JP2009001509A - Composition for regenerating tissue by using adipose tissue-originated stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique by which the transplantation of adipose tissue-originated stem cells (ASCs) in mass at once is enabled, and to provide applications thereof. <P>SOLUTION: The composition for regenerating the tissue is obtained by combining the adipose tissue-originated stem cell with heparin. The composition for regenerating the tissue contains the adipose tissue-originated stem cell and the heparin. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a tissue regeneration composition. Specifically, the present invention relates to a tissue regeneration composition using adipose tissue-derived stem cells and heparin and its use. The composition for tissue regeneration of the present invention is effective for the treatment or prevention of liver damage, for example.

Regenerative medicine is the most recognized medical field among new medical technologies. Beginning with the regeneration of skin and bone and cartilage, research subjects have expanded to include cardiovascular, cranial nerves, and hepatopancreatic diseases. Cell sources used in regenerative medicine include embryonic stem cells (ES cells) and somatic stem cells. Currently, clinical trials using somatic stem cells are ahead.
Bone marrow-derived stem cells are well known as somatic stem cells. However, considering clinical application, when bone marrow-derived stem cells are used, there are problems of relatively high invasiveness and acquisition of the required number of cells. Therefore, adipose tissue-derived stem cells (hereinafter also referred to as “ASCs”. ASCs is an abbreviation for Adipose tissue-derived Stem Cells) are attracting attention recently. Extraction of stem cells from adipose tissue is minimally invasive and simple, and many cells can be obtained. Therefore, ASCs can be said to be very promising somatic stem cells in clinical application.
Recently, several research groups have reported that adipose tissue is promising as a source of pluripotent stem cells (Non-patent Document 1). On the other hand, Kitagawa et al. Reported that it is possible to prepare a large number of cell populations showing multipotency from adipose tissue by a simple operation, and that the obtained cells have the ability to differentiate into adipose tissue. And it was shown that it is effective for the reconstruction of adipose tissue (Patent Document 1). A new application of ASCs has also been studied (Patent Document 2).
International Publication No. 2006 / 006692A1 Pamphlet Special Table 2007-507202 Secretion of Angiogenic and Antiapoptotic Factors by Human Adipose Stromal Cells. Circulation 109: 1292-1298, 2004

So far, our research group has confirmed that liver injury can be improved by transplanting ASCs from the tail vein into mice with acute liver failure. By the way, the level of the effect (degree of regeneration) in the treatment involving cell transplantation depends largely on the number of cells to be transplanted. Therefore, to obtain a high therapeutic effect, it is desired to transplant a large amount of cells. However, the present inventors have examined that mice transplanted with a large amount of ASCs at a time (1 × 10 ⁶ cells / mouse / 150 μL or more) frequently cause pulmonary infarction or liver infarction, resulting in death. A problem was found.
Therefore, an object of the present invention is to provide a technique that enables transplantation of a large amount of ASCs at a time and a use thereof in order to provide a good therapeutic effect.

In view of the above problems, the present inventors have focused on heparin, which is an anticoagulant. Then, a model mouse for acute hepatitis was prepared, and the effect of coadministering heparin at the time of transplantation of ASCs was examined. As a result, a decrease in the liver damage markers AST (GOT) and ALT (GPT) was observed in the group administered with heparin, and LDH was compared to the control group (group administered with physiological saline). A significant decrease was confirmed. In addition, it should be noted that in the group administered with ASCs alone, 4 out of 6 animals developed lung infarction and liver infarction and died immediately after transplantation (survival rate 33%), whereas in the group administered with heparin in combination. There was no infarct after transplantation and the survival rate was 100%. Thus, it became clear that infarction caused by cell transplantation can be prevented by combined use of heparin. This means that if heparin is used in combination, it is possible to administer a large amount of ASCs at a time, thereby improving the therapeutic effect (regeneration effect).
The present invention has been completed mainly based on the above findings, and provides the following tissue regeneration composition, preparation method and use thereof.
[1] A composition for tissue regeneration comprising a combination of adipose tissue-derived stem cells and heparin.
[2] The tissue regeneration composition according to [1], comprising adipose tissue-derived stem cells and heparin.
[3] The composition for tissue regeneration according to [1], which is a kit comprising a first component containing adipose tissue-derived stem cells and a second component containing heparin.
[4] The composition for tissue regeneration according to [1], comprising adipose tissue-derived stem cells, wherein heparin is administered together during administration.
[5] The composition for tissue regeneration according to [1], comprising heparin, wherein adipose tissue-derived stem cells are administered in combination at the time of administration.
[6] The adipose tissue-derived stem cell is
(1) Adherent cells or passage cells thereof included in a sedimented cell population that precipitates when a cell population separated from adipose tissue is centrifuged under conditions of 800 to 1500 rpm for 1 to 10 minutes,
(2) cells grown when the precipitated cell population is cultured under low serum conditions, or (3) cells grown when the cell population separated from adipose tissue is cultured under low serum conditions,
The composition for tissue regeneration according to any one of [1] to [5], wherein
[7] The composition for tissue regeneration according to [6], wherein the low serum condition is a condition where the serum concentration in the culture solution is 5% (V / V) or less.
[8] The cell population separated from the adipose tissue contains a sedimented cell population that precipitates when the cell population is centrifuged at 800 to 1500 rpm for 1 to 10 minutes, and heparin. A composition for tissue regeneration.
[9] The composition for tissue regeneration according to any one of [1] to [8], which is used for treatment or prevention of liver damage.
[10] The composition for tissue regeneration according to [9], wherein the liver disorder is hepatitis, cirrhosis, liver cancer or postoperative liver failure.
[11] The composition for tissue regeneration according to any one of [1] to [10], wherein the adipose tissue is human adipose tissue.
[12] A method for preparing a tissue regeneration composition comprising the following steps:
(1) preparing adipose tissue-derived stem cells and heparin;
(2) A step of mixing the adipose tissue-derived stem cells and the heparin.
[13] Use of adipose tissue-derived stem cells and heparin for producing a composition for tissue regeneration.
[14] A tissue regeneration method comprising administering a therapeutically effective amount of adipose tissue-derived stem cells and heparin to a patient having a tissue for regeneration.
[15] A method for treating or preventing liver damage, comprising administering a therapeutically effective amount of adipose tissue-derived stem cells and heparin to a patient suffering from or likely to suffer from liver damage.

  1st aspect of this invention is related with the composition (composition for structure | tissue regeneration) used for the reproduction | regeneration of a structure | tissue. One of the features of the composition for tissue regeneration of the present invention is that adipose tissue-derived stem cells (ASCs) and heparin are used in combination. In the present specification, “using ASCs and heparin in combination” or “combining ASCs and heparin” means that ASCs and heparin are used in combination. The specific mode of combined use will be described in detail in the column “(5) Formulation” below.

  In the present invention, “adipose tissue-derived stem cells (ASCs)” refers to somatic stem cells contained in adipose tissue. As long as pluripotency is maintained, the somatic stem cells are cultured (passaged). The cells obtained from the above are also “adipose tissue-derived stem cells (ASCs)”. As described later, ASCs are prepared in an isolated state as cells constituting the SVF fraction or as cells grown by culture using the SVF fraction (details of the method for preparing ASCs will be described later). 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

  Heparin is one of the anticoagulants and is used for the treatment and prevention of recurrence of thromboembolism and disseminated intravascular coagulation syndrome (DIC), artificial dialysis, and prevention of blood coagulation in extracorporeal circulation. A type of heparan sulfate, a glycosaminoglycan (β-D-glucuronic acid or α-L-iduronic acid and D-glucosamine, which are widely present in the body such as small intestine, muscle, lung, spleen, and mast cells. High molecular compound). Heparin is often prepared from the intestinal mucosa of domestic animals (pigs and cattle). Low molecular weight heparin (Andersson LO, Barrowcliffe TW, et al: Anticoagulant properties of heparin fractionated by affinity chromatography on matrix bound antithrombin III and by gel filtration. Thromb. Res. 9; 575-683, 1976.) It is used as an anticoagulant. Low molecular weight heparin has few side effects of bleeding and has been used frequently in recent years. In this specification, “normal heparin” is used as a term for heparin that is not “small molecule heparin”. In addition, the term “heparin” in the present specification is used to encompass normal heparin and low molecular weight heparin.

  As listed below, many commercial products are sold as normal heparin and low molecular weight heparin. If such commercially available heparin is used, the composition for tissue regeneration of the present invention can be prepared by a simpler operation. In addition, you may decide to use together 2 or more types of heparin instead of using only 1 type of heparin.

(1) Heparin sodium formulation Novo heparin injection 10,000 units (trade name, Mochida Pharmaceutical), Novo heparin injection 1000 (brand name, Mochida Pharmaceutical), heparin mochida (trade name, Mochida Pharmaceutical), heparin sodium injection N "Ajinomoto (Trade name, Ajinomoto) Delibadex 100 unit syringe (trade name, Shiono Chemical), Hepaflash 100 unit / mL syringe 10 mL (trade name, Terumo), Hepaflash 100 unit / mL syringe 5 mL (trade name, Terumo), Heparin Na500 Unit / mL syringe “NP” (trade name, Nipro Pharma), heparin Na lock 100 syringe (trade name, Mitsubishi Pharma), 100 unit / mL syringe “Otsuka” for heparin Na lock 10 mL (trade name, Otsuka Pharmaceutical Factory), heparin 250 units / mL syringe 20 for Na dialysis mL "AT" (trade name, Taiyo Pharmaceutical), 500 units / mL syringe for heparin Na dialysis 10 mL "AT" (trade name, Taiyo Pharmaceutical), Pemilock 100 units / mL syringe (trade name, Taiyo Pharmaceutical).

(2) Heparin calcium preparation Heparin calcium injection solution (trade name, Ajinomoto), caprocin for subcutaneous injection (trade name, Sawai Pharmaceutical).

(3) Dalteparin sodium preparation (low molecular weight heparin)
Dalteparin sodium intravenous injection 1000 units / mL (trade name, Merck Pharmaceutical) Fragmin intravenous injection (trade name, Pfizer), Rizalmin Injection 1000 (trade name, Ito Life Science), Fluzepamine intravenous injection 1000 units / mL (trade name, Taiyo Pharmaceuticals), Hepagumin IV 1000 units / mL (trade name, Sawai Pharmaceutical), Hepaclon Injection 5000 (trade name, Sankyo Ale), Daltepan IV 5000 (trade name, Nichi-Iko), Dalteparin Na IV 1000 units / mL “HK” 5 mL (trade name, photopharmaceutical), dalteparin Na syringe 5000 “HK” (trade name, photopharmaceutical).

(4) Parnaparin sodium preparation (low molecular weight heparin)
Minihepa Note 500 (brand name, Ito Life Science), Lowhepa Note 500 (brand name, Ajinomoto).

(Applicable)
The composition for tissue regeneration of the present invention is used for regeneration (reconstruction) of damaged tissues. Here, “disorder” comprehensively means that the tissue is not in a normal state, such as a state in which a part of the tissue is necrotic or missing. The “tissue” refers to a structure formed by assembling one or several types of cells in a predetermined pattern. On the other hand, an “organ” is formed by gathering a plurality of tissues. Therefore, in this specification, “for tissue regeneration” means that the tissue is used for regeneration of a tissue forming an organ or a tissue not forming an organ. Tissues are roughly classified into epithelial tissues (for example, skin epithelium), connective tissues (for example, dermis, adipose tissue), muscle tissues (for example, striated muscle, smooth muscle), and nerve tissues. On the other hand, examples of organs include the esophagus, stomach, duodenum, cecum, small intestine, large intestine, rectum, colon, liver, pancreas, tongue, pharynx, tonsils, blood vessels, heart, adrenal gland, spleen, lymphatic vessels, lymph nodes, kidneys, bladder, Mention may be made of the ureter, urethra, prostate, vas deferens, testis, fallopian tube, ovary, trachea, lung, nose, skin, breast, diaphragm.

  The composition for tissue regeneration of the present invention is preferably used for tissue regeneration for the purpose of treating or preventing liver damage. That is, in a preferred embodiment of the present invention, a tissue regeneration composition is used as a pharmaceutical composition (cell preparation) for liver damage. “Liver disorders” include hepatitis, cirrhosis, liver cancer, and postoperative liver failure. “Hepatitis” includes acute hepatitis, fulminant hepatitis, chronic hepatitis and the like. “Hepatitis” is classified into viral hepatitis, alcoholic hepatitis, autoimmune hepatitis, and drug-induced hepatitis depending on the cause.

  The use of the composition for tissue regeneration of the present invention is not limited to the treatment or prevention of liver damage. For example, pancreatic diseases (such as diabetes), refractory neurological diseases (such as Alzheimer's disease, Parkinson's disease, retinal degeneration), osteochondral diseases, immune diseases (including those caused by transplantation), renal disorders (acute renal failure, chronic Renal failure, hemolytic uremic syndrome, acute tubular necrosis, interstitial nephritis, acute papillary necrosis, glomerulonephritis, diabetic nephropathy, nephritis associated with collagen disease, nephropathy associated with vasculitis, pyelonephritis, nephrosclerosis , Drug-induced nephropathy, renal disorder associated with transplantation, etc.), ischemic disease (obstructive arteriosclerosis such as obstructive arteriosclerosis of the lower limbs), ischemic heart disease such as myocardial infarction and angina, brain such as cerebral infarction It is also expected that the composition for tissue regeneration of the present invention is applied as a pharmaceutical composition for treating or preventing vascular disorders or wounds.

(Subject and method of administration)
Subjects to which the composition for tissue regeneration of the present invention is administered include humans or non-human mammals (including pet animals, farm animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, Pigs, goats, sheep, dogs, cats, etc.). Preferably, the composition for tissue regeneration of the present invention is used for humans.

  The route of administration of the composition for tissue regeneration of the present invention is not particularly limited as long as it is delivered to the target tissue. For example, the composition for tissue regeneration of the present invention is administered by intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Local administration may be used instead of systemic administration. As local administration, direct injection or application to a target tissue can be exemplified. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the gender, age, weight, disease state, etc. of the subject (recipient) can be considered.

(Method for preparing ASCs)
Hereinafter, an example of a method for preparing ASCs will be described.
(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 from the same individual as the subject (recipient) to which the composition for tissue regeneration 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 100 g. When humans are used as donors, 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.

  Enzymatic treatment is performed by digesting adipose tissue with enzymes such as collagenase, trypsin, dispase and the like. Such enzyme treatment may be performed by techniques and conditions known to those skilled in the art (see, for example, 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 the centrifugation process 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.

  The “SVF fraction” obtained here contains ASCs. Therefore, the tissue regeneration composition of the present invention can be prepared using the SVF fraction. That is, the SVF fraction is contained in one embodiment of the composition for tissue regeneration of the present invention. 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
In addition to ASCs, the SVF fraction contains other cell components (endothelial cells, stromal cells, blood cells, progenitor cells thereof, etc.). 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 composition for tissue regeneration of the present invention.

  First, the SVF fraction is suspended in an appropriate medium, 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.), DMED: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Research Institute), etc. can be used. A medium supplemented with serum (fetal calf 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 manner, a composition for tissue regeneration 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 are used as ASCs in the composition for tissue regeneration 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 can be used in the low serum culture method, it is possible to use the serum of the subject (recipient) who administers the tissue regeneration composition of the present invention. 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.
Serum is not limited to fetal bovine serum, and human serum, sheep serum and the like can be used. Preferably, human serum, more preferably serum of a subject to which the composition for tissue regeneration of the present invention is applied (that is, autologous serum) is used.

  As a medium, a normal medium for animal cell culture can be used on the 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.), DMED: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Research Institute), etc. can be used.

  By culturing by the above method, multipotent stem cells (ASCs) can be selectively proliferated. In addition, pluripotent stem cells (ASCs) that proliferate under the above culture conditions have a high proliferative activity, so that the number of cells required for the tissue regeneration composition of the present invention can be easily prepared by subculture. be able to. 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 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), it is possible to prepare a tissue regeneration composition containing ASCs with high purity.

(5) Formulation
Cells obtained from the SVF fraction, the cells obtained as a result of the selective culture (3), or the cells obtained as a result of the low serum culture (4) (hereinafter, these cells are collectively referred to as “cells of the present invention”). The composition for tissue regeneration of the present invention is obtained by using a combination of heparin and heparin. Typically, the composition for tissue regeneration of the present invention will be provided as a combination drug in which the cells of the present invention and heparin are mixed. For example, heparin may be mixed with a cell suspension obtained by suspending the cells of the present invention in physiological saline or an appropriate buffer (for example, phosphate buffer).

  In the above method, a composition for tissue regeneration is constructed using cells grown by low-serum culture of the SVF fraction, but the cell population obtained from the adipose tissue is directly (centrifuged to obtain the SVF fraction). A composition for tissue regeneration may be prepared using cells proliferated by low serum culture as ASCs (without going through the above). 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.

  In a preferred embodiment, the composition for tissue regeneration of the present invention contains no cellular components other than ASCs. In other words, the cell component contains only ASCs. Thus, containing ASCs with high purity is advantageous for exhibiting the regeneration effect expected for ASCs, but is effective for preventing unexpected side effects due to other cell components.

  On the other hand, for example, the composition for tissue regeneration of the present invention can be provided in the form of a kit comprising a first component containing ASCs and a second component containing heparin. In this case, both elements will be administered to the subject simultaneously or at a predetermined time interval. Preferably both elements will be administered simultaneously. This is because the effect of heparin found by the present inventors, which makes it possible to safely administer a large amount of cells, is maximized. “Simultaneous” here does not require strict simultaneity. Therefore, when both elements are administered under the condition that there is no time difference, for example, both elements are mixed and then administered to the subject, both elements are immediately administered after one is administered. The case where the administration is performed under conditions without a substantial time difference is also included in the concept of “simultaneous” herein. On the other hand, when the other is administered at a predetermined time difference after one administration, it is preferable to set the time difference as short as possible so that the effect of the heparin is satisfactorily achieved. For example, the other is administered within 15 minutes, preferably within 10 minutes, more preferably within 5 minutes after administration of one.

  A composition for tissue regeneration containing ASCs may be used, and heparin may be administered in combination at the time of administration. The timing of administration of the tissue regeneration composition and heparin in this case is the same as in the case of the kit form described above. That is, both are preferably administered at the same time, but they may be administered at a predetermined time difference. In contrast to the above-described embodiment, a tissue regeneration composition containing heparin may be used, and ASCs may be administered in combination at the time of administration. The timing of administration in this case conforms to the case of the above embodiment.

For example, 1 × 10 ⁵ to 1 × 10 ¹⁰ ASCs may be contained in the tissue regeneration composition as a single dose so that a desired regeneration effect is exhibited. About heparin, it is good to make the composition for tissue regeneration contain, for example, 100 units to 10,000 units as a single dose. The amount of ASCs and heparin can be appropriately adjusted in consideration of the purpose of use, gender of the subject to be applied (recipient), age, weight, affected state, cell state, 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 included in the tissue regeneration composition 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 composition for structure | tissue regeneration of this invention.

<ASCs and heparin co-administration study in acute hepatitis mice>
The following experiment was conducted for the purpose of examining the effect of using heparin in combination with administration of adipose tissue-derived stem cells (ASCs).

1. Preparation of ACSs ASCs were prepared from mouse fat by the following series of operations.
(1) Collagenase was dissolved in Hank's buffer at a concentration of 1 mg / ml, then passed through a 0.45 μm filter and sterilized (collagenase solution). The collagenase solution was stored at 4 ° C until use. Incubated at 37 ° C just prior to use.
(2) C57BL / 6 Cr SLC mice (female, 14 weeks old, Japan SLC Co., Ltd.) were immersed in EtOH after dislocation of the cervical spine.
(3) The epidermis was peeled up and down from the abdomen, the skin was wrapped around the foot well, and adipose tissue was collected. The collected adipose tissue was lightly washed with PBS and then immersed in FD medium (F12: DMEM = 1: 1) on ice.
(4) After removing unnecessary connective tissue, blood vessels, and other tissues (muscles), the fat tissue was shredded with a knife.
(5) The adipose tissue was washed again with PBS.
(6) The weight of the adipose tissue was measured, a double volume collagenase solution was added, and then shaken in a 37 ° C. incubator for 60 minutes.
(7) After adding 10 ml of FD medium, the mass was loosened by pipetting.
(8) After passing through a cell strainer (70 μm mesh), a new centrifuge tube was transferred.
(9) The cell strainer was rinsed with 10 ml of FD medium, and the cells remaining on the cell strainer were collected and added to the centrifuge tube of (8).
(10) The centrifuge tube was centrifuged at 1200 rpm for 5 minutes at room temperature, and then the supernatant was removed.
(11) The sediment was suspended in 10 ml of FD medium.
(12) The operations of (9) to (11) were repeated (three times).
(13) A cell suspension was obtained by suspending the finally obtained sediment in 5 ml of FD medium, and then the number of cells was counted.
(14) Cells were seeded in a culture flask to 2 × 10 ⁵ cells / 25 cm ² and cultured overnight at 37 ° C.
(15) Non-adherent cells were removed by exchanging the medium. Thereafter, the medium was changed every 3 days.
As a result of the above operation, about 5 × 10 ⁶ cells of ASCs could be recovered from 1 g of adipose tissue.

2. Transplantation experiment 2-1. Mouse preparation
A total of 28 C57BL / 6 Cr SLC mice (male, 6 weeks old, Japan SLC Co., Ltd.) were prepared, normal group (3 animals), raw food administration group (6 animals), heparin administration group (7 animals), ASCs The group was divided into an administration group (6 mice) and a heparin ASCs combination administration group (6 mice). The conditions for each group are as follows.
Normal group: A group that does not induce hepatitis by CCl ₄ Saline group (control group): A group that induces hepatitis by CCl ₄ followed by intravenous administration of physiological saline (150 μl) Heparin group: Hepatitis by CCl ₄ After inducing heparin (5 μl Novo-heparin injection 1000 mixed with 145 μl physiological saline) intravenously
ASCs administration group: after inducing hepatitis by CCl _4, ASCs group heparin ASCs combination administering (ASCs prepared above procedure (1 × 10 ⁶⁾ those suspended in physiological saline 150 [mu] l) intravenously administered Group: After inducing hepatitis due to CCl _4, a mixture of heparin and ASCs (ASCs (1 × 10 ⁶ ) prepared in the above procedure) was suspended in 150 μl of physiological saline, and then 5 μl of novo-heparin injection 1000 was added. Group to be administered intravenously

2-2. Test method (1) Hepatitis-inducing mice (saline-administered group, heparin-administered group, ASCs-administered group, heparin-ASCs combined-administered group) were treated with CCl ₄ (0.5 mL / kg of a 10-fold diluted solution of CCl ₄ with olive oil). It was administered intraperitoneally.
(2) Four hours after the administration of CCl ₄ , saline was given to mice in the saline group, heparin was given to mice in the heparin intravenous group, ASCs were given to mice in the ASCs intravenous group, and heparin ASCs were intravenously injected. Heparin ASCs mixture was administered to each group of mice via the tail vein.
(3) Sampling (blood and liver tissue) was performed from each mouse 24 hours after administration of CCl ₄ .
(4) The liver injury markers AST (GOT), ALT (GPT), and LDH were measured using the sampled samples, and the measured values were compared and evaluated between groups. In recent years, GOT (glutamate oxaloacetate transaminase) is called AST (aspartate aminotransferase) and GPT (glutamate pyruvate transaminase) is called ALT (alanine aminotransferase), which is becoming an international standard. LDH is an abbreviation for lactate dehydrogenase.

2-3. Results The measurement results of each liver injury marker are shown in FIG. FIG. 1a is a graph comparing the measured values of AST, b is a graph comparing the measured values of ALT, and c is a graph comparing the measured values of LDH. In each graph, the measurement value of the normal group, the measurement value of the saline administration group, the measurement value of the heparin administration group, the measurement value of the ASCs administration group, and the measurement value of the heparin ASCs combination administration group are shown in order from the left. FIG. 1d is a graph comparing the survival rates of the ASCs administration group and the heparin ASCs combination administration group. FIG. 2 shows the results of comparing and evaluating the measured values of the saline-administered group and the measured values of the heparin ASCs administered group by t-test for each hepatic disorder marker (a: AST value test result, b: ALT value test result, c: Test result of LDH value).

2-4. Discussion Compared with the saline-administered group, a decreasing tendency of the AST value can be confirmed in the heparin ASCs combined administration group (p = 0.052) (FIGS. 1a and 2a). Although the AST value is slightly lower in the ASCs administration group than in the heparin ASCs combination administration group, only 2 mice (out of 6) survived in the ASCs administration group (FIG. 1d).
Compared to the saline-administered group, a decreasing tendency of the ALT value can be confirmed in the heparin ASCs combined administration group (FIGS. 1b and 2b).
Compared to the saline-administered group, a significant decrease in the LDH value can be confirmed in the heparin ASCs combined administration group (p = 0.029) (FIGS. 1c and 2c).
In the ASCs administration group, 4 out of 6 animals developed lung infarction and hepatic infarction, and death was confirmed immediately after transplantation (survival rate 33%), whereas in the heparin ASCs administration group, infarction was observed in any mouse There was no death (survival rate 100%).
As described above, when ASCs and heparin were administered in combination, a high therapeutic effect was obtained. In addition, it has been found that the infarction caused by large doses of ASCs can be prevented by combined use of heparin, that is, if combined with heparin, a large amount of ASCs can be safely administered.

<Acquisition of ASCs by low serum culture>
1. Preparation of sedimented cell population (SVF fraction) from adipose tissue The SVF fraction is prepared from human adipose tissue by the following procedure.
(1) Human adipose tissue (subcutaneous fat or visceral fat) obtained by excision several times (for example, 3 times) in DMEM / F12 solution (medium (Sigma) in which Dulbecco's modified Eagle medium and F12 medium are mixed in equal amounts) ) Wash and remove the attached blood.
(2) Adipose tissue is cut into pieces with a scalpel in a sterile culture dish.
(3) Put adipose tissue in a centrifuge tube and measure its weight.
(4) After adding the required amount of collagenase type1 solution to the above centrifuge tube, the solution is shaken at 37 ° C. for a predetermined time (for example, 1 to 2 hours). For example, the shaking condition is 120 times / min.
(5) Subsequently, a predetermined amount of DMEM / F12 solution is put into a centrifuge tube and pipetted.
(6) The cell suspension after pipetting is filtered through a filter having a pore size of 100 μm.
(7) The obtained filtrate is centrifuged at 1200 rpm for 5 minutes at room temperature. Collect the sediment and use it as the SVF fraction.
In the case of using sucked fat, the operations (1) and (2) can be omitted.

2. Low serum culture of SVF fraction Follow the procedure below to culture the SVF fraction in low serum.
(1) Suspend nucleated cells in the SVF fraction in a low-serum culture medium and seed them in a fibronectin-coated 25 cm flask. The low serum culture solution was prepared as follows (a to e).
(A) DMEM (Nissui Pharmaceutical) 5.7 g, MCDB201 (Sigma) 7 g, L-glutamine (Sigma) 0.35 g, NaHCO ₃ (Sigma Aldrich Japan) 1.2 g, 0.1 mM ascorbic acid (Wako Pure Chemical Industries) 1 ml, antibiotics Dissolve 0.5 ml of the substance (100,000 units / ml penicillin and 100 mg / ml streptomycin) in 980 ml of distilled water.
(B) Adjust the pH to 7.2 with 10N NaOH.
(C) Filter and sterilize.
(D) 10 ml of linoleic acid-albumin (sigma) and 10 ml of 100 × ITS (insulin 10 mg, transferrin 5.5 mg, sodium selenite 5 μg, sigma) are added.
(E) Add 1 μl of 100 μg / ml bFGF (peprotech) (final concentration 10 ng / ml).

(2) Replace the entire medium every 2 days.
(3) After reaching confluence, after washing with 1 mM EDTA-containing PBS, the cells are detached and collected by treatment with 0.05 to 0.25% trypsin solution, and the collected cells are collected at a predetermined density (for example, 8 × 10 ³ cells / cm ^2). ) To seed fibronectin coated plate.
(4) Repeat the above subcultures as necessary.

  According to the present invention, a large amount of ASCs can be safely administered during tissue regeneration. The present invention provides an effective means of overcoming the current problem that there is no choice but to suppress the dose from the viewpoint of safety, although it is desired to administer a large amount of ASCs for the purpose of improving the therapeutic effect, Its clinical significance is great. The present invention can be used, for example, as a medicament for the treatment or prevention of liver damage.

The present invention is not limited to the description of the embodiments and examples of the invention described above. 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.

Measurement results of each liver injury marker. (A) is the graph which compared the measured value of AST, (b) is the graph which compared the measured value of ALT, (c) is the graph which compared the measured value of LDH. In each graph, the measurement value of the normal group, the measurement value of the saline administration group, the measurement value of the heparin administration group, the measurement value of the ASCs administration group, and the measurement value of the heparin ASCs combination administration group are shown in order from the left. (D) is a graph comparing the survival rates of the ASCs administration group and the heparin ASCs combination administration group. ● is the survival rate of the ASCs administration group, and ▲ is the survival rate of the heparin ASCs combination administration group. Results of comparing and evaluating the measured values of the saline-administered group and the measured values of the heparin ASCs administered group by t-test for each liver injury marker. (A) is the test result of the AST value, (b) is the test result of the ALT value, and (c) is the test result of the LDH value.

Claims (15)

  A composition for tissue regeneration comprising a combination of adipose tissue-derived stem cells and heparin.   The composition for tissue regeneration according to claim 1, comprising adipose tissue-derived stem cells and heparin.   The composition for tissue regeneration according to claim 1, which is a kit comprising a first component containing adipose tissue-derived stem cells and a second component containing heparin.   The composition for tissue regeneration according to claim 1, comprising adipose tissue-derived stem cells, and heparin being administered in combination at the time of administration.   The composition for tissue regeneration according to claim 1, comprising heparin, wherein adipose tissue-derived stem cells are administered in combination at the time of administration. The adipose tissue-derived stem cells are
(1) Adherent cells or passage cells thereof included in a sedimented cell population that precipitates when a cell population separated from adipose tissue is centrifuged under conditions of 800 to 1500 rpm for 1 to 10 minutes,
(2) cells grown when the precipitated cell population is cultured under low serum conditions, or (3) cells grown when the cell population separated from adipose tissue is cultured under low serum conditions,
The composition for tissue regeneration according to any one of claims 1 to 5, wherein   The composition for tissue regeneration according to claim 6, wherein the low serum condition is a condition in which a serum concentration in a culture solution is 5% (V / V) or less.   The tissue regeneration according to claim 1, comprising a precipitated cell population and heparin that precipitate when the cell population separated from adipose tissue is centrifuged under conditions of 800 to 1500 rpm for 1 to 10 minutes. Composition.   The composition for tissue regeneration according to any one of claims 1 to 8, wherein the composition is used for treatment or prevention of liver damage.   The composition for tissue regeneration according to claim 9, wherein the liver disorder is hepatitis, cirrhosis, liver cancer or postoperative liver failure.   The composition for tissue regeneration according to any one of claims 1 to 10, wherein the adipose tissue is human adipose tissue. A method of preparing a tissue regeneration composition comprising the following steps:
(1) preparing adipose tissue-derived stem cells and heparin;
(2) A step of mixing the adipose tissue-derived stem cells and the heparin.   Use of adipose tissue-derived stem cells and heparin for producing a composition for tissue regeneration.   A tissue regeneration method comprising administering a therapeutically effective amount of adipose tissue-derived stem cells and heparin to a patient having a tissue for regeneration.   A method for treating or preventing a liver disorder, comprising administering a therapeutically effective amount of adipose tissue-derived stem cells and heparin to a patient suffering from or possibly suffering from a liver disorder.

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