JP6583830B2 - Cell sheet containing fibroblasts with peripheral blood mononuclear cells or factors secreted from peripheral blood mononuclear cells - Google Patents

Description

  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. However, there is a problem that when the drug is used, it is necessary to have a minimum blood flow in the lesion to obtain a therapeutic effect, so that the therapeutic effect on ischemic ulcer is not always sufficient. is there.

  Artificial dermis, on the other hand, 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.

  Thus, in the existing treatment methods, there are many cases in which sufficient improvement cannot be obtained for ischemic ulcers and cases where it takes a long time to heal ulcers, especially in the treatment methods using artificial dermis. However, there is also an adverse effect such as immune rejection, and it is desired to establish an effective treatment method that clears these problems.

  Cell transplantation therapy is attracting attention as an effective treatment for various diseases and tissue damage. The inventors have been studying cell transplantation therapy using bone marrow cells for cardiovascular disease for many years. In 1999, 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.

  However, it has been pointed out that mere transplantation of transplanted cells does not necessarily have a high engraftment rate with respect to the tissue. In order to obtain a higher effect of transplantation, it has recently been desired as a biological material for transplantation. 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.

  The cell sheet has been studied as a technique for improving the cell engraftment rate at the target site in various surgical fields. For example, in the heart field, high engraftment in the infarcted heart and accompanying promotion of myocardial regeneration have been reported. (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).

  However, the use of a cell sheet instead of normal cultured cells for the treatment of intractable skin ulcers has not been reported so far, and the effect as a therapeutic method for the disease has been unknown. In particular, when used for the treatment of diseases such as ischemic ulcers with poor blood flow, in order to achieve sufficient improvement / healing of the ulcers, the blood flow at the ulcer site is somehow formed using a transplanted cell sheet. Improvement induction is necessary, and avoiding the occurrence of immune rejection as observed in the treatment with artificial dermis is also an important issue. Thus, although there have been various reports so far on the transplantation of cell sheets, regarding the function of the cell sheet, the constitution suitable for the treatment of intractable skin ulcer as a biological material for transplantation and the production conditions thereof It has not yet been fully elucidated.

Japanese Patent No. 4716479

Esato et al. 2002, Cell Transplant. Vol. 11, pp. 747-752 Miyahara et al. 2006, Nature Medicine Vol. 12, pp. 459-465 Alshammary et al. 2013, Surg. Today. Vol. 43 pp. 970-976

  The present invention provides a cell sheet effective for treating intractable skin ulcers and a method for producing the cell sheet. In particular, by 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. Provided are a cell sheet and a method for producing the same.

  As a result of earnest research on the cell sheet and the production method thereof, 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.

In the present 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.
In particular, because it uses 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.

  Furthermore, 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. As research progresses, transforming growth factor (TGF) or platelet-derived growth factor (PDGF) produced from coexisting peripheral blood mononuclear cells increases the production amount of the blood vessel growth factor I found out that it is guiding.

  By applying this phenomenon, instead of adding peripheral blood mononuclear cells to fibroblasts, 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.

  According to the present invention, 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. .

Accordingly, 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.
(2) The cell sheet according to (1), wherein the fibroblast is obtained from an individual suffering from intractable skin ulcer, which is a subject to be treated.
(3) The cell sheet according to (1) or (2), wherein stimulation with the transforming growth factor or the platelet-derived growth factor is started simultaneously with seeding of the fibroblasts.
(4) A transplant material for treating refractory skin ulcer comprising the cell sheet according to any one of (1) to (3).
(5) 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).
(8) 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. The cell sheet according to (10), wherein the co-culture is performed by seeding at step ² .
(12) The cell sheet according to (10) or (11), wherein the co-culture is started by simultaneously seeding peripheral blood mononuclear cells and fibroblasts.
(13) The cell sheet according to any one of (8) to (12), wherein the cell sheet is cultured for a predetermined period under low temperature and low oxygen conditions.
(14) A transplant material for treating refractory skin ulcer, comprising the cell sheet according to any one of (8) to (13).
(15) 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 cell sheet that significantly increases the production amount of blood vessel growth factor that plays an important role in angiogenesis by the present invention, and induces a significant increase in the healing rate of the wound site by transplanting it to the skin ulcer site of a diabetic mouse. Can be provided.
Therefore, according to the present invention, as a biomaterial for transplantation that can be used for the treatment of refractory skin ulcers, particularly ischemic ulcers, blood flow improvement by angiogenesis is realized, and the patient's own cells are used for By providing a therapeutic tool by producing a large amount of cell sheets having the advantage of not causing rejection and / or fibroblast sheets derived from other families and recombinant proteins of transforming growth factor or platelet-derived growth factor, It will be possible to establish a breakthrough treatment method for refractory skin ulcers.

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. Cell sheet residual test: Cell sheets containing peripheral blood mononuclear cells and fibroblasts derived from male C57BL / 6 mice were transplanted into diabetic female C57BL / 6 mice lacking all layers of the back skin (No. 1-4). The detection of male-derived genes (SRY, ZFY1, ZFY2) at the site around 3 weeks after transplantation was performed. Blood vessel growth factor producing ability (A) and therapeutic effect (B) in a cell sheet containing peripheral blood mononuclear cells and fibroblasts derived from rabbits. A: Blood vessel growth factor concentration in culture supernatant of peripheral blood mononuclear cell culture, cell sheet containing fibroblasts and cell sheet containing peripheral blood mononuclear cells and fibroblasts. 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. Induction of blood vessel growth factor production from cultured fibroblasts by peripheral blood mononuclear cell culture supernatant (A) and transforming growth factor secreted from cultured peripheral blood mononuclear cells or cultured fibroblasts (B) Alternatively, quantification of platelet-derived growth factor (C). Induction of vascular growth factor production of transforming growth factor and platelet-derived growth factor (A) and attenuation of vascular growth factor production in peripheral blood mononuclear cell culture supernatant by anti-transforming growth factor antibody and anti-platelet-derived growth factor antibody (B ). The therapeutic effect by the cell sheet containing the fibroblast stimulated by the transforming growth factor or the platelet-derived growth factor. The wound area healing rate 14 days after transplantation is shown. Preparation of cell sheet containing human-derived peripheral blood mononuclear cells and fibroblasts (A) and confirmation of vascular growth factor production ability (B). Examination of the ability to produce blood vessel growth factor by stimulating activating factor candidates in cell sheets containing human-derived fibroblasts. The administration concentration of each factor and the blood vessel growth factor concentration in the culture supernatant after administration are shown. Dose dependence of vascular growth factor production ability (A) and cell growth ability (B) stimulated by transforming growth factor, platelet-derived growth factor and basic fibroblast growth factor using cell sheets containing human-derived fibroblasts Sexual examination.

The first aspect of the present invention is a cell sheet containing peripheral blood mononuclear cells and fibroblasts.
In the present invention, 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.

  Here, “peripheral blood mononuclear cells (PBMNC)” 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. As 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. However, for example, 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. Here, 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.
In the co-culture, 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 [ Preferably, the peripheral blood mononuclear cells are 5.0 × 10 ⁴ cells / cm ^{2 to} 1.5 × 10 ⁶ cells / cm ² and the fibroblasts are 1.0 × 10 ⁴ cells / cm ² It may be seeded at 1.5 × 10 ⁵ cells / cm ² , and more preferably, peripheral blood mononuclear cells are 5.5 × 10 ⁴ cells / cm ^{2 to} 1.0 × 10 ⁶ cells / cm ² and fibroblasts are 1. It may be seeded at ⁵ × 10 ⁴ cells / cm ^{2 to} 1.0 × 10 ⁵ cells / cm ² , and more preferably, the peripheral blood mononuclear cells are 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.

Here, in the step (d), 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. Alternatively, it can be carried out under conditions ordinarily practiced in the art suitable for the peripheral blood mononuclear cells and fibroblasts used. For example, 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%, and the O ₂ concentration is atmospheric oxygen concentration (approximately 20%). However, this condition is not limited, and culture temperature, CO ₂ concentration, and O ₂ concentration can be appropriately selected. For example, in a cell sheet formed using peripheral blood mononuclear cells prepared from mouse blood and fibroblasts prepared from mouse tail, the culture temperature is 37 ° C., the CO ₂ concentration is 5%, and 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, and may be, for example, about 10 hours to 240 hours, preferably 12 hours to 168. The culture time is about an hour, and more preferable results are obtained in 48 hours to 96 hours.
In addition, 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. Specifically, as described above, preferably, 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 It 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 are 5.0 × 10 ⁵ cells / cm ^{2 to} 1.0 × 10 ⁶ cells / cm ^2. Then, the fibroblasts may be seeded at 6.0 × 10 ⁴ cells / cm ^{2 to} 1.0 × 10 ⁵ cells / cm ² . Moreover, 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.

Here, 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. Examples of 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.
Furthermore, 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. When coating the culture surface of the cell culture substrate with these components, the concentration of the solution used to coat the culture surface differs depending on each component. Depending on the method that can be considered, an appropriate solution concentration can be determined for the coating of each component.

  As a medium used in the method for producing a cell sheet according to the present invention, 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. For example, 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. Preferably, RPMI-1640 may be used for peripheral blood mononuclear cells and DMEM for fibroblasts, but is not limited thereto.

In particular, when culturing human-derived peripheral blood mononuclear cells and fibroblasts, AIM V (registered trademark) medium CTS ^™ may be used without limitation.

  Furthermore, you may use for a culture medium, adding an appropriate additive as needed. Examples of 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, other animal sera such as fetal bovine serum (FBS), horse serum, etc. Etc. may be included. Moreover, you may add a buffering agent (for example, PBS, HEPES, MES, HANK'S etc.) to a culture medium suitably. Furthermore, cell growth factors and the like may be added as appropriate according to the origin and purpose of the cells to be cultured.

  In particular, in the case of culturing human-derived peripheral blood mononuclear cells and fibroblasts, blood is collected from a subject from which the cells are collected, serum is prepared from the blood, and this is used. Good.

  As an example, as a medium used for a cell sheet composed of peripheral blood mononuclear cells and fibroblasts, 10% FBS is added to RPMI-1640, which is a culture medium for peripheral blood mononuclear cells, and penicillin 100 Unit / ml, streptomycin 100 μg / ml and 1 mM L-glutamine added, and fibroblast medium, DMEM to 20% FBS, penicillin 100 units / ml, streptomycin 100 μg / ml and What added 1 mM L-glutamine and what was 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. For example, 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. Alternatively, as long as the sheet-like structure is not damaged, 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. Alternatively, 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.
When a cell culture substrate whose surface is coated with a temperature-responsive material is used, 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.

In addition, before the step (d) of the method for producing a cell sheet including the steps (d) to (e) described above,
(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. By adding this step, 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.

  According to a third aspect of the present invention, there is provided a cell sheet and a method for producing the cell sheet, wherein the cell sheet according to the first aspect is cultured for a predetermined period under low temperature and low oxygen conditions.

Until now, 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. When transplanted into a failing heart of an old myocardial infarction model mouse, it was found that significant improvement in cardiac function (left ventricular ejection fraction and left ventricular diameter shortening rate) occurred compared to transplantation with an untreated cell sheet. Are (already been filed as Japanese Patent Application No. 2014-188322).
Even in the cell sheet comprising peripheral blood mononuclear cells and fibroblasts according to the first aspect of the present invention, the effect of co-culturing peripheral blood mononuclear cells and fibroblasts by applying hypoxic preconditioning In addition, a significant increase in the production amount of blood vessel growth factor was confirmed (Example [0075]), and the effect was greatly exceeded.

The method for producing a cell sheet comprising peripheral blood mononuclear cells and fibroblasts subjected to such hypoxic preconditioning is not particularly limited.
Between the steps (d) and (e) of the method for producing a cell sheet including the steps (d) to (e) according to the second aspect of the present invention described above,
(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 a cell sheet comprising peripheral blood mononuclear cells and fibroblasts may be used.

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. Here, 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. For example, under low oxygen conditions, 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. to 34 ° C., more preferably 33 ° C., and the treatment period is 12 hours to 72 hours, preferably 24 hours. .
In addition, after the treatment period has elapsed, 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. Here, 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.

Furthermore, the 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 with a transforming growth factor or a platelet-derived growth factor.

  Transforming growth factor (TGF) is one of the growth factors, and is known to play an important role in tissue development, cell differentiation, embryo development and the like. 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. On the other hand, 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). As a result, the increase in the production amount of the blood vessel growth factor is that the 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 herein is preferably a beta type, and particularly preferably a beta 1 subtype, but is not limited thereto.

  Platelet-derived growth factor (PDGF) 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 disulfide bond to form a homo- or hetero-dimer structure, and three isoforms (PDGF- AA, AB, BB).

  As in the case of transforming growth factor, the inventors activate 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 was induced (FIGS. 8, 9, 12, and 13). Moreover, it discovered that PDGF-AA was contained in the factor which the secretion increased specifically only in the cell mixing sheet | seat of a human origin peripheral blood mononuclear cell and a fibroblast (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.

In the present invention, “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.
Here, 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.

  According to the present invention, 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. Although it is not limited, a stimulus that starts simultaneously with sowing may be preferable.

  Further, the fibroblasts may be obtained from an individual suffering from refractory skin ulcer, which is a subject to be treated. By producing a cell sheet from fibroblasts obtained from the individual, it becomes possible to suppress immune rejection at the time of transplantation.

  The origin, preparation method, seeding density on the culture substrate and the like of the fibroblasts according to the fourth aspect of the present invention are the same as those of the first aspect of the present invention, and an example is given 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.

  Here, in the step (a), 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, the part about a fibroblast is mentioned among the description regarding the 2nd aspect of this invention described in [0068]-[0069].

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. By adding the step, 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.
In this case, 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. However, 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. In platelet-derived growth factor, it is 5 ng / mL or more, preferably 10 ng / mL or more.

  In the step (a), 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]. .

  According to a sixth aspect of the present invention, there is provided 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 of the present invention. Is a transplant material for treating refractory skin ulcer containing the cell sheet produced by

  As described in the following Examples, when the cell sheet was transplanted into a full-thickness skin defect in the back of a diabetes model animal (mouse / rabbit) corresponding to a disease model of refractory skin ulcer, it was prominent compared to the control group A therapeutic effect (increase in wound area healing rate) has been confirmed (FIGS. 3, 5, 7, and 10). Therefore, 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 | seat.

As described above, the inventors have confirmed that 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. For example, refractory typified by pressure ulcer, obstructive arteriosclerosis, diabetes, venous insufficiency, collagen disease, vasculitis, etc. Examples include skin ulcers.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Materials and reagents]
1) Purchased from mouse C57BL / 6NCrSlc (Japan SLC, Inc.).
2) Penicillin--added to a medium for peripheral blood mononuclear cells RPMI-1640 (Gibco) so that FBS (Gibco) is 10%, and the final concentration is 100 units / mL for penicillin and 100 μg / mL for streptomycin. Streptomycin, Liquid (Gibco) is added.
3) Add the FBS (Gibco) to 20% to the fibroblast medium DMEM (Gibco) so that the non-essential amino acids (Gibco) and final concentrations are 100 units / mL for penicillin and 100 μg / mL for streptomycin. Penicillin-Streptomycin, Liquid (Gibco) is added.

[Separation of mononuclear cells from peripheral blood]
A 26 G needle was attached to a 1 mL syringe, about 0.10 mL of heparin was taken, and blood was collected from the abdominal vena cava. 3 mL of Lympolyte-Mammal (CEDARLANE) was added to a 15 mL tube, blood diluted twice with PBS was added, and centrifugation was performed at 800 g for 20 minutes with no brake. After centrifugation, the mononuclear cell layer was transferred to a new 15 mL tube, PBS was added to 10 mL, and centrifugation was performed at 2000 g for 5 minutes with no brake. Washing with PBS was performed twice in total, followed by washing once with 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.

[Separation of fibroblasts]
The mouse tail was wiped with Kimwipe sprayed with 70% ethanol, the tip was cut about 1 cm, soaked in fibroblast medium (500 U / mL for penicillin and 500 μg / mL for streptomycin) for 10 minutes, and then rinsed with PBS. . Then, a collagenase (Wako Pure Chemical Industries) was dissolved in a fibroblast medium medium and adjusted to 500 U / mL, and a tail finely cut using a disposable scalpel was added and cultured at 37 ° C. overnight. The finely chopped tail was transferred to a 15 mL tube and centrifuged at 1200 rpm for 2 minutes. After centrifugation, the supernatant was removed by suction, the pellet was transferred to one well of a 6-well plate, 5 mL of fibroblast medium was added, and cultured at 37 ° C., 5% CO ₂ for 2-4 days. Cells were detached with Trypsin-EDTA (Gibco), passed through a 40 μm Cell Strainer (BD Falcon), and centrifuged at 1200 rpm for 2 minutes. After centrifugation, the supernatant was removed by aspiration, the pellet was transferred to a 10 cm dish, 10 mL of fibroblast medium was added and cultured at 37 ° C., 5% CO ₂ . For cell sheet production, fibroblasts were prepared at 1.25 × 10 ⁵ cells / mL using a fibroblast medium.

[Preparation of cell sheet (fibroblasts alone / fibroblasts + peripheral blood mononuclear cells) and culture of peripheral blood mononuclear cells alone]
1) Cell sheet of fibroblasts alone (examination of culture conditions)
In one well of UpCell 24-well plate (Cellseed Co., Ltd.), fibroblasts separated by the above method using a fibroblast medium were obtained as No. 1 in Table 1. Each cell number described in 1 to 8 was added and cultured for 48 hours at 37 ° C. under 20% O ₂ and 5% CO ₂ conditions, and then the formation of the cell sheet was confirmed.
No. When fibroblasts seeded under 5-8 conditions were observed overnight in each well of an UpCell 24-well plate, a gap remained in the wells at 1.25 × 10 ⁵ or more (No. 5). It wasn't.
In any case, since the formation of the cell sheet was confirmed, the peeling state of the sheet was observed with a microscope while removing the cell sheet. In the cell sheet formed under each of the conditions 1 to 4, it was observed that the sheet was cleaved when peeled from the plate.
Based on the above results, for production of a cell sheet containing fibroblasts, the cells were seeded under conditions of 1.25 × 10 ⁵ cells / well (= 6.58 × 10 ⁴ cells / cm ² ). That is, 1 mL of the cell suspension adjusted to 1.25 × 10 ⁵ cells / mL and 1 mL of the peripheral blood mononuclear cell medium were added to make a total of 2 mL.

2) Cell sheet of fibroblasts alone (collection of culture supernatant under each condition)
A group of fibroblasts seeded under the above conditions for 48 hours at 37 ° C., 20% O ₂ , 5% CO ₂ , and then further cultured for 24 hours under the same conditions (Normo condition group) The conditions were changed to 33 ° C., 2% O ₂ , 5% CO ₂ and the cells were divided into two groups: a group cultured for 24 hours (Hypo condition group). The culture supernatant of each well was collected and centrifuged at 3000 rpm for 5 minutes, and blood vessel growth factor (VEGF) contained in the supernatant was measured by ELISA.

3) Preparation of cell sheet containing peripheral blood mononuclear cells and fibroblasts (conditional study)
Next, we will examine the conditions to determine how many cells of peripheral blood mononuclear cells can be seeded together with fibroblasts under the above seeding conditions to form a cell sheet containing stable peripheral blood mononuclear cells and fibroblasts. went.
Peripheral blood mononuclear cells separated by the above method using a peripheral blood mononuclear cell culture medium in one well of the UpCell 24-well plate, together with fibroblasts (1.25 × 10 ⁵ cells / well), No. 2 Each cell number described in 1 to 6 was added and cultured under conditions of 37 ° C., 20% O ₂ , 5% CO ₂ .
The above culture conditions and No. After overnight culture with 1 to 6 cells, the formation of the cell sheet was confirmed. As a result, it was confirmed that the cell sheet was formed in all combinations and the sheet could be peeled off.
Based on the above results, for the production of cell sheets containing peripheral blood mononuclear cells and fibroblasts, the same number as the number of cells (1.25 × 10 ⁵ cells / well) to be seeded at the time of cell sheet production with fibroblasts alone. Peripheral blood mononuclear cells were seeded together with fibroblasts under conditions of ² × 10 ⁶ cells / well (= 10.53 × 10 ⁵ cells / cm ² ). That is, 1 mL of a cell suspension prepared by using a peripheral blood mononuclear cell medium to a concentration of 2 × 10 ⁶ cells / mL using a peripheral blood mononuclear cell medium, and 1.25 × 10 ⁵ fibroblasts using a fibroblast medium. 1 mL of the cell suspension prepared to ⁵ cells / mL was added to one well of the UpCell 24-well plate to make a total of 2 mL.

4) Preparation of cell sheet containing peripheral blood mononuclear cells and fibroblasts (collection of culture supernatant under each condition )
Peripheral blood mononuclear cells and fibroblasts seeded under the above conditions were cultured for 48 hours at 37 ° C., 20% O ₂ , 5% CO ₂ and then further cultured for 24 hours under the same conditions. The condition was changed to 33 ° C., 2% O ₂ , 5% CO ₂ and the group was cultured for 24 hours (Hypo condition group) and divided into two groups. The culture supernatant of each well was collected and centrifuged at 3000 rpm for 5 minutes, and VEGF contained in the supernatant was measured by ELISA.
In addition, 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.

5) 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 adjusted to a peripheral blood mononuclear cell concentration of 2 × 10 ⁶ cells / mL using peripheral blood mononuclear cell medium To 2 mL in total.
A group in which peripheral blood mononuclear cells seeded under the above conditions were cultured for 48 hours at 37 ° C., 20% O ₂ , 5% CO ₂ and then further cultured for 24 hours under the same conditions (Normo condition group) ) And 33 ° C., 2% O ₂ , 5% CO ₂ , and the group was divided into two groups: a group cultured for 24 hours (Hypo condition group). The culture supernatant of each well was collected and centrifuged at 3000 rpm for 5 minutes, and VEGF contained in the supernatant was measured by ELISA.
In addition, 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.

2) Results and Discussion In each supernatant of A) peripheral blood mononuclear cell single culture, B) fibroblast single cell sheet and C) cell sheet containing peripheral blood mononuclear cells and fibroblasts in Normo condition group When the concentration of VEGF contained was measured, a significant increase in VEGF concentration was observed in C) compared to B), and VEGF was not detected in the supernatant of A) (FIG. 2).
Therefore, the cell sheet consisting of peripheral blood mononuclear cells and fibroblasts plays a more important role in angiogenesis than cultured cells and cell sheets of peripheral blood mononuclear cells and fibroblasts alone. It became clear that the production amount of the factor was greatly increased.
Furthermore, in each of A), B), and C), the change in VEGF concentration in the Hypo condition group relative to the Normo condition group was measured. As in the Normo condition group, the change was below the detection limit, but B) In C and C), a significant increase in VEGF concentration was confirmed with respect to the Normo condition group, and in particular, a significant increase in concentration was observed in C) (FIG. 2).
From this, a cell sheet containing peripheral blood mononuclear cells and fibroblasts is cultured for a predetermined period of time under low temperature and low oxygen conditions (hypoxic preconditioning), so that the preconditioning treatment is not performed. In comparison, it became clear that the production amount of blood vessel growth factor increased significantly.

[Wound area healing rate by cell sheet transplantation]
1) 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.

2) Cell Sheet Transplantation An about 8 mm full-thickness skin defect was created on the back of a diabetic mouse by a surgical technique, and a cell sheet was transplanted to the site. From above, it was covered with ABSOCURE (registered trademark) -UND (Nitto Medical) and fixed with an adhesive bandage (Nichiban). 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).

  The 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.

3) Wound healing evaluation As described above, a skin full-thickness defect was prepared by a surgical technique, and the wound surface was photographed with a digital camera before cell sheet transplantation, and this was defined as day 0. After transplanting the cell sheet, the wound surface was photographed 7 or 14 days later, the wound area was calculated using ImageJ (NIH), and the healing rate was evaluated based on the wound surface at 0 day.

4) Results and discussion At both time points 7 days and 14 days after transplantation, in c), a significant increase in wound healing rate occurred for both a) and b) (FIG. 3A).

  Thus, in 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) In addition to the group of single use, 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.

  On the other hand, in comparison between the positive control fiblast spray group and the peripheral blood mononuclear cells according to the present invention and the group of cell sheets containing fibroblasts, 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.

[Comparison of the pathological image of the treatment site in peripheral blood mononuclear cells and fibroblast cell sheet transplantation and fiblast spray treatment]
In cell sheet transplantation including peripheral blood mononuclear cells and fibroblasts and fibroblast spray treatment, which is an existing treatment method, when the wound area healing rate was used as an index, no clear difference could be confirmed between the two groups. Therefore, the pathological image of the treatment site in both treatments was compared.

1) Pathological image analysis Since the skin defect site was closed on the 17th day after the start of treatment, the mice treated with both treatments were sacrificed, and the tissue at the treatment site was collected and fixed with 10% neutral formalin. The tissue was embedded in paraffin, and the tissue was sliced and then stained with HE according to a conventional method.

2) Results and discussion In both groups, a pathological image in which the tissue of the skin defect wound was healed was confirmed (FIG. 4). However, in the cell sheet transplantation including peripheral blood mononuclear cells and fibroblasts, the tissue was a natural healing tissue, but in the tissue in the fiblast spray treatment group, poor granulation and infiltrating inflammatory cells were observed. .
In this way, in the group using the cell sheet containing peripheral blood mononuclear cells and fibroblasts according to the present invention for treatment, the apparent cure rate compared to the treatment group using fiblast spray which is clinically used in practice. Although there was a similar improvement, there was a significant qualitative difference.
Therefore, the cell sheet containing peripheral blood mononuclear cells and fibroblasts according to the present invention is extremely effective in diseases such as intractable skin ulcers as compared with the existing treatment (treatment by fiblast spray). It was suggested to be a therapeutic material.

[Allo transplantation evaluation using mice]
When transplanting a cell sheet containing peripheral blood mononuclear cells and fibroblasts to a patient as a therapeutic material, is there any problem such as rejection in the case of transplanting another person's cells into the patient (allotransplantation)? It was confirmed.

1) 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]. As a diabetes model animal, 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].

2) Results and discussion Even when a cell sheet containing peripheral blood mononuclear cells and fibroblasts was prepared from C57BL / 6 mice, and the cell sheet was transplanted to the back of a diabetes model by C3H mice as other systems, The effect of the wound area healing rate (FIG. 3) by transplantation between mice of the same strain was confirmed (FIGS. 5A and 5B).
Based on the above, it was suggested that even if a cell sheet containing peripheral blood mononuclear cells and fibroblasts not derived from the patient itself was used for treatment, immune rejection would not necessarily occur.

[Assessment of survival of transplanted cell sheet in tissue]
When a cell sheet containing peripheral blood mononuclear cells and fibroblasts was transplanted, it was examined whether the sheet engrafted in the transplanted tissue.

1) Preparation of cell sheet derived from male mouse and transplantation to female mouse diabetes model [0067]-[0068] After preparing peripheral blood mononuclear cells and fibroblasts from male C57BL / 6 mice, A cell sheet containing was prepared [0071]. In addition, a diabetes model was prepared using C57BL / 6 mice (female) according to [0076], and the skin full-thickness defect made on the back of the diabetes mouse according to the method described in [0077] Cell sheets were transplanted.

2) Detection of Y-chromosome-specific gene in DNA extracted from surrounding tissue after transplantation Three weeks after transplantation, 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.

3) Results and Discussion According to the above method, a cell sheet containing peripheral blood mononuclear cells and fibroblasts derived from male mice was transplanted against the full thickness skin defect on the back of a diabetes model derived from the same strain of female mice. ACTB and ZFX were detected in the tissue around the transplanted site after a week, but the gene derived from the cell sheet (Y chromosome) was not detected (FIG. 6). This revealed that the cell sheet was not engrafted in the transplanted tissue.

[Cell sheet VEGF producing ability including peripheral blood mononuclear cells and fibroblasts derived from rabbits and therapeutic effect by transplantation]
In order to confirm that a cell sheet containing peripheral blood mononuclear cells and fibroblasts is beneficial also in animals other than mice, the rabbit-derived cell sheet was prepared and examined for its ability to produce VEGF and the therapeutic effect of transplantation. did.

1) Preparation of peripheral blood mononuclear cells and fibroblasts derived from rabbits Fibroblasts were prepared from tissues collected from rabbit ears according to the method of [0068]. Peripheral blood mononuclear cells were collected from a rabbit ear artery by attaching a G winged needle to a 20 mL syringe in which about 1 to 1.5 mL of heparin had been collected in advance, 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].

2) Single culture of peripheral blood mononuclear cells, preparation of a cell sheet containing only fibroblasts and a cell sheet containing both cells 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 can be cultured in a single well of UpCell 6-well plate after 4 mL of the above peripheral blood mononuclear cell suspension is added, and then 4 mL of mouse fibroblast medium is added to make a total of 8 mL. To start.
For a cell sheet containing only fibroblasts, 4 mL of the fibroblast suspension is taken into one well of UpCell 6-well plate, and then 4 mL of mouse peripheral blood mononuclear cell culture medium is added to make a total of 8 mL. It started with that.
A cell sheet containing peripheral blood mononuclear cells and fibroblasts is added to 4 mL of the peripheral blood mononuclear cell suspension and 4 mL of the fibroblast suspension in one well of the UpCell 6-well plate for a total of 8 mL. And started.

3) Quantification of VEGF in each culture supernatant The above three types of cultured cells were cultured under the conditions of Hypo described in [0072]. The culture supernatant of the cells was collected in a 1.5 mL tube and centrifuged at 3000 rpm for 5 minutes, and then VEGF contained in the supernatant was analyzed using a Human VEGF Quantikine ELISA Kit (R & D systems). ] By the ELISA method based on.

4) Cell sheet transplantation Alloxan (SIGMA-ALDRICH) dissolved in physiological saline was administered to New Zealand white rabbit at 120 mg / kg to prepare alloxan-induced diabetic rabbits. An about 4 cm full-thickness skin defect was treated by a surgical technique on the back of the diabetic rabbit. Subsequent operations were in accordance with [0077].

5) Results and discussion With this study, it is possible to prepare a cell sheet containing peripheral blood mononuclear cells and fibroblasts not only in mice but also in rabbits. Is significantly higher in VEGF production than the cell sheet of fibroblasts alone (FIG. 7A), and when this cell sheet is transplanted into the skin full-thickness defect site on the back of a diabetic rabbit, it has a marked improvement effect. Was confirmed (FIG. 7B).

[Elucidation of the cause of increased VEGF production in cell sheets containing peripheral blood mononuclear cells and fibroblasts]
In order to elucidate the mechanism by which the cell sheet containing peripheral blood mononuclear cells and fibroblasts enhances the ability to produce VEGF as compared to the cell sheet of fibroblasts alone, It was verified whether the same effect could be obtained by adding the cultured cell supernatant to the cell sheet of fibroblasts alone. Furthermore, it was confirmed whether or not TGF-beta1 and PDGF-BB, which are candidate factors for inducing increased VEGF production ability, were contained in the culture cell supernatants of peripheral blood mononuclear cells alone and fibroblasts alone.

1) Examination of the presence or absence of VEGF-producing ability enhancing factor in the culture supernatant of peripheral blood mononuclear cells alone Mouse peripheral blood mononuclear cells were prepared to 2 × 10 ⁶ cells / mL in peripheral blood mononuclear cell medium [0066]. Then, 1 mL was added to the 24 well plate, followed by culturing for 48 hours under Normo conditions (37 ° C., 20% O ₂ 5% CO ₂ ). After culturing, the culture solution was collected into a 1.5 mL tube, centrifuged at 3000 rpm for 5 minutes, the supernatant was collected into a new 1.5 mL tube, and used as the culture supernatant in the following experiments.
According to the previously reported [0068], mouse 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. Using the cell preparation and peripheral blood mononuclear cell culture 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.
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].

2) Quantification of TGF-beta1 in cultured cell supernatants of peripheral blood mononuclear cells alone and fibroblasts alone FBS used for 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 ^™ 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 ^™ AIM V (registered trademark) Medium,
Mouse fibroblast group: fibroblast 1.25 × 10 ⁵ cells / 2 mL CTS ^™ AIM V® Medium
Mouse peripheral blood mononuclear cell group: peripheral blood mononuclear cells 2 × 10 ⁶ cells / 2 mL CTS ^™ AIM V (registered trademark) Medium
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.
After culturing for 48 hours, 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 Mouse / Rat / Porcine / Canine TGF-beta 1 Quantikine ELISA Kit.

3) Quantification of PDGF-BB in cultured cell supernatants of peripheral blood mononuclear cells alone and fibroblasts alone According to the previously reported [0067]-[0068], fibroblasts and peripheral blood mononuclear cells were obtained from mice. Each cell preparation is collected so that the former becomes 1.25 × 10 ⁵ cells / 1 mL in the fibroblast medium and the latter becomes 2 × 10 ⁶ cells / 1 mL in the peripheral blood mononuclear cell medium. Prepare. Using the cell preparation and peripheral blood mononuclear cell culture medium or fibroblast culture medium,
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.
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 then 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).

4) Results and discussion In order to elucidate the mechanism by which a cell sheet containing peripheral blood mononuclear cells and fibroblasts enhances the ability to produce VEGF as compared with a cell sheet of fibroblasts alone, peripheral blood mononuclear cells When the culture supernatant of a single sphere culture was added to a cell sheet of fibroblasts alone (peripheral blood mononuclear cell Conditioned medium group), a significant increase in the amount of VEGF was confirmed with respect to the control group (control medium group). (FIG. 8A). Furthermore, when it was measured whether 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 alone 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 of the enhanced VEGF production ability from fibroblasts is TGF-beta1 secreted from peripheral blood mononuclear cells and / or It was speculated that it was due to PDGF-BB.

[Effects of TGF-beta1 and PDGF-BB on VEGF production in cell sheets of fibroblasts alone and fibroblasts alone induced by peripheral blood mononuclear cell culture supernatant by neutralizing antibodies against both factors Of VEGF on the ability to produce VEGF in cell sheets]
TGF-beta1 or PDGF-BB was added to a cell sheet of fibroblasts alone, and the effect on the amount of VEGF produced from the cell sheet was examined. In addition, by adding a neutralizing antibody against TGF-beta1 or PDGF-BB to the culture supernatant of peripheral blood mononuclear cell single culture, VEGF production from the cell sheet of fibroblasts alone contained in the supernatant It was examined whether the induction effect was suppressed.

1) Measurement of the amount of VEGF produced from a cell sheet of fibroblasts alone under the conditions of varying the dose of TGF-beta1 or PDGF-BB TGF-beta1 (R & D systems) recombinant protein was added to peripheral blood mononuclear cells Diluted to 500, 1000, 2000, 10000, 20000, and 40000 pg / mL with the medium [0066], and each 500 mL was dispensed into each well of a 24 well plate.
PDGF-BB (Sigma-Aldrich) recombinant protein was diluted to 500, 1000, 2000, 10000, 20000, and 40000 pg / mL using a peripheral blood mononuclear cell culture medium, 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]. prepare. 500 mL of the prepared solution was added to each well of a 24 well plate containing TGF-beta 1 concentration solutions to make a total of 1 mL, and then cultured under Normo conditions for 48 hours.
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].

2) Measurement of the amount of VEGF produced in cell sheet of fibroblasts alone with the addition of peripheral blood mononuclear cell culture supernatant when neutralizing antibody against TGF-beta1 or PDGF-BB was added. Peripheral blood mononuclear cells were collected, and a cell preparation solution was prepared so as to be 2 × 10 ⁶ cells / 1 mL in a medium for peripheral blood mononuclear cells. The suspension was seeded on a 24-well plate at 1 mL / well, and then cultured under Normo conditions for 48 hours. After culturing for 48 hours, 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 μL + anti-ATM antibody (1 mg / mL abcam ab78) 5 μL
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.
In accordance with the previous report [0068], 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. 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].

3) Results and discussion When TGF-beta1 or PDGF-BB was added to a cell sheet of fibroblasts alone at different doses, the dose-dependent increase in VEGF production from the cell sheet was observed for any factor. Was confirmed (FIG. 9A).
Further, when neutralizing antibody against TGF-beta1 or PDGF-BB is added to the culture supernatant of peripheral blood mononuclear cell single culture, the medium originally has a fibroblast single cell sheet. The effect of inducing VEGF production was suppressed in any antibody (FIG. 9B).
From the above, in the cell sheet containing peripheral blood mononuclear cells and fibroblasts, the mechanism of enhanced VEGF production ability from fibroblasts is TGF-beta1 secreted from peripheral blood mononuclear cells and / or The possibility by PDGF-BB was strongly suggested.
In addition, 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.

[Therapeutic effect of cell sheet containing fibroblasts stimulated by transforming growth factor or platelet-derived growth factor]
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 In order to clarify whether it can be an effective therapeutic transplant material in skin ulcers, we investigated the therapeutic effect of transplanting to the full-thickness skin defect on the back of diabetic mice.

1) Cell sheet transplantation and wound healing evaluation Preparation of diabetic mice and wound healing evaluation were based on [0076]-[0078]. The treatment groups for the skin full-thickness defect site on the back of the diabetic mouse were as follows:
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; group TGF-beta1 + fibroblast sheet group transplanted with a cell sheet prepared by adding 10 ng / mL PDGF-BB at the time of fibroblast seeding; A group transplanted with a cell sheet prepared by adding 5 ng / mL TGF-beta1 at the time of fibroblast seeding

2) Results and discussion When a cell sheet containing fibroblasts stimulated by TGF-beta1 or PDGF-BB was transplanted to the skin full-thickness defect site on the back of a diabetic mouse, peripheral blood mononuclear cells and fibroblasts were transplanted. Similar to the cell sheet containing, good results were confirmed in the wound area healing rate (FIG. 10).
This suggests that a cell sheet containing fibroblasts stimulated by the factor is a very effective therapeutic material for diseases such as intractable skin ulcers.

[Method for preparing cell sheet containing human-derived peripheral blood mononuclear cells and fibroblasts and comparing VEGF production ability]
If the cell sheet which concerns on this invention can be used as a therapeutic material with respect to a human disease, it will be very useful. Therefore, a method for producing a cell sheet containing human-derived peripheral blood mononuclear cells and fibroblasts was established, and the VEGF production ability of the cell sheet was also examined.

1) Method for producing human-derived peripheral blood mononuclear cells alone, fibroblast-only cell sheets and cell sheets containing peripheral blood mononuclear cells and fibroblasts In culturing human-derived cell lines, the medium is AIM. Using V Medium CTS (Life Technologies), we prepared our own autologous serum. Specifically, serum was prepared from self-collected blood using CellAid (JM Co., Ltd.).
Human-derived fibroblasts were prepared by the following method. AIM V Medium CTS (10 mL) and Penicillin-Streptomycin, Liquid (200 μL) were placed in a 50 mL tube, and the collected oral tissues were placed in the 50 mL tube. The tissue is moved to a 6 cm-dish with tweezers, the tissue is shredded with a disposable scalpel, the shredded tissue piece is pressed and adhered to a 6-well plate, and conditions of 37 ° C. and 5% CO ₂ for 10 minutes. Incubated with Thereafter, in order to prevent drying, 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 μL of autoserum, and 200 μL of Penicillin-Streptomycin, Liquid were added to 1 well of 6-well plate, and cultured at 37 ° C., 5% CO ₂ for 3-4 weeks. The cells were detached with Trypsin-EDTA (Gibco), permeated through 40 μm 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 .

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. 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. In addition, nuclear staining with DAPI was performed.

Separation of human-derived peripheral blood mononuclear cells was performed by the following method. About 4 mL of blood was collected in a BD vacutina blood collection tube and centrifuged at 3000 rpm for 20 minutes with no brake. After centrifugation, the mononuclear cell layer was collected in a new 15 mL tube, added with PBS to 10 mL, and centrifuged again at 1500 rpm for 3 minutes with no brake. Then, it was washed once with AIM V Medium CTS. For cell sheet preparation or single culture, 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).

2) Results and Discussion This study revealed that fibroblasts with higher purity than human oral tissues can be collected and cell sheets can be prepared (FIG. 11A).
Further, in the cell sheet containing the peripheral blood mononuclear cells similarly derived from the fibroblasts, more VEGF is produced than the cell sheet of the fibroblasts alone, and the VEGF producing ability is determined under the Hypo condition. Further remarkable enhancement was observed by (culturing at 33 ° C., 2% O ₂ , 5% CO _{2 for} 24 hours) (FIG. 11B).

[Search for factors that activate VEGF production in cell sheets of human-derived fibroblasts alone]
Regarding cells derived from mice already, 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.
If 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. In addition, for example, when culturing a large amount of cell sheets of fibroblasts derived from another family (other than the patient), 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. Specifically, a 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.

1) Collection of used cells and preparation of samples Separation of human-derived peripheral blood mononuclear cells and fibroblasts was performed according to the previously reported [0112] and [0114], the former being 1 × 10 ⁶ cells / mL and the latter being 1.25 × 10 ^5. cells / mL of cell suspension (9.5 mL of AIM V Medium CTS + 0.5 mL of autoserum was used as the medium).
Samples were prepared in the following four groups using the cell suspension and media:
Medium group: 2 mL of medium (AIM V Medium CTS 9.5 mL + autologous serum 0.5 mL) was added to one well of 24-well plate and cultured under Normo conditions.
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 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.

2) Measurement of factors in culture supernatant Proteome Profiler Human XL Cytokine Array Kit (R & D systems) and Proteome Profiler Human Angiogenesis Array Kit (R & D systems) were used to measure each factor in the above samples. An Amersham Imager 600 (GE Healthcare) was used for band detection.

3) Results and discussion When the above-mentioned four groups of samples were measured with the above Array Kit and the factors secreted only by peripheral blood mononuclear cells were identified,
The factors detected in the Cytokine Array Kit are IL-1ra, CXCL1, CXCL5, CXCL10, CCL3, CCL4, and the factors detected in the 6 factor Angiogenesis Array Kit are PDGF-AA, HB-EGF, CXCL16, CCL2, There were 4 factors of CCL3.
A total of 12 factors including TGF-beta and PDGF-BB, which are likely to work as activators in mouse-derived cell lines, were used as activator candidates.

[Investigation of VEGF-producing ability in fibroblasts as candidate activators 1]
Based on the above evaluation, twelve kinds of candidates (activators) that induce VEGF production were found by activating cell sheets of fibroblasts alone secreted from peripheral blood mononuclear cells. Therefore, it was examined whether the amount of VEGF production was increased by adding the activator candidate to a cell sheet of fibroblasts alone.

1) Treatment of activating factor candidate to cell sheet of fibroblasts alone and VEGF quantification Separation of human-derived fibroblasts was performed according to the previous report [0112], and a cell suspension (medium) of 1.25 × 10 ⁵ cells / mL AIM V Medium CTS 9.0 mL + autologous serum 1 mL was used), and 0.5 mL of the cell suspension was dispensed into each well of a 48-well plate.
Using media (AIM V Medium CTS), 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) ) Prepared to 20 ng / mL, 0.5 mL each was added to each well of the above 48-well plate from which the cell suspension was previously prepared to make a total of 1 mL, and then cultured under Normo conditions for 72 hours did. As a control, 0.5 mL of the above medium was added to one well of a 48-well plate from which a cell suspension was collected to make a total of 1 mL, and similarly 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].

2) Results and discussion Among the 12 activator candidates evaluated, PDGF-BB and TGFbeta1 showed a significant increase in VEGF concentration, and PDGF-AA, HB-EGF and CXCL16 showed a tendency to increase the concentration. (FIG. 12).

[Investigation of VEGF-producing ability in fibroblasts as candidate activators 2]
Among the 12 candidate factors (activators) that are secreted from human-derived peripheral blood mononuclear cells and activate fibroblast-only cell sheets to induce VEGF production, the induction activity is particularly high in the above evaluation. In order to examine the more detailed effects of PDGF-BB and TGFbeta1 showing the above, the dose correlation with the VEGF production inducing activity was examined. In addition, in order to confirm that the VEGF production inducing activity by the factor is not due to the proliferation of fibroblasts, the cell proliferation ability of the factor was also examined.

1) Dose correlation study of VEGF production ability in PDGF-BB and TGFbeta1 Separation of human-derived fibroblasts was performed according to the previous report [0112], and a cell suspension of 1.25 × 10 ⁵ cells / mL (AIM as a medium) V Medium CTS 9.0 mL + autologous serum 1 mL was used), and 0.5 mL of the cell suspension was dispensed into each well of a 48-well plate.
In AIM V Medium CTS, TGF-beta1 (R & D systems), PDGF-BB (eBioscience), bFGF (Sigma-Aldrich) each factor is 40, 20, 15, 10, 5, 2, 1.5, 1, After adjusting to 0.5, 0.2, 0.1 ng / mL, and adding 0.5 mL to each well of the 48-well plate from which the cell suspension was collected, 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. As 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 collected 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].

2) Cell proliferation ability of PDGF-BB and TGFbeta1 in fibroblasts per TGF-beta1 (R & D systems), PDGF-BB (eBioscience), bFGF (Sigma-Aldrich) recombinant protein is 5, 7.5 per mL. 10, 20 ng / mL, human-derived fibroblast cell concentration is 1 × 10 ⁴ cells / mL, prepared by AIM V Medium CTS to 5% autoserum, and 100 μL per well of 96-well plate And incubated for 3 days at 37 ° C., 5% CO ₂ . On the third day after the start of culture, 10 μL of CellTiter 96 (registered trademark) AQueous One Solution Cell Proliferation Assay was administered to each well, and cultured for 4 hours at 37 ° C., 5% CO ₂ , and then to 2030 ArvoX4 (Perkin Elmer). The absorbance was measured. The data is corrected for the absorbance obtained by the control.

3) Results and discussion As in the case of mice, even in a cell sheet of human-derived fibroblasts alone, both PDGF-BB and TGFbeta1 were treated with this cell sheet, and thus dose-dependent VEGF production-inducing activity was obtained. It became clear to have (FIG. 13A).
Further, both factors did not have the ability to proliferate against fibroblasts (FIG. 13B), and it was confirmed that the VEGF production inducing activity seen in FIG. 13A was not due to the proliferation of fibroblasts.
From the above, even in human-derived fibroblasts, a cell sheet containing fibroblasts stimulated by transforming growth factor or platelet-derived growth factor is a very useful invention as a transplant material for treatment of intractable skin ulcer. It became clear.

  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. To the medical field related to transplantation, especially in intractable skin ulcers, and in addition, 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.

Claims (9)

  A transplant material for treating refractory skin ulcer, comprising a cell sheet containing peripheral blood mononuclear cells and fibroblasts.   The cell sheet according to claim 1, wherein the peripheral blood mononuclear cells and / or the fibroblasts are obtained from an individual suffering from intractable skin ulcer, which is a subject to be treated. A transplant material for the treatment of refractory skin ulcers.   A transplant material for the treatment of intractable skin ulcer, comprising the cell sheet according to claim 1 or 2, which is produced by co-culturing peripheral blood mononuclear cells and fibroblasts.   Peripheral blood mononuclear cells 5.0 x 10 ⁴ Piece / cm ² ~ 1.5x10 ⁶ Piece / cm ² And seeded fibroblasts with 1.0 × 10 ⁴ Piece / cm ² ~ 1.5x10 ⁵ Piece / cm ² The transplant material for treatment of refractory skin ulcer containing the cell sheet according to claim 3, wherein the co-culture is carried out by seeding with a cell.   5. The transplant material for refractory skin ulcer treatment containing the cell sheet according to claim 3 or 4, wherein the co-culture is started by simultaneously seeding peripheral blood mononuclear cells and fibroblasts.   The transplant material for treating refractory skin ulcer containing the cell sheet according to any one of claims 1 to 5, wherein the cell sheet is cultured for a predetermined period of time under low temperature and low oxygen conditions. 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 containing the cells,
(E) Step of peeling the cell sheet from the culture substrate 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;
The method of manufacturing the cell sheet of Claim 7 containing this. 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;
The method of manufacturing the cell sheet of Claim 7 or 8 containing this.