JP2017104106A - Single species stem cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide single species stem cells without containing miscellaneous stem cells which have a remarkable effect in the treatment of a wide variety of diseases and on regeneration in regenerative medicine.SOLUTION: A single species stem cell is obtained by the following steps: extracting a middle layer bone marrow fluid 23 of a middle layer from a raw material bone marrow fluid 19 which is separated in layers; culturing the middle layer bone marrow fluid 23 with a culture medium to fix first stem cells to the bottom surface of a first culture vessel; extracting the first stem cells from the fist culture vessel when the total flat surface area of the first stem cells reaches a first target ratio to the bottom surface area of the first culture vessel; extracting the second stem cells of an uppermost layer from the first stem cells separated in layers and culturing the second stem cells with a culture medium to fix the second stem cells to the bottom surface of the second culture vessel; and extracting the second stem cells from the second culture vessel when the total flat surface area of the second stem cells reaches a second target ratio to the bottom surface area of the second culture vessel.SELECTED DRAWING: Figure 4

Description

  The present invention relates to single-type stem cells made from raw bone marrow fluid collected from a donor.

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

JP-A-2015-186465

  Since various stem cells are present in the tissue or cells collected from the donor, even if stem cells existing in the tissue or cells are cultured, the various stem cells will proliferate and only a single type of stem cell of a specific type will be cultured I can't. Stem cells are used for the treatment of various diseases (cardiovascular diseases, central nervous system diseases, etc.), regenerative medicine, and non-therapeutic applications, but the variegated stem cells cultured from only a single type of stem cell Compared to the case, the effect of treatment for various diseases and the effect of regeneration in regenerative medicine are small, the probability of completely curing various diseases is low, and the probability of regenerating various tissues and various organs is low. In addition, in the stem cell culture method disclosed in Patent Document 1, various stem cells are cultured, and it is not possible to culture only a specific type of single-type stem cells. Stem cells produced by the stem cell culturing method disclosed in Patent Document 1 vary in treatment effect and regeneration effect in regenerative medicine, and are established to completely cure various diseases and regenerate various tissues and organs. Is low.

  The object of the present invention is that it does not contain various stem cells, has a large therapeutic effect on various diseases and a regenerative effect in regenerative medicine, is highly established to completely cure various diseases, and various tissues and various organs. It is to provide a single-type stem cell having a high probability of regenerating.

  The premise of the present invention to solve the above problems is a single-type stem cell made from a raw bone marrow fluid collected from a donor.

  The single-type stem cell of the present invention in the above premise is obtained by separating a raw material bone marrow fluid collected from a donor into a layered form, extracting an intermediate layer bone marrow fluid located in an intermediate layer of the separated raw material bone marrow fluid, and having a predetermined volume In addition, the intermediate layer bone marrow fluid and the culture solution are injected into a first culture vessel having a bottom surface of a predetermined area 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 cells are After fixing on the bottom surface of one culture vessel, a new culture solution is poured into the first culture vessel while discharging the culture solution in the first culture vessel to proliferate the first stem cells. After the total planar area of one stem cell reaches the first target ratio with respect to the bottom area of the first culture container, the first culture means for extracting the first stem cells from the first culture container and the first culture means are used for extraction. First stem cells are centrifuged in layers and separated into layers Extracting the second stem cell located in the lowest layer of the cells, and having the second stem cell in a second culture container having a predetermined volume and a predetermined area of a bottom surface and a larger capacity and a larger bottom area than the first culture container A new culture solution is injected to fix the second stem cells on the bottom surface of the second culture vessel. After the second stem cells are fixed to the bottom surface of the second culture vessel, the culture solution in the second culture vessel is discharged. A new culture solution is injected into the second culture vessel to proliferate the second stem cells, the second stem cells expand, and the total planar area of the second stem cells becomes the second target ratio with respect to the bottom area of the second culture vessel. And a second culturing means for extracting the second stem cells from the second culture container.

  As an example of the present invention, single-type stem cells are produced by performing the first culturing means once and performing the second culturing means at least twice. In the second culturing means from the second time, a predetermined volume and A third culture container having a bottom surface with a predetermined area and a larger capacity and a larger bottom surface area than the second culture container is used, and the second stem cells extracted from the second culture container by the first second culture means are used as the first culture cells. The second stem cells are allowed to grow in the third culture container while being fixed in the three culture containers.

  As another example of the present invention, in the first culture means, 2 to 3 cc of raw bone marrow fluid is collected from a donor, and 2 to 3 cc of raw bone marrow fluid is injected into a separation container extending in the vertical direction. The intermediate layer located in the intermediate layer of the raw bone marrow fluid separated into layers in the separation container by allowing the raw material bone marrow fluid to stand statically for a predetermined time at substantially the same temperature to separate the raw material bone marrow fluid in layers in the vertical direction. Bone marrow fluid is extracted by syringe or pipette.

  As another example of the present invention, the capacity of the first culture vessel is about 20 to 30 cc. In the first culture means, the intermediate layer bone marrow fluid and the culture solution are injected into the first culture vessel, and then the first culture is performed. While the container is inclined at a predetermined angle, the first culture container is statically left at a temperature substantially the same as the body temperature for 12 to 24 hours, and the first culture is performed at intervals of about 1 to 2 hours between 12 and 24 hours. When the deformation of the first stem cell in the container from the initial planar shape is observed and the first stem cell is deformed from the initial planar shape to a predetermined planar shape, it is determined that the first stem cell has settled on the bottom surface of the first culture container. .

  As another example of the present invention, the initial planar shape of the first stem cell is a substantially circular shape, and the planar shape after the deformation of the first stem cell is a flat shape in which the first stem cell extends in one direction indefinitely with a substantially circular shape as a nucleus. The first culture means determines that the first stem cells have settled on the bottom surface of the first culture container when the first stem cells are deformed into an irregular flat shape.

  As another example of the present invention, in the first culture means, the first stem cells are fixed on the bottom surface of the first culture vessel, and the new culture solution is discharged to the first culture vessel while discharging the culture solution in the first culture vessel. After the injection, the first culture container is statically left at a temperature substantially the same as the body temperature for 36 to 48 hours with the first culture container tilted at a predetermined angle, and about 1 to about 36 to 48 hours. The total planar area with respect to the bottom area of the first culture vessel of the first stem cells fixed on the bottom surface of the first culture vessel is observed at intervals of 2 hours.

  As another example 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 present invention, in the second culture means, the first stem cells are injected into a separation container, the separation container is placed in a centrifuge, the first stem cells are centrifuged, and the layers are centrifuged in layers in the separation container. The 2nd stem cell located in the lowest layer among the 1st stem cells which were done is extracted with a syringe or a pipette.

  As another example 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 second culture means uses the second and third culture containers. After injecting the second stem cells and a new culture medium into the second and third culture vessels, the second and third culture vessels are inclined at a predetermined angle, and the second and third culture vessels are at a temperature substantially the same as the body temperature for 36 to 48 hours. While standing statically, 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 shape is changed to a predetermined planar shape, it is determined that the second stem cells have settled on the bottom surfaces of the second and third culture vessels.

  As another example of the present invention, the initial planar shape of the second stem cell is a substantially circular shape, and the planar shape after the deformation of the second stem cell is a flat shape in which the second stem cell extends in an indefinite shape in one direction with a substantially circular shape as a nucleus. The second culture means determines that the second stem cells have settled on the bottom surfaces of the second and third culture containers when the second stem cells are deformed into an irregular flat shape.

  As another example of the present invention, in the second culture means, the second stem cells are fixed on the bottom surfaces of the second and third culture vessels, and a new culture solution is discharged while discharging the culture solution in the second and third culture vessels. Is injected into the second and third culture vessels, and the second and third culture vessels are statically kept at the same temperature as the body temperature for 36 to 48 hours with the second and third culture vessels inclined at a predetermined angle. The total planar area of the second stem cells fixed to 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 present invention, the second target ratio of the total planar area of the second stem cells to the bottom area of the second and third culture vessels is 88 to 92%.

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

  According to the single-type stem cell according to the present invention, the intermediate layer bone marrow fluid located in the intermediate layer of the raw material 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 cell. 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, the first stem cells are extracted from the first culture container and layered. The second stem cells located in the lowermost layer of the first stem cells separated into the second stem cells are cultured, and the second stem cells are cultured together with the culture solution so that the second stem cells are fixed on the bottom surface of the second culture vessel. When the total plane area of the second culture container reaches the second target ratio with respect to the bottom surface area of the second culture container, it is made from extracting the second stem cells from the second culture container, so that various stem cells are included. Never contain miscellaneous stem cells Compared to cases, there is a high probability of complete cure of various diseases and a high probability of regeneration of various tissues and organs, which can be used favorably for the treatment of various diseases. It can be preferably used. Single-species stem cells are extracted by extracting specific intermediate layer bone marrow fluid from the raw material bone marrow fluid separated in layers, and by extracting specific second stem cells from the first stem cells separated in layers, unnecessary stem cells are removed. Because pure stem cells do not contain a variety of stem cells in a single type of stem cell, the effect of treating various diseases and the effect of regeneration in regenerative medicine are stable, and various diseases can be treated early and reliably. It can be completely cured, and various tissues and various organs can be regenerated early and reliably.

  A single-type stem cell is produced by performing the first culturing means once and the second culturing means at least twice, and the second culturing means from the second time has a bottom surface with a predetermined volume and a predetermined area. A third culture vessel having a larger capacity and a larger bottom area than the second culture vessel is used, and the second stem cells extracted from the second culture vessel by the first second culture means are fixed in the third culture vessel. The single-type stem cells for proliferating the second stem cells in the third culture vessel are subjected to the second culture means at least twice, and a large amount of single-type stem cells are used in the second culture means from the second time using the third culture vessel. It can be used for the complete cure of diseases of many patients and can be used for regeneration of tissues and organs of many patients.

  In the first culturing means, 2 to 3 cc of raw bone marrow fluid is collected from a donor, and 2 to 3 cc of raw bone marrow fluid is injected into a separation container extending in the vertical direction, and the separation container is kept at a temperature substantially the same as the body temperature for a predetermined time. The bone marrow fluid is separated into layers in the vertical direction in the separation container, and the middle layer bone marrow fluid located in the middle layer of the raw material bone marrow fluid separated into layers in the separation container is extracted with a syringe or pipette Single-species stem cells consist of a specific intermediate layer bone marrow from the source bone marrow fluid by separating the raw bone marrow fluid containing various stem cells statically at a temperature substantially the same as the body temperature for a predetermined time and separating it in layers in the vertical direction. Since the fluid is reliably extracted and unnecessary stem cells are removed, the effect of treating various diseases and the effect of regeneration in regenerative medicine are stabilized, and various diseases can be cured quickly and reliably. It can be regenerated seed organs early and reliably. Since single-type stem cells are made from a small amount of raw bone marrow fluid of 2-3 cc collected from the donor, the burden on the donor can be minimized when collecting the raw bone marrow fluid.

  The first culture vessel has a capacity of about 20 to 30 cc, and in the first culture means, after the intermediate layer bone marrow fluid and the culture solution are injected into the first culture vessel, the first culture vessel is inclined at a predetermined angle. The initial culture plane of the first stem cells in the first culture container at intervals of about 1 to 2 hours between 12 and 24 hours while the first culture container is statically left at a temperature substantially the same as the body temperature for 12 to 24 hours. When the first stem cell is observed to be deformed from the shape and the first stem cell is deformed from the initial planar shape to the predetermined planar shape, it is determined that the first stem cell is fixed on the bottom surface of the first culture vessel. When the capacity of the first stem cell exceeds 30 cc, the first stem cell becomes difficult to settle on the bottom surface of the first culture container and the growth of the first stem cell is slowed down. By using the first culture container of the capacity, the first stem cell Quickly and easily settles on the bottom of the first culture vessel By first stem cells in the first culture vessel to quickly grow, because a single type stem cells are made in a short period, it can be utilized in a timely manner for the treatment of various diseases. The single-type stem cells are the first stem cells in the first culture container at intervals of about 1 to 2 hours between 12 and 24 hours while the first culture container is statically left at a temperature substantially the same as the body temperature for 12 to 24 hours. After the deformation from the initial planar shape of the first stem cells was observed, the first stem cells were properly fixed on the bottom surface of the first culture container, and after confirming the first stem cells were fixed, the culture medium in the first culture container was discharged. By injecting a new culture solution into the first culture vessel, the growth of the first stem cells is surely promoted, and single-type stem cells are produced in a short period of time, so that they can be used in a timely manner for the treatment of various diseases. be able to. Single-type stem cells can be firmly fixed on the bottom surface of the first culture container by statically leaving the first culture container in a state where the first culture container is inclined at a predetermined angle for a predetermined time.

  The initial planar shape of the first stem cell is a substantially circular shape, and the planar shape after the deformation of the first stem cell is a flat shape in which the first stem cell extends in an indefinite shape in one direction with the substantially circular shape as a nucleus. The single-type stem cell that determines that the first stem cell has settled on the bottom surface of the first culture container when the first stem cell is deformed into an irregular flat shape is accurately fixed to the bottom surface of the first culture container. After confirming the establishment of the first stem cells, the growth of the first stem cells is surely promoted by injecting a new culture solution into the first culture vessel while discharging the culture solution in the first culture vessel. Since single-type stem cells are produced in a short period of time, it can be used in a timely manner for the treatment of various diseases.

  In the first culture means, the first stem cells are fixed on the bottom surface of the first culture container, and after the culture medium in the first culture container is discharged, a new culture liquid is injected into the first culture container, and then the first culture container The first culture vessel is allowed to stand at a temperature substantially the same as the body temperature for 36 to 48 hours while being inclined at a predetermined angle, while the first culture vessel is about 1 to 2 hours apart during 36 to 48 hours. The single-type stem cell that observes the total planar area of the first stem cells that have settled on the bottom surface of the first culture cell with respect to the bottom area of the first culture vessel observes the total planar area at intervals of about 1 to 2 hours. Since the total planar area with respect to the bottom surface area of the culture container is accurately confirmed, the second stem cell from which unnecessary stem cells are removed is extracted by centrifuging the first stem cells extracted from the first culture container in a single layer. Stem cells may contain a variety of stem cells Ku, the effect of regeneration in effect and regenerative medicine treatment of various diseases is stable, the various diseases early and reliably can be cured, it is possible to reproduce the various tissues and various organs early and reliably. After the first stem cells have settled on the bottom surface of the first culture vessel, the single-type stem cells are poured into the first culture vessel while discharging the culture solution in the first culture vessel, and the first culture vessel is The static growth of the first stem cell can be surely promoted by statically leaving it for a predetermined time in a state inclined at a predetermined angle.

  Single-type stem cells 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% has a total planar area of the first stem cells of 80% to the bottom area of the first culture container. When the first stem cells proliferate beyond this, 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, By extracting the first stem cell from the first culture vessel, the activity of the first stem cell is retained, so that the single seed stem cell does not lose its activity, and by using the active single seed stem cell, various diseases Can be cured completely, and various tissues and organs can be reliably regenerated.

  In the second culturing means, the first stem cells are injected into a separation container, the separation container is placed in a centrifuge, the first stem cells are centrifuged, and the first stem cells are centrifuged in layers in the separation container. The single stem cell from which the second stem cell located in the lowest layer among the stem cells is extracted by a syringe or pipette is separated into layers by centrifugation of the first stem cells containing unnecessary stem cells, and the first stem cells centrifuged in layers Since the second stem cell located in the lowermost layer is extracted, a single kind of stem cell does not contain a variety of stem cells, the effect of treating various diseases and the effect of regeneration in regenerative medicine are stable, and various diseases Can be cured quickly and reliably, and various tissues and organs can be regenerated early and reliably.

  The volume of the second culture vessel is about 40 to 60 cc, the volume of the third culture vessel is about 70 to 80 cc, and in the second culturing means, the second stem cell and a new culture solution are added to the second and third culture vessels. And injecting the second and third culture vessels statically at a temperature substantially the same as the body temperature for 36 to 48 hours with the second and third culture vessels inclined at a predetermined angle, During the 48 hours, the deformation of the second stem cells in the second and third culture vessels from the initial planar shape was observed at intervals of about 1 to 2 hours, and the second stem cells were deformed from the initial planar shape to a predetermined planar shape. In the case, the single-type stem cell that determines that the second stem cell has settled on the bottom surface of the second and third culture containers, when the capacity of the second culture container exceeds 60 cc and the capacity of the third culture container exceeds 80 cc, The second stem cell of the second and third culture vessel It becomes difficult to settle on the surface and the growth of the second stem cell is slow, but by using the second and third culture vessels having the above-mentioned capacity, the second stem cell can be quickly and easily attached to the bottom surfaces of the second and third culture vessels. Since the second stem cells proliferate quickly in the second and third culture vessels and single seed stem cells are produced in a short period of time, they can be used in a timely manner for the treatment of various diseases. Single-type stem cells are obtained by second and third cultures at intervals of about 1 to 2 hours between 36 and 48 hours, while the second and third culture vessels are statically left for 36 to 48 hours at approximately the same temperature as the body temperature. After observing the deformation of the second stem cells in the container from the initial planar shape, the second stem cells are appropriately fixed on the bottom surfaces of the second and third culture containers, and the second stem cells are confirmed to be fixed. Injecting a new culture solution into the second and third culture vessels while draining the culture solution in the third culture vessel surely promotes the growth of the second stem cells, so that single-type stem cells can be produced in a short period of time. Therefore, it can be used in a timely manner for the treatment of various diseases. The single-type stem cells can be firmly fixed on the bottom surface of the second culture container by leaving the second culture container statically for a predetermined time with the second culture container inclined at a predetermined angle.

  The initial planar shape of the second stem cell is a substantially circular shape, and the deformed planar shape of the second stem cell is a flat shape in which the second stem cell extends in an indefinite shape in one direction with the substantially circular shape as a nucleus. The single-type stem cell that determines that the second stem cell has settled on the bottom surfaces of the second and third culture vessels when the second stem cell is deformed into an irregular flat shape is the second and third culture vessels of the second stem cell. After the establishment of the second stem cells has been confirmed, the new culture solution is injected into the second and third culture vessels while discharging the culture solution in the second and third culture vessels. As a result, the proliferation of the second stem cells is surely promoted, and single-type stem cells are produced in a short period of time, so that it can be used in a timely manner for the treatment of various diseases.

  In the second culture means, the second stem cells are fixed on the bottom surfaces of the second and third culture vessels, and the new culture solution is discharged while the second and third culture vessels are discharged. 36-48, while the second and third culture vessels are tilted at a predetermined angle, the second and third culture vessels are statically left at the same temperature as the body temperature for 36-48 hours. A single-type stem cell that observes 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 over time relative to the bottom area of the second and third culture vessels is about By observing the total planar area at intervals of 1 to 2 hours, the total planar area with respect to the bottom area of the second and third culture vessels of the second stem cells can be accurately confirmed, and unnecessary stem cells from the second and third culture vessels Because the second stem cell from which A variety of stem cells are not included in one type of stem cell, the effect of treatment of various diseases and the effect of regeneration in regenerative medicine are stable, and various diseases and organs can be cured quickly and reliably. It can be regenerated early and reliably. After the second stem cells have settled on the bottom surface of the second culture vessel, the single-type stem cells are poured into the second culture vessel while discharging the culture solution in the second culture vessel, and the second culture vessel is The static growth of the second stem cell can be surely promoted by statically leaving it for a predetermined time in a state inclined at a predetermined angle.

  A single-type stem cell in which the second target ratio of the total planar area of the second stem cells to the bottom area of the second and third culture vessels is 88 to 92% is the second stem cell relative to the bottom area of the second and third culture vessels. When the total planar area exceeds 92% and the second stem cells proliferate, the activity of the second stem cells is gradually lost, but the total planar area of the second stem cells is 88 with respect to the bottom area of the second and third culture vessels. Since the activity of the second stem cell is retained by extracting the second stem cell from the second and third culture vessels when proliferating to ˜92%, the single-type stem cell does not lose its activity, and the activity is maintained. By utilizing the single-type stem cells, various diseases can be completely cured, and various tissues and organs can be reliably regenerated.

  Single-species stem cells in which the first and second stem cells are mesenchymal stem cells are identified from the first mesenchymal stem cells that have been extracted from the intermediate bone marrow fluid that has been separated into layers and then separated into layers. By extracting the second mesenchymal stem cells, unnecessary mesenchymal stem cells are removed, and a single kind of stem cell does not contain a variety of mesenchymal stem cells. The effect of regeneration in medicine is stable, various diseases can be cured quickly and reliably, and various tissues and organs can be regenerated early and reliably.

The schematic block diagram of the stem cell culture system shown as an example. Explanatory drawing which shows an example of a bone marrow fluid separation means. Explanatory drawing of the bone marrow fluid separation means which continues from FIG. Explanatory drawing of the bone marrow fluid separation means which continues from FIG. The perspective view of a 1st flat culture container (1st culture container). The side view of a 1st flat culture container (1st 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 means. The perspective view of a glass test tube. The perspective view of a 2nd flat culture container (2nd culture container). The side view of a 2nd flat culture container (2nd 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 the preservation | save of a 2nd mesenchymal stem cell. The side view of a 3rd flat culture container (3rd culture container).

  The details of the single-type stem cell 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 stem cell culture system 10 shown as an example. 2 is an explanatory view showing an example of the bone marrow fluid separating means forming the first culture means, and FIG. 3 is an explanatory view of the bone marrow fluid separating means continuing from FIG. FIG. 4 is an explanatory view of the bone marrow fluid separating means continuing from FIG.

  The single-type stem cells are produced by performing the first culture means and the second culture means by the stem cell culture system 10 using raw bone marrow fluid collected from a plurality of donors (people). The first culture means is formed from bone marrow fluid separation means, bone marrow fluid extraction means, stem cell first observation means, stem cell second observation means, and stem cell first extraction means. The second culturing means is formed from stem cell separating means, stem cell second extracting means, stem cell third observing means, stem cell fourth observing means, and stem cell third extracting means. The system 10 cultures (manufactures) a specific type of single-type mesenchymal stem cells (single-type stem cells) from among a plurality of types of mesenchymal stem cells contained in the raw bone marrow fluid.

  The stem cell culture 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 bone marrow culture 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 means forming the first culture means, the raw material bone marrow fluid 19 collected from the donor is separated into layers. In bone marrow fluid collection, 2 to 3 cc (2 to 3 ml) of raw bone marrow fluid 19 is collected from the sternum or iliac bone (pelvis) of these donors. The raw bone marrow fluid 19 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 the system 10 in the computer 11 simultaneously with the collection of the raw bone marrow fluid 19, and uses the input device such as the keyboard 14 and the mouse 15 to transfer the donor data to the computer 11. input. Also, donor data (donor identification information) can be managed by an IC tag (IC chip).

  The computer 11 generates a unique donor identifier that identifies each donor each time donor data is input (every time the raw bone marrow fluid 19 is collected from the donor), and stores the donor data in association with the donor identifier. Store (store) in the area (donor data storage means). The computer 11 converts the input 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 and second codes on which the QR code is printed The sheets 18a and 18b are output (two-dimensional code (QR code) output means), and 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 means).

  The QR code printed on the first code sheet 18a printed with NO1 includes the first culture means (bone marrow fluid separating means, bone marrow fluid extracting means, bone marrow fluid separating means, bone marrow fluid extracting means, stem cell first observation). No. 1 of the means, stem cell second observation means, stem cell first extraction means) is stored, and the second culture means (stem cell separation means, stem cells) is included in the QR code printed on the second code sheet 18b on which NO2 is printed. No. 2 of second extraction means, stem cell third observation means, stem cell fourth observation means, stem cell third extraction means) is stored. The computer 11 compares the donor data stored in the storage area with the donor data indicated by the QR code read by the barcode reader 12 every time each means from the bone marrow fluid separating means to the third stem cell extracting means is executed. .

As shown in FIG. 2, 2 to 3 cc of raw bone marrow fluid 19 collected from a donor is injected (accommodated) into a glass test tube 20 (separation container) extending in the vertical direction. The 2 to 3 cc of raw bone marrow fluid 19 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 18a is attached to the outer peripheral surface of the glass test tube 20 into which the raw bone marrow fluid 19 is injected. As shown in FIG. 3, the glass test tube 20 into which the raw bone marrow fluid 19 has been injected is set in a test tube stand 21 and accommodated in a thermostatic chamber 22 together with the test tube stand 21.

  A person in charge such as a doctor, a nurse, or a researcher attaches the first code sheet 18 a on which the QR code is printed on the outer peripheral surface of the glass test tube 20, and then attaches the QR code of the glass test tube 20 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 the first culture first display screen (not shown) on the display 16. indicate. On the first culture 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 extraction button, and logout A button is displayed. When not performing the first culture, click the logout button. 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, then injects the raw bone marrow fluid 19 from the syringe into the glass test tube 20, and sets the glass test tube 20 into which the raw material bone marrow fluid 19 is injected into the test tube holder 21. Insert (set) into. As shown in FIG. 3, the person in charge accommodates the test tube stand 21 in a thermostat 22 and statically holds the glass test tube 20 into which the raw bone marrow fluid 19 has been injected in the thermostat 22 for a predetermined time (about 2 hours). Leave it alone (don't move it quietly). The temperature in the thermostat 22 is maintained at approximately 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 18 a on the display 16, and displays a bone marrow separation in progress message and a bone marrow separation end button on the display 16.

  By leaving the glass test tube 20 statically in the thermostat 22 for a predetermined time (about 2 hours), the raw bone marrow fluid 19 injected into the test tube 20 is vertically moved in the test tube 20 as shown in FIG. Separate into several layers (three layers). Stem cell culture usually requires 150 to 200 cc (150 to 200 ml) of bone marrow fluid, but the stem cell culture system 10 is a single-species stem cell from a small amount of 2-3 cc of raw bone marrow fluid 19 collected from a donor. Therefore, it is only necessary to collect a small amount of 2-3 cc of the raw bone marrow fluid 19 from the donor, and the burden on the donor when the raw material bone marrow fluid 19 is collected can be minimized.

  Bone marrow fluid extraction means is performed after the bone marrow fluid separation means in which the raw bone marrow fluid 19 is separated into layers. In the bone marrow fluid extraction means, the intermediate layer bone marrow fluid 23 is extracted from the raw material bone marrow fluid 19 separated into layers. The person in charge confirms that the raw material bone marrow fluid 19 has been separated into layers, and then clicks a 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 first culture second display screen (not shown) on the display 16. The first culture second display screen displays number 1 and donor data, and includes 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 extraction button. Is displayed.

  The person in charge clicks on the bone marrow extract button on the first culture second display screen to cause the barcode reader 12 to read the QR code of the glass test tube 20. 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 holder 21 from the thermostatic chamber 22, pulls out the glass test tube 20 from the test tube holder 21, and extracts the intermediate layer bone marrow fluid 23 existing in a specific layer of the raw material bone marrow fluid 19 separated into layers. To do. The person in charge extracts (suctions) the intermediate layer bone marrow fluid 23 having a layer thickness of 3 to 4 mm located in the intermediate layer of the raw material bone marrow fluid 19 separated into layers using a syringe (not shown). Alternatively, an intermediate layer bone marrow fluid 23 having a layer thickness of 3 to 4 mm located in the intermediate layer of the raw material bone marrow fluid 19 separated into layers using a pipette (not shown) is extracted (sucked). In the stem cell culture system 10, the raw bone marrow fluid 19 containing various mesenchymal stem cells is separated into layers in the vertical direction, and then a specific intermediate layer bone marrow fluid is separated from the raw bone marrow fluid 19 by using a syringe or pipette. 23 can be reliably extracted, and unnecessary mesenchymal stem cells contained in the raw bone marrow fluid 19 can be removed.

  FIG. 5 is a perspective view of the first flat culture vessel 25 (first culture vessel), and FIG. 6 is a side view of the first flat culture vessel 25 (first culture vessel). FIG. 7 is a partially enlarged view showing an example of the planar shape of the first mesenchymal stem cell 27, and FIG. 8 is a partially enlarged view showing another example of the planar shape of the first mesenchymal stem cell 27. 7 and 8 show enlarged images of the planar shape of the first mesenchymal stem cell 27 photographed by the electron microscope 13.

  After the bone marrow fluid extraction means for extracting the specific middle layer bone marrow fluid 23 located in the middle layer from the raw material bone marrow fluid 19, the stem cell first observation means is performed. In the first stem cell observation means, the intermediate layer bone marrow fluid 23 and the culture fluid 24 are injected (contained) into the first flat culture vessel 25 (first culture vessel), and the temperature inside the culture vessel 25 is substantially the same as the body temperature (about 36). The intermediate layer bone marrow fluid in the culture vessel 25 is allowed to stand statically (without moving) for 12 to 24 hours while being held at ~ 37 ° C, and at intervals of about 1 to 2 hours during 12 to 24 hours. 23, the deformation of the first mesenchymal stem cell 27 (first stem cell) contained in the initial planar shape of the first mesenchymal stem cell 27 is observed with the electron microscope 13 to determine whether the stem cell 27 has settled on the bottom surface 28 of the culture vessel 25 or not. A first code sheet 18a on which a QR code specifying a donor is printed is attached to the bottom surface 28 (outer surface of the bottom wall) of the culture vessel 25 into which the intermediate layer bone marrow fluid 23 and the culture solution 24 are injected.

  A person in charge such as a doctor, a nurse, or a researcher extracts a specific intermediate layer bone marrow fluid 23 from the raw material bone marrow fluid 19 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 first culture third display screen (not shown) on the display 16. Number 1 and donor data are displayed on the first culture third display screen, bone marrow fluid separation end message, bone marrow fluid extraction end message, stem cell first observation button, stem cell second observation button, stem cell first extraction button Is displayed.

  The person in charge affixes the first code sheet 18a on which the QR code is printed to the bottom surface 28 (bottom wall outer surface) of the first flat culture vessel 25. Next, the stem cell first observation button on the first culture third 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 2 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 ongoing 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 23 sucked into the syringe or pipette into the culture container 25 and injects (accommodates) the culture medium 24 sucked into the syringe or pipette into the culture container 25. The first flat culture vessel 25 (first culture vessel) is a flat vessel made of transparent glass or transparent plastic and having a bottom surface 28 with a small capacity and a predetermined area and having a substantially square shape. The first flat culture vessel 25 has a top portion 32, a central portion 33 located between the bottom portion 34 and the top bottom portions 32, 34, and an inlet 35 formed in the top portion 32. The injection port 35 is watertightly closed by a lid 36. The first flat culture vessel 25 used in the first stem cell observation means has a capacity of about 20 to 30 cc (preferably 25 cc) and a bottom area of about 25 to 36 mm ² . The culture container 25 has a side length of 5 to 6 mm. In addition, as the first flat culture vessel 25, a flat vessel having a small volume and a bottom surface 28 having a predetermined area can be used.

  The person in charge removes the lid 36 from the injection port 35 and injects (accommodates) the intermediate layer bone marrow fluid 23 sucked into the syringe or pipette from the injection port 35 of the first flat culture vessel 25 into the culture vessel 25. The culture solution 24 sucked into the syringe or pipette is injected (stored) into the inside of the culture vessel 25 from the injection port 35 of the culture vessel 25, and the injection port 36 is closed with a lid 36.

  The culture solution 24 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 27 contained in the intermediate layer bone marrow fluid 23 injected into the first flat culture vessel 25 are cultured with the culture solution 24 while being fixed on the bottom surface 28 of the culture vessel 25 over time. It gradually grows (differentiates) on the bottom surface 28 of the container 25 to form a colony.

  The person in charge injects the intermediate layer bone marrow fluid 23 and the culture solution 24 into the first flat culture vessel 25 and then installs (sets) the culture vessel 25 in the sample holder 37 of the electron microscope 13. A spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the bottom 34 of the first flat culture vessel 25, and the bottom 34 of the culture vessel 25 is held in a state where it is lifted by the spacer 39. The culture vessel 25 is held at a predetermined angle so that the bottom 34 of the vessel 25 is on top and the top 32 (injection port 35) of the culture vessel 25 is on the bottom. A spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the top portion 32 of the first flat culture vessel 25, and the top portion 32 of the culture vessel 25 is held in a state lifted by the spacer 39, thereby culturing. You may hold | maintain the culture container 25 in the state inclined to the predetermined angle so that the top part 32 of the container 25 may become the upper side and the bottom part 34 of the culture container 25 may become the lower side. The inclination angle α1 of the first flat culture vessel 25 with respect to the upper surface 38 of the sample holder 37 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

  The stem cell culture system 10 cultivates the intermediate layer bone marrow fluid 23 and the culture solution 24 in the first flat culture vessel 25 by inclining the first flat culture vessel 25 at the inclination angle with respect to the upper surface 38 of the sample holder 37. The pressure is biased toward the top 32 side (or the bottom 34 side) of the container 25, and the water pressure between the intermediate layer bone marrow fluid 23 and the culture solution 24 increases on the top 32 side (or the bottom 34 side) of the culture container 25. One mesenchymal stem cell 27 concentrates on the bottom surface 28 side of the culture vessel 25, thereby increasing the activity of the first mesenchymal stem cells 27, and the first mesenchymal stem cell 27 is easily formed on the bottom surface 28 of the culture vessel 25. And it can be fixed quickly.

  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 an enlarged image of the planar shape of the first mesenchymal stem cell 27 contained in the intermediate layer bone marrow fluid 23 injected into the first flat culture vessel 25 at intervals of about 1 to 2 hours, and the taken stem cells 27 enlarged images having a planar shape are 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 the keyboard 14 or the mouse 15.

  The computer 11 stores (stores) the planar enlarged image of the first mesenchymal stem cell 27 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 storage means). . 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 27 displayed on the display 16 at intervals of about 1 to 2 hours during 12 to 24 hours, and determines the stem cells 27 contained in the intermediate layer bone marrow fluid 23. Observe the change in planar shape. The person in charge may directly observe the change in the planar shape of the first mesenchymal stem cell 27 from the observation window of the electron microscope 13 at intervals of about 1 to 2 hours during 12 to 24 hours.

  The initial planar shape of the first mesenchymal stem cell 27 is approximately circular, and when the planar shape of the stem cell 27 is approximately circular, the stem cell 27 is not fixed on the bottom surface 28 (bottom wall inner surface) of the first flat culture vessel 25. In other words, the stem cell 27 has not started proliferation (differentiation). The planar shape after the deformation of the first mesenchymal stem cell 27 is a flat shape in which the stem cell 27 is expanded (expanded) in one direction (predetermined direction) with a substantially circular shape before fixing as a nucleus, and the stem cell 27 is a culture vessel. The stem cells 27 have started to proliferate (differentiate).

  As shown in FIG. 7, the person in charge observes the enlarged image of the planar shape of the first mesenchymal stem cell 27 displayed on the display 16 as a substantially circular shape as a result of the observation in the first stem cell observation means. It is determined that the stem cells 27 are not settled on the bottom surface 28 (bottom wall inner surface) of the culture vessel 25, and the change in the planar shape of the stem cells 27 is continuously observed at intervals of about 1 to 2 hours. As shown in FIG. 8, as a result of observation by the first stem cell observation means, the person in charge has a flat shape in which the planar shape of the first mesenchymal stem cell 27 displayed on the display 16 is from an approximately circular shape to an approximately circular shape as a nucleus. When the shape is deformed, it is determined that the stem cell 27 has settled on the bottom surface 28 of the culture vessel 25.

When a large culture container having a capacity exceeding 30 cc and a bottom area exceeding 36 mm ² is used when the first mesenchymal stem cell 27 is established, the stem cell 27 becomes difficult to settle on the bottom face of the container and the proliferation of the stem cell 27 is slow. However, the stem cell culture system 10 can easily fix the stem cells 27 to the bottom surface 28 of the culture container 25 by using the first flat culture container 25 having the capacity and the bottom area. Stem cells 27 can be rapidly proliferated.

  The stem cell culture system 10 allows the first flat culture vessel 25 to stand statically at a temperature substantially the same as the body temperature for 12 to 24 hours, while the first flat culture vessel 25 is left in the culture vessel 25 at intervals of about 1 to 2 hours during 12 to 24 hours. Since the deformation of the mesenchymal stem cell 27 from the initial planar shape is observed, the deformation of the stem cell 27 is not missed, and the fixation of the stem cell 27 to the bottom surface 28 of the culture vessel 25 can be confirmed accurately.

  FIG. 9 is a partially enlarged view showing another example of the planar shape of the first mesenchymal stem cell 27. FIG. 9 shows an enlarged image of the planar shape of the first mesenchymal stem cell 27 photographed by the electron microscope 13. As a result of observation by the first stem cell observation means, the first mesenchymal stem cell 27 (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 of the stem cells 27 The stem cell second observation means is performed after the stem cell first observation means that has confirmed the fixation on the bottom surface 28 of the flat culture container 25 (first culture container).

  In the stem cell second observation means, the culture solution 24 injected into the first flat culture vessel 25 is discharged from the culture vessel 25 and a new culture solution 24 is injected (accommodated) into the culture vessel 25. Next, the first flat culture vessel 25 is left to stand for 36 to 48 hours at a temperature substantially the same as the body temperature (about 36 to 37 ° C.) (for a quiet period without moving), and for 36 to 48 hours. The total planar area of the first mesenchymal stem cells 27 fixed to the bottom surface 28 of the culture vessel 25 with respect to the bottom surface area of the culture vessel 25 is observed with an electron microscope 13 at intervals of about 1 to 2 hours. It is determined whether or not the first target ratio has been reached with respect to the bottom surface area of the container 25. The first target ratio of the total planar area of the first mesenchymal stem cell 27 to the bottom surface area of the culture vessel 25 is 70 to 80% (70 to 80% confluent).

  After confirming that the first mesenchymal stem cells 27 contained in the intermediate layer bone marrow fluid 23 are fixed on the bottom surface 28 of the culture vessel 25, a doctor, nurse, researcher, or other person in charge of the stem cells 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 first culture fourth display screen (not shown) on the display 16. On the first culture fourth 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 button, stem cell first extraction A button is displayed.

  The person in charge clicks the second stem cell observation button on the first culture fourth display screen, removes the culture container 25 from the sample holder of the electron microscope 16, and causes the barcode reader 12 to read the QR code of the culture container 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 2 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 the culture solution 24 injected into the culture vessel 25 in the first stem cell observation means from the culture vessel 25 using a syringe or pipette, and the new culture solution 24 sucked into the syringe or pipette is supplied to the culture vessel 25. Inject (contain). The new culture solution 24 is the same as that injected in the stem cell first observation means. The person in charge injects a new culture solution 24 into the culture vessel 25 and then installs (sets) the culture vessel 25 in the sample holder 37 of the electron microscope 13.

  A spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the bottom 34 of the first flat culture vessel 25, and the bottom 34 of the culture vessel 25 is held in a state where it is lifted by the spacer 39. The culture vessel 25 is held at a predetermined angle so that the bottom 34 of the vessel 25 is on the top and the top 32 (injection port 35) of the culture vessel 25 is on the bottom (see FIG. 6). A spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the top portion 32 of the first flat culture vessel 25, and the top portion 32 of the culture vessel 25 is held in a state lifted by the spacer 39, thereby culturing. You may hold | maintain the culture container 25 in the state inclined to the predetermined angle so that the top part 32 of the container 25 may become the upper side and the bottom part 34 of the culture container 25 may become the lower side. The inclination angle α1 of the first flat culture vessel 25 with respect to the upper surface 38 of the sample holder 37 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

  The stem cell culture system 10 confirms the establishment of the first mesenchymal stem cells 27 and then injects a new culture solution 24 into the culture vessel 25 while discharging the culture solution 24 in the culture vessel 25, thereby proliferating the stem cells 27. Can be surely promoted. The stem cell culture system 10 causes the first mesenchymal stem cell 27 and the culture solution 24 to be placed in the culture container 25 by inclining the first flat culture container 25 at the inclination angle with respect to the upper surface 38 of the sample holder 37. 25, and the water pressure between the first mesenchymal stem cell 27 and the culture solution 24 increases on the top 32 side (or the bottom 34 side) of the culture vessel 25. The first mesenchymal stem cells 27 are concentrated on the bottom surface 28 side of the culture vessel 25, thereby increasing the activity of the first mesenchymal stem cells 27, and the first mesenchymal stem cells 27 are placed on the bottom surface 28 of the culture vessel 25. It 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 27 in the first flat culture vessel 25 at an interval of about 1 to 2 hours, and an enlarged image of the planar shape of the taken stem cell 27 is about 1 Transmit to the computer 11 at intervals of ~ 2 hours. The computer 11 stores (stores) the enlarged image of the planar shape of the stem cell 27 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 storage means). The computer 11 displays the enlarged image of the planar shape of the stem cell 27 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 27 displayed on the display 16 at intervals of about 1 to 2 hours during 36 to 48 hours, and the first flat culture container. While observing the total planar area of the stem cells 27 fixed to the bottom surface 28 of the culture container 25 with respect to the bottom surface area of the culture container 25, the total planar area of the stem cells 27 is the first target ratio (70 to 80%) It is determined whether or not confluence has been reached. The person in charge directly observes the total planar area of the first mesenchymal stem cell 27 with respect to the bottom 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 27 has reached the first target ratio (70 to 80% confluent) with respect to the bottom area of the culture vessel 25.

  As a result of the observation by the second stem cell observation means, the person in charge has a total plane area of the first mesenchymal stem cell 27 displayed on the display 16 with respect to the bottom area of the first flat culture vessel 25 as shown in FIG. When the target ratio (70 to 80% confluent) has not been reached, the total planar area of the stem cells 27 with respect 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 first mesenchymal stem cell 27 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 25 of the stem cell 27 It is assumed that the area has reached the first target ratio.

  As a result of observation by the second stem cell observation means, the first mesenchymal stem cell 27 grows on the bottom surface 28 (bottom wall inner surface) of the culture vessel 25 to form a colony, and the planar shape of the stem cell 27 expands. 9, when the total planar area of the stem cells 27 displayed on the display 16 with respect to the bottom surface area of the culture container 25 reaches the first target ratio (70 to 80% confluent), the stem cells are removed from the culture container 25. First stem cell extraction means for extracting 27 is performed. In the first stem cell extraction means, the first mesenchymal stem cells 27 grown (differentiated) in the culture vessel 25 are extracted from the culture vessel 25.

  After confirming that the total planar area of the first mesenchymal stem cell 27 with respect to the bottom area of the first flat culture vessel 25 has reached the first target ratio, a person in charge such as a doctor, a nurse, or a researcher displays the display 16 Click the button for ending stem cell second observation displayed in. When the stem cell second observation end button is clicked, the computer 11 displays a first culture fifth display screen (not shown) on the display 16. On the first culture 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 Extract button is displayed.

  The person in charge clicks the first stem cell extraction button on the first culture fifth 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 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 extraction in progress message, and a stem cell 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 discharges the culture solution 24 at the time of the second observation of the stem cells injected into the first flat culture vessel 25 from the culture vessel 25 using a syringe or pipette, and after washing the inside of the culture vessel 25 with PBS, The trypsin solution sucked by the syringe or pipette is injected into the culture container 25. When the trypsin solution is injected into the culture vessel 25, the first mesenchymal stem cells 27 fixed on the bottom surface 28 of the culture vessel 25 are detached from the bottom surface 28 by the trypsin solution and float on the water surface of the trypsin solution. The person in charge uses the pipette to aspirate the stem cells 27 and accommodates the stem cells 27 in the pipette.

  The stem cell culture system 10 can accurately confirm the total plane area with respect to the bottom area of the first flat culture vessel 25 of the first mesenchymal stem cell 27 by observing the total plane area at intervals of about 1 to 2 hours. It is possible to reliably grasp that the total planar area of the stem cells 27 has reached the first target ratio with respect to the bottom surface area of the culture vessel 25.

  FIG. 10 is an explanatory view showing an example of the stem cell separating means forming the second culturing means. After the first stem cell extraction means for extracting the first mesenchymal stem cells 27 from the culture vessel 25, the stem cell separation means for forming the second culture means is performed. In the stem cell separation means, the first mesenchymal stem cells 27 extracted by the stem cell first extraction means are centrifuged in layers by a centrifuge 29. A person in charge, such as a doctor, nurse, or researcher, aspirates the first mesenchymal stem cell 27 from the culture vessel 25 into the pipette, and then clicks the stem cell first extraction end button displayed on the display 16.

  When the stem cell first extraction end button is clicked, the computer 11 displays a second culture first display screen (not shown) on the display 16. On the second culture first display screen, number 1 and donor data are displayed, a stem cell separation button, a stem cell second extraction button, a stem cell third observation button, a stem cell fourth observation button, a stem cell third extraction button, a first 1 Culture resume button and logout button are displayed.

  When the first culture means is performed again, the first culture resumption button is clicked, and the bone marrow fluid separation means, bone marrow fluid extraction means, stem cell first observation means, stem cell second observation means described above shown in FIGS. The stem cell first extraction means is performed again. Click the logout button if you do not want to perform the second culture. When the logout button is clicked, the system 10 stops in the computer 11.

  The person in charge affixes the second code sheet 18b on which the QR code is printed to the outer peripheral surface of the glass test tube 30 into which the first mesenchymal stem cells 27 are injected. Next, the stem cell separation button on the second culture first display screen is clicked, and the barcode reader 12 reads the QR code of the glass test tube 30. 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 3 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 27 in the pipette into the glass test tube 30, and installs (sets) the glass test tube 30 in the centrifuge 29. The person in charge centrifuges the stem cells 27 with the centrifuge 29 for a predetermined time, and then removes the glass test tube 30 from the centrifuge 29. The first mesenchymal stem cell 27 in the glass test tube 30 is separated into two layers in the vertical direction by the centrifuge 29.

  FIG. 11 is a perspective view of a glass test tube 30 used for the second stem cell extraction means. The stem cell second extracting means is performed after the stem cell separating means for separating the first mesenchymal stem cells 27 in layers. The second stem cell extraction means extracts the second mesenchymal stem cell 31 located in the lower layer (lowermost layer) from the first mesenchymal stem cell 27 separated into layers. A doctor, nurse, researcher, or other person in charge uses the centrifuge 29 to separate the first mesenchymal stem cells 27 in the vertical direction, and then removes the glass test tube 30 from the centrifuge 29 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 second culture second display screen (not shown) on the display 16. On the second culture second display screen, number 2 and donor data are displayed, and a stem cell separation end message, a stem cell second extraction button, a stem cell third observation button, a stem cell fourth observation button, and a stem cell third extraction button are displayed. Is displayed.

  The person in charge clicks the stem cell second extraction button on the second culture second display screen, and causes the barcode reader 12 to read the QR code of the glass test tube 30. 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 2 and the donor data are displayed on the display 16, and a stem cell separation end message, a stem cell second extraction in progress message, and a stem cell second extraction 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 extracts the second mesenchymal stem cell 31 present in the lower layer (lowermost layer) of the first mesenchymal stem cells 27 separated into layers in the glass test tube 30. The person in charge extracts (sucks) the second mesenchymal stem cell 31 located in the lower layer (lowermost layer) of the first mesenchymal stem cells 27 separated into layers using a syringe. Alternatively, the second mesenchymal stem cells 31 located in the lower layer (lowermost layer) of the first mesenchymal stem cells 27 separated into layers using a pipette are extracted (sucked).

  The stem cell culture system 10 includes a first mesenchymal stem cell 27 containing unnecessary stem cells, which is centrifuged in a centrifuge 29 to be separated into layers in the vertical direction, and the lower layer (lowermost layer) of the stem cells 27 centrifuged in layers. 2), the specific second mesenchymal stem cell 31 can be reliably extracted from the stem cell 27, and unnecessary mesenchymal stem cells are removed from the stem cell 27. be able to.

  When the total planar area of the first mesenchymal stem cell 27 with respect to the bottom surface area of the first flat culture vessel 25 (first culture vessel) exceeds 80% and the stem cell 27 proliferates, the stem cell culture system 10 activates the stem cell 27. Although the stem cells 27 are extracted from the culture vessel 25 when the total planar area of the stem cells 27 grows to 70 to 80% of the bottom surface area of the culture vessel 25, the activity of the stem cells 27 is retained. The stem cells 27 can be grown while maintaining the activity, and the second mesenchymal stem cells 31 having activity can be extracted from the stem cells 27.

  FIG. 12 is a perspective view of the second flat culture vessel 26 (second culture vessel), and FIG. 13 is a side view of the second flat culture vessel 26 (second culture vessel). FIG. 14 is a partially enlarged view showing an example of the planar shape of the second mesenchymal stem cell 31, and FIG. 15 is a partially enlarged view showing another example of the planar shape of the second mesenchymal stem cell 31. 14 and 15 show enlarged images of the planar shape of the second mesenchymal stem cell 31 taken by the electron microscope 13.

  After the second stem cell extraction means for extracting specific second mesenchymal stem cells 31 located in the lower layer (lowermost layer) from the first mesenchymal stem cell 27, the third stem cell observation means is performed. In the third stem cell observation means, the second mesenchymal stem cell 31 and the culture solution 24 are injected (contained) into the second flat culture vessel 26 (second culture vessel), and the culture vessel 26 is heated to a temperature substantially equal to the body temperature (about The second mesenchymal stem cells in the culture vessel 26 at intervals of about 1 to 2 hours during 36 to 48 hours while statically standing (36 to 37 ° C.) for 36 to 48 hours. The deformation from the initial planar shape of 31 (second stem cell) is observed with the electron microscope 13 to determine whether the stem cell 31 has settled on the bottom surface 28 of the culture vessel 26. A second code sheet 18b on which a QR code specifying a donor is printed is attached to the bottom surface 28 (outer surface of the bottom wall) of the culture vessel 26 into which the second mesenchymal stem cells 31 and the culture solution 24 are injected.

  A person in charge such as a doctor, a nurse, or a researcher extracts a specific second mesenchymal stem cell 31 from the first mesenchymal stem cell 27 and then clicks a stem cell second extraction end button displayed on the display 16. . When the stem cell second extraction end button is clicked, the computer 11 displays a second culture third display screen (not shown) on the display 16. On the second culture third display screen, number 2 and donor data are displayed, stem cell separation end message, stem cell second extraction end message, stem cell third observation button, stem cell fourth observation button, stem cell third extraction button Is displayed.

  The person in charge attaches the second code sheet 18b on which the QR code is printed to the bottom surface 28 (outer surface of the bottom wall) of the second flat culture vessel 26. Next, the stem cell third observation button on the second culture third display screen is clicked to cause the barcode reader 12 to read the QR code of the culture vessel 26. 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 2 and the donor data are displayed on the display 16, and the stem cell separation end message, the stem cell second extraction 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 31 sucked into the syringe or pipette into the second flat culture container 26 and injects the culture solution 24 sucked into the syringe or pipette into the culture container 26 ( Contain). The second flat culture container 26 (second culture container) is a flat container having a bottom surface 40 having a small capacity and a predetermined area and made of transparent glass or transparent plastic. The area is larger than that of the first flat culture vessel 25 (first culture vessel), and the area of the bottom surface 40 is about twice that of the first flat culture vessel 25. The second flat culture vessel 26 has a top portion 41 and a central portion 42 located between the bottom portion 43 and the top bottom portions 41, 43, and an inlet 44 formed in the top portion 41. The injection port 44 is watertightly closed by a lid 45. As the second flat culture vessel 26, a flat vessel having a small volume and a bottom surface 40 having a predetermined area and having a circular or oval planar shape may be used.

The second flat culture vessel 26 (second culture vessel) used in the stem cell third observation means has a capacity of about 40 to 60 cc (preferably 50 cc) and a bottom surface area of about 50 to 72 mm ² . . The culture container 26 has a length of one side of about 7 to 8.5 mm. The culture solution 24 is the same as that injected in the first observation of stem cells. The second mesenchymal stem cells 31 injected into the culture vessel 26 are cultured with the culture solution 24 while being fixed on the bottom surface 28 of the culture vessel 26 over time, and gradually grow (differentiate) on the bottom surface 28 of the culture vessel 26. To form colonies.

  The person in charge removes the lid 45 from the injection port 44 and injects (accommodates) the second mesenchymal stem cell 31 sucked into the pipette from the injection port 44 of the second flat culture vessel 26 into the culture vessel 26. The culture solution 24 sucked into the syringe or pipette is injected (accommodated) into the culture vessel 26 from the injection port 44 of the culture vessel 26, and the injection port 44 is closed with a lid 45. The person in charge injects the second mesenchymal stem cell 31 and the culture solution 24 into the second flat culture vessel 26 and then installs (sets) the culture vessel 26 in the sample holder 37 of the electron microscope 13.

  A spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the bottom 43 of the second flat culture vessel 26, and the bottom 43 of the culture vessel 26 is held up by the spacer 39. The culture vessel 26 is held in a state inclined at a predetermined angle such that the bottom portion 43 of the culture vessel is on the top and the top portion 41 (injection port 44) of the culture vessel 26 is on the bottom. Note that a spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the top 41 of the second flat culture vessel 26, and the top 41 of the culture vessel 26 is held in a state of being lifted by the spacer 39. You may hold | maintain the culture container 26 in the state inclined by the predetermined angle so that the top part 41 of the container 26 may become upper and the bottom part 43 of the culture container 26 may become lower. The inclination angle α2 of the second flat culture vessel 26 with respect to the upper surface 38 of the sample holder 37 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

  The stem cell culture system 10 inclines the second flat culture vessel 26 with respect to the upper surface 38 of the sample holder 37 at the inclination angle, so that the second mesenchymal stem cells 31 and the culture solution 24 are cultured in the culture vessel 26. 26, and the water pressure between the second mesenchymal stem cell 31 and the culture solution 24 is increased on the top 41 side (or the bottom 43 side) of the culture vessel 26. The second mesenchymal stem cells 31 concentrate on the bottom surface 40 side of the culture vessel 34, thereby increasing the activity of the second mesenchymal stem cells 31, and the second mesenchymal stem cells 31 are added to the bottom surface 40 of the culture vessel 26. It can be fixed easily and quickly.

  The electron microscope 13 takes a magnified image of the planar shape of the second mesenchymal stem cell 31 injected into the culture vessel 26 at intervals of about 1 to 2 hours, and captures a magnified image of the planar shape of the photographed stem cell 31 by about 1 to 2 hours. It transmits to the computer 11 at intervals of 2 hours. The computer 11 stores (stores) in the storage area the enlarged image of the planar shape of the second mesenchymal stem cell 31 and the imaging time transmitted from the electron microscope 13 in a state associated with the donor identifier (stem cell image storage means). . The computer 11 displays the enlarged image of the planar shape of the stem cell 31 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 31 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 31. The person in charge may directly observe the change in the planar shape of the stem cell 31 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 31 is substantially circular. When the planar shape of the stem cell 31 is approximately circular, the stem cell 31 is not fixed on the bottom surface 40 (bottom wall inner surface) of the culture vessel 26, and the stem cell. 31 has not started to proliferate (differentiate). The planar shape after the deformation of the second mesenchymal stem cell 31 is a flat shape in which the stem cell 31 extends in an indefinite shape in one direction with a substantially circular shape before fixation as a nucleus, and the stem cell 31 is a bottom surface 40 (bottom wall) of the culture vessel 26. The stem cells 31 have started to proliferate (differentiate).

  As shown in FIG. 14, when the person in charge observes the planar shape of the second mesenchymal stem cell 31 displayed on the display 16 as a substantially circular shape, as shown in FIG. It is judged that the culture vessel 26 has not settled on the bottom surface 40 (bottom wall inner surface), and changes in the planar shape of the stem cells 31 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, as shown in FIG. 15, has a planar shape of the second mesenchymal stem cell 31 displayed on the display 16 having an irregular shape with a substantially circular shape to a substantially circular shape as a nucleus. When the shape is deformed, it is determined that the stem cell 31 has settled on the bottom surface 40 of the culture vessel 26.

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 31 is fixed, the stem cell 31 is difficult to settle on the bottom surface of the container and the proliferation of the stem cell 31 is slow. However, the stem cell culture system 10 can easily fix the stem cells 31 to the bottom surface 40 of the culture container 26 by using the second flat culture container 26 having the capacity and the bottom surface area. Stem cells 31 can be rapidly proliferated.

  The stem cell culture system 10 allows the second flat culture vessel 26 to be statically left at a temperature substantially the same as the body temperature for 36 to 48 hours, and within the culture vessel 26 at intervals of about 1 to 2 hours during 36 to 48 hours. Since the deformation of the second mesenchymal stem cell 31 from the initial planar shape is observed, the deformation of the stem cell 31 is not missed, and the fixation of the stem cell 31 to the bottom surface 40 of the culture vessel 26 can be confirmed accurately.

  FIG. 16 is a partially enlarged view showing another example of the planar shape of the second mesenchymal stem cell 31, and FIG. 17 is a diagram showing an example of preservation of the second mesenchymal stem cell 31. FIG. 16 shows an enlarged image of the planar shape of the second mesenchymal stem cell 31 photographed by the electron microscope 13. As a result of the observation by the third means for observing stem cells, the second mesenchymal stem cell 31 (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 second of the stem cells 31 The stem cell fourth observation means is performed after the stem cell third observation means that has confirmed the establishment of the flat culture container 26 (second culture container) on the bottom surface 40.

  In the fourth stem cell observation means, the culture solution 24 injected into the culture vessel 26 is discharged from the culture vessel 26 and a new culture solution 24 is injected (accommodated) into the culture vessel 26. Next, the culture vessel 26 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 about 1 to about 36 to 48 hours. The total planar area of the second mesenchymal stem cells 31 fixed on the bottom surface 40 of the culture container 26 with respect to the bottom surface area of the culture container 26 is observed with the electron microscope 13 at intervals of 2 hours. It is determined whether the second target ratio has been reached with respect to the bottom surface area. The second target ratio of the total planar area of the second mesenchymal stem cell 31 to the bottom surface area of the culture vessel 26 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 31 has settled on the bottom surface 40 of the culture vessel 26 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 second culture fourth display screen (not shown) on the display 16. On the second culture fourth display screen, number 2 and donor data are displayed, stem cell separation end message, stem cell second extraction end message, stem cell third observation end message, stem cell fourth observation button, stem cell third extraction A button is displayed.

  The person in charge clicks the fourth stem cell observation button on the second culture fourth display screen, removes the culture container 26 from the sample holder of the electron microscope 13, and causes the barcode reader 12 to read the QR code of the culture container 26. 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 2 and the donor data are displayed on the display 16, and the stem cell separation end message, the stem cell second extraction 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 the culture solution 24 injected into the culture vessel 26 in the stem cell third observation means from the culture vessel 26 using a syringe or pipette, and the new culture solution 24 sucked into the syringe or pipette is supplied to the culture vessel. 26 is injected (accommodated). The new culture solution 24 is the same as that injected in the stem cell first observation means. The person in charge injects a new culture solution 24 into the culture vessel 26 and then installs (sets) the culture vessel 26 in the sample holder 37 of the electron microscope 13.

  A spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the bottom 43 of the second flat culture vessel 26, and the bottom 43 of the culture vessel 26 is held up by the spacer 39. The culture container 26 is held in a state inclined at a predetermined angle so that the bottom part 43 of the culture medium is on the top and the top part 41 (inlet 44) of the culture container 26 is on the bottom (see FIG. 13). Note that a spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the top 41 of the second flat culture vessel 26, and the top 41 of the culture vessel 26 is held in a state of being lifted by the spacer 39. You may hold | maintain the culture container 26 in the state inclined by the predetermined angle so that the top part 41 of the container 26 may become upper and the bottom part 43 of the culture container 26 may become lower. The inclination angle α2 of the second flat culture vessel 26 with respect to the upper surface 38 of the sample holder 37 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 cells 31, the stem cell culture system 10 injects a new culture solution 24 into the culture vessel 26 while discharging the culture solution 24 in the culture vessel 26, thereby The proliferation of the leaf stem cells 31 can be surely promoted. The stem cell culture system 10 inclines the second flat culture vessel 26 with respect to the upper surface 38 of the sample holder 37 at the inclination angle, so that the second mesenchymal stem cells 31 and the culture solution 24 are cultured in the culture vessel 26. 26, and the water pressure between the second mesenchymal stem cell 31 and the culture solution 24 is increased on the top 41 side (or the bottom 43 side) of the culture vessel 26. The second mesenchymal stem cells 31 are concentrated on the bottom surface 40 side of the culture vessel 26, thereby increasing the activity of the second mesenchymal stem cells 31, and the second mesenchymal stem cells 31 are added to the bottom surface 40 of the culture vessel 26. It 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 31 in the second flat culture vessel 26 at intervals of about 1 to 2 hours, and an enlarged image of the taken planar shape of the stem cell 31 is about 1 Transmit to the computer 11 at intervals of ~ 2 hours. The computer 11 stores (stores) in the storage area the enlarged image of the planar shape of the second mesenchymal stem cell 31 and the imaging time transmitted from the electron microscope 13 in a state associated with the donor identifier (stem cell image storage means). . The computer 11 displays on the display 16 the enlarged image of the planar shape of the second mesenchymal stem cell 31 transmitted from the electron microscope 13 and the imaging time.

  The person in charge checks (views) the enlarged image of the planar shape of the second mesenchymal stem cell 31 displayed on the display 16 at intervals of about 1 to 2 hours during 36 to 48 hours, and the bottom surface 40 of the culture vessel 26. The total planar area of the stem cells 31 reaches the second target ratio (82-92% confluent) with respect to the bottom area of the culture vessel 26 while observing the total planar area of the stem cells 31 settled on the bottom surface area of the culture vessel 26. Determine whether or not. The person in charge directly observes the total planar area of the second mesenchymal stem cell 31 with respect to the bottom surface area of the culture vessel 26 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 31 has reached the second target ratio (88-92% confluent) with respect to the bottom area of the culture vessel 26.

  As a result of the observation by the stem cell fourth observation means, the person in charge, as shown in FIG. 15, determines that the total planar area of the second mesenchymal stem cell 31 displayed on the display 16 with respect to the bottom area of the culture vessel 26 is the second target ratio. If it has not reached (88 to 92% confluent), the total planar area of the stem cells 31 relative to the bottom area of the culture vessel 26 is continuously observed at intervals of about 1 to 2 hours. In addition, when the total plane area of the second mesenchymal stem cell 31 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 26 of the stem cell 31 It is assumed that the area has reached the second target ratio.

  As a result of the observation by the stem cell fourth observation means, the second mesenchymal stem cell 31 grows on the bottom surface 40 (bottom wall inner surface) of the second flat culture vessel 26 (second culture vessel), and the stem cell 31 forms a colony, As the planar shape of the stem cell 31 expands, as shown in FIG. 16, the total planar area of the stem cell 31 displayed on the display 16 with respect to the bottom surface area of the culture vessel 26 becomes the second target ratio (88 to 92% confluent). When it reaches, the 3rd stem cell extraction process which extracts the stem cell 31 from the culture container 26 is performed. In the third stem cell extraction step, the second mesenchymal stem cells 31 grown (differentiated) in the culture vessel 26 are extracted from the culture vessel 26.

  The stem cell culture system 10 can accurately confirm the total plane area with respect to the bottom area of the culture vessel 26 of the second mesenchymal stem cell 31 by observing the total plane area at intervals of about 1 to 2 hours. It can be ascertained that the total planar area of the stem cells 31 has reached the second target ratio with respect to the bottom surface area of the container 26.

  A person in charge such as a doctor, nurse or researcher confirms that the total planar area of the second mesenchymal stem cell 31 with respect to the bottom area of the culture vessel 26 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 second culture fifth display screen (not shown) on the display 16. On the second culture fifth display screen, number 2 and donor data are displayed, and a stem cell separation end message, stem cell second extraction end message, stem cell third observation end message, stem cell fourth observation end message, stem cell third Extract button is displayed.

  The person in charge clicks the third stem cell extraction button on the second culture fifth display screen, and causes the barcode reader 12 to read the QR code of the culture vessel 26. 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 2 and the donor data are displayed on the display 16, and the stem cell separation end message, the stem cell second extraction end message, the stem cell third observation end message, and the stem cell fourth observation An end message and a stem cell third 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 discharges the culture fluid 24 injected into the culture vessel 26 in the fourth stem cell observation means from the culture vessel 26 using a syringe or pipette, and after washing the inside of the culture vessel 26 with PBS, The aspirated trypsin solution is injected into the culture vessel 26. When the trypsin solution is injected into the culture vessel 26, the second mesenchymal stem cells 31 fixed on the bottom surface 40 of the culture vessel 26 are detached from the bottom surface 40 by the trypsin solution and float on the water surface of the trypsin solution.

  The person in charge uses the pipette to aspirate the second mesenchymal stem cell 31 and injects (accommodates) the stem cell 31 from the pipette into the storage container 32. The second mesenchymal stem cell 31 injected into the storage container 32 is a specific type of single-type mesenchymal stem cell 33 (single-type stem cell) to be cultured having an activity from which unnecessary mesenchymal stem cells are removed. The person in charge stores the first code sheet 18a or the second code sheet 18b on which the QR code is printed after injecting the second mesenchymal stem cell 31 (single seed mesenchymal stem cell 33) from the pipette into the storage container 32. Affixed to the outer peripheral surface of the container 32.

  The person in charge clicks the stem cell third extraction end button on the second culture / fifth display screen displayed on the display 16 and causes the barcode reader 12 to read the QR code of the storage container 32, and then the second mesenchymal system. A storage container 32 into which the stem cells 31 are injected is stored in a refrigerator 33. The second mesenchymal stem cell 31 (single seed mesenchymal stem cell 33) is stored in the refrigerator 33 at about 3 to 4 ° C.

  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 2 and the donor data are displayed on the display 16, and the stem cell separation end message, the stem cell second extraction end message, the stem cell third observation end message, and the stem cell fourth observation An end message, a stem cell third extraction end message, a culture end button, and a second culture restart button are displayed on the display 16. To end the second culture, click the culture end button. When the culture end button is clicked, the system 10 stops in the computer 11.

  When the total planar area of the second mesenchymal stem cell 31 with respect to the bottom surface area of the second flat culture vessel 26 (second culture vessel) exceeds 92% and the stem cell 31 proliferates, the stem cell culture system 10 activates the stem cell 31. Although the stem cells 31 are extracted from the culture vessel 26 when the total planar area of the stem cells 31 grows to 88 to 92% with respect to the bottom surface area of the culture vessel 26, the activity of the stem cells 31 is retained. The stem cells 31 can be grown in a state where the activity is maintained.

  FIG. 18 is a side view of the third flat culture vessel 46 (third culture vessel). When the second culturing means is performed again (when the second culturing means is performed twice or more), the second culturing restart button is clicked, and the already described stem cell third observation means and stem cell fourth observation shown in FIGS. Means and stem cell third extraction means are carried out again. Single-type stem cells are produced by performing the first culture means once, performing the second culture means once, or performing the second culture means twice or more. When the second culturing means is performed again, in the second and subsequent second culturing means, the third flat culture vessel 46 (third) having a bottom surface with a predetermined volume and a predetermined area and having a capacity larger than that of the second flat culture vessel 26. Culture vessels) are used.

  The person in charge injects (accommodates) the second mesenchymal stem cell 31 cultured (manufactured) by the first second culture means into the third flat culture vessel 46 and injects (accommodates) the culture solution 24 into the culture vessel 46. ) The third flat culture vessel 46 is a flat vessel made of transparent glass or transparent plastic, having a small volume and having a bottom surface 47 with a predetermined area and having a substantially rectangular plane shape. The area of the bottom surface 47 is the second flat culture. It is larger than that of the container 26 (second culture container). The third flat culture vessel 46 has a top portion 48 and a central portion 49 located between the bottom portion 50 and the top bottom portions 48 and 50, and an inlet 51 formed in the top portion 48. The injection port 51 is watertightly closed by a lid 52. As the third flat culture container 46, a flat container having a small volume and a bottom surface 47 having a predetermined area and having a circular or elliptical planar shape may be used.

The third flat culture vessel 46 (third culture vessel) has a capacity of about 70 to 80 cc (preferably 75 cc) and a bottom surface 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 24 is the same as that injected in the first observation of stem cells. The second mesenchymal stem cells 31 injected into the culture vessel 46 are cultured with the culture solution 24 while being fixed on the bottom surface 47 of the culture vessel 46 over time, and gradually grow (differentiate) on the bottom surface 47 of the culture vessel 46. To form colonies.

  The person in charge removes the lid 52 from the injection port 51 and injects (accommodates) the second mesenchymal stem cell 31 sucked into the pipette into the culture vessel 46 from the injection port 51 of the third flat culture vessel 46. The culture solution 24 sucked into the syringe or pipette is injected (stored) into the culture vessel 46 from the injection port 51 of the culture vessel 46, and the injection port 51 is closed with a lid 52. The person in charge injects the second mesenchymal stem cell 31 and the culture solution 24 into the third flat culture vessel 46 and then installs (sets) the culture vessel 46 in the sample holder 37 of the electron microscope 13.

  A spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the bottom 50 of the third flat culture vessel 46, and the bottom 50 of the culture vessel 46 is held up by the spacer 39. The culture vessel 46 is held in a state inclined at a predetermined angle so that the bottom portion 50 of the culture vessel 50 is on the top and the top portion 48 (injection port 51) of the culture vessel 46 is on the bottom. In addition, a spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the top 48 of the third flat culture vessel 46, and the top 48 of the culture vessel 46 is held in a state lifted by the spacer 39. You may hold | maintain the culture container 46 in the state inclined by the predetermined angle so that the top part 48 of the container 46 may become the upper side and the bottom part 50 of the culture container 46 may become the lower side. The inclination angle α2 of the third flat culture vessel 46 with respect to the upper surface 38 of the sample holder 37 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

  The stem cell culture system 10 causes the second mesenchymal stem cell 31 and the culture solution 24 to be cultivated in the culture container 46 by inclining the third flat culture container 46 at the inclination angle with respect to the upper surface 38 of the sample holder 37. 46, the water pressure between the second mesenchymal stem cell 31 and the culture solution 24 increases on the top 48 side (or the bottom 50 side) of the culture vessel 46. The second mesenchymal stem cells 31 concentrate on the bottom surface 47 side of the culture vessel 34, thereby increasing the activity of the second mesenchymal stem cells 31, and the second mesenchymal stem cells 31 are added to the bottom surface 470 of the culture vessel 46. It can be fixed easily and quickly.

  The electron microscope 13 takes a magnified image of the planar shape of the second mesenchymal stem cell 31 injected into the culture vessel 46 at intervals of about 1 to 2 hours, and captures a magnified image of the planar shape of the photographed stem cell 31 by about 1 to 2 hours. It transmits to the computer 11 at intervals of 2 hours. The computer 11 stores (stores) in the storage area the enlarged image of the planar shape of the second mesenchymal stem cell 31 and the imaging time transmitted from the electron microscope 13 in a state associated with the donor identifier (stem cell image storage means). . The computer 11 displays the enlarged image of the planar shape of the stem cell 31 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 31 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 31.

  The initial planar shape of the second mesenchymal stem cell 31 is substantially circular. When the planar shape of the stem cell 31 is substantially circular, the stem cell 31 is not fixed on the bottom surface 47 (bottom wall inner surface) of the culture vessel 46, and the stem cell. 31 has not started to proliferate (differentiate). The planar shape after deformation of the second mesenchymal stem cell 31 is a flat shape in which the stem cell 31 extends in an indefinite shape in one direction with a substantially circular shape before fixation as a nucleus, and the stem cell 31 is a bottom surface 47 (bottom wall) of the culture vessel 46. The stem cells 31 have started to proliferate (differentiate).

  When the person in charge observes the second mesenchymal stem cell 31 displayed on the display 16 as a substantially circular shape as a result of the observation by the third means for observing the stem cells (see FIG. 14), the stem cell 31 is cultured in the culture vessel. 46 is determined not to have settled on the bottom surface 47 (bottom wall inner surface), and the change in the planar shape of the stem cells 31 is 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 31 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. 15), it is determined that the stem cells 31 have settled on the bottom surface 47 of the culture vessel 46.

When a large culture container with a capacity exceeding 80 cc and a bottom area exceeding 108 mm ² is used when the second mesenchymal stem cells 31 are fixed, the stem cells 31 are difficult to settle on the bottom of the container and the growth of the stem cells 31 is slow. However, the stem cell culture system 10 can easily fix the stem cells 31 to the bottom surface 47 of the culture container 46 by using the third flat culture container 46 having the capacity and the bottom surface area. Stem cells 31 can be rapidly proliferated.

  The stem cell culture system 10 allows the third flat culture vessel 46 to be statically left at a temperature substantially the same as the body temperature for 36 to 48 hours, while the culture in the culture vessel 46 is repeated at intervals of about 1 to 2 hours during 36 to 48 hours. Since the deformation of the second mesenchymal stem cell 31 from the initial planar shape is observed, the deformation of the stem cell 31 is not missed, and the fixation of the stem cell 31 to the bottom surface 47 of the culture vessel 46 can be confirmed accurately.

  As a result of the observation by the third stem cell observation means, the second mesenchymal stem cell 31 (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 of the stem cells 31 The stem cell fourth observation means is performed after the stem cell third observation means that has confirmed the fixation on the bottom surface 47 of the flat culture container 46 (third culture container).

  In the fourth stem cell observation means, the culture solution 24 injected into the culture vessel 46 is discharged from the culture vessel 46 and a new culture solution 24 is injected (accommodated) into the culture vessel 46. Next, the culture vessel 46 is statically left at 36 ° C. for 36 to 48 hours at a temperature substantially the same as the body temperature (about 36 to 37 ° C.). The total planar area of the second mesenchymal stem cells 31 fixed on the bottom surface 47 of the culture vessel 46 at two-hour intervals with respect to the bottom surface area of the culture vessel 46 is observed with the electron microscope 13, and the total planar area of the stem cells 31 is It is determined whether the second target ratio has been reached with respect to the bottom surface area. The second target ratio of the total planar area of the second mesenchymal stem cell 31 to the bottom surface area of the culture vessel 46 is 88 to 92% (88 to 92% confluent).

  After confirming that the second mesenchymal stem cells 31 have settled on the bottom surface 47 of the culture vessel 46, a person in charge such as a doctor, a nurse, or a researcher 24, the culture solution 24 injected into the culture vessel 46 by the third stem cell observation means. Is discharged from the culture vessel 26 using a syringe or pipette, and a new culture solution 24 sucked into the syringe or pipette is injected (accommodated) into the culture vessel 46. The new culture solution 24 is the same as that injected in the stem cell first observation means. The person in charge injects the new culture solution 24 into the culture vessel 46 and then installs (sets) the culture vessel 46 in the sample holder 37 of the electron microscope 13.

  A spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the bottom 50 of the third flat culture vessel 46, and the bottom 50 of the culture vessel 46 is held up by the spacer 39. The culture vessel 46 is held in a state inclined at a predetermined angle so that the bottom 50 of the tube is on the top and the top 48 (inlet 51) is on the bottom. In addition, a spacer 39 is interposed between the upper surface 38 of the sample holder 37 of the electron microscope 13 and the top 48 of the third flat culture vessel 46, and the top 48 of the culture vessel 46 is held in a state lifted by the spacer 39. The culture vessel 46 may be held at a predetermined angle so that the top 48 of the vessel 46 is on the top and the bottom 50 is on the bottom. The inclination angle α2 of the third flat culture vessel 46 with respect to the upper surface 38 of the sample holder 37 is in the range of 2 to 5 °, and preferably in the range of 2 to 3 °.

  The stem cell culture system 10 confirms the establishment of the second mesenchymal stem cells 31, and then injects a new culture solution 24 into the culture vessel 46 while discharging the culture solution 24 in the culture vessel 46, so that the second cell The proliferation of the leaf stem cells 31 can be surely promoted. The stem cell culture system 10 causes the second mesenchymal stem cell 31 and the culture solution 24 to be cultivated in the culture container 46 by inclining the third flat culture container 46 at the inclination angle with respect to the upper surface 38 of the sample holder 37. 46, the water pressure between the second mesenchymal stem cell 31 and the culture solution 24 increases on the top 48 side (or the bottom 50 side) of the culture vessel 46. The second mesenchymal stem cells 31 concentrate on the bottom surface 47 side of the culture vessel 46, thereby increasing the activity of the second mesenchymal stem cells 31, and the second mesenchymal stem cells 31 are added to the bottom surface 47 of the culture vessel 46. It can be propagated (differentiated) easily and rapidly.

  The electron microscope 13 takes a magnified image of the planar shape of the second mesenchymal stem cell 31 in the third flat culture vessel 46 at intervals of about 1 to 2 hours, and the magnified image of the planar shape of the photographed stem cell 31 is approximately 1 Transmit to the computer 11 at intervals of ~ 2 hours. The computer 11 stores (stores) in the storage area the enlarged image of the planar shape of the second mesenchymal stem cell 31 and the imaging time transmitted from the electron microscope 13 in a state associated with the donor identifier (stem cell image storage means). . The computer 11 displays on the display 16 the enlarged image of the planar shape of the second mesenchymal stem cell 31 transmitted from the electron microscope 13 and the imaging time.

  The person in charge checks (views) the enlarged image of the planar shape of the second mesenchymal stem cell 31 displayed on the display 16 at intervals of about 1 to 2 hours during 36 to 48 hours, and the bottom surface 47 of the culture vessel 46. The total planar area of the stem cells 31 reaches the second target ratio (82 to 92% confluent) with respect to the bottom area of the culture vessel 46 while observing the total planar area of the stem cells 31 settled on the bottom surface area of the culture vessel 46. Determine whether or not.

  As a result of the observation by the stem cell fourth observation means, the person in charge is the second target ratio (88 to 92% confluent) with respect to the total planar area of the second mesenchymal stem cell 31 displayed on the display 16 with respect to the bottom surface area of the culture vessel 46. If it has not reached (supported in FIG. 15), the total planar area of the stem cell 31 with respect to the bottom area of the culture vessel 46 is continuously observed at intervals of about 1 to 2 hours. In addition, when the total plane area of the second mesenchymal stem cell 31 reaches the second target ratio with respect to the total area of the enlarged image displayed on the display 16, the total plane with respect to the bottom area of the culture vessel 46 of the stem cell 31 It is assumed that the area has reached the second target ratio.

  As a result of the observation by the stem cell fourth observation means, the second mesenchymal stem cell 31 grows on the bottom surface 47 (bottom wall inner surface) of the third flat culture vessel 46 to form a colony, and the planar shape of the stem cell 31 is When the total planar area of the stem cells 31 displayed on the display 16 with respect to the bottom surface area of the culture vessel 46 reaches the second target ratio (88-92% confluent) by expanding (from FIG. 16), the culture vessel 46 A third stem cell extraction step for extracting the stem cells 31 is performed. In the third stem cell extraction step, the second mesenchymal stem cell 31 grown (differentiated) in the culture vessel 46 is extracted from the culture vessel 46.

  The stem cell culture system 10 can accurately confirm the total planar area with respect to the bottom area of the culture vessel 46 of the second mesenchymal stem cell 31 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 31 has reached the second target ratio with respect to the bottom area of the container 46.

  The person in charge discharges the culture solution 24 injected into the culture vessel 46 in the fourth stem cell observation means from the culture vessel 46 using a syringe or pipette, and after washing the inside of the culture vessel 46 with PBS, The aspirated trypsin solution is injected into the culture vessel 46. When the trypsin solution is injected into the culture vessel 46, the second mesenchymal stem cells 31 fixed on the bottom surface 47 of the culture vessel 46 are detached from the bottom surface 47 by the trypsin solution and float on the water surface of the trypsin solution.

  The person in charge uses the pipette to aspirate the second mesenchymal stem cell 31 and injects (accommodates) the stem cell 31 from the pipette into the storage container 32. The second mesenchymal stem cell 31 injected into the storage container 32 is a specific type of single-type mesenchymal stem cell 33 (single-type stem cell) to be cultured having an activity from which unnecessary mesenchymal stem cells are removed. The person in charge stores the first code sheet 18a or the second code sheet 18b on which the QR code is printed after injecting the second mesenchymal stem cell 31 (single seed mesenchymal stem cell 33) from the pipette into the storage container 32. The storage container 32 that is affixed to the outer peripheral surface of the container 32 and into which the second mesenchymal stem cells 31 are injected is stored in the refrigerator 33. The second mesenchymal stem cell 31 (single seed mesenchymal stem cell 33) is stored in the refrigerator 33 at about 3 to 4 ° C.

  The stem cell culture system 10 activates the stem cell 31 when the total planar area of the second mesenchymal stem cell 31 with respect to the bottom surface area of the third flat culture vessel 46 (third culture vessel) exceeds 92% and the stem cell 31 proliferates. Although the stem cells 31 are extracted from the culture vessel 46 when the total planar area of the stem cells 31 grows to 88 to 92% with respect to the bottom surface area of the culture vessel 46, the activity of the stem cells 31 is retained. The stem cells 31 can be grown in a state where the activity is maintained.

  Single-type mesenchymal stem cells 33 (single-type stem cells) produced by the stem cell culture system 10 extract the intermediate layer bone marrow fluid 23 located in the intermediate layer of the raw material bone marrow fluid 19 separated in layers, and the intermediate layer The bone marrow fluid 23 is cultured with the culture solution 24 to fix the first stem cells 27 to the bottom surface 28 of the first culture vessel 25, and the total planar area of the first stem cells 27 is the first relative to the bottom surface area of the first culture vessel 25. When the target ratio is reached, the first stem cells 27 are extracted from the first culture vessel 25, and the second stem cells 31 located in the lower layer (lowermost layer) of the first stem cells 27 separated into layers are extracted, The second stem cells 31 are cultured together with the culture medium 24 to fix the second stem cells 31 to the bottom surface 28 of the second culture container 26, and the total planar area of the second stem cells 31 is the second relative to the bottom area of the second culture container 26. 2 Target ratio reached In this case, since the second stem cell 31 is extracted from the second culture vessel 26, various kinds of stem cells are not included, and various kinds of stem cells are included. Highly established to completely cure the disease, and highly established to regenerate various tissues and various organs, can be suitably used for treatment of various diseases, and can be suitably used for regeneration of various tissues and organs .

  The single-species mesenchymal stem cell 33 produced by the stem cell culture system 10 is obtained by extracting a specific intermediate layer bone marrow fluid 23 from the raw material bone marrow fluid 19 separated in a layered manner and specifying a specific portion from the first stem cell 27 separated in a layered manner. By extracting the second stem cell 31, it is a pure (pure) stem cell from which unnecessary stem cells have been removed, and various kinds of stem cells are not included in the single-species mesenchymal stem cell 33. And the effect of regeneration in regenerative medicine can be stabilized, various diseases can be cured quickly and reliably, and various tissues and organs can be regenerated early and reliably.

  When the single-type mesenchymal stem cell 33 produced by the stem cell culture system 10 is produced by performing the first culture means once and performing the second culture means at least twice, the second culture from the second time is performed. In the means, a third culture vessel having a predetermined volume and a bottom area with a predetermined area and a capacity larger than the second culture vessel 26 is used, and a large amount of single-type mesenchymal stem cells 33 can be produced by the third culture vessel. It can be used for the complete cure of diseases of many patients and can be used for regeneration of tissues and organs of many patients.

DESCRIPTION OF SYMBOLS 10 Stem cell culture system 11 Computer 12 Bar code reader 13 Electron microscope 14 Keyboard 15 Mouse 16 Display 18a 1st code paper 18b 2nd code paper 19 Raw material bone marrow fluid 20 Glass test tube 21 Test tube stand 22 Incubator 23 Middle layer bone marrow fluid 24 Culture solution 25 First flat culture vessel (first culture vessel)
26 Second flat culture vessel (second culture vessel)
27 First mesenchymal stem cell (first stem cell)
28 Bottom surface 29 Centrifuge 30 Glass test tube 31 Second mesenchymal stem cell (second stem cell)
32 Top 33 Central part 34 Bottom 35 Inlet 36 Lid 37 Sample holder 38 Upper surface 39 Spacer 40 Bottom 41 Top 42 Central part 43 Bottom 44 Inlet 45 Lid 46 Third flat culture vessel (third culture vessel)
47 Bottom 48 Top 49 Central 50 Bottom 51 Inlet 52 Lid

Claims (13)

In single-type stem cells made from raw bone marrow fluid collected from donors,
The single-type stem cells separate the raw material bone marrow fluid collected from the donor into layers, and extract the intermediate layer bone marrow fluid located in the intermediate layer of the raw material bone marrow fluid separated into layers, with a predetermined volume and a predetermined area Injecting the intermediate layer bone marrow fluid and the culture solution into a first culture vessel having a bottom surface of the first layer causes the first stem cells contained in the intermediate layer bone marrow fluid to settle on the bottom surface of the first culture vessel, 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 to proliferate the first stem cells. A first culture in which the first stem cells are extracted from the first culture container after the stem cells have expanded and the total planar area of the first stem cells has reached a first target ratio with respect to the bottom area of the first culture container. And the first stem cell extracted by the first culture means The second stem cells located in the lowermost layer of the first stem cells separated by layered centrifugation are extracted, and have a bottom surface with a predetermined volume and a predetermined area and are larger than the first culture vessel. The second stem cells and a new culture solution are injected into a second culture vessel having a large volume and a bottom area to fix the second stem cells on the bottom surface of the second culture vessel, and the second stem cells are in the second culture. After fixing on the bottom surface of the container, a new culture solution is poured into the second culture vessel while discharging the culture solution in the second culture vessel to proliferate the second stem cells, and the second stem cells expand. And a second culturing means for extracting the second stem cells from the second culture container after the total planar area of the second stem cells reaches a second target ratio with respect to the bottom area of the second culture container. Single species characterized by being made from Cell.   The single-type stem cell is produced by performing the first culturing means once and performing the second culturing means at least twice. In the second culturing means from the second time, a predetermined volume and a predetermined area are obtained. A third culture vessel having a bottom and a larger capacity and a larger bottom area than the second culture vessel is used, and the second stem cells extracted from the second culture vessel by the second culture means for the first time are used. The single-type stem cell according to claim 1, wherein the single stem cell is allowed to settle in the third culture container and the second stem cell is proliferated in the third culture container.   In the first culturing means, 2 to 3 cc of raw bone marrow fluid is collected from the donor, and the 2 to 3 cc of raw bone marrow fluid is injected into a separation container extending in the vertical direction. The raw bone marrow fluid is allowed to stand statically for a predetermined time in the separation container to be separated into layers in the vertical direction, and the intermediate layer located in the intermediate layer of the raw material bone marrow fluid separated into layers in the separation container The single-species stem cell according to claim 1 or 2, wherein the layered bone marrow fluid is extracted by a syringe or a pipette.   The first culture vessel has a capacity of about 20-30 cc. In the first culture means, the intermediate layer bone marrow fluid and the culture solution are injected into the first culture vessel, and then the first culture vessel is used. While the first culture vessel is statically left at a temperature substantially the same as the body temperature for 12 to 24 hours while being inclined at a predetermined angle, the first culture container is about 1 to 2 hours apart during the 12 to 24 hours. When the deformation of the first stem cell in the culture vessel from the initial planar shape is observed and the first stem cell is deformed from the initial planar shape to a predetermined planar shape, the first stem cell is fixed on the bottom surface of the first culture vessel. The single-type stem cell according to any one of claims 1 to 3, which is judged to have been obtained.   The initial planar shape of the first stem cell is a substantially circular shape, and the planar shape after deformation of the first stem cell is a flat shape in which the first stem cell extends in an indefinite shape in one direction with the substantially circular shape as a nucleus. The single species according to claim 4, wherein the first culturing means judges that the first stem cells have settled on the bottom surface of the first culture container when the first stem cells are deformed into the irregular flat shape. Stem cells.   In the first culturing means, after the first stem cells have settled on the bottom surface of the first culture vessel and a new culture solution is injected into the first culture vessel while discharging the culture solution in the first culture vessel. In the state where the first culture vessel is inclined at a predetermined angle, the first culture vessel is statically left at a temperature substantially the same as the body temperature for 36 to 48 hours, and about 1 to 3 for the 36 to 48 hours. The single-species stem cell according to any one of claims 1 to 5, wherein a total planar area of the first stem cell fixed on the bottom surface of the first culture container with respect to the bottom surface area of the first culture container is observed at intervals of 2 hours.   The single-type stem cell according to claim 6, wherein the first target ratio of the total planar area of the first stem cell to the bottom area of the first culture container is 70 to 80%.   In the second culturing means, the first stem cells are injected into a separation container, the separation container is placed in a centrifuge, the first stem cells are centrifuged, and the first stem cells are centrifuged in layers in the separation container. The single-type stem cell according to any one of claims 1 to 7, wherein the second stem cell located in the lowermost layer of the one stem cell is extracted by a syringe or a pipette.   The volume of the second culture vessel is about 40 to 60 cc, the volume of the third culture vessel is about 70 to 80 cc, and the second culture means includes the second and third culture vessels with the second and third culture vessels. 2 After injecting the stem cells and a new culture solution, the second and third culture vessels are tilted at a predetermined angle, and the second and third culture vessels are at a temperature substantially the same as the body temperature for 36 to 48 hours. While standing statically, the deformation of the second stem cells in the second and third culture vessels from the initial planar shape is observed at intervals of about 1-2 hours during the 36-48 hours, and the second stem cells The single-species stem cell according to any one of claims 2 to 8, wherein the second stem cell is determined to have settled on the bottom surfaces of the second and third culture vessels when the cell is deformed from an initial planar shape to a predetermined planar shape. .   The initial planar shape of the second stem cell is a substantially circular shape, and the planar shape after deformation of the second stem cell is a flat shape in which the second stem cell extends in one direction indefinitely with the substantially circular shape as a nucleus. The second culturing means determines that the second stem cells have settled on the bottom surfaces of the second and third culture vessels when the second stem cells are deformed into the irregular flat shape. Single-species stem cells.   In the second culture means, the second stem cells are fixed on the bottom surfaces of the second and third culture vessels, and a new culture solution is discharged while discharging the culture solution in the second and third culture vessels. And injecting into the third culture container, the second and third culture containers are statically kept at a temperature substantially equal to the body temperature for 36 to 48 hours with the second and third culture containers inclined at a predetermined angle. The total of the second stem cells fixed on the bottom surfaces of the second and third culture vessels at an interval of about 1 to 2 hours during the 36-48 hours with respect to the bottom area of the second and third culture vessels. The single-species stem cell according to any one of claims 2 to 10, wherein a planar area is observed.   The single-species stem cell according to any one of claims 2 to 11, wherein a second target ratio of a total planar area of the second stem cells to a bottom area of the second and third culture vessels is 88 to 92%. The single-type stem cell according to any one of claims 1 to 12, wherein the first and second stem cells are mesenchymal stem cells.

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