Method for producing mature adipocyte-containing composition

Abstract

To provide a method for producing a mature adipocyte-containing composition which has high revascularization ability and low possibility of inducing inflammation.SOLUTION: A method for producing mature adipocyte-containing composition includes: a cutting and filtering step of cutting and filtering an adipose tissue to which enzyme treatment is not performed to obtain micro-fat; a micro-fat culturing step of culturing micro-fat by using a culture medium after the cutting and filtering step to obtain a mature adipocyte; and a mature adipocyte-containing composition acquisition step of obtaining a mature adipocyte-containing composition containing the mature adipocyte and a conditioned medium obtained by subjecting micro-fat to the micro-fat culturing step, where the culture medium contains self serum or contains FBS, hydrocortisone and FGF-2.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to a method for producing a mature adipocyte-containing composition. More specifically, it relates to a method for producing a mature adipocyte-containing composition containing mature adipocytes and a conditioned medium (e.g., supernatant of mature adipocytes) that promotes revascularization of adipose tissue for transplantation and improves the survival rate of fat transplantation. [Background technology]

[0002] Fat transplantation as a breast reconstruction procedure after breast cancer resection is considered safe and does not promote cancer metastasis. For this reason, breast reconstruction is frequently performed in the fields of plastic surgery and cosmetic surgery. However, due to the large amount of tissue in the breast, simply transplanting fat tissue fragments obtained by aspirating fat tissue from the body has only achieved a 30-45% survival rate (Zhou et al., Non-Patent Document 1).

[0003] The main reason for the low survival rate of aspirated lipoaspirates in the recipient environment is the lack of vascular supply. Because transplanted adipose tissue fragments are separated from their original vascular supply, they become malnourished and suffer from necrosis. It is well known that adipose tissue fragments are recognized as foreign bodies after transplantation, resulting in absorption, cyst formation, and calcification. To improve engraftment rates, a method has been reported in which adipose tissue is transplanted together with adipose-derived stem cells (ASCs) present in the small vascular fraction (SVF) in the stromal vascular compartment surrounding the blood vessels of adipose tissue (cell-assisted lipotransfer: CAL (Eto et al., Non-Patent Document 2)). This method involves enzymatically treating the aspirated lipoaspirate to remove fat components, and then extracting SVF preadipocytes, cells present around the blood vessels, which are then transplanted together with the aspirated lipoaspirate. SVF is characterized by its high self-renewal capacity and multipotency (Zuk PA, et al., Non-Patent Document 3).

[0004] Zhou et al. systematically reviewed 17 articles including 387 cases (Zhou et al., Non-Patent Document 1). The fat survival rate was significantly higher in the CAL group than in the single liposuction and lipotransfer group (60% vs. 45%, p = 0.0096). CAL significantly improved facial fat survival (19%) and reduced the incidence of multiple surgeries (13.6%). However, in breast fat grafting, the improvement in fat survival was only 9%, which was not statistically significant.

[0005] On the other hand, cell-assisted lip transfer (CAL) in breast cases (Eto et al., Non-Patent Document 2) had a higher incidence of complications than facial cases (p <0.001). CAL is a method that utilizes adipose-derived stem cells (ASCs) present in the stromal vascular fraction (SVF) obtained by enzymatically treating adipose tissue, and transplants ASCs, cells that regenerate blood vessels, together with adipose tissue. However, most CAL procedures end in failure due to absorption of the transplanted fat. This is thought to be because the transplanted adipose tissue is separated from its original nutrient vessels, and while some (fat under 300 microns in size) remains, much of it becomes malnourished and suffers from necrosis.

[0006] To date, two cells have been successfully developed for vascular regeneration: adipose stem cells (ASCs), which are fat precursor cells, and dedifferentiated fat cells (DFATs).

[0007] ASCs are fat precursor cells, but they are also stem cells with the multipotency to differentiate not only into adipocytes but also into bone cells, muscle cells, chondrocytes, and other cells (Zuk PA, et al., Non-Patent Document 3). Furthermore, because they have been reported to promote cancer metastasis, they are unsuitable for fat transplantation, which is frequently performed in breast reconstruction surgery after breast cancer resection. Furthermore, ASCs induce large amounts of TNF-α in hypoxic conditions, which can cause inflammation, and because absorption due to inflammation after transplantation cannot be suppressed, they are unsuitable for transplantation.

[0008] Furthermore, the factors that cause DFAT cells to induce blood vessels have not been identified, and the unclear mechanism is also a factor hindering their application to humans. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Zhou Y, Wang J, Li H, et al. Efficacy and Safety of Cell-Assisted Lipotransfer: A Systematic Review and Meta-Analysis. Plast Reconstr Surg 2016: 137: 44e-57e. [Non-patent document 2] Eto H, et al.: The fate of adipocytes after non vascularized fat grafting: Evidence of early death and replacement of adipocytes. Plast Reconstr Surg. 2012;129(5):1081-92. [Non-patent document 3] Zuk PA. et al.: Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2001; 13:4279-95. Summary of the Invention [Problem to be solved by the invention]

[0010] Given the above background, there has been a demand for a mature adipocyte-containing composition with high vascular regeneration ability, as well as a mature adipocyte-containing composition with low potential for inflammation.

[0011] Furthermore, there was a demand for vascular regeneration promoters containing such mature adipocyte-containing compositions, compositions to be used as substitutes for subcutaneous adipose tissue, and fat-containing agents for use in breast augmentation, breast reconstruction, or tissue recession surgery. [Means for solving the problem]

[0012] The present invention is basically based on the finding from examples that adipose tissue is cut and filtered, microadipose tissue is cultured using a culture medium to obtain mature adipocytes, and the composition containing the conditioned medium (culture supernatant) obtained during the culture contains a large amount of chemokines / cytokines that promote angiogenesis, such as IL-8, GRO, MCP-1 or VEGF (vascular endothelial growth factor), and that breast regeneration surgery, for example, was extremely successful when this composition was used.

[0013] The first invention described in this specification relates to a method for producing a mature adipocyte-containing composition, which includes a cutting and filtering step, a micro-adipose culturing step, and a mature adipocyte-containing composition obtaining step. The cutting and filtration step is a step in which adipose tissue that has not been subjected to enzyme treatment is cut and filtered to obtain minute fat particles. The microfat culture step is a step for culturing the microfat in a culture medium after the cutting and filtration step to obtain mature adipocytes. The culture medium contains hydrocortisone and FGF-2. The mature adipocyte-containing composition obtaining step is a step for obtaining a mature adipocyte-containing composition containing the mature adipocytes and a conditioned medium obtained through the microadipocyte culture step.

[0014] A preferred example of adipose tissue is liposuction that has been washed and then blood components removed.

[0015] A preferred example of the cutting and filtering step includes a step of performing centrifugation to remove precipitated stromal vascular cells (SVF).

[0016] Preferred examples of mature adipocytes are cells that express the chemokines / cytokines IL-8, GRO, and MCP-1 at levels four times or more (preferably ten times or more, and more preferably twenty times or more) than IL-6. The conditioned medium preferably contains these chemokines / cytokines. The conditioned medium preferably contains vascular endothelial growth factor (VEGF) at levels between 0.5 and 1.5 times higher than IL-6.

[0017] A preferred example of the use of the mature adipocyte-containing composition is as a subcutaneous adipose tissue substitute. The mature adipocyte-containing composition may also be used in breast augmentation, breast reconstruction, or tissue cupping surgery.

[0018] The second invention described in this specification relates to a composition used as a substitute for subcutaneous adipose tissue. This composition is a mature adipocyte-containing composition produced by any of the above-mentioned methods for producing a mature adipocyte-containing composition, and is used as a substitute for subcutaneous adipose tissue. This composition is used, for example, in breast augmentation surgery, breast reconstruction surgery, or tissue cupping surgery.

[0019] The third invention described in this specification relates to a vascular regeneration promoter. This vascular regeneration promoter includes, for example, a mature adipocyte-containing composition obtained by any of the above-mentioned methods for producing a mature adipocyte-containing composition. The mature adipocyte-containing composition includes vascular endothelial growth factor receptor (VEGFR) 2 or positive cells and platelet-derived growth factor (PDGFR) β. [Effects of the Invention]

[0020] The mature adipocyte-containing composition obtained by the first invention usually highly expresses IL-8, GRO, MCP-1, or VEGF, which have high angiogenesis-promoting properties, and therefore has high vascular regeneration potential.

[0021] Furthermore, as mentioned above, ASCs induce large amounts of TNF-α under hypoxic conditions, which may cause inflammation and make them unsuitable for transplantation. On the other hand, the mature adipocyte-containing composition obtained by the first invention highly expresses IL-8, GRO, and MCP-1, which promote angiogenesis, while hardly expressing TNF-α, IL-1β, or INF-γ. Therefore, the mature adipocyte-containing composition obtained by the above method is less likely to cause inflammation.

[0022] As described above, the second invention contains a mature adipocyte-containing composition, which can be used as a substitute for subcutaneous adipose tissue, as well as for breast augmentation, breast reconstruction, or tissue depression surgery. Furthermore, the mature adipocyte-containing composition highly expresses IL-8, GRO, and MCP-1, which promote angiogenesis, and can therefore be used as a vascular regeneration promoter. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a photograph, in lieu of a drawing, showing the state of finely divided, filtered, and crushed microfat particles placed in a culture flask. [Figure 2] FIG. 2 is a phase contrast micrograph, in place of a drawing, showing mature adipocytes in culture. [Figure 3] Figure 3 is a photograph, instead of a drawing, of mature adipocytes in culture stained with Sudan III. [Figure 4] FIG. 4 is a graph instead of a drawing showing the results of cytokine / chemokine analysis of conditioned media. [Figure 5] FIG. 5 is a photograph, in place of a drawing, showing a specimen excised 6 months after transplantation of a human lipoaspirate piece alone into the abdomen of a nude mouse. [Figure 6] FIG. 6 is a photograph, in place of a drawing, showing an excised specimen six months after transplantation of human adipose tissue, cultured adipocytes, and conditioned medium from the abdominal region of a nude mouse. [Figure 7] FIG. 7 is a photograph, in place of a drawing, showing an excised specimen six months after transplantation of human adipose tissue, cultured adipocytes, and conditioned medium from the abdominal region of a nude mouse. [Figure 8]Figure 8 is a photograph in lieu of a drawing showing an example of breast reconstruction surgery after left breast cancer surgery. [Figure 9] FIG. 9 is a photograph, instead of a drawing, showing the results of immunostaining of VEGFR2 and PDGFRβ on the membrane of cultured adipocytes. [Figure 10] FIG. 10 is a graph, instead of a drawing, showing the number of adipocytes that can be passaged and PDL. [Figure 11] FIG. 11 is a photograph, instead of a drawing, showing cultured adipocytes on day 4 of P2 culture in α-MEM alone (control). [Figure 12] FIG. 12 is a photograph, in place of a drawing, showing cultured adipocytes on day 4 of culture in mesenchymal stem cell serum-free medium P2. [Figure 13] FIG. 13 is a photograph, instead of a drawing, showing adipocytes cultured in a medium supplemented with α-MEM and 10% fetal calf serum (FCS). DETAILED DESCRIPTION OF THE INVENTION

[0024] The following describes embodiments for carrying out the present invention. The present invention is not limited to the embodiments described below, and also includes appropriate modifications of the embodiments below within the scope obvious to those skilled in the art.

[0025] The first invention described in this specification relates to a method for producing a mature adipocyte-containing composition. A mature adipocyte-containing composition is, for example, a composition containing mature adipocytes derived from humans. For example, Patent No. 5991687 describes a method for isolating mature adipocytes. Thus, mature adipocytes are publicly known. The size of mature adipocytes (the length of the part giving the maximum length) may be 60 to 100 μm, or preferably 1 μm to 60 μm, or may be 5 μm to 40 μm.

[0026] This method includes a cutting and filtering step, a microfat culture step, and a step of obtaining a composition containing mature adipocytes.

[0027] Cutting and filtration process The cutting and filtration step is a step for cutting and filtering adipose tissue that has not been subjected to enzyme treatment to obtain microfat. A preferred example of adipose tissue is that from which blood components have been removed after washing aspirated fat. The adipose tissue from which blood components have been removed may be sterilized with antibiotics and then filtered to finely crush the cells. The cutting and filtration step preferably includes a step of centrifuging to remove the precipitated stromal vascular cells (SVF). In this case, only the adipose components that float to the top of the liquid phase obtained by centrifugation are collected to obtain microfat. A known method may be used as appropriate for centrifugation.

[0028] Microfat culture process The microfat culture step is a step for culturing the microfat using a culture medium after the cutting and filtration step to obtain mature adipocytes. These mature adipocytes are included in the cultured cell group. The culture medium is a medium containing hydrocortisone and FGF-2. A preferred culture medium is a medium containing autologous serum or a medium containing FBS, hydrocortisone, and FGF-2. The culture medium may also be a medium containing autologous serum, hydrocortisone, and FGF-2.

[0029] The medium of the present invention can be prepared by adding necessary elements to a basal medium as appropriate. Examples of basal media include α-MEM medium, Eagle's basal medium, and DMEM. Known reagents used in culture media can be added as appropriate. Examples of reagents include fetal bovine serum (FBS), HC (hydrocortisone), FGF2, IGF (insulin-like growth factor), insulin, PDGF (platelet-derived growth factor), ACTH (adrenocorticotropic hormone), LIF (leukemia inhibitory factor), TGFβ, BMP, steroids, fatty acids, soybean trypsin inhibitor, ascorbic acid, hyaluronic acid, proline, dexamethasone, insulin, transferrin, and selenite. When adding ascorbic acid or the like to the medium, it may be added in the form of a salt, such as 2-phosphate. Each should be added to the medium at a concentration of 0.1 ng / mL to 20 μg / mL (or 0.2 ng / mL to 10 μg / mL). These can be added after adjusting appropriately depending on the degree of purification and the required amount. Autologous serum may be added instead of FBS. A serum-free medium may also be used. In any case, it is preferable to add HC (hydrocortisone) and FGF-2 to the medium.

[0030] An example of a culture medium is α-MEM medium supplemented with 1-10% fetal bovine serum (FBS), 20 ng / ml to 100 ng / ml hydrocortisone, and 5 ng / ml to 20 ng / ml (or 50 ng / ml) FGF2 (Fibroblast Growth Factor 2). 1-10% autologous serum may be added in addition to or instead of the 1-10% fetal bovine serum (FBS). The amounts of these may be adjusted appropriately. For example, the medium may contain 0.1% to 20% FBS and autologous serum.

[0031] The microfat cells can be cultured and grown under standard culture conditions. The cell volume can range from around 10% to 100% confluence, and they can also be cultured at high densities, even exceeding 100% confluence, in a layered state. They can be placed in a hypoxic state immediately after transplantation, or after being left to stand for a while. Hypoxic culture can be achieved using a hypoxic incubator that lowers the oxygen partial pressure by mixing commercially available nitrogen gas, or by blowing nitrogen gas into an appropriate space to lower the oxygen partial pressure.

[0032] The culture conditions are preferably 3% to 20% carbon dioxide and 1% to 10% oxygen, or alternatively 5% to 20% carbon dioxide and 2% to 10% oxygen, or 8% to 12% carbon dioxide and 3% to 7% oxygen.

[0033] Process for obtaining mature adipocyte-containing composition The step of obtaining a composition containing mature adipocytes is a step for obtaining a composition containing mature adipocytes, which comprises mature adipocytes and a conditioned medium obtained through a microadipocyte culture step. The composition containing mature adipocytes may comprise mature adipocytes and a culture supernatant.

[0034] Preferred examples of mature adipocytes are cells that express the chemokines / cytokines IL-8, GRO, and MCP-1 at levels four times higher than IL-6. They may also express one or more of IL-8, GRO, and MCP-1 at levels ten times higher than IL-6, or at levels twenty times higher than IL-6, or at levels twenty times higher than IL-6. By containing cytokines / chemokines with high vascular regeneration-promoting ability and low inflammatory potential, mature adipocyte-containing compositions exhibit high vascular regeneration-promoting ability without causing inflammation when transplanted, making them suitable for transplantation. It is particularly preferred that this mature adipocyte-containing composition contains fat collected from the patient to be transplanted.

[0035] Preferred examples of mature adipocytes are those that express one or more of IL-8, GRO and MCP-1 at a level 10 times or more compared to TNF-α, preferably those that express it at a level 50 times or more compared to TNF-α, preferably those that express it at a level 100 times or more compared to TNF-α, and preferably those that express it at a level 500 times or more compared to TNF-α.

[0036] Preferred examples of mature adipocytes are those that express one or more of IL-8, GRO, and MCP-1 at a level 10 times or more compared to IL-1-β, preferably at a level 50 times or more compared to IL-1-β, preferably at a level 100 times or more compared to IL-1-β, and preferably at a level 500 times or more compared to IL-1-β.

[0037] Preferred examples of mature adipocytes are those that express one or more of IL-8, GRO and MCP-1 at a level 10 times or more compared to INF-γ, preferably at a level 50 times or more compared to INF-γ, preferably at a level 100 times or more compared to INF-γ, and preferably at a level 500 times or more compared to INF-γ.

[0038] The mature adipocyte-containing composition may contain an appropriate amount of mature adipocytes and conditioned medium (or culture supernatant) depending on the purpose. This composition may be stored in a required container in the same way as a normal composition, and may be used as needed. The amount of mature adipocytes may be, for example, 1 x 10 per use. 4 More than 1 x 10 cells 10 Cells are acceptable, or 1 x 10 per use 5 More than 1 x 10 cells 8 It may also be a cell.

[0039] Agents containing a culture supernatant as an active ingredient are publicly known, as disclosed in, for example, JP 2013-18756 A, Japanese Patent No. 5139294, and Japanese Patent No. 5526320. Therefore, a composition containing a conditioned medium can be produced using a publicly known method.

[0040] Examples of mature adipocyte culture supernatants include a processed product obtained by freeze-drying the culture supernatant, which is the supernatant component obtained by solid-liquid separation of the culture supernatant by centrifugation, to remove water; a processed product obtained by concentrating the culture supernatant under reduced pressure using an evaporator or the like; a processed product obtained by concentrating the culture supernatant using an ultrafiltration membrane or the like; a processed product obtained by solid-liquid separation of the culture supernatant using a filter; or the undiluted culture supernatant before the above-mentioned processes. Furthermore, for example, the supernatant obtained by culturing the mature adipocytes of the present invention may be centrifuged (e.g., 1,000 × g, 10 minutes), fractionated with ammonium sulfate (e.g., 65% saturated ammonium sulfate), the precipitate suspended in an appropriate buffer, dialyzed, and filtered through a syringe filter (e.g., 0.2 μm) to obtain a sterile culture supernatant. The collected culture supernatant may be used as is, or may be frozen and thawed prior to use. Alternatively, a pharmaceutically acceptable carrier may be added and dispensed into sterile containers to obtain a liquid volume that is easy to handle. Furthermore, as a countermeasure against the risk of infectious pathogens, the culture supernatant may be treated with a virus clearance filter or by ultraviolet irradiation. The mature adipocyte-containing composition preferably contains 1 mL to 1,000 mL of culture supernatant as a single dosage unit, and more preferably 30 mL to 300 mL.

[0041] A preferred example of the use of the mature adipocyte-containing composition is as a subcutaneous adipose tissue substitute. This specification also provides a method for subcutaneous adipose tissue substitution, which includes the step of administering a mature adipocyte-containing composition to a human patient. In this case, the mature adipocyte-containing composition preferably further contains the patient's adipose tissue in addition to mature adipocytes and conditioned medium. The mature adipocyte-containing composition may also be used in breast augmentation surgery, breast reconstruction surgery, or tissue depression surgery. For example, breast reconstruction surgery includes the step of injecting a mature adipocyte-containing composition into a breast lacking fat components.

[0042] The second invention described in this specification relates to a composition used as a substitute for subcutaneous adipose tissue. This composition is a mature adipocyte-containing composition produced by any of the above-mentioned methods for producing a mature adipocyte-containing composition, and is used as a substitute for subcutaneous adipose tissue. This composition is used, for example, in breast augmentation surgery, breast reconstruction surgery, or tissue cupping surgery.

[0043] The third invention described in this specification relates to a vascular regeneration promoter. This vascular regeneration promoter includes, for example, a mature adipocyte-containing composition obtained by any of the above-mentioned methods for producing a mature adipocyte-containing composition. In other words, this vascular regeneration promoter is the same as the above-mentioned mature adipocyte-containing composition. The vascular regeneration promoter may be, for example, an injectable agent. The above-mentioned mature adipocyte-containing composition may be contained in a syringe, mixed with the patient's tissue as needed, and transplanted into the required location on the patient. [Example]

[0044] Culture method Composition of culture medium for cultured fat After obtaining informed consent, adipose tissue was aspirated from the abdomen, thigh, buttocks, and upper arm using a cannula with a diameter of 2 mm or less (diameter not limited). The aspirated fat was washed to remove blood components. This tissue was then sterilized with antibiotics, then finely filtered to crush the fat into small pieces, and centrifuged to culture only the fat components that floated to the top of the liquid phase. SVF precipitated as a pellet at the bottom when centrifuged, so it was removed.

[0045] This microfat was placed in a culture flask and culture began. Figure 1 is a photograph, in place of a drawing, showing the state of the finely filtered and crushed microfat placed in a culture flask. As shown in Figure 1, the SVF has been removed and only the fat component remains. The amount of medium used during culture was kept small enough so that the tissue did not float. After five days of culture, it was confirmed that cells had adhered to the tissue fragments, so additional medium was added and culture continued.

[0046] The culture medium used was α-MEM supplemented with 5% fetal bovine serum (FBS), 40 ng / ml hydrocortisone, and 10 ng / ml fibroblast growth factor 2 (FGF2). The culture conditions were 10% carbon dioxide and 5% oxygen.

[0047] On day 12 of primary culture, 2–3 × 10 6 The cultured cells were harvested. Some of these cells were frozen, and the other cells were frozen at 4–5 × 10 4 / cm 2 The cells were seeded at a density of 1000 and cultured for 5 days. After 6 days, they were washed three times with PBS(-) and cultured for 2 days in a medium containing the above medium components but excluding FBS. This medium was used as conditioned medium for analysis.

[0048] Figure 2 is a phase-contrast micrograph, in place of a drawing, showing mature adipocytes in culture. As shown in Figure 2, the adipocytes contained many fat granules. Sudan III staining was performed to confirm the mature adipocytes in culture. Mature adipocytes also stain specifically red with Sudan III, a special stain for fat. The results are shown in Figure 3. Figure 3 is a photograph, in place of a drawing, of mature adipocytes in culture stained with Sudan III. As shown in Figure 3, it can be seen that the medium contains many fat granules. [Example]

[0049] Cytokine / chemokine analysis of conditioned medium from cultured mature adipocytes was performed using the antibody-immobilized magnetic bead method. The conditioned medium sample was centrifuged at 13,000 G at 4°C for 5 minutes, and the supernatant was used for measurement. The concentrations of 40 target proteins in the conditioned medium were measured using the Luminex® system. 25 μL of pretreated sample was used per well, and measurements were performed in triplicate. One standard solution was added according to the manual, and seven 5-fold dilution series were prepared and measured in triplicate. The 40 target proteins are EGF, FGF-2, eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, fractalkine, IFNα2, IFNγ, GRO, IL-10, MCP-3, IL-12P40, MDC, IL-12P70, PDGF-AA, IL-13, PDGF-AB / They were BB, IL-15, sCD40L, IL-17A, IL-1RA, IL-1α, IL-9, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, TNFα, TNFβ, and VEGF.

[0050] Figure 4 is a graph (in place of a drawing) showing the results of cytokine / chemokine analysis of the conditioned medium. As shown in Figure 4, it was demonstrated that the conditioned medium of cultured mature adipocytes produces large amounts (ng / ml) of IL-8, GRO, and MCP-1, cytokines with high vascular regeneration potential. On the other hand, these cells produce very little TNF-α, which increases in adipose stem cells and causes inflammation, or IL-1-β and INF-γ, which have strong inflammation-inducing properties, and therefore are safe. [Example]

[0051] Fat transplantation experiment into nude mice - Mouse transplantation experiments and examination of the transplanted tissue images - After obtaining informed consent, adipose tissue was aspirated from the abdomen. The cells were sterilized and inoculated in the same manner as above. On the 12th day of primary culture, 2 × 10 6 Cultured adipocytes were collected from 100 cells. These cells were temporarily frozen and stored. Before transplantation, the cells were thawed and diluted to 5 × 10 4 cells / cm 2 The cultured adipocytes were seeded at a density of 3 × 10 for 0.6-0.7 g of fat to be transplanted. 6 The cells were added to the mixture and the mixture was transplanted subcutaneously into the backs of nude mice the following day. Mice in the control group were transplanted with fat alone. Six months after transplantation, the tissue was excised and subjected to histological staining. When only human adipose tissue was transplanted, the adipose tissue was almost completely absorbed, but when cultured cells and human fat were co-transplanted, the adipose tissue remained intact. Furthermore, blood vessels were observed to form in the center.

[0052] A human lipoaspirate piece was transplanted alone into the abdomen of a nude mouse, and the excised specimen was stained with Oil Red O 6 months after transplantation. Figure 5 is a photograph, in place of a drawing, showing a specimen excised 6 months after transplantation of a human lipoaspirate piece alone into the abdomen of a nude mouse. The magnification of Figure 5 is 100x. Although some areas where adipose tissue has formed can be seen around the periphery, the hollow central area is the area where fat was absorbed after transplantation. Scar tissue and some adipose tissue have formed around it.

[0053] Human adipose tissue, cultured adipocytes, and conditioned medium were co-transplanted into the abdomen of a nude mouse, and the excised specimen was stained with Oil Red O 6 months after transplantation. Figure 6 is a photograph in place of a drawing showing an excised specimen 6 months after co-transplantation of human adipose tissue, cultured adipocytes, and conditioned medium into the abdomen of a nude mouse. The magnification of Figure 6 is 100x. Adipose tissue was formed in more than 95% of the specimen tissue. Vascular tissue was observed inside.

[0054] Human adipose tissue, cultured adipocytes, and conditioned medium were co-transplanted into the abdomen of nude mice. To confirm that the adipose tissue was of human origin, it was stained using fluorescent immunostaining (Human nuclear antigen antibody 231-1 [Alexa Fluor® 488] Novus, Southpark Way, USA). Figure 7 is a photograph (in lieu of a drawing) showing a specimen excised 6 months after co-transplantation of human adipose tissue, cultured adipocytes, and conditioned medium into the abdomen of a nude mouse. Adipose tissue was formed throughout the specimen tissue. Vascular-like tissue was also observed within. [Example]

[0055] Fat transplantation into humans (total mastectomy reconstruction) Example 4-1: Cultured mature adipocytes, conditioned medium, and autologous fat transplantation for breast reconstruction after left breast cancer surgery A 46-year-old woman underwent surgery for left breast cancer. A tissue expander was placed in the chest at the time of breast cancer resection to expand the subcutaneous tissue and pectoralis major muscle, creating a bulge in the chest. Later, at the patient's request, this procedure was scheduled. 2 cc of fat was aspirated from the abdomen, sterilized, and seeded and cultured. On the 14th day of primary culture, 3 × 10 6 The cultured adipocytes were collected from the cells. These cultured adipocytes were temporarily frozen and stored. Before transplantation, the cells were thawed and placed in a 150 cm2 basal area. 2 The cells were cultured for 6 days in 12 flasks. The aspiration fat tissue to be transplanted was mixed with mature cultured cells and conditioned medium and transplanted. The first transplantation was performed around the subcutaneous tissue expander, with a total volume of 252 ml (17.16 × 10 7 The cells were immediately transplanted subcutaneously into the left chest. When the tissue expander was removed approximately 6 months later, a total of 258 ml (11.44 × 10 cells) of eight flasks was left in the lumen and surrounding area. 7 cells) were immediately transplanted subcutaneously into the left chest.

[0056] The medium used for primary culture and for culturing thawed cells was supplemented with 10% autologous serum, 40 ng / ml hydrocortisone, and 210 ng / ml FGF. It was confirmed that similar results could be obtained by using FBS instead of 10% autologous serum.

[0057] MRI image analysis of the transplanted fat tissue one year after transplantation showed that the transplanted fat tissue was not absorbed and maintained its original size. The results are shown in Figure 8. Figure 8 is a photograph in place of a drawing showing an example of breast reconstruction surgery after left breast cancer surgery. The image shows a horizontal MRI view from below, with the mammary glandular tissue nipple visible on the healthy side. On the reconstructed side, the transplanted fat was not absorbed and was engrafted at nearly 100%. The adipose tissue is bright white, and it can be seen that fat has formed throughout the entire layer. Numerous blood vessels can also be seen running inside. No fat necrosis, cysts, calcification, or lumps (induration) were observed. Stable fat formation was observed (the same results were obtained in the other 10 cases). Because the image was taken in the prone position, the healthy side is drooping.

[0058] Fat transplantation into humans (breast-conserving reconstruction) Example 4-2: Case of cultured mature adipocytes, conditioned medium, and autologous fat transplantation into a patient with left breast cancer after breast-conserving surgery and right breast atrophy and hypoplasia A 44-year-old woman who had undergone left breast cancer surgery was found to have significant breast depression (deformation of more than half) after breast-conserving surgery, and hypoplasia and atrophy of the right breast. This procedure was planned at her request. 2 cc of fat was aspirated from the abdomen, sterilized, and seeded and cultured. On the 14th day of primary culture, 3.4 × 10 6 The cultured cells were harvested and temporarily frozen. Before transplantation, the thawed cells were placed in a 150 cm2 tube with a base area of ​​150 cm. 2 The cells were cultured for 7 days in 15 flasks. The medium used for primary culture and thawed cell culture was a medium supplemented with 10% autologous serum, 40 ng / ml hydrocortisone, and 210 ng / ml FGF. The aspirated fat tissue to be transplanted, cultured adipocytes, mature cultured cells, and conditioned medium were mixed and transplanted. The total transplant volume was 310 ml (21.45 × 107 A total of 220 ml was injected and transplanted into the entire subcutaneous tissue of the left breast cancer-preserving area. 90 ml was injected and transplanted into the atrophic and hypoplastic area of ​​the right breast. One year after transplantation, the adipose tissue had not been absorbed and maintained its size, no lumps (hardening) were observed, and the external appearance was good.

[0059] Fat transplantation to humans (facial depression deformity surgery) Example 4-3: Transplantation of cultured mature adipocytes, conditioned medium, and autologous fat into facial atrophy A 68-year-old woman who had undergone surgery for left breast cancer presented with a depressed deformity due to atrophy of the right face. Furthermore, she had undergone abdominal and thigh liposuction at other hospitals, making standard fat collection difficult. At her request, this method was planned. 2 cc of fat was aspirated from the abdomen, sterilized, and seeded and cultured. On the 16th day of primary culture, 4.4 × 10 6 The cultured cells were harvested and frozen. Before transplantation, the cells were thawed and placed in a 175 cm2 tube. 2 The cells were cultured for 7 days in five flasks. The medium used for primary culture and for culturing the thawed cells was a medium containing 10% autologous serum, 40 ng / ml hydrocortisone, and 10 ng / ml FGF. The aspirated fat tissue, cultured cells, mature cultured cells, and conditioned medium were mixed and transplanted. The total volume of transplantation was 35 ml (8.35 x 10 7 One year after transplantation, the adipose tissue was not absorbed and maintained its size, no lumps (induration) were observed, and the external appearance was good.

[0060] Furthermore, since pectus excavatum is a chest wall depression deformation, similar to Example 4-2, it can be considered a severe depression deformation medically, and is therefore a disease suitable for this method.

[0061] Consideration The conventional concept was that SVF containing adipose stem cells (ASCs) promotes angiogenesis, improving the survival rate of grafts when co-transplanted with fat. Animal studies have shown that the viable area of ​​the graft is approximately 1.5-0.5 mm from the margin, with 60% of the surviving adipocytes dead. Even when adipose-derived stromal cells (ASCs) contained in SVF are added to adipocytes, the survival zone remains narrower than 300 μm. Human studies have also reported that breast fat grafting using cell-assisted lipotrasfer (CAL) is no different from conventional fat grafting, with one finding that conventional fat grafting achieved a 39% success rate and CAL achieved a 65% success rate (Toyserkani MN, Quaade ML, and Sorensen JA. Cell-Assisted Lipotransfer: A Systematic Review of Its Efficacy. Aesthetic Plast Surg. 2016; 40:309-318). The survival rate of CAL to the breast was reported in 5 of 10 studies, and was unstable, at 51.8%, 47%, 40%-80%, 54%, and 50%. (Cell-assisted lipotransfer in breast augmentation and reconstruction: A systematic review of safety, efficacy, use of patient-reported outcomes and study quality. Arshad Z, Karmen L, Choudhary R, ​​Smith JA, Branford OA, Brindley DA, Pettitt D and Davies BM. JPRAS OPEN. 2016 Dec;10:5-20)

[0062] GRO has been shown to have angiogenic potential in the literature (Caunt M,1 Liang Hu,1 Thomas Tang,1 Peter C. Brooks,2 Sherif Ibrahim,3 and Simon Karpatkin Growth-regulated Oncogene Is Pivotal in Thrombin-Induced Angiogenesis. Cancer Res 2006;66(8):4125 -32, Keglowich1 L, Roth M, Philippova1 M, Resink T, Tjin G, Bronchial Smooth Muscle Cells of Asthmatics Promote Angiogenesis Through Elevated Secretion of CXC-chemokines (ENA-78, GRO-α, and IL-8) PLoS One.2013 ;8(12):e81494. doi: 10.1371 / journal.pone.0081494).

[0063] The angiogenic potential of IL-8 has also been demonstrated (Mikula-Pietrasik J, Kuczmarska A, Kucin´ska M, Muriaset M. et al. Resveratrol and its synthetic derivatives exert opposite effects on mesothelial cell-dependent angiogenesis via modulating secretion of VEGF and IL-8 / CXCL8. Angiogenesis 2012; 15:361-376 and Keglowich et al. supra).

[0064] MCP-1 has been reported as an angiogenic chemokine and its angiogenic potential has been demonstrated in another paper (6. Niu J, Azfer A, Zhelyabovska O, Fatma S, and Kolattukudy PE. Monocyte Chemotactic Protein (MCP)-1 Promotes Angiogenesis via a Novel Transcription Factor, MCP-1-induced Protein (MCPIP). J Biol Chem 2008 May 23;283(21): 14542-51. doi: 10.1074 / jbc.M802139200). Furthermore, it has been shown in literature that MCP-1 mediates the angiogenic activity of VEGF (3. Hong KH, Ryu J, Han KH. Monocyte Chemoattractant protein-1-induced Angiogenesis Is Mediated by Vascular Endothelial Growth Factor-A. Blood 2005; 105:1405-1407 DOI:10,1182 / blood-2004-08-3178). Macrophage recruitment is essential for angiogenesis, and MCP-1 is an essential factor for this.

[0065] Therefore, it was shown that all four cytokines (MCP-1, IL-8, GRO, VEGF) present in the conditioned medium secreted by the cultured adipocytes in the examples have angiogenic potential. [Example]

[0066] 1x10 mature cultured adipocytes P2 4 / 6well(8cm 2 / well) to create a group of mature cultured adipocytes alone and a co-culture group of mature cultured adipocytes + aspirated fat. These were cultured for 5 days and immunostained for VEGFR2 (Vascular endothelial growth factor receptor 2) and PDGFRβ (platelet-derived growth factor beta). The reagents used were anti-VEGFR2 polyclonal antibody (Funakoshi: bs-10412), anti-PDGFRbeta polyclonal antibody [Y92]-C-terminal (ab32570), goat anti-rabbit IgG H&L (HRP) (ab205718), and DAB substrate kit (Funakoshi: CSK-4100, VEC). The results are shown in Figure 9. Both groups showed the presence of cells with strong VEGFR2 and PDGFRβ expression. VEGFR2 is a primary responder of VEGF signaling and is expressed only in vascular endothelial cells and their precursor cells, indicating its role in angiogenesis. PDGFRβ is expressed when PPARγ in adipose precursor cells is stimulated by fatty acids, etc. Therefore, the results suggest that adipose precursor cells are also present. Since both types of cells are present, it is thought that angiogenesis and adipogenesis are simultaneously promoted after transplantation. Furthermore, since the previously mentioned angiogenic factors (MCP-1, IL-8, GRO, VEGF) are also expressed in large amounts, angiogenesis after transplantation becomes a ring of two rings: cell type + factor, and it was found to have very strong angiogenic effects. Furthermore, strong expression of VEGFR2 and PDGFRβ was also observed in groups co-cultured with mature adipocytes and aspirated fat. [Example]

[0067] Tumorous cells have the characteristic of continuing to grow without stopping their proliferation. In order to confirm the safety of the cultured adipocytes created in this study, we conducted an aging test on the adipocytes. This test involves culturing adipocytes and continuing to passage them to see when their proliferation stops. Adipocytes from six individuals were cultured for three months and the PDL (CPD) until proliferation stopped was measured. This indicates the number of times they divided. The results are shown in Figure 10. Figure 10 is a graph in place of a drawing showing the number of possible passages of adipocytes and PDL. As shown in Figure 10, it was confirmed that proliferation stopped due to aging. P14 = total 102-day culture: vertical axis CDL (= PDL). 49 years old (stopped at 102 days), 40 years old (stopped at 95 days), 60 years old (stopped at 67 days), 54 years old (stopped at 61 days) 52 years old (stops at 62 days), 50 years old (stops at 67 days) [Example]

[0068] An experiment was conducted to determine whether cultured adipocytes could be cultured in serum-free medium, and it was found that they could proliferate in a commercially available serum-free medium for mesenchymal stem cells (Lonza PT-3001: Lonza.KK), although the proliferation rate was lower than that in fetal bovine serum. Basal medium: A: α-MEM only, no serum added (control) The concentrations of HC and FGF added are the same as those in clinical trials. B: Mesenchymal stem cell serum-free medium (Lonza PT-3001) added C: α-MEM supplemented with fetal calf serum (FCS) to a concentration of 10% (positive control). The above were compared and examined. The results are shown in Figures 11 to 13. Figure 11 is a photograph, instead of a drawing, showing cultured adipocytes on day 4 of P2 culture in α-MEM only (control). Figure 12 is a photograph, instead of a drawing, showing cultured adipocytes on day 4 of P3 culture in serum-free medium (Lonza PT-3001). Figure 13 is a photograph, instead of a drawing, showing cultured adipocytes in medium supplemented with α-MEM and 10% fetal calf serum (FCS). Table 1 shows the cell numbers in each medium.

[0069] [Table 1] [Industrial Applicability]

[0070] This invention can be used in the field of medical equipment, etc.

Claims

1. A cutting and filtration process in which non-enzyme-treated adipose tissue is cut and filtrated to obtain microfat; After the cutting and filtration step, a microfat culture step of culturing the microfat using a culture medium to obtain mature adipocytes; and a mature adipocyte-containing composition obtaining step of obtaining a mature adipocyte-containing composition comprising the mature adipocytes and a conditioned medium obtained through the microadipocyte culture step. A method for producing a mature adipocyte-containing composition, The cutting and filtering step includes a step of centrifuging to remove precipitated stromal vascular cells (SVF) and recovering the microfat, which is a fat component that floats to the top of the liquid phase; The culture medium comprises: Serum-free medium containing hydrocortisone and FGF-2 A method for producing a composition containing mature adipocytes.

2. A method for producing a mature adipocyte-containing composition according to claim 1, A method for producing a composition containing mature adipocytes, wherein the adipose tissue is obtained by washing aspirated fat and removing blood components.

3. A method for producing a mature adipocyte-containing composition according to claim 2, The method for producing a mature adipocyte-containing composition, wherein the cutting and filtering step further includes a step of recovering fat components that float to the top of the liquid phase obtained by centrifugation, thereby obtaining the microcells.

4. A method for producing a mature adipocyte-containing composition according to any one of claims 1 to 3, wherein the mature adipocyte-containing composition is used as a substitute for subcutaneous adipose tissue.

5. A method for producing a mature adipocyte-containing composition according to any one of claims 1 to 3, wherein the mature adipocyte-containing composition is used for breast augmentation surgery, breast reconstruction surgery, or tissue depression formation surgery.

6. A method for producing a vascular regeneration promoter, comprising a mature adipocyte-containing composition obtained by the method for producing a mature adipocyte-containing composition according to any one of claims 1 to 3.

7. The method for producing a vascular regeneration promoter according to claim 8, wherein the mature adipocyte-containing composition comprises: The method includes vascular endothelial growth factor receptor (VEGFR) 2 or positive cells and platelet-derived growth factor (PDGFR) beta.

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