JP5706081B2 - Implantable biomaterial collection device - Google Patents

Description

  The present invention relates to an implantable in-vivo functional substance collection device capable of collecting a substance present in a very small amount in a living body tissue, for example, a functional cell such as a bone marrow stem cell, with low invasiveness and high efficiency.

  Conventionally, as a method of collecting highly functional cells from a living body, for example, for hematopoietic medullary stem cells, a needle is directly inserted into the bone marrow of a long bone or pelvic bone, and the bone marrow fluid is collected and centrifuged, followed by For peripheral blood stem cells, G-CSF is administered in advance to mobilize hematopoietic stem cells into peripheral blood, and then peripheral blood is collected to isolate hematopoietic stem cells. In the case of mesenchymal stem cells, directly culture bone marrow fluid and collect adherent proliferating cells, surgically collect peripheral tissues such as adipose tissue, and isolate and culture mesenchymal stem cells present in them Was known.

Transplantation of hematopoietic stem cells from the peripheral blood.J Cell Mol Med. 2005; 9: 37-50 Role of mesenchymal stem cells in regenerate medicine: application to bone and cartilage repair.Expert Opin Biol Ther. 2008; 8: 255-268

In the above prior art, the collection of bone marrow fluid from the bone marrow is highly invasive, the burden on the subject is large, and there is a risk of osteomyelitis, so very strict medical management is necessary, and frequent enforcement is difficult . Surgical removal of peripheral tissues carries similar risks. Hematopoietic stem cell mobilization by G-CSF is difficult to perform frequently because it is economically expensive and takes a long time to treat.
Therefore, as a method for collecting functional cells in vivo more efficiently and safely, in the present invention, a technique for collecting and collecting in vivo functional cells with high efficiency by inserting a minimally invasive container into the living body is developed. did.

The configuration of the present invention is as described below.
(1) An in-vivo implantable substance collecting device for recovering a target substance existing in a living tissue, a flat container having a central hollow part between a side wall part, an upper wall part, and a lower wall part. A substance collecting device comprising a flat container in which one or more openings are provided in the upper wall part and / or the lower wall part, and the collected target substance can be held in the central hollow part.
(2) The upper wall portion and / or the lower wall portion of the flat container have any one of a flat surface, a convex curved surface, a concave curved surface, and a shape in which a central portion is recessed in a concave shape. Material collection device.
(3) The planar shape of the flat container has a shape selected from a polygon, a substantially polygon, a circle, a substantially circle, an ellipse, a substantially ellipse, a trapezoid and a substantially trapezoid (1) or (2) The substance collection device described in 1.
(4) The substance collection according to any one of (1) to (3) above, wherein joint portions between the upper wall portion and the side wall portion of the flat container and between the lower wall portion and the side wall portion are curved. device.
(5) The substance collecting device according to any one of (1) to (4), wherein the flat container is configured by combining a component including an upper wall portion and a component including a lower wall portion.
(6) The substance collection device according to any one of (1) to (5), wherein the flat container is made of a silicone resin material.
(7) The substance collection device according to any one of (1) to (6), wherein the flat container is inserted between an epithelium and a dermis of a living body.
(8) The substance collection device according to any one of (1) to (7), wherein the target substance is a bone marrow stem cell.
(9) The substance collection device according to (8) above, wherein the bone marrow stem cells are pluripotent stem cells.
(10) The above (8) or (9), wherein the bone marrow stem cells are mobilized by a substance or composition containing at least one selected from the following groups (a) to (r) inserted into the flat container: ) Substance collection device:
(A) HMGB1 (High mobility group box 1) protein (b) Cells secreting HMGB1 protein (c) Vector inserted with DNA encoding HMGB1 protein (d) HMGB2 (High mobility group box 2) protein (e) Cells that secrete HMGB2 protein (f) Vector inserted with DNA encoding HMGB2 protein (g) HMGB3 (High mobility group box 3) protein (h) Cells that secrete HMGB3 protein (i) DNA encoding HMGB3 protein (J) S100A8 protein (k) S100A8 protein secreting cell (l) S100A8 protein DNA vector (m) S100A9 protein (n) S100A9 protein secreting cell (o) Vector inserted with DNA encoding S100A9 protein (p) Hyaluronic acid (q) Cell or tissue extract (r) Cell or Heparin-binding fraction of the extract of woven.
(11) The substance or composition is in at least one medium selected from the group consisting of a polymer or copolymer of polyethylene glycol (PEG), cyclodextrin, gelatin, albumin, chitosan, sodium carboxymethylcellulose, and physiological saline. The substance collection device according to (10), which is present in
(12) A kit comprising the substance collection device according to (8) or (9) above, further comprising a substance or composition comprising at least one selected from the following groups (a) to (r): :
(A) HMGB1 protein (b) Cell that secretes HMGB1 protein (c) Vector inserted with DNA encoding HMGB1 protein (d) HMGB2 protein (e) Cell that secretes HMGB2 protein (f) Encodes HMGB2 protein Vector inserted with DNA (g) HMGB3 protein (h) Cells secreting HMGB3 protein (i) Vector inserted with DNA encoding HMGB3 protein (j) S100A8 protein (k) Cells secreting S100A8 protein (l ) Vector inserted with DNA encoding S100A8 protein (m) S100A9 protein (n) Cell secreting S100A9 protein (o) Vector inserted with DNA encoding S100A9 protein (p) Hyaluronic acid (q) Cell or Tissue extract (r) Heparin-binding fraction of cell or tissue extract.
(13) The above (12), further comprising at least one medium selected from the group consisting of a polymer or copolymer of polyethylene glycol (PEG), cyclodextrin, gelatin, albumin, chitosan, sodium carboxymethylcellulose, and physiological saline. A kit comprising the substance collection device according to 1.
(14) A method for producing an implantable substance collection device for recovering bone marrow stem cells present in tissue in a living body,
A flat container having a central hollow part between a side wall part, an upper wall part, and a lower wall part, wherein one or more openings are provided in the upper wall part and / or the lower wall part, and recovered bone marrow stem cells Prepare a flat container that can be held in the central hollow portion,
A production method comprising a step of inserting a substance or composition containing at least one selected from the following groups (a) to (r) into the flat container:
(A) HMGB1 protein (b) Cell that secretes HMGB1 protein (c) Vector inserted with DNA encoding HMGB1 protein (d) HMGB2 protein (e) Cell that secretes HMGB2 protein (f) Encodes HMGB2 protein Vector inserted with DNA (g) HMGB3 protein (h) Cells secreting HMGB3 protein (i) Vector inserted with DNA encoding HMGB3 protein (j) S100A8 protein (k) Cells secreting S100A8 protein (l ) Vector inserted with DNA encoding S100A8 protein (m) S100A9 protein (n) Cell secreting S100A9 protein (o) Vector inserted with DNA encoding S100A9 protein (p) Hyaluronic acid (q) Cell or Tissue extract (r) Heparin-binding fraction of cell or tissue extract.
(15) The substance or composition is in at least one medium selected from the group consisting of a polymer or copolymer of polyethylene glycol (PEG), cyclodextrin, gelatin, albumin, chitosan, sodium carboxymethylcellulose, and physiological saline. The production method according to (14), which exists in

The substance collection device of the present invention can be safely implanted in a living body by a relatively simple operation, and can be taken out after a certain period of time to efficiently recover the target substance. There are few risks and there is little economic burden.
Specifically, a conventional cylindrical cylindrical container made of silicon is easy to manufacture, but the number of cells that can be collected and stored per container is small, and it is difficult to recover the cells stored in the container. The recovery rate was also limited. In the present invention, the number of holes that pass when cells are accommodated, the degree of freedom of the position is increased, and a container having a flat shape, preferably a disc shape, is adopted, so that a layered structure such as between the epithelium and the dermis is adopted. It can be easily inserted into the gaps between living tissues, has a low sense of foreign matter during mounting, is easily pulled out from the living body, and is less invasive. Furthermore, since the volume of the hollow portion is large, the recovery efficiency is excellent.
The container of the present invention that can be divided into halves in an assembling type enhances work efficiency while facilitating work when inserting bone marrow pluripotent cell mobilization factor into the container or recovering cells contained in the container Is possible.

  By utilizing the substance collection device of the present invention, pluripotent stem cells useful for regenerative medicine can be collected from a treatment target with minimal invasiveness and high efficiency, so that it has been difficult to treat until now. Can also provide effective regenerative medicine. In that case, when a bone marrow stem cell mobilization factor including cells classified as bone marrow pluripotent stem cells, bone marrow mesenchymal stem cells, or bone marrow stromal stem cells is inserted into the substance collection device of the present invention, peripheral blood flow A small amount of bone marrow stem cells circulating inside can be efficiently collected. The collected cells can be directly administered to a damaged tissue requiring regenerative medicine for treatment. Bone marrow stem cells are known to regenerate a variety of cells such as bone, cartilage, and fat as well as fibroblasts, nerves, and epithelia as pluripotent stem cells. Diseases such as refractory skin ulcers such as skin ulcers, anti-aging medicine), osteochondral diseases (refractory fractures, etc.), cranial nerve diseases (cerebral infarction, etc.) can be treated.

It is an example of the container which has an opening part in the upper wall part of the implantable substance collection device of this invention. It is an example of the container which has an opening part in the upper wall part and lower wall part of the implantable substance collection device of this invention. It is sectional drawing of components containing the upper wall part in which the opening part of the example of FIG.1 and FIG.2 was provided. FIG. 4 is a cross-sectional view of a component that is not provided with an opening, and that can constitute the container of FIG. 1 in combination with the component of FIG. FIG. 4 is a cross-sectional view of a part provided with an opening, which can constitute the container of FIG. 2 in combination with the part of FIG. 3. It is the shape at the time of insertion in the body of the substance collection device of this invention shown in FIG. 1 and 2 and the conventional tube-type substance collection device. It is the shape at the time of cell collection of the device shown in FIG. 6A. It is a photograph which shows the GFP positive cell collect | recovered in the substance collection device of this invention with which the center hollow part was filled with HMGB-1 and hyaluronic acid. It is a photograph which shows the GFP positive cell collect | recovered in the substance collection device of this invention with which the center hollow part was filled with hyaluronic acid. It is a photograph 24 hours after the start of culture of TECs (cells accumulated in a container). The photo on the left shows a bright field image of fibroblast-like cells and epithelial cell-like cells that are proliferating attached to a cultured plastic petri dish, and the photo on the right shows a GFP fluorescence image in the dark field. It is the photograph which evaluated the osteoblast differentiation ability of TECs collect | recovered from the substance collection device of this invention. As a result of culturing the cells collected from the container in an osteoblast differentiation induction medium, differentiation into alizarin red staining positive osteoblasts was confirmed in about 2 weeks. It is the photograph which evaluated the adipocyte differentiation ability of TECs collect | recovered from the substance collection device of this invention. As a result of culturing the cells collected from the container in the adipocyte differentiation-inducing medium, differentiation into oil red staining positive adipocytes was confirmed in about 2 weeks. It is the photograph which evaluated the epidermal cell differentiation ability of TECs collect | recovered from the substance collection device of this invention. As a result of culturing the cells collected from the container in the epidermal cell differentiation-inducing medium, differentiation into keratinocytes expressing epidermal cell-specific keratin 5 was confirmed in about 2 weeks. It is the figure which examined the expression in TECs of PDGFR (alpha) and CD44. It was confirmed that PDGFRα-positive and CD44-positive cells were collected in the substance collection device of the present invention. It is the photograph which detected the HMGB family in the newborn mouse skin extract using Western blot method. It is a figure of HMGB1 expression vector. It is a photograph which shows the result of Western blotting of the purified recombinant Flag tag-HMGB family fusion protein expressed in HEK293 cells. It is a figure which shows the bone marrow mesenchymal stem cell migration activity of recombinant HMGB1, HMGB2, and HMGB3 using the Boyden chamber. All the recombinant proteins showed migration activity compared to the control group. It is a figure which shows the treatment result by the HMGB family in a mouse | mouth skin ulcer treatment model. All of HMGB1, HMGB2, and HMGB3 showed a significant effect of reducing the ulcer area compared to the control group. It is the photograph which confirmed the activity which human HMGB1 and a human skin extract migrate a human bone marrow origin mesenchymal stem cell using the Boyden chamber. It is the photograph which refine | purified the bone marrow mesenchymal stem cell induction active substance in a mouse heart, a mouse brain, and a mouse skin extract with a heparin column, and confirmed the activity using a Boyden chamber. It is the photograph which confirmed the bone marrow mesenchymal stem cell migration activity of cultured cell line HEK293 and HeLa extract using Boyden chamber method. All cultured cell lines showed human bone marrow mesenchymal stem cell migration activity. A is a photograph in which a mouse was fixed to a stereotaxic apparatus, a midline incision was made in the head with a scalpel, and burr was made using a drill. B is a photograph in which a negative pressure is applied to the brain using a syringe to partially suck the brain tissue. C is a photograph after injecting 5 μl of the purified heparin column fraction of skin extract dissolved in fibrin glue preparation (fibrinogen) and then injecting 5 μl of fibrin glue preparation (thrombin). D and E are photographs 2 weeks after the brain injury model treatment. Compared to control D, accumulation of GFP positive cells was observed in E of the treatment group using the purified fraction of the skin extract heparin column. F and G are photographs 6 weeks after the brain injury model treatment. Compared with control F, accumulation of GFP positive cells was observed in G of the treatment group using the purified fraction of the skin extract heparin column. It is a photograph which shows the bone marrow origin mesenchymal stem cell migration activity activity measurement result of the skin extract using Boyden chamber. Bone marrow mesenchymal stem cells that have passed through the micropores of the polycarbonate membrane membrane with a micropore of 8 μm from the upper tank of the Boyden chamber, migrate to the lower tank side containing the skin extract, and adhere to the submembrane tank side Is an image dyed with a blue pigment. The lower layer was filled with skin extracts collected from 2-day-old mice and 6-week-old mice. It is the figure which confirmed the presence of the protein of S100A8 and S100A9 in a skin extract by Western blot method. It is a photograph which shows the result of having eluted heparin binding protein in a skin extract from a heparin affinity column with a NaCl concentration gradient. Proteins of each fraction were fractionated by SDS-PAGE and detected by silver staining. It is a photograph which shows the bone marrow origin mesenchymal stem cell migration activity activity measurement result of the skin extract using Boyden chamber. Bone marrow mesenchymal stem cells that migrate from the upper tank of the Boyden chamber through the micropores on the membrane to the heparin-bound fractions of the skin extract (lower tank side) and adhere to the submembrane tank side Is an image dyed with a blue pigment. It is a photograph showing the results of detecting the presence of S100A8 and S100A9 proteins in each heparin-bound fraction of the skin extract using Western blotting. It is a figure of S100A8, S100A9 expression vector. It is a photograph which shows the bone marrow origin mesenchymal stem cell migration activity activity measurement result of the skin extract using Boyden chamber. Bone marrow mesenchyme that has migrated from the upper tank of the Boyden chamber to the lower layer containing the recombinant GST-S100A8, GST-S100A9, and skin extract through the fine holes on the membrane, and adhered to the submembrane tank It is the image which dye | stained the stem cell with the blue pigment | dye. A is the figure which administered GST-S100A8 and GST-S100A9 from the tail vein of the mouse | mouth, and performed FACS of CD44, PDGFR (alpha), and PDGFR (beta) with respect to the fraction of the CD45 negative cell in a peripheral blood 12 hours afterward. B quantitatively graphed the appearance of CD45 negative CD44 positive PDGFRα positive cells or CD45 negative CD44 positive PDGFRβ positive cells in peripheral blood 12 hours after administration of GST-S100A8 and GST-S100A9 based on the results of FACS. FIG. It is a figure which shows the therapeutic effect of the skin ulcer by the cell mobilized in the device by the skin extract and the peripheral blood extract. It is the figure which detected the bone marrow stem cell mobilized in the device with the heparin affinity column binding component of the peripheral blood extract with the fluorescence microscope. It is the graph which detected the bone marrow stem cell mobilized in the device with the heparin affinity column binding component of the peripheral blood extract with a fluorescence microscope, and quantified the number of cells using image processing software. It is the figure which detected the bone marrow origin cell (GFP positive cell) mobilized in the device using each of S100A8, HMGB1, HMGB2, and HMGB3 with the fluorescence microscope (A: S100A81, B: HMGB1, C: HMGB2, D: HMGB3, E: negative control). It is a figure which shows the therapeutic effect with respect to the skin ulcer produced in BALB / cAJcl-nu / nu by the bone marrow origin cell mobilized in the device using each of S100A8, HMGB1, and HMGB2. It is a figure of HMGB1 expression vector. It is a figure which administers a skin extract (SE) from a mouse tail vein, and collects peripheral blood. It is the figure which labeled the mouse | mouth peripheral blood mononuclear cell fraction 12 hours after administering skin extract (SE) with the anti-mouse PDGFR (alpha) antibody and the anti-mouse CD44 antibody, and fractionated by flow cytometry. The upper three are the negative control PBS administration group (n = 3) and the lower three are the skin extract (SE) administration group (n = 3). The vertical axis represents the expression level of CD44, and the horizontal axis represents the expression level of PDGFRα. The part surrounded by the line shows the CD44-positive and PDGFRα-positive cell group, which is increased in the skin extract administration group (SE) compared to the PBS group. It is a figure which administers HMGB1 and collects peripheral blood from a mouse tail vein. It is the figure which carried out the fluorescence labeling of the mouse peripheral blood mononuclear cell fraction 12 hours after HMGB1 administration with the anti-mouse PDGFR (alpha) antibody and the anti-mouse CD44 antibody, and fractionated by flow cytometry. The left is a negative control PBS-administered mouse, and the right is a HMGB1-administered mouse. The vertical axis represents the expression level of CD44, and the horizontal axis represents the expression level of PDGFRα. The part surrounded by a line shows a CD44-positive and PDGFRα-positive cell group, which is increased in HMGB1-administered mice compared to PBS-administered mice. A is a figure which shows the result of the flow cytometry showing the existence frequency of the cell which has CD44 and PDGFR (alpha). Cell groups of both PDGFRα-positive and CD44-positive cells and PDGFRα-positive and CD44-negative cells in peripheral blood are increased by HMGB1 administration. B shows PDGFRα-positive and CD44-positive cells, and C shows PDGFRα-positive and CD44-negative cells in the PBS administration group and the HMGB1 administration group, respectively. In any cell group, there is a statistically significant increase in the HMGB1 administration group.

  The flat container provided in the implantable substance collecting device of the present invention is a flat container having a central hollow portion between a side wall portion, an upper wall portion, and a lower wall portion, and the upper wall portion and / or the lower wall. The part is provided with one or more openings, and has a shape capable of collecting the target substance existing in the in vivo tissue and holding it in the central hollow part.

The size of the flat container may be any size that can be inserted subcutaneously. For example, in the case of a circular planar shape, the diameter may be 5 to 20 mm, the thickness is 2 to 5 mm, and the volume is 40 to 1570 mm ^3. It is not something.
The thickness of the flat container side wall is not particularly limited as long as it has a thickness necessary for maintaining sufficient strength, and may be, for example, about 0.3 to 0.9 mm. .

The volume of the central hollow part may be sufficient to hold the collected target substance and, if necessary, may be sufficient to install a cell mobilization factor. Examples thereof include 20 to 1000 mm ^3, but are not limited thereto.
The shape of the opening may be any shape as long as the target substance flows into the central hollow portion and can be collected, and an arbitrary number, size, and arrangement can be selected. For example, you may be comprised from several circular holes.

  The upper wall portion and / or the lower wall portion of the flat container has any one of a flat surface, a convex curved surface, a concave curved surface, and a shape in which a central portion is recessed in a concave shape. When placed in a living body, it does not move in the body without causing inflammation, and since the embedded device has less foreign material feeling and is easy and inexpensive to manufacture, it is preferably centered in a flat, concave curved surface or concave shape. It is a shape with a recessed part.

  The flat shape of the flat container has a shape selected from a polygon, a substantially polygon, a circle, a substantially circle, an ellipse, a substantially ellipse, a trapezoid and a substantially trapezoid. In order to be held in the living body, a shape including a curve with a large curvature radius (small curvature) is preferable, and an appropriate shape can be selected according to the insertion position in the body. For the same reason, it is preferable that the joint portion between the upper wall portion and the side wall portion and between the lower wall portion and the side wall portion of the flat container exhibits a curved surface having a large curvature radius.

  The flat container may be integrally formed, or may be configured by combining a part including an upper wall part and a part including a lower wall part. In that case, the number, size, and shape of the opening can be selected as appropriate according to the combination, and a substance that is difficult to insert from the opening can be installed and assembled in the central hollow portion.

  The material of the flat container may be any material as long as it is biocompatible and does not decompose for a certain period in the body. For example, polydimethylsiloxane (silicone resin), polyester, polyamide, fluorocarbon, polyethylene, polypropylene (PP), polycarbonate, polyurethane, and polystyrene, vinyl chloride, polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), There are various materials such as carbon materials, carbon composite materials, ceramics, glass, and metals. Among these materials, when silicone resin is used as a material, it is inexpensive, less invasive, and less uncomfortable during use. Furthermore, it is not preferable that the bone marrow stem cells contained in the peripheral blood flow develop and organize the cells collected in the flat container of the present invention. Since silicone resin has a hydrophobic surface and low affinity for living tissue, cells collected in a flat container develop and do not organize inside and outside the container, so there is no difficulty in collection.

  The flat container may be inserted into any place in the living body where the target substance can be collected, and preferably between the epithelium and the dermis. When a flat container is inserted between the epithelium and the dermis, the opening provided in the upper wall and / or lower wall directly faces the epithelium and / or dermis, so that it is included in the peripheral blood flow through the capillaries The substance is easy to flow into the container and the collection efficiency is high.

  Examples of the target substance of the present invention include bone marrow stem cells. Bone marrow stem cells targeted in the present invention are cells other than hematopoietic stem cells and leukocytes, erythrocytes, and platelets derived therefrom, and are classified as bone marrow pluripotent stem cells, bone marrow mesenchymal stem cells, or bone marrow stromal stem cells. A small amount of bone marrow stem cells circulating in the peripheral blood stream can be efficiently collected by inserting a certain bone marrow stem cell mobilization factor. Bone marrow stem cells are contained in minute amounts in peripheral blood, and it is known that pluripotent stem cells regenerate a variety of cells such as fibroblasts, nerves, and epithelia in addition to bone, cartilage, and fat. Accordingly, the collected bone marrow stem cells can be directly administered to damaged tissues that require regenerative medicine.

  Preferred bone marrow stem cells for the purpose of the present invention are derived from bone marrow, collected from peripheral blood, and can be cultured and propagated as adherent cells (made of plastic or glass) on a culture dish. It is a mesenchymal stem cell having the ability to differentiate into leaf tissue, or a pluripotent stem cell having the ability to differentiate into skeletal muscle, cardiac muscle, and further nerve tissue and epithelial tissue. The bone marrow stem cells can be differentiated into epithelial tissues such as keratinocytes that make up the skin and nervous system tissues that make up the brain by administering cells cultured and expanded in a culture dish to the damaged part of the living body. It also has the characteristic of having.

  Bone marrow stem cells for the purpose of the present invention are preferably osteoblasts (identifiable by calcium deposition when differentiation is induced), chondrocytes (identifiable by positive Alcian blue staining, positive Safranin-O staining, etc.) Have the ability to differentiate into mesenchymal cells such as adipocytes (positive for Zudan III staining), fibroblasts, smooth muscle cells, stromal cells, tendon cells, and also neurons, epithelial cells, and vascular endothelial cells . However, the differentiated cells are not limited to the above-mentioned cells, and also include parenchymal organ cells such as liver, kidney, and pancreas. However, the differentiated cells are not limited to the above cells.

  Bone marrow stem cells targeted by the present invention may contain tissue progenitor cells that are undifferentiated cells having unidirectional differentiation ability to specific tissue cells other than the blood system. Tissue progenitor cells include the above-described undifferentiated cells having the ability to differentiate into mesenchymal tissue, epithelial tissue, neural tissue, parenchymal organ, and vascular endothelium.

  Examples of bone marrow stem cells targeted by the present invention include, but are not limited to, bone marrow stem cells that are positive for at least one of cell surface markers CD44, PDGFRα, and PDGFRβ.

When implanting the in vivo implantable substance collecting device of the present invention under the skin, for example, after performing general (or local) anesthesia, cut a few mm of skin with a scalpel and use a metal rod with a round tip (such as a mosquito pair). Then, a necessary space is created in the subcutaneous adipose tissue by blunt dissection, and the substance collection device of the present invention is embedded in the space, and finally the incised skin is sutured or stapled.
Other methods for placing the device under the skin include inserting the device folded along the guide after the incision, removing the guide, and then injecting the drug solution into the hollow center of the flat container to enlarge it. A method such as detention is conceivable.

  Examples of the target for embedding the container include humans and non-human animals. For example, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, etc. can be exemplified, but humans are preferred.

One or more substances or mixtures described in any of (a) to (r) below are inserted into the central hollow part of the in vivo implantable substance collecting device of the present invention, and bone marrow stem cells are mobilized in a flat container. can do.
(A) HMGB1 protein (b) Cell that secretes HMGB1 protein (c) Vector inserted with DNA encoding HMGB1 protein (d) HMGB2 protein (e) Cell that secretes HMGB2 protein (f) Encodes HMGB2 protein Vector inserted with DNA (g) HMGB3 protein (h) Cells secreting HMGB3 protein (i) Vector inserted with DNA encoding HMGB3 protein (j) S100A8 protein (k) Cells secreting S100A8 protein (l ) Vector inserted with DNA encoding S100A8 protein (m) S100A9 protein (n) Cell secreting S100A9 protein (o) Vector inserted with DNA encoding S100A9 protein (p) Hyaluronic acid (q) Cell or Tissue extract (r) Heparin-binding fraction of cell or tissue extract

  In addition, a partial peptide of the HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein, which has a bone marrow stem cell inducing activity, a cell that secretes the partial peptide, or a vector into which a DNA encoding the partial peptide is inserted Can also be used.

  Examples of the HMGB1 protein in the present invention include, but are not limited to, proteins containing the amino acid sequence set forth in SEQ ID NO: 1, 3 or 5. The HMGB1 protein of the present invention also includes a protein functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 3 or 5. Examples of such proteins include 1) an amino acid sequence in which one or a plurality of amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in SEQ ID NO: 1, 3, or 5. An isolated protein functionally equivalent to the protein comprising the amino acid sequence of No. 1, 3 or 5, and 2) a DNA comprising the base sequence of SEQ ID No .: 2, 4 or 6 and a stringent And an isolated protein that is functionally equivalent to a protein that is encoded by DNA that hybridizes under various conditions and includes the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5.

  Examples of the HMGB2 protein in the present invention include, but are not limited to, a protein comprising the amino acid sequence set forth in SEQ ID NO: 7, 9, or 11. The HMGB2 protein of the present invention also includes proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 7, 9 or 11. Examples of such a protein include 1) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in SEQ ID NO: 7, 9 or 11. An isolated protein that is functionally equivalent to the protein comprising the amino acid sequence of No. 7, 9 or 11, and 2) a DNA comprising the base sequence of SEQ ID No .: 8, 10 or 12, and a stringent And an isolated protein that is functionally equivalent to a protein that is encoded by a DNA that hybridizes under various conditions and includes the amino acid sequence set forth in SEQ ID NO: 7, 9, or 11.

  Examples of the HMGB3 protein in the present invention include, but are not limited to, a protein containing the amino acid sequence set forth in SEQ ID NO: 13 or 15. The HMGB3 protein of the present invention includes a protein functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 13 or 15. Examples of such a protein include 1) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in SEQ ID NO: 13 or 15. An isolated protein functionally equivalent to the protein comprising the amino acid sequence described in 13 or 15, and 2) hybridizing under stringent conditions with the DNA comprising the base sequence described in SEQ ID NO: 14 or 16 And an isolated protein functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NO: 13 or 15.

  Examples of the S100A8 protein in the present invention include, but are not limited to, a protein containing the amino acid sequence set forth in SEQ ID NO: 17, 19, or 21. The S100A8 protein of the present invention also includes proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 17, 19, or 21. Examples of such a protein include 1) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in SEQ ID NO: 17, 19, or 21. An isolated protein that is functionally equivalent to the protein comprising the amino acid sequence of No. 17, 19, or 21; and 2) a DNA and stringent comprising the base sequence of SEQ ID No. 18, 20, or 22. And an isolated protein that is functionally equivalent to a protein that is encoded by DNA that hybridizes under various conditions and includes the amino acid sequence set forth in SEQ ID NO: 18, 20, or 22.

  Examples of the S100A9 protein in the present invention include, but are not limited to, a protein comprising the amino acid sequence set forth in SEQ ID NO: 23, 25, or 27. The S100A9 protein of the present invention includes proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 23, 25 or 27. Examples of such a protein include 1) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in SEQ ID NO: 23, 25, or 27. An isolated protein that is functionally equivalent to the protein comprising the amino acid sequence of No. 23, 25 or 27, and 2) a DNA and stringent comprising the base sequence of SEQ ID No. 24, 26 or 28 And an isolated protein that is functionally equivalent to a protein encoded by a DNA that hybridizes under various conditions and comprising the amino acid sequence set forth in SEQ ID NO: 23, 25, or 27.

  An isolated protein functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 , SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. The protein may be a homolog or a paralog of the amino acid sequence. A protein functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 is obtained by those skilled in the art. It can be isolated by a known method.

  As a protein functionally equivalent to the protein comprising the amino acid sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, bone marrow stem cells And a protein having an inducing activity.

  In the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, one or more amino acids are substituted, deleted, inserted, And / or a protein comprising the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. Equivalent proteins include naturally occurring proteins. In general, eukaryotic genes have a polymorphism, as is known for interferon genes and the like. One or more amino acids may be substituted, deleted, inserted and / or added due to a change in the base sequence caused by this polymorphism. Thus, it is a naturally occurring protein and 1 in the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27 Or a plurality of amino acids have an amino acid sequence substituted, deleted, inserted and / or added, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, Proteins functionally equivalent to the protein consisting of the amino acid sequence described in 23, 25 or 27 are included in the HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein.

  Moreover, as long as it is a protein functionally equivalent to the protein which consists of an amino acid sequence as described in sequence number: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27 Artificially produced mutant proteins are also included in the present invention. As a method for randomly mutating a given base sequence, for example, substitution of base pairs by nitrite treatment of DNA is known.

  The number of amino acids to be modified is typically within 50 amino acids, preferably within 30 amino acids, and more preferably within 5 amino acids (eg, 1 amino acid).

  When an amino acid is artificially substituted, it is considered that the activity of the original protein is easily maintained if the amino acid is substituted with an amino acid having similar properties. The protein of the present invention is a protein in which a conservative substitution is added in the above amino acid substitution, and SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, A protein functionally equivalent to a protein comprising the amino acid sequence described in 25 or 27 is included. Conservative substitution is considered to be important, for example, when substituting amino acids of domains important for protein activity. Such amino acid conservative substitutions are well known to those skilled in the art.

Examples of amino acid groups corresponding to conservative substitutions include basic amino acids (eg, lysine, arginine, histidine), acidic amino acids (eg, aspartic acid, glutamic acid), uncharged polar amino acids (eg, glycine, asparagine, glutamine, serine, Threonine, tyrosine, cysteine), nonpolar amino acids (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched amino acids (eg threonine, valine, isoleucine), and aromatic amino acids (eg tyrosine, phenylalanine) , Tryptophan, histidine) and the like.
It is also conceivable to increase the activity of the protein by non-conservative substitution (for example, including a permanently activated protein).

  In addition, a protein functionally equivalent to the protein comprising the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 is obtained. Examples of the method include a method using hybridization. That is, the HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein according to the present invention as shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28 Using the encoded DNA or a fragment thereof as a probe, DNA that can hybridize with this is isolated. If hybridization is carried out under stringent conditions, DNA with high homology is selected as the base sequence, and the resulting isolated protein is functionally equivalent to the HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein. The possibility that a new protein is included increases. A highly homologous base sequence can show, for example, 70% or more, desirably 90% or more identity.

The stringent conditions can specifically include conditions such as 6 × SSC, 40% formamide, hybridization at 25 ° C., and washing at 1 × SSC, 55 ° C. Stringency depends on conditions such as salt concentration, formamide concentration, or temperature, but those skilled in the art will readily set these conditions to obtain the required stringency.
By utilizing hybridization, for example, HMGB1 other than a protein containing the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 DNA encoding a homologue of HMGB2, HMGB3, S100A8 or S100A9 protein can be isolated.

  A protein functionally equivalent to a protein comprising the amino acid sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 is usually a sequence. No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, and has high homology. High homology refers to sequence identity of at least 30% or more, preferably 50% or more, more preferably 80% or more (eg, 95% or more). The identity of base sequences and amino acid sequences can be performed using a homology search site using the Internet [for example, homology such as FASTA, BLAST, PSI-BLAST, and SSEARCH in Japan DNA Data Bank (DDBJ). Search is available. In addition, a search using BLAST can be performed in the National Center for Biotechnology Information (NCBI).

  For example, the amino acid sequence identity in Advanced BLAST 2.1 is calculated using blastp as the program, Expect value is 10, Filter is all OFF, BLOSUM62 is used for Matrix, Gap existence cost, Per residue gap cost, and Lambda ratio Can be set to 11, 1, 0.85 (default value), respectively, and a search can be performed to obtain an identity value (%).

  The protein according to the present invention or a functionally equivalent protein thereof is a protein to which various modifications such as physiological modifications such as sugar chains, labels such as fluorescence and radioactive substances, or fusion with other proteins are added. be able to. In the gene recombinants described below, there may be differences in modification with sugar chains depending on the host to be expressed. However, even if there is a difference in the modification of the sugar chain, any of the HMGB1, HMGB2, HMGB3, S100A8 or S100A9 proteins disclosed in the present specification can be used as long as they exhibit similar properties. HMGB2, HMGB3, S100A8 or S100A9 protein, or a functionally equivalent protein.

  The HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein can be obtained not only as a biomaterial but also as a gene recombinant by incorporating a gene encoding it into an appropriate expression system. In order to obtain HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein by genetic engineering techniques, the DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein described above is incorporated into an appropriate expression system and expressed. Just do it. Examples of host / vector systems applicable to the present invention include expression vector pGEX and E. coli. Since pGEX can express a foreign gene as a fusion protein with glutathione S-transferase (GST) (Gene, 67: 31-40, 1988), the gene encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein The incorporated pGEX is introduced into E. coli strains such as BL21 by heat shock, and after an appropriate incubation time, isopropylthio-β-D-galactoside (IPTG) is added and GST-fused HMGB1, GST-fused HMGB2, GST-fused HMGB3, Induces expression of fusion S100A8 or GST fusion S100A9 protein. Since GST according to the present invention is adsorbed to glutathione sepharose 4B, the expression product can be easily separated and purified by affinity chromatography.

  In addition to this, the following can be applied as a host / vector system for obtaining a recombinant of HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein. When a bacterium is used as a host, a fusion protein expression vector using a histidine tag, an HA tag, a FLAG tag or the like is commercially available. In yeast, it is known that yeasts of the genus Pichia are effective for the expression of proteins with sugar chains. In terms of glycosylation, an expression system using a baculovirus vector hosted by insect cells is also useful (Bio / Technology, 6: 47-55, 1988). Furthermore, transfection of vectors using promoters such as CMV, RSV, or SV40 has been performed using mammalian cells, and these host / vector systems are all HMGB1, HMGB2, HMGB3, S100A8. Alternatively, it can be used as an expression system for S100A9 protein. In addition, the gene can be introduced using a viral vector such as a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector.

  The protein obtained by the above-described method can be isolated from the inside of the host cell or outside the cell (such as a medium) and purified as a substantially pure and homogeneous protein. The separation and purification of the protein may be performed using the separation and purification methods used in normal protein purification, and is not limited at all. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected, When combined, proteins can be separated and purified.

  Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography. These chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC.

  The protein used in the present invention is preferably a substantially purified protein. Here, “substantially purified” means that the degree of protein purification (the ratio of the target protein in the entire protein component) is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more , 95% or more, 100% or close to 100%. The upper limit close to 100% depends on the purification technique and analysis technique of those skilled in the art, but is 99.999%, 99.99%, 99.9%, 99%, etc.

  Moreover, as long as it has said purity, what was refine | purified by what kind of purification method is contained in the substantially purified protein. For example, the above chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. Although substantially purified protein can be illustrated by selection or combination, it is not limited to these.

  The cells that release or secrete HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein in the present invention basically correspond to all tissue-derived cells in vivo. Examples of cells that can be easily collected and cultured include fibroblasts (for example, normal skin fibroblasts and cell lines derived therefrom), but are not limited thereto. In addition, cells secreting HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein are DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein, or DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein. A DNA encoding a secretory signal (ATG CAG ACA GAC ACA CTC CTG CTA TGG GTA CTG CTG CTG TGG GTT CCA GGT TCC ACT GGT GAC; SEQ ID NO: 29) is combined with a known expression vector or gene therapy vector. It can also be prepared by introducing a vector prepared by insertion into a mammalian cell such as a fibroblast (for example, normal skin fibroblast and a cell line derived therefrom), an insect cell, or other cells. it can. Examples of the DNA encoding the secretion signal include, but are not limited to, DNAs having the above-described sequences. In addition, the animal species from which these cells are derived is not particularly limited, but the cells of the subject animal species to which the vector is administered, the subject's own cells, or cells derived from the relatives of the subject to whom the vector is administered are used. It is preferable.

  The DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein in the present invention may be cDNA or genomic DNA as long as it encodes HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein, It may be natural DNA or artificially synthesized DNA. DNA encoding HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein is usually administered in a state of being inserted into a vector.

  Examples of vectors in the present invention include, but are not limited to, plasmid vectors, retrovirus vectors, lentivirus vectors, adenovirus vectors, adenoassociate virus vectors, Sendai virus vectors, Sendai virus envelope vectors, papilloma virus vectors, and the like. It is not something. The vector may include a promoter DNA sequence that effectively induces gene expression, a factor that controls gene expression, and a molecule necessary to maintain the stability of the DNA. When a vector or DNA is inserted into the device of the present invention, macrophages, dermal fibroblasts, adipocytes or other cells existing in the vicinity of the device come into contact with the vector or DNA in the device, and target proteins such as HMGB1 and S100A8 It is considered that the same effect as when a protein is inserted into the device of the present invention occurs.

  Hyaluronic acid is a glycosaminoglycan (mucopolysaccharide) in which N-acetylglucosamine and glucuronic acid disaccharides are linked and linked, and is also called hyaluronan. The chemical name is represented by [→ 3] -2acetamiod-2deoxy-β-D-glucopyranosyl- (1 → 4) β-D-glucopyranosyluronic acid- [1 →] n. Most of natural hyaluronic acid is high molecular hyaluronic acid having a molecular weight of several hundreds of thousands or more. On the other hand, low molecular hyaluronic acid having 2 to 14 sugars can be artificially produced. For example, in the medical field, it is used as sodium hyaluronate for use in injection into a joint cavity. As a production method, there are a method of extracting and purifying hyaluronic acid produced by Streptococcus zooepidemicus, which is a kind of lactic acid bacteria, and a method of extracting and purifying it from chicken breasts. Hyaluronic acid can be subjected to various modifications such as esterification, periodate oxidation, isourea coupling, and sulfation. It is also possible to carry out ether crosslinking and ester crosslinking. By performing such a modification, it is possible to add a property of stabilizing against decomposition, insolubility, shaping into a sponge, or slow release of a substance. CD44 is a receptor for hyaluronic acid, and hyaluronic acid has a migration activity for mesenchymal stem cells having CD44 on the cell surface. As a result of the experiment, when hyaluronic acid alone was compared with (physiological) phosphate buffer (PBS) as a negative control, hyaluronic acid showed a significantly more significant effect of mobilizing cells than PBS. .

The cell or tissue extract to be inserted into the central hollow portion of the flat container of the present invention can be produced by a method including a step of immersing the cells or tissue in a solvent.
The cell or tissue immersed in the solvent is not particularly limited, but is tissue-derived cell, a cell line established from the tissue-derived cell (for example, HeLa, HEK293 can be exemplified, but is not limited thereto), isolation Examples thereof include cells that have been isolated, cells that have not been isolated (for example, cells that are present in an isolated tissue), cells into which DNA encoding HMGB1, HMGB2, HMGB3, S100A8, or S100A9 protein has been introduced. The tissue may be any tissue, for example, living skin tissue, body biopsy (surgical) tissue (brain, lung, heart, liver, stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, Testis, blood, etc.) can be exemplified, but not limited thereto.

  Examples of the solvent include physiological saline, PBS (Phosphate-buffered saline), and TBS (Tris-buffered saline), but are not limited thereto. The time for immersing cells and tissues in a solvent is necessary and sufficient for cell necrosis to be induced, that is, 1 to 48 hours (for example, 6 to 48 hours), preferably 12 to 24 hours. However, it is not limited to this time. Therefore, “the step of immersing the cells in the solvent” can be rephrased as “the step of immersing the cells in the solvent for a necessary and sufficient time to induce necrosis” and “the step of necrosing the cells”. Examples of the temperature at which cells and tissues are immersed in a solvent include 4 to 25 ° C. (eg, 4 to 8 ° C.), preferably 4 ° C., but are not limited thereto. Examples of the pH for immersing cells and tissues in a solvent include pH 7 to 8, preferably pH 7.5, but are not limited thereto. Examples of the buffer component include phosphate buffer (PBS) having a concentration of 10 mM to 50 mM, preferably 10 to 20 mM, but are not limited thereto.

  In the present invention, the cells and tissues can be removed from the solvent containing the cells and tissues after the cells and tissues are immersed in the solvent by the above procedure. The method for removing cells and tissues from the solvent is not particularly limited as long as it is a method well known to those skilled in the art. For example, cells and tissues can be removed from the solvent by centrifuging at 4 ° C. to 25 ° C. (for example, 4 ° C.) and gravity acceleration of 10 G to 100000 G (for example, 440 G) and collecting the supernatant. Not limited. The supernatant can be used as a cell or tissue extract.

Examples of the cell or tissue extract to be inserted into the central hollow portion of the flat container of the present invention include, but are not limited to, a skin extract and a peripheral blood mononuclear cell extract (peripheral blood extract). .
The peripheral blood extract is prepared by collecting blood using a syringe or the like, then freezing the cells in a freezer, liquid nitrogen, dry ice, etc., and then re-thawing at a temperature of 0 ° C. or higher. Furthermore, in order to remove insoluble components of cells, for example, centrifugation is performed at 4 ° C. to 25 ° C. (for example, 4 ° C.) or gravitational acceleration of 10 G to 100000 G (for example, 440 G), and the supernatant is separated to remove cells from the solvent. The insoluble component can be removed, but is not limited thereto. The supernatant can be used as a cell or tissue extract. In order to remove insoluble components of cells, insoluble components can be removed by passing through a nitrocellulose filter having a minute pore of 0.45 μm instead of centrifugation. In addition, by placing the collected peripheral blood at 4 ° C. for 3 to 48 hours, cell necrosis can be induced and intracellular components can be secreted from the cells in the peripheral blood. Thereafter, centrifugation is performed at a gravitational acceleration of 10 G to 100,000 G (for example, 440 G), and the supernatant is collected, whereby the insoluble components of the cells can be removed from the solvent. However, the present invention is not limited thereto. The supernatant can be used as a cell or tissue extract. In order to remove insoluble components of cells, insoluble components can be removed by passing through a nitrocellulose filter having a minute pore of 0.45 μm instead of centrifugation.

The heparin-binding fraction of the cell or tissue extract to be inserted into the hollow center of the flat container of the present invention can be produced by a method comprising the following steps.
(A) a step of immersing cells or tissues in a solvent;
(B) a step of bringing the extract obtained in step (a) into contact with immobilized heparin; and (c) an immobilized heparin-to-heparin-binding fraction (which can also be expressed as a heparin purified fraction or a heparin column purified fraction). Step of eluting Immobilized heparin is obtained by covalently binding heparin to an insoluble carrier. Examples of the insoluble carrier include Sepharose beads (Sepharose 4B, Sepharose 6B, etc .: GE Healthcare), but are not limited thereto. In the present invention, commercially available immobilized heparin (Hitrap heparin HP column: GE Healthcare) may be used.
Examples of contact conditions between cell and tissue extracts and immobilized heparin are about pH 7 to 8 (preferably pH 7.5), and salt concentration is 0 to 200 mM, preferably about 100 to 200 mM. Not. Although the time for which the extract and the immobilized heparin are in contact is not particularly limited, it is preferably maintained for 5 minutes or more from the viewpoint of sufficiently adsorbing the heparin-binding fraction to the immobilized heparin. Moreover, as temperature, although 4-8 degreeC, Preferably 4 degreeC is mentioned, It is not restrict | limited to these. Furthermore, examples of elution conditions for the heparin-binding fraction adsorbed on the immobilized heparin include a pH of about 7 to 8 and a salt concentration of 200 to 1000 mM (preferably about 1000 mM), but are not limited thereto.

  Examples of the mixture of two or more substances described in the above (a) to (r) inserted into the central hollow part of the flat container of the present invention include hyaluronic acid and HMGB1 protein, hyaluronic acid and HMGB2 protein, and hyaluron. Acid and HMGB3 protein, hyaluronic acid and S100A8 protein, hyaluronic acid and S100A9 protein, hyaluronic acid and HMGB1 protein and HMGB2 protein, hyaluronic acid and HMGB2 protein and HMGB3 protein, hyaluronic acid and HMGB1 protein and HMGB3 protein, hyaluronic acid and HMGB3 protein HMGB2 protein and HMGB3 protein, hyaluronic acid and S100A8 protein and S100A9 protein, hyaluronic acid and HMGB1 protein and S100A8 protein, hyaluronic acid and HMGB2 protein and S100A8 protein, hyaluronic acid and HMGB3 protein and S100A8 protein, hyaluronic acid and HMGB1 protein Protein and S100A9 protein, hyaluronic acid and HMGB2 protein and S100A9 protein, hyaluronic acid and HMGB3 protein and S100A9 protein, cell or tissue extract and hyaluronic acid, cell or tissue extract heparin binding fraction and hyaluronic acid, etc. Combinations may be mentioned, and a cell or tissue extract and a mixed solution of hyaluronic acid are preferable, but not limited thereto. The mixing ratio is 1 in which the smallest component in volume ratio is 1 when dissolved in a solvent, and other components can be mixed up to 10,000 times, but preferably the smallest component is 1. The other components can be added in an amount of 1 to 10 times.

  Extracts inserted into the hollow center of the flat container of the present invention, heparin binding fraction, HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein species of animal species include human or non-human animals, for example Humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs and the like can be exemplified, but the animal species is preferably the same as the animal species to which the extract or the like is administered.

  In order to insert a substance or composition containing at least one selected from the group (a) to (r) into the flat container of the present invention, for example, a solution dissolved in physiological saline, PBS, TBS or the like as a solvent And the solution can be mixed with physiological saline and injected. As the injection method, a known method such as manual injection using a syringe or pipette, or mechanical injection at the time of device manufacture can be adopted. Instead of the physiological saline, at least one selected from the group consisting of a polymer or copolymer of polyethylene glycol (PEG), cyclodextrin, gelatin, albumin, chitosan, and sodium carboxymethyl cellulose may be used as a medium.

The concentration of the solution of the substance or composition mixed with the physiological saline or the like can be appropriately selected, and the concentration of the mixture with the physiological saline or the like can also be appropriately selected.
Further, the amount of insertion into the flat container of the present invention can be appropriately determined according to the volume of the central hollow portion of the container. For example, in the case of a mouse, about 10 ml can be inserted when placed in the abdominal subcutaneous fat, and several ml or less may be used when placed in other subcutaneous. The total amount of the substance or composition inserted into the container can also be appropriately selected.

For the insertion, the extract, heparin-binding fraction, HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein can be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA), may include both pharmaceutically acceptable carriers and additives. For example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, flavoring agents However, it is not limited to these, and other commonly used carriers can be used as appropriate. Specifically, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, Examples thereof include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts.
Although the present invention is not limited by theory, the substances (a) to (r) used in the present invention are released from the container into the blood, and the bone marrow stem cells in the bone marrow are mobilized into the blood. It is considered that the effect of increasing the number of bone marrow stem cells recovered from the blood terminal is exhibited.

  Upon recovery of bone marrow stem cells to the in vivo implantable substance collection device of the present invention, the extract of the present invention, heparin-binding fraction, HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein, or a mixture of any two or more substances May be administered to blood vessels or muscles. The timing of administration of the mixture may be any time before implantation of the device, immediately after implantation, or before removal of the device. The administration method is parenteral administration into blood vessels or muscles, and specifically includes injection administration. For example, by intravascular injection (intraarterial injection, intravenous injection, etc.), intramuscular injection, subcutaneous injection, etc., the drug of the present invention is blood vessel, muscle, or subcutaneous (for example, in a container embedded in the skin or in the vicinity of the container). Can be administered. It can also be absorbed in a controlled manner by means of a transdermally absorbable patch or an external preparation.

  Moreover, an administration method can be appropriately selected depending on the age and symptoms of the administration subject. When HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein is administered, for example, the dose can be selected in the range of 0.0000001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.00001 to 100,000 mg / body per subject. Even when administering HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein secreting cells or HMGB1, HMGB2, HMGB3, S100A8 or gene therapy vectors inserted with DNA encoding S100A9 protein, HMGB1, HMGB2, HMGB3, S100A8 Or it can administer so that the quantity of S100A9 protein may become in the said range. However, the dosage of the extract, heparin-binding fraction, HMGB1, HMGB2, HMGB3, S100A8 or S100A9 protein in the present invention is not limited to these dosages.

  Upon administration, like the device insertion composition of the present invention, it can be formulated according to a conventional method and may contain both pharmaceutically acceptable carriers and additives.

  The step of recovering the cell population from the in-vivo implantable substance collecting device of the present invention includes the step of recovering the cell population from the container implanted subcutaneously, or the cell population removed from the subcutaneously removed container. It may be either of the steps of recovering. For example, in the embodiment of the present invention, a flat container embedded under the skin is collected by skin incision, and cells are aspirated and collected from the container by a pipette, but other methods may be used.

  To recover the cell population from a container that is implanted subcutaneously, fix the tube with the tube inserted into the opening of the container that is implanted subcutaneously, fix the syringe to the tube when collecting cells, and remove the cells by negative pressure. Can be sucked and collected. Thereafter, by reinjecting the cell inducing solution such as HMGB1 solution into a tube placed in the living body, the cells in the container that has been repeatedly implanted subcutaneously can be recovered. In that case, the recovery amount can be increased while minimizing invasiveness.

In the present invention, the step of isolating bone marrow stem cells from the recovered cell population is performed, for example, by isolating adherent cells that adhere to the culture dish from the recovered cell population.
Other isolation methods include, for example, reacting cells collected by pipetting from a container removed from the body with at least one of cell surface markers CD44, PDGFRα, PDGFRβ and reacting with antibodies labeled with different fluorescent species. Then, using a cell sorter, a cell population having a specific cell surface marker can be sorted using the presence or absence of each fluorescence as an index.

  In addition, as another isolation method, for example, an antibody having metal particles immobilized thereon instead of fluorescence is similarly reacted with each cell surface marker, and the cells bound to the metal-attached antibody are magnetically absorbed in the tube. There is also a method (MACS) in which the target cell adsorbed in the tube is recovered by removing the magnetic force after the antibody and non-reactive cells are sufficiently eluted by adsorbing and fixing to one wall.

The present invention provides a cell population recovered by the method of recovering the cell population.
Cell populations or bone marrow stem cells that can be collected by the device of the present invention include hereditary diseases, skin diseases (burns, skin ulcers, etc.), cranial nerve diseases (cerebral infarction, Alzheimer's disease, spinal cord injury, brain injury, etc.), cardiovascular diseases. It can be administered and treated for diseases associated with tissue damage such as (myocardial infarction, cardiomyopathy, arterial embolism, etc.), osteochondral diseases (fractures, rheumatism, etc.). The collected cells can be directly administered into a circulatory blood flow such as intravenously or intraarterially, or directly to a damaged tissue site to regenerate the tissue. Or after adding culture | cultivation operation using a culture dish and a culture flask, a tissue can be reproduced | regenerated by administering in the state which spread | diffused the cell, the state shape | molded in the sheet form, and the state which formed the cell mass. Dosage for therapy may be administered from one cell 10 ^14, preferably may be administered from 10 ² 10 ¹⁰ a. Therefore, the present invention also provides a tissue regeneration agent containing a cell population recovered by the method of recovering the cell population or a bone marrow stem cell recovered by the method of recovering the bone marrow stem cell.

  The tissue to be regenerated is not particularly limited, and may be any tissue as long as it is damaged, for example, living skin tissue, biopsy (surgical) tissue (brain, lung, heart, liver, Examples include stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, testis and blood. In particular, the regenerative agent of the present invention is effectively used to regenerate tissues (brain, heart, etc.) where it is difficult to administer the drug directly from outside the body. In the present invention, examples of the damaged tissue include tissues damaged by various pathological conditions causing ischemia, ischemia / hypoxia, trauma, burn, inflammation, autoimmunity, gene abnormality, etc., but are not limited to these causes. It is not something. Injured tissues also include necrotic tissues.

  The tissue regenerated in the present invention is not particularly limited as long as bone marrow-derived cells can be differentiated. For example, skin tissue, bone tissue, cartilage tissue, muscle tissue, adipose tissue, myocardial tissue, nervous system tissue Examples include all tissues in the living body such as lung tissue, gastrointestinal tissue, liver / bile / pancreatic tissue, urinary / genital organs, and the like. Moreover, by using the above tissue regenerative agent, in addition to skin diseases such as refractory skin ulcers, skin wounds, blistering and alopecia, tissue damage such as cerebral infarction, myocardial infarction, fracture, lung infarction, gastric ulcer, enteritis Can be treated. Examples of animal species to which the tissue regenerative agent is administered include humans and non-human animals, and examples include, but are not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, and guinea pigs. Is not to be done. The regenerative agent of the present invention can be administered to diabetic patients. Refractory skin ulcers, which are skin complications in diabetes, are known to be difficult to heal than normal skin ulcers, but the regenerative agent of the present invention is effective for such diabetic patients. Used.

The tissue regenerating agent of the present invention can regenerate a tissue by administering the cells dispersed in physiological saline after the collection into a circulating blood flow such as intravenously or intraarterially. Or it can reproduce | regenerate a tissue by apply | coating and sticking etc. on the surface of a damaged tissue site | part. Furthermore, the tissue can be regenerated by injecting the inside of the damaged site using a syringe or the like. In addition, after adding a culture operation using a culture dish or culture flask, the tissue can be regenerated by administering it to the damaged site in a diffused state, in a sheet shape, or in a state where a cell mass is formed. it can. The dosage can be administered from one cell 10 ^14, preferably may be administered from 10 ² 10 ¹⁰ a.
The entirety of all prior art documents cited in the present specification is incorporated herein by reference. In addition, the entire application documents of the international application PCT / JP2009 / 058525 are incorporated herein by reference.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[Example 1]
Purpose: To recover functional cells such as stem cells, which are present in extremely small numbers in living tissues, with minimal invasiveness and high efficiency.
Method: The following method was studied for the above purpose.
1) After irradiating 8-week-old mice (male) with radiation (10Gy), bone marrow stem cells derived from green fluorescent protein (GFP) transgenic mice (5x10 ⁶ cells / 0.1ml PBS pH7.4) were transplanted from the tail vein. Bone marrow transplanted mice were created.

2) Flat container (no opening on one side, center) with parts of size and shape shown in Fig. 3 and Fig. 4 manufactured using Toray's trade name Dow Corning Economy type hardness 30 (silicone resin) Four hollow part volumes of 580 μl) were prepared. As a comparative example, a tube-type container having a hollow portion having a cylindrical shape with an outer diameter of 0.3 mm and a length of 10 mm, which is often used, and a medium hole inner diameter of 0.15 mm and a hole depth of 0.85 mm was prepared. As examples, (1) 580 μl of PBS containing 1100 μg of purified HMGB secreted into the culture supernatant and (2) MP Biomedicals, trade name Hyaluronic Acid (hyaluronic acid) 580 μl (1) (Final concentration 1 mg / ml), (3) 580 μl of skin extract, and (4) 580 μl of PBS (pH 7.4), respectively. The comparative container was also filled with 580 μl of PBS containing (1) 1100 μg of purified HMGB.
In the comparative example, the left and right dorsal skins of GFP bone marrow transplanted mice are subcutaneously contacted with the opening of the device of the present invention on the fascia side. /) Transplanted. The maximum number of mice that can be inserted subcutaneously is 4 for each of the comparative container and the flat container of the present invention.

  In the above (3) skin extract, the skin of one 2-day-old C57 / Bl6 mouse was immersed in 1 ml of PBS (manufactured by nacalai) and incubated at 4 ° C. for 24 hours. After removing the skin from the solution, centrifugation was performed at 4 ° C. and a gravitational acceleration of 440 G, and the supernatant was collected to remove cells and tissues from the solvent, and the supernatant was used as an extract of cells and tissues.

3) The container was taken out after 2 weeks. FIG. 6A shows the shape of the device of the present invention and the conventional device when inserted into the body, and FIG. 6B shows the shape of the device when collecting cells. It can be understood that the split device of the present invention is easy to collect cells. Cells (tube-entrapping cells; TECs) accumulated in the container were collected by suction with a negative pressure using a pipette. After collection, the cells were cultured in Dulbecco medium (D-MEM) containing 10% fetal calf serum at 37 ° C. and a carbon dioxide gas concentration of 5%.

4) When the culture density of TECs reached about 80% of the culture dish bottom area, the medium was replaced with a bone differentiation induction medium, a fat differentiation induction medium, or an epidermal differentiation induction medium, and the culture was further continued. Two weeks after the start of culturing in each differentiation induction medium, bone differentiation was evaluated by alizarin red staining, fat differentiation was oil red staining, and epidermal differentiation was evaluated by immunostaining for keratin 5.

5) The ratio of platelet-derived growth factorα (PDGFRα positive and CD44 positive cells in TECs was analyzed by flow cytometry.

Results: A silicone container filled with HMGB1, hyaluronic acid, and skin extract was transplanted subcutaneously into GFP bone marrow transplanted mice. As a result, there were many GFP-positive bone marrow-derived cells (TECs) in one week regardless of which solution was filled. Accumulated in the container.
Specifically, in the examples of the present invention, there are 4968 GFP positive cells, GFP positive and 960 PDGFRα cells, whereas in the tube type comparative example, 595 GFP positive cells and 77 GFP positive cells and 77 PDGFRα cells. There was a marked difference (the evaluation procedure is shown below). PDGFRα is a kind of bone marrow pluripotent stem cell marker that is a kind of bone marrow stem cell. FIG. 7 shows images of GFP positive cells accumulated in the container of the present invention filled with HMGB-1 and hyaluronic acid. On the other hand, in the container of the present invention filled with only hyaluronic acid, the number of collected adhesive TECs was smaller than that of HMGB-1 and hyaluronic acid, but significantly higher than that of the blank (FIG. 8). As a result of culturing the cells collected from the HMGB1 and hyaluronic acid-filled silicone container of the present invention, adherent TECs that adhere to the culture dish and proliferate 24 hours after the start of the culture were observed (FIG. 9). Adhesive TECs that migrated in these HMGB1 and hyaluronic acid-filled silicone containers were cultured in bone differentiation, adipose differentiation, and epidermal differentiation induction media, resulting in alizarin red positive osteoblasts, oil red positive adipocytes, and keratin 5 positive epidermis, respectively. It was shown that there are cell populations that show differentiation potential into cells and have mesenchymal and epithelial differentiation potential in adherent TECs (FIGS. 10 to 12). In addition, as a result of examining the expression of PDGFRα and CD44, which are known to be expressed on the surface of mesenchymal stem cells, in TECs by flow cytometry, about 60% of TECs may be positive for PDGFRα and CD44. Was shown (FIG. 13).

  In addition, the container of the present invention filled with hyaluronic acid at 100 ng / ml (PBS) was transplanted subcutaneously to the back of the GFP bone marrow transplanted mouse, and the surface markers of the cells mobilized in the container were analyzed by FACS after 2 weeks. As a result, similar to the TECs obtained from the HMGB1 container, approximately 60% were CD44-positive and PDGFRα-positive P44 cells, which showed the ability to differentiate into mesenchymal and epithelial cells such as osteoblasts and adipocytes. .

DISCUSSION: The present inventors have succeeded in transplanting subcutaneously the device of the present invention filled with HMGB1, hyaluronic acid, and skin extract, respectively, so that bone marrow having bone differentiation ability, fat differentiation ability, and epidermal differentiation ability is very efficiently obtained. It was found that TECs derived from TEC could be recovered. This is because in the comparative example, the opening of the tube-type container faces the joint between the epithelium and the dermis, so that it is difficult to access the blood end, whereas the flat container of the present invention is transplanted so that the opening is in contact with the fascia. Therefore, it is possible to directly contact the blood end, and it is considered that the recovery of cells from peripheral blood vessels is promoted. Furthermore, in the comparative example, the angular end angle of the transplanted part of the subject is almost right, so that when the transplanted part comes into contact with a foreign substance outside the body, the skin surface and the body tissue are susceptible to irritation and the wearability is inferior. Since the container of the present invention has a gentle curve at the end of the transplanted portion, the burden on the subject is small. Furthermore, cell recovery from the device of the present invention can be easily recovered from the inside by removing a part of the device formed by combining a plurality of components.
Many of the recovered bone marrow-derived TECs express PDGFRα and CD44, which are known to be expressed on the surface of mesenchymal stem cells, and HMGB1, hyaluronic acid, and skin extract all have mesenchymal stem cell mobilization activity In combination with TECs, bone marrow-derived mesenchymal stem cells are preferentially mobilized by TECs. By selecting a solution to be filled in the silicone container, it is possible to collect specific in vivo functional cells selectively and with high efficiency according to the specific function cell mobilization active substance contained in the solution. By using this new invention technology, avoiding highly invasive methods such as inserting a needle into the bone marrow, collecting functional cells in vivo as needed, and designing customized regenerative medicine according to the purpose It becomes possible to do. In addition, the collected cells can be provided as materials for many basic researches including stem cell research, and greatly contribute to the progress of basic and clinical research, drug discovery, and medical technology.

[Example 2]
Objective: Evaluation of skin ulcer treatment effect of bone marrow-derived cells mobilized to the device of the present invention 1) The same implantable device of the present invention as in Example 1 was produced.

2) In order to prepare a skin tissue extract containing bone marrow pluripotent stem cell mobilization factor, free skin pieces obtained from 2 C57 / BL6 newborn mice (2 days old) were placed in 2 ml of PBS (pH 7.4). After soaking and incubating at 4 ° C. for 24 hours, in order to remove the tissue, the supernatant was collected by centrifugation at 440 G for 10 minutes at 4 ° C. to prepare a skin extract.
In addition, peripheral blood whole blood was collected from two 4-week-old C57 / BL6 mice, and the whole volume was diluted to 4 mL with PBS. 3 mL of Ficoll-Paque Plus (GE) solution was inserted into the centrifuge tube, and diluted blood was layered thereon. Centrifugation was performed at 100 G (18 ° C.) for 10 minutes, and the intermediate layer containing mononuclear cells was collected in a new centrifuge tube. To remove the Ficoll-Paque Plus solution from the recovered intermediate layer, add 45 mL of PBS, centrifuge at 440G (18 ° C) for 5 minutes, remove the supernatant, add 45 mL of PBS again, and add 440G (18 ° C) for 5 minutes. The supernatant was removed by centrifugation. 200 μl of PBS was added to the precipitated cells and suspended. The cell suspension was frozen for 30 minutes in a -80 ° C freezer and thawed on ice from the freezer. This freeze-thaw operation was repeated three times. The supernatant was further collected by centrifugation at 440 G (4 ° C.) for 15 minutes to obtain a peripheral blood mononuclear cell extract.

3) C57 / BL6 male mice (6-8 weeks old) were irradiated with lethal dose radiation (10 Gy), and immediately after that, GFP transgenic mouse-derived bone marrow stem cells (5 × 10 ⁶ cells / 0.1 ml PBS (pH 7.4) from the tail vein )) Transplanted. Only mice that survived until 8 weeks after transplantation were used in subsequent experiments.

4) Three devices of the present invention were inserted with (1) skin extract prepared in 2), (2) peripheral blood extract, and (3) PBS 40 μl for negative control. This device was inserted one by one on the left and right sides (total of 2 / mouse) so that the openings of the device were in contact with the left and right dorsal subcutaneous fascia side of the mouse prepared in 3). Two weeks later, the device was removed from the mouse.

5) After removing C.B-17 / lcr-scid / scidJcl 8-week-old (obtained from Charles River Japan) hair on the back, circular skin ulcers with a diameter of 6 mm were prepared on the left and right sides of the back. In order to prevent the skin of the mouse from contracting, a disc made of silicone with an outer diameter of 10 mm, an inner diameter of 6 mm and a thickness of 1 mm was applied to the ulcer using double-sided adhesive tape and the medical adhesive product name “Aron Alpha A” (manufactured by Sankyo) Glued. Cells were collected from inside the device on one side of the left and right devices taken out in 3) and administered to the skin ulcer. In order to prevent drying of the ulcer and bacterial infection, the ulcer was covered with a silicone disk having a diameter of 10 mm and a thickness of 1 mm. In order to protect the ulcer part, it was covered with the trade name “Tegaderm” (manufactured by 3M). After 7 days, the area of the ulcer was measured. (Fig. 31)

  As a result, the ulcer area of the negative control did not close even after 7 days, whereas in the ulcer administered with the cells collected with the blood extract, a decrease in the ulcer area was observed on the 5th day. Moreover, closure of the ulcer area was observed on the 7th day in the ulcer administered with the cells collected with the skin extract (FIG. 31).

  According to this example, the effect of treating ulcer was confirmed by administering bone marrow-derived cells mobilized in an implantable device containing a tissue extract to the skin ulcer portion. Rather than taking bone marrow stem cells from the bone marrow, there is an unprecedented new treatment method that mobilizes the device in the body and uses the cells for treatment.

Example 3
1) A skin extract and a peripheral blood extract were prepared by the method described in Example 2 above.
A HiTrap heparin HP 1 mL column (GE) was equilibrated with 10 mL of 10 mM PBS (pH 7.5). The skin extract and peripheral blood extract were each diluted with 10 volumes of 10 mM PBS (pH 7.5) and bound to an equilibrated column. The column was washed with 10 mL of 10 mM PBS (pH 7.5) to wash away nonspecifically adsorbed components. The adsorbed components were eluted using 1000 mM NaCl-containing 10 mM PBS (pH 7.5), and 120 μl each was dispensed into a plastic container. The amount of protein was quantified using Protein assay kit (manufactured by Bio-Rad), and three fractions with the highest concentration were collected (tissue extract heparin adsorption).

2) 0.1% hyaluronic acid-containing PBS was prepared, 10 μl of this hyaluronic acid solution and 40 μl of tissue extract were mixed and inserted into the device of the present invention in Example 1. As a negative control, a mixed solution of 1000 mM NaCl-containing 10 mM PBS (pH 7.5) 5 and hyaluronic acid was used.

3) A GFP bone marrow transplanted mouse was prepared in the same manner as in Example 2, and the device prepared in 2) was inserted subcutaneously into the back of the mouse. Ten days after insertion, the device was removed from the skin and the cells were collected. The collected cells were cultured in IMDM (Invitrogen) containing 10% Fetal bovine serum using a culture dish in a 37 ° C., 5% CO ₂ environment. One day after the start of the culture, the medium was changed to remove non-adherent cells. Thereafter, the medium was changed every 3 days. One week after cell recovery, the cells were observed using a fluorescence microscope to detect bone marrow-derived cells (GFP positive cells) (FIG. 32). Also. The number of GFP positive cells was counted using image processing software (Image J) (FIG. 33).

  In the heparin-binding fraction mixed solution of hyaluronic acid and tissue extract (blood extract (Fig. 32C), skin extract (Fig. 28D)), compared with hyaluronic acid alone (Fig. 32B) or PBS (Fig. 32A) It was observed that there was a strong mobilization activity against bone marrow-derived adherent cells in the liver (FIGS. 32 and 35).

  This example confirmed that the bone marrow stem cell mobilization factor in the tissue extract can be purified with a heparin column. The skin extract contains HMGB1, HMGB2, HMGB3, S100A8, and S100A9, and these components bind to the heparin column, so the recruitment of bone marrow stem cells may involve these cells. It is suggested. In addition, since many cytokines such as growth factors also bind to the heparin column, it may be due to the comprehensive effect of these multiple factors.

Example 4
1) RNA was extracted from newborn mouse skin using the trade name Trizol (manufactured by Invitrogen), and cDNA was synthesized using the trade name SuperScript III cDNA synthesis kit (manufactured by Invitrogen). Using this cDNA as a template, the S100A8 cDNA is amplified using PCR, and the protein is expressed in mammalian cells so as to express a protein with the Flag tag and 6XHis tag sequences added to the N-terminus of the amino acid sequence for purification. The plasmid vector pCAGGS was inserted. Human fetal kidney cell-derived HEK293 cultured cell line was transfected with pCAGGS-Flag-His-S100A8 using polyethyleneimine (PEI), and after 48 hours, cells and culture supernatant were collected. The cells and culture supernatant were centrifuged at 4400 G for 5 minutes at 4 ° C., and the supernatant and cells were separated and collected. The collected supernatant was further passed through a cellulose acetate filter (Nalgene) having a pore of 0.8 μm in diameter, and then passed through a nitrocellulose filter (Corning) having a pore of 0.45 μm to prepare a sample from which the insoluble fraction was removed. . This sample was inserted into 5 mL HisTrap FF (GE) equilibrated with 50 mL of 50 mM Tris HCl (pH 8.0) containing 50 mM NaCl, and the adsorbed component was further washed with 50 mM TriCl HCl (pH 8.0) containing 10 mM imidazole. Non-specific adsorption components were removed. Specific adsorption components were eluted from the column with 50 mM NaCl-containing 50 mM Tris HCl (pH 8.0) containing 100 mM imidazole. Each of the adsorbed fractions was fractionated into 500 μl silicone-coated plastic containers, and the protein-containing fractions were combined and mixed with anti-Flag antibody M2 beads (Sigma), and reacted at 4 ° C. for 12 hours with gentle mixing. After the reaction, the beads were centrifuged at 440 G for 5 minutes, and the supernatant was removed. Then, the beads were suspended in PBS and centrifuged in the same manner to remove the supernatant. The beads were inserted into a column with a volume of 3 mL and an inner diameter of 1 cm, and the adsorbed protein was eluted from the beads with 100 mM Glycine-pH 3.5, and neutralized with one-tenth amount of 500 mM Tris HCl (pH 7.5). The amount of purified protein was quantified using Protein assay kit (manufactured by Bio-Rad).

2) RNA was extracted from newborn mouse skin using Trizol (invitrogen), and cDNA was further synthesized using SuperScript III cDNA synthesis kit (Invitrogen). Using this cDNA as a template, the cDNA of HMGB1 is amplified using PCR, and the protein is expressed in mammalian cells so that the protein with the Flag tag and 6XHis tag sequences added to the N-terminus of the amino acid sequence for purification. The plasmid vector pCAGGS was inserted. Human fetal kidney cell-derived HEK293 cultured cell line was transfected with pCAGGS-GST-His-HMGB1 using polyethyleneimine (PEI), and cells and culture supernatant were collected after 48 hours. The cells and culture supernatant were centrifuged at 4400 g for 5 minutes at 4 ° C., and the supernatant and cells were separated and collected. The collected supernatant was further passed through a cellulose acetate filter having a 0.8 μm diameter hole, and then passed through a nitrocellulose filter having a 0.45 μm hole to prepare a sample from which the insoluble fraction was removed. This sample was inserted into 5 mL HisTrap FF (GE) equilibrated with 50 mL of 50 mM NaCl-containing 50 mM Tris HCl (pH 8.0), and the adsorbed component was further added with 50 mM NaCl-containing 50 mM NaCl 50 mM Tris HCl (pH 8.0). Washing was performed to remove non-specifically adsorbed components. Specific adsorption components were eluted from the column with 50 mM NaCl containing 50 mM Tris HCl (pH 8.0) containing 100 mM imidazole. Each of the adsorbed fractions was fractionated in a 500 μl silicone-coated plastic container, and the protein-containing fractions were collected, and then imidazole was removed using a desalting column PD10 (GE), and 50 mM Tris HCl (pH 7.5), Elute with 150 mM NaCl. HRV3C (Novagen) was added to the eluted sample and reacted at 4 ° C. for 3 hours. After cleavage, the sample was bound to 50 mM Tris HCl8 (pH 7.5, 150 mM NaCl equilibrated HiTrap heparin 1 mL column (GE), the column interior was washed with 50 mM Tris HCl (pH 7.5), 150 mM NaCl, and then 50 mM Tris HCl. The bound protein was eluted with 1000 mM NaCl (pH 7.5), and the eluted sample was dispensed in 500 μl aliquots into a silicone-coated plastic container.

3) 40 μg each of S100A8, HMGB1, HMGB2, and HMGB3 was inserted into the device of the present invention used in Example 1, and inserted so that the holes were in contact with the fascia under the left and right dorsal skin of GFP bone marrow transplanted mice. Ten days after the insertion, the device was taken out subcutaneously and the cells accumulated in the device were collected. The cells were cultured in IMDM (Invitrogen) containing 10% Fetal bovine serum using a culture dish in an environment of 37 ° C. and 5% CO ₂ . One day after the start of the culture, the medium was changed to remove non-adherent cells. Thereafter, the medium was changed every 3 days. One week after cell recovery, the cells were observed using a fluorescence microscope, and bone marrow-derived cells (GFP positive cells) were detected (FIG. 34).

4) BALB / cAJcl-nu / nu 8 week-old (obtained from CLEA Japan, Inc.) circular skin ulcers with a diameter of 6 mm were prepared on the left and right sides of the back. In order to prevent the mouse skin from contracting, a disk made of silicone having an outer diameter of 10 mm, an inner diameter of 6 mm, and a thickness of 1 mm was adhered to the ulcer using double-sided adhesive tape and medical adhesive Aron Alpha A (Sankyo).

5) Treat the cells of 3) with 0.5 g / l-Trypsin / 0.53 mmol / l-EDTA solution (Nacalai), release them from the culture dish, inactivate trypsin with IMDM containing 10% FBS, After centrifuging at 440 G at 4 ° C. for 5 minutes to remove the supernatant, the ulcer prepared in 4) was administered. To prevent local dryness and bacterial infection, the ulcer was covered with a silicone disk 10 mm in diameter and 1 mm in thickness. Furthermore, in order to protect an ulcer part, it covered with tegaderm (3M). After 7 days, the size of the ulcer was measured (FIG. 35).

Compared to the negative control (E), mobilization activity of bone marrow stem cells was observed in any of S100A8, HMGB1, HMGB2, and HMGB3 (FIG. 34-A; S100A8, B; HMGB1, C; HMGB2, D; HMGB3). In particular, strong mobilization activity was observed for HMGB1 and HMGB2.
In addition, the bone marrow-derived cells mobilized by HMGB1, HMGB2, and S100A8 were administered, and as a result of treating the skin ulcer, an ulcer reduction effect was observed compared to the negative control (PBS administration) (FIG. 35).

  In this example, mobilization activity of bone marrow-derived adherent cells into the device was observed in any of S100A8, HMGB1, HMGB2, and HMGB3. The main cells in the bone marrow are blood cells such as erythrocytes and blood cells, but most of these cells are non-adherent cells. Mesenchymal cells such as mesenchymal stem cells are known as adherent cells, and pluripotent cells that can differentiate into epithelial and nervous systems are considered to be included. Since the therapeutic effect of the skin ulcer was recognized by the cells mobilized in the device by S100A8, HMGB1, and HMGB2 according to this example, the cells mobilized in the device are bone marrow-derived cells that can induce treatment of tissue damage. . There is also a report that bone marrow mesenchymal stem cells are administered to skin ulcer sites to show therapeutic effects (Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Wu Y, Chen L, Scott PG, Tredget EE. Stem Cells. 2007 Oct; 25 (10): 2648-59. Epub 2007 Jul 5.), suggesting the involvement of bone marrow mesenchymal stem cells in the therapeutic effect in this example. In addition, bone marrow-derived cells such as bone marrow mesenchymal stem cells are known to differentiate into nerve cells, adipocytes, bone cells, chondrocytes, and epithelial cells. The cells collected by this device can be applied to new regeneration-inducing medicine for supplying and treating these cells in damaged tissues.

[Reference Example 1]
Objective: Identification of HMGB1 family in skin extract and examination of bone marrow mesenchymal stem cell inducing activity: The presence of HMGB protein family contained in newborn mouse skin extract was confirmed using Western blot.
As a sample, free skin pieces obtained from 400 newborn mice were immersed in 400 ml of PBS (pH 7.4), incubated at 4 ° C. for 24 hours, and then 440G for 10 minutes at 4 ° C. to remove tissue. 10 μl of the skin extract obtained by centrifuging and recovering the supernatant was electrophoresed using SDS-PAGE, and the protein separated in the gel was transferred to a PVDF membrane using a blotting apparatus (ATTO). Rabbit anti-mouse HMGB1 antibody, rabbit anti-mouse HMGB2 antibody, rabbit anti-mouse HMGB3 antibody diluted 1000-fold with ST-PBS after 1 hour incubation in PBS containing 3% skim milk 0.1% Tween20 (ST-PBS) Each was reacted at 4 ° C. for 16 hours. After the reaction, the PVDF membrane was washed 5 times with ST-PBS for 5 minutes, and then the PVDF membrane was washed at 25 ° C. with a peroxidase-labeled goat anti-rabbit IgG antibody (GE Healthcare) diluted 2000 times with ST-PBS. Incubated for hours. Furthermore, after washing with ST-PBS for 5 minutes 5 times, the ECL Western Blotting Detection System (GE Healthcare) was reacted with the PVDF membrane, the ECL film was exposed to light and developed to detect the presence of HMGB1, HMGB2, and HMGB3 proteins. .

  RNA was extracted from newborn mouse skin using Trizol (invitrogen), and cDNA was further synthesized using SuperScript III cDNA synthesis kit (Invitrogen). Using this cDNA as a template, cDNAs of HMGB1, HMGB2, and HMGB3 were amplified by PCR, and a Flag tag sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Lys; SEQ ID NO: It was inserted into a plasmid vector pCAGGS for expressing the protein in mammalian cells so that the protein added with 30) was expressed. These plasmid vectors were transfected into HEK293 (human embryonic kidney cell-derived cultured cell line) and cultured for 48 hours to express the protein. Cells and culture supernatants expressing HMGB1, HMGB2, and HMGB3 proteins were incubated at 4 ° C. for 16 hours, and then centrifuged at 4400 g for 5 minutes to collect the supernatant. 100 μl of Anti Flag antibody Gel (manufactured by Sigma) per 50 mL of the supernatant was mixed and incubated at 4 ° C. for 16 hours. The gel was collected by centrifugation and washed 5 times with PBS. Further, elution was carried out using 3X Flag peptide (final 100 μg / ml). Recombinant protein expression was confirmed by Western blot using a mouse anti-Flag antibody diluted 1000-fold with S-T-PBS and a peroxidase-labeled anti-mouse IgG antibody (GE Healthcare) diluted 2000-fold with S-T-PBS. The migration activity of mouse purified bone marrow mesenchymal stem cells of these purified recombinant proteins was evaluated using a Boyden chamber. In addition, in order to observe the in vivo efficacy of the HMGB family, the skin of the back of 8 week-old C57BL / 6 mice was excised into a circular shape with a diameter of 8 μm, and a skin ulcer model was prepared, and purified HMGB1, HMGB2, HMGB3 Each 100 ng / μl concentration solution was mixed with an equal volume of 1 g / 100 mL PBS hyaluronic acid solution, and 100 μl was administered to the ulcer surface. The ulcer surface was covered with an adhesive transparent wound covering / protective material Tegaderm (manufactured by 3M Healthcare) so as not to dry, and the wound area was measured over time to determine the healing effect.

Furthermore, human bone marrow mesenchymal stem cells were evaluated using a Boyden chamber in order to examine whether the human skin extract and human purified HMGB1 have the activity of migrating. Human skin having an area of 1 cm ² was soaked in 1 ml of PBS, incubated at 4 ° C. for 16 hours, and then centrifuged at 440 G for 10 minutes at 4 ° C. Only the supernatant was collected and used as a human skin extract. In addition, human bone marrow mesenchymal stem cells (Cambrex) were used as cells to be placed in the upper part of the Boyden chamber. (This cell has been determined to be CD105 positive, CD166 positive, CD29 positive, CD44 positive, CD34 negative, CD45 negative as a result of cell surface antigen analysis by flow cytometry. In addition, human HMGB1 was diluted 10-fold with 100 ng / well (R & D) and PBS at the bottom of the chamber, and PBS was used as a control.

Results: As a result of Western blotting, HMGB2 and HMGB3 bands were detected in addition to the HMGB1 band. Therefore, it was confirmed that the newborn mouse skin extract contained HMGB2 and HMGB3, which are family proteins, in addition to HMGB1 (FIG. 14). Expression vectors of HMGB1, HMGB2, and HMGB3 were prepared by adding a Flag tag to the N-terminus of each protein (FIG. 15). An expression vector was introduced into HEK293 cells, and the expressed protein was purified using Flag tag, and then the protein was confirmed using Western blotting (FIG. 16). When the migration activity of mouse bone marrow mesenchymal stem cells using these purified proteins was measured, the activity of any protein was confirmed (FIG. 17). When the ulcer area produced on the back of the mouse was measured every 7 days, the treatment group with HMGB 1, 2 and 3 significantly confirmed the effect of reducing the ulcer area compared to the non-treatment group (FIG. 18). As in the case of mice, it was revealed that human HMGB1 and human skin extract have an activity of migrating human bone marrow mesenchymal stem cells (FIG. 19).

Discussion: HMGB2 and HMGB3 are known as proteins having high homology with HMGB1. These proteins are also expected to have the same properties as HMGB1. Accordingly, it was confirmed that HMGB2 and HMGB3, which are family of HMGB1, were also produced from the free skin piece extract. Furthermore, recombinant proteins of HMGB1, HMGB2, and HMGB3 were prepared, bone marrow mesenchymal stem cell migration activity was confirmed in vitro, and skin ulcer treatment effect was confirmed in vivo. The HMGB family (HMGB1, HMGB2, HMGB3) and the recombinant HMGB family in newborn mouse free skin have the activity to induce bone marrow mesenchymal stem cells and stem cells that can differentiate into bone marrow-derived epithelial systems, It was revealed that these induced bone marrow-derived cell groups differentiated into various cells such as epidermal keratinocytes, hair follicles and fibroblasts in the damaged tissue, and promoted healing of the damaged tissue. In addition, since bone marrow mesenchymal stem cells are pluripotent stem cells, the HMGB family can be administered systemically or locally to treat other tissue damage conditions such as brain damage, myocardial infarction, fractures, etc. Be confident that you can expect a therapeutic effect.

  In addition, human and mouse HMGB1 have 98% (213/215) homology in the amino acid sequence constituting each, and HMGB2 has 96% (202/210) homology in the amino acid sequence constituting each, It is known that HMGB3 has 97% (195/200) homology in the amino acid sequence constituting each. Therefore, it is considered that human HMGB has the same activity as mouse HMGB, but from this result, human skin extract and HMGB1 were used as mouse skin extract and bone marrow mesenchymal stem cells as in HMGB1. It became clear that there was activity to induce.

[Reference Example 2]
Objective: Establishment of bone marrow mesenchymal stem cell inducer tissue extract preparation method: 6 weeks old C57BL6 brain, heart, intestine, kidney, liver and newborn mouse skin PBS (pH 7.4) After immersing in 1 ml and incubating at 4 ° C. for 24 hours, in order to remove the tissue, the supernatant was collected by centrifugation at 440 G for 10 minutes at 4 ° C. to obtain a tissue extract. In order to confirm the presence of bone marrow mesenchymal stem cell-inducing activity in the obtained extract, the Boyden chamber was used to examine the migration activity against bone marrow-derived mesenchymal stem cells. Further, the concentration of HMGB1 contained in the same sample was measured using HMGB1 ELISA kit (manufactured by Shinotest). Further, brain, heart and skin tissue extracts were bound to a heparin affinity column, and the bone marrow mesenchymal stem cell inducing activity of the bound fraction protein was confirmed using a Boyden chamber.

Results: The mouse brain extract contained HMGB1 equivalent to the newborn mouse skin extract. Furthermore, the inducing activity of bone marrow mesenchymal stem cells was observed in the mouse brain as well as the skin. The mouse intestinal extract and mouse heart extract contained almost no HMGB1, but bone marrow mesenchymal stem cell-inducing activity was observed. Further, the heparin column-binding fraction of mouse brain and mouse heart had the activity of inducing bone marrow mesenchymal stem cells in the same manner as the heparin column-binding fraction of mouse skin (FIG. 20). Table 1 shows the results of measuring the HMGB1 concentration and the bone marrow mesenchymal stem cell inducing activity of each mouse tissue extract.

Discussion: We have developed a simple method for extracting HMGB1 by simply immersing organs in physiological buffer, not only in the skin but also in the brain. This method is the same for other organs such as the liver and kidney. The extract from heart and intestine showed bone marrow mesenchymal stem cell-inducing activity despite containing almost no HMGB1. This may be due to the fact that the extract contains other bone marrow mesenchymal stem cell inducers different from HMGB1. Substances contained in these extracts are originally present in each tissue, and are physiologically considered to induce bone marrow mesenchymal stem cells to the damaged tissue at the time of tissue damage. As described above, a novel method for functionally and easily extracting a plurality of bone marrow mesenchymal stem cell inducers including HMGB1 from various organs has been developed. Furthermore, in order to purify the bone marrow mesenchymal stem cell inducer from the tissue extract, a method for binding to a heparin column was developed. In addition, these components having bone marrow mesenchymal stem cell inducing activity can be purified from the brain and heart using a heparin column in the same manner as for skin. The purified component obtained can be inserted into the device of the present invention and used to recover bone marrow stem cells.

[Reference Example 3]
Objective: To establish a method for extracting mesenchymal stem cell migration active substances from cultured cells.
Method: Human fetal kidney-derived cultured cell line HEK293 and human cervical cancer cell line HeLa were each cultured in D-MEM (manufactured by nacalai) containing 10% fetal calf serum. After washing each cell with PBS, 10 ⁷ cells were immersed in 5 ml PBS (manufactured by nacalai) at 4 ° C. for 16 hours. Centrifugation was performed at 4 ° C. for 5 minutes at a gravitational acceleration of 440 G, and the supernatant was collected. Human bone marrow mesenchymal stem cells were placed in the upper layer of the Boyden chamber, and a cell extract diluted 5-fold with DMEM was placed in the lower layer to confirm human bone marrow mesenchymal stem cell migration activity.
Results: Both HEK293 extract and HeLa extract showed activity to migrate bone marrow mesenchymal stem cells (FIG. 21).
Discussion: We succeeded in extracting active substances that migrate bone marrow mesenchymal stem cells by a simple method of immersing cultured cells in PBS. The resulting active substance can be inserted into the device of the present invention and used to recover bone marrow stem cells.

[Reference Example 4]
Objective: To create a mouse brain defect model, administer the skin extract heparin column purified fraction at the site of local injury, and administer stem cells contained in the own bone marrow system to the site of local injury. Consider whether cell regeneration can be induced.
Method:
(1) Preparation of skin extract heparin column purified fraction Skin extract extracted from excised newborn mouse skin by incubation in PBS (1 animal / ml) at 4 ° C for 16 hours at 9 ° C 20 mM PBS (4 ° C) The mixture was diluted 10 times using pH 7.5). In advance, 20 mM PBS (pH 7.5) (30 ml) was poured into a HiTrap heparin HP column (column volume: 5 ml, GE Healthcare) to equilibrate the column. Furthermore, the diluent was bound to the column. Thereafter, the column was washed with 20 mM PBS (pH 7.5) and 100 mM NaCl (30 ml). In order to elute the adsorbed protein, 20 mM PBS (pH 7.5) and 1000 mM NaCl were flowed into the column, and the eluate was fractionated into containers. Each of the adsorbed fractions was evaluated for migration activity of a mouse bone marrow-derived cell line using the Boyden chamber method, and fractions having migration ability were collected. The solution having this activity was used in the following reference examples as a purified fraction of skin extract heparin.

(2) Preparation of bone marrow-suppressed mice Mice were irradiated with 10 Gy X-rays once to prepare bone marrow-suppressed mice.

(3) GFP mouse bone marrow transplantation into myelosuppressed mice
Bone marrow stem cells were collected from bilateral femurs and lower leg bones of GFP mice. This was administered from the tail vein of myelosuppressed mice 24 hours after irradiation. The administration was performed under inhalation anesthesia with isoflurane.

(4) Creation of a mouse brain injury (brain tissue defect) model
Myelosuppressed mice transplanted with bone marrow stem cells of GFP mice were subjected to inhalation anesthesia with isoflurane and pentobarbital (45 mg / kg) was injected intraperitoneally. The mouse was fixed to a stereotaxic apparatus, and a midline incision was made in the head with a scalpel. A burr was drilled from the bregma to the right outside 2.5 mm and the front 1 mm using a drill (FIG. 22A). An outer cylinder of a 20G Surfflow needle was inserted and fixed with the tip at a position 3 mm deep from this site. Here, negative pressure was applied using a syringe to partially suck brain tissue (FIG. 22B).

(5) Administration of the purified fraction of skin extract heparin column to the brain tissue defect site Using the Hamilton syringe and 26G syringe, the fibrin glue preparation (fibrinogen) (Bolheel (manufactured by Kakenken)) 5 μl of the dissolved skin extract heparin column purified fraction was injected, and then 5 μl of fibrin glue preparation (thrombin) (Bolheel (Kakekenken)) was injected (FIG. 22C). By this operation, the effect of the skin extract heparin column purified fraction as a sustained release agent was aimed.

(6) Evaluation of neural cell regeneration effect in brain tissue defect site The control group and the treatment group mice were evaluated. Appropriate course settings were determined (over time), and the mice were perfused and fixed with 4% paraformaldehyde, and then the brains were excised. Furthermore, 4% paraformaldehyde was externally fixed. After dehydration with sucrose with a gradient of 15% and 30%, frozen sections were prepared.
Nuclear staining was performed with DAPI (4 ′, 6-Diamidino-2-phenylindole, dihydrochloride) solusion, and the mixture was encapsulated with a photobleaching inhibitor. Accumulation of GFP-positive cells at the damaged site (brain tissue defect) was evaluated with a confocal laser microscope.
Results: Qualitatively shows GFP positive cell accumulation after 2 weeks and 6 weeks after administration. After 2 weeks (control; FIG. 22D, purified fraction of skin extract heparin column; FIG. 22E) and after 6 weeks (control; FIG. 22F purified fraction of skin extract heparin column; FIG. 22G) compared to the control group. There was a tendency for the accumulation of GFP-positive cells to be high at the injury site in the treatment group.
Discussion: Bone marrow-derived cells accumulated in the brain tissue defect site after administration of the purified fraction of skin extract heparin column, indicating the morphology of nerve cells. Bone marrow-derived mesenchymal stem cells are known to differentiate into nerve cells, and it was clarified from this result that nerve cell regeneration at the brain injury site can be induced by the purified fraction of skin extract heparin column. This can also be applied to nerve regeneration at a brain tissue injury site in cerebral ischemic disease or cerebral contusion.

[Reference Example 5]
Objective: Identification method of bone marrow-derived tissue stem cell inducer present in skin tissue extract: To identify bone marrow mesenchymal stem cell mobilization factor that is expected to be released from resected skin in an anemic state I proceeded with the research.
1) To obtain mouse bone marrow-derived mesenchymal stem cells, bone marrow stem cells of C57BL / 6 mice were collected from the femur or lower leg bones, and D-MEM (Nacalai) containing 10% fetal bovine serum was used as the cell culture medium. As a cell culture dish and cultured under conditions of 37 ° C. and carbon dioxide concentration of 5%. When the area occupied by the cells grew from 70 to 100% of the culture dish bottom area, the cells were detached from the culture dish using 0.25% trypsin 1 mM EDTA (manufactured by Nacalai) and further subcultured under the same conditions. The passage work was repeated at least 5 times. Furthermore, these adherent cells were isolated and cultured, and cell surface antigens were analyzed by flow cytometry, and confirmed to be Lin negative, CD45 negative, CD44 positive, Sca-1 positive, and c-kit negative. It was confirmed that these cells can be differentiated into bone cells and adipocytes and have the properties of bone marrow mesenchymal stem cells.

2) Immerse free skin pieces from 5 newborn mice (2 days old) in 5 ml of PBS (pH 7.4), incubate at 4 ° C for 24 hours, and then remove the tissue under 4 ° C conditions. The supernatant was collected by centrifugation at 440 G for 10 minutes to prepare a skin extract. Similarly, free skin pieces obtained from one 6-week-old mouse were immersed in 5 ml of PBS (pH 7.4), incubated at 4 ° C. for 24 hours, and then removed at 10 ° C. under 4 ° C. conditions to remove the tissue. The supernatant was collected by centrifugation at 440G for a minute to prepare a skin extract.
3) In order to confirm that bone marrow mesenchymal stem cell-inducing activity exists in the obtained skin extract, Boyden chamber was used, and the C57BL6 mouse bone marrow-derived mesenchymal system already established by the present inventors was established. The migration activity against stem cells was examined. Specifically, a polycarbonate membrane with 8 μm fine holes is inserted into the lower tank (volume 25 μl) of the Boyden chamber with a mixture of 2 day or 6 week old mouse skin extract (5 μl) and DMEM (20 μl). In addition, place the Boyden chamber upper tank (50 μl in volume) in contact with this, and place bone marrow-derived mesenchymal stem cell suspension (5 × 10 ⁴ cells / 50 ml culture medium: DMEM / 10% fetal bovine serum) Then, the cells were cultured at 37 ° C. for 4 to 24 hours in a CO ₂ incubator. After culturing, the upper tank of the chamber was removed, the silicone thin film was taken out, and the number of bone marrow-derived mesenchymal stem cells that had migrated to the lower tank through the fine holes was quantitatively examined by staining (FIG. 23).

4) About 2 cm ² of the skin of each of the 2-day-old mice and the 6-week-old mice was collected, rapidly frozen in liquid nitrogen, and pulverized using a mortar. RNA was extracted and purified from these samples using RNeasy (Qiagen). Using the purified RNA, mRNA expressed more in 2-day-old mice was screened by microarray assay. There were 767 genes that scored more than twice in 2-day-old mice. Among these genes, a protein with high affinity to heparin, a protein that may be secreted, and a gene with a score 6 times or more higher in 2 day-old mice, S100A9 exists as the top 57 gene. did. Therefore, the presence of S100A8, which is known to form a heterodimer with S100A9, in a 2-day-old skin extract was detected by Western blot. Specifically, 5 μl of 2-day-old skin extract and 5 μl of SDS-PAGE sample buffer (Bio-Rad) were mixed, heated in a heat block at 98 ° C. for 5 minutes, and then cooled to 25 ° C. This sample was applied to e-PAGEL (ATTO) of 12.5% acrylamide gel and electrophoresed at 40 mA for 75 minutes using an electrophoresis apparatus (ATTO). After electrophoresis, the gel was collected, and the protein in the gel was transferred to a PVDF membrane (manufactured by Millipore) 7 cm in length and 9 cm in length, which was previously treated with 100% methanol using a blotting apparatus (ATTO). Transcription was performed at 120 mA for 75 minutes. After completion of the transfer, the PVDF membrane was recovered and shaken in 4% skim milk-containing PBS (nacalai) for 30 minutes at room temperature. Thereafter, the recovered PVDF membrane was immersed in a solution obtained by diluting 5 μl of anti-S100A8 antibody (manufactured by R & D) or anti-S100A9 (R & D) in 10 mL of PBS containing 4% skim milk, and shaken at room temperature for 60 minutes. After removing the antibody solution, the membrane was washed with 30 mL of 0.1% Tween20-containing PBS for 5 minutes at room temperature. Washing was repeated 5 times. After washing, the membrane was placed in a solution obtained by diluting 5 μl of HRP-labeled anti-goat IgG antibody (GE healthcare) in 10 mL of 4% skim milk-containing PBS and shaken at room temperature for 45 minutes. After removing the antibody solution, the membrane was washed with 30 mL of 0.1% Tween20-containing PBS for 5 minutes at room temperature. Washing was repeated 5 times. The membrane was exposed to light with an ECL detection kit (GE healthcare) to expose the film. The film was developed with a developing device, and S100A8 and S100A9 protein signals were obtained (FIG. 24).

5) Heparin affinity column chromatography was performed in order to purify the factor having bone marrow-derived mesenchymal stem cell mobilization activity in the skin extract. The following operations were performed using an FPLC apparatus (GE healthcare). First, a 2-day-old mouse skin extract was diluted 10-fold with 9-fold volume 20 mM PBS (pH 7.5) at 4 ° C. (Dilution A). In advance, 20 mM PBS (pH 7.5) (300 ml) was poured into HiPrep16 / 10 heparin FF (GE Healthcare) to equilibrate the column. Further, Diluent A was bound to the column. Thereafter, the column was washed with 20 mM PBS (pH 7.5), (300 ml). In order to elute the adsorbed protein, (A solution) 20 mM PBS (pH 7.5), 10 mM NaCl and (B solution) 20 mM PBS (pH 7.5), 500 mM NaCl were prepared. First, the solution A was sent at 100% / solution B 0%, the ratio of solution B was gradually increased, and finally solution A was sent at 0% / solution B 100%. The total liquid feeding amount was 150 mL. The eluate was fractionated into 3 mL portions in silicone-coated containers. 5 μl of each fractionated sample and 5 μl of SDS-PAGE sample buffer (Bio-Rad) were mixed, heated in a heat block at 98 ° C. for 5 minutes, and then cooled to 25 ° C. This sample was applied to e-PAGEL (ATTO) of (5-20% gradient) acrylamide gel, and electrophoresed at 40 mA for 75 minutes using an electrophoresis apparatus (ATTO). After electrophoresis, the migrated protein was detected using Dodeca silver stain kit (Bio-Rad) (FIG. 25).
Each of the fractionated samples was evaluated for migration activity using a Boyden chamber in the same manner as described above (FIG. 26).
The fractionated samples were detected for the presence of S100A8 and S100A9 proteins using the Western blot method in the same manner as described above (FIG. 27).

6) RNA was extracted from newborn mouse skin using Trizol (invitrogen), and cDNA was further synthesized using SuperScript III cDNA synthesis kit (Invitrogen). Using this cDNA as a template, the S100A8 and S100A9 cDNAs were amplified using the PCR method, and the GST tag sequence (SEQ ID NO: 31 (amino acid sequence), SEQ ID NO: 32 (DNA sequence)) was added to the N-terminus of the amino acid sequence. In order to express the obtained protein, it was inserted into a plasmid vector pCAGGS for expressing the protein in mammalian cells. (FIG. 28) Human fetal kidney cell-derived HEK293 cultured cell line was transfected with pCAGGS-GST-S100A8 or pCAGGS-GST-S100A9 using a lipofection reagent (Invitrogen), and after 48 hours, cells and culture supernatant were collected. The cells and culture supernatant were centrifuged at 4400 g for 5 minutes at 4 ° C., and the supernatant (supernatant A) and cells were separated and collected. Cells were disrupted by adding 0.1% Tween20-containing PBS and applying ultrasonic waves for 30 seconds under ice-cooling. Further, the supernatant was collected by centrifugation at 4400 g for 5 minutes at 4 ° C. (Supernatant B). Supernatant A and supernatant B were mixed and added to a HiTrap GST FF column (GE healthcare, 5 mL) whose buffer was replaced with 30 mL of PBS in advance. After the addition, the column was washed with 100 mL of PBS, and the adsorbed protein was eluted with 20 mM PBS (pH 8) containing reduced glutathione. Bone marrow mesenchymal stem cell migration activity was studied using recombinant S100A8 and S100A9 Boyden chambers. In the lower layer of the Boyden chamber, a sample prepared by adjusting purified S100A8 and S100A9 proteins to a concentration of 0.1 ng / μl and dissolving in DMEM, or a sample of 2-day-old mouse skin extract diluted with 4 volumes of DMEM was inserted. . For negative control, proteins were extracted from cells transfected with control vectors into which S100A and S100A9 cDNA had not been inserted, and fractions eluted from the HiTrap GST FF column were used in the same manner. After inserting the sample into the lower layer, place a polycarbonate membrane with 8 μm fine holes, and in contact with it, place the Boyden chamber upper tank (capacity 50 μl) in it, and bone marrow-derived mesenchymal stem cell suspension (5 × 10 ⁴ Pieces / 50 ml culture solution: DMEM / 10% fetal bovine serum) and cultured in a CO ₂ incubator at 37 ° C. for 4 to 24 hours. After culturing, the upper tank of the chamber was removed, the silicone thin film was taken out, and the number of bone marrow-derived mesenchymal stem cells that had migrated to the lower tank through the microhole was quantitatively examined by staining (FIG. 29).

7) 250 μl (1 ng / μl) of the aforementioned purified GST-S100A8 and S100A9 recombinant proteins were injected from the tail vein of 8-week-old male mice. Twelve hours later, under inhalation anesthesia with isoflurane, 1 mL of peripheral blood was collected from the heart of a mouse using a 1 mL syringe coated with heparin, mixed with 3 mL of PBS, and then placed on 3 mL of Ficol (GE healthcare). Layered quietly. Using a centrifuge, centrifugation was performed at 25 ° C. for 400 g for 40 minutes. The cells in the middle layer that were cloudy were collected as the mononuclear cell fraction. 1 mL of hemolytic agent HLBsolution (manufactured by Immunobiological Laboratories) was added to the collected cells and incubated at room temperature for 5 minutes. This hemolysis operation was repeated twice. 10 mL of PBS was added, centrifuged at 440 g for 5 minutes at 25 ° C., and the supernatant was removed to recover the cells. Anti-mouse PE-labeled PDGFRα antibody (manufactured by e-Bioscience), PE-labeled anti-mouse PDGFRβ antibody (e-Bioscience), FITC-labeled anti-mouse CD45 antibody (BD biosciences), PerCy5-labeled anti-mouse CD44 antibody (BD) (biosciences) Each was diluted 100-fold with PBS and incubated for 20 minutes at room temperature. Thereafter, the cells were centrifuged at 25 ° C. and 440 g for 5 minutes to remove the supernatant. 400 μl of PBS containing 1% paraformaldehyde was added to prepare a sample for flow cytometry analysis. The antibody was used in the combination of (I) PDGFRα / CD45 / CD44 (II) PDGFβ / CD45 / CD44. As a result of analysis, the ratio of cells expressing PDGFRα (or β) and CD44 to CD45 weak positive-negative cells was examined. (Fig. 30A, Fig. 30B)

Results: The migration activity of bone marrow mesenchymal stem cells in excised 2-day-old mouse skin and 6-week-old mouse skin was examined, and a stronger activity was found in the skin extract of 2-day-old mice than in 6-week-old mice. From the results of DNA microarray, strong expression of S100A9 was observed in the skin of 2-day-old mice. A correlation was observed between the mesenchymal stem cell migration activity of the sample obtained by roughly purifying the skin extract with a heparin column and the inclusion of S100A9 and S100A8. Expression vectors for these proteins were prepared, and recombinant proteins were produced and purified using HEK293. These purified S100A8 and S100A9 products had bone marrow mesenchymal stem cell migration activity in an assay using Boyden chamber. Moreover, the activity which mobilizes PDGFR (alpha) and a CD44 positive cell group in a peripheral blood was recognized also by mouse | mouth intravenous administration (FIG. 30).

Discussion: The present inventors have now found for the first time in the world that free skin pieces produce S100A8 and S100A9, and the produced S100A8 and S100A9 have a strong activity of migrating bone marrow-derived mesenchymal stem cells. Bone marrow mesenchymal stem cells are known to be pluripotent stem cells that differentiate into bone tissue, adipose tissue, cartilage tissue, fibroblasts, etc. It has also been pointed out that pluripotent stem cells that differentiate into tissues such as cells and epidermal cells also exist. Since the epidermal cells, hair follicle cells, and subcutaneous fibroblasts of the skin graft are composed of bone marrow-derived cells, S100A8 and S100A9 mobilize bone marrow-derived tissue stem cells to the skin graft, and the function of the damaged tissue. It is thought that it induces sexual repair. Since S100A8 and S100A9 can mobilize bone marrow mesenchymal stem cells in peripheral blood by intravenous injection, they can be administered to deep tissues (brain, heart, spinal cord, etc.) that are difficult to administer via peripheral circulation . More efficient recovery of bone marrow stem cells can be achieved by inserting S100A8 and S100A9 into the central hollow portion of the device of the present invention, or by administering to all nerves to allow mobilization of bone marrow stem cells in peripheral blood.

[Reference Example 6]
Objective: Method of mobilizing bone marrow tissue stem cells to peripheral blood by bone marrow-derived tissue stem cell inducer present in skin tissue extract: The above method was studied by the following method.
1) Preparation of bone marrow-derived tissue stem cell inducer: In order to remove tissue after free skin pieces obtained from 25 newborn mice (2 days old) were immersed in 25 ml of PBS (pH 7.4) and incubated at 4 ° C. for 24 hours Then, the mixture was centrifuged at 440 G for 10 minutes at 4 ° C., and the supernatant was collected to prepare a skin extract (SE).
Further, RNA was extracted from C57 / Bl6 newborn mouse skin using Trizol (invitrogen), and cDNA was further synthesized using SuperScript III cDNA synthesis kit (Invitrogen). Using this cDNA as a template, the cDNA of HMGB1 was amplified by PCR, and the Flag tag sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Lys; SEQ ID NO: 30) was added to the N-terminus of the amino acid sequence. To express the protein, it was inserted into a plasmid vector pCAGGS for expressing the protein in mammalian cells (FIG. 36). These plasmid vectors were transfected into HEK293 (human embryonic kidney cell-derived cultured cell line) and cultured for 48 hours to express the protein. Cells and culture supernatants each expressing HMGB1 protein were incubated at 4 ° C. for 16 hours, and then centrifuged at 4400 g for 5 minutes to recover the supernatant. 100 μl of Anti Flag antibody Gel (Sigma) per 50 mL of the supernatant was mixed and incubated at 4 ° C. for 16 hours. The gel was collected by centrifugation and washed 5 times with PBS. Further, elution was performed using 3X Flag peptide (final 100 μg / ml). The concentration of the eluted protein was confirmed using HMGB1 ELISA kit (Sinotest), freeze-dried, and adjusted to 200 μg / mL using PBS.

2) From the tail vein of 8-week-old male mice (C57 / Bl6), 500 μl of the aforementioned skin extract (SE) or 500 μl of PBS as a negative control group was administered using a syringe equipped with a 30G1 / 2 needle (FIG. 37). . 6/12/24/48 hours after administration, under inhalation anesthesia with isoflurane, 1 mL of peripheral blood was collected from the heart of the mouse using a 1 mL syringe coated with heparin, mixed with 3 mL of PBS, and then 3 mL of Ficol (GE layered gently over healthcare). Using a centrifuge, centrifugation was performed at 25 ° C. for 400 g for 40 minutes. The cells in the middle layer that were cloudy were collected as the mononuclear cell fraction. 1 mL of HLB solution (Immuno-Biological Laboratories), which is a hemolytic agent, was added to the collected cells and incubated at room temperature for 5 minutes. This hemolysis operation was repeated twice. 10 mL of PBS was added, centrifuged at 440 g for 5 minutes at 25 ° C., and the supernatant was removed to recover the cells. Anti-mouse PE-labeled PDGFRα antibody (e-Bioscience), PE-labeled anti-mouse PDGFRβ antibody (e-Bioscience), and PerCy5-labeled anti-mouse CD44 antibody (BD biosciences) were each diluted 100-fold with PBS for 1,000,000 cells and incubated at room temperature for 20 minutes. Incubated with. Thereafter, the cells were centrifuged at 25 ° C. and 440 g for 5 minutes to remove the supernatant. 400 μl of PBS containing 1% paraformaldehyde was added to prepare a sample for flow cytometry analysis.
From the tail vein of 8-week-old male mice (C57 / Bl6), 250 μl (1 μg / μl) of mouse HMGB1 or 250 μl of PBS as a negative control group was administered using a syringe equipped with a 30G1 / 2 needle (FIG. 39). 12 hours after administration, under inhalation anesthesia with isoflurane, 1 mL of peripheral blood was collected from the heart of a mouse using a 1 mL syringe coated with heparin, mixed with 3 mL of PBS, and then gently over 3 mL of Ficol (GE healthcare). Layered. Using a centrifuge, centrifugation was performed at 25 ° C. for 400 g for 40 minutes. The cells in the middle layer that were cloudy were collected as the mononuclear cell fraction. To the collected cells, 1 mL of hemolytic agent HLBsolution (Immuno-Biological Laboratories) was added and incubated at room temperature for 5 minutes. This hemolysis operation was repeated twice. 10 mL of PBS was added, centrifuged at 440 g for 5 minutes at 25 ° C., and the supernatant was removed to recover the cells. Anti-mouse PE-labeled PDGFRα antibody (e-Bioscience) and PerCy5-labeled anti-mouse CD44 antibody (BD biosciences) were each diluted 100-fold with PBS and incubated for 20 minutes at room temperature. Thereafter, the cells were centrifuged at 25 ° C. and 440 g for 5 minutes to remove the supernatant. 400 μl of PBS containing 1% paraformaldehyde was added to prepare a sample for flow cytometry analysis.

Results: It was confirmed that PDGFRα-positive and CD44-positive cells were mobilized significantly in peripheral blood 12 hours after injection of the skin extract (SE) (FIG. 38). It was confirmed that PDGFRα-positive and CD44-positive cells were mobilized significantly in peripheral blood 12 hours after the injection of HMGB1 (FIG. 40).

[Reference Example 7]
Objective: To confirm whether mesenchymal stem cells are mobilized into peripheral blood by intravenous administration of recombinant HMGB1 protein.
Method: C57BL6 mice (8-10 weeks old, male) were administered 400 μl (40 μg HMGB1) or 400 μl of physiological saline from the tail vein of recombinant HMGB1 protein / saline (100 μg / ml). After 12 hours, mouse peripheral blood was collected and PBS was added to dilute the total volume to 4 mL. 3 mL of Ficoll-Paque Plus (GE) solution was inserted into the centrifuge tube, and diluted blood was layered thereon. Centrifugation was performed at 100 G (18 ° C.) for 10 minutes, and the intermediate layer containing mononuclear cells was collected in a new centrifuge tube, 45 mL of PBS was added, and centrifugation was performed at 440 G (18 ° C.) for 5 minutes to remove the supernatant. Further, 45 mL of PBS was added once more and centrifuged at 440 G (18 ° C.) for 5 minutes to remove the supernatant. The resulting mononuclear cells were reacted with Phycoerythrobilin (PE) -labeled anti-mouse PDGFRα antibody and Allophycocyanin (APC) -labeled anti-mouse CD44 antibody, followed by flow cytometry (Facscan; manufactured by Becton, Dickinson and Company). The frequency of PDGFRα positive / CD44 positive cells in the fraction was evaluated.

Results: 12 hours after administration of HMGB1, it was revealed that PDGFRα-positive and CD44-positive cells and PDGFRα-positive and CD44-negative cells in the peripheral blood mononuclear cell fraction were significantly increased (FIG. 41). That is, it was shown that HMGB1 has an activity of mobilizing PDGFRα-positive cells known as markers of mesenchymal stem cells from the bone marrow to the peripheral blood.

Discussion: PDGFRα and CD44 are known as surface markers for bone marrow mesenchymal stem cells that represent bone marrow-derived pluripotent stem cells. Bone marrow mesenchymal stem cells are pluripotent stem cells that can differentiate into bone cells, chondrocytes, and adipocytes, and can also differentiate into nerve cells, epithelial cells, and the like. In addition, since the skin pieces used in this experiment are ischemic, the tissue gradually becomes necrotic, and intracellular proteins such as nuclei are released from the proteins on the surface of the cells to the surroundings. HMGB1 is a protein contained in the skin extract. In skin grafts, these proteins act as signals to mobilize bone marrow-derived tissue stem cells into the skin graft, reconstruct bone marrow stem cell-derived epidermis, subcutaneous tissue, hair follicle tissue, etc. within the skin graft to regenerate functional skin it seems to do. This experiment is the first discovery that has succeeded in mobilizing bone marrow-derived tissue stem cells in the peripheral circulation by intravenous administration of such skin extract or HMGB1. By this discovery, bone marrow-derived pluripotent stem cells can be mobilized in peripheral blood, and more efficient recovery of bone marrow stem cells can be achieved by the device of the present invention.

  The device of the present invention has enabled the minimally invasive recovery of functional cells in vivo, which has been difficult in the past. As a result, progress in basic research using these functional cells derived from living organisms and progress in regenerative medicine using recovered cells is expected, and it is expected to become an innovative technology for providing new medical care to patients suffering from intractable diseases Is done. The device of the present invention is a seed for the development of new medical materials, and is expected to contribute to industrial progress in this field.

Claims (12)

An in-vivo implantable substance collecting device for collecting a target substance existing in a living tissue, a flat container having a central hollow part between a side wall part, an upper wall part, and a lower wall part, 1 or more openings are provided in the wall and / or the lower wall, and is a substance collection device provided with a flat container capable of holding the collected target substance in the central hollow part ,
The target substance is bone marrow stem cells;
A substance collection device in which the bone marrow stem cells are mobilized by a substance or composition containing at least one selected from the following groups (a) to (r) inserted into the flat container:
(A) HMGB1 (High mobility group box 1) protein
(B) Cells secreting HMGB1 protein
(C) A vector into which DNA encoding HMGB1 protein has been inserted
(D) HMGB2 (High mobility group box 2) protein
(E) Cells that secrete HMGB2 protein
(F) A vector into which DNA encoding HMGB2 protein has been inserted
(G) HMGB3 (High mobility group box 3) protein
(H) Cells secreting HMGB3 protein
(I) A vector into which DNA encoding HMGB3 protein has been inserted
(J) S100A8 protein
(K) Cells secreting S100A8 protein
(L) A vector into which DNA encoding S100A8 protein has been inserted
(M) S100A9 protein
(N) Cells that secrete S100A9 protein
(O) A vector into which DNA encoding S100A9 protein has been inserted
(P) Hyaluronic acid
(Q) Skin or peripheral blood mononuclear cell or tissue extract
(R) A heparin-binding fraction of a cell or tissue extract of skin or peripheral blood mononuclear cells .   2. The substance collection device according to claim 1, wherein the upper wall portion and / or the lower wall portion of the flat container has any one of a flat surface, a convex curved surface, a concave curved surface, and a shape in which a central portion is recessed in a concave shape.   3. The substance collection device according to claim 1, wherein the flat shape of the flat container has a shape selected from a polygon, a substantially polygon, a circle, a substantially circle, an ellipse, a substantially ellipse, a trapezoid, and a substantially trapezoid. .   The substance collection device according to any one of claims 1 to 3, wherein joint portions between the upper wall portion and the side wall portion of the flat container and between the lower wall portion and the side wall portion have a curved shape.   The said flat container is a substance collection device of any one of Claims 1-4 comprised by combining the components containing an upper wall part and the components containing a lower wall part.   The substance collection device according to claim 1, wherein the flat container is made of a silicone resin material.   The said flat container is a substance collection device of any one of Claims 1-6 inserted between the epithelium and dermis of a biological body. The substance or composition is present in at least one medium selected from the group consisting of a polymer or copolymer of polyethylene glycol (PEG), cyclodextrin, gelatin, albumin, chitosan, sodium carboxymethylcellulose, and physiological saline. The substance collection device according to claim 1 . A kit comprising the substance collection device according to claim 1 , further comprising a substance or a composition comprising at least one selected from the following groups (a) to (r):
(A) HMGB1 protein (b) Cell that secretes HMGB1 protein (c) Vector inserted with DNA encoding HMGB1 protein (d) HMGB2 protein (e) Cell that secretes HMGB2 protein (f) Encodes HMGB2 protein Vector inserted with DNA (g) HMGB3 protein (h) Cells secreting HMGB3 protein (i) Vector inserted with DNA encoding HMGB3 protein (j) S100A8 protein (k) Cells secreting S100A8 protein (l ) Vector inserted with DNA encoding S100A8 protein (m) S100A9 protein (n) Cell secreting S100A9 protein (o) Vector inserted with DNA encoding S100A9 protein (p) Hyaluronic acid (q) Skin or cell or tissue extract of peripheral blood mononuclear cells (r) of the skin or peripheral blood mononuclear cells or tissues Hepa of extract of Down bound fraction. The substance according to claim 9 , further comprising at least one medium selected from the group consisting of a polymer or copolymer of polyethylene glycol (PEG), cyclodextrin, gelatin, albumin, chitosan, sodium carboxymethylcellulose, and physiological saline. Kit containing collection device. A method for producing an implantable substance collection device for recovering bone marrow stem cells present in in vivo tissue,
A flat container having a central hollow part between a side wall part, an upper wall part, and a lower wall part, wherein one or more openings are provided in the upper wall part and / or the lower wall part, and recovered bone marrow stem cells Prepare a flat container that can be held in the central hollow portion,
A production method comprising a step of inserting a substance or composition containing at least one selected from the following groups (a) to (r) into the flat container:
(A) HMGB1 protein (b) Cell that secretes HMGB1 protein (c) Vector inserted with DNA encoding HMGB1 protein (d) HMGB2 protein (e) Cell that secretes HMGB2 protein (f) Encodes HMGB2 protein Vector inserted with DNA (g) HMGB3 protein (h) Cells secreting HMGB3 protein (i) Vector inserted with DNA encoding HMGB3 protein (j) S100A8 protein (k) Cells secreting S100A8 protein (l ) Vector inserted with DNA encoding S100A8 protein (m) S100A9 protein (n) Cell secreting S100A9 protein (o) Vector inserted with DNA encoding S100A9 protein (p) Hyaluronic acid (q) Skin or cell or tissue extract of peripheral blood mononuclear cells (r) of the skin or peripheral blood mononuclear cells or tissues Hepa of extract of Down bound fraction. The substance or composition is present in at least one medium selected from the group consisting of a polymer or copolymer of polyethylene glycol (PEG), cyclodextrin, gelatin, albumin, chitosan, sodium carboxymethylcellulose, and physiological saline. The manufacturing method according to claim 11 .