WO2016068217A1 - Feuillet de cellules contenant des cellules mononucléaires du sang périphérique ou des fibroblastes associés au facteur sécrété par les cellules mononucléaires du sang périphérique - Google Patents

Feuillet de cellules contenant des cellules mononucléaires du sang périphérique ou des fibroblastes associés au facteur sécrété par les cellules mononucléaires du sang périphérique Download PDF

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WO2016068217A1
WO2016068217A1 PCT/JP2015/080465 JP2015080465W WO2016068217A1 WO 2016068217 A1 WO2016068217 A1 WO 2016068217A1 JP 2015080465 W JP2015080465 W JP 2015080465W WO 2016068217 A1 WO2016068217 A1 WO 2016068217A1
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cell sheet
fibroblasts
peripheral blood
blood mononuclear
cells
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PCT/JP2015/080465
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English (en)
Japanese (ja)
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濱野公一
細山徹
上野耕司
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国立大学法人山口大学
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Priority to JP2016556612A priority Critical patent/JP6583830B2/ja
Publication of WO2016068217A1 publication Critical patent/WO2016068217A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses

Definitions

  • the present invention relates to a cell sheet containing fibroblasts with peripheral blood mononuclear cells or factors secreted from peripheral blood mononuclear cells and a method for producing the cell sheet.
  • Intractable skin ulcers are wounds that are normal in wounds that have been cured, but have become intractable ulcers due to abnormal factors such as infection, vascular disorders, and sensory disturbances. Examples include pressure ulcers, obstructive arteriosclerosis, diabetes, venous insufficiency, collagen disease, vasculitis and the like. In Japan, there are many patients who suffer from refractory skin ulcers, and the annual number of patients with refractory skin ulcers is 1.3 million. Currently, fiblast spray and artificial dermis are mainly used as a treatment for the ulcer.
  • Fiblast spray is a drug made by purifying FGF-2, which is a kind of growth factor related to wound healing, and can be treated simply by spraying the affected area.
  • FGF-2 a kind of growth factor related to wound healing
  • Artificial dermis is an artificial dermis-like structure consisting of a two-layer structure of collagen sponge and silicone sheet made from porcine or bovine collagen as a raw material. It is known to work. However, the structure may cause immune rejection in some patients, and is often accompanied by infection in ischemic ulcers. The frequency of actual use is extremely low due to the fact that it is not suitable for use at the site of infection and is also expensive.
  • Non-patent document 1 we conducted autologous bone marrow cell transplantation treatment for severe limb ischemia for the first time in the world ( Non-patent document 1). After that, because bone marrow cells are too invasive, the cell type used for transplantation is changed from bone marrow cells to peripheral blood mononuclear cells that can be easily obtained by blood collection for the purpose of minimizing the treatment. In particular, the effectiveness has been confirmed in basic experiments.
  • transplanted cells does not necessarily have a high engraftment rate with respect to the tissue.
  • Development of cell sheets in which these cells are cultured in sheet form is underway.
  • the cell sheet is capable of fixing a large amount of desired cells at the damaged site, and also enables transplantation of a moderately organized cell population according to the characteristics of the recipient tissue. It is a useful therapeutic material.
  • Non-Patent Documents 2 and 3 as an application to regeneration of epidermis and mucosal tissue, for example, “corneal regeneration epithelial sheet” and “esophageal regeneration epithelial sheet” aiming at tissue regeneration after removal of esophageal cancer, etc. It is considered (Patent Document 1).
  • the present invention provides a cell sheet effective in treating intractable skin ulcer and a method for producing the cell sheet.
  • a cell sheet effective in treating intractable skin ulcer and a method for producing the cell sheet.
  • transplanting the cell sheet for an ischemic ulcer with insufficient blood circulation sufficient healing of the ulcer site can be achieved without causing immune rejection as induced in an artificial dermis or the like.
  • the inventors have found that in a cell sheet containing peripheral blood mononuclear cells and fibroblasts, the peripheral blood mononuclear cells and fibroblasts are each cultured cells alone. In comparison, the inventors have found that the amount of vascular growth factor that plays an important role in angiogenesis is greatly increased. Further, the inventors transplanted a cell sheet containing peripheral blood mononuclear cells and fibroblasts to a skin ulcer site prepared on the back of a diabetic mouse or diabetic rabbit, and only the wound covering material for skin defect (control group). ) And transplantation with cell sheets of fibroblasts alone (peripheral blood mononuclear cells alone cannot form cell sheets), by finding significant improvements in wound site healing rates, Completed the invention.
  • immune rejection in transplantation can be suppressed by using both cells obtained from the patient with refractory skin ulcer to be transplanted as the peripheral blood mononuclear cells and fibroblasts according to the present invention.
  • peripheral blood mononuclear cells that can be obtained by an easy and expensive method such as blood collection, it is a regenerative medicine using its own cells, but it is possible to carry out minimally invasive and safe treatment at low cost Became possible.
  • the inventors co-cultured peripheral blood mononuclear cells and fibroblasts on a culture substrate to form a cell sheet, and then cultured the sheet under a low temperature and low oxygen condition for a predetermined period.
  • Hypoxic preconditioning and then the cell sheet obtained by peeling the sheet from the culture substrate is an vascular growth factor that plays an important role in angiogenesis compared to the case where the preconditioning treatment is not performed. It was found not only in mice but also in human-derived cells that the production was greatly increased.
  • the inventors of the present invention have further described the cause of the increase in the amount of vascular growth factor produced from the cell sheet in the cell sheet obtained by adding peripheral blood mononuclear cells to fibroblasts compared to the culture sheet of fibroblasts alone.
  • transforming growth factor Transformation Growth Factor: TGF
  • PDGF platelet-derived growth factor
  • the inventors developed transforming growth factor or platelet-derived growth on cell sheets containing fibroblasts that do not contain peripheral blood mononuclear cells. It was found that the same effect (increase in the production amount of blood vessel growth factor) can be reproduced by applying stimulation with factors, and the present invention has been completed.
  • the possibility of industrialization as a therapeutic tool is expected by preparing a cell sheet of non-patient derived fibroblasts and a recombinant protein of transforming growth factor or platelet-derived growth factor. .
  • the present invention includes the following (1) to (17).
  • (1) A cell sheet containing fibroblasts stimulated by a transforming growth factor or a platelet-derived growth factor.
  • a transplant material for treating refractory skin ulcer comprising the cell sheet according to any one of (1) to (3).
  • a method for producing a cell sheet comprising the following steps (a) to (c): (A) seeding fibroblasts on a culture substrate and forming a cell sheet containing the cells; (B) stimulating the cell sheet with a transforming growth factor or a platelet-derived growth factor; (C) The step of peeling the cell sheet from the culture substrate (6) The method of producing the cell sheet according to (5), wherein the step (a) and the step (b) are performed simultaneously. (7) Before the step (a), (A0) a step of obtaining fibroblasts from an individual suffering from intractable skin ulcer, which is a subject to be treated; A method for producing the cell sheet according to (5) or (6).
  • a cell sheet containing peripheral blood mononuclear cells and fibroblasts (9) The cell according to (8), wherein the peripheral blood mononuclear cell and / or the fibroblast is obtained from an individual suffering from intractable skin ulcer, which is a subject to be treated. Sheet. (10) The cell sheet according to (8) or (9), which is produced by co-culturing peripheral blood mononuclear cells and fibroblasts. (11) Peripheral blood mononuclear cells are seeded at 5.0 ⁇ 10 4 cells / cm 2 to 1.5 ⁇ 10 6 cells / cm 2 , and fibroblasts are 1.0 ⁇ 10 4 cells / cm 2 to 1.5 ⁇ 10 5 cells / cm 2.
  • a transplant material for treating refractory skin ulcer comprising the cell sheet according to any one of (8) to (13).
  • a method for producing a cell sheet comprising the following steps (d) to (e): (D) co-culturing peripheral blood mononuclear cells and fibroblasts on a culture substrate to form a cell sheet comprising the cells; (E) Step of peeling the cell sheet from the culture substrate (16) Before the step (d), (D0) a step of obtaining peripheral blood mononuclear cells and / or fibroblasts from an individual suffering from intractable skin ulcer, which is a subject to be treated; A method for producing the cell sheet according to (15), comprising: (17) Between the steps (d) and (e), (D2) a step of culturing the cell sheet of step (d) for a predetermined period under low temperature and low oxygen conditions; A method for producing the cell sheet according to (15) or (16).
  • a biomaterial for transplantation that can be used for the treatment of intractable skin ulcers, particularly ischemic ulcers, blood flow improvement by angiogenesis is achieved, and immunity is achieved by using patient's own cells.
  • Cell sheet image containing peripheral blood mononuclear cells and fibroblasts Flow of peripheral blood mononuclear cell culture, cell sheet containing fibroblasts and cell sheet containing peripheral blood mononuclear cells and fibroblasts (Normo & Hypo conditions) and the ability to produce each blood vessel growth factor.
  • Negative control positive control (fibrous spray formulation group), change in wound area healing rate by transplantation of cell sheets containing fibroblasts and cell sheets containing peripheral blood mononuclear cells and fibroblasts (A) and negative controls, Wound healing state of positive control and cell sheet transplanted group containing peripheral blood mononuclear cells and fibroblasts (B).
  • Pathological image analysis of cell sheet transplanted group including peripheral blood mononuclear cells and fibroblasts and fiblast spray prescription group. Allo-transplantation experiment in mice; transplantation of cell sheets containing peripheral blood mononuclear cells and fibroblasts prepared from C57BL / 6 mice to the back of C3H mice.
  • A Wound healing state (days 0, 7, 14) of cell sheet transplanted group (No. 1, No. 2) and fiblast spray prescription group containing peripheral blood mononuclear cells and fibroblasts.
  • B Wound area healing rate of a control group, a cell sheet transplanted group containing peripheral blood mononuclear cells and fibroblasts, and a fiblast spray prescription group.
  • B Wound healing state on day 0 and day 21 of the control group and the cell sheet transplantation group containing peripheral blood mononuclear cells and fibroblasts.
  • the wound area healing rate 14 days after transplantation is shown.
  • the administration concentration of each factor and the blood vessel growth factor concentration in the culture supernatant after administration are shown.
  • the first aspect of the present invention is a cell sheet containing peripheral blood mononuclear cells and fibroblasts.
  • the “cell sheet” is a general term for a culture of cells in which cells are combined in a sheet form, and the cell sheet is composed of one cell layer or two or more cell layers. It may be.
  • peripheral blood mononuclear cells is a generic name of white blood cells composed of lymphocytes and monocytes contained in blood collected from peripheral blood vessels, and many cells having nuclei close to a circle are included. Because it has, it is called by the generic name.
  • typical constituent cells and constituent ratios about 70-80% of all cells are lymphocytes, and the remaining 20-30% are composed of monocytes, macrophages, dendritic cells, etc.
  • Stem cells derived from the cells may be present, but are not limited to the constituent cells and the constituent ratios, and may be a group of cells obtained under conditions normally prepared as a peripheral blood mononuclear cell fraction in the art. It may be appropriately changed depending on the preparation conditions and the individual to be collected. A specific preparation method is described in Example [0067], but is not limited thereto.
  • the “fibroblast” according to the present invention is a cell unique to the tissue constituting the connective tissue. It does not have a particularly remarkable function in normal tissues, but when a bruise is applied, it migrates to the bruise and secretes collagen, elastin, hyaluronic acid, etc., and starts production of the extracellular matrix. It has a function to update. Besides this, it plays an important role in the wound healing process, such as inducing wound contraction.
  • the method for preparing the cells is described in, for example, Example [0068], but is not limited thereto, and may be a cell group obtained under conditions normally prepared as a fibroblast fraction in the technical field. That's fine.
  • the peripheral blood mononuclear cells and fibroblasts used to form the cell sheet of the present invention may be derived from any animal species from which the cells can be collected, preferably mammals, particularly preferably In addition to humans, pet animals such as dogs, cats and rabbits, livestock animals such as cows, pigs, sheep and horses, and more preferably humans. Further, the peripheral blood mononuclear cells and fibroblasts according to the present invention may be obtained from any animal individual, but may preferably be obtained from an individual suffering from refractory skin ulcer, which is a subject to be treated. Good. By using the cells of the individual to be treated in this way, it is possible to suppress immune rejection that occurs during transplantation.
  • the cell sheet according to the present invention is a cell sheet containing the peripheral blood mononuclear cells and the fibroblasts.
  • “Including peripheral blood mononuclear cells and fibroblasts” means that both types of cell groups may be contained in the same cell sheet as main constituent cells, and the production method is not particularly limited.
  • the peripheral blood mononuclear cells and the fibroblasts are prepared as a peripheral blood mononuclear cell fraction and a fibroblast fraction with a certain degree of purity, and are produced by co-culturing them. Also good.
  • co-culture means culturing two or more different cells together, and in the present invention, it means culturing at least peripheral blood mononuclear cells and fibroblasts together.
  • how to seed the peripheral blood mononuclear cells and fibroblasts is not particularly limited, but it may be preferably started by seeding both cells simultaneously.
  • the seeding density of each of the peripheral blood mononuclear cells and fibroblasts according to the present invention on the culture substrate is not particularly limited, but the seeding conditions examined by the inventors (Examples [0069] and [ [0071] preferably) peripheral blood mononuclear cells at 5.0 ⁇ 10 4 cells / cm 2 to 1.5 ⁇ 10 6 cells / cm 2 and fibroblasts from 1.0 ⁇ 10 4 cells / cm 2
  • the seed may be seeded at 1.5 ⁇ 10 5 cells / cm 2 , and more preferably, the peripheral blood mononuclear cells are 5.5 ⁇ 10 4 cells / cm 2 to 1.0 ⁇ 10 6 cells / cm 2 and the fibroblasts are 1.
  • the seed may be seeded at 5 ⁇ 10 4 cells / cm 2 to 1.0 ⁇ 10 5 cells / cm 2 , and more preferably, peripheral blood mononuclear cells at 5.0 ⁇ 10 5 cells / cm 2 to 1.0 ⁇ 10 6 cells / cm 2 . And 6.0 ⁇ 10 4 fibroblasts / cm 2 to 1.0 ⁇
  • the seeding may be performed at 10 5 pieces / cm 2 .
  • the second aspect of the present invention is a method for producing a cell sheet containing peripheral blood mononuclear cells and fibroblasts.
  • the invention may be any method as long as it is a method for producing a cell sheet containing peripheral blood mononuclear cells and fibroblasts, and is not particularly limited.
  • a method for producing a cell sheet comprising the steps (d) to (e), wherein the steps (d) and (e) are: (D) a step of co-culturing peripheral blood mononuclear cells and fibroblasts on a culture substrate to form a cell sheet comprising the cells; (E)
  • a method for producing a cell sheet, which is a step of peeling the cell sheet from the culture substrate, may be used.
  • any method can be used as long as it is possible to form a cell sheet comprising the cells by co-culturing peripheral blood mononuclear cells and fibroblasts on a culture substrate.
  • it can be carried out under conditions ordinarily practiced in the art suitable for the peripheral blood mononuclear cells and fibroblasts used.
  • the culture temperature is 30 to 40 ° C., preferably 36 to 38 ° C.
  • the CO 2 concentration is 0 to 10%, preferably 4 to 6%
  • the O 2 concentration is atmospheric oxygen concentration (approximately 20%).
  • this condition is not limited, and culture temperature, CO 2 concentration, and O 2 concentration can be appropriately selected.
  • the culture temperature is 37 ° C.
  • the CO 2 concentration is 5%
  • the O 2 concentration is the atmospheric oxygen concentration. (Approximately 20%).
  • the culture time is not particularly limited as long as it is a time necessary for forming a desired cell sheet.
  • the culture time may be about 10 hours to 240 hours, and preferably 12 hours to 168.
  • the culture time is about an hour, and more preferable results are obtained in 48 hours to 96 hours.
  • the cell density initially seeded to form a cell sheet is not particularly limited as long as it is a condition normally performed in cell culture, but in order to produce a cell sheet in good condition, It is preferable that the state is substantially confluent at the time of sowing.
  • peripheral blood mononuclear cells are 5.0 ⁇ 10 4 cells / cm 2 to 1.5 ⁇ 10 6 cells / cm 2 and fibroblasts are 1.0 ⁇ 10 4 cells / cm 2. ⁇ 1.5 ⁇ 10 5 cells / cm 2 , more preferably 5.5 ⁇ 10 4 peripheral blood mononuclear cells / cm 2 to 1.0 ⁇ 10 6 cells / cm 2 and 1 fibroblast cell.
  • the state of the cells after the formation of the cell sheet is not particularly limited as long as it is in a healthy state, but may preferably be in a confluent state.
  • the “culture substrate” may be any cell as long as cells can form a cell sheet on the surface thereof, and at least includes a flat portion to which cells can adhere, Typically, it is a cell culture dish or a cell culture bottle (or flask), and a commercially available culture dish or the like can be used, and the material is not particularly limited.
  • the culture substrate material include polyethylene, polypropylene, polyethylene terephthalate, and the like.
  • the culture surface of the “culture substrate” is made of a material whose physical properties change due to a temperature change or the like (temperature responsive material), or the culture surface of the culture substrate is layered by the temperature responsive material. It may be coated.
  • a cell adhesion component and / or a cell adhesion inhibitory component may be present on the culture surface of the main culture substrate.
  • the cell adhesion component may be any component that is usually used for adhering cells to the culture surface in cell culture technology, such as collagen, fibronectin, laminin, heparan sulfate proteoglycan, cadherin, gelatin, Examples include fibrinogen, fibrin, poly L lysine, hyaluronic acid, platelet-rich plasma, and polyvinyl alcohol.
  • the cell adhesion-inhibiting component may be any component that is usually used for inhibiting cell adhesion to the culture surface in the cell culture technique, and examples thereof include albumin and globulin.
  • a medium suitable for the origin and culture conditions of peripheral blood mononuclear cells and fibroblasts to be cultured can be appropriately selected and used.
  • MEM, DMEM, F12, IMEM, IMDM, RPMI-1640, Neurobasal, etc. can be mentioned as media that can be generally used. You may purchase and use these culture media. Moreover, these culture media may be used independently or may be used in combination of 2 or more types.
  • RPMI-1640 may be used for peripheral blood mononuclear cells and DMEM for fibroblasts, but is not limited thereto.
  • AIM V (registered trademark) medium CTS TM may be used without limitation.
  • an appropriate additive may be added to the medium as necessary.
  • the additive include L-type amino acids (for example, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-trionine, L-tryptophan, L-tyrosine, etc.), vitamins (eg, folic acid, riboflavin, thiamine, etc.), D-glucose, and other animal sera such as fetal bovine serum (FBS), horse serum, etc. Etc. may be included.
  • L-type amino acids for example, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-trionine, L-trypto
  • a buffering agent for example, PBS, HEPES, MES, HANK'S etc.
  • a cell growth factor for example, PBS, HEPES, MES, HANK'S etc.
  • blood is collected from a subject from which the cells are collected, serum is prepared from the blood, and this is used. Good.
  • RPMI-1640 which is a culture medium for peripheral blood mononuclear cells, is added to 10% of FBS, and penicillin 100 Unit / ml, streptomycin 100 ⁇ g / ml and 1 mM L-glutamine added, and fibroblast medium, DMEM to 20% FBS, penicillin 100 units / ml, streptomycin 100 ⁇ g / ml and What added 1 mM L-glutamine and mixed suitably may be used.
  • the step (e) is a step of peeling the cell sheet from the culture substrate after culturing under the above conditions. Peeling of the cell sheet from the culture substrate can be carried out by a method that does not damage the sheet-like structure.
  • the sheet-like cell culture is directly picked with tweezers and peeled off from the culture surface, or Alternatively, a physical technique such as peeling the cells from the culture surface by pipetting may be used.
  • an enzyme treatment such as trypsin or collagenase may be performed, and an appropriate method can be selected according to the properties of the cells.
  • the cell sheet can be peeled and collected by covering the upper surface of the cell sheet with a substrate having affinity for cells, such as a PVDF membrane or a nitrocellulose membrane, and copying the cells onto the membrane.
  • a substrate having affinity for cells such as a PVDF membrane or a nitrocellulose membrane
  • the above-described cell detachment and recovery may be performed after the temperature of the container is lowered to, for example, about 0 to 30 ° C. Good.
  • step (D0) a step of obtaining peripheral blood mononuclear cells and / or fibroblasts from an individual suffering from intractable skin ulcer, which is a subject to be treated; May be included.
  • the immune rejection at the time of transplantation can be suppressed by the cell sheet containing peripheral blood mononuclear cells and fibroblasts produced by the production method.
  • a third aspect of the present invention is a cell sheet and a method for producing the cell sheet, wherein the cell sheet according to the first aspect is cultured under a low temperature and low oxygen condition for a predetermined period of time.
  • the inventors have been studying not only intractable skin ulcers but also methods for producing cell sheets useful for the treatment of other diseases such as ischemic heart disease. After culturing cells and forming a cell sheet, the sheet is cultured for a predetermined period of time under low temperature and low oxygen conditions (low oxygen preconditioning), and then the sheet is peeled from the culture substrate.
  • the cell sheet obtained in this manner has a significantly increased production amount of vascular growth factor that plays an important role in angiogenesis compared to the case where the preconditioning treatment is not performed.
  • the method for producing a cell sheet comprising peripheral blood mononuclear cells and fibroblasts subjected to such hypoxic preconditioning is not particularly limited.
  • a step of culturing the cell sheet of step (d) for a predetermined period under low temperature and low oxygen conditions may be sufficient.
  • Step (d2) is a step of culturing the cell sheet prepared in step (d) for a predetermined period under low temperature and low oxygen conditions.
  • the low-temperature and low-oxygen conditions and the treatment period vary depending on the origin and culture conditions of peripheral blood mononuclear cells and fibroblasts constituting the cell sheet, and can be easily examined by those skilled in the art such as preliminary experiments. Appropriate conditions can be determined for each cell sheet by a method that can be performed.
  • the O 2 concentration is 0% to 8%, preferably 0.1% to 5%, more preferably 2%.
  • the low temperature condition is that the culture temperature is 30 ° C. to 36 ° C., preferably 32 ° C.
  • the cell sheet may be immediately transferred to the step (e), or may be transferred to the step (e) after returning to normal culture conditions for a certain period.
  • the conditions can be set for each cell sheet, and the fixed period when returning to normal culture conditions for a fixed period is not particularly limited, but may be, for example, 0 to 12 hours, preferably It may be 0 to 6 hours, more preferably 0 hour (not returning to normal culture conditions).
  • the cell sheet containing the peripheral blood mononuclear cells and fibroblasts according to the present invention can be used as a biological material for transplantation for the purpose of improving functional disorders of various animal tissues, organs, organs and the like. I can do it.
  • the “animal” is not particularly limited, but is preferably an animal whose function is expected to be improved by transplantation. Specifically, in addition to humans, pet animals such as dogs, cats and rabbits, cattle , Livestock animals such as pigs, sheep and horses, preferably humans.
  • tissue, organ, organ and the like to which the cell sheet produced according to the present invention is transplanted are not particularly limited, and examples thereof include skin, skeletal muscle, blood vessel and the like, preferably skin.
  • the disease when targeting the skin is not particularly limited, but includes intractable skin ulcers represented by pressure ulcer, obstructive arteriosclerosis, diabetes, venous insufficiency, collagen disease, vasculitis and the like.
  • a fourth aspect of the present invention is a cell sheet containing fibroblasts stimulated by a transforming growth factor or a platelet-derived growth factor.
  • TGF Transforming growth factor
  • the types are broadly divided into two types, alpha and beta, and the former has been reported to be involved in overexpression in several cancers and induction of epithelial development in macrophages, brain cells, keratinocytes, and the like.
  • the latter is produced by various cells such as kidney, bone marrow, and platelets, and there are mainly five types (beta1 to beta5).
  • the inventors have made extensive studies to elucidate the cause of the increase in the production amount of blood vessel growth factor in a cell sheet containing fibroblasts with peripheral blood mononuclear cells (first aspect of the present invention).
  • the increase in the production amount of the blood vessel growth factor is caused by the fact that transforming growth factor (particularly TGF-beta1) secreted from peripheral blood mononuclear cells activates fibroblasts constituting the cell sheet. (Fig. 8, 9, 12, 13).
  • the transforming growth factor is preferably a beta type, particularly preferably a beta 1 subtype, but is not limited thereto.
  • Platelet-derived growth factor is a growth factor mainly involved in the regulation of migration and proliferation of mesenchymal cells (fibroblasts, smooth muscle cells, glial cells, etc.). It belongs to the VEGF family. In addition to being produced mainly by megakaryocytes, it is also contained in the alpha granules of platelets, and is also produced in various cells such as epithelial cells and endothelial cells. There are at least four types of PDGF, PDGF-A, B, C, and D. A chain and B chain form a homo- or hetero-dimer structure by forming a disulfide bond, and three types of isoforms (PDGF- AA, AB, BB).
  • fibroblasts constituting the cell sheet by platelet-derived growth factor (particularly PDGF-BB) secreted from peripheral blood mononuclear cells, thereby vascular growth factor It was clarified that an increase in the production amount of was induced (FIGS. 8, 9, 12, and 13). In addition, it was found that PDGF-AA is contained in the factor that is specifically secreted and elevated only in the cell mixed sheet of human-derived peripheral blood mononuclear cells and fibroblasts (FIG. 12).
  • the transforming growth factor in the present invention is not particularly limited, but is preferably PDGF-AA or PDGF-BB, and particularly preferably PDGF-BB.
  • “stimulated by a transforming growth factor or platelet-derived growth factor” means that the factor is directly contacted with a cell sheet containing a fibroblast that is a target for the stimulation, thereby The information transmission system related to the receptor is activated by the binding of the factor to the receptor for each factor.
  • a method for transmitting a stimulus by the factor is not particularly limited, but the factor is administered to a medium in which the cell sheet is cultured, and a substance that releases the factor gradually is placed in the vicinity of the cell sheet. Examples include adding, co-culturing the cells producing the factor and the like with the cell sheet, and the like, and a method of directly administering to the medium is preferable.
  • the stimulation by the transforming growth factor or the platelet-derived growth factor may be started simultaneously with the seeding of the fibroblasts, and may be applied after the cell sheet has been formed.
  • a stimulus that starts simultaneously with sowing may be preferable.
  • the fibroblasts may be obtained from an individual suffering from an intractable skin ulcer, which is a subject to be treated.
  • the fibroblasts may be obtained from an individual suffering from an intractable skin ulcer, which is a subject to be treated.
  • the origin of the fibroblasts according to the fourth aspect of the present invention, the preparation method, the seeding density on the culture substrate, etc. are in accordance with the first aspect of the present invention, and an example is provided in Examples [0068]-[0069 However, the present invention is not limited to this.
  • a fifth aspect of the present invention is a method for producing a cell sheet containing fibroblasts stimulated with a transforming growth factor or a platelet-derived growth factor, and the invention is not particularly limited.
  • a method for producing a cell sheet comprising the steps (a) to (c), wherein the steps (a) to (c) are: (A) seeding fibroblasts on a culture substrate and forming a cell sheet containing the cells; (B) stimulating the cell sheet with a transforming growth factor or a platelet-derived growth factor; (C)
  • a method for producing a cell sheet, which is a step of peeling the cell sheet from the culture substrate, may be used.
  • any method may be used as long as it is possible to form a cell sheet containing the cells by culturing fibroblasts on a culture substrate. It can be performed under conditions ordinarily practiced in the technical field suitable for cells. As a specific example, among the descriptions related to the second aspect of the present invention described in [0068] to [0069], there is a portion regarding fibroblasts.
  • step (A0) Prior to step (a) (A0) a step of obtaining fibroblasts from an individual suffering from intractable skin ulcer, which is a subject to be treated; May be included.
  • the cell sheet containing fibroblasts produced by the production method can suppress immune rejection at the time of transplantation according to the present invention described in [0037]. This is the same as the step (d0) in the second embodiment.
  • the step (b) is a step of stimulating the cell sheet formed in the step (a) with a transforming growth factor or a platelet-derived growth factor, and there is no particular limitation on the stimulation method.
  • the cell sheet may be administered to the culture medium.
  • the concentration of the transforming growth factor or platelet-derived growth factor to be administered activates the receptor signal transduction system existing in the fibroblasts constituting the formed cell sheet.
  • it is not particularly limited, and for example, it is 0.25 ng / mL or more, preferably 2.5 mg / mL or more for transforming growth factor.
  • platelet-derived growth factor it is 5 ng / mL or more, preferably 10 ng / mL or more.
  • the step (b) may be performed simultaneously. Specifically, in order to form a cell sheet containing fibroblasts, fibroblasts are seeded on a culture substrate. At the same time, a predetermined amount of transforming growth factor or platelet-derived growth factor may be added.
  • the step (c) is a step of peeling the cell sheet from the culture substrate after the step (b), and conforms to the step (e) in the second aspect of the present invention described in [0041]. .
  • a method for producing a cell sheet according to any one of the first aspect or the fourth aspect of the present invention or the second aspect, the third aspect, or the fifth aspect. Is a transplant material for treating refractory skin ulcer containing the cell sheet produced by
  • the cell sheet according to the present invention can be used as a transplant material for treatment of intractable skin ulcer.
  • the seventh aspect of the present invention is the above-described cell sheet according to the first aspect or the fourth aspect of the present invention, or the cell according to any one of the second aspect, the third aspect or the fifth aspect of the present invention. It is the treatment method using the cell sheet manufactured by the manufacturing method of a sheet
  • the cell sheet is effective for the treatment of the disease by using it in an animal model of intractable skin ulcer. It has been found that the sheet can be used in a treatment method for a predetermined disease.
  • the predetermined disease is not particularly limited, but is preferably a disease that develops in the skin.
  • refractory typified by pressure ulcer, obstructive arteriosclerosis, diabetes, venous insufficiency, collagen disease, vasculitis, etc. Examples include skin ulcers.
  • washing with PBS was performed twice in total, followed by washing once with a peripheral blood mononuclear cell culture medium.
  • a peripheral blood mononuclear cell culture medium For cell sheet preparation and culture, cells were counted and the concentration of peripheral blood mononuclear cells was adjusted to 2 ⁇ 10 6 cells / mL using a peripheral blood mononuclear cell culture medium.
  • fibroblasts were prepared at 1.25 ⁇ 10 5 cells / mL using a fibroblast medium.
  • 1 mL of the cell suspension prepared to 5 cells / mL was added to 1 well of the UpCell 24-well plate to make a total of 2 mL.
  • FIG. 1 shows a state 72 hours after seeding both cells. It can be seen that a cell sheet is formed by round peripheral blood mononuclear cells and flat fibroblasts.
  • Peripheral blood mononuclear cell culture (collection of culture supernatant under each condition ) In 1 well of UpCell 24-well plate, 1 mL of cell suspension and 1 mL of fibroblast medium were prepared by adjusting the concentration of peripheral blood mononuclear cells to 2 ⁇ 10 6 cells / mL using peripheral blood mononuclear cells. To 2 mL in total.
  • peripheral blood mononuclear cells since many of the cells constituting the peripheral blood mononuclear cells are lymphocytes that are floating cells that are difficult to adhere to the culture substrate, no cell sheet was formed in the peripheral blood mononuclear cell culture alone. There has been no example of producing a cell sheet by combining peripheral blood mononuclear cells and other cells so far, and a cell sheet that cannot be formed by the cell alone can only be obtained by combining it with fibroblasts (FIG. 1). This is one of the remarkable effects that greatly exceeds the range that can be predicted by those skilled in the art.
  • VEGF production capacity of cell sheet 1) Measurement of VEGF concentration contained in supernatant by ELISA method VEGF concentration contained in cell culture supernatant was measured using Mouse VEGF Quantikine ELISA Kit (R & D systems). For absorbance measurement and concentration calculation, iMark Microplate Reader (BIO-RAD) and MPM6. exe (BIO-RAD) was used.
  • Streptozotocin SIGMA-ALDRICH dissolved in physiological saline of diabetic mice was administered to C57BL / 6NCrSlc mice at 200 mg / kg to prepare streptozotocin-induced diabetic mice.
  • the treatment groups were the following 4 groups; a) control group; no cell sheet transplantation + absocure (registered trademark) -und b) fibroblast sheet group; fibroblast single cell sheet transplantation + absocure (registered trademark) -undo c) peripheral blood mononuclear cells + fibroblasts Cell sheet group; cell sheet transplantation including peripheral blood mononuclear cells and fibroblasts + absocure (registered trademark) -und d) fiblast spray group; fiblast spray which is a current therapeutic agent for diseases such as refractory skin ulcers (Kaken Pharmaceutical Co., Ltd .; spray type therapeutic agent mainly composed of basic fibroblast insulting factor (bFGF) recombinant protein) was used as a positive target. Fiblast spray was sprayed on the entire skin defect area, covered with non-adhesive gauze after 1 minute, and fixed with an adhesive bandage (Nichiban).
  • bFGF basic fibroblast insulting factor
  • fibroblast single cell sheet and the cell sheet containing peripheral blood mononuclear cells and fibroblasts used here were cell sheets of the Hypo condition group.
  • the group using the cell sheet containing peripheral blood mononuclear cells and fibroblasts according to the present invention clinically used hydrocolloid dressing material (by maintaining the moist environment of the wound part, Material that promotes healing of wounds only with the body's inherent healing power)
  • the group of fibroblasts alone which is a category that can be easily conceived by those skilled in the art based on the prior art
  • the cell sheet containing peripheral blood mononuclear cells and fibroblasts according to the present invention is extremely effective for diseases such as refractory skin ulcers. It was suggested to be a therapeutic material.
  • the peripheral blood mononuclear cells + Since a wound area healing rate comparable to that of the fibroblast sheet group was confirmed (FIGS. 3A and 3B), a clear difference between the two groups using at least the wound area healing rate as an index was not confirmed.
  • a cell sheet containing peripheral blood mononuclear cells and fibroblasts was prepared from allo-transplanted C57BL / 6 mice, and the cell sheets were transplanted to the back of a diabetic model of C3H mice as another system.
  • a cell sheet containing peripheral blood mononuclear cells and fibroblasts was prepared according to [0071].
  • C3H mice were prepared according to [0076], and the cell sheet was transplanted and fiblast spray treatment (positive control) was performed according to [0077] and [0081].
  • the wound treatment evaluation (wound area healing rate) on the 14th day after treatment was in accordance with [0078].
  • genomic DNA is extracted from the transplanted surrounding tissue and should be contained only in the transplanted cell sheet (derived from male). PCR was performed using primers specific to the genomic DNA of SRY, ZFY1, ZFY2 encoded on the Y chromosome and ZFX encoded on the X chromosome. Specifically, 3 weeks after transplantation, tissues around the transplantation were collected, extracted from RNAlater (registered trademark) Solution (Ambion) and stored at 4 ° C., and DNA was extracted using AllPrepDNA / RNAMiniKit (Qiagen). did.
  • PCR was performed on the DNA using primers for KOD FX (ToYoBo) and SRY, ZFY1, ZFY2 (Y chromosome specific gene) ZFX (X chromosome specific gene) and ACTB (beta-actin).
  • the PCR reaction was performed at 94 ° C for 2 minutes, followed by 40 cycles of 98 ° C for 10 seconds, 60 ° C for 30 seconds, and 68 ° C for 30 seconds.
  • Fibroblasts were prepared from tissues collected from rabbit ears according to the method of [0068].
  • Peripheral blood mononuclear cells were collected from a rabbit otic artery by attaching a G winged needle to a 20 mL syringe in which heparin had been collected in an amount of about 1 to 1.5 mL, and this was transferred to a 15 mL tube. Later, 3 mL of Lympolyte-M (CEDARLANE) was added. Subsequent methods were in accordance with [0067].
  • Peripheral blood mononuclear cells were prepared at 2 ⁇ 10 6 cells / mL using a mouse peripheral blood mononuclear cell culture medium.
  • the fibroblasts were prepared to 1.25 ⁇ 10 5 cells / mL using a mouse fibroblast medium.
  • Peripheral blood mononuclear cells were cultured alone by separating 4 mL of the peripheral blood mononuclear cell suspension into one well of the UpCell 6-well plate and then adding 4 mL of mouse fibroblast medium to a total of 8 mL. To start.
  • fibroblast suspension For the cell sheet containing only fibroblasts, 4 mL of the fibroblast suspension is dispensed into one well of UpCell 6-well plate, and then 4 mL of mouse peripheral blood mononuclear cell medium is added to make a total of 8 mL. It started with that.
  • a cell sheet containing peripheral blood mononuclear cells and fibroblasts was added to 4 mL of the above peripheral blood mononuclear cell suspension and 4 mL of the above fibroblast suspension in one well of the UpCell 6-well plate for a total of 8 mL. And started.
  • mice fibroblasts are collected, and a cell preparation solution is prepared so that it becomes 1.25 ⁇ 10 5 cells / 500 microL in a fibroblast medium.
  • Control medium group Peripheral blood mononuclear cell culture medium 500 microL + fibroblast 1.25 ⁇ 10 5 cells / 500 microL
  • Peripheral blood mononuclear cell Conditioned medium group peripheral blood mononuclear cell culture supernatant 500 microL + fibroblast 1.25 ⁇ 10 5 cells / 500 microL 2 groups of cell suspensions were prepared, and the suspensions were seeded at 1 mL / well on a 24 well plate and then cultured for 48 hours under Normo conditions.
  • the culture solution of each group was transferred to a 1.5 mL tube, centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected into a new 1.5 mL tube, and the VEGF concentration in the supernatant was determined by ELISA [ 0074].
  • TGF-beta1 Quantification of TGF-beta1 in cultured cell supernatants of peripheral blood mononuclear cells alone and fibroblasts alone FBS used in normal culture contains a large amount of TGF-beta1, -Mouse / Rat / Portine / Canine TGF-beta 1 Quantikine ELISA Kit (R & D systems) used for beta 1 measurement detects TGF-beta 1 contained in FBS. Therefore, CTS TM AIM V (registered trademark) Medium (Life Technologies), which is xeno-free, was used as a medium for measuring TGF-beta1.
  • Peripheral blood mononuclear cells and fibroblasts were collected from mice according to the previously reported [0067]-[0068], and using CTS TM AIM V (registered trademark) Medium, Mouse fibroblast group: fibroblast 1.25 ⁇ 10 5 cells / 2 mL CTS TM AIM V® Medium
  • Mouse peripheral blood mononuclear cell group peripheral blood mononuclear cells 2 ⁇ 10 6 cells / 2 mL CTS TM AIM V (registered trademark)
  • 2 groups of cell suspensions were prepared, and the suspensions were seeded at a rate of 2 mL / well on a 24 well plate and then cultured for 48 hours under Normo conditions.
  • the culture solution of each group was transferred to a 1.5 mL tube and centrifuged at 3000 rpm for 5 minutes, and then the supernatant was separated into a new 1.5 mL tube, and the TGF-beta1 concentration in the supernatant was Was measured by ELISA using a Mouse / Rat / Portine / Canine TGF-beta 1 Quantikine ELISA Kit.
  • Mouse fibroblast group fibroblasts 1.25 ⁇ 10 5 cells / 1 mL (medium for fibroblasts) + medium for peripheral blood mononuclear cells 1 mL
  • Mouse peripheral blood mononuclear cells peripheral blood mononuclear cells 2 ⁇ 10 6 cells / 1 mL (peripheral blood mononuclear cell culture medium) + fibroblast medium 1 mL 2 groups of cell suspensions were prepared, and the suspensions were seeded at a rate of 2 mL / well on a 24 well plate and then cultured for 48 hours under Normo conditions.
  • the culture solution of each group was transferred to a 1.5 mL tube, centrifuged at 3000 rpm for 5 minutes, and the supernatant was separated into a new 1.5 mL tube, and the PDGF-BB concentration in the supernatant was determined. Measurement was performed by ELISA using Mouse / Rat PDGF-BB Quantikine ELISA Kit (R & D systems).
  • TGF-beta1 and PDGF-BB which are candidate factors for inducing increased VEGF production ability, were contained in the cultured cell supernatant of peripheral blood mononuclear cells and fibroblasts alone. Both factors were expressed only in the culture cell supernatant of peripheral blood mononuclear cells alone (FIGS. 8B and 8C). From the above events, in the cell sheet containing peripheral blood mononuclear cells and fibroblasts, the mechanism for enhancing the ability to produce VEGF from fibroblasts is TGF-beta1 secreted from peripheral blood mononuclear cells and / or It was speculated that it was due to PDGF-BB.
  • PDGF-BB (Sigma-Aldrich) recombinant protein was diluted to 500, 1000, 2000, 10000, 20000, and 40000 pg / mL with peripheral blood mononuclear cells, and 500 mL each was dispensed into each well of a 24 well plate.
  • Peripheral blood mononuclear cells and fibroblasts were collected from mice according to the previously reported [0067]-[0068], and a cell preparation solution was prepared so as to be 1.25 ⁇ 10 5 cells / 500 mL in fibroblast medium [0066].
  • the culture solution was transferred to a 1.5 mL tube and centrifuged at 3000 rpm for 5 minutes, and then the supernatant was separated into a new 1.5 mL tube, and the supernatant was used as a conditioned medium for the following experiment. .
  • the following three media were prepared using the conditioned medium and various antibodies; Control; conditioned medium 250 microL + anti-ATM antibody (1 mg / mL abcam ab78) 5 microL Alpha-TGF-beta 1; conditioned medium 250 microL + anti-TGF-beta 1 antibody (1 mg / mL abcam ab64715) 5 microL Alpha-PDGF-BB; conditioned medium 250 microL + anti-PDGF-BB antibody (0.2 mg / mL R & D AF-220-NA) 20 microL After the above three types of media were reacted in a 1.5 mL tube at 4 ° C. for 90 minutes, the entire amount of each medium was transferred to one well of 48 well plate.
  • mouse fibroblasts were collected, and a cell preparation solution was prepared so that it would be 1.25 ⁇ 10 5 cells / mL in a medium for fibroblasts, and 250 mL was added to each well containing the above three types of media. After the addition, the cells were cultured for 48 hours under Normo conditions. After culturing for 48 hours, the culture solution of each group was transferred to a 1.5 mL tube, centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected into a new 1.5 mL tube, and the VEGF concentration in the supernatant was determined by ELISA [ 0074].
  • the mechanism for enhancing the ability to produce VEGF from fibroblasts is TGF-beta1 secreted from peripheral blood mononuclear cells and / or The possibility of PDGF-BB was strongly suggested.
  • the cell sheet of fibroblasts alone stimulated by the factor like the cell sheet containing peripheral blood mononuclear cells and fibroblasts, in the medical field related to transplantation, particularly in intractable skin ulcers, It is expected to be an effective therapeutic transplant material.
  • Cell sheets containing fibroblasts stimulated by transforming growth factor or platelet-derived growth factor are similar to cell sheets containing peripheral blood mononuclear cells and fibroblasts, especially in the medical field related to transplantation, particularly refractory
  • Control group Group that does not give any treatment to the skin full-thickness defect site on the back of diabetic mice
  • Peripheral blood mononuclear cell + fibroblast sheet group Transplant a cell sheet containing peripheral blood mononuclear cells and fibroblasts to the above site
  • Group PDGF-BB + fibroblast sheet group Transplant a cell sheet containing peripheral blood mononuclear cells and fibroblasts to the above site
  • Group PDGF-BB + fibroblast sheet group Group to which a cell sheet prepared by adding 10 ng / mL PDGF-BB at the time of fibroblast seeding is transplanted to the above site
  • TGF-beta1 + fibroblast sheet group Group transplanted with cell sheet prepared by adding 5 ng / mL TGF-beta1 at the time of fibroblast seeding
  • AIM V Medium CTS was sprayed around the tissue, and the culture was continued at 37 ° C., 5% CO 2 for 4 hours. Thereafter, 5 mL of AIM V Medium CTS, 250 ⁇ L of autoserum, and 200 ⁇ L of Penicillin-Streptomycin, Liquid were added to 1 well of 6-well plate and cultured at 37 ° C. and 5% CO 2 for 3-4 weeks.
  • the cells were detached with Trypsin-EDTA (Gibco), permeated through 40 ⁇ m Cell Strainer (BD Falcon), and then centrifuged at 1200 rpm for 2 minutes. After centrifugation, the supernatant was removed by suction, the pellet was transferred to a 10 cm dish, 10 mL of medium (9.5 mL of AIM V Medium CTS + 0.5 mL of autoserum) was added, and the mixture was cultured at 37 ° C. and 5% CO 2 .
  • fibroblast marker In order to confirm that the cells isolated and cultured from the oral tissue were fibroblasts, immunostaining was performed using Vimentin, a fibroblast marker, according to the following method. 1 mL of human-derived fibroblasts prepared to 1.25 ⁇ 10 5 cells / mL in a medium (AIM V Medium CTS 9.5 mL + autologous serum 0.5 mL) was dispensed into one well of 24-well plate, After culturing at 5% CO 2 , fluorescent immunostaining was performed.
  • a medium AIM V Medium CTS 9.5 mL + autologous serum 0.5 mL
  • Vimentin (D21H3) XP (registered trademark) Rabbit mAb (Cell Signaling) is used as the primary antibody, and anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor (registered trademark) 488 conjugate (liged) is used as the secondary antibody.
  • DAPI nuclear staining with DAPI was performed.
  • the number of cells was counted, and the peripheral blood mononuclear cells were prepared to 2 ⁇ 10 6 cells / mL using a medium (AIM V Medium CTS 9.5 mL + autologous serum 0.5 mL). .
  • the cell sheet preparation schedule was the same cell concentration and culture period as those derived from mice, and the Normo and Hypo conditions were the same, but the medium used was all of AIM V Medium CTS plus 5% autologous serum. .
  • the VEGF concentration in the culture supernatant was measured according to [0074] using a Human VEGF Quantikine ELISA Kit (R & D systems).
  • TGF-beta1 secreted from peripheral blood mononuclear cells in the mechanism of enhancement of VEGF production ability from fibroblasts in a cell sheet containing peripheral blood mononuclear cells and fibroblasts. And / or PDGF-BB has been shown to be involved. And the cell sheet of fibroblasts alone stimulated by the factor is similar to the cell sheet containing peripheral blood mononuclear cells and fibroblasts, in the medical field related to transplantation, particularly in intractable skin ulcers, It has shown potential as an effective therapeutic implant.
  • activators such as TGF-beta1 and PDGF-BB found in these mouse-derived cells can also be found in human-derived cells, they are very useful for use as therapeutic materials for human diseases.
  • activators such as TGF-beta1 and PDGF-BB found in these mouse-derived cells
  • they are very useful for use as therapeutic materials for human diseases.
  • a desired amount of refractory skin ulcer can be treated by adding the activator as a recombinant protein.
  • Cell sheet production may be possible. From the above, a search was made for a factor that activates VEGF production in a cell sheet of human-derived fibroblasts alone.
  • peripheral blood mononuclear cell culture alone a cell sheet of fibroblasts alone, and a culture supernatant of a cell sheet containing peripheral blood mononuclear cells and fibroblasts, the peripheral blood mononuclear cells only Identified the factors secreted by.
  • Peripheral blood mononuclear cell group 1 mL of the cell suspension of the above peripheral blood mononuclear cells and 1 mL of the above medium were mixed in one well of 24-well plate to make a total of 2 mL, and cultured under Hypo conditions.
  • Fibroblast group 1 mL of the above-mentioned cell suspension of fibroblasts and 1 mL of the above medium were mixed in one well of a 24-well plate to make a total of 2 mL, and cultured under Hypo conditions.
  • Peripheral blood mononuclear cell + fibroblast group 1 mL of the above-mentioned peripheral blood mononuclear cell suspension and 1 mL of the above-mentioned fibroblast cell suspension are mixed in one well of a 24-well plate to make a total of 2 mL. Culture under Hypo conditions. Each of the culture broths was transferred to a 1.5 mL tube and centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected as a sample.
  • PDGF-BB (eBioscience) is 200 ng / mL
  • TGF-beta1 (R & D systems) is 20 ng / mL
  • PDGF-AA (Wako) is 200 ng / mL
  • HB-EGF (R & D systems) is 200 ng / mL
  • CXCL16 (Pepro Tech) is 200 ng / mL
  • CXCL1 (R & D systems) is 600 ng / mL
  • CCL2 (eBioscience) is 200 ng / mL
  • IL-1ra (R & D systems) is 80 ng / mL
  • CCL4 (R & D systems) is 20 ng / mL
  • CXCL5 (Gene Tex) is 200 ng / mL
  • CXCL10 (Gene Tex) is 100 ng / mL
  • CCL3 (R & D systems)
  • VEGF concentration contained in the supernatant was measured by ELISA according to the previous report [0115].
  • VEGF-producing ability in fibroblasts as candidate activators 2
  • the induction activity is particularly high in the above evaluation.
  • the dose correlation with the VEGF production inducing activity was examined.
  • the cell proliferation ability of the factor was also examined.
  • each factor of TGF-beta1 (R & D systems), PDGF-BB (eBioscience), bFGF (Sigma-Aldrich) was changed to 40, 20, 15, 10, 5, 2, 1.5, 1, After adjusting to 0.5, 0.2, 0.1 ng / mL, 0.5 mL each was added to each well of the 48-well plate from which the cell suspension was separated to make a total of 1 mL. The culture was performed under normal conditions for a period of time. Since bFGF is a main component of fiblast spray as a control agent, it was added to the evaluation target.
  • a control 0.5 mL of AIM V Medium CTS was added to one well of a 48-well plate from which the cell suspension was separated to make a total of 1 mL, and cultured under Normo conditions for 72 hours.
  • the cultured medium was transferred to a 1.5 mL tube, centrifuged at 3000 rpm for 5 minutes, and the VEGF concentration contained in the supernatant was measured by ELISA according to the previous report [0115].
  • TGF-BB and TGFbeta1 Cell growth ability of PDGF-BB and TGFbeta1 in fibroblasts per 1 mL of TGF-beta1 (R & D systems), PDGF-BB (eBioscience), bFGF (Sigma-Aldrich) recombinant protein is 5, 7.5, 10, 20 ng / mL, human-derived fibroblast cell concentration of 1 ⁇ 10 4 cells / mL, prepared by AIM V Medium CTS to 5% autoserum, 100 ⁇ L per well of 96-well plate And incubated for 3 days at 37 ° C., 5% CO 2 .
  • the present invention provides a cell sheet that significantly increases the production amount of blood vessel growth factor that plays an important role in angiogenesis, and induces a significant increase in the healing rate of the wound site by transplantation to the skin wound site.
  • blood vessel growth factor that plays an important role in angiogenesis
  • peripheral blood mononuclear cells and fibroblasts from patients undergoing transplantation both can be collected minimally invasively
  • the produced cell sheet is extremely useful in that it does not cause immune rejection and greatly contributes to the development of the medical field.

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Abstract

　La présente invention concerne un feuillet de cellules se révélant utile en vue du traitement d'ulcères cutanés réfractaires, ainsi qu'un procédé de production dudit feuillet de cellules. L'invention concerne, plus précisément, un feuillet de cellules capable d'améliorer suffisamment la cicatrisation d'un ulcère ischémique dans lequel la circulation sanguine se fait mal par greffe du feuillet de cellules au niveau du site de l'ulcère, et ce, sans provoquer de réaction de rejet immunitaire, telle qu'une réaction induite par un derme artificiel ou équivalent ; et un procédé de production dudit feuillet de cellules. La présente invention concerne, donc, un feuillet de cellules qui contient des cellules mononucléaires du sang périphérique et des fibroblastes, et un feuillet de cellules qui contient des fibroblastes qui ont été stimulés par un facteur de croissance transformant ou un facteur de croissance dérivé des plaquettes. La quantité de facteur de croissance des vaisseaux sanguins, qui joue un rôle important dans l'angiogenèse, produite dans ledit feuillet augmente de manière significative, et suite à la greffe dudit feuillet au niveau du site d'un ulcère cutané créé sur le dos d'une souris diabétique, la vitesse de guérison du site de la plaie est significativement améliorée par rapport à ce que l'on observe suite à la greffe du feuillet de cellules ne comprenant que des fibroblastes. En outre, en utilisant des cellules prélevées chez un patient en tant que cellules mononucléaires du sang périphérique et fibroblastes de la présente invention, il est possible de supprimer toute réaction de rejet immunitaire suite à la greffe.
PCT/JP2015/080465 2014-10-29 2015-10-28 Feuillet de cellules contenant des cellules mononucléaires du sang périphérique ou des fibroblastes associés au facteur sécrété par les cellules mononucléaires du sang périphérique WO2016068217A1 (fr)

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JP2021503923A (ja) * 2017-11-29 2021-02-15 フィジーン、エルエルシーＦｉｇｅｎｅ， Ｌｌｃ 活性化のための線維芽細胞と免疫細胞との相互作用及びそれらの使用
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US11311579B2 (en) 2016-08-30 2022-04-26 Niigata University Cell preparation and method for producing cell preparation
JP7416367B2 (ja) 2017-09-01 2024-01-17 国立大学法人鳥取大学 線維化抑制作用を有する細胞シート
JP2021503923A (ja) * 2017-11-29 2021-02-15 フィジーン、エルエルシーＦｉｇｅｎｅ， Ｌｌｃ 活性化のための線維芽細胞と免疫細胞との相互作用及びそれらの使用
JP7387603B2 (ja) 2017-11-29 2023-11-28 フィジーン、エルエルシー 活性化のための線維芽細胞と免疫細胞との相互作用及びそれらの使用
US11866483B2 (en) 2018-06-08 2024-01-09 Kanoncure, Inc. Fibrosis-inhibiting composition, cells producing same, and cell sheet comprising said cells
WO2021131261A1 (fr) * 2019-12-23 2021-07-01 学校法人順天堂大学 Groupe de cellules et son procédé d'acquisition
CN114901806A (zh) * 2019-12-23 2022-08-12 学校法人顺天堂大学 细胞群以及其取得方法
EP4197547A1 (fr) * 2021-12-20 2023-06-21 Aposcience AG Composition pour le traitement ou la prévention de la vasculite et des maladies associées à la vasculite

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