JP2005287479A - Method for extracting tissue stem cell and device using the method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily extracting tissue stem cells, and to provide a device using the method. <P>SOLUTION: The method for obtaining specific tissue stem cells from tissue pieces extracted from a biological tissue with a puncturing needle comprises, in a closed system, the step (1) of passing the tissue pieces through a filter 200-300 μm in pore size to remove a hemocyte component therefrom, the step (2) of cleansing the resultant tissue pieces with an antibiotic-containing phosphate buffer solution, the step (3) of subjecting the resultant tissue pieces to enzyme treatment into a cell suspension, the step (4) of separating the aimed cells from cells of smaller specific gravity by making the lighter cells float, the step (5) of passing a suspension containing the aimed cells through a filter 250-300 μm in pore size again, the step (6) of further passing the suspension through a filter 20-40 μm in pore size, and the step (7) of either centrifuging or passing the suspension through a filter 400-500 nm in pore size to obtain the objective cells from the cell suspension. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a tissue stem cell collection method using cultured cells in fields such as biology and medicine, and an apparatus using the same.

  Regenerative medicine is an epoch-making process that restores and restores the functions of human cells and tissues that have been greatly damaged or lost due to various diseases and traumas that were previously considered impossible or difficult. As a medical treatment, it is currently receiving great attention, and its early practical application is strongly desired. However, ES cells need to overcome some major technical as well as ethical problems for medical applications. On the other hand, there is a possibility that stem cells exist in the constituent cells of all organs and tissues, and it is expected that these stem cells can be identified, freely replicated, and applied.

  Among them, the adipose tissue has been reported that the inventor has stem cells in the adipose tissue and is expected to have a very high regenerative ability to secrete a large amount of protein (Maeda K, et al. Gene May 1997; 190 (2): 227-35, Kazuhisa Maeda et al. Annual Review 2004 Endocrine, Metabolism p15-19). In addition, liposuction itself is one of the most frequently performed operations in the world, and it is an ideal tissue extraction without any complications or pain to the person.

  However, adipose tissue has been extremely difficult to handle after collection, and so far it has been rarely treated from a research and clinical standpoint, and innovative techniques have been strongly desired.

  Under such circumstances, Japanese Patent Application Laid-Open No. 2002-80377 discloses a method of recovering target cells from a cell suspension collected from a living body using a nonwoven fabric and using them as cells for tissue regeneration. The technology is used in systems where cells such as bone marrow are floating, and separation is used in an open system. Therefore, development of simpler and more efficient cell collection methods is strongly desired. It was.

  The present invention has been made with the intention of solving the problems of the prior art as described above. That is, an object of the present invention is to provide a novel tissue stem cell collection method based on an idea completely different from the prior art. Moreover, an object of this invention is to provide the apparatus using it.

  In order to solve the above-mentioned problems, the present inventors have studied and developed from various angles. As a result, surprisingly, (1) a closed system circuit that can efficiently wash out and remove mixed red blood cells by establishing a filter on the syringe itself used in the liposuction method, and (2) the closed system circuit itself is established. It was discovered that stem cells can be separated from matured adipocytes suspended by inversion. The present invention has been completed based on such findings.

That is, when obtaining a specific tissue stem cell from a tissue piece collected from a biological tissue using a puncture needle, (1) a step of removing blood cell components from the tissue piece by passing through a filter having a pore diameter of 250 to 300 μm in a closed system;
(2) a step of washing with a phosphate buffer containing an antibiotic,
(3) A step of subjecting the obtained tissue piece to enzyme treatment to obtain a cell suspension,
(4) a process of separating cells of low specific gravity from the target cells by floating;
(5) a step of passing the target cell suspension again through a filter having a pore size of 250 to 300 μm;
(6) a step of further passing through a filter having a pore size of 8 to 25 μm,
There is provided a tissue stem cell collection method characterized by undergoing centrifugation and / or a step of obtaining a target cell from a cell suspension by passing through a 450-500 nm filter.
The present invention also provides a tissue stem cell collection device using the tissue stem cell collection method.

  Although clinical application of tissue-derived stem cells is a very new field, even if a person who has become accustomed to handling adipose tissue has performed a series of operations in the past, the tissue removed from the operating field should be cleaned. It has been stored in a clean container and carried to a laboratory equipped with a clean bench (CPC is essential in the current Ministry of Health and Welfare's idea) and cell separation has been performed. The prior art required the same level of cleanliness as transporting the heart during a heart transplant. The present invention facilitates the collection of fat from the adipose tissue, enables removal of contaminants such as blood, decomposition of adipocytes, and isolation of adipose tissue-derived stem cells in a single tube. This is an epoch-making way to prevent it from happening at all. If this system is used for cell extraction from tissue in the field of regenerative medicine, which is expected in the future, tissue stem cells can be extracted safely and cleanly. Furthermore, if this system is used, separation in a transfer means or a laboratory is facilitated, and cell separation can be performed at the clinic level.

  The present invention will be described below. Throughout this specification it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  As used herein, “adipocyte” is a general term for fat storage tissues. Among the loose connective tissues, there are particularly fatty ones and subcutaneous fat tissues. Each fat cell is surrounded by lattice fibers, and capillaries are densely distributed between the cells to form a fat body. As used herein, any adipose tissue can be a source. A fat body is an almost constant mass or tufted adipose tissue independent of other tissues, and in vertebrates, it exists in the abdominal cavity in contact with the kidneys and gonads. Often white, yellow or orange.

  As used herein, a “cell” is defined in the same way as the broadest meaning used in the art, and is a structural unit of a tissue of a multicellular organism that is enclosed in a membrane structure that isolates the outside world, Refers to a living organism having self-regenerative ability and having genetic information and its expression mechanism. Any cell can be targeted in the method of the present invention. The number of “cells” used in the present invention can be counted through an optical microscope. When counting through an optical microscope, counting is performed by counting the number of nuclei. The tissue is used as a tissue slice, and hematoxylin-eosin (HE) staining is performed, whereby the extracellular matrix (for example, elastin or collagen) and the nucleus derived from the cells are dyed with a dye. This tissue piece can be examined with an optical microscope, and the number of nuclei per specific area (for example, 200 μm × 200 μm) can be estimated and counted. The cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells). The source of cells can be, for example, a single cell culture, or such as cells from a normally grown transgenic animal embryo, blood, or body tissue, or a normally grown cell line Examples include but are not limited to cell mixtures. Moreover, such a supply source can also be used as a cell as it is.

  The cell used in the present invention may be a cell derived from any organism (for example, larvae, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.) as long as there are adipocytes or their counterparts. Can be used. Preferably, such cells are mammalian (eg, monoporous, marsupial, rodent, winged, winged, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed , Rodents, scales, sea cattle, cetaceans, primates, rodents, rabbits, etc.). In one embodiment, cells derived from primates (eg, chimpanzees, Japanese monkeys, humans), particularly cells derived from humans, are used, but are not limited thereto.

  As used herein, “stem cell” refers to a cell having a self-renewal ability and having pluripotency (ie, “pluripotency”). Stem cells are usually able to regenerate the tissue when the tissue is damaged. As used herein, stem cells can be embryonic stem (ES) cells or tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells or somatic stem cells), but are not limited thereto. In addition, as long as it has the above-mentioned ability, an artificially produced cell (for example, a fusion cell described herein, a reprogrammed cell, etc.) can also be a stem cell. Embryonic stem cells refer to pluripotent stem cells derived from early embryos. Embryonic stem cells were established for the first time in 1981 and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and are being used in regenerative medicine. Unlike embryonic stem cells, tissue stem cells are cells in which the direction of differentiation is limited, are present at specific positions in the tissue, and have an undifferentiated intracellular structure. Therefore, tissue stem cells have a low level of pluripotency. Tissue stem cells have a high nucleus / cytoplasm ratio and poor intracellular organelles. Tissue stem cells generally have pluripotency, have a slow cell cycle, and maintain proliferative ability over the life of the individual. As used herein, stem cells can be tissue stem cells, preferably mesenchymal stem cells, although embryonic stem cells can also be used depending on the situation.

  In the present specification, the term “stem cell” refers to a tissue aggregate containing at least a certain amount of stem cells. Therefore, in this specification, for example, stem cells collected from adipocytes after collagenase treatment (such as stem cells used in the Examples) can be used as stem cells, but not limited thereto.

  When classified by the site of origin, tissue stem cells are classified into, for example, the skin system, digestive system, myeloid system, nervous system, and the like. Examples of skin tissue stem cells include epidermal stem cells and hair follicle stem cells. Examples of digestive tissue stem cells include pancreatic (common) stem cells and hepatic stem cells. Examples of myeloid tissue stem cells include hematopoietic stem cells and mesenchymal stem cells. Examples of nervous system tissue stem cells include neural stem cells and retinal stem cells.

  As used herein, “adipose stem cell” refers to a stem cell derived from an adipose tissue. Some of the methods for isolating such stem cells are known. For example, adipose stem cells can be obtained from WO00 / 53795; WO03 / 022988; WO01 / 62901; Zuk, P. et al. A. , Et al. Tissue Engineering, Vol. 7, 211-228, 2001; Zuk, P .; A. , Et al. , Molecular Biology of the Cell Vol. , 13, 4279-4295, 2002, or the like, or modifications thereof. Specifically, for example, (1) The lipoaspirate is sufficiently washed with physiological saline using a separatory funnel; (2) It is confirmed that the lipoaspirate is sufficiently separated in the upper layer and the physiological saline is sufficiently separated in the lower layer. And discard the lower layer. Repeat this until the saline is almost clear with the naked eye; (3) Add the same amount of 0.075% collagenase / PBS as the lipoaspirate and incubate for 30 minutes at 37 ° C. with good agitation; 4) Add an equal volume of 10% serum-added DMEM to the above sample; (5) Centrifuge the above sample at 1200 g for 10 minutes; (6) Add the PBS to the pellet and suspend it at room temperature as appropriate (eg, (7) The sample can be prepared by suction filtration using a 100 μm caliber mesh; and (8) Centrifugation of the filtrate at 1200 g for 5 minutes. Here, it is within the technical scope of those skilled in the art to scale up or down the protocol according to the adjustment amount.

  Examples of the cell marker include CD4, CD13, CD34, CD36, CD49d, CD71, CD90, CD105, CD117, CD151; or a cell surface selected from the group consisting of CD105, CD73, CD29, CD44 and Sca-1 Examples include, but are not limited to, markers. Alternatively, the surface antigens of mesenchymal stem cells are considered to be CD105 (+), CD73 (+), CD29 (+), CD44 (+), CD14 (−), CD34 (−), CD45 (−). It is understood that cells that exhibit at least any one of these, preferably two or more, more preferably all of these properties are preferred as the cells used in the present invention.

  Stem cells isolated by this system are not only differentiated into multi-lineage cells, but are also expected to be applied to feeder cells in corneal sheet regeneration culture, which is currently being studied for clinical application. This is because mouse fibroblasts were conventionally used as a feeder for corneal cells, but when this system is put to practical use, stem cells or fibroblasts can be easily separated from their own adipose tissue. However, the needs are considered to be extremely high.

  As used herein, “fibroblast” refers to a cell that supplies the fiber component of the supporting tissue and forms an important component of the fibrous connective tissue. Tissue sections have a flat, long outline and often show irregular protrusions. The cytoplasm includes mitochondria, Golgi apparatus, centrosome, small fat globules, etc., but does not show special differentiation. The nucleus is elliptical and often exists in close proximity to the collagen fibers. It is said that fibroblasts isolated from adipose tissue often contain stem cells.

  As used herein, “differentiated cells” refers to cells with specialized functions and morphology (eg, muscle cells, nerve cells, etc.), and unlike stem cells, there is no or almost no pluripotency. . Examples of differentiated cells include epidermal cells, pancreatic parenchymal cells, pancreatic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, neurons, vascular endothelial cells, pigment cells, Examples include smooth muscle cells, fat cells, bone cells, chondrocytes. As used in the present invention, the differentiated cells may be in the form of a population or tissue.

  Any culture solution can be used for the cells of the present invention as long as the cells are maintained or differentiated into desired differentiated cells. Examples of such a culture solution include, but are not limited to, DMEM, P199, MEM, HBSS, Ham's F12, BME, RPMI 1640, MCDB104, MCDB153 (KGM), and mixtures thereof. Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate-2-phosphate, ascorbic acid and its derivatives, glycerophosphate, estrogen and its derivatives, Progesterone and its derivatives, Androgen and its derivatives, aFGF, bFGF, EGF, IGF, TGFβ, ECGF, BMP, PDGF and other growth factors, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, fetal bovine Serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (such as sodium selenite), linolenic acid, 3-isobutyl-1-methyl chloride Demethylating agents such as Nthin and 5-Azancytidine, histone deacetylating agents such as trichostatin, cytokines such as activin, LIF and IL-2 / IL-6, hexamethylenebisacetamide (HMBA), dimethylacetamide (DMA) , Dibutyl cAMP (dbcAMP), dimethyl sulfoxide (DMSO), iododeoxyuridine (IdU), hydroxyurea (HU), cytosine arabinoside (AraC), mitomycin C (MMC), sodium butyrate (NaBu), polybrene, selenium, Cholera toxin and the like may be included as one or a combination thereof.

  As used herein, “in vivo” or “in vivo” refers to the interior of an organism. In a particular context, “in vivo” refers to the location where the target tissue or organ is to be placed.

  As used herein, “in vitro” refers to a state in which a part of a living body is removed or released “in vitro” (eg, in a test tube) for various research purposes. A term that contrasts with in vivo.

  In the present specification, “ex vivo” refers to a series of operations ex vivo when a target cell for gene transfer is extracted from a subject, a therapeutic gene is introduced in vitro, and then returned to the same subject again.

In one embodiment, the conditions for culturing in the regenerative treatment method of the present invention, and the conditions for providing a culture of a desired organ, tissue or cell part used in the present invention or a stem cell capable of differentiating into the cells are the cells. Any of those usually used for culturing tissues or organs can be used. As an example of such culture conditions, for example, the culture conditions can usually use 37 ° C. and 5% CO ₂ . Any medium can be used as the medium to be used. For example, a medium containing DMEM / Ham12 (1: 1), 10% FCS, insulin / colotoxin and the like can be used. Further, if necessary, a culture solution containing differentiation factors (for example, EGF (10 ng / ml) in advance) may be used, but is not limited thereto.

In one preferred embodiment, differentiation factors can be added for culturing in the methods of the invention. Examples of such differentiation factors include DNA demethylating agents (such as 5-azacytidine), histone deacetylating agents (such as trichostatin), nuclear receptor ligands (such as retinoic acid (ATRA), vitamin D ₃ , T3 etc.), cell growth factor (activin, IGF-1, FGF, PDGF, TGF-β, BMP2 / 4 etc.), cytokine (LIF, IL-2, IL-6 etc.) hexamethylenebisacetamide, dimethylacetamide, dibutyl Examples include, but are not limited to, cAMP, dimethylthiol sulfoxide, iododeoxyuridine, hydroxylurea, cytosine arabonoside, mitomycin C, sodium butyrate, aphidicolin, fluorodeoxyuridine, polybrene, sesin and the like. For differentiation into the cornea, a differentiation factor called EGF can be added. Any other differentiation factor described herein can also be used for other differentiated cells.

  The regenerative medicine field is said to reach a scale of several tens of trillions in a few years. Currently, bone marrow is most frequently used as an autologous cell therapy source, but many other human tissues have been named as alternative sources. Unfortunately, however, many of these stem cell sources have 1) limited volume, 2) some painful treatment is required, and 3) the amount of stem cells produced from these sources is itself limited. Therefore, what is required as an ideal source of stem cells is 1) easy culturing, 2) abundant 3) collection without pain, etc. Adipose tissue is considered to be the most powerful tissue that satisfies this condition.

  In Europe and the United States, many liposuctions have been performed due to the spread of cosmetic surgery. Usually, at least a few liters and a few tens of liters of adipose tissue containing human-derived mesenchymal cells are removed. It is one of the most frequent surgeries in the world. Of course, this procedure itself was performed to remove unwanted fat tissue from the human body, but it is actually an ideal method for culturing self-regenerating tissue for cell-based tissue engineering. The adipose-derived stem cells isolated by this method do not require special serum or the like for growth, unlike 1) bone marrow-derived mesenchymal cells. 2) Stable proliferation ability after several passages. 3) Easy to obtain. With local anesthesia and minimal treatment, it can be easily obtained from several liters to several milliliters. 4) Over 1 million cells can usually be obtained with 250 to 500 milliliters of LPA. 5) Furthermore, it has stable multipotency even after gene introduction.

  The marketability of this system is immeasurable due to its simplicity in the rehabilitation medical source established from adipose tissue. In addition, a fat bank that is not a bone marrow bank is born, and it may be possible to easily collect adipose tissue-derived stem cells from the abdomen during childhood and store them in preparation for tissue repair in the middle age. Absent.

  Although this system has been used only for adipose tissue from the research stage, it is also possible to isolate stem cells from other tissues. In addition, if the stem cells do not necessarily have to be cultured immediately, they can be stored as they are. Conversely, in the cosmetic surgery field, a healthy liposuction (in the sense that only fat can be removed without removing stem cells) is performed by reinjecting the extracted fat cells into the fat portion. It is also possible.

  In the present invention, a tissue piece collected from a living tissue is first passed through a filter having a pore size of 200 to 300 μm to remove blood components, other cells, and substances from the tissue piece. At that time, the pore diameter is preferably 200 to 300 μm, preferably 200 to 280 μm, more preferably 250 to 270 μm. If it is 200 μm or less, it is not possible to sufficiently divide what is to be removed, and conversely if it is 300 μm or more, the target cell passes and is not efficient and not preferable. The material of the filter is not particularly limited, and examples thereof include usually used cellulose, Teflon, hydrophilic Teflon, polyethylene terephthalate, and polycarbonate.

  In the present invention, thereafter, the obtained tissue piece is subjected to an enzyme treatment to obtain a cell suspension. The enzyme is not particularly limited, and examples thereof include commonly used proteolytic enzymes such as trypsin, dispase, collagenase, and pepsin. The method for suspending is not particularly limited, and may be followed by a conventional method.

  In the present invention, the cell suspension thus obtained is again passed through a filter having a pore size of 200 to 300 μm to filter out undesired cells. The filter at that time may be the same as described above.

  In the present invention, the cells are further passed through a filter having a pore size of 20 to 40 μm, and undesired cells are filtered out. At that time, the pore diameter is preferably 20 to 40 μm, preferably 25 to 35 μm, more preferably 25 to 30 μm. If it is 20 μm or less, the target cell cannot be passed, and if it is 40 μm or more, the non-target cell passes and is not preferable because it is not efficient. The material of the filter is not particularly limited, and examples thereof include usually used cellulose, Teflon, hydrophilic Teflon, polyethylene terephthalate, and polycarbonate.

  In the present invention, target cells are obtained from the cell suspension thus obtained. At that time, the method for concentrating the cells is not particularly limited, and examples thereof include a method of centrifuging, a method of passing through a 400 to 500 nm filter, and the like.

  The present invention also shows a tissue stem cell collection device according to the method described above. The device comprises a collection device and a separation device, wherein the device is preferably a closed system.

  The collection device is not particularly limited in its form and mechanism as long as it can collect tissue from a biological tissue. For example, a syringe like a puncture needle for collecting a tissue piece from a biological tissue and a filter having a pore diameter of 200 to 300 μm is attached. The apparatus is preferable because it is simple and easy to carry. The material of the apparatus at that time is not particularly limited, and examples thereof include commonly used polyethylene, polypropylene, polyethylene terephthalate, and polycarbonate.

  The separation device of the present invention is not particularly limited as long as the target cells can be recovered, but a centrifuge-like device is equipped with a pore diameter 200-300 μm filter on the opening side, and a pore diameter 20-40 μm below it. A filter-mounted one is preferable because it is simple and easy to carry. The material of the apparatus at that time is not particularly limited, and examples thereof include commonly used polyethylene, polypropylene, polyethylene terephthalate, and polycarbonate.

  In the separation apparatus of the present invention, a filter having a pore diameter of 400 to 500 nm may be further attached to the lower part of the above-mentioned pore diameter 20 to 40 μm filter in order to collect target cells. At that time, the pore diameter is preferably 400 to 500 μm, preferably 420 to 480 nm, and more preferably 450 to 470 nm. If the thickness is 400 nm or less, it is not possible to sufficiently separate what is desired to be removed. Conversely, if the thickness is 500 nm or more, the target cell passes and is not efficient and is not preferable. The material of the filter is not particularly limited, and examples thereof include usually used cellulose, Teflon, hydrophilic Teflon, polyethylene terephthalate, and polycarbonate.

  By using the technique shown in the present invention, tissue stem cells can be easily obtained from human biological tissue. For example, if human adipose tissue is targeted, adipose tissue stem cells that can differentiate into various cells can be obtained. The stem cells thus obtained are extremely useful as cells for cell gene therapy in which genes are introduced and returned to the body.

  Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

  The reagents used in the following examples were obtained from Wako Pure Chemicals and Sigma unless otherwise specified. When human subjects were used, experiments were conducted after obtaining prior consent in accordance with the standards of the Ministry of Health, Labor and Welfare.

(Adjustment of cells including tissue stem cells)
In this example, cells were first prepared from adipose tissue from humans who gave consent to this experiment. After local anesthesia (using a solution obtained by diluting 1% E xylocaine 4 times with saline), approximately 20 cc of physiological saline was placed in a Terumo 50 cc syringe equipped with a 14G needle, and approximately 20 cc of liposuction was performed. .

  As shown in FIG. 1, the collected tissue piece was passed through a 250 μm filter while maintaining the cleanness in an open biohazard cabinet, thereby removing the liquid component from the tissue piece. The obtained product was thoroughly washed with physiological saline. This was repeated until the physiological saline was almost transparent with the naked eye. In this experimental example, the test was performed seven times.

  Thereafter, the same amount of 0.075% collagenase / PBS (Gibco) was added to the obtained tissue piece, and the mixture was incubated at 37 ° C. with good agitation for 1 hour. The suspension was again passed through a filter having a pore size of 250 μm. It was.

  The filtrate was centrifuged at room temperature and the supernatant was discarded. Further, DMEM was added to the precipitate, suspended with a pipette, passed through a filter with a pore size of 25 μm, and centrifuged to obtain a target tissue stem cell from the cell suspension.

(Differentiation of cells including tissue stem cells)
Next, the cells in the cells prepared in Example 1 were first cultured, subcultured, and then differentiated into mature adipocytes. Specifically, cells containing tissue stem cells grown to confluence were treated with an adipocyte differentiation medium every two days. On the first day of differentiation and on the second day, the cells were treated with 10% CCS-DMEM medium containing 0.5 mM IBMX, 1 μM dexamethasone, 1 μM triglitazone, 5 μg / ml insulin. On the 4th day, the cells were treated with 10% CCS-DMEM medium containing triglitazone and insulin, and after the 6th day, they were treated with 10% CCS-DMEM medium containing 500 ng / ml insulin. The obtained results are shown in FIG.

(Differentiation of cells including tissue stem cells into cells other than fat)
The cell group used in Example 1 was confirmed to differentiate into cells other than fat cells. For differentiation into neurons, 1 mM beta-mercaptoethanol was added to DMEM medium supplemented with 20% serum and cultured for 24 hours. The next day, after washing twice with PBS, 5 mM beta-mercaptoethanol was added to serum-free DMEM medium. (Woodbury et al., J Neurosci Res 61; 364-370, 2000).
For differentiation into vascular endothelium, the cells obtained in Example 1 were selected with an anti-PECAM-1 antibody and cultured in a medium containing VEGF. When stem cells obtained from adipose tissue were cultured on a collagen-coated culture dish in a medium supplemented with VEGF, FGF2 and EGF, a part of this cell group differentiated into endothelial cells. According to another report, 30% of this cell group has the characteristics of CD34 positive cells, that is, hematopoietic stem cells, and can differentiate into α-smooth muscle actin positive cells, which are markers of smooth muscle cells. This is considered to be a group of cells that can be mobilized for revascularization. In addition, the secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) from this cell group is extremely high as compared with vascular endothelium and smooth muscle cells. It was also revealed that cell proliferation and migration were significantly promoted. It has been recognized that the contribution of endothelial growth factor secretion from mesenchymal stem cells is important as a mechanism of angiogenesis using bone marrow cells in recent years, and this cell group can also function as so-called support cells. Suggests that. The obtained results are shown in FIG.

(Gene transfer into cells including tissue stem cells)
In addition, the cells of Example 1 were successfully introduced using a non-viral vector HVJ-E (Sendai virus envelope vector, Mol Ther. 2001). In the collection and isolation device using the syringe according to the present invention, it is also possible to perform a gene introduction kit using HVJ-E (such as GENOME-ONE manufactured by Ishihara Sangyo Co., Ltd.) in the final collection system.

  However, HVJ-E has been developed on the premise of clinical application to humans, and because of its low toxicity, there has been no report of introduction efficiency, particularly in vivo efficacy. The present invention proposes that after in vitro gene introduction into adipose stem cells, it is possible to inject self-derived cells that are not immune-rejected in vivo, and to compensate for the drawbacks of HVJ-E itself. It is valid.

  If the method described in the present invention is used to extract cells from tissues in the field of regenerative medicine, tissue stem cells can be extracted safely and cleanly. Furthermore, if this system is used, separation in a transfer means or a laboratory is facilitated, and cell separation can be performed at the clinic level. Therefore, the present invention is extremely useful in the fields of medicine, biology and the like.

FIG. 6 is a diagram schematically showing the operation shown in the first embodiment. It is a figure which shows the fat cell, the vascular endothelial cell, and the nerve cell which were isolated by the method according to this invention shown in Example 2, 3, and contain the adipose tissue stem cell, and differentiation induction from them.

Claims (10)

When obtaining specific tissue stem cells from a tissue piece collected from a living tissue using a puncture needle, (1) a step of removing blood cell components from the tissue piece by passing through a filter having a pore diameter of 200 to 300 μm in a closed system;
(2) a step of washing with a phosphate buffer containing an antibiotic,
(3) A step of subjecting the obtained tissue piece to enzyme treatment to obtain a cell suspension,
(4) a process of separating cells of low specific gravity from the target cells by floating;
(5) a step of passing the target cell suspension again through a filter having a pore size of 250 to 300 μm;
(6) a step of passing through a filter having a pore size of 20 to 40 μm,
(7) Step of obtaining the target cells from the cell suspension by centrifugation and / or passing through a 400-500 nm filter,
A tissue stem cell collection method characterized by passing through. The tissue stem cell collection method according to claim 1, wherein the living tissue is adipose tissue, liver, or muscle. The method for collecting tissue stem cells according to any one of claims 1 and 2, wherein the specific tissue stem cells are adipose tissue stem cells. The method for collecting tissue stem cells according to any one of claims 1 to 3, wherein the cells are human. A tissue stem cell collection device according to the tissue stem cell collection method of claim 1. The tissue stem cell collection device according to claim 5, wherein the device comprises a collection device and a separation device. The tissue stem cell collection device according to claim 6, wherein the collection device is a syringe-like device equipped with a puncture needle for collecting a tissue piece from a living tissue and a fine pore diameter 200-300 µm filter. The separation device is a filter having a pore diameter of 200 to 300 μm attached to the opening side of the centrifugal separator, a pore diameter of 20 to 40 μm attached to the lower part thereof, and a pore diameter of 400 to 500 nm filter attached to the lower part thereof. The tissue stem cell collection device according to claim 6. The tissue stem cell collection device according to any one of claims 5 to 8, which is intended to collect adipose tissue stem cells from adipose cells of a living tissue. The tissue stem cell collection device according to any one of claims 5 to 9, wherein the cells are human.

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