in one direction with a substantially circular nucleus, the first stem cell was fixed on the bottom surface of the first culture vessel. Therefore, it is possible to accurately grasp the fixation of the first stem cell to the bottom surface of the first culture container by the change in the planar shape of the first stem cell without missing the fixation of the first stem cell to the bottom surface of the first culture container. And after confirming the establishment of the first stem cells on the bottom surface of the first culture container, by injecting a new culture medium into the first culture container while discharging the culture medium in the first culture container, Encourage the proliferation of primary stem cells It can be, it is possible to make the culture product liquor containing only a predetermined metabolites secreted by utilizing the first stem cells from a single type of stem cell to ensure.

In the first stem cell collection step, the bottom surface of the first culture container is left at about 1 to 2 hours between 36 and 48 hours while the first culture container is statically left at a temperature substantially the same as the body temperature for 36 to 48 hours. The culture product producing method for observing the total planar area of the first stem cells settled on the first culture vessel with respect to the bottom area of the first culture vessel observes the total planar area at intervals of about 1 to 2 hours. The total plane area relative to the bottom area of the culture vessel can be accurately confirmed, and the total plane area of the first stem cells has reached the first target ratio with respect to the bottom area of the first culture container. The first stem cell can be collected from the first culture container while maintaining the activity of the first stem cell without mistaking the timing of collecting the first stem cell from the first culture container. Using a single species of trunk It can be made to ensure the culture product liquor containing only a predetermined metabolites secreted from cells.

In the culture product production method in which the first target ratio of the total planar area of the first stem cells to the bottom area of the first culture container is 70 to 80%, the total planar area of the first stem cells relative to the bottom area of the first culture container is When the first stem cells proliferate in excess of 80%, the activity of the first stem cells is gradually lost, but when the total planar area of the first stem cells grows to 70-80% with respect to the bottom area of the first culture vessel In addition, since the first stem cells are collected from the first culture vessel, the activity of the first stem cells can be maintained, the first stem cells can be propagated while maintaining the activity, and the first stem cells are used. Thus, a culture product solution containing only a predetermined metabolite secreted from a single kind of stem cell can be reliably produced.

In the stem cell separation step, the first stem cells are injected into a separation container, the separation container is placed in a centrifuge and the first stem cells are centrifuged. In the first stem cell collection step, the first stem cells are centrifuged in layers in the separation container. A method for producing a culture product solution for collecting second stem cells located in the lowermost layer of one stem cell includes separating first stem cells containing unnecessary stem cells into layers by centrifugation, and centrifuging the first stem cells into layers. By collecting the second stem cells located in the lowermost layer, specific second stem cells can be reliably extracted from the first stem cells, and unnecessary stem cells can be removed from the first stem cells. The culture product production method can culture only a specific type of second stem cells to be cultured, and includes only a predetermined metabolite secreted from a single type of stem cell using the first stem cells. Can be made reliably.

The capacity of the second culture vessel is about 40 to 60 cc, the capacity of the third culture vessel is about 70 to 80 cc, the initial planar shape of the second stem cell is substantially circular, and the planar shape after deformation of the second stem cell Is a flat shape in which the second stem cell is indefinitely elongated in one direction with a substantially circular nucleus, and the second culture vessel and the third culture vessel are substantially the same as the body temperature in the second stem cell fixing step and the third stem cell fixing step. While standing statically for 36 to 48 hours at a temperature of 36 to 48 hours, the deformation from the initial planar shape of the second stem cells in the second and third culture vessels was observed at intervals of about 1 to 2 hours during 36 to 48 hours. When the second stem cell is deformed from the initial planar shape to an indeterminate flat shape, the culture product producing method for determining that the second stem cell has settled on the bottom surfaces of the second and third culture vessels Volume exceeds 60cc, 3rd culture When the capacity of the vessel exceeds 80 cc, the second stem cells are difficult to settle on the bottom surfaces of the second and third culture vessels and the growth of the second stem cells is slow, but the second and third culture vessels having the above-mentioned capacities are used. As a result, the second stem cells are quickly and easily fixed on the bottom surfaces of the second and third culture vessels, and the second stem cells proliferate quickly in the second and third culture vessels. A large amount of a culture product containing only a predetermined metabolite secreted from a single type of second stem cell from which unnecessary stem cells have been removed can be produced in a short period of time. The production method of the culture product comprises the second and third culture vessels statically left at a temperature substantially the same as the body temperature for 36 to 48 hours, while the second and third culture containers are spaced at intervals of about 1 to 2 hours between 36 and 48 hours. When the deformation of the second stem cell in the third culture vessel from the initial planar shape is observed and the second stem cell is deformed into a flat shape extending in an irregular shape in one direction with a substantially circular shape as the nucleus, the second stem cell is second By determining that the second stem cells have settled on the bottom surface of the third culture container, the second stem cells are not missed by the second stem cells due to the change in the planar shape of the second stem cells. The second and third culture vessels can be accurately fixed on the bottom surfaces of the second and third culture vessels, and the second stem cells are confirmed to be fixed on the bottom surfaces of the second and third culture vessels. New medium while draining By injecting the nutrient solution into the second and third culture vessels, the proliferation of the second stem cell can be surely promoted, and a predetermined secreted from a single type of second stem cell using the second stem cell. A culture product containing only metabolites can be produced reliably.

In the second and third stem cell collection steps, the second and third culture vessels are statically left at the same temperature as the body temperature for 36 to 48 hours, and between about 36 and 48 hours, about 1-2 hours. The method for producing a culture solution for observing the total planar area of the second stem cells fixed on the bottom surfaces of the second and third culture vessels with respect to the bottom surface area of the second and third culture vessels at intervals of about 1 to 2 hours By observing the planar area, the total planar area with respect to the bottom area of the second and third culture vessels of the second stem cells can be confirmed accurately, and the second stem cell is compared with the bottom areas of the second and third culture vessels. While it is possible to reliably grasp that the total planar area of the stem cells has reached the second and third target ratios, the second stem cells can be collected from the second and third culture vessels without mistaking the timing. Retains stem cell activity The second stem cells can be collected from the second and third culture vessels while the second stem cells are used to reliably produce a culture solution containing only a predetermined metabolite secreted from a single kind of stem cells. Can do.

The second target ratio of the total planar area of the second stem cells to the bottom area of the second culture container is 88 to 92%, and the third target ratio of the total planar area of the second stem cells to the bottom area of the third culture container is 88. In the method for producing a culture product solution of ˜92%, the activity of the second stem cells is increased when the total planar area of the second stem cells with respect to the bottom area of the second and third culture vessels exceeds 92% and the second stem cells proliferate. Although gradually lost, the second stem cells are collected from the second and third culture vessels when the total planar area of the second stem cells grows to 88-92% with respect to the bottom area of the second and third culture vessels. The second stem cell can retain the activity, and the second stem cell can be proliferated while retaining the activity, and the second stem cell is used to secrete the second stem cell from a single type of second stem cell. Contains only the metabolites of Nutrient product solution can be made to ensure.

The method for producing a culture product in which the first and second stem cells are mesenchymal stem cells uses a variety of mesenchymal stem cells that are secreted from a variety of mesenchymal stem cells by using a single type of cultured second mesenchymal stem cells. It does not contain a variety of metabolites, contains only certain metabolites secreted from a single type of second mesenchymal stem cell, and is used for certain diseases such as skin care, hair care, and body care. It is possible to make a culture product solution that exhibits only a specific effect, to make a culture product solution that can be used effectively for treatment of a given disease, and to be used effectively for a given beauty. Possible culture products can be made. In the culture product production method, a culture product solution is made using pure second mesenchymal stem cells from which unnecessary stem cells have been removed, and a single type of second mesenchymal stem cells is used as the culture product solution. Because it can contain only certain metabolites secreted from, it can produce a culture product that has a high therapeutic effect on a given disease and is highly established to completely cure the disease. It is possible to produce a culture product solution having a high effect on the above.

The schematic block diagram of the culture solution manufacturing system shown as an example. Explanatory drawing which shows an example of a bone marrow fluid separation process. Explanatory drawing of the bone marrow fluid separation process continued from FIG. Explanatory drawing of the bone marrow fluid separation process following FIG. Explanatory drawing which shows an example of a stem cell 1st observation process. The side view of a 1st flat culture container. The partial enlarged view which shows an example of the planar shape of a 1st mesenchymal stem cell. The elements on larger scale which show another example of the planar shape of a 1st mesenchymal stem cell. The elements on larger scale which show another example of the planar shape of a 1st mesenchymal stem cell. Explanatory drawing which shows an example of a stem cell isolation | separation process. Explanatory drawing which shows an example of a stem cell 2nd extraction process. Explanatory drawing which shows an example of a stem cell 3rd observation process. The side view of a 2nd flat culture container. The elements on larger scale which show an example of the planar shape of a 2nd mesenchymal stem cell. The elements on larger scale which show another example of the planar shape of a 2nd mesenchymal stem cell. The elements on larger scale which show another example of the planar shape of a 2nd mesenchymal stem cell. The figure which shows an example of preservation | save of the extracted culture product liquid and a 2nd mesenchymal stem cell. Explanatory drawing which shows an example of a stem cell 5th observation process. The side view of a 3rd flat culture container.

The details of the culture product production method according to the present invention will be described below with reference to the accompanying drawings such as FIG. 1 which is a schematic configuration diagram of the culture product production system 10 shown as an example. 2 is an explanatory view showing an example of the bone marrow fluid separation step, and FIG. 3 is an explanatory view of the bone marrow fluid separation step continued from FIG. FIG. 4 is an explanatory diagram of a bone marrow fluid separation process continued from FIG.

The culture product production method is a method of culturing a specific type of single mesenchymal stem cell (single seed stem cell) from a plurality of types of mesenchymal stem cells contained in bone marrow fluid, A culture product containing a secreted predetermined metabolite is produced. In the culture product production method, bone marrow fluid (raw material bone marrow fluid) collected from a plurality of donors (people) is used as a raw material, and the bone marrow fluid separation step, bone marrow fluid extraction step, stem cell first using the culture product production system 10. Manufactured through a fixing process, a stem cell first culturing process, a stem cell first collecting process, a stem cell separating process, a stem cell second collecting process, a stem cell second fixing process, a stem cell second culturing process, and a culture product solution first extracting process. The Furthermore, it is manufactured by using the culture product production system 10 through a stem cell second collection step, a stem cell third fixing step, a stem cell third culture step, and a culture product second extraction step.

The stem cell first fixing step includes a stem cell first observation step, and the stem cell second collection step includes a stem cell second observation step. The stem cell second fixing step includes a stem cell third observation step, and the culture product solution first extraction step includes a stem cell fourth observation step. The stem cell third fixing step includes a stem cell fifth observation step, and the culture product solution second extraction step includes a stem cell sixth observation step.

The culture product production system 10 includes a computer 11, a barcode reader 12, and an electron microscope 13. 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). An input device such as a keyboard 14 and a mouse 15 and an output device such as a display 16 and a printer (not shown) are connected to the computer 11 via an interface (wireless or wired).

In the culture product production system 10, donor data (donor identification information) is managed using a QR code (registered trademark). Donor data includes the donor's name, address, telephone number, date of birth, sex, blood type, height, weight, email address, and the like. In the system 10, a QR code is used as a two-dimensional code, but in addition to the QR code, a matrix type SP code, VeriCode (MaxiCode), CP code, DataMatrix, Code1, AztecCode, Intercode Kuta code and card e can be used. The two-dimensional code used in the system 10 includes all that will be developed in the future.

In the bone marrow fluid separation step, the bone marrow fluid 18 collected from the donor is separated into layers. In the bone marrow fluid collection, 2 to 3 cc (2 to 3 ml) of bone marrow fluid 18 is collected from the sternum or iliac bone (pelvis) of these donors. The bone marrow fluid 18 is collected by “bone marrow puncture” (Marc), in which the donor is locally anesthetized and then the bone marrow is punctured to suck the bone marrow fluid (bone marrow blood). A doctor, nurse, researcher, or other person in charge starts up the system 10 in the computer 11 simultaneously with the collection of the bone marrow fluid 18 and inputs donor data into the computer 11 using an input device such as a keyboard 14 or a mouse 15. To do.

The computer 11 generates a unique donor identifier that identifies each donor each time donor data is input (every time the bone marrow fluid 18 is collected from the donor), and stores the storage area in a state in which the donor data is associated with the donor identifier. (Donor data storage step). The computer 11 converts the inputted donor data into a QR code (two-dimensional code) by a two-dimensional code writer function (two-dimensional code (QR code) conversion means), and the first code sheet 17 on which the QR code is printed In addition to outputting (two-dimensional code (QR code) output step), the QR code is stored (stored) in a storage area in association with a donor identifier that identifies each donor (two-dimensional code (QR code) storage step). . When the second donor data is input, the second code sheet on which the QR code is printed is output. When the third donor data is input, the third code sheet on which the QR code is printed is output. Thus, the first to nth code sheets are output according to different donors.

The QR code printed on the first code sheet 17 printed with NO1 includes a bone marrow fluid separation step, a bone marrow fluid extraction step, a stem cell first observation step, a stem cell first colonization step, a stem cell first culture step, a stem cell. Second observation step, stem cell first collection step, stem cell separation step, stem cell second collection step, stem cell third observation step, stem cell second colonization step, stem cell second culture step, stem cell fourth observation step, culture product solution first Extraction step, stem cell second collection step, stem cell third colonization step, stem cell third culture step, culture product solution second extraction step, stem cell second collection step, stem cell fifth observation step, stem cell third colonization step, stem cell third The number 1 indicating the culture step, the sixth stem cell observation step, and the culture product second extraction step is stored. Each time the computer 11 performs the steps from the bone marrow fluid separation step to the culture product first extraction step, the computer 11 stores the donor data stored in the storage area and the donor data indicated by the QR code read by the barcode reader 12. Compare.

As shown in FIG. 2, 2 to 3 cc of bone marrow fluid 18 collected from the donor is injected (accommodated) into a glass test tube 19 (separation container) extending vertically. The 2-3 cc bone marrow fluid 18 contains 0.5 to 1 ml (about 5 × 10 ⁷ (cells / ml)) of a plurality of types of mesenchymal stem cells. A first code sheet 17 is attached to the outer peripheral surface of the glass test tube 19 into which the bone marrow fluid 18 is injected. As shown in FIG. 3, the glass test tube 19 into which the bone marrow fluid 18 has been injected is set in a test tube stand 20 and accommodated in a thermostatic chamber 21 together with the test tube stand 20.

A person in charge such as a doctor, nurse, researcher or the like attaches the first code sheet 17 on which the QR code is printed on the outer peripheral surface of the glass test tube 19, and then attaches the QR code of the glass test tube 19 to the barcode reader 12. Let me read. The barcode reader 12 is connected to the computer 11 via an interface (wired or wireless). The bar code reader 12 transmits the read QR code to the computer 11.

The computer 11 extracts the number 1 and the donor data indicated by the QR code transmitted from the barcode reader 12 from the storage area and reads them into the cache memory, and displays a process first display screen (not shown) on the display 16. . On the process first display screen, number 1 and donor data are displayed, and a bone marrow fluid separation button, a bone marrow fluid extraction button, a stem cell first observation button, a stem cell second observation button, a stem cell first collection button, and a logout button Is displayed. When the logout button is clicked, the system 10 stops in the computer 11.

The person in charge clicks the bone marrow fluid separation button displayed on the display 16, injects the bone marrow fluid 18 into the glass test tube 19 from the syringe, and inserts the glass test tube 19 into which the bone marrow fluid 18 is injected into the test tube stand 20. (set. As shown in FIG. 3, the person in charge accommodates the test tube stand 20 in a thermostatic chamber 21 and statically leaves the glass test tube 19 into which the bone marrow fluid 18 has been injected in the thermostatic chamber 21 for a predetermined time (about 2 hours). (Leave quietly without moving). The temperature in the thermostatic chamber 21 is maintained at about 36 to 37 ° C., which is substantially the same as the body temperature. The computer 11 displays the number 1 and the donor data indicated by the QR code printed on the first code sheet 17 on the display 16, and displays a bone marrow fluid separation in progress message and a bone marrow fluid separation end button on the display 16.

By leaving the glass test tube 19 statically in the thermostatic chamber 21 for a predetermined time (about 2 hours), the bone marrow fluid 18 injected into the test tube 20 is vertically moved in the test tube 20 as shown in FIG. Separated into several layers (bone marrow fluid separation step). Stem cell culture usually requires 150 to 200 cc (150 to 200 ml) of bone marrow fluid, but the production method of the culture product is based on a single type of stem cell from a small amount of 2 to 3 cc of bone marrow fluid 18 collected from a donor. Therefore, a small amount of 2 to 3 cc of bone marrow fluid 18 may be collected from the donor, and the burden on the donor when collecting bone marrow fluid 18 can be minimized.

The bone marrow fluid extraction step is performed after the bone marrow fluid separation step in which the bone marrow fluid 18 is separated into layers. In the bone marrow fluid extraction step, the intermediate layer bone marrow fluid 22 is extracted from the bone marrow fluid 18 separated into layers. The person in charge confirms that the bone marrow fluid 18 has been separated into layers, and then clicks the bone marrow fluid separation end button displayed on the display 16. When the bone marrow fluid separation end button is clicked, the computer 11 displays a process second display screen (not shown) on the display 16. On the process second display screen, number 1 and donor data are displayed, and a bone marrow fluid separation end message, a bone marrow fluid extraction button, a stem cell first observation button, a stem cell second observation button, and a stem cell first collection button are displayed. The

The person in charge clicks the bone marrow fluid extraction button on the process second display screen, and causes the barcode reader 12 to read the QR code of the glass test tube 19. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the donor data match, the number 1 and the donor data are displayed on the display 16 and a bone marrow fluid separation end message, a bone marrow fluid extraction in progress message, and a bone marrow fluid extraction end button are displayed on the display. . If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge takes out the test tube stand 20 from the thermostatic chamber 21, pulls out the glass test tube 19 from the test tube stand 20, and extracts the intermediate layer bone marrow fluid 22 existing in a specific layer of the bone marrow fluid 18 separated into layers. (Bone marrow fluid extraction process). The person in charge extracts (suctions) the intermediate layer bone marrow fluid 22 having a layer thickness of 2 to 4 mm located in the intermediate layer of the bone marrow fluid 18 separated into layers using a syringe (not shown). Alternatively, the intermediate layer bone marrow fluid 22 having a layer thickness of 2 to 4 mm located in the intermediate layer of the bone marrow fluid 18 separated into layers using a pipette (not shown) is extracted (suctioned).

In the culture product production method, bone marrow fluid 18 containing various mesenchymal stem cells is separated into layers in the vertical direction, and then a specific intermediate layer bone marrow fluid 22 is obtained from bone marrow fluid 18 by using a syringe or pipette. Can be reliably extracted, and unnecessary mesenchymal stem cells contained in the bone marrow fluid 18 can be removed.

FIG. 5 is an explanatory view showing an example of the first stem cell observation step, and FIG. 6 is a side view of the first flat culture vessel. FIG. 7 is a partially enlarged view showing an example of the planar shape of the first mesenchymal stem cell 26, and FIG. 8 is a partially enlarged view showing another example of the planar shape of the first mesenchymal stem cell 26. 7 and 8 show enlarged images of the planar shape of the first mesenchymal stem cell 26 taken by the electron microscope 13.

The stem cell first observation step is performed after the bone marrow fluid extraction step of extracting a specific middle layer bone marrow fluid 22 located in the middle layer from the bone marrow fluid 18. In the first stem cell observation step, the intermediate layer bone marrow fluid 22 and the culture solution 23 are injected (contained) into the first flat culture vessel 24 (first culture vessel), and the temperature inside the culture vessel 24 is substantially the same as the body temperature (about 36). The culture vessel 24 is statically left for 12 to 24 hours (slowly left unmoved) while being held at 37 ° C).

A person in charge such as a doctor, nurse, or researcher extracts a specific intermediate layer bone marrow fluid 22 from the bone marrow fluid 18 and then clicks a bone marrow fluid extraction end button displayed on the display 16. When the bone marrow fluid extraction end button is clicked, the computer 11 displays a process third display screen (not shown) on the display 16. On the process third display screen, number 1 and donor data are displayed, and a bone marrow separation end message, a bone marrow extraction end message, a stem cell first observation button, a stem cell second observation button, and a stem cell first collection button are displayed. Is done.

The person in charge attaches the first code sheet 17 on which the QR code is printed to the bottom surface 27 (the outer surface of the bottom wall) of the first flat culture vessel 24. Next, the stem cell first observation button on the process third display screen is clicked to cause the barcode reader 12 to read the QR code of the culture vessel 24. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. If the donor data match, the number 1 and the donor data are displayed on the display 16, and the bone marrow fluid separation end message, bone marrow fluid extraction end message, stem cell first observation in progress message, stem cell first Display the observation end button on the display. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge injects (accommodates) the intermediate layer bone marrow fluid 22 sucked into the syringe or pipette into the culture container 24 and injects (accommodates) the culture medium 23 into the culture container 24 using the syringe or pipette. The first flat culture vessel 24 (first culture vessel) is a flat vessel made of transparent glass or transparent plastic and having a bottom surface 27 with a small capacity and a predetermined area and having a substantially square shape. The first flat culture vessel 24 has a top 40 and a bottom 38 and an inlet 41 formed in the top 40. The injection port 41 is watertightly closed by a lid 42. As the first flat culture container 24, a flat container having a small volume and a bottom surface 27 having a predetermined area and having a circular or elliptical planar shape may be used. The first flat culture vessel 24 used in the first stem cell observation means has a capacity of about 20-30 cc (preferably 25 cc) and a bottom area of about 25-36 mm ² . The culture container 24 has a side length of 5 to 6 mm.

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 first mesenchymal stem cells 26 contained in the intermediate layer bone marrow fluid 22 injected into the culture vessel 24 are cultured in the culture solution 23 while being fixed on the bottom surface 27 of the culture vessel 24 over time. It gradually grows (differentiates) on the bottom surface 27 to form a colony.

In addition, Dulbecco's Modified ａ Eagle's Medium (DMEM), Grasgo Minimum Essential Medium (GMEM), RPMI 640, and the like can be used as the culture solution. In the culture medium, insulin, transferrin, ethanolamine, selenium, 2-mercaptoethanol, L-alanyl-L-glutamine, sodium pyruvate, L-alanine, L-asparagine, L-aspartic acid, glycine, L-proline L-serine or the like can also be added.

The person in charge injects the intermediate layer bone marrow fluid 22 and the culture solution 23 into the first flat culture vessel 24 and then installs (sets) the culture vessel 24 in the sample holder of the electron microscope 13. A spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the bottom portion 38 of the first flat culture vessel 24, and the bottom portion 38 of the culture vessel 24 is held in a state of being lifted by the spacer 39. The culture vessel 24 is held at a predetermined angle so that the bottom 38 of the vessel 24 is on top and the top 40 (inlet 41) of the culture vessel 24 is on the bottom. In addition, a spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the top 40 of the first flat culture vessel 24, and the top 40 of the culture vessel 24 is held in a state of being lifted by the spacer 39. You may hold | maintain the culture container 24 in the state inclined by the predetermined angle so that the top part 40 of the container 24 may become the upper side and the bottom part 38 of the culture container 24 may become the lower side. The inclination angle α1 of the first flat culture vessel 24 with respect to the upper surface 37 of the sample holder 36 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

In the culture product production method, the first flat culture vessel 24 is inclined at the inclination angle with respect to the upper surface 37 of the sample holder 36 so that the intermediate bone marrow fluid 22 and the culture solution 23 are contained in the culture vessel 24 in the culture vessel 24. The first mesenchymal system is biased toward the top 40 (or the bottom 38), and the water pressure between the intermediate bone marrow fluid 22 and the culture fluid 23 increases on the top 49 side (or the bottom 38 side) of the culture vessel 24. The stem cells 26 are concentrated on the bottom 38 side of the culture vessel 24, thereby increasing the activity of the first mesenchymal stem cells 26, and the first mesenchymal stem cells 26 are easily and quickly fixed on the bottom surface 27 of the culture vessel 24. Can be made.

The electron microscope 13 is connected to the computer 11 via an interface (wired or wireless). 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. The electron microscope 13 takes magnified images of the planar shape of the first mesenchymal stem cells 26 contained in the intermediate layer bone marrow fluid 22 injected into the culture vessel 24 at intervals of about 1 to 2 hours, and the planar surface of the photographed stem cells 26 is taken. The enlarged image of the shape is transmitted to the computer 11 at intervals of about 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 first mesenchymal stem cell 26 transmitted from the electron microscope 13 and the imaging time in the storage area in a state associated with the donor identifier (first storage of the stem cell image) Process). The computer 11 displays on the display 16 an enlarged image of the planar shape of the stem cell 28 transmitted from the electron microscope 13 and the imaging time. The person in charge confirms (views) the enlarged image of the planar shape of the stem cell 26 displayed on the display 16 at intervals of about 1 to 2 hours during 12 to 24 hours, and determines the stem cells 26 contained in the intermediate layer bone marrow fluid 22. A change in planar shape is observed (stem cell first observation step). The person in charge may directly observe the change in the planar shape of the first mesenchymal stem cell 26 from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 12 to 24 hours.

In the culture product production method, the first mesenchymal stem cell 26 is fixed to the bottom surface 27 of the first flat culture vessel 24 (first culture vessel) while culturing the first mesenchymal stem cell 26 contained in the intermediate layer bone marrow fluid. (Stem cell first fixing step). The initial planar shape of the first mesenchymal stem cell 26 is substantially circular. When the planar shape of the stem cell 26 is substantially circular, the stem cell 26 is not fixed on the bottom surface 27 (bottom wall inner surface) of the culture vessel 24, and the stem cell. 26 has not started to proliferate (differentiate). The planar shape after the deformation of the first mesenchymal stem cell 26 is a flat shape in which the stem cell 26 is stretched (expanded) in one direction (predetermined direction) with a substantially circular shape before fixing as a nucleus, and the stem cell 26 is a culture vessel. 24 has settled on the bottom surface 27 (inner surface of the bottom wall), and the stem cells 26 have started to grow (differentiate).

As a result of the observation in the first stem cell observation step, the person in charge, as shown in FIG. 6, when the enlarged image of the planar shape of the first mesenchymal stem cell 26 displayed on the display 16 is observed in a substantially circular shape, It is determined that the stem cells 26 are not settled on the bottom surface 27 (bottom wall inner surface) of the culture vessel 24, and the change in the planar shape of the stem cells 26 is continuously observed at intervals of about 1 to 2 hours. As a result of the observation in the first stem cell observation step, the person in charge, as shown in FIG. 7, has a flat shape in which the planar shape of the first mesenchymal stem cell 26 displayed on the display 16 is from an approximately circular shape to an approximately circular shape. When the shape is deformed, it is determined that the stem cell 26 has settled on the bottom surface 27 of the culture vessel 24.

If a large culture container with a capacity exceeding 30 cc and a bottom area exceeding 36 mm ² is used when the first mesenchymal stem cell 26 is established, the stem cell 26 becomes difficult to settle on the bottom face of the container and the proliferation of the stem cell 26 is slow. However, in the culture product production method, the stem cell 26 can be easily fixed on the bottom surface 27 of the culture container 24 by using the first flat culture container 24 having the above-described capacity and the bottom surface area. The stem cell 26 can be rapidly proliferated.

The culture product producing method is such that the culture container 24 is left to stand at a temperature substantially the same as the body temperature for 12 to 24 hours, and the first interval in the culture container 24 is about 1 to 2 hours between 12 and 24 hours. Since the deformation of the leaf stem cells 26 from the initial planar shape is observed, the deformation of the stem cells 26 is not missed, and the establishment of the stem cells 26 on the bottom surface 27 of the culture vessel 24 can be confirmed accurately.

FIG. 9 is a partially enlarged view showing another example of the planar shape of the first mesenchymal stem cell 26. FIG. 9 shows an enlarged image of the planar shape of the first mesenchymal stem cell 26 photographed by the electron microscope 13. As a result of the observation in the first stem cell observation step, the first mesenchymal stem cell 26 (first 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, and the first stem cell 26 has a first shape. After the stem cell first fixing step in which the fixation on the bottom surface 27 of the flat culture vessel 24 (first culture vessel) is confirmed, the stem cell first culturing step and the stem cell second observation step are performed.

In the stem cell first culture step and the stem cell second observation step, the culture solution 23 injected into the culture vessel 24 is discharged (discarded) from the culture vessel 24, and a new culture solution 23 is injected (accommodated) into the culture vessel 24. ) Next, the culture vessel 24 is statically left (i.e., silently left unmoved) for 36 to 48 hours at a temperature substantially the same as the body temperature (about 36 to 37 ° C.), and the first mesenchyme relative to the bottom surface area of the culture vessel 24 is left. The stem cells 26 are cultured until the total planar area of the stem cells 26 reaches the first target ratio (stem cell first culture step).

The total planar area of the first mesenchymal stem cells 26 fixed to the bottom surface 27 of the culture vessel 24 at intervals of about 1 to 2 hours in a standing time of 36 to 48 hours is observed with the electron microscope 13 with respect to the bottom surface area of the culture vessel 24. It is determined whether or not the total planar area of the stem cells 26 has reached the first target ratio with respect to the bottom area of the culture vessel 24. The first target ratio of the total planar area of the first mesenchymal stem cell 26 to the bottom surface area of the culture vessel 24 is 70 to 80% (70 to 80% confluent).

A person in charge such as a doctor, nurse, or researcher confirms that the first mesenchymal stem cell 26 contained in the intermediate layer bone marrow fluid 22 has settled on the bottom surface 27 of the culture vessel 24 and then displays the stem cell number displayed on the display 16. 1 Click the observation end button. When the stem cell first observation end button is clicked, the computer 11 displays a process fourth display screen (not shown) on the display 16. On the process fourth display screen, number 1 and donor data are displayed, and a bone marrow fluid separation end message, a bone marrow fluid extraction end message, a stem cell first observation end message, a stem cell second observation button, and a stem cell first collection button are displayed. Is displayed.

The person in charge clicks the stem cell second observation button on the process fourth display screen, removes the culture container 24 from the sample holder of the electron microscope 16, and causes the barcode reader 12 to read the QR code of the culture container 24. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the donor data match, the number 1 and the donor data are displayed on the display 16, and the bone marrow fluid separation end message, bone marrow fluid extraction end message, stem cell first observation end message, stem cell second observation An ongoing message and a stem cell second observation end button are displayed on the display 16. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge discharges (discards) the culture solution 23 injected into the culture vessel 24 in the stem cell first observation step from the culture vessel 24 and injects (accommodates) the new culture solution 23 into the culture vessel 24. The new culture solution 23 is the same as that injected in the stem cell first observation step. The person in charge injects a new culture solution 23 into the culture vessel 24 and then installs (sets) the culture vessel 24 in the sample holder of the electron microscope 13.

A spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the bottom portion 38 of the first flat culture vessel 24, and the bottom portion 38 of the culture vessel 24 is held in a state of being lifted by the spacer 39. The culture vessel 24 is held at a predetermined angle so that the bottom 38 of the vessel 24 is on top and the top 40 (inlet 41) of the culture vessel 24 is on the bottom (see FIG. 6). In addition, a spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the top 40 of the first flat culture vessel 24, and the top 40 of the culture vessel 24 is held in a state of being lifted by the spacer 39. You may hold | maintain the culture container 24 in the state inclined by the predetermined angle so that the top part 40 of the container 24 may become the upper side and the bottom part 38 of the culture container 24 may become the lower side. The inclination angle α1 of the first flat culture vessel 24 with respect to the upper surface 37 of the sample holder 36 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

After confirming the establishment of the first mesenchymal stem cell 26, the culture product production method injects a new culture solution 23 into the culture vessel 24 while discharging the culture solution 23 in the culture vessel 24. Proliferation can be reliably promoted. In the culture product production method, the first mesenchymal stem cell 26 and the culture solution 23 are cultured in the culture vessel 24 by inclining the first flat culture vessel 24 at the inclination angle with respect to the upper surface 37 of the sample holder 36. It is biased toward the top 40 side (or the bottom 38 side) of the container 24, and the water pressure between the first mesenchymal stem cell 26 and the culture solution 23 increases on the top 40 side (or the bottom 38 side) of the culture container 24. Thus, the first mesenchymal stem cells 26 concentrate on the bottom 38 side of the culture vessel 24, thereby increasing the activity of the first mesenchymal stem cells 26, and the first mesenchymal stem cells 26 are formed on the bottom surface 27 of the culture vessel 24. Can be propagated (differentiated) easily and rapidly.

The electron microscope 13 takes an enlarged image of the planar shape of the first mesenchymal stem cell 26 in the culture vessel 24 at intervals of about 1 to 2 hours, and takes an enlarged image of the planar shape of the photographed stem cell 26 for about 1 to 2 hours. It transmits to the computer 11 at intervals. The computer 11 stores (stores) the enlarged image of the planar shape of the stem cell 26 transmitted from the electron microscope 13 and the imaging time in the storage area in a state associated with the donor identifier (stem cell image second storage step). The computer 11 displays the enlarged image of the planar shape of the stem cell 26 transmitted from the electron microscope 13 and the imaging time on the display 16.

The person in charge confirms (views) the enlarged image of the planar shape of the first mesenchymal stem cell 26 displayed on the display 16 at intervals of about 1 to 2 hours during 36 to 48 hours. While observing the total planar area of the stem cells 26 settled on the bottom surface area of the culture container 24, the total planar area of the stem cells 26 reaches the first target ratio (70 to 80% confluent) with respect to the bottom surface area of the culture container 24. It is determined whether or not (stem cell second observation step). The person in charge directly observes the total planar area of the first mesenchymal stem cell 26 with respect to the bottom surface area of the culture vessel 24 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 26 has reached the first target ratio (70 to 80% confluent) with respect to the bottom area of the culture vessel 24.

As a result of the observation in the second stem cell observation step, the person in charge shows that the total planar area with respect to the bottom area of the culture vessel 24 of the first mesenchymal stem cell 26 displayed on the display 16 is the first target ratio as shown in FIG. If it has not reached (70 to 80% confluence), the total planar area of the stem cells 26 relative to the bottom area of the culture vessel 24 is continuously observed at intervals of about 1 to 2 hours. In addition, when the total plane area of the first mesenchymal stem cell 26 reaches the first target ratio with respect to the entire area of the enlarged image displayed on the display 16, the total plane with respect to the bottom area of the culture vessel 24 of the stem cell 26 It is assumed that the area has reached the first target ratio.

As a result of the observation in the second stem cell observation step, the first mesenchymal stem cell 26 grows on the bottom surface 27 (bottom wall inner surface) of the culture vessel 24 to form a colony, and the planar shape of the stem cell 26 expands. As shown in FIG. 8, when the total planar area of the stem cells 26 displayed on the display 16 with respect to the bottom surface area of the culture vessel 24 reaches the first target ratio (70 to 80% confluent), the stem cells are removed from the culture vessel 24. A first stem cell collection step for extracting 26 is performed. In the first stem cell collection step, when the total planar area of the first mesenchymal stem cells 26 reaches the first target ratio, the first mesenchymal stem cells 26 grown (differentiated) in the culture vessel 24 are cultured in the culture vessel 24. Taken from.

A person in charge, such as a doctor, nurse, or researcher, confirms that the total planar area of the first mesenchymal stem cell 26 relative to the bottom area of the culture vessel 24 has reached the first target ratio, and then is displayed on the display 16. Click the button for ending stem cell second observation. When the stem cell second observation end button is clicked, the computer 11 displays a process fifth display screen (not shown) on the display 16. On the process fifth display screen, number 1 and donor data are displayed, bone marrow fluid separation end message, bone marrow fluid extraction end message, stem cell first observation end message, stem cell second observation end message, stem cell first collection button Is displayed.

The person in charge clicks the stem cell first collection button on the process fifth display screen, and causes the barcode reader 12 to read the QR code of the culture vessel 24. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the donor data match, the number 1 and the donor data are displayed on the display 16, and the bone marrow fluid separation end message, bone marrow fluid extraction end message, stem cell first observation end message, stem cell second observation An end message, a stem cell first collection execution message, and a stem cell first collection end button are displayed on the display 16. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge discharges (discards) the culture solution 23 in the second observation of the stem cells injected into the culture vessel 24 from the culture vessel 24 using a syringe or pipette, and after washing the inside of the culture vessel 24 with PBS, The trypsin solution sucked into the syringe or pipette is injected into the culture container 24. When the trypsin solution is injected into the culture vessel 24, the first mesenchymal stem cells 26 fixed on the bottom surface 27 of the culture vessel 24 are detached from the bottom surface 27 by the trypsin solution and float on the water surface of the trypsin solution. The person in charge collects (suctions) the stem cells 26 using a pipette, and stores the stem cells 26 in the pipette (stem cell first collection step).

By observing the total planar area at intervals of about 1 to 2 hours, the culture product production method can accurately confirm the total planar area with respect to the bottom area of the culture vessel 24 of the first mesenchymal stem cell 26, It can be ascertained that the total planar area of the stem cells 26 has reached the first target ratio with respect to the bottom surface area of the culture vessel 24.

FIG. 10 is an explanatory diagram showing an example of the stem cell separation step. After the first stem cell collection step in which the first mesenchymal stem cells 26 are collected from the culture vessel 24, a stem cell separation step is performed. In the stem cell separation step, the first mesenchymal stem cells 26 collected by the first collection means are centrifuged in layers by the centrifuge 28. A person in charge, such as a doctor, nurse, or researcher, aspirates the first mesenchymal stem cell 26 from the culture vessel 24 into the pipette, and then clicks the stem cell first collection end button displayed on the display 16.

When the stem cell first collection end button is clicked, the computer 11 displays a process sixth display screen (not shown) on the display 16. On the process sixth display screen, number 1 and donor data are displayed, and a stem cell separation button, a stem cell second extraction button, a stem cell third observation button, a stem cell fourth observation button, and a first extraction button are displayed. The person in charge affixes the first code sheet 17 on which the QR code is printed to the outer peripheral surface of the glass test tube 29 into which the first mesenchymal stem cells 26 are injected.

Next, the stem cell separation button on the process sixth display screen is clicked, and the QR code of the glass test tube 29 is read by the barcode reader 12. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the data are compared with each other and the donor data match, the number 1 and the donor data are displayed on the display 16, and a stem cell separation in progress message and a stem cell separation end button are displayed on the display 16. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge injects (accommodates) the first mesenchymal stem cell 26 in the pipette into the glass test tube 29 and installs (sets) the glass test tube 29 in the centrifuge 28. The person in charge centrifuges the stem cells 26 with the centrifuge 28 for a predetermined time, and then removes the glass test tube 29 from the centrifuge 28. The first mesenchymal stem cells 26 in the glass test tube 29 are separated into several layers in the vertical direction by the centrifuge 28 (stem cell separation step).

FIG. 11 is an explanatory diagram showing an example of the second stem cell collection step. After the stem cell separation step in which the first mesenchymal stem cells 26 are separated into layers, a stem cell second collection step is performed. In the second stem cell collection step, second mesenchymal stem cells 30 located in the lower layer (lowermost layer) are collected from the first mesenchymal stem cells 26 separated into layers. A doctor, nurse, researcher, or other person in charge uses the centrifuge 28 to separate the first mesenchymal stem cells 26 in layers in the vertical direction, and then removes the glass test tube 29 from the centrifuge 28 and displays the display 16. Click the stem cell separation end button displayed in.

When the stem cell separation end button is clicked, the computer 11 displays a process seventh display screen (not shown) on the display 16. On the process seventh display screen, number 1 and donor data are displayed, and a stem cell separation end message, a stem cell second collection button, a stem cell third observation button, a stem cell fourth observation button, and a culture product solution first extraction button are displayed. Is displayed.

The person in charge clicks the stem cell second collection button on the process seventh display screen, and causes the barcode reader 12 to read the QR code of the glass test tube 29. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the donor data match with each other, the number 1 and the donor data are displayed on the display 16, and a stem cell separation end message, a stem cell second collection in progress message, and a stem cell second collection end button are displayed on the display 16. indicate. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge collects the second mesenchymal stem cell 30 present in the lower layer (lowermost layer) of the first mesenchymal stem cells 26 separated into layers in the glass test tube 29 (second stem cell collecting step). The person in charge collects (sucks) the second mesenchymal stem cells 30 located in the lower layer (lowermost layer) of the first mesenchymal stem cells 26 separated into layers using a syringe. Alternatively, the second mesenchymal stem cells 30 located in the lower layer (lowermost layer) of the first mesenchymal stem cells 26 separated into layers using a pipette are collected (sucked).

The production method of the culture product is obtained by centrifuging the first mesenchymal stem cells 26 containing unnecessary stem cells with a centrifuge 28 and separating them in layers in the vertical direction. By collecting the second mesenchymal stem cell 30 located in the lower layer, a specific second mesenchymal stem cell 30 can be reliably collected from the stem cell 26, and unnecessary mesenchymal stem cells are removed from the stem cell 26. can do.

When the total planar area of the first mesenchymal stem cell 26 with respect to the bottom surface area of the first flat culture vessel 24 (first culture vessel) exceeds 80% and the stem cell 26 proliferates, Although the activity is gradually lost, the stem cell 26 is collected from the culture vessel 24 when the total planar area of the stem cell 26 grows to 70 to 80% of the bottom surface area of the culture vessel 24, so that the activity of the stem cell 26 is retained. The stem cells 26 can be proliferated while maintaining the activity, and the second mesenchymal stem cells 30 having activity can be collected from the stem cells 26.

FIG. 12 is an explanatory view showing an example of the third observation process of stem cells included in the second fixing means, and FIG. 13 is a side view of the second flat culture vessel. FIG. 14 is a partially enlarged view showing an example of the planar shape of the second mesenchymal stem cell 30, and FIG. 15 is a partially enlarged view showing another example of the planar shape of the second mesenchymal stem cell 30. 14 and 15 show enlarged images of the planar shape of the second mesenchymal stem cell 30 photographed by the electron microscope 13.

The stem cell third observation step and the stem cell second colonization step are performed after the stem cell second collection step in which specific second mesenchymal stem cells 30 located in the lower layer (lowermost layer) are collected from the first mesenchymal stem cell 26. . In the stem cell third observation step and the stem cell second fixing step, the second mesenchymal stem cell 30 and the culture solution 23 are injected (contained) into the second flat culture vessel 25 (second culture vessel), and the culture vessel 25 is heated to the body temperature. It is left to stand statically (without moving) for 36 to 48 hours at substantially the same temperature (about 36 to 37 ° C.).

Deformation from the initial planar shape of the second mesenchymal stem cell 30 (second stem cell) in the culture vessel 25 is observed with the electron microscope 13 at intervals of about 1 to 2 hours in a standing time of 36 to 48 hours. It is determined whether or not the bottom surface 27 of the culture vessel 25 is fixed. A first code sheet 17 on which a QR code specifying a donor is printed is attached to the bottom surface 27 (outer surface of the bottom wall) of the culture vessel 25 into which the second mesenchymal stem cells 30 and the culture solution 23 are injected.

A person in charge such as a doctor, a nurse, or a researcher collects a specific second mesenchymal stem cell 30 from the first mesenchymal stem cell 26 and then clicks a stem cell second collection end button displayed on the display 16. . When the stem cell second collection end button is clicked, the computer 11 displays a process eighth display screen (not shown) on the display 16. On the process eighth display screen, number 1 and donor data are displayed, a stem cell separation end message, a stem cell second collection end message, a stem cell third observation button, a stem cell fourth observation button, and a culture solution first extraction button Is displayed.

The person in charge attaches the first code sheet 17 on which the QR code is printed to the bottom surface 27 (outer surface of the bottom wall) of the second flat culture vessel 25. Next, the third stem cell observation button on the process eighth display screen is clicked to cause the barcode reader 12 to read the QR code of the culture vessel 25. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the donor data match, the number 1 and the donor data are displayed on the display 16, and the stem cell separation end message, the stem cell second collection end message, the stem cell third observation in progress message, the stem cell third An observation end button is displayed on the display 16. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge injects (accommodates) the second mesenchymal stem cell 30 sucked into the syringe or pipette into the culture container 25 and injects (accommodates) the culture solution 23 into the culture container 25. The second flat culture vessel 25 (second culture vessel) is a flat vessel made of transparent glass or transparent plastic and having a bottom surface 27 having a predetermined capacity and a predetermined area and having a substantially square shape. The area is about twice that of the first flat culture vessel 24 (first culture vessel). The second flat culture vessel 25 has a top portion 43 and a bottom portion 44 and an injection port 45 formed in the top portion 43. The injection port 45 is watertightly closed by a lid 46. As the second flat culture vessel 25, a flat vessel having a circular shape or an elliptical shape having a bottom surface 27 having a predetermined capacity and a predetermined area may be used.

The second flat culture vessel 25 (second culture vessel) used in the third stem cell observation 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 25 has a side length of about 7 to 8.5 mm. The culture solution 23 is the same as that injected in the first observation of stem cells. The second mesenchymal stem cells 30 injected into the culture vessel 25 are cultured with the culture solution 23 while being fixed on the bottom surface 27 of the culture vessel 25 as time passes, and gradually grow (differentiate) on the bottom surface 27 of the culture vessel 25. To form colonies.

The person in charge injects the second mesenchymal stem cell 30 and the culture solution 23 into the second flat culture vessel 25 and then installs (sets) the culture vessel 25 in the sample holder 36 of the electron microscope 13. A spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the bottom portion 44 of the second flat culture vessel 25, and the bottom portion 44 of the culture vessel 25 is held in a state lifted by the spacer 39. The culture vessel 25 is held in a state inclined at a predetermined angle such that the bottom portion 44 of the culture vessel is on the top and the top portion 43 (injection port 45) of the culture vessel 25 is on the bottom. In addition, a spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the top 43 of the second flat culture vessel 25, and the top 43 of the culture vessel 25 is held in a state lifted by the spacer 39. You may hold | maintain the culture container 25 in the state inclined to the predetermined angle so that the top part 43 of the container 25 may become the upper side and the bottom part 44 of the culture container 25 may become the lower side. The inclination angle α2 of the second flat culture vessel 25 with respect to the upper surface 37 of the sample holder 36 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 mesenchymal stem cell 30 and the culture solution 23 are cultured in the culture vessel 25 by inclining the second flat culture vessel 25 at the inclination angle with respect to the upper surface 37 of the sample holder 36. It is biased toward the top 43 side (or the bottom 44 side) of the container 25, and the water pressure between the second mesenchymal stem cell 30 and the culture solution 23 increases on the top 43 side (or the bottom 44 side) of the culture container 25. Thus, the second mesenchymal stem cells 30 are concentrated on the bottom 44 side of the culture vessel 25, thereby increasing the activity of the second mesenchymal stem cells 30, and the second mesenchymal stem cell 30 on the bottom surface 27 of the culture vessel 25. Can be fixed easily and quickly.

The electron microscope 13 takes a magnified image of the planar shape of the second mesenchymal stem cell 30 injected into the culture vessel 25 at intervals of about 1 to 2 hours, and the magnified image of the planar shape of the photographed stem cell 30 is approximately 1 to 2 times. It transmits to the computer 11 at intervals of 2 hours. The computer 11 stores (stores) the planar enlarged image of the second mesenchymal stem cell 30 transmitted from the electron microscope 13 and the imaging time in a storage area in a state associated with the donor identifier (stem cell image third memory). Process). 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 16. The person in charge checks (views) the enlarged image of the planar shape of the stem cell 30 displayed on the display 16 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 ( Stem cell third observation step). 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 second mesenchymal stem cell 30 is substantially circular. When the planar shape of the stem cell 30 is approximately circular, the stem cell 30 is not fixed on the bottom surface 27 (bottom wall inner surface) of the culture vessel 25, and the stem cell. 30 has not started to proliferate (differentiate). The planar shape after deformation of the second mesenchymal stem cell 30 is a flat shape in which the stem cell 30 extends in an indefinite shape in one direction with a substantially circular shape before fixation as a nucleus, and the stem cell 30 is a bottom surface 27 (bottom wall) of the culture vessel 25. The stem cell 30 has started to proliferate (differentiate).

As a result of the observation in the third stem cell observation step, the person in charge looks at the stem cell 30 when the planar shape of the second mesenchymal stem cell 30 displayed on the display 16 remains substantially circular as shown in FIG. It is determined that the bottom surface 27 (inner wall inner surface) of the culture vessel 25 has not settled, 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. 13, as a result of observation by the stem cell third observation means, the person in charge has an irregular flat shape in which the planar shape of the second mesenchymal stem cell 30 displayed on the display 16 is from a substantially circular shape to a substantially circular shape as a nucleus. When the shape is deformed, it is determined that the stem cell 30 has settled on the bottom surface 27 of the culture vessel 25 (stem cell second fixing step).

When a large culture container having a capacity exceeding 60 cc and a bottom area exceeding 72 mm ² is used when the second mesenchymal stem cell 30 is fixed, the stem cell 30 becomes difficult to settle on the bottom surface of the container and the proliferation of the stem cell 30 is slow. However, in the method for producing a culture product solution, the stem cell 30 can be easily fixed on the bottom surface 27 of the culture container 25 by using the second flat culture container 25 having the above-mentioned capacity and the bottom surface area. The stem cell 30 can be rapidly proliferated.

The method for producing a culture product solution is to leave the culture vessel 25 statically at a temperature substantially the same as the body temperature for 36 to 48 hours, and at intervals of about 1 to 2 hours between 36 and 48 hours. Since the deformation of the leaf stem cells 30 from the initial planar shape is observed, the deformation of the stem cells 30 is not overlooked, and the establishment of the stem cells 30 on the bottom surface 27 of the culture vessel 25 can be confirmed accurately.

FIG. 16 is a partially enlarged view showing another example of the planar shape of the second mesenchymal stem cell 30, and FIG. 17 shows an example of preservation of the extracted culture product solution 31 and the second mesenchymal stem cell 30. FIG. FIG. 16 shows an enlarged image of the planar shape of the second mesenchymal stem cell 30 photographed by the electron microscope 13. As a result of the observation in the third stem cell observation step, the second 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, and the second stem cell 30 The stem cell second culture step and the stem cell fourth observation step are performed after the stem cell third observation step in which the fixation to the bottom surface 27 of the flat culture vessel 25 (second culture vessel) is confirmed.

In the stem cell second culture step and the stem cell fourth observation step, the culture solution 23 injected into the culture vessel 25 is discharged (discarded) from the culture vessel 25, and a new culture solution 23 is injected (accommodated) into the culture vessel 25. Is done. The culture vessel 25 is left to stand statically (without moving) for 36 to 48 hours at a temperature substantially the same as the body temperature (about 36 to 37 ° C.), and the second mesenchymal stem cell 30 is cultured in the culture vessel 25. (Stem cell second culture step). The total planar area of the second mesenchymal stem cells 30 fixed on the bottom surface 27 of the culture vessel 25 at intervals of about 1 to 2 hours in the standing time of 36 to 48 hours is observed with the electron microscope 13 with respect to the bottom surface area of the culture vessel 25; It is determined whether or not the total planar area of the stem cells 30 has reached the second target ratio with respect to the bottom area of the culture vessel 25. The second target ratio of the total planar area of the second mesenchymal stem cell 30 to the bottom surface area of the culture vessel 25 is 88 to 92% (88 to 92% confluent).

A person in charge, such as a doctor, nurse, or researcher, confirms that the second mesenchymal stem cell 30 has settled on the bottom surface 27 of the culture vessel 25, and then clicks the stem cell third observation end button displayed on the display 16. When the stem cell third observation end button is clicked, the computer 11 displays a process eighth display screen (not shown) on the display 16. On the process eighth display screen, number 1 and donor data are displayed, a stem cell separation end message, a stem cell second collection end message, a stem cell third observation end message, a stem cell fourth observation button, and a culture product solution first extraction A button is displayed.

The person in charge clicks the stem cell fourth observation button on the process eighth display screen, removes the culture vessel 25 from the sample holder of the electron microscope 13, and causes the barcode reader 12 to read the QR code of the culture vessel 25. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data displayed on the display 16 (donor data read into the memory) and the donor data indicated by the QR code read by the barcode reader. When the donor data match, the number 1 and the donor data are displayed on the display 16, and the stem cell separation end message, the stem cell second collection end message, the stem cell third observation end message, and the stem cell fourth observation are performed. A middle message and a stem cell fourth observation end button are displayed on the display 16. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge discharges (discards) the culture solution 23 injected into the culture vessel 25 in the stem cell second culture step and the stem cell fourth observation step, and injects (accommodates) the new culture solution 23 into the culture vessel 25. ) The new culture solution 23 is the same as that injected in the stem cell first observation means. The person in charge injects a new culture solution 23 into the culture vessel 25 and then installs (sets) the culture vessel 25 on the sample holder of the electron microscope 13. A spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the bottom portion 44 of the second flat culture vessel 25, and the bottom portion 44 of the culture vessel 25 is held in a state lifted by the spacer 39. The culture vessel 25 is held in a state inclined at a predetermined angle such that the bottom portion 44 of the culture vessel is on the top and the top 43 (injection port 45) of the culture vessel 25 is on the bottom. In addition, a spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the top 43 of the second flat culture vessel 25, and the top 43 of the culture vessel 25 is held in a state lifted by the spacer 39. You may hold | maintain the culture container 25 in the state inclined to the predetermined angle so that the top part 43 of the container 25 may become the upper side and the bottom part 44 of the culture container 25 may become the lower side. The inclination angle α2 of the second flat culture vessel 25 with respect to the upper surface 37 of the sample holder 36 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

After confirming the establishment of the second mesenchymal stem cell 30, the culture product production method is configured to inject a new culture solution 23 into the culture vessel 25 while discharging the culture solution 23 in the culture vessel 25. The proliferation of the mesenchymal stem cell 30 can be surely promoted. In the culture product production method, the second mesenchymal stem cell 30 and the culture solution 23 are cultured in the culture vessel 25 by inclining the second flat culture vessel 25 at the inclination angle with respect to the upper surface 37 of the sample holder 36. It is biased toward the top 43 side (or the bottom 44 side) of the container 25, and the water pressure between the second mesenchymal stem cell 30 and the culture solution 23 increases on the top 43 side (or the bottom 44 side) of the culture container 25. Thus, the second mesenchymal stem cells 30 are concentrated on the bottom 44 side of the culture vessel 25, thereby increasing the activity of the second mesenchymal stem cells 30, and the second mesenchymal stem cell 30 on the bottom surface 27 of the culture vessel 25. Can be propagated (differentiated) easily and rapidly.

The electron microscope 13 takes an enlarged image of the planar shape of the second mesenchymal stem cell 30 in the culture vessel 25 at intervals of about 1 to 2 hours, and takes an enlarged image of the planar shape of the photographed stem cell 30 for about 1 to 2 hours. It transmits to the computer 11 at intervals. The computer 11 stores (stores) the enlarged image of the planar shape of the second mesenchymal stem cell 30 and the imaging time transmitted from the electron microscope 13 in a storage area in a state associated with the donor identifier (stem cell image fourth memory). Process). The computer 11 displays the enlarged image of the planar shape of the second mesenchymal stem cell 30 transmitted from the electron microscope 13 and the imaging time on the display 16.

The person in charge confirms (views) the enlarged image of the planar shape of the second mesenchymal stem cell 30 displayed on the display 16 at intervals of about 1 to 2 hours during 36 to 48 hours. While observing the total planar area of the stem cells 30 settled on the bottom surface area of the culture vessel 25, the total planar area of the stem cells 30 reaches the second target ratio (82 to 92% confluent) with respect to the bottom surface area of the culture vessel 25. It is determined whether or not (stem cell fourth observation means). The person in charge directly observes the total planar area of the second mesenchymal stem cell 30 with respect to the bottom surface area of the culture vessel 25 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 plane area of 30 has reached the second target ratio (88-92% confluent) with respect to the bottom area of the culture vessel 25.

As a result of the observation in the fourth stem cell observation step, the person in charge is, as shown in FIG. 13, the total plane area of the second mesenchymal stem cell 30 displayed on the display 16 with respect to the bottom area of the culture vessel 25 is the second target ratio. If it has not reached (88 to 92% confluence), the total planar area of the stem cells 30 relative to the bottom area of the culture vessel 25 is continuously observed at intervals of about 1 to 2 hours. In addition, when the total plane area of the second mesenchymal stem cell 30 reaches the second target ratio with respect to the entire area of the enlarged image displayed on the display 16, the total plane with respect to the bottom area of the culture vessel 25 of the stem cell 30 It is assumed that the area has reached the second target ratio.

As a result of the observation in the stem cell fourth observation step, the second mesenchymal stem cell 30 grows on the bottom surface 27 (bottom wall inner surface) of the second flat culture vessel 25 (second culture vessel), and the stem cell 30 forms a colony, As the planar shape of the stem cell 30 expands, as shown in FIG. 14, the total planar area of the stem cell 30 displayed on the display 16 with respect to the bottom surface area of the culture vessel 25 becomes the second target ratio (88 to 92% confluent). When it reaches, a single kind of second mesenchymal stem cell 30 is proliferated in the culture vessel 25, and a culture product solution 31 containing a predetermined metabolite secreted from the active single kind of stem cell 30 is generated. Has been.

When the total planar area of the second mesenchymal stem cell 30 exceeds 92% with respect to the bottom surface area of the second flat culture vessel 25 (second culture vessel), the stem cell 30 is proliferated. Although the activity is gradually lost, the stem cell 30 is extracted from the culture vessel 25 when the total planar area of the stem cell 30 grows to 88 to 92% of the bottom surface area of the culture vessel 25, so that the activity of the stem cell 30 is retained. The stem cell 30 can be proliferated while maintaining the activity.

When the total planar area of the stem cells 30 with respect to the bottom surface area of the culture vessel 25 reaches the second target ratio, the culture product solution first extraction step for extracting the culture product solution 31 from the culture vessel 25 is performed. In the culture product solution first extraction step, a culture product solution 31 containing a predetermined metabolite secreted from the active second mesenchymal stem cell 30 is extracted from the culture vessel 25. The method for producing a culture product solution can accurately confirm the total planar area with respect to the bottom area of the culture vessel 25 of the second mesenchymal stem cell 30 by observing the total planar area at intervals of about 1 to 2 hours. It can be ascertained that the total planar area of the stem cells 30 has reached the second target ratio with respect to the bottom surface area of the culture vessel 25.

A person in charge such as a doctor, nurse, or researcher confirms that the total planar area of the second mesenchymal stem cell 30 with respect to the bottom area of the culture vessel 25 has reached the second target ratio, and then is displayed on the display 16. Click the stem cell fourth observation end button. When the stem cell fourth observation end button is clicked, the computer 11 displays a process ninth display screen (not shown) on the display 16. On the process 9th display screen, number 1 and donor data are displayed, stem cell separation end message, stem cell second collection end message, stem cell third observation end message, stem cell fourth observation end message, culture product first Extract button is displayed.

The person in charge clicks the culture solution first extraction button on the process ninth display screen, and causes the barcode reader 12 to read the QR code of the culture vessel 25. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the donor data match, the number 1 and the donor data are displayed on the display 16, and the stem cell separation end message, the stem cell second collection end message, the stem cell third observation end message, and the stem cell fourth observation An end message and a culture product first extraction end button are displayed on the display 16. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge aspirates the culture product solution 31 changed from the culture solution 23 injected into the culture vessel 25 in the fourth stem cell observation step from the culture vessel 25 using a syringe or pipette (culture product solution first extraction step). Then, the culture product solution 31 is injected (stored) in the storage container 32. The culture product solution 31 injected into the storage container 32 contains a predetermined metabolite secreted from a specific type of single-type mesenchymal stem cells (single-type stem cells) having an activity from which unnecessary mesenchymal stem cells have been removed. It is the culture product solution 31 containing.

The person in charge injects the culture product solution 31 into the storage container 32, and then attaches the first code sheet 17 on which the QR code is printed to the outer peripheral surface of the storage container 32. The person in charge clicks the culture solution first extraction end button on the ninth display screen displayed on the display 16, causes the barcode reader 12 to read the QR code of the storage container 32, and then injects the culture product 31. The stored storage container 32 is stored in the refrigerator 34. The culture product solution 31 is stored at about 3 to 4 ° C. in the refrigerator 34.

When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the donor data match, the number 1 and the donor data are displayed on the display 16, and the stem cell separation end message, the stem cell second collection end message, the stem cell third observation end message, and the stem cell fourth observation An end message, a culture product extraction end message, a culture product manufacture end button, and a culture product manufacture continue button are displayed on the display 16. When the production of the culture product is finished, the culture product production end button is clicked. When the culture product production end button is clicked, the system 10 stops in the computer 11.

When the culture production solution production end button is clicked, a stem cell collection step of collecting the stem cells 30 from the culture vessel 25 is performed. In the stem cell collection step, the second mesenchymal stem cells 30 that have proliferated (differentiated) in the culture vessel 25 are collected from the culture vessel 25. After the culture vessel 25 is washed with PBS, a trypsin solution is injected into the culture vessel 25. When the trypsin solution is injected into the culture vessel 25, the second mesenchymal stem cells 30 fixed on the bottom surface 27 of the culture vessel 25 are detached from the bottom surface 27 by the trypsin solution and float on the water surface of the trypsin solution. The person in charge sucks the second mesenchymal stem cell 30 using a syringe or pipette, and injects (accommodates) the stem cell 30 into the storage container 33 from the syringe or pipette.

The second mesenchymal stem cell 30 injected into the storage container 33 is a specific kind of single mesenchymal stem cell (single seed stem cell) having an activity from which unnecessary mesenchymal stem cells are removed. The person in charge injects the second mesenchymal stem cell 30 (single mesenchymal stem cell) into the storage container 33, and then attaches the first code sheet 17 on which the QR code is printed to the outer peripheral surface of the storage container 33. The storage container 33 into which the second mesenchymal stem cells 30 are injected is stored in the refrigerator 34. The second mesenchymal stem cell 30 (single mesenchymal stem cell) is stored in the refrigerator 34 at about 3 to 4 ° C.

In the production method of the culture solution, the intermediate layer bone marrow fluid located in the intermediate layer of the bone marrow fluid separated into layers is extracted, and the intermediate layer bone marrow fluid is cultured together with the culture solution 23 to obtain the first mesenchymal stem cells 26 (first 1 stem cell) is fixed to the bottom surface 27 of the first culture vessel 24, and the stem cell 26 is cultured while the total planar area of the stem cell 26 reaches the first target ratio with respect to the bottom surface area of the first culture vessel 24. Stem cells 26 are collected from the container 24, and the second mesenchymal stem cells 30 (second stem cells) located in the lower layer (lowermost layer) of the stem cells 26 separated in layers are collected, and the stem cells 30 are added to the culture solution 23. And the stem cells 30 are fixed to the bottom surface 27 of the second culture vessel 25, a new culture solution 23 is injected into the culture vessel 25, and the stem cells 30 are cultured while the total planar area of the stem cells 30 is that of the culture vessel 25. For the bottom area 2. When the target ratio is reached, the culture product solution 31 containing a predetermined metabolite secreted from a single kind of stem cell 30 grown in the course of culturing the stem cell 30 is extracted from the culture vessel 25 and thus has a cultured activity. By using the second stem cell 30 of a single species, a wide variety of metabolites secreted from a variety of stem cells are not included, and only a predetermined metabolite secreted from a single species of stem cell 30 having activity is included. Can be produced, and can be used effectively for the treatment of a predetermined disease, and can produce a culture solution 31 that exhibits only a specific effect on a predetermined disease, skin care, hair care, body care, etc. Can be produced, and the culture product 31 that can be used effectively for a predetermined beauty can be produced.

In the culture product production method, a culture product solution 31 is produced using pure second pure mesenchymal stem cells 30 (second stem cells) from which unnecessary stem cells have been removed. Since only a predetermined metabolite secreted from a kind of second stem cell can be contained, a culture product solution 31 having a high therapeutic effect for a predetermined disease and having a high probability of completely curing the disease is produced. The culture product solution 31 having a high effect on a predetermined beauty can be made. The culture product solution 31 produced by the culture product production method can be used for the treatment of skin diseases such as burns and eczema, for example, by applying it to the skin. In addition to being used as eye drops, it can be used for the treatment of hay fever by injecting it into the nose, and it can be used for the treatment of scalp disease by applying it to the scalp. In addition, it can be used as a measure against rough skin, breakouts and sunburn, and can be used for beauty such as whitening and removal of wrinkles.

FIG. 18 is an explanatory view showing an example of the fifth stem cell observation step, and FIG. 19 is a side view of the third flat culture vessel. When the production of the culture product solution is continued, a second stem cell collecting step for collecting the second mesenchymal stem cells 30 proliferated (differentiated) in the culture vessel 25 from the culture vessel 25 is performed. In the second stem cell collection step, the second mesenchymal stem cell 30 proliferated (differentiated) in the culture vessel 25 is collected from the culture vessel 25.

The person in charge, such as a doctor, nurse, or researcher, clicks the culture product production continuation button displayed on the display 16. When the culture product production continuation button is clicked, the computer 11 displays a process tenth display screen (not shown) on the display 16. In the process 10th display screen, number 1 and donor data are displayed, and a stem cell second collection button, a stem cell fifth observation button, a stem cell sixth observation button, and a culture product second extraction button are displayed.

The person in charge clicks the stem cell second collection button on the process tenth display screen, and causes the barcode reader 12 to read the QR code of the culture vessel 25. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the donor data match with each other, the number 1 and the donor data are displayed on the display 16 and the second stem cell collection in progress message, the stem cell second collection end button, the stem cell fifth observation button, the stem cell number 1 A 6 observation button and a culture product solution second extraction button are displayed on the display 16. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge wash | cleans the inside of the culture container 25 with PBS, inject | pours a trypsin liquid in the culture container 25, and collect | recovers the 2nd mesenchymal stem cell 30 which floated on the water surface of a trypsin liquid using a syringe or a pipette ( The stem cells 26 are accommodated in a syringe or pipette (second stem cell collection means). The person in charge collects the stem cells 30 from the culture vessel 25 and then clicks the stem cell second collection end button. When the stem cell second collection end button is clicked, the computer 11 displays a process eleventh display screen (not shown) on the display 16. On the process eleventh display screen, number 1 and donor data are displayed, and a stem cell second collection end message, a stem cell fifth observation button, a stem cell sixth observation button, and a culture product second extraction button are displayed.

After the second stem cell collecting step in which the second mesenchymal stem cells 30 are collected from the culture vessel 25, the fifth stem cell observation step and the third stem cell fixing step are performed. In the stem cell fifth observation step and the stem cell third fixing step, the second mesenchymal stem cell 30 and the culture solution 23 are injected (contained) into the third flat culture vessel 35 (third culture vessel), and the culture vessel 35 is heated to the body temperature. It is left to stand statically (without moving) for 36 to 48 hours at substantially the same temperature (about 36 to 37 ° C.).

Deformation from the initial planar shape of the second mesenchymal stem cells 30 (second stem cells) in the culture vessel 35 is observed with an electron microscope 13 at intervals of about 1 to 2 hours in a standing time of 36 to 48 hours. It is determined whether or not the bottom surface 27 of the culture vessel 35 is fixed. A first code sheet 17 on which a QR code specifying a donor is printed is attached to the bottom surface 27 (outer surface of the bottom wall) of the culture vessel 35 into which the second mesenchymal stem cells 30 and the culture solution 23 are injected.

A person in charge such as a doctor, nurse, researcher, etc. collects the second mesenchymal stem cell 30 from the culture container 25 and then uses the first code sheet 17 on which the QR code is printed as the bottom surface 27 of the third flat culture container 35. Affix to (outer surface of bottom wall). Next, the fifth button for observing stem cells on the process eleventh display screen is clicked to cause the barcode reader 12 to read the QR code of the culture vessel 25. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the donor data match with each other, the number 1 and the donor data are displayed on the display 16, and the stem cell second collection end message, the stem cell fifth observation in progress message, the stem cell fifth observation end button, the stem cell A sixth observation button and a culture product second extraction button are displayed on the display 16. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge injects (accommodates) the second mesenchymal stem cell 30 sucked into the syringe or pipette into the culture vessel 35 and injects (accommodates) the culture solution 23 into the culture vessel 35. The third flat culture vessel 35 (third culture vessel) is a flat vessel made of transparent glass or transparent plastic and having a bottom surface 27 having a predetermined capacity and a predetermined area and having a substantially square shape. The area is larger than the second flat culture vessel 25 (second culture vessel). The third flat culture vessel 35 has a top 47 and a bottom 48 and an injection port 49 formed in the top 47. The injection port 49 is watertightly closed by the lid 50. As the third flat culture container 35, a flat container having a circular shape or an elliptical shape having a bottom surface 27 having a predetermined capacity and a predetermined area may be used.

The third flat culture vessel 35 (third culture vessel) used in the fifth stem cell observation step has a capacity of about 70 to 80 cc (preferably 75 cc) and a bottom area of about 75 to 108 mm ² . . The third flat culture vessel has a side length of about 8.7 to 10.4 mm. The culture solution 23 is the same as that injected in the first observation of stem cells. The second mesenchymal stem cells 30 injected into the culture vessel 35 are cultured on the bottom surface 27 of the culture vessel 35 with the passage of time, are cultured with the culture solution 23, and gradually grow (differentiate) on the bottom surface 27 of the culture vessel 35. To form colonies.

The person in charge injects the second mesenchymal stem cell 30 and the culture solution 23 into the culture container 35, and then installs (sets) the culture container 35 in the sample holder of the electron microscope 13. A spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the bottom 48 of the third flat culture vessel 35, and the bottom 48 of the culture vessel 35 is held up by the spacer 39. The culture vessel 35 is held in a state inclined at a predetermined angle such that the bottom portion 48 of the culture vessel 25 is on the top and the top portion 47 (injection port 49) of the culture vessel 25 is on the bottom. Further, a spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the top 48 of the third flat culture vessel 35, and the top 47 of the culture vessel 35 is held in a state where it is lifted by the spacer 39. You may hold | maintain the culture container 35 in the state inclined to the predetermined angle so that the top part 47 of the container 35 may become the upper side and the bottom part 49 of the culture container 35 may become the lower side. The inclination angle α2 of the third flat culture vessel 35 with respect to the upper surface 37 of the sample holder 36 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 mesenchymal stem cell 30 and the culture solution 23 are cultured in the culture vessel 35 by inclining the third flat culture vessel 35 at the inclination angle with respect to the upper surface 37 of the sample holder 36. The pressure is biased toward the top 47 (or the bottom 48) of the container 35, and the water pressure between the second mesenchymal stem cell 30 and the culture solution 23 increases on the top 47 (or the bottom 48) of the culture container 35. Thus, the second mesenchymal stem cells 30 concentrate on the bottom 48 side of the culture vessel 35, thereby increasing the activity of the second mesenchymal stem cells 30, and the second mesenchymal stem cell 30 is formed on the bottom surface 27 of the culture vessel 35. Can be fixed easily and quickly.

The electron microscope 13 takes a magnified image of the planar shape of the second mesenchymal stem cell 30 injected into the culture vessel 35 at intervals of about 1 to 2 hours, and the magnified image of the planar shape of the photographed stem cell 30 is approximately 1 to 2 hours. It transmits to the computer 11 at intervals of 2 hours. The computer 11 stores (stores) the planar enlarged image of the second mesenchymal stem cell 30 transmitted from the electron microscope 13 and the imaging time in the storage area in a state associated with the donor identifier (stem cell image fifth memory). Process). 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 16. The person in charge checks (views) the enlarged image of the planar shape of the stem cell 30 displayed on the display 16 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 ( Stem cell fifth observation step). 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 second mesenchymal stem cell 30 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 27 (bottom wall inner surface) of the culture vessel 35, and the stem cell. 30 has not started to proliferate (differentiate). The planar shape after the deformation of the second mesenchymal stem cell 30 is a flat shape in which the stem cell 30 extends in an indefinite shape in one direction with the substantially circular shape before fixing as a nucleus, and the stem cell 30 is a bottom surface 27 (bottom wall) of the culture vessel 35. The stem cell 30 has started to proliferate (differentiate).

In the case where the planar shape of the second mesenchymal stem cell 30 displayed on the display 16 is observed as a substantially circular shape as a result of the observation in the fifth observation step of the stem cells (see FIG. 12 for assistance), 35 is determined not to have settled on the bottom surface 27 (inner surface of the bottom wall), and changes in the planar shape of the stem cells 30 are continuously observed at intervals of about 1 to 2 hours. As a result of the observation by the third stem cell observation means, the person in charge changes the planar shape of the second mesenchymal stem cell 30 displayed on the display 16 from an approximately circular shape to an indeterminate flat shape with the approximately circular shape as a nucleus (see FIG. 13), it is determined that the stem cells 30 have settled on the bottom surface 27 of the culture vessel 35 (stem cell third fixing step).

When a large culture container having a capacity exceeding 80 cc and a bottom area exceeding 108 mm ² is used when the second mesenchymal stem cell 30 is fixed, the stem cell 30 becomes difficult to settle on the bottom surface of the container and the proliferation of the stem cell 30 is slow. However, in the method for producing a culture product solution, the stem cell 30 can be easily fixed on the bottom surface 27 of the culture container 35 by using the third flat culture container 35 having the above-described capacity and the bottom surface area. The stem cell 30 can be rapidly proliferated.

The method for producing a culture product solution is to leave the culture vessel 35 statically at a temperature substantially the same as the body temperature for 36 to 48 hours, and at intervals of about 1 to 2 hours between 36 and 48 hours. Since the deformation of the leaf stem cells 30 from the initial planar shape is observed, the deformation of the stem cells 30 is not overlooked, and the establishment of the stem cells 30 on the bottom surface 27 of the culture vessel 35 can be confirmed accurately.

As a result of the observation in the fifth stem cell observation step, the second mesenchymal stem cell 30 (second stem cell) 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 third stem cell 30 The stem cell third culturing step and the stem cell sixth observing step are performed after the stem cell third fixing step in which the fixation to the bottom surface 27 of the flat culture vessel 35 (third culture vessel) is confirmed.

In the stem cell third culture step and the stem cell sixth observation step, the culture solution 23 injected into the culture vessel 35 is discharged (discarded) from the culture vessel 35, and a new culture solution 23 is injected (accommodated) into the culture vessel 35. Is done. The culture vessel 35 is left to stand statically (without being moved) for 36 to 48 hours at a temperature substantially the same as the body temperature (about 36 to 37 ° C.), and the second mesenchymal stem cell 30 is cultured in the culture vessel 35. (Stem cell third culture step). The total planar area of the second mesenchymal stem cells 30 fixed on the bottom surface 27 of the culture vessel 35 at intervals of about 1 to 2 hours in the standing time of 36 to 48 hours is observed with the electron microscope 13 with respect to the bottom surface area of the culture vessel 35. It is determined whether or not the total planar area of the stem cells 30 has reached the third target ratio with respect to the bottom area of the culture vessel 35. The third target ratio of the total planar area of the second mesenchymal stem cell 30 to the bottom surface area of the culture vessel 35 is 88 to 92% (88 to 92% confluent).

A person in charge, such as a doctor, nurse, or researcher, confirms that the second mesenchymal stem cell 30 has settled on the bottom surface 27 of the culture vessel 35, and then clicks the stem cell fifth observation end button displayed on the display 16. When the stem cell fifth observation end button is clicked, the computer 11 displays a process twelfth display screen (not shown) on the display 16. On the process twelfth display screen, number 1 and donor data are displayed, and a stem cell second collection end message, a stem cell fifth observation end message, a stem cell sixth observation button, and a culture product second extraction button are displayed. .

The person in charge clicks the sixth stem cell observation button on the process twelfth display screen, removes the culture vessel 35 from the sample holder of the electron microscope 13, and causes the barcode reader 12 to read the QR code of the culture vessel 35. When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data displayed on the display 16 (donor data read into the memory) and the donor data indicated by the QR code read by the barcode reader. When the donor data match, the number 1 and the donor data are displayed on the display 16, and the stem cell second collection end message, the stem cell fifth observation end message, the stem cell sixth observation in progress message, the stem cell number 6 An observation end button and a culture product second extraction button are displayed on the display 16. If the donor data do not match, the computer 11 displays an error message and a culture stop message on the display 16.

The person in charge discharges (discards) the culture solution 23 injected into the culture vessel 35 in the fifth stem cell observation step, and injects (accommodates) the new culture solution 23 into the culture vessel 35. The new culture solution 23 is the same as that injected in the stem cell first observation means. The person in charge injects a new culture solution 23 into the culture vessel 35 and then installs (sets) the culture vessel 35 in the sample holder 36 of the electron microscope 13.

A spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the bottom 48 of the third flat culture vessel 35, and the bottom 48 of the culture vessel 35 is held up by the spacer 39. The culture vessel 35 is held in a state inclined at a predetermined angle such that the bottom portion 48 of the culture vessel 25 is on the top and the top portion 47 (injection port 49) of the culture vessel 25 is on the bottom. Further, a spacer 39 is interposed between the upper surface 37 of the sample holder 36 of the electron microscope 13 and the top 48 of the third flat culture vessel 35, and the top 47 of the culture vessel 35 is held in a state where it is lifted by the spacer 39. You may hold | maintain the culture container 35 in the state inclined to the predetermined angle so that the top part 47 of the container 35 may become the upper side and the bottom part 49 of the culture container 35 may become the lower side. The inclination angle α2 of the third flat culture vessel 35 with respect to the upper surface 37 of the sample holder 36 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

After confirming the establishment of the second mesenchymal stem cell 30, the culture product production method is configured to inject a new culture solution 23 into the culture vessel 35 while discharging the culture solution 23 in the culture vessel 35. The proliferation of the mesenchymal stem cell 30 can be surely promoted. In the culture product production method, the second mesenchymal stem cell 30 and the culture solution 23 are cultured in the culture vessel 35 by inclining the third flat culture vessel 35 at the inclination angle with respect to the upper surface 37 of the sample holder 36. The pressure is biased toward the top 47 (or the bottom 48) of the container 35, and the water pressure between the second mesenchymal stem cell 30 and the culture solution 23 increases on the top 47 (or the bottom 48) of the culture container 35. Thus, the second mesenchymal stem cells 30 concentrate on the bottom 48 side of the culture vessel 35, thereby increasing the activity of the second mesenchymal stem cells 30, and the second mesenchymal stem cell 30 is formed on the bottom surface 27 of the culture vessel 35. Can be propagated (differentiated) easily and rapidly.

The electron microscope 13 takes an enlarged image of the planar shape of the second mesenchymal stem cell 30 in the culture vessel 35 at intervals of about 1 to 2 hours, and takes an enlarged image of the taken planar shape of the stem cell 30 for about 1 to 2 hours. It transmits to the computer 11 at intervals. The computer 11 stores (stores) the planar enlarged image of the second mesenchymal stem cell 30 transmitted from the electron microscope 13 and the imaging time in the storage area in a state associated with the donor identifier (stem cell image sixth memory). Process). The computer 11 displays the enlarged image of the planar shape of the second mesenchymal stem cell 30 transmitted from the electron microscope 13 and the imaging time on the display 16.

The person in charge confirms (views) the enlarged image of the planar shape of the second mesenchymal stem cell 30 displayed on the display 16 at intervals of about 1 to 2 hours during 36 to 48 hours. The total planar area of the stem cells 30 reaches the third target ratio (82 to 92% confluent) with respect to the bottom area of the culture container 35 while observing the total planar area of the stem cells 30 settled on the culture container 35 with respect to the bottom area. It is determined whether or not (stem cell sixth observation step). The person in charge directly observes the total planar area of the second mesenchymal stem cell 30 with respect to the bottom surface area of the culture vessel 35 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 plane area of 30 has reached the third target ratio (88-92% confluent) with respect to the bottom area of the culture vessel 35.

As a result of the observation in the stem cell sixth observation step, the person in charge has the third target ratio (88 to 92% confluent) as the total planar area of the second mesenchymal stem cell 30 displayed on the display 16 with respect to the bottom area of the culture vessel 35. If it has not reached (supported in FIG. 13), the total planar area of the stem cell 30 with respect to the bottom area of the culture vessel 35 is continuously observed at intervals of about 1 to 2 hours. In addition, when the total plane area of the second mesenchymal stem cell 30 reaches the third target ratio with respect to the entire area of the enlarged image displayed on the display 16, the total plane with respect to the bottom area of the culture vessel 35 of the stem cell 30 It is assumed that the area has reached the third target ratio.

As a result of the observation in the stem cell sixth observation step, the second mesenchymal stem cell 30 grows on the bottom surface 27 (bottom wall inner surface) of the third flat culture vessel 35 (third culture vessel), and the stem cell 30 forms a colony, When the planar shape of the stem cell 30 is expanded, the total planar area of the stem cell 30 displayed on the display 16 with respect to the bottom surface area of the culture vessel 35 reaches the third target ratio (88 to 92% confluent) (see FIG. 14). ) In the culture vessel 35, a single type of second mesenchymal stem cell 30 is proliferating, and a culture product solution 31 containing a predetermined metabolite secreted from the active single type of stem cell 30 is generated. Yes.

When the total planar area of the second mesenchymal stem cell 30 exceeds 92% with respect to the bottom surface area of the third flat culture container 35 (third culture container) and the stem cell 30 proliferates, Although the activity is gradually lost, the stem cell 30 is extracted from the culture vessel 35 when the total planar area of the stem cell 30 grows to 88 to 92% with respect to the bottom surface area of the culture vessel 35, so that the activity of the stem cell 30 is retained. The stem cell 30 can be proliferated while maintaining the activity.

When the total planar area of the stem cells 30 with respect to the bottom surface area of the culture vessel 35 reaches the third target ratio, a culture product second extraction step for extracting the culture product 31 from the culture vessel 35 is performed. In the culture product solution second extraction step, the culture product solution 31 containing a predetermined metabolite secreted from the active second mesenchymal stem cell 30 is extracted from the culture vessel 35. The method for producing a culture product solution can accurately confirm the total planar area of the second mesenchymal stem cell 30 relative to the bottom area of the culture vessel 35 by observing the total planar area at intervals of about 1 to 2 hours. It can be ascertained that the total planar area of the stem cells 30 has reached the third target ratio with respect to the bottom surface area of the culture vessel 35.

A person in charge such as a doctor, a nurse, or a researcher confirms that the total planar area of the second mesenchymal stem cell 30 with respect to the bottom area of the culture vessel 35 has reached the third target ratio, and then is displayed on the display 16. Click the button for ending stem cell sixth observation. When the stem cell sixth observation end button is clicked, the computer 11 displays a process thirteenth display screen (not shown) on the display 16. On the process 13th display screen, number 1 and donor data are displayed, and a stem cell second collection end message, a stem cell fifth observation end message, a stem cell sixth observation end message, and a culture product second extraction button are displayed. The

The person in charge aspirates the culture product solution 31 changed from the culture solution 23 injected into the culture vessel 35 in the stem cell sixth observation step from the culture vessel 35 using a syringe or pipette (culture product solution second extraction step). Then, the culture product solution 31 is injected (stored) in the storage container 32. The culture product solution 31 injected into the storage container 32 contains a predetermined metabolite secreted from a specific type of single-type mesenchymal stem cells (single-type stem cells) having an activity from which unnecessary mesenchymal stem cells have been removed. It is the culture product solution 31 containing.

The person in charge injects the culture product solution 31 into the storage container 32, and then attaches the first code sheet 17 on which the QR code is printed to the outer peripheral surface of the storage container 32. The person in charge clicks the culture solution second extraction end button on the thirteenth display screen displayed on the display 16, causes the barcode reader 12 to read the QR code of the storage container 32, and then injects the culture product 31. The stored storage container 32 is stored in the refrigerator 34. The culture product solution 31 is stored at about 3 to 4 ° C. in the refrigerator 34.

When the QR code is transmitted from the barcode reader 12 to the computer 11, the computer 11 displays the donor data (donor data read into the memory) displayed on the display 16 and the donor indicated by the QR code read by the barcode reader 12. When the data are compared and the donor data match, the number 1 and the donor data are displayed on the display 16, and the culture product extraction end message and the culture product preparation end button are displayed on the display 16. The person in charge clicks the culture production solution production end button. When the culture product production end button is clicked, the system 10 stops in the computer 11.

When the culture production solution production end button is clicked, a stem cell collection step of collecting the stem cells 30 from the culture vessel 25 is performed. In the stem cell collection step, the second mesenchymal stem cells 30 that have proliferated (differentiated) in the culture vessel 35 are collected from the culture vessel 35. The person in charge cleans the inside of the culture container 35 with PBS, injects a trypsin solution into the culture container 35, and aspirates the second mesenchymal stem cells 30 floating on the water surface of the trypsin solution using a syringe or pipette. The stem cell 30 is injected (accommodated) into the storage container 33 from a syringe or pipette.

In the culture product production method, the culture product solution 31 is extracted from the second culture vessel 25, the second stem cells 30 are collected from the second culture vessel 25, the stem cells 30 are cultured together with the culture solution 23, and the stem cells 30 are extracted. 2. Fix to the bottom surface 27 of the third culture container 35 having a larger capacity than the culture container 25, inject a new culture solution 23 into the culture container 35, and culture the stem cells 30, while the total planar area of the stem cells 30 is the culture container. When the third target ratio is reached with respect to the bottom surface area of 35, the culture product solution 31 containing a predetermined metabolite secreted from a single type of second stem cell 30 having an activity proliferated in the course of culturing the stem cell 30 is cultured. Since it is extracted from the container 35, a variety of stem cells are not cultured in the culture process of the stem cell 30, and a variety of metabolites secreted from the variety of stem cells are included. And producing a culture product solution 31 that contains only a predetermined metabolite secreted from a single type of active second stem cell 30 and exhibits a specific effect on a predetermined disease or a predetermined beauty. The culture product solution 31 that can be effectively used for the treatment of a predetermined disease can be manufactured, and the culture product solution 31 that can be used effectively for a predetermined beauty can be manufactured. .

DESCRIPTION OF SYMBOLS 10 Culture production | generation production system 11 Computer 12 Bar code reader 13 Electron microscope 14 Keyboard 15 Mouse 16 Display 17 1st code paper 18 Raw material bone marrow 19 Glass test tube 20 Test tube stand 21 Constant temperature bath 22 Middle layer bone marrow 23 Culture solution 24 First culture vessel (first flat culture vessel)
25 Second culture vessel (second flat culture vessel)
26 First mesenchymal stem cells (first stem cells)
27 Bottom surface 28 Centrifuge 29 Glass test tube 30 Second mesenchymal stem cell (second stem cell)
31 Culture Product 32 Storage Container 33 Storage Container 34 Refrigerator 35 Third Culture Container (Third Flat Culture Container)
36 Sample holder 37 Upper surface 38 Bottom 39 Spacer 40 Top 41 Inlet 42 Lid 43 Top 44 Bottom 45 Inlet 46 Lid 47 Top 48 Bottom 49 Inlet 50 Lid

Claims

In a culture product production method for producing a culture product using stem cells made from bone marrow fluid collected from a donor,
The culture product production method includes a bone marrow fluid separation step in which bone marrow fluid collected from the donor is separated into layers, and an intermediate layer bone marrow fluid located in an intermediate layer of the bone marrow fluid separated into layers by the bone marrow fluid separation step A bone marrow fluid extraction step for extracting the bone marrow fluid, and the intermediate layer bone marrow fluid extracted by the bone marrow fluid extraction step and a predetermined culture solution are injected into a first culture vessel having a predetermined volume and a bottom area, and the first culture A first stem cell fixing step in which the container is allowed to stand statically for a predetermined time to fix the first stem cells contained in the intermediate layer bone marrow fluid to the bottom surface of the first culture vessel, and the first stem cell by the first stem cell fixing step. Is fixed to the bottom surface of the first culture vessel, and a new culture solution is poured into the first culture vessel while discharging the culture solution in the first culture vessel, and the first culture vessel is allowed to stand for a predetermined time. Leave the first culture A first stem cell culturing step of culturing the first stem cells until the total planar area of the first stem cells with respect to the bottom surface area of the vessel reaches a first target ratio, and a culturing process of the first stem cells in the first stem cell culturing step When the total planar area of the first stem cells reaches the first target ratio, the stem cell first collection step for collecting the first stem cells from the first culture vessel, and the stem cell first collection step, Stem cell separation step for centrifuging first stem cells in layers, stem cell second collection step for collecting second stem cells located in the lowest layer of the first stem cells separated in layers by the stem cell separation step, and the stem cells The second stem cell collected in the second collection step and a predetermined culture solution are poured into a second culture vessel having a bottom having a larger volume and a larger bottom area than the first culture vessel. The second stem cell is allowed to stand statically for a predetermined time to establish the second stem cell on the bottom surface of the second culture vessel, and the second stem cell is fixed by the stem cell second colonization step. After fixing on the bottom surface of the second culture vessel, a new culture solution is poured into the second culture vessel while discharging the culture solution in the second culture vessel, and the second culture vessel is statically fixed for a predetermined time. In the stem cell second culturing step of culturing the second stem cell until the total planar area of the second stem cell with respect to the bottom surface area of the second culture container reaches a second target ratio; In the culturing process, when the total planar area of the second stem cells reaches the second target ratio, the culture product solution containing a predetermined metabolite secreted from the single stem cell that has proliferated is added to the second stem cell. Culture production extracted from culture vessels It has a liquid 1st extraction process, The culture production liquid manufacturing method characterized by the above-mentioned.
In the stem cell first fixing step and the stem cell first culturing step, the first culture vessel is statically left for a predetermined time in a state inclined at a predetermined angle, and the stem cell second fixing step and the stem cell second culturing step. The method for producing a culture product solution according to claim 1, wherein the second culture vessel is statically left for a predetermined time in a state where the second culture container is inclined at a predetermined angle.
The culture product production method extracts the culture product solution from the second culture vessel in the culture product solution first extraction step, and then collects the second stem cell from the second culture vessel. And the second stem cells collected by the second harvesting step of the stem cells in a third culture container having a bottom surface with a predetermined volume and a predetermined area and a larger capacity and a larger bottom area than the second culture container. A third stem cell fixing step, in which the third culture vessel is statically left for a predetermined time and the second stem cells are fixed on the bottom surface of the third culture vessel, and the third stem cell fixing step. After fixing the second stem cells on the bottom surface of the third culture vessel, a new culture solution is poured into the third culture vessel while discharging the culture solution in the third culture vessel, and the third culture is performed. Keep the container for a specified time In the stem cell third culture step of culturing the second stem cells until the total planar area of the second stem cells with respect to the bottom surface area of the third culture container reaches a third target ratio, In the culturing process, when the total planar area of the second stem cells reaches the third target ratio, the culture product solution containing a predetermined metabolite secreted from the single stem cell that has proliferated is added to the third culture. The culture product solution production method according to claim 1, further comprising a culture product solution second extraction step of extracting from the culture vessel.
The method for producing a culture product solution according to claim 3, wherein, in the stem cell third fixing step and the stem cell third culture step, the third culture vessel is statically left for a predetermined time in a state where the third culture vessel is inclined at a predetermined angle.
The bone marrow fluid separation step collects 2 to 3 cc of bone marrow fluid from the donor, injects the 2 to 3 cc of bone marrow fluid into a separation container extending in the vertical direction, and the separation container is predetermined at a temperature substantially equal to the body temperature. The bone marrow fluid is left standing statically for time and separated into layers in the vertical direction in the separation container, and the bone marrow fluid extraction step is located in an intermediate layer of the bone marrow fluid separated in layers in the separation container. The method for producing a culture product solution according to any one of claims 1 to 4, wherein the intermediate layer bone marrow fluid is extracted.
The capacity of the first culture vessel is about 20-30 cc, the initial planar shape of the first stem cells is substantially circular, and the planar shape after deformation of the first stem cells is the center of the substantially circular shape as the nucleus. The first stem cell has a flat shape in which the first stem cell is irregularly extended in one direction. In the first stem cell fixing step, the first culture vessel is left statically at a temperature substantially the same as the body temperature for 12 to 24 hours, The deformation of the first stem cells in the first culture vessel from the initial planar shape is observed at intervals of about 1 to 2 hours during 12 to 24 hours, and the first stem cells are deformed from the initial planar shape to the irregular flat shape. The method for producing a culture product solution according to any one of claims 1 to 5, wherein the first stem cells are determined to have settled on the bottom surface of the first culture container when deformed to.
In the first stem cell collection step, the first culture vessel is statically left at a temperature substantially the same as the body temperature for 36 to 48 hours, while the first culture container is spaced at about 1 to 2 hours between the 36 and 48 hours. The method for producing a culture product solution according to any one of claims 1 to 6, wherein the total planar area of the first stem cells fixed on the bottom surface of the culture vessel with respect to the bottom surface area of the first culture vessel is observed.
The culture product production method according to claim 7, wherein the first target ratio of the total planar area of the first stem cells to the bottom area of the first culture vessel is 70 to 80%.
In the stem cell separation step, the first stem cells are injected into a separation container, the separation container is placed in a centrifuge to centrifuge the first stem cells, and the stem cell first collection step is performed in the separation container. The method for producing a culture product solution according to any one of claims 1 to 8, wherein the second stem cells located in the lowermost layer of the first stem cells centrifuged in a layered manner are collected.
The volume of the second culture vessel is about 40-60 cc, the volume of the third culture vessel is about 70-80 cc, the initial planar shape of the second stem cell is substantially circular, The planar shape after deformation of the stem cell is a flat shape in which the second stem cell extends indefinitely in one direction with the substantially circular shape as a nucleus. In the stem cell second fixing step and the stem cell third fixing step, The second and third culture vessels are statically left at the same temperature as the body temperature for 36 to 48 hours, while the second and third culture vessels are spaced at about 1 to 2 hours between the 36 and 48 hours. When the deformation of the second stem cell in the container from the initial planar shape is observed and the second stem cell is deformed from the initial planar shape to the irregular flat shape, the second stem cell is converted into the second and third culture containers. Claim to have settled on the bottom of 3 through claim 9 culture product solution producing method according to any one.
In the stem cell second collection step and the stem cell third collection step, the second culture vessel and the third culture vessel are statically left at the same temperature as the body temperature for 36 to 48 hours, while the 36 to 48 hours. The total planar area of the second stem cells fixed on the bottom surfaces of the second and third culture vessels at a time interval of about 1 to 2 hours is observed with respect to the bottom area of the second and third culture vessels. The method for producing a culture product solution according to claim 10.
The second target ratio of the total planar area of the second stem cells to the bottom area of the second culture container is 88 to 92%, and the second target ratio of the total planar area of the second stem cells to the bottom area of the third culture container is The method for producing a culture product solution according to any one of claims 3 to 11, wherein the 3 target ratio is 88 to 92%.
The method for producing a culture product solution according to any one of claims 1 to 12, wherein the first and second stem cells are mesenchymal stem cells.