WO2014112607A1 - Cell preparation and method for enhancing cell activity - Google Patents

Abstract

The present invention addresses the problem of providing a means for enhancing the activity of mesenchymal stem cells derived from a fat tissue, and a cell preparation and a therapeutic method utilizing or applying the aforesaid means. Provided is a cell preparation which contains mesenchymal stem cells derived from a fat tissue and a low concentration of mannose. In another embodiment, broadband near-infrared radiation is used as a substitute for a low concentration of mannose or in addition thereto.

Description

Cell preparation and method for enhancing cell activity

The present invention relates to a cell preparation and use thereof, and a method for enhancing cell activity. This application claims priority based on Japanese Patent Application No. 2013-008355 filed on Jan. 21, 2013, the entire contents of which are incorporated by reference.

Adipose tissue is rich in mesenchymal stem cells. Because of this fact and the fact that it can be collected in large quantities with simple operation and the burden on patients during collection is small, adipose tissue is considered promising as a source of stem cells for regenerative medicine. Yes. In fact, adipose tissue-derived mesenchymal stem cells (Adipose-derived stem cells: ASCs, ADSCs, Adipose-derived regeneration cells: ADRCs, Adipose-derived mesenchymal stem cells: AT-MSCs, AD-MSCs, etc. Aiming at the clinical application of derived mesenchymal stem cells (sometimes abbreviated as “ASCs”), research on various diseases as therapeutic targets is underway (see, for example, Patent Documents 1 to 3).

International Publication No. 2008/018450 A1 Pamphlet JP 2009-001509 A Special table 2010-528107 gazette JP 2008-185378 A Special table 2010-528107 gazette

The therapeutic effect depends on the activity of ASCs (pluripotency, growth factor secretion ability, etc.). Although the activity of ASCs depends on the isolation conditions and origin (individual and individual differences), it is effective and important to increase the activity of ASCs in order to maximize the potential of ASCs and achieve high therapeutic effects. It is. Therefore, an object of the present invention is to provide means for increasing the activity of ASCs, and cell preparations and treatment methods using or applying the means.

As shown in the examples described later, as a result of intensive studies by the present inventors, the ability to produce cytokines of ASCs is improved when a low concentration of mannose coexists by an experiment using cultured cells (in vitro experiment), That is, it became clear that the activity of ASCs increased. In addition, when ASCs and mannose were mixed at a low concentration and administered to a rat model of renal ischemia (in vivo study), a marked improvement in microcirculation (capillary blood flow) and a recovery of renal function were observed. Concomitant concentrations of mannose have been shown to be effective in enhancing the therapeutic effects of ASCs.

On the other hand, attention was paid to broadband near-infrared rays (for example, refer to Patent Document 4, hereinafter sometimes abbreviated as “WNIR”), and the effectiveness was examined. As a result, it was found that WNIR is effective as a means of increasing the activity of ASCs by experiments using cultured cells (in vitro experiments). In addition, mannose was applied to the skin and irradiated with WNIR (in vivo experiment) in cases with reduced skin microcirculation due to hyperlipidemia, and it was clear that blood flow in the skin capillaries improved It became. This result indicates that the activity of ASCs present in subcutaneous fat was increased by WNIR, and angiogenesis and cytokine secretion were promoted, in other words, as a means for increasing the activity of ASCs and improving its therapeutic effect. This suggests that WNIR irradiation is effective and that the combined use of mannose is effective in improving the therapeutic effect. Furthermore, in consideration of the results of in vitro experiments, in order to improve the therapeutic effect of ASCs, a method of irradiating ASCs before administration with WNIR and irradiating ASCs after administration with WNIR It can be said that any of the methods can be adopted.

As a further study, experiments using human umbilical vein endothelial cells (HUVEC) and bone marrow-derived mesenchymal stem cells (BM-MSCs) were performed. As a result, for each of the low concentrations of mannose and WNIR irradiation, a tendency to improve the cytokine production ability of the cells is recognized, and the use of low concentrations of mannose and WNIR becomes a general-purpose means for enhancing the activity of the cells. It became clear.

On the other hand, as a result of further studies on the effectiveness of mannose, it was found that even mannose alone exerts an improvement effect on blood flow.

Furthermore, experiments using model animals revealed that the combination of mesenchymal stem cells (ASCs or BM-MSCs) and low concentrations of mannose has an excellent therapeutic effect on interstitial cystitis. It became.

The present invention shown below is mainly based on the above results and considerations. Incidentally, Patent Document 5 (Japanese Patent Publication No. 2010-528107) discloses a cell composition for treating ischemic disease using ASCs and mannose in combination, and the concentration of mannose therein is high (2%). And is distinct from the present invention. In addition, mannose is used as an excipient, and there is no mention or suggestion regarding the action characteristic of the present invention.
[1] A cell preparation that contains adipose tissue-derived mesenchymal stem cells and is used in combination with a low concentration of mannose.
[2] The cell preparation according to [1], comprising adipose tissue-derived mesenchymal stem cells and a low concentration of mannose.
[3] The cell preparation according to [1], comprising adipose tissue-derived mesenchymal stem cells treated with a low concentration of mannose.
[4] The cell preparation according to any one of [1] to [3], wherein the low concentration is a concentration of 0.8% (w / v) or less.
[5] The cell preparation according to any one of [1] to [3], wherein the low concentration is 0.2% (w / v) to 0.8 (w / v).
[6] The cell preparation according to [1], which contains adipose tissue-derived mesenchymal stem cells and is administered with mannose at the same time when administered to a treatment target.
[7] A cell preparation containing adipose tissue-derived mesenchymal stem cells and irradiated with broadband near-infrared rays before or after administration to a treatment target.
[8] The cell preparation according to [7], wherein the near-infrared wavelength of the broadband has a maximum emission intensity in a range of 750 to 1200 nm and a half-value width of an emission spectrum is 70 to 200 nm.
[9] The cell preparation according to [7] or [8], further containing a low concentration of mannose.
[10] The cell preparation according to [7] or [8], wherein mannose is administered simultaneously with administration to a treatment subject.
[11] The cell preparation according to any one of [1] to [10], which is for treatment of a disease or disorder in which improvement in blood flow has a therapeutic effect.
[12] The cell preparation according to [11], which is used for treatment of ischemic disease or hyperlipidemia.
[13] The cell preparation according to [12], wherein the ischemic disease is ischemic renal failure.
[14] The cell preparation according to any one of [1] to [10], which is used for treatment of interstitial cystitis.
[15] A method for enhancing the activity of a cell, comprising the following steps (1) and / or (2):
(1) contacting the cells with a low concentration of mannose;
(2) A step of irradiating a cell with broadband near infrared rays.
[16] The method according to [15], wherein the cells are mesenchymal stem cells or endothelial cells.
[17] The method according to [16], wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells, and the endothelial cells are umbilical vein endothelial cells.
[18] The method according to any one of [15] to [17], wherein steps (1) and (2) are performed ex vivo.
[19] A cell preparation containing cells whose activity has been enhanced by the method according to [18].
[20] A blood flow improving agent containing mannose.
[21] The blood flow improving agent according to [20], which is for skin care, face care, body care or hair care.

Promotion of secretion of various cytokines by adding low concentrations of mannose. Mannose was added to the medium at a low concentration, and ASCs were cultured. Each cytokine level in the culture supernatant on the third day of culture was examined and compared with a control (no mannose added). In addition, each cytokine level was compared between a culture under normal oxygen concentration conditions (left) and a culture under low oxygen concentration conditions (right). Promotion of secretion of various cytokines by adding low concentrations of mannose. The relationship between mannose concentration and VEGF-A secretion was examined. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Promotion of secretion of various cytokines by adding low concentrations of mannose. The relationship between mannose concentration and HGF secretion was examined. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Promotion of secretion of various cytokines by adding low concentrations of mannose. The relationship between mannose concentration and NGF-β secretion was examined. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Promotion of secretion of various cytokines by adding low concentrations of mannose. The relationship between mannose concentration and SDF-1 secretion was examined. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Cell activation by broadband near infrared (WNIR). ASCs were irradiated with WNIR before sowing (culture day 0 and day 3). Various cytokine levels in the culture supernatants on the 3rd and 6th day of culture were examined and compared with the control (non-irradiated with WNIR). In addition, each cytokine level was compared between a culture under normal oxygen concentration conditions (left) and a culture under low oxygen concentration conditions (right). The therapeutic effect of ASCs combined with mannose and / or WNIR. ASCs were administered to a rat ischemic rat model under predetermined conditions (mannose admixture, WNIR irradiation, mannose admixture and WNIR irradiation), and the therapeutic effects (upper: renal function, lower: renal damage score) were compared. Cont is no treatment, Sham is unilateral nephrectomy, Isch is unilateral ischemia, A is administration of ASCs, M is mannose admixed, and W is WNIR irradiation. The therapeutic effect of ASCs combined with mannose and / or WNIR. ASCs were administered to a renal ischemic rat model under predetermined conditions (mixed mannose, irradiated with WNIR, mixed with mannose and irradiated with WNIR), and the blood flow volume of capillaries around the tubules was compared. Sham is unilateral nephrectomy, Isch is unilateral renal ischemia, A is administration of ASCs, M is mannose admixture, and W is WNIR irradiation. Integrated imaging of ASCs in a rat model of renal ischemia. Isch represents single kidney ischemia, A represents administration of ASCs, and W represents WNIR irradiation. Effects of mannose skin application and WNIR irradiation on hyperlipidemia cases. Mannose was applied to the hyperlipidemic patient's skin and irradiated with WINS. Subcutaneous capillary imaging was performed before and after WINR irradiation to quantify blood flow. Effects of mannose skin application and WNIR irradiation on hyperlipidemia cases. The imaging results before and after treatment (mannose application and WINS irradiation) were shown. An example of a WNIR light source. Blood flow improvement effect by mannose. The imaging results before mannose application (left) and after mannose application (right) are shown. Blood flow improvement effect by mannose. The blood flow velocity before mannose application (left) and after mannose application (right) was compared. Effect of low concentration mannose addition on HUVEC. The relationship between mannose concentration and VEGF-A secretion was examined. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Effect of low concentration mannose addition on HUVEC. The relationship between mannose concentration and HGF secretion was examined. The result at the time of culture | cultivating on low oxygen concentration conditions is shown. Effect of low concentration mannose addition on HUVEC. The relationship between mannose concentration and NGF-β secretion was examined. The result at the time of culture | cultivating on low oxygen concentration conditions is shown. Effect of low concentration mannose addition on HUVEC. The relationship between mannose concentration and SDF-1 secretion was examined. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Effect of low concentration mannose addition on BM-MSCs. The relationship between mannose concentration and VEGF-A secretion was examined. The results when cultured under normal oxygen concentration conditions are shown. Effect of low concentration mannose addition on BM-MSCs. The relationship between mannose concentration and NGF-β secretion was examined. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Effect of WNIR irradiation on HUVEC. It was investigated whether the secretion amount of Adrenomedullin was changed by irradiation with WNIR. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Effect of WNIR irradiation on BM-MSCs. It was investigated whether the secretion amount of VEGF-A was changed by irradiation with WNIR. The results when cultured under normal oxygen concentration conditions are shown. Effect of WNIR irradiation on BM-MSCs. It was investigated whether the amount of HGF secretion changed by irradiation with WNIR. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Effect of WNIR irradiation on BM-MSCs. It was examined whether the amount of NGF-β secretion changed by irradiation with WNIR. The left is the result when cultured under normal oxygen concentration conditions, and the right is the result when cultured under low oxygen concentration conditions. Effect of WNIR irradiation on BM-MSCs. It was investigated whether the secretion amount of Adrenomedullin was changed by irradiation with WNIR. The results when cultured under normal oxygen concentration conditions are shown. A therapeutic effect on interstitial cystitis by ASCs or a combination of ASCs and mannose. After inducing cystitis by intraperitoneal injection of cyclophosphamide (CYP), intravesical injection of rat ASCs mixed with saline, rat ASCs, or low concentration mannose (0.2% (w / v)), Two days later, intravesical pressure (CMG) was measured. Upper left: urination interval of normal group (n = 6), upper right: urination interval of rats infused with physiological saline after CYP intraperitoneal injection (CYP-saline urinary injection group: n = 6), lower left: ASCs after intraperitoneal injection of CYP Urination interval of rats (CYP-ASCs urinary injection group: n = 6), lower right: rats injected with CSC-MN-mixed ASCs urinary injection group (n = 6) Urination interval. A therapeutic effect on interstitial cystitis by ASCs or a combination of ASCs and mannose. After cystitis was induced by intraperitoneal injection of CYP, rat ASCs mixed with physiological saline, rat ASCs, or low-concentration mannose (0.2% (w / v)) were injected into the urinary bladder. It used for HE (hematoxylin eosin) dyeing | staining. Upper left: normal group, upper right: CYP-raw bladder injection group, lower left: CYP-ASCs bladder injection group, lower right: CYP-MN mixed ASCs bladder injection group. Integrated imaging of ASCs in a rat model of interstitial cystitis. Left: Before ASCs bladder injection, middle: 2 days after ASCs bladder injection, right: 2 days after MN-mixed ASCs bladder injection. Therapeutic effect on interstitial cystitis by the combination of BM-MSCs or BM-MSCs and mannose. The result of intravesical pressure measurement is shown. Upper row: urination interval of rats (n = 3) injected with BM-MSCs after intraperitoneal injection of CYP, lower row: urination interval of rats (n = 3) injected with low concentration mannose-mixed BM-MSCs after intraperitoneal injection of CYP.

<First aspect: Cell preparation and use thereof>
The present invention relates to cell preparations applied to specific diseases and uses thereof. The cell preparation of the present invention contains adipose tissue-derived mesenchymal stem cells (ASCs). In the present invention, “adipose tissue-derived mesenchymal stem cells (ASCs)” refers to somatic stem cells contained in adipose tissue. As long as pluripotency is maintained, the somatic stem cells are cultured ( Cells obtained by subculture) also correspond to “adipose tissue-derived mesenchymal stem cells (ASCs)”. Normally, ASCs are prepared in an “isolated state” as cells constituting a cell population (including cells other than ASCs derived from adipose tissue) using adipose tissue separated from a living body as a starting material. The “isolated state” as used herein means a state extracted from its original environment (that is, a state constituting a part of a living body), that is, a state different from the original existence state by an artificial operation. Means that Adipose tissue-derived mesenchymal stem cells are also called ADSCs, ADRCs, AT-MSCs, AD-MSCs, and the like. In the present specification, the following terms, that is, adipose tissue-derived mesenchymal stem cells, ASCs, ADSCs, ADRCs, AT-MSCs, and AD-MSCs are used interchangeably.

(Method for preparing ASCs)
ASCs are prepared through steps such as separation, washing, concentration, and culture of stem cells from adipose matrix. A method for preparing ASCs is not particularly limited. For example, a known method (Fraser JK et al. (2006), Fat tissue: an underappreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr; 24 (4): 150-4. Epub 2006 Feb 20. Review .; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells.Molecular Biology of the Cell; Dec; 13 (12): 4279-95 .; Zuk PA et al. (2001), Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr; 7 (2): 211-28. An apparatus for preparing ASCs from adipose tissue (for example, Celution (registered trademark) apparatus (Cytory Therapeutics, Inc., San Diego, USA)) is also commercially available, and ASCs are prepared using the apparatus. You may decide. By using this apparatus, cells positive for cell surface markers CD29 and CD44 can be separated from adipose tissue. Hereinafter, specific examples of methods for preparing ASCs will be shown.

(1) Preparation of cell population from adipose tissue Adipose tissue is collected from animals by means such as excision and suction. The term “animal” herein includes humans and non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, Dogs, cats, etc.). In order to avoid the problem of immune rejection, it is preferable to collect adipose tissue (self-adipose tissue) from the same individual as the subject (patient) to which the cell preparation of the present invention is applied. However, this does not preclude the use of adipose tissue of the same species (other family) or adipose tissue of different species.

Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, subcutaneous fat can be collected very easily under local anesthesia, so that the burden on the patient at the time of collection is small and it can be said that it is a preferable cell source. Usually, one type of adipose tissue is used, but two or more types of adipose tissue can be used in combination. In addition, adipose tissue collected in multiple times (not necessarily the same type of adipose tissue) may be mixed and used for subsequent operations. The amount of adipose tissue collected can be determined in consideration of the type of donor, the type of tissue, or the amount of ASCs required, for example, about 0.5 to 500 g. When a human is used as a donor, the amount collected at a time is preferably about 10 to 20 g or less in consideration of the burden on the donor. The collected adipose tissue is subjected to the following enzyme treatment after removal of blood components adhering to it and fragmentation as necessary. The blood component can be removed by washing the adipose tissue in an appropriate buffer or culture solution.

Enzyme treatment is performed by digesting adipose tissue with enzymes such as collagenase, trypsin, dispase and the like. Such enzyme treatment may be carried out by methods and conditions known to those skilled in the art (for example, see RI Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). . Preferably, the enzyme treatment here is performed according to the methods and conditions described in the Examples described later. The cell population obtained by the above enzyme treatment includes multipotent stem cells, endothelial cells, stromal cells, blood cells, and / or precursor cells thereof. The type and ratio of the cells constituting the cell population depend on the origin and type of the adipose tissue used.

(2) Acquisition of sedimented cell population (SVF fraction: stroma vascular fractions) The cell population is subsequently subjected to centrifugation. The sediment by centrifugation is collected as a sedimented cell population (also referred to herein as “SVF fraction”). The conditions for centrifugation vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes and 800 to 1500 rpm. Prior to centrifugation, the cell population after the enzyme treatment is preferably subjected to filtration or the like, and the enzyme undigested tissue contained therein is preferably removed.

“SVF fraction” obtained here includes ASCs. Therefore, the cell preparation of the present invention can be prepared using the SVF fraction. That is, in one embodiment of the cell preparation of the present invention, the SVF fraction is contained. The type and ratio of cells constituting the SVF fraction depend on the origin and type of the adipose tissue used, the conditions for enzyme treatment, and the like. Further, the SVF fraction is characterized by including a CD34-positive and CD45-negative cell population and a CD34-positive and CD45-negative cell population (WO 2006 / 006692A1 pamphlet).

(3) Selective culture of adherent cells (ASCs) and cell recovery The SVF fraction contains other cell components (endothelial cells, stromal cells, blood cells, progenitor cells thereof, etc.) in addition to ASCs. . Therefore, in one embodiment of the present invention, the following selective culture is performed to remove unnecessary cell components from the SVF fraction. The resulting cells are used as ASCs in the cell preparation of the present invention.

First, after suspending the SVF fraction in an appropriate medium, it is seeded on a culture dish and cultured overnight. Suspension cells (non-adherent cells) are removed by medium exchange. Thereafter, the culture is continued while appropriately changing the medium (for example, once every 3 days). Subculture as necessary. The passage number is not particularly limited. As the culture medium, a normal animal cell culture medium can be used. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd.), α-MEM (Dainippon Pharmaceutical Co., Ltd.), DMEM: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Laboratory), etc. can be used. A medium supplemented with serum (fetal bovine serum, human serum, sheep serum, etc.) or a serum substitute (Knockout serum replacement (KSR), etc.) may be used. The addition amount of serum or serum replacement can be set, for example, within a range of 5% (v / v) to 30% (v / v).

By the above operation, adherent cells selectively survive and proliferate. Subsequently, the proliferated cells are collected. The collection operation may be carried out in accordance with a conventional method. For example, the cells after enzyme treatment (trypsin or dispase treatment) can be easily collected by detaching them with a cell scraper or pipette. In addition, when sheet culture is performed using a commercially available temperature-sensitive culture dish or the like, it is also possible to recover the cells as they are without performing enzyme treatment. By using the cells (ASCs) collected in this way, a cell preparation containing ASCs with high purity can be prepared.

(4) Low-serum culture (selective culture in low-serum medium) and cell recovery In one embodiment of the present invention, the following low-serum culture is performed instead of or after the operation of (3) above. I do. The resulting cells can be used as ASCs in the cell preparation of the present invention.

In low serum culture, the SVF fraction (if this step is carried out after (3), the cells collected in (3) are used) are cultured under low serum conditions and the desired multipotent stem cells (ie ASCs) ) Selectively. Since a small amount of serum is used in the low serum culture method, it is possible to use the serum of the subject (patient) to whom the cell preparation of the present invention is administered. That is, culture using autoserum becomes possible. By using autologous serum, a cell preparation is provided that is capable of excluding foreign animal material from the manufacturing process, and is expected to have high safety and high therapeutic effect. Here, “under low serum conditions” is a condition containing 5% or less of serum in the medium. The cells are preferably cultured in a culture solution containing 2% (V / V) or less of serum. More preferably, the cells are cultured in a culture solution containing 2% (V / V) or less of serum and 1 to 100 ng / ml of fibroblast growth factor-2 (bFGF).

Serum is not limited to fetal bovine serum, and human serum or sheep serum can be used. Preferably, human serum, more preferably, serum of a subject to which the cell preparation of the present invention is applied (that is, autoserum) is used.

As a medium, a normal medium for animal cell culture can be used on condition that the amount of serum contained in use is low. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd.), α-MEM (Dainippon Pharmaceutical Co., Ltd.), DMEM: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Laboratory), etc. can be used.

ASCs can be selectively proliferated by culturing by the above method. In addition, since ASCs proliferating under the above culture conditions have high proliferative activity, the number of cells required for the cell preparation of the present invention can be easily prepared by subculture. Cells selectively proliferating by low serum culture of the SVF fraction are CD13, CD90 and CD105 positive and CD31, CD34, CD45, CD106 and CD117 negative (International Publication No. 2006 / 006692A1 pamphlet). .

Subsequently, the cells selectively proliferated by the above low serum culture are collected. The collection operation may be performed in the same manner as in the above (3). By using the collected cells (ASCs), a cell preparation containing ASCs with high purity can be prepared.

The cells obtained by the above method, that is, not the cells grown by culturing the SVF fraction in low serum but directly the cell population obtained from the adipose tissue (without going through the centrifugation to obtain the SVF fraction) Cells grown by serum culture may be used as ASCs. That is, in one embodiment of the present invention, cells that proliferate when a cell population obtained from adipose tissue is cultured in low serum are used as ASCs. Further, instead of ASCs obtained by selective culture (above (3) and (4)), an SVF fraction (containing adipose tissue-derived mesenchymal stem cells) may be used as it is. The cell preparation of this embodiment comprises (a) a precipitated cell population (SVF fraction) recovered as a sediment by subjecting adipose tissue to protease treatment, followed by filtration, and then centrifuging the filtrate, or (b) fat After the tissue is treated with protease, it contains a sedimented cell population (SVF fraction) that is collected as a sediment by centrifuging without filtration. Here, “use as it is” means to use it as an active ingredient of a cell preparation without undergoing selective culture.

(Formulation)
For the preparation, ASCs or a cell population containing ASCs (SVF fraction cells, cells obtained as a result of the selective culture (3), cells obtained as a result of the low serum culture (4), or commercially available devices. (For example, cells obtained by a Celution (registered trademark) apparatus) are suspended in physiological saline, Ringer's solution (acetated Ringer's solution, lactated Ringer's solution, etc.), buffer (eg, phosphate buffer), or the like. For example, 1 × 10 ⁵ to 1 × 10 ¹⁰ cells may be contained as a single dose so that a therapeutically effective amount of cells is administered. The content of the cells can be appropriately adjusted in consideration of the purpose of use, the target disease, the sex of the application target (recipient), age, weight, the state of the affected area, the state of the cells, and the like.

Dimethyl sulfoxide (DMSO), serum albumin, etc. for the purpose of cell protection, antibiotics, etc. for the purpose of preventing bacterial contamination, and various components for the purpose of cell activation, proliferation or differentiation induction, etc. (Vitamins, cytokines, growth factors, steroids, etc.) may be contained in the cell preparation of the present invention. Examples of cytokines are interleukin (IL), interferon (IFN), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF) and erythropoietin (EPO), activin, oncostatin M (OSM). CSF, G-CSF, EPO, etc. are also growth factors. Examples of growth factors are hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF, FGF2), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) Transforming growth factor (TGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). In addition, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) You may make it contain in the cell formulation of this invention.

The cell preparation of the present invention can be provided in a state of being stored or filled in an appropriate container, for example, a syringe, ampoule, vial, plastic container, infusion bag or the like.

(Applicable disease)
The cell preparation of the present invention is applicable to various diseases (target diseases) in which therapeutic or prophylactic effects are brought about by administration of ASCs. The therapeutic effect includes alleviation of symptoms or associated symptoms characteristic of the target disease (mildness), prevention or delay of deterioration of symptoms, and the like. The latter can be regarded as one of the preventive effects in terms of preventing the seriousness. In this way, the therapeutic effect and the preventive effect are partially overlapping concepts, and it is difficult to clearly distinguish them from each other, and there is little benefit in doing so. A typical preventive effect is to prevent or delay the recurrence of symptoms characteristic of the target disease.

Examples of target diseases include ischemic diseases, renal dysfunction, wounds, urinary incontinence, osteoporosis (for example, refer to WO 2008/018450 A1 for the above application examples), hyperlipidemia, tumors It is. Ischemia is caused by cessation of blood flow to organs and tissues and reduction of blood flow. If the ischemic time is short, the organ function is restored by resuming blood flow (reperfusion). If the ischemic time is long, the organs and the like are irreversibly damaged (ischemic reperfusion injury) due to reperfusion, resulting in malfunction. Such a disease caused by ischemia or ischemia reperfusion is referred to as “ischemic disease”. For example, ischemic kidney injury (eg ischemic renal failure), obstructive arteriosclerosis (eg lower limb arteriosclerosis), ischemic heart disease (myocardial infarction, angina etc.), cerebrovascular disorder (cerebral infarction, etc.) ), Ischemic injury of the liver, etc. correspond to ischemic diseases. One of the preferable target diseases of the cell preparation of the present invention is such an ischemic disease.

The cell preparation of the present invention is particularly effective for treating diseases or disorders in which improvement of blood flow has a therapeutic effect. For example, an ischemic disease corresponds to the disease or disorder.

Another example of target disease is interstitial cystitis. Interstitial cystitis is an organic disease of the bladder that causes symptoms such as frequent urination, increased urinary urgency, urgency, inflammation, and bladder pain. In severe cases, the quality of life is significantly reduced. Although the underlying cause of interstitial cystitis has not yet been clarified, three pathophysiological mechanisms (epithelial dysfunction, mast cell activation, neuropathitis) have been suggested. Although there are effective treatments for some patients, there is a great need for treatments for interstitial cystitis.

The cell preparation of the present invention is usually used for the prevention or treatment of target diseases. Therefore, typically, the cell preparation of the present invention will be administered to a patient suffering from or developing a target disease or a potential patient (a person who is likely to suffer or develop). The cell preparation of the present invention can also be used for experimental purposes such as confirmation and verification.

(Applicable)
Subjects to which the cell preparation of the present invention is administered include humans or non-human mammals (pet animals, domestic animals, laboratory animals. Specific examples include mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. Sheep, dogs, cats, etc.). Preferably, the cell preparation of the present invention is used for humans.

(Method of administration)
The administration route of the cell preparation of the present invention is not particularly limited. For example, the cell preparation of the present invention is administered by intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Preferably, the cell preparation of the present invention is administered by local injection into the affected area. That is, the cell preparation of the present invention is particularly suitable for local treatment (focal therapy). Examples of the dosage of the cell preparation are 0.1 ml to 100 ml, preferably 5 ml to 60 ml, for example.

The administration schedule may be created taking into account the gender, age, weight, disease state, etc. of the subject of application (patient). In addition to single administration, multiple administration may be performed continuously or periodically. The administration interval for multiple administrations is not particularly limited, and is, for example, 1 day to 1 month. Moreover, the frequency | count of administration is not specifically limited. An example of the administration frequency is 2 to 10 times.

In the cell preparation of the present invention, mannose and / or WNIR are used in combination in order to increase the activity of ASCs. Details of the combined use will be described below.

(First embodiment: containing a low concentration of mannose)
The cell preparation of the first embodiment contains a low concentration of mannose in addition to ASCs. That is, the cell preparation of this embodiment is characterized by containing ASCs and a low concentration of mannose. In the present invention, “low concentration” means a concentration of 0.8 (w / v) or less. Therefore, the cell preparation of the first embodiment contains mannose at a concentration of 0.8% (w / v) or less. The lower limit of the concentration is not particularly limited as long as the effect characteristic of the present invention, that is, the effect of enhancing the activity of ASCs can be exhibited. For example, the mannose concentration is 0.2% (w / v) to 0.8 (w / v). Mannose can be produced by hydrolyzing (acid degradation, enzymatic degradation, etc.) galactomannan contained in glucomannan, guar gum and the like contained in wood, konjac and the like. It can also be produced from coconut oil extraction residue (Japanese Patent Laid-Open Nos. 11-137288 and 2010-22267). On the other hand, a method for producing from glucose using molybdate as a catalyst and a production method using an enzyme derived from a microorganism have also been reported (Japanese Patent Laid-Open No. 2001-231592).

(Second aspect: treatment of ASCs with low concentration of mannose)
In the cell preparation of the second embodiment, ASCs previously treated with a low concentration of mannose are used. The cell preparation is a cell preparation containing ASCs whose activity is increased by treatment with a low concentration of mannose. “Pre-treatment with a low concentration of mannose” means contacting ASCs with a low concentration of mannose prior to formulation. Typically, ASCs satisfying the above conditions are obtained by culturing ASCs (or a cell population containing ASCs) under conditions where low concentrations of mannose are present in the culture medium at or after the preparation stage. Can do. Here, “low concentration mannose” refers to a mannose concentration of 0.8% (w / v) or less. For example, a mannose concentration of 0.2% (w / v) to 0.8 (w / v) is employed. The treatment time or culture time is not particularly limited, but is, for example, 1 hour to 3 weeks. The subculture may be performed while maintaining the condition in which a low concentration of mannose is present. In view of the experimental results shown in the examples described later, it is preferable to culture under low oxygen concentration conditions. The combined use of hypoxic conditions can be expected to further improve the activity of ASCs.

(Third embodiment: simultaneous administration of mannose)
The cell preparation of the second aspect is characterized in that it does not contain mannose per se, but mannose is administered simultaneously with the administration. “Simultaneous” here does not require strict simultaneity. For example, mannose is administered immediately after administration of the cell preparation, as well as when the administration of the cell preparation and mannose is performed under conditions that do not cause a time difference, such as administration to a subject after mixing mannose with the cell preparation. The concept of “simultaneous” is also included in the case where the cell preparation and mannose are administered under conditions with no substantial time difference, such as administration of cell preparation and mannose in the reverse order. . In the latter case, it is preferable to set the time difference between the administration of the two (cell preparation and mannose) as short as possible so that the effect of mannose to enhance the activity of ASCs is exhibited well. For example, the other is administered within 10 minutes, preferably within 5 minutes, more preferably within 1 minute after one administration.

In the present invention, in order to increase the activity of ASCs, it is important that a state in which a low concentration of mannose contacts ASCs is formed. Therefore, if the cell preparation and mannose are mixed prior to administration, the mannose concentration in the mixture will be 0.8% (w / v) or less, for example 0.2% (w / v) to 0.8 (w / v). It is preferable to adjust the amount of mannose used. When the cell preparation and mannose are administered separately, the mannose concentration should be set high, taking into account diffusion and circulation in the living body (for example, 0.2% (w / v) to 2% (w / v)) Mannose solution).

(Fourth aspect: Broadband near-infrared irradiation before administration)
The cell preparation of the fourth aspect is characterized in that broadband near-infrared radiation is irradiated before administration to a treatment subject. In the present invention, broadband near-infrared (WNIR) is light having a wavelength with a maximum emission intensity in the range of 750 to 1200 nm and a half-value width of the emission spectrum of 70 to 200 nm. Preferably, light having a maximum emission intensity in the range of 900 to 1100 nm, more preferably light having a half-value width of the emission spectrum of 70 to 100 nm is used as WNIR. A light source for generating WNIR will be described in detail with reference to FIG. The light source 20 is an incoherent light source. The emission spectrum of light emitted from the light source 20 has a wavelength at which the emission intensity is maximum within a range of 750 to 1200 nm (more preferably 900 to 1100 nm), and a half width of 70 to 200 nm (more preferably). 70 to 100 nm). Such near-infrared light is less scattered and has high biological permeability (not easily absorbed by the living body), so that it has little influence when irradiated on the living body, and is considered useful for, for example, living body imaging. However, as shown in the examples described later, a phenomenon was observed that overturned the original expectation that when the skin was irradiated, the activity of ASCs in adipose tissue was increased. This fact is important in understanding the features and significance of the present invention.

The emission spectrum of light emitted from the light source 20 preferably has a continuous wavelength range. Here, the continuous wavelength range means that there is no missing wavelength in the wavelength range of the light emitted from the light source 20. Since the emission spectrum of light emitted from the light source 20 has a continuous wavelength region, it is possible to prevent light having a wavelength useful for enhancing the activity of ASCs from being lost.

As shown in FIG. 12, the light source 20 includes a semiconductor light emitting element 28 disposed on a substrate 26. The substrate 26 is formed of a material that does not have transparency. For example, a material having high reflectivity and good thermal conductivity can be used. A known semiconductor light emitting element can be used as the semiconductor light emitting element 28. For example, a light emitting diode, a super luminescent diode, a laser diode, or the like can be used. By using the semiconductor light emitting element 28, the amount of heat generated by the light source 20 can be reduced as compared with a light source such as a halogen lamp.

The periphery of the semiconductor light emitting element 28 is surrounded by a metal body 24 fixed on the substrate 26. That is, a through hole 24a is formed in the metal body 24, and the semiconductor light emitting element 28 is disposed in the through hole 24a. For this reason, the light from the semiconductor light emitting element 28 is irradiated to the opposite side of the substrate 26 through the through hole 24 a of the metal body 24. For the metal body 24, for example, an aluminum block can be used.

In addition, an infrared glass phosphor 30 is disposed in the through hole 24 a of the metal body 24. The infrared glass phosphor 30 is located on the light emitting surface side of the semiconductor light emitting element 28 (that is, the side opposite to the substrate 26), and the outer periphery thereof is in contact with the metal body 24. The infrared glass phosphor 30 has a function of converting light emitted from the semiconductor light emitting element 28 into light in the near infrared region (wavelength 700 to 2500 nm) and expanding the wavelength region of the emission spectrum. Yes. That is, when light from the semiconductor light emitting element 28 (for example, light in the visible light region) enters the infrared glass phosphor 30, the infrared glass phosphor 30 emits light in the near infrared region. The half width of the spectrum of the irradiated light is wider than the half width of the spectrum of the light irradiated from the semiconductor light emitting element 28. As a result, the emission spectrum of the light emitted from the light source 20 has the characteristics described above.

The infrared glass phosphor 30 can be formed, for example, by containing fluorescent ions in glass (amorphous) that is a base material. For example, it can be formed by adding Yb ions in glass, or can be formed by adding Yb ions and Nd ions in glass. When Yb ions are added to the glass, Yb ₂ O ₃ may be added to the glass. Also, when adding Yb ions and Nd ions in the glass, it may be added _Yb 2 _{O 3} and _Nd 2 _{O 3} in the glass. For example, glass made of Bi ₂ O ₃ and B ₂ O ₃ can be used as the base glass. A specific method for manufacturing the infrared glass phosphor 30 is disclosed in detail in Japanese Patent Application Laid-Open No. 2008-185378.

As shown in FIG. 12, a filter 22 can be disposed on the upper surface side (the side on which light is irradiated) of the infrared glass phosphor 30. The filter 22 can be fixed to the upper surface of the metal body 24. The filter 22 has a function of blocking light having a wavelength in the visible light region of the light irradiated from the infrared glass phosphor 30.

As described above, the cell preparation of this embodiment is irradiated with broadband near-infrared rays before being administered to a treatment target. In the case of irradiation before administration, the prepared cell preparation is irradiated with light at an intensity of 0.1 to 1 mW for 1 minute to 1 hour, for example. You may irradiate light in several steps.

(Fifth embodiment: WNIR irradiation after administration)
The cell preparation of the fifth aspect is characterized in that WNIR is irradiated after administration to a treatment subject. That is, in the present invention, two irradiation modes, irradiation before administration (fourth embodiment) and irradiation after administration (fifth embodiment), can be employed. In the case of irradiation after administration, after the cell preparation is administered by a predetermined administration method (for example, intravenous injection, local injection), at the administration site or in the vicinity thereof, or at the target site (injured site), for example, 0.1 to Irradiate light with an intensity of 1 mW for 1 minute to 1 hour. In addition, about the detail of WNIR, the description in a 4th aspect is used.

(Sixth embodiment: combined use of mannose and WNIR)
In the fourth aspect or the fifth aspect, one or more of the characteristics of the first aspect to the third aspect (containing low mannose, simultaneous administration of mannose, treatment of ASCs with low mannose) are used in combination. Also good.

<Second aspect: Method for enhancing cell activity>
The second aspect of the present invention provides a method for enhancing cell activity based on the finding that the use of low concentrations of mannose and WNIR becomes a general-purpose means for enhancing cell activity. The method of the present invention is characterized by including the following step (1) and / or step (2).
(1) A step of bringing a cell into contact with a low concentration of mannose (2) A step of irradiating the cell with broadband near infrared rays

The cell in this aspect is not particularly limited. Examples of cells include cardiomyocytes, smooth muscle cells, adipocytes, fibroblasts, bone cells, chondrocytes, osteoclasts, parenchymal cells, epidermal keratinocytes, epithelial cells (skin epidermal cells, corneal epithelium) Cells, conjunctival epithelial cells, oral mucosal epithelium, follicular epithelial cells, oral mucosal epithelial cells, airway mucosal epithelial cells, intestinal mucosal epithelial cells, etc.), endothelial cells (corneal endothelial cells, vascular endothelial cells, etc.), nerve cells, Schwann cells , Glial cells, spleen cells, pancreatic β cells, mesangial cells, Langerhans cells, hepatocytes, their precursor cells or stem cells, adipose tissue-derived mesenchymal stem cells (ASCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), Artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic germ cells (EG cells), and embryonic tumor cells (EC cells). Passage cells, cells induced to differentiate into specific cell lineages, cell lines (eg, HeLa cells, CHO cells, Vero cells, HEK293 cells, HepG2 cells, COS-7 cells, NIH3T3 cells, Sf9 cells) Can also be used. Preferably, adult stem cells (tissue stem cells, somatic stem cells) or endothelial cells, more preferably ASCs, BM-MSCs or Huvec are employed as the cells of the present invention.

Step (1) and step (2) are performed ex vivo (in vitro or ex vivo), for example. In this case, typically, step (1) can be carried out under the same method and conditions as in the second embodiment of the first aspect (treatment of ASCs with low concentration of mannose). That is, the cells in the preparation stage or after preparation may be cultured for a predetermined time (for example, 1 hour to 3 weeks) under the condition that a low concentration of mannose is present in the culture solution. Step (2) is typically performed under the same method and conditions as the treatment method in the fourth embodiment (irradiation of WNIR before administration) of the first aspect. Alternatively, a part of primary culture or subculture may be performed in a state where WNIR is irradiated, and WNIR may be irradiated to cells in the preparation stage.

Step (1) and step (2) can also be performed in vivo. In this case, as step (1), for example, cells and mannose may be simultaneously administered to a living body, similarly to the third embodiment of the first aspect (simultaneous administration of mannose). On the other hand, even when the cell preparation of the first embodiment (containing a low concentration of mannose) of the first aspect is administered to a living body, step (1) is performed in the living body. As step (2), for example, WNIR may be irradiated after cells are administered to a living body in the same manner as in the fifth embodiment of the first aspect (irradiation of WNIR after administration).

The cells obtained by carrying out the method of the present invention in vitro, that is, cells with enhanced activity are useful as active ingredients of cell preparations. Improvement in the therapeutic effect can be expected by enhancing the activity.

<Third aspect: blood flow improving agent>
As shown in Examples described later, as a result of examining the effectiveness of mannose, it was found that even mannose alone exerts an improvement effect on blood flow. Based on this finding, the third aspect of the present invention provides a blood flow improving agent containing mannose. The content of mannose is not particularly limited as long as the expected action / effect, that is, improving blood flow at or near the application site when applied to a living body (specifically, for example, skin) is exhibited. . For example, the content of mannose can be set within a range of 0.1% (w / v) to 95% (w / v). The blood flow improving agent of the present invention can be used for skin care, face care, body care or hair care, for example. Therefore, typically, the blood flow improving agent of the present invention is in the form of a cosmetic composition (eg, emulsion, lotion, lotion, lip balm, pack, facial cleansing foam, body soap, hand soap, shampoo, rinse, conditioner). , Hair spray, hair restorer). The cosmetic composition comprises mannose, which is an active ingredient, and ingredients / bases usually used in cosmetics (for example, various oils and fats, mineral oil, petrolatum, squalane, lanolin, beeswax, denatured alcohol, dextrin palmitate, glycerin, (Glycerin fatty acid ester, ethylene glycol, paraben, camphor, menthol, various vitamins, zinc oxide, titanium oxide, benzoic acid, edetic acid, chamomile oil, carrageenan, chitin powder, chitosan, fragrance, coloring agent, etc.) be able to. On the other hand, it is also possible to provide the blood flow improving agent of the present invention in the form of a pharmaceutical composition or a quasi-drug composition.

In the course of conducting research aimed at clinical application of regenerative therapy using ASCs, we focused on mannose and near-infrared rays and examined their regenerative support effects.

1. Changes in cell activity by mannose (in vitro experiments)
The effect of mannose on the activity of ASCs was investigated.
(1) Method 6-well plate (BD Falcon) is seeded with human ASCs (Lonza) (1.5 x 10 ⁵ cells / well) and cultured under specified conditions (20% or 5% O ₂ , 37 ° C) did. Stem containing 2% FBS with or without mannose added (0.2% (w / v), 0.4% (w / v), 0.8% (w / v)) Pro MSC SFM (Invitrogen) (registered trademark) was used. The cells were passaged on the 3rd and 6th days of culture. During the passage, the culture supernatant was sampled and various cytokines were measured.

(2) Results The measurement results for each cytokine are shown in FIGS. As shown in FIG. 1 (mannose addition concentration 0.4 (w / v)), the secretion of each cytokine was promoted by the addition of a low concentration of mannose. In addition, the amount of cytokine secretion is higher at the low oxygen concentration (5%, FIG. 1 right) than the normal oxygen concentration (20%, FIG. 1 left). On the other hand, secretion of various cytokines was generally promoted in the mannose concentration range of 0.2 (w / v) to 0.8 (w / v) (FIGS. 2 to 5).

(3) Summary As a method for enhancing the activity of ASCs, it has become clear that culturing ASCs in the presence of mannose at a low concentration (0.2 (w / v) to 0.8 (w / v)) is effective. The increased amount of cytokine secretion under the low oxygen concentration condition means that culture under the low oxygen concentration condition is preferable for preparing highly active ASCs. In addition, as a means for enhancing the activity of ASCs, it is suggested that it is effective to form a state in which mannose contacts ASCs in a living body having a low oxygen concentration environment.

2. Changes in cellular activity by broadband near infrared (WNIR) (in vitro experiments)
The effect of WNIR on the activity of ASCs was investigated.
(1) Method 6-well plate (BD Falcon) is seeded with human ASCs (Lonza) (1.5 x 10 ⁵ cells / well) and cultured under specified conditions (20% or 5% O ₂ , 37 ° C) did. Stem Pro MSC SFM (Invitrogen) (registered trademark) containing 2% FBS was used as the medium. The cells were passaged on the 3rd and 6th days of culture. The cells before seeding (Day 0 and Day 3) were irradiated with WNIR under the condition of 0.27 mW for 15 minutes. During the passage, the culture supernatant was sampled and various cytokines were measured.

(2) Results FIG. 6 shows the measurement results for each cytokine. Secretion of each cytokine was promoted by irradiation with WNIR (culture day 3). In addition, the amount of cytokine secretion is higher at the low oxygen concentration (5%, right of FIG. 6) than at the normal oxygen concentration (20%, left of FIG. 6).

(3) Summary It has become clear that WNIR irradiation is effective as a method for increasing the activity of ASCs. Since the amount of cytokine secretion increased under the low oxygen concentration condition, it can be said that irradiating ASCs with WNIR in vivo in a low oxygen concentration environment is also effective as a means for enhancing the activity of ASCs.

3. Action on rat model of renal ischemia (in vivo experiment)
Using a rat model of renal ischemia, the effects of low concentrations of mannose and WNIR on the therapeutic effects of ASCs (renal protective action in ischemic renal failure) were examined.
(1) Method Collagenase treatment (1 to 3% collagenase, 37 ° C., 60 to 90 minutes) and centrifugation treatment (1200 rpm, 5 minutes) from the tail vein of a rat model of single kidney 45 minutes ischemic renal failure (WO / 2008 / Rat ASCs prepared by 018450 (method described in PCT / JP2007 / 065431) (ASCs administration group), rat ASC 0.4% (w / v) mannose (derived from palm fruit, purity 99.7%) 1 hour culture (MesenPRO medium, 37 ° C) (mannose admixture group), rat ASCs irradiated with WNIR for 15 min (WNIR irradiation group), rat ASC 0.4% (w / v) mannose, 1 hour culture (MesenPRO medium, 37 ° C) followed by irradiation with WNIR for 15 minutes (Mannose WNIR combination group) or physiological saline (control group) 24 hours later, renal function, peritubular blood flow, And the pathological tissues were compared. The dose of ASCs was about 1 × 10 ⁶ cells. WNIR irradiation was performed for 15 minutes under the condition of 0.27 mW. As an indicator of renal function, blood creatinine level (Cre) and kidney injury score (Noiri E, Peresleni T, Miller F, and Goligorsky MS. Antisense oligonucleotides to the inducible NOS prevent tubular cell death in ischemic acute renal failure. J Clin Invest 97: 2377-2383, 1996). The peritubular blood flow was evaluated by the following method. That is, the microcirculation was visualized using a pencil probe type CCD microscope system (PPVM) for the cortical superficial tubule capillaries (PTC: n = 6) exfoliated and exposed in anesthetized rats (n = 6), Recorded as a digital image. The red blood cell velocity was calculated from data obtained by subjecting the moving image to spatiotemporal image processing (see Japanese Patent Application Laid-Open No. 2010-187925). The flow rate was calculated from the red blood cell velocity and the blood vessel diameter.

(2) Results The results are shown in FIGS. Blood Cr after 24 hours was significantly lower in the mannose admixture group (lsch + A + M) (n = 6) than in the untreated group (Isch), and in the WNIR irradiation group (lsch + A + WNIR) ( n = 6) and the mannose WNIR combination group (lsch + A + M + WNIR) (n = 6) are even lower (the difference from the untreated group is large) (upper FIG. 7).

The score of kidney damage decreased in the mannose admixture group (Isch + A + M) and WNIR irradiation group (Isch + A + WNIR), and further decreased in the mannose WNIR combination group (Isch + A + M + WNIR) (FIG. 7). under).

Comparing the amount of peritubular capillary blood flow (PTC), compared with the control group (Cont), mannose admixture group (Isch + A + M), WNIR irradiation group (Isch + A + WNIR), mannose WNIR combination There was a significant improvement in blood flow in the group (Figure 8). The degree of improvement increases in the order of the mannose admixture group (Isch + A + M), the WNIR irradiation group (Isch + A + WNIR), and the mannose WNIR combination group (Isch + A + M + WNIR). (Figure 8).

Furthermore, accumulation of ASCs at the site of renal ischemia was observed in the ASCs administration group (Isch + A), and accumulation of ASCs was enhanced in the WNIR irradiation group (Isch + A + WNIR) (FIG. 9).

(3) Summary The nephroprotective effect of ASCs was enhanced by mixing mannose (Isch + A + M) and WNIR irradiation (Isch + A + WNIR). It was also found that the enhancement effect was further enhanced when mannose mixing and WNIR irradiation were combined (Isch + A + M + WNIR). Mannose is presumed to enhance secretion of tissue repair cytokines secreted from ASCs. On the other hand, it is speculated that irradiation with WNIR promoted the accumulation of ASCs in the renal ischemic site and enhanced the renal protection support action.

4). Skin microcirculation improvement for hyperlipidemia (in vivo experiment)
The effects of mannose and WNIR on hyperlipidemia cases (n = 6) were investigated.
(1) Method Hyperlipidemia cases (n = 6: hypertension, 3 cases with diabetes, 4 cases of smoking, 3 cases of obesity) were treated. After applying 2% (w / v) mannose (derived from coconut, purity 99.7%) to nails, lips and scalp, WNIR was irradiated for 20 minutes under the condition of 0.27 mV. This treatment (application of mannose and irradiation with WNIR) was performed for 3 days, and before and after that, subcutaneous capillary blood flow was compared and evaluated by the following method. Subcutaneous capillary blood flow was visualized using a pari-hocal video microscope (VFVMS) and recorded as a digital image. The red blood cell velocity was calculated from data obtained by subjecting the moving image to spatiotemporal image processing (see Japanese Patent Application Laid-Open No. 2010-187925). The flow rate was calculated from the red blood cell velocity and the blood vessel diameter.

(2) Results The whole-body subcutaneous capillary blood flow was visualized and the blood flow was quantified (FIG. 10). Subcutaneous capillary blood flow was clearly reduced in the hyperlipidemia group compared to the normal group. Application of mannose and irradiation with WNIR significantly increased capillary blood flow compared to before treatment (FIGS. 10 and 11).

(3) Summary Significant improvement in subcutaneous capillary blood flow in the hyperlipidemic group was observed by the combined use of mannose and WNIR. Adipose tissue-derived mesenchymal stem cells (ASCs) are abundant in the stroma of subcutaneous fat. It is presumed that capillary blood flow was improved as a result of the activity of the ASCs being enhanced by mannose and WWIN that had penetrated into the skin and reached a low concentration, and promoted angiogenesis and cytokine secretion.

5. Mannose improves skin microcirculation (in vivo experiments)
(1) Method 2% (w / v) mannose (derived from coconut, purity 99.7%) was applied to the lips of a human (healthy person), and capillary blood flow was measured 4 days later. The blood flow measurement is 4. The same experiment was performed.

(2) Results The blood flow of the lips was visualized and the blood flow was quantified (FIG. 13). Application of mannose significantly increased capillary blood flow (FIGS. 13 and 14).

(3) Summary Significant improvement in subcutaneous capillary blood flow was observed by application of mannose. Application of mannose alone proved effective in improving subcutaneous capillary blood flow.

6). Effects of low-concentration mannose on various cells Human umbilical vein endothelial cells (HUVEC) and bone marrow-derived mesenchymal systems with the expectation that culture in the presence of low-concentration mannose is a versatile means of enhancing cell activity Experiments using stem cells (BM-MSCs) were performed.
(1) Method 6-well plate (BD Falcon) is seeded with HUVEC (Kurashiki Spinning Co., Ltd.) (2.0 x 10 ⁵ cells / well) under the specified conditions (20% or 5% O ₂ , 37 ° C) Cultured. The medium contains HUVEC medium (Kurashiki Spinning) with or without mannose (derived from coconut, purity 99.7%) (0.2% (w / v), 0.4% (w / v), 0.8% (w / v)) Used). It was subcultured on the third day of culture. During the passage, the culture supernatant was sampled and various cytokines were measured.

On the other hand, BM-MSCs (Lonza) was seeded on a 6-well plate (BD Falcon) (1.0 x 10 ⁵ cells / well), and cultured under predetermined conditions (20% or 5% O ₂ , 37 ° C). . Stem Pro MSC SFM (Invitrogen) (registered trademark) containing 2% FBS with or without addition of mannose (derived from coconut, purity 99.7%) (0.4% (w / v)) or not added was used as the medium. It was subcultured on the third day of culture. During the passage, the culture supernatant was sampled and various cytokines were measured.

(2) Results The measurement results of each cytokine for HUVEC are shown in FIGS. The addition of a low concentration of mannose tends to promote the secretion of each cytokine. In general, the amount of cytokine secretion is higher at the low oxygen concentration (5%) than at the normal oxygen concentration (20%). As for BM-MSCs, secretion of VEGF-A and NGF-β was promoted by addition of a low concentration of mannose (FIGS. 19 and 20).

(3) Summary It was revealed that the activity of HUVEC and BM-MSCs can be enhanced when cultured in the presence of a low concentration of mannose. Based on this result and the results of the experiment using ASCs (1. above), it can be said that culturing in the presence of a low concentration of mannose is highly versatile as a means for enhancing cell activity.

7). The effect of broadband near-infrared (WNIR) on various cells. Experiments using HUVEC and BM-MSCs were conducted with the expectation that WNIR is a versatile means for enhancing cell activity.
(1) Method 6-well plate (BD Falcon) is seeded with HUVEC (Kurashiki Spinning Co., Ltd.) (2.0 x 10 ⁵ cells / well) under the specified conditions (20% or 5% O ₂ , 37 ° C) Cultured. HUVEC medium (Kurashiki Boseki Co., Ltd.) was used as the medium. It was subcultured on the third day of culture. The cells before seeding (Day 0 and Day 3) were irradiated with WNIR under the condition of 0.27 mV for 15 minutes. During the passage, the culture supernatant was sampled and various cytokines were measured.

On the other hand, BM-MSCs (Lonza) was seeded on a 6-well plate (BD Falcon) (1.0 x 10 ⁵ cells / well), and cultured under predetermined conditions (20% or 5% O ₂ , 37 ° C). . Stem Pro MSC SFM (Invitrogen) (registered trademark) containing 2% FBS was used as the medium. It was subcultured on the third day of culture. The cells before seeding (Day 0 and Day 3) were irradiated with WNIR under the condition of 0.27 mV for 15 minutes. During the passage, the culture supernatant was sampled and various cytokines were measured.

(2) Results FIG. 21 shows the cytokine measurement results for HUVEC. WNIR irradiation significantly promoted Adrenomedullin secretion. In addition, the secretion amount of Adrenomedullin is much higher at the low oxygen concentration (5%, FIG. 21 right) than the normal oxygen concentration (20%, FIG. 21 left). On the other hand, the measurement results of each cytokine for BM-MSCs are shown in FIGS. Secretion of each cytokine was promoted by irradiation with WNIR.

(3) Summary It became clear that the activity of HUVEC and BM-MSC can be enhanced by irradiation with WNIR. Based on this result and the result of the experiment using ASCs (2. above), it can be said that WNIR irradiation is highly versatile as a means for enhancing cell activity. On the other hand, the promotion of the secretion of Adrenomedullin, which exhibits a vasodilatory action (FIGS. 21 and 25), confirms that irradiation with WNIR is effective in improving blood flow.

8). Action on rat model of interstitial cystitis (in vivo experiment)
As an animal model of interstitial cystitis, a cystitis model using cyclophosphamide (CYP) which is an immunosuppressant is widely used. This model is the main symptom of bladder inflammation, increased frequency of micturition, and bladder pain peaking in hours after administration and recovering in just a few days thereafter (Arms L, Girard BM, Malley SE, Vizzard MA. Expression and function of CCL2 / CCR2 in rat micturition reflexes and somatic sensitivity with urinary bladder inflammation. Am J Physiol Renal Physiol. 2013 Jul 1; 305 (1): F111-22. 2013 Apr 17).

(1) Method Collagenase treatment (1 to 3% collagenase, 37 ° C., 60 to 90 minutes) and centrifugation treatment (1200 rpm, 5 minutes) 5 hours after CYP administration in which symptoms of cystitis appear (WO / 2008/018450 ( Intravesical injection of rat ASCs prepared by the method described in PCT / JP2007 / 065431)) or rat ASCs mixed with low-concentration mannose (0.2% (w / v)) and cultured for 1 hour (MesenPRO medium, 37 ° C) Two days later, intravesical pressure (CMG) was measured and the pathological tissue was examined. The dose of ASCs was about 2 × 10 ⁶ cells. Rats not administered CYP (normal group: n = 6), rats injected with physiological saline after intraperitoneal injection of CYP (CYP-live food bladder injection group: n = 6), rats injected with ASCs after intraperitoneal injection of CYP (CYP -ASCs urinary injection group: n = 6), rats with CYP-MN admixture ASCs urinary injection group (n = 6) were compared after intraperitoneal injection of CYP. Moreover, about the rat model which performed the same process using Dir label ASCs, fluorescence was detected 2 days after the process and image analysis was carried out.

(2) Results FIG. 26 shows the results of measuring the intravesical pressure. As shown in the figure, the urination interval was remarkably shortened in the CYP-meal urinary bladder injection group (5.6 ± 5.1 minutes) compared to the normal group (34.4 ± 4.1 minutes). On the other hand, the urination interval was prolonged in the CYP-ASCs urinary injection group (15.4 ± 3.1 min) compared with the CYP-raw diet urinary injection group. In the CYP-MN-mixed ASCs urinary injection group (21.4 ± 2.8 minutes), the urination interval was further extended. Thus, in the interstitial cystitis model, it has been clarified that administration of ASCs brings about a therapeutic effect and that the therapeutic effect is enhanced by the combined use of a low concentration of mannose.

Fig. 27 shows the results of HE staining (examination of pathological tissue). Compared with the normal group, the CYP-live urinary bladder group had marked mucosal edema and inflammatory cell infiltration reflecting inflammatory lesions. In the CYP-ASCs urinary injection group, the degree of mucosal edema and inflammatory cell infiltration was reduced. In the CYP-MN mixed ASCs urinary injection group, mucosal edema and inflammatory cell infiltration were further reduced.

In an experiment using Dir-labeled ASCs (ASCs bioimaging), in the CYP-ASCs bladder injection group (2 days after ASCs urinary injection), the fluorescence of Dir-labeled ASCs was detected in accordance with the bladder. A stronger signal of ASCs was detected in the CYP-MN-mixed ASCs bladder injection group (FIG. 28).
(3) Summary As described above, it was shown that intravesical injection of ASCs is an effective treatment for interstitial cystitis. Further, it has been clarified that the combined use of mannose enhances the therapeutic effect, that is, the combined use of ASCs and mannose provides an excellent therapeutic effect.

(4) Use of bone marrow-derived mesenchymal stem cells (BM-MSCs) The therapeutic effect on interstitial cystitis when rat BM-MSCs were used instead of rat ASCs was evaluated in the same experimental system. Intravesical injection of BM-MSCs (n = 3) (FIG. 29, upper row), urination interval was extended (18.4 ± 2.1 minutes) compared to control (CYP-live urinary bladder injection group), and mannose was combined (FIG. 29). The effect was enhanced (25.2 ± 3.4 minutes). Thus, like ASCs, BM-MSCs have been shown to be effective in the treatment of interstitial cystitis.

In the cell preparation of the present invention, ASCs whose activity is enhanced by the action of low concentration of mannose and / or broadband near infrared (WNIR) are active ingredients. Therefore, the cell preparation of the present invention can be applied to the treatment of various diseases for which administration of ASCs is effective. In particular, application to diseases or disorders in which improvement of blood flow has a therapeutic effect is assumed.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

20: Light source 28: Semiconductor light emitting element 30: Infrared glass phosphor

Claims (21)

1. A cell preparation containing adipose tissue-derived mesenchymal stem cells and a low concentration of mannose.
2. The cell preparation according to claim 1, comprising adipose tissue-derived mesenchymal stem cells and a low concentration of mannose.
3. The cell preparation according to claim 1, comprising adipose tissue-derived mesenchymal stem cells treated with a low concentration of mannose.
4. The cell preparation according to any one of claims 1 to 3, wherein the low concentration is a concentration of 0.8% (w / v) or less.
5. The cell preparation according to any one of claims 1 to 3, wherein the low concentration is 0.2% (w / v) to 0.8 (w / v).
6. The cell preparation according to claim 1, which contains adipose tissue-derived mesenchymal stem cells, and mannose is administered simultaneously when administered to a treatment target.
7. A cell preparation that contains adipose tissue-derived mesenchymal stem cells and is irradiated with broadband near-infrared rays before or after administration to a treatment target.
8. The cell preparation according to claim 7, wherein the wavelength of the emission intensity of broadband near-infrared light is in the range of 750 to 1200 nm and the half width of the emission spectrum is 70 to 200 nm.
9. The cell preparation according to claim 7 or 8, further comprising a low concentration of mannose.
10. The cell preparation according to claim 7 or 8, wherein mannose is administered at the same time as administration to a treatment subject.
11. The cell preparation according to any one of claims 1 to 10, which is used for treatment of a disease or disorder in which improvement of blood flow brings a therapeutic effect.
12. The cell preparation according to claim 11, which is used for treatment of ischemic disease or hyperlipidemia.
13. The cell preparation according to claim 12, wherein the ischemic disease is ischemic renal failure.
14. The cell preparation according to any one of claims 1 to 10, which is used for treatment of interstitial cystitis.
15. A method for increasing the activity of a cell, comprising the following steps (1) and / or step (2):
    (1) contacting the cells with a low concentration of mannose;
    (2) A step of irradiating a cell with broadband near infrared rays.
16. The method according to claim 15, wherein the cells are mesenchymal stem cells or endothelial cells.
17. The method according to claim 16, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells, and the endothelial cells are umbilical vein endothelial cells.
18. The method according to any one of claims 15 to 17, wherein steps (1) and (2) are performed ex vivo.
19. A cell preparation containing cells whose activity has been enhanced by the method according to claim 18.
20. A blood flow improving agent containing mannose.
21. 21. The blood flow improving agent according to claim 20, which is used for skin care, face care, body care or hair care.

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