JP2012100662A - Cell proliferation method, and medicine for repairing and regenerating tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly and massively proliferate cells in vitro, which have been collected from a living body and to provide a safe and effective medicine for repairing and regenerating the tissue of the living body.SOLUTION: The method, for culturing and proliferating cells in a sample collected from a living body in a culture medium, which contains the same kind of serum and has been tested to be negative to a serum cancer marker and/or an infection factor, controls the amount of an anticoagulant (for example, heparin, a heparin derivative or their salts) added to the collected sample to <5 U/mL based on the volume of the sample, or controls the amount of the anticoagulant in the culture medium on the start of the culture to <0.5 U/mL.

Description

  The present invention relates to a method for rapidly and massively proliferating cells collected from a living body in vitro, a cell proliferated by such a method, a medicine for repairing and regenerating a living tissue containing said cell, and a method for producing such a medicine. On how to do. This method is particularly suitable for autologous transplantation of mesenchymal stem cells and can be applied especially for the repair and regeneration of nervous system tissue.

  Traditionally, it has been thought that it is very difficult to recover the function of damaged nerve tissue, but based on the recent discovery of neural stem cells that retain self-proliferation and pluripotency in the mature brain. In the central nervous system, research on regenerative medicine is being conducted energetically. Cell therapy that replaces damaged cells is considered to be the most practical regenerative medicine using stem cells. In cell therapy, cells provided from a donor (hereinafter referred to as donor cells) are cultured, expanded, and / or differentiated in vitro and administered to the recipient's body in an appropriate form. Replenish damaged tissue cells.

  For cranial nerve diseases, attempts have been made in ischemia models, trauma models, etc., to extract cells from tissues that have the ability to differentiate into nervous system cells, culture them, and transplant them into individuals. For example, the present inventors have already found that a cell fraction containing cells that can differentiate into nerve cells exists in bone marrow cells, and further, when these cells are transplanted into a rat spinal cord demyelination model, demyelination occurs. It has been confirmed that the regenerated myelons are remyelinated (Patent Document 1).

  When used for the treatment of a disease, particularly for the treatment of a disease having acute symptoms such as ischemic brain disease due to cerebral infarction, it is important to proliferate donor cells rapidly and in large quantities. In order to proliferate cells in vitro, various attempts have been made to improve cell survival rate and increase proliferation rate.

  For example, in order to improve the cell growth rate, a medium supplemented with various growth promoting substances is used. For example, Patent Document 2 discloses that leukocyte inhibitory factors increase the rate of cell growth, and Patent Document 3 discloses a medium containing recombinant human serum albumin for growing hematopoietic cells. ing. Patent Document 4 discloses a method of culturing mesenchymal stem cells in a medium containing vitamin C and basic fibroblast growth factor. The use of other growth factors (eg, epidermal growth factor, nerve cell growth factor, hepatocyte growth factor, thrombopoietin, interleukin, etc.) is also known.

  Culture substrates that further increase the cell growth rate have been developed. For example, Patent Document 5 discloses a method of culturing mesenchymal stem cells on a basement membrane extracellular matrix.

  Serum is also commonly used as a substance that promotes growth. Conventionally, in stem cell culture, a medium supplemented with heterogeneous animal serum such as fetal bovine serum (FBS) of about 10 to 20% as a cell growth factor has been widely used. However, animal serum such as FBS has a large compositional difference between lots, and there is a problem of contamination with pathogens such as viruses and prions.

  In order to cope with such a problem, a medium not containing serum has also been developed (see, for example, Patent Document 2, Patent Document 3 and the like). However, in the current situation, it is difficult to obtain the same growth as the serum-added medium in the culture in the serum-free medium.

  On the other hand, in an attempt to use human serum, since it is difficult to use fetal serum from an ethical viewpoint, adult human serum is used (see, for example, Patent Documents 6 to 8). An advantage of using adult serum is that it is possible to use the serum of the same individual as the individual from which the donor cells were collected, which is very preferable from the viewpoint of compatibility and safety.

  A problem when using adult serum is, for example, low growth-promoting activity when compared with FBS. Adult serum alone does not show sufficient cell growth promoting activity, and in order to obtain the same level of growth as FBS, it is indispensable to add FBS (Patent Document 6) or other growth factors. Patent Document 7). However, even when an effect equivalent to that of FBS is obtained by adding growth factors or the like, rapid and large-scale growth that can cope with the treatment of the acute phase disease as described above has not been obtained. .

  On the other hand, in the conventional method of culturing and proliferating cells collected from a living body, heparin is added to prevent blood coagulation when a tissue or cells containing blood components are collected from a donor (for example, patents). (See Document 9, Non-Patent Document 1, Non-Patent Document 2, and the like). In a typical case (for example, bone marrow cell collection in normal bone marrow transplantation), the amount of heparin with respect to the volume of cell fluid is about several tens of U / mL (about 20 to 40 U / mL). 8 discloses adding a heparin / buffer solution containing heparin in the range of about 5 to 15 U / mL to the bone marrow fluid. Patent Document 2 discloses a method in which heparin is further contained in the culture medium.

  Heparin is also used as a growth aid in addition to the above-described use for preventing blood coagulation. For example, in Patent Document 4, heparin includes basic fibroblast growth factor (bFGF), its receptor, It has been disclosed that it has an effect of increasing the affinity of. Patent Document 10 discloses a modified form of sulfated glycosaminoglycan containing heparin as a neural stem cell growth aid. However, even in these methods using heparin, it is still impossible to achieve a sufficiently rapid and large growth rate.

  By the way, when the living body is damaged, it has a mechanism for autonomously repairing the damaged part, and if it is damaged to some extent, it can be repaired without leaving a functional failure. . However, if the degree of damage is large, the underlying repair mechanism alone may not be sufficient, the recovery may be prolonged, or the damage may not be completely repaired, leaving a functional failure. Conventionally, it has been thought that it is very difficult to repair such damaged biological tissues, particularly nerve tissues, etc., but recently, the damaged cells were triggered by the discovery of multipotent stem cells. Attempts have been made to compensate for this with stem cells. For example, Patent Document 11 shows that when a mesenchymal stem cell was administered to a cerebral infarction model rat in the acute phase of the disease (3 to 24 hours after induction of an ischemic state), a remarkable therapeutic effect was obtained. Are listed.

  However, there is still no report on an effective method for recovering the lost function after the subacute period after the injury has passed and the damaged site has been stabilized to some extent.

International Publication No. WO02 / 00849A1 Pamphlet Special Table 2002-518990 Japanese Patent Laid-Open No. 2005-204539 JP 2006-136281 JP JP 2003-52360 A Japanese Patent Laid-Open No. 10-179148 JP 2003-235548 JP 2006-55106 A International Publication No. WO01 / 48147A1 Pamphlet JP 2005-218308 A International Publication No. WO2005 / 007176 Pamphlet

Fumiaki Takahisa, "Bone marrow transplantation manual", first edition, Ichigai, Inc., 1996, p. 86 Miura Tadashi, “Blood Stem Cell Culture Method”, 2nd revised edition, 1 edition, Ichigai, 1989, p. 38

  Therefore, the object of the present invention is to solve the problems of the prior art when proliferating cells in vitro for cell transplantation, obtain a higher proliferation rate than the conventional method, and rapidly and proliferate cells in large quantities There is. It is another object of the present invention to provide a highly practical tissue repair aid that has a therapeutic effect even when administered after the subacute phase of injury using expanded cells.

  In order to solve the above-mentioned problems, the present inventors conducted research while focusing on heparin, which is usually used for the purpose of preventing blood coagulation, while verifying various conditions and procedures in cell culture. The finding that heparin has a significant inhibitory effect on cell proliferation was obtained. As a result of further research, if cells are cultured under conditions that do not come into contact with heparin, cells that have excellent growth efficiency (fast growth) even in a culture method using allogeneic serum instead of FBS In addition, the present inventors have found that the obtained cells are safe with high differentiation ability, and have completed the present invention.

  That is, the present invention is a method of culturing and proliferating cells in a sample collected from a living body in a medium, and culturing the cells in a medium containing allogeneic serum in a state where they are not substantially in contact with an anticoagulant. On how to do. Here, the allogeneic serum is preferably tested negative for serum cancer markers and / or infectious agents, and is preferably the subject's own autoserum from which the cells were collected.

  Here, as serum cancer markers, for example, ferritin, CEA, AFP, BFP, CA125, CA15-3, CA19-9, CA72-4, STN, DUPAN-2, SLX, ST-439, SPAN-1, SCC , PSA, G-seminoprotein, TPA, Shifura, PAP, NSE, C-peptide, PIVKA, Pro-GRP, HCGβ, elastase, β2 microglobulin, S-NTX, anti-p53 antibody, HER2, etc. . Examples of infectious agents include HIV, ATL, HB, HC, syphilis, and human parvovirus B19.

  The present invention also relates to the above method, wherein the amount of the anticoagulant added to the collected sample is less than 5 U / mL with respect to the volume of the sample.

  Furthermore, the present invention relates to the method, wherein the amount of the anticoagulant in the medium at the start of culture is less than 0.5 U / mL.

In addition, the present invention relates to the cell proliferation method, wherein the anticoagulant is heparin, a heparin derivative or a salt thereof.
The present invention relates to the method, wherein the cells are grown in a medium containing serum.

  The present invention also relates to the method, wherein the cells are grown in a medium containing the serum of an animal individual of the same species as the animal from which the cells are derived.

Furthermore, the present invention relates to the method, wherein the cells are grown in a medium containing autologous serum.
The present invention also relates to the method, wherein the serum content in the medium is 1 to 20% by volume.
The present invention relates to the method, wherein stem cells are grown.

The present invention also relates to the method, wherein mesenchymal stem cells are grown.
Furthermore, the present invention relates to the method, wherein human cells are grown.
The present invention relates to the method, wherein the stem cells are grown in an undifferentiated state.

The present invention also relates to the above-mentioned method, wherein the subculture is performed when the density of mesenchymal stem cells in the medium reaches 5,500 cells / cm 2 or more.
Furthermore, the present invention relates to the method, wherein the medium is changed at least once a week.

  The present invention relates to the method, wherein the subculture is repeated until the total number of cells reaches 100,000,000 or more.

  The present invention relates to an isolated cultured human cell, which is a cell cultured by any one of the methods described above, and does not express CD24.

  The present invention also provides a cell cultured by any of the above methods, wherein at least 90% of the positive CD antigen group described in Table 1 is expressed and at least 90% of the negative CD antigen group is expressed. It also relates to isolated human cultured cells, characterized in that they are not.

  The cells further include various cancer-related genes whose expression in humans is not preferable, such as EWI-FLI-1, FUS-CHOP, EWS-ATF1, SYT-SSX1, PDGFA, FLI-1, FEV, ATF-1, WT1, NR4A3, CHOP / DDIT3, FUS / TLS, BBF2H7, CHOP, MDM2, CDK4, HGFR, c-met, PDGFα, HGF, GFRA1, FASN, HMGCR, RGS2, PPARγ, YAP, BIRC2, lumican, caldesmon, ALCAM, Jam-2, Jam-3, cadherin II, DKK1, Wnt, Nucleostemin, Neurofibromin, RB, CDK4, p16, MYCN, telomere, hTERT, ALT, Ras, TK-R, CD90, CD105, CD133, VEGFR2, CD99, ets , ERG, ETV1, FEV, ETV4, MYC, EAT-2, MMP-3, FRINGE, ID2, CCND1, TGFBR2, CDKNIA, p57, p19, p16, p53, IGF-1, c-myc, p21, cyclin D1, None of the genes of p21 is abnormally expressed. Abnormal expression means expression that is significantly higher than the expression level of healthy subjects.

The present invention relates to a method for producing a medicament for tissue repair / regeneration using the cells.
The present invention also relates to a tissue repair / regeneration medicament containing the cells.

  In the present invention, the cells are mesenchymal stem cells, and are administered intravenously, lumbar puncturely, intracerebrally, intracerebroventricularly, locally or intraarterially, to assist in repairing the damaged site, or to add The present invention relates to the medicine for assisting in repairing a corridor site due to age. The tissue is not particularly limited, for example, nervous system tissue including brain and spinal cord, kidney, pancreas, liver, intestine, stomach, digestive organs, lung, heart, spleen, blood vessels, blood, skin, bone, cartilage, teeth, And prostate. The cell is preferably an autologous cell.

  In a preferred embodiment, the tissue repair / regeneration medicament of the present invention is administered after the subacute phase of a disease or disorder, and assists the repair of the damaged site by cytokine secretion, angiogenesis and / or nerve regeneration.

  In one embodiment, the damaged site is the kidney and the medicament promotes repair of the damaged site by improving BUN and / or creatinine levels.

  In one embodiment, the damaged site is the pancreas and the medicament promotes repair of the damaged site by improving blood glucose level, blood Glu A1 concentration and / or blood HbA1C concentration.

  In one embodiment, the damaged site is the heart, and the medicament promotes repair of the damaged site by improving blood prostaglandin D synthase concentration and / or blood homocysteine concentration.

  In one embodiment, the damaged site is liver, and the medicament promotes repair of the damaged site by improving GOT value, GPT value and / or γ-GTP value.

  In one embodiment, the damaged site is the brain, and the medicament may improve a SLTA value as an index of aphasia and a WAIS-R value as an index of intelligence recovery, and treat aphasia and dementia.

  In one embodiment, the damaged site is the prostate and the medicament facilitates repair of the damaged site by improving PSA levels.

  Specific examples of diseases and disorders targeted by the tissue repair / regeneration pharmaceutical of the present invention include renal disorders, liver disorders, pancreatic disorders including diabetes, enlarged prostate, hyperlipidemia, aphasia and dementia. Examples include, but are not limited to, higher brain dysfunction, post-resuscitation encephalopathy, heart disease, spinal cord injury, and the like.

  The present invention also relates to a kit for preparing the cell of the present invention, comprising a container filled with a medium characterized in that the anticoagulant is less than 0.5 U / mL. The kit may contain other reagents and equipment necessary for cell preparation, and the anticoagulant (if included) is, for example, heparin.

  Furthermore, the present invention includes a step of culturing cells isolated from a subject according to the method of the present invention, a step of examining the expression of cancer-related genes for the cells obtained in the step, and the test confirming safety. And cell therapy for tissue repair and regeneration, including the step of administering the obtained cells to the subject. Here, examples of the cancer-related gene to be examined include those described above.

  In a preferred embodiment, in the cell therapy, the tissue is a nervous system tissue, the cell is an autologous mesenchymal stem cell, and is administered intravenously, lumbar puncture administration, intracerebral administration, intracerebroventricular administration, or intraarterial administration. For the treatment of ischemic neurological diseases.

  The present invention adds a very small amount of an anticoagulant that has been conventionally considered to be essential or useful for cell growth, or cultures without substantially adding an anticoagulant, thereby producing a heterogeneous serum (FBS). When using allogeneic serum instead of (for example, the subject's own serum from which the cells were collected), a significant improvement in the growth rate is achieved, and cells with excellent growth efficiency (fast growth) can be obtained. , It has a surprising effect that cannot be predicted at all from conventional common sense. Compared with the conventional culture method using FBS, the obtained cells are remarkably excellent in safety and differentiation ability in the culture method not containing autoserum.

  The medicament of the present invention has a therapeutic effect even when administered after the subacute phase of a disease whose efficacy has not been envisaged conventionally, so it is necessary to prepare cells to be administered before onset However, since it is sufficient to collect and culture from the subject after onset, the burden on the subject can be greatly reduced. In addition, since the medicament of the present invention can assist in the repair of any damaged site in the body by intravenous injection, there is less invasiveness to the subject, and there is no need for surgery, and it can be administered in a normal treatment room, etc. The burden on the provider and medical costs can be reduced. Furthermore, since the medicament of the present invention has a repair assisting effect on a plurality of different tissues, it has a wide range of applications, and in particular, can effectively treat a subject having a plurality of different damaged sites simultaneously.

FIG. 1 is a graph showing the proliferation of human mesenchymal stem cells when the amount of heparin added to the bone marrow fluid is 0.1 U / mL (♦) or 267 U / mL (■). FIG. 2 is a graph showing the number of cells 4 days after the start of culture when a very small amount (left) or 2 mL (right) of heparin is added to the bone marrow fluid. By adding a very small amount of heparin to be added, the number of cells increased about 40 times. FIG. 3 is a graph showing proliferation of mesenchymal stem cells in a medium supplemented with human adult serum (♦) or FBS (■) with an addition amount of heparin of 0.1 U / mL. FIG. 4 is a graph showing the growth rate when rat mesenchymal stem cells are cultured in a medium containing 10% FBS with various amounts of heparin added (in order from the top, 1 U / mL, 10 U / mL, 100 U / mL, 1000 U / mL). FIG. 5 shows the case where rat mesenchymal stem cells are cultured in a medium supplemented with heparin (♦), or when the same amount of heparin is added to bone marrow fluid and then cultured in the medium (■). It is a graph which shows a proliferation rate. FIG. 6 is a graph showing the growth of human mesenchymal stem cells in αMEM medium supplemented with either human adult autologous serum (solid line) or FBS (dotted line) with an addition amount of heparin of 0.1 U / mL. is there. FIG. 7 is a graph showing the proliferation of human mesenchymal stem cells when the amount of heparin added is less than 0.1 U / mL and glutamine is added (♦) or when glutamine is not added (■). FIG. 8 is a graph showing the proliferation rate when rat mesenchymal stem cells after heparin-untreated (♦), heparin-treated (■), and heparin + protamine-treated (▲) are cultured in a medium containing FBS. FIG. 9 is a diagram showing a therapeutic mechanism of the medicine of the present invention. The vertical axis represents the symptom severity, the horizontal axis represents the elapsed time from the onset, and the arrow represents the administration time of the medicament of the present invention. On the right side from the arrow, the upper curve shows the change of the symptom of the subject who received the administration agent, and the lower curve shows the change of the symptom of the subject who did not receive the administration. FIG. 10 is a brain MRI image of a patient with ischemic neurological disease before (left) and 2 weeks after administration (right) of the medicament of the present invention. The damaged site is shown as a white part. FIG. 11 shows cerebral infarction levels (NIHSS: National Institutes of Health Stroke Scale (●), JSS: Stroke Scale (■), MRS: Correction, before and after administration of the pharmaceutical agent of the present invention to patients with ischemic neurological diseases. It is a graph which shows transition of Rankine scale (▲). Arrows indicate the time of cell administration. FIG. 12 is a thermographic image of a patient with ischemic neurological disease before administration (left) and 1 week after administration (right) of the medicament of the present invention. The dark area showing high temperature is remarkably reduced one week after administration. FIG. 13 is a graph showing changes in blood glucose level (BS: ●), blood Glu A1 concentration (■), and blood HbA1C concentration (▲) of diabetic patients before and after administration of the pharmaceutical of the present invention. . The vertical axis shows mg / dl on the left, the right shows%, and the horizontal axis shows the number of days when the administration day is 0 day. Arrows indicate the time of administration of cells (medicine of the present invention). FIG. 14 is a graph showing changes in PSA values of patients with benign prostatic hyperplasia before and after administration of the medicament of the present invention. Arrows indicate the time of administration of cells (medicine of the present invention). FIG. 15 is a graph showing changes in γ-GTP values (left), GOT (right ◆), and GPT (right ■) of patients with hepatic disorder before and after administration of the medicament of the present invention. Arrows indicate the time of administration of cells (medicine of the present invention). FIG. 16 is a graph showing the transition of β2-microglobulin value in patients with renal disorder before and after administration of the medicament of the present invention. Arrows indicate the time of administration of cells (medicine of the present invention). FIG. 17 is a graph showing the transition of neutral fat in hyperlipidemic patients before and after administration of the medicament of the present invention. Arrows indicate the time of administration of cells (medicine of the present invention). FIG. 18 is a graph showing changes in SLTA test results and WAIS-R test results of patients with higher brain dysfunction (dementia and aphasia) before and after administration of the medicament of the present invention. FIG. 19 shows the results of comparing the therapeutic effects of the administration of the pharmaceutical agent of the present invention in the rat cerebral infarction model in terms of MRI and angiogenesis (from the left to (i) non-transplanted group, (ii) Cells (1.0 × 106) IV group, (iii) Angiopoietin gene-introduced cells (1.0 × 106) IV group, (iv) VEGF gene-introduced cells (1.0 × 106) IV group, (v) Angiopoietin and VEGF Gene-introduced cells (1.0 × 106) intravenous group: * p <0.05, ** p <0.01). FIG. 20 shows the results of comparison of the therapeutic effect of the administration of the pharmaceutical agent of the present invention in the rat cerebral infarction model in each group from the behavioral side ((i) non-transplanted group, (ii) cells ( 1.0 × 106) IV group, (iii) Angiopoietin gene-introduced cells (1.0 × 106) IV group, (iv) VEGF gene-introduced cells (1.0 × 106) IV group, (v) Angiopoietin and VEGF gene-introduced Cells (1.0 × 10 6) intravenous injection group: * p <0.05, ** p <0.01). FIG. 21 shows the results of evaluation of the therapeutic effect of administration of the medicament of the present invention in a rat cardiopulmonary arrest model (post-resuscitation encephalopathy) by the number of Tunnel positive cells (A) and the number of nerve cells (B) (left: control group, Right: Cell administration group: * p <0.05). FIG. 22 shows the results of evaluation of the therapeutic effect of the administration of the medicament of the present invention in a rat cardiopulmonary arrest model using the Morris water maze test (* p <0.05). FIG. 23 is a graph in which recovery of motor function of spinal cord injury model rats transplanted with human mesenchymal stem cells according to the present invention was evaluated by a treadmill test (in the figure, 6 hours, 1 day, and 3 days from the left). 7 and 14 days after transplantation).

  This specification is based on Japanese Patent Application No. 2007-235436 (filed on September 11, 2007), Japanese Patent Application No. 2007-236499 (filed on September 12, 2007), and Japanese Patent Application No. 2007-267211, which are the basis of the priority of the present application. No. (filed Oct. 12, 2007), Japanese Patent Application No. 2007-278049 (filed Oct. 25, 2007), and Japanese Patent Application No. 2007-278083 (filed Oct. 25, 2007) Is included.

Hereinafter, the cell growth method according to the present invention will be described in detail.
The method of the present invention is a method in which cells in a sample collected from a living body are cultured and grown in a medium, and the cells are not substantially contacted with an anticoagulant in a medium containing allogeneic serum. It is characterized by culturing. The allogeneic serum is preferably tested negative for serum cancer markers and / or infectious agents, and is preferably the subject's own autoserum from which the cells were collected.

  Examples of serum cancer markers to be tested in the present invention include ferritin, CEA, AFP, BFP, CA125, CA15-3, CA19-9, CA72-4, STN, DUPAN-2, SLX, ST-439, SPAN- 1, SCC, PSA, G-seminoprotein, TPA, Shifura, PAP, NSE, C-peptide, PIVKA, Pro-GRP, HCGβ, elastase, β2 microglobulin, S-NTX, anti-p53 antibody, HER2 Can do. Examples of infectious agents include HIV, ATL, HB, HC, syphilis, and human parvovirus B19.

  The sample used in the present invention is a body fluid and / or tissue having proliferative capacity and / or containing cells useful for tissue repair / regeneration, such as bone marrow fluid, blood (peripheral blood or Umbilical cord blood) or body fluid such as lymph, muscle tissue, bone tissue, skin, lymphoid tissue, vascular or digestive organ tissue, or embryo (except human embryo). A sample collected from a living body is processed as it is or as needed (for example, removal of unnecessary components, purification of a specific cell fraction, enzyme treatment, etc.), and the method according to the present invention is applied. Provide.

  As used herein, “cells are not substantially in contact with an anticoagulant” means that the amount of anticoagulant used is substantially reduced at any point in time from the collection of cells to the entire culture period. For example, an anticoagulant is added to such an extent that the inner wall of a cell collection vessel (such as a blood collection tube) is wetted with an anticoagulant solution, or not added at all, or in the sample at the start of culture. It means a state obtained when the anticoagulant is substantially removed.

  In order to obtain cell growth more rapidly and in large quantities, it is preferable that the amount of anticoagulant added when collecting a sample is small, and the collected cells are collected immediately after collection (for example, 30 to avoid clotting). Within a minute). More preferably, the cells are not substantially contacted with the anticoagulant. By the above operation, a surprising growth rate of about 3 to 100 times the conventional one can be obtained.

  In a preferred embodiment of the present invention, the amount of the anticoagulant added to a sample collected from a living body (that is, added in advance to a blood collection tube in which the collected sample is stored) is 5 U with respect to the volume of the sample. / ML, preferably less than 2 U / mL, more preferably less than 0.2 U / mL.

  In another preferred embodiment of the present invention, when culturing cells in a sample collected from a living body, the amount of the anticoagulant present in the medium is less than 0.5 U / mL with respect to the volume of the medium, Preferably it is less than 0.2 U / mL, most preferably less than 0.02 U / mL. More specifically, the anticoagulant administered in advance to the blood collection tube for sampling so that the amount of the anticoagulant at the start of culture is less than 0.5 U / mL with respect to the volume of the medium. Reduce the amount and / or adjust the amount of anticoagulant added to the medium.

  In the present specification, the term “anticoagulant” means that when present in a body fluid or a medium, it binds to the cell surface and interacts with a protein having an anticoagulant action present in an extracellular matrix, And extracellular matrix, a substance that inhibits adhesion between cells or between a cell and a substrate. Typically, for example, heparin and heparin derivatives (for example, heparin disclosed in JP-A-2005-218308A) And the like, or a salt thereof.

  The medium for growing cells in the method of the present invention is not particularly limited as long as it is a medium usually used in the field of cell culture, but a serum-containing medium is preferable in order to obtain more rapid and large-scale growth. As the serum-containing medium, a medium supplemented with serum in an amount of less than 12% based on a standard medium as described elsewhere in this specification is used. The amount of serum is preferably as small as possible considering the burden on the individual providing the serum, but it is 1% to 20% by volume in view of the fact that the desired rapid cell growth promoting action is obtained. Preferably, it is 3 to 12%, more preferably 5 to 10%.

  The serum used in the method of the present invention is mammalian serum, and is the serum of an individual from which the cultured cells are derived (autologous serum). However, when the cells to be cultured are human cells, it is difficult to collect autologous sera, even if different animal sera (for example, FBS) or sera from other individuals of the same species (cross-family sera) are used, the cells High growth efficiency can be achieved as long as the cells are cultured in substantially no contact with the coagulant. However, for example, when human serum is used, the effect of the present invention by not adding an anticoagulant appears more conspicuously than FBS. The serum may be serum derived from peripheral blood or serum derived from cord blood.

  The cells to be proliferated by the method of the present invention may be cells prepared from a sample containing a blood component, attached, and cultured. For example, mesenchymal cells, mesenchymal stem cells, hematopoietic stem cells, umbilical cord blood stem cells, cornea In addition to somatic stem cells such as stem cells, hepatic stem cells and pancreatic stem cells, embryonic stem cell mononuclear cells such as fetal stem cells (excluding human embryos), osteoblasts, fibroblasts, ligament cells, epithelial cells, vascular endothelial cells, etc. However, it is not limited to these.

  In one embodiment, the method of the present invention is suitable for growing stem cells, and is used, for example, for growing mesenchymal stem cells.

  Mesenchymal stem cells are pluripotent and self-replicating stem cells that are present in minute amounts in stromal cells of mesenchymal tissue and differentiate into connective tissue cells such as bone cells, chondrocytes, and adipocytes In recent years, it has been found that it has the ability to differentiate into nerve cells and cardiomyocytes.

  In one aspect, the methods of the invention may be used to grow human cells.

  In another embodiment of the present invention, cells to be proliferated are non-human animals (eg, rodents such as mice, rats, guinea pigs, hamsters, primates such as chimpanzees, artiodactyles such as cows, goats, sheep, horses, etc. May be cells of the territorial form, rabbits, dogs, cats, etc.).

  Furthermore, in one embodiment of the method of the present invention, the stem cells may be grown in an undifferentiated state.

In general, stem cells have a higher proliferation rate and survival rate after introduction into a living body in an undifferentiated state. For example, when rapid and large-scale cell growth is required, such as for the treatment of ischemic brain disease, the necessary number of cells can be obtained in a short time by growing the collected stem cells in an undifferentiated state. be able to.
Alternatively, if cells that have differentiated into a certain cell type are desired, stem cells or blasts are proliferated in large quantities in an undifferentiated state and then added with known growth factors that induce differentiation into the desired cell type or A large number of differentiated cells may be obtained by inducing differentiation by introduction of a gene having such properties.

  In one embodiment of the present invention, it is preferable that subculture is performed when the density of mesenchymal stem cells in the medium reaches 5,500 cells / cm 2 or more.

  The density of the cells in the medium affects the nature of the cells and the direction of differentiation. For example, when culturing mesenchymal stem cells, if the density of the cells in the medium exceeds 8,500 cells / cm 2, the properties of the cells change, and therefore, the subculture is performed at a maximum of 8,500 cells / cm 2 or less. The culture is preferably performed, and more preferably, the subculture is performed at 5,500 cells / cm 2 or more.

  In a preferred embodiment of the present invention, the medium is changed at least once a week.

  Medium exchange supplies nutrients, growth factors, growth factors, etc. necessary for cell culture and growth, and also removes waste products such as lactic acid produced by cell metabolism, keeping the pH of the medium constant. Is necessary for. The period of medium exchange is selected depending on the cell type, culture conditions, etc. In particular, when using a medium containing human serum, it is desirable that the number of times is as small as possible in consideration of the burden on the serum donor. For example, when mesenchymal stem cells are cultured by the method of the present invention, the medium is changed at least once a week, more preferably once or twice a week. According to the method of the present invention, the number of days for culturing until the required number of cells is obtained is shortened, so that the amount of serum used by medium exchange can be suppressed.

  In a preferred embodiment of the method of the present invention, subculture can be repeated until the total number of cells reaches 100,000,000 or more.

  By culturing cells using the method of the present invention, a growth rate 3 to 100 times higher than usual can be obtained, so that a large amount of cells can be obtained in a short time. The number of cells required may vary depending on the purpose for which the cells are used, but for example, the number of mesenchymal stem cells required for transplantation for the treatment of ischemic brain disease due to cerebral infarction is It is considered to be 10,000,000 or more. When the method of the present invention is used, typically 10,000,000 mesenchymal stem cells can be obtained in 12 days. Such rapid cell growth has not been realized so far, and has been made possible for the first time by the method of the present invention. The cells themselves obtained by this method also have excellent proliferation efficiency. It is extremely surprising for those skilled in the art that such rapid cell growth and high-efficiency (rapid growth) cells can be obtained by culturing in a serum-containing medium substantially without heparin. It is.

  The cells obtained by the method of the present invention have high differentiation potential and are safe, and the inventors have named them “somatic stem cells”. In the “somatic stem cells” obtained by the method of the present invention, the expression level of a specific gene is changed (expressed / non-expressed, reduced / increased) as compared to the culture using heterologous serum (eg, FBS). ing.

  For example, Table 1 shows a cell surface antigen (CD antigen) group expressed in autoserum cultured cells and a CD antigen group not expressed in autoserum cultured stem cells. The cells obtained by the method of the present invention are characterized in that at least 90% of the positive CD antigen groups listed in Table 1 are expressed and at least 90% of the negative CD antigen groups are not expressed. The cells obtained by the method of the present invention are characterized in that the differentiation marker CD24 expressed in heterologous serum (for example, FBS) culture is not expressed, and a more undifferentiated state is maintained.

  Table 3 shows cytokines that were increased or decreased in common in the five cases. Further, Table 4 shows gene groups that have a difference of two times or more in expression level in common in five cases. As shown in these tables, the cells obtained by the method of the present invention express a series of growth factors, and particularly for EGF, its expression is compared to a culture method using a heterologous serum (for example, FBS). High level. This suggests the possibility that the cells of the present invention may be advantageous for growth.

  Furthermore, Table 2 shows growth factor-related factors that were increased or decreased in common in five cases in FBS cultured cells and autologous serum cultured cells. As shown in this table, in the cells obtained by the method of the present invention, the expression or expression level of the cancer-related gene group is low compared to the culture method using heterologous serum (for example, FBS). The undesirable cancer-related genes include EWI-FLI-1, FUS-CHOP, EWS-ATF1, SYT-SSX1, PDGFA, FLI-1, FEV, ATF-1, WT1, NR4A3, CHOP / DDIT3, FUS / DDIT3 TLS, BBF2H7, CHOP, MDM2, CDK4, HGFR, c-met, PDGFα, HGF, GFRA1, FASN, HMGCR, RGS2, PPARγ, YAP, BIRC2, lumican, caldesmon, ALCAM, Jam-2, Jam-3, cadherin II , DKK1, Wnt, Nucleostemin, Neurofibromin, RB, CDK4, p16, MYCN, telemere, hTERT, ALT, Ras, TK-R, CD90, CD105, CD133, VEGFR2, CD99, ets, ERG, ETV1, FEV, ETV4, MYC EAT-2, MMP-3, FRINGE, ID2, CCND1, TGFBR2, CDKNIA, p57, p19, p16, p53, IGF-1, c-myc, p21, cyclin D1, p21 and the like.

  In this specification, “the expression level is reduced (or increased)” means that “Signal Log Ratio” based on an algorithm well known in the art (GeneChip Algorithm (AFFYMETRIX)) is “−1 ≦” or “1 ≦ (That is, the ratio of gene expression levels is 2 times or more (Table 5)), more preferably “−2 ≦” or “2 ≦” (that is, the ratio of gene expression levels is 4 times or more) ) Is intended. As used herein, “genes described in the table” is intended to include genes (including mutants that retain the function of the gene) specified by the “Probe Set ID” described in the table. . These genes correspond to the gene indicated by Gene Symbol. Gene Symbol is a name uniquely associated with each gene by the US NCBI. Details of the correspondence between Probe Set ID and Gene Symbol are described in the AFFYMETRIX NetAffix database and can be easily understood by those skilled in the art. When used for a gene, “mutant” is used to be able to replace “gene mutant”, and (i) one or several bases are deleted, substituted or added to the base sequence of the specified gene. From a base sequence, (ii) From a base sequence that can hybridize to a specified gene under stringent conditions, or (iii) From a base sequence that is at least 80% identical to the base sequence of the specified gene Is intended, and in any case, retains the function of the identified gene. As used herein, “gene function” is used interchangeably with “protein function encoded by the gene”. The protein encoded by the “gene listed in the table” is a protein whose function is well known, and an assay system for confirming the function is well known in the art. Therefore, those skilled in the art can easily produce a gene mutant as described above and confirm its function easily by using a well-known technique in the art. For example, for specific hybridization procedures and “stringent” hybridization conditions, see “Molecular Cloning: A Laboratory Manual 3rd edition, edited by J. Sambrook and DW Russll, Cold Spring Harbor Laboratory, NY (2001)” ( It can be carried out according to well-known methods such as those described in the present specification.

  In the present invention, “expressed” or “not expressed” of a CD antigen group is intended to be a result when using the Analysis mode of Genechip Operating Software (GCOS) of Affymetrix. Moreover, the protein encoded by the gene variant which retained the function of the gene described in the table may be a variant of the protein encoded by the gene described in the table. When used for a protein, a “variant” is used interchangeably with a “protein variant”, wherein one or several amino acids are deleted, substituted or added to the amino acid sequence of the protein encoded by the identified gene. It is intended to consist of an amino acid sequence made up of. A person skilled in the art can also easily produce a protein variant as described above and easily confirm its function by using a well-known technique in the art.

  The cell according to the present invention is a cell having a proliferative ability that can be used for tissue repair and regeneration. Donor cells used for tissue repair and regeneration include autologous or allogeneic tissue stem cells or somatic stem cells or embryonic stem cells. Therefore, the cells according to the present invention can be cells derived from tissue stem cells, somatic stem cells or embryonic stem cells. Considering ethical issues, the risk of infection, or the need to use immunosuppressants, autologous cells, especially somatic stem cells that can secure donor cells non-invasively (for example, bone marrow cells) It is preferably a cell derived from.

  Tissue stem cells, somatic stem cells or embryonic stem cells can be supplied from tissues or body fluids of mammalian individuals. Preferred tissues for the source of these cells include, for example, muscle tissue, bone tissue, adipose tissue, skin, lymphatic system Examples include tissues, vessels, digestive organs, hair roots, dental pulp, and embryos (excluding human embryos). Preferred body fluids for the source include bone marrow fluid, blood (peripheral blood or umbilical cord blood), lymph fluid, and the like. Can be mentioned. In particular, when the target of repair and regeneration is a nervous system tissue, examples of the source of donor cells include bone marrow, peripheral blood, umbilical cord blood, fetal embryo, and brain, and the target of repair and regeneration is hematopoietic tissue. , Bone marrow, peripheral blood, umbilical cord blood, fetal embryo and the like. A person skilled in the art can easily prepare a target cell by using a technique well known in the art.

  Since the cell according to the present invention is a cell that can be used for tissue repair and regeneration, it is preferably an adhesive cell, such as a mesenchymal cell, a mesenchymal stem cell, a hematopoietic stem cell, an umbilical cord blood stem cell, Somatic stem cells such as corneal stem cells, hepatic stem cells, pancreatic stem cells, embryonic stem cell mononuclear cells such as fetal stem cells (excluding human embryos), osteoblasts, fibroblasts, ligament cells, epithelial cells, vascular endothelial cells More preferably derived from cells such as In addition, when intended for use in the treatment of acute stage neurological diseases, the cells according to the present invention are preferably derived from stem cells, particularly mesenchymal stem cells. Mesenchymal stem cells are pluripotent and self-replicating stem cells that are present in minute amounts in stromal cells of mesenchymal tissue and differentiate into connective tissue cells such as bone cells, chondrocytes, and adipocytes In recent years, it has been found that it has the ability to differentiate into nerve cells and cardiomyocytes. In addition, since the proliferation rate and the survival rate after introduction | transduction in a living body are higher in the undifferentiated state of a stem cell, it is preferable that the cell which concerns on this invention is a cell of the undifferentiated state derived from a stem cell.

  The cells according to the present invention are preferably cells derived from humans, but mammals other than humans (eg, rodents such as mice, rats, guinea pigs, hamsters, primates such as chimpanzees, cows, goats, sheep) The cells may be derived from cloven-hoofed animals such as horseshoes, horseshoes such as horses, rabbits, dogs, cats, etc.).

  As used herein, autoserum is the serum of an individual from which cells are derived (autoserum), but when it is difficult to use autoserum, high growth rates can be obtained using other adult human sera. be able to.

  As described above, the cell according to the present invention is a cell having a high differentiation potential in the same species, particularly in the presence of autologous serum, which is higher than the growth rate in the presence of heterologous serum, and in which a safe and more undifferentiated state is maintained. It is. As the medium used for culturing the cells according to the present invention, various standard media known in the art (for example, depending on the kind of cells, the direction and level of desired differentiation, the required growth rate, etc.) Dulbecco's modified Eagle medium (DMEM), NPBM, αMEM, etc.) are appropriately selected, and DMEM is preferably used.

  In one aspect, the cells cultured by the method of the present invention may be used in the manufacture of a medicament for tissue repair / regeneration.

  By administering a therapeutic agent containing cells obtained by the proliferation method of the present invention as an active ingredient to a subject, the tissue of the subject whose function has been lost can be repaired and regenerated. In particular, when mesenchymal stem cells are used, it is possible to repair and regenerate brain tissue of ischemic brain disease (see International Publication WO02 / 00849A1). As referred to in this specification, tissue repair and regeneration are synonymous with functional repair and regeneration. As a therapeutic effect, for example, when nerve tissue is repaired and regenerated, a nerve protective action (for example, , Axonal remyelination), neurotrophic effects (eg, glial cell recruitment), cerebrovascularization, nerve regeneration, and the like. That is, the substance of the therapeutic effect of the medicine containing the cells grown by the method of the present invention means the repair and regeneration of the tissue, and the phenomenon means the repair and regeneration of the dysfunction of the tissue. When proliferating cells are used for tissue repair / regeneration, it is confirmed beforehand by peripheral blood that the source of donor cells is not infected with HIV, ATL, HB, HC, syphilis, human parvovirus B19, etc. It is preferable that

  In one embodiment, the cells grown by the method of the present invention can also be used for diagnosis of disease or pathogen infection. For example, the risk of carcinogenesis can be diagnosed by examining the state of a cancer-related gene contained in cells collected from a subject and grown outside the body.

  In addition, when the subject is infected with prion disease, it is difficult to detect by a normal test method, but by proliferating the cells by the method of the present invention, the abnormal prion is rapidly amplified to a detection sensitivity or higher, A diagnosis of prion disease can be made.

  Alternatively, cells grown by the methods of the present invention may be used for in vivo or in vitro experiments.

  Examples of the medicament for tissue repair / regeneration comprising cells grown by the method of the present invention include, for example, cells ex vivo amplified using the method of the present invention and pharmaceutically acceptable diluents, excipients and / or Or injection consisting of a base material (for example, injection including neural progenitor cells, hematopoietic stem cells, hepatocytes, pancreatic cells, lymphocyte cells, etc.) and an implant for transplantation (for example, cardiomyocyte sheet, artificial skin, artificial cornea, artificial Tooth roots, artificial joints, etc.), but is not limited thereto.

  The medicine containing the cells cultured by the method of the present invention may be used for repair / regeneration of nervous system tissue. For example, autologous mesenchymal stem cells may be proliferated and used as a medicament for the treatment of ischemic neurological diseases. In a preferred embodiment, the cells contained in the medicament are cells (autologous cells) derived from the subject to which the cells are administered.

  Nervous system diseases that are the subject of such cell therapy include, for example, central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke (including cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage), brain tumor , Higher dysfunction including dementia, mental illness, epilepsy, traumatic nervous system diseases (including head trauma, brain contusion, spinal cord injury), spinal cord infarction, and Creutzfeldt-Jakob disease, Kourou disease, bovine spongiform encephalopathy And prion diseases such as scrapie, but are not limited thereto.

  The cells grown by the method of the present invention are also useful in the treatment of diseases other than nervous system diseases. For example, hematopoietic stem cells may be amplified in vitro and transplanted into bone marrow for the treatment of acute leukemia. In normal bone marrow transplantation, an autologous transplant typically requires 2 × 10 8 cells per body weight of the recipient and 4 × 10 8 cells in the case of another transplant, and bone marrow collected from a cell donor Although the volume of the liquid can reach 1000 mL, the physical burden of the cell donor can be reduced by rapidly growing the cells in vitro using the method of the present invention. In addition, T lymphocytes collected from a patient's peripheral blood may be amplified in vitro using the method of the present invention and transplanted into the same patient for the treatment of viral infection and the like.

  The source of donor cells used to replenish cells for tissue repair / regeneration can be derived from autologous or other tissue stem cells or somatic stem cells or embryonic stem cells, but ethical issues, risk of infection, Or, considering difficulties such as the necessity of using an immunosuppressive agent, autotransplantation therapy using autologous cells, particularly somatic stem cells (for example, bone marrow cells) that can secure donor cells noninvasively is desirable. When autotransplantation is difficult, it is possible to use cells derived from other people or other animals. If the amount of the anticoagulant contained in the sample at the start of culture is less than 5 U / mL, the donor cell is a cryopreserved cell even if it is contained in the sample collected immediately before the culture. It may be. For example, autologous cells may be proliferated in advance, cryopreserved, and administered at the time of disease, as in a treatment model using the therapeutic cell delivery support system described in International Publication WO 2005 / 001732A1.

  The source of the cells should preferably include cells that are already known to differentiate into the cell type of the specific tissue to be repaired / regenerated, for example, cells of the same germ layer or totipotent stem cells, but to some extent It has also been found that stem cells differentiated into germ layers (eg, fetal liver cells) redifferentiate into cells of other tissues such as nervous system cells (eg, cells described in WO 02 / 00849A1) ), A tissue containing different germ layer cells may be used as long as it can induce differentiation into a desired cell type using a known differentiation-inducing factor or the like.

  When the tissue to be repaired is the nervous system, examples of the source of donor cells include cells derived from bone marrow, peripheral blood, umbilical cord blood, fetal embryo, brain, and the like. When the tissue to be repaired is a hematopoietic tissue, hematopoietic stem cells, umbilical cord blood stem cells and the like contained in bone marrow, peripheral blood, umbilical cord blood, fetal embryo and the like can be mentioned.

  When using bone marrow-derived mesenchymal stem cells for the repair of nervous system tissues, there are the following advantages: 1) A significant effect can be expected 2) Low risk of side effects 3) Sufficient 4) Non-invasive treatment and autotransplantation is possible, 5) Low risk of infection, 6) No fear of immune rejection, 7) Ethical issues 8) Easily accepted by society, 9) Easily established as general medical care. Furthermore, bone marrow transplantation is a treatment that has already been used in clinical practice, and its safety has been confirmed. In addition, bone marrow-derived stem cells are highly migratory and can reach the target damaged tissue not only by local transplantation but also by intravenous administration, and a therapeutic effect can be expected.

  Bone marrow fluid can be collected, for example, by anesthetizing animals (including humans) as a collection source (local or general anesthesia), inserting a needle into the sternum or iliac bone, and aspirating with a syringe. In addition, it has become an established technique to puncture a baby with a needle directly into the umbilical cord, suck it with a syringe, and collect and store the umbilical cord blood. In the conventional method, an anticoagulant is used to prevent blood components from coagulating in the collected bone marrow fluid, but in the method of the present invention, the anticoagulant is not used as described above. It is.

  As a method for delivering a drug containing cells grown by the method of the present invention to damaged tissue, for example, local transplantation by surgical means, intravenous administration, lumbar puncture administration, local injection administration, subcutaneous administration, intradermal administration, abdominal cavity Intramuscular administration, intramuscular administration, intracerebral administration, intraventricular administration, intravenous administration, and the like are possible. In addition, cells grown by the method of the present invention may be contained or seeded in an implant, a cell sheet substrate, an artificial joint, or the like and transplanted into a living body.

  For example, when transplantation by injection of cells into a patient is used for repair of the nervous system, the cells to be transplanted are suspended in an artificial cerebrospinal fluid or physiological saline, and stored in a syringe to perform surgery. Can be performed by exposing the damaged nerve tissue and directly injecting the damaged tissue with an injection needle. In the case of a cell that has a high migration ability to move within the tissue (for example, a cell described in WO 02 / 00849A1), the cell may be transplanted in the vicinity of the damaged site, or in cerebrospinal fluid The effect can be expected by injecting into the tube. In this case, since cells can be injected by normal lumbar puncture, there is no need for the patient's surgery, and only local anesthesia is required, which is preferable in that the patient can be treated in a hospital room. Furthermore, an effect can be expected by intravenous injection. Therefore, it is preferable in that transplantation can be performed in the manner of normal blood transfusion and transplantation operation in a ward is possible.

  A suitable medium for cell growth according to the present invention is selected depending on the cell type, the desired direction and level of differentiation, the required growth rate, and the like. For example, as a medium suitable for growing mesenchymal stem cells for use in repair of the nervous system, in addition to Dulbecco's modified Eagle medium (DMEM) shown below, neural progenitor cell standard medium (NPBM: manufactured by Clontech), Examples include, but are not limited to, αMEM medium. Based on such a standard medium, serum is added as described above, and further, nutrient factors such as amino acids, antibiotics, growth factors and / or growth factors are added as necessary.

Specific examples of the standard medium include Dulbecco's modified medium containing the following components at the following concentrations (mg / L):
CaCl2 (anhydride): 160-240
KCL: 320-480
Fe (NO3) 3.9H2O: 0.08 to 1.2
MgSO4 (anhydrous): 80-120
NaCl: 5120-7680
NaHCO3: 2960-4440
NaH2PO4 · H2O: 100-150
D-glucose: 3600-5400
Phenol red: 12-18
Sodium pyruvate: 88-132
L-arginine / HCl: 67 to 101
L-cystine 2HCl: 50 to 76
L-histidine / HCl / H 2 O: 34-50
L-isoleucine: 84-126
L-leucine: 84-126
L-Lysine / HCl: 117-175
L-methionine: 24-36
L-phenylalanine: 53-79
L-serine: 34-50
L-threonine: 76-114
L-tryptophan: 13-19
L-tyrosine (disodium salt): 83-125
L-valine: 75-113
Choline chloride: 3.2 to 4.8
D-Ca-pantothenic acid: 3.2 to 4.8
Folic acid: 3.2 to 4.8
i-inositol: 5.8-8.6
Niacinamide: 3.2 to 4.8
Pyridoxal / HCl: 3.2 to 4.8
Riboflavin: 0.3-0.5
Thiamine / HCl: 3.2 to 4.8

  If desired, antibiotics commonly used in the field of cell culture (eg, penicillin, streptomycin, etc.) may be used alone or in combination. It is preferable to use a plurality of antibiotics in combination. For example, when penicillin and streptomycin are used in combination, the amount is 0.5 to 2% by volume, preferably 0.8 to 1.2% by volume, based on the medium. .

  The low molecular amino acids contained in the medium include L-alanine, L-asparagine sun, L-cysteine, L-glutamine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-serine, L- Examples include threonine, L-tyrosine, L-valine, L-ascorbic acid, and L-glutamic acid. These amino acids are contained as nutrients in a medium usually used in the field of cell culture.

  Furthermore, the present inventors added that glutamine is added at 0.1 to 2% (weight / volume) of the total amount of the medium, which is indispensable for the rapid proliferation of mesenchymal stem cells. It has been found that supplementation to maintain 0.1-2% (weight / volume) further promotes rapid growth.

  A growth / growth factor and / or a differentiation-inducing factor may be added to the standard medium as necessary. Growth / growth factors and differentiation-inducing factors are selected according to the desired direction and level of differentiation, the required proliferation rate, and the like. For example, vitamins such as ascorbic acid and nicotinamide, neurotrophic factors such as NGF and BDNF, bone morphogenetic factors such as BMP, epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor, IL-2, etc. However, it is not limited to these.

The cell culture method of the present invention is specifically performed as follows, for example.
1. A sample collected from a living body with a syringe whose inner wall has been wetted with a heparin solution as described above is diluted 100 times or less, preferably 10 times or less, more preferably about 2 to 6 times. Add to medium previously maintained at 37 ± 0.5 ° C., inoculate in culture dishes and incubate in 5% carbon dioxide at 37 ± 0.5 ° C. The medium is changed at least once a week, typically once or twice a week. The medium is prepared by adding serum and necessary auxiliaries to a suitable standard medium, sterilized by a filter sterilizer, and aliquoted and stored in a 4 ° C. refrigerator at 37 ± 0.5 ° C. Keep and use. When the temperature exceeds 37.5 ° C, dead cells increase, and when the temperature is less than 36.5 ° C, the growth is slow. The carbon dioxide gas concentration is preferably in the range of 5 ± 1%. Keeping the solution that contacts the cells in all steps in a similar temperature range facilitates rapid growth.

  2. After the cells adhere to the culture dish substrate, the medium and blood cell components floating in the medium are aspirated and separated and removed. Next, the attached stem cell surface is washed using phosphate buffered saline as a washing solution.

  3. Passage is 60-80% of confluence, preferably 65-75 so that the cell density does not exceed 8,500 cells / cm 2, with reference to the time point when the number of cells in the dish reaches 5,500 cells / cm 2 or more. When the percentage is reached. At the time of passage, 3 mL of a release agent mainly composed of trypsin and, if necessary, EDTA (ethylenediaminetetraacetic acid) was added per dish, and adhered after incubation at 37 ± 5 ° C. for 3 to 5 minutes. Confirm that the stem cells have detached. The separation solution is replaced with the culture medium by the gradient method, the cells in the culture medium are transferred to a predetermined centrifuge tube, and the cells are spun down by centrifugation and passaged. The cycle of culture, medium exchange, and passage is repeated until at least the total number of mesenchymal stem cells is 100,000,000 or more. The medium is changed at least once a week.

  By repeating the above cycle, the desired number of cells can be quickly obtained (for example, in the case of mesenchymal stem cells, 1 × 10 8 cells can be obtained within 2 weeks).

  If desired, a cell scaffold that facilitates detachment of cells after growth can be used. Examples of suitable scaffolds include, but are not limited to, porous inorganic ceramics, micropillars (for example, nanopillar cell culture sheets manufactured by Hitachi, Ltd.), non-woven fabrics, and honeycomb membrane films.

  In one aspect of the present invention, a cell to be cultured may have been previously introduced with a gene that induces proliferation and differentiation, such as a BDNF gene, a PLGF gene, a GDNF gene, or an IL-2 gene. Alternatively, the cells to be cultured may be immortalized cells into which an immortalizing gene such as a telomerase gene has been introduced. Such gene introduction is disclosed in, for example, WO03 / 038075A1.

  Selecting a combination that induces the desired growth rate and / or differentiation into a specific cell type from media, scaffolds, auxiliaries, growth factors and / or gene transfer as exemplified above Is within the ability of one skilled in the art.

  The cells grown by the method of the present invention can be administered as they are for the repair and regeneration of tissues as they are, but in order to improve the treatment efficiency, as a composition to which various drugs are arbitrarily added or It may be modified by gene transfer and administered or transplanted. For example, the addition of a substance that improves the further growth rate of the cells grown by the method of the present invention in the tissue, a substance that promotes differentiation into a desired cell, or a substance that improves the survival rate in the tissue, and And / or introduction of a gene having such an effect; addition of a substance having an effect of preventing the transplanted cell from being adversely affected by damaged living tissue, and / or introduction of such a gene; addition of a substance that extends the life of the donor cell And / or introduction of a gene having such an effect; addition of a substance that regulates the cell cycle; and / or introduction of a gene having such an effect; addition of a substance for the purpose of suppressing immune cells; and / or such an effect. Introduction of a gene having an effect; addition of a substance that activates energy metabolism and / or introduction of a gene having such an effect; The addition of a substance that improves the ability to migrate in the tissues of and / or the introduction of a gene having such an effect; the addition of a substance that improves the blood flow and / or the introduction of a gene having such an effect, etc. It is not limited to these.

  The cells are preferably cultured in a cell preparation center “CPC (Cell Processing Center)” based on GMP standards. Preparation of “clinical grade cells” to be administered to a subject is a facility specially designed to manipulate cells in a sterile condition, more specifically air conditioning control, room pressure control, temperature / humidity control, particle counter, HEPA It is preferable to use CPC with cleanliness guaranteed by a filter or the like. In addition to the CPC facility itself, all devices used in the CPC are guaranteed to have performance through validation, and it is preferable to monitor and record their functions as needed. It is desirable to strictly manage and record according to the “procedure manual”.

  The medicament of the present invention may contain cell types other than mesenchymal stem cells as cell components, but those having a high proportion of mesenchymal stem cells in the cell components are preferred. Therefore, in a preferred embodiment of the medicament of the present invention, the ratio of the number of mesenchymal stem cells to the total number of cells contained in the medicament is 50% or more, preferably 75% or more, more preferably 80% or more, and still more preferably 90%. %, More preferably 95% or more, particularly preferably 98% or more, and most preferably substantially free of cell types other than mesenchymal stem cells, such as hematopoietic stem cells. The proportion of mesenchymal stem cells occupying the cell component is, for example, that the cells contained in the pharmaceutical are one or more markers specific to mesenchymal stem cells (for example, CD105, CD73, CD166, CD9, CD157, etc. It can be easily determined by labeling with a labeled antibody against the surface antigen) and analyzing by a flow cytometry method or the like.

  The larger the number of mesenchymal stem cells contained in the medicament of the present invention, the better. However, in view of the administration time to the subject and the time required for culture, it is practical that the amount is minimal as long as it is effective. Therefore, in a preferred embodiment of the medicament of the present invention, the number of mesenchymal stem cells is 107 or more, preferably 5 × 10 7 or more, more preferably 108 or more, and further preferably 5 × 10 8 or more.

  As a cell type other than the above mesenchymal stem cells, when it is intended to assist the repair of nerve tissue, for example, it can differentiate into neural cells obtained from bone marrow, umbilical cord blood, peripheral blood or fetal liver. Cells, which are Lin−, Sca-1 +, CD10 +, CD11D +, CD44 +, CD45 +, CD71 +, CD90 +, CD105 +, CDW123 +, CD127 +, CD164 +, fibronectin +, ALPH +, collagenase-1 + or AC133 + However, the present invention is not limited to these, and any other cell type that can differentiate into neural cells can be used.

  The stromal cells can be obtained, for example, by selecting cells having a cell surface marker such as CD45 from cell fractions obtained by centrifugation from bone marrow cells and cord blood cells. In addition, bone marrow cells and umbilical cord blood cells collected from vertebrates are subjected to density gradient centrifugation in the solution for a time sufficient for separation according to the specific gravity at 800 g. After centrifugation, the specific gravity is 1.07 to 1.1 g / ml. It can also be prepared by collecting a cell fraction having a specific gravity included in the range. Here, “a sufficient time for separation according to the specific gravity” means a time sufficient for the cells to occupy a position according to the specific gravity in the solution for density gradient centrifugation. About a minute. The specific gravity of the cell fraction to be collected is preferably in the range of 1.07 to 1.08 g / ml, for example, 1.077 g / ml. As a solution for density gradient centrifugation, Ficol liquid or Percol liquid can be used, but is not limited thereto.

  Specifically, first, bone marrow fluid or umbilical cord blood collected from a vertebrate is mixed with an equal amount of solution (PBS + 2% BSA + 0.6% sodium citrate + 1% penicillin-streptomycin), and 5 ml of the solution is Ficol + Paque solution. (1.077 g / ml) and centrifugation (800 g for 20 minutes) to extract the mononuclear cell fraction. This mononuclear cell fraction was subjected to culture solution (αMEM, 12.5% FBS, 12.5% horse serum, 0.2% i-inositol, 20 mM folic acid, 0.1 mM 2-mercaptoethanol, Mix in 2 mM L-glutamine, 1 μM hydrocortisone, 1% anti-biotic-antimycotic solution) and centrifuge (2000 rpm, 15 minutes). Then, after removing the supernatant after centrifugation, the precipitated cells are collected and cultured (37 ° C., 5% CO 2).

  The AC133 + cells can be obtained, for example, by selecting cells having a cell surface marker of AC133 from cell fractions obtained by centrifugation from bone marrow cells, umbilical cord blood cells, or peripheral blood cells. In another embodiment, fetal hepatocytes collected from vertebrates are subjected to density gradient centrifugation in a solution at 2000 rotations for a time sufficient for separation according to the specific gravity. After centrifugation, the specific gravity is 1.07 to 1. It can also be prepared by collecting a cell fraction contained in the range of 1 g / ml and collecting cells having the characteristics of AC133 + from this cell fraction. Here, “a sufficient time for separation according to the specific gravity” means a time sufficient for the cells to occupy a position according to the specific gravity in the solution for density gradient centrifugation. About a minute. As a solution for density gradient centrifugation, Ficol liquid or Percol liquid can be used, but is not limited thereto.

  Specifically, first, liver tissue collected from a vertebrate is washed in an L-15 solution, and treated with an enzyme (L-15 + 0.01% DNase I, 0.25% trypsin, 0.1% collagenase). In solution for 30 minutes at 37 ° C.) and pipetted to single cells. The fetal liver cells that have become single cells are centrifuged. The cells thus obtained are washed, and AC133 + cells are recovered from the washed cells using the AC133 antibody. As a result, cells capable of differentiating from fetal liver cells to nervous system cells can be prepared. Recovery of AC133 + cells using antibodies can be performed using magnetic beads or using a cell sorter (such as FACS).

  The medicament of the present invention is preferably a parenteral preparation, more preferably a parenteral systemic preparation, particularly an intravenous preparation. Examples of dosage forms suitable for parenteral administration include, but are not limited to, injections such as solution injections, suspension injections, emulsion injections, injections prepared at the time of use, and grafts. . Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions. Specifically, for example, a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, culture medium, physiological buffer such as PBS, vegetable oil, emulsifier, suspending agent, surfactant, Appropriate combinations with stabilizers, excipients, vehicles, preservatives, binders and the like can be formulated into suitable unit dosage forms.

  Examples of aqueous solutions for injection include, but are not limited to, isotonic solutions containing, for example, physiological saline, culture media, physiological buffers such as PBS, glucose and other adjuvants, such as D-sorbitol, D-mannose, and D-mannitol. , Sodium chloride and the like, and used in combination with a suitable solubilizer such as alcohol, specifically ethanol, polyalcohol such as propylene glycol, polyethylene glycol, nonionic surfactant such as polysorbate 80, HCO-50, etc. May be.

  In the present invention, the term “damage” means that a living body undergoes some kind of damage systemically or locally due to internal and / or external factors. Accordingly, the damage in the present invention includes various conditions such as various traumas, infarctions, degenerative lesions, tissue destruction. Injured sites also include various tissues throughout the body, such as the brain, nerves, kidneys, pancreas, liver, heart, skin, bone, cartilage and the like. The damaged part may be one place or a plurality of places. The medicament of the present invention is effective for various tissues, and can repair a plurality of damaged sites at a time by a single administration, and thus is particularly effective for treatment of a subject having a plurality of damaged sites. Factors that cause damage include physical external forces (accidents, burns, exposure, etc.), various ischemic diseases (ischemic neurological diseases such as cerebral infarction and spinal cord infarction, ischemic heart diseases such as myocardial infarction) , Various inflammations, diabetes, various infections, autoimmune diseases, tumors, toxic exposure, central and peripheral demyelinating diseases, central and peripheral degenerative diseases, cerebral hemorrhage, subarachnoid hemorrhage, brain tumor Examples include, but are not limited to, higher-order dysfunction including dementia, mental illness, epilepsy, Creutzfeldt-Jakob disease, Kourou disease, bovine spongiform encephalopathy, and scrapie. Injuries in the present invention typically refer to those with a loss and / or decrease in tissue function.

  Examples of loss and / or decrease in tissue function include, but are not limited to, for example, nerve tissue, pain, wrinkles, sensory disturbances such as numbness, paralysis, slippery, half body instability, lightheadedness, gait disturbance, slow movement and awkwardness. Autonomic nerves such as movement disorders such as headache, memory disorder, consciousness disorder, language disorder, convulsion, tremor, brain dysfunction such as dementia, hallucinations, abnormal behavior, dizziness, dizziness, fainting, dysuria, sweating disorder In the case of disorders such as kidney, excretion function, regulation of body fluid balance such as electrolyte / water balance, loss and / or decrease in endocrine function, etc. In the case of pancreas, loss and / or decrease in exocrine function and endocrine function, etc. In the case of liver, substance metabolism function, substance synthesis function, loss and / or decrease of exocrine function, etc., in the case of heart, blood pumping function, endocrine function Loss and / or reduction can be mentioned. The specific loss and / or reduction of function and associated symptoms that occur when a tissue is damaged are known to those skilled in the art.

  In addition, the repair and regeneration of the damaged site is synonymous with the repair and regeneration of the function, and the therapeutic effect is, for example, when the nervous system tissue is repaired / regenerated, the protective action of the nerve (for example, the restoration of axons) Including myelination), neurotrophic effects (eg, recruitment of glial cells), cerebrovascularization, nerve regeneration and the like. That is, the substance of the therapeutic effect of the medicament of the present invention means the repair and regeneration of the tissue, and the phenomenon means the repair and regeneration of the dysfunction of the tissue. For example, in the case of brain damage, the substance of therapeutic effect is reduction of edema, axon remyelination, increase of glial cells, angiogenesis, nerve regeneration, etc. Appears as recovery of paralysis, recovery of paralysis, relief of pain and numbness. These effects can be confirmed by physical examination of damaged tissues, for example, X-ray examination, CT examination, MRI examination, ultrasonic examination, endoscopy, biopsy, and various hematological examinations. It can also be confirmed by biochemical tests, endocrinological tests, motor function tests, brain function tests, cognitive function tests, and the like.

  When the medicament of the present invention “helps repair” a damaged site, typically, by administration of the medicament of the present invention, the components of the agent assist and assist the body's repair mechanism, and repair the damaged site. However, this is not limited to this, and it is not limited to this, but it can prevent the damage from spreading and seriousness, and prevent ongoing damage. And also to lead this to recovery. By administration of this agent, a suitable environment is created at the site of injury because the repair mechanism of the living body functions. Specifically, the repair assistance provided by the medicament of the present invention can be brought about by, for example and without limitation, cytokine secretion, angiogenesis and / or tissue regeneration (see FIG. 9).

  In addition, according to the medicament of the present invention, for example, improvement of renal function index such as BUN value and / or creatinine value in damaged kidney with renal failure, insulin secretion in damaged pancreas with reduced insulin secretion function of pancreatic islets of Langerhans Or improvement of diabetes index such as blood glucose level, blood Glu A1 concentration and / or blood HbA1C concentration, blood prostaglandin D synthase concentration and / or blood homocysteine concentration in heart damaged by ischemic heart disease In an injured liver accompanied by liver failure, improvement of liver function indicators such as GOT value, GPT value and / or γ-GTP value can be brought about.

  In a preferred embodiment of the medicament of the present invention, the medicament is administered after the treatment of the injury site. Here, treatment of the damaged site typically means treatment in the hyperacute phase or acute phase of damage, and includes, for example, treatment for suppressing the spread of damage and treatment for repairing damage surgically. It is. In another preferred embodiment, the medicament of the present invention is administered for the purpose of assisting the autonomous repairing power provided in the living body.

  In addition, the medicament of the present invention is preferably administered after the subacute phase of injury. The sub-acute phase refers to a recovery phase that shifts after the exacerbation of symptoms due to injury (hyperacute phase or acute phase). For example, in the case of cerebral infarction, it refers to a period of 1 to 2 months after onset.

  Preferred mesenchymal stem cells in the medicament of the present invention are autologous mesenchymal stem cells derived from cells collected from a subject having an injury site. By using autologous cells, risks such as rejection and infection can be avoided. In addition, the autologous mesenchymal stem cells may be collected before the occurrence of damage or after the occurrence of damage. When it is collected before the occurrence of damage, it is usually difficult to predict the occurrence of damage, and thus it is necessary to arbitrarily purify and proliferate the collected cells and store them by a technique such as cryopreservation. When collecting after the occurrence of damage, after purifying and proliferating the collected cells, it can be administered to the subject as it is, or stored by a technique such as cryopreservation (for example, in a -152 ° C deep freezer) It is also possible to administer appropriately according to the timing. Moreover, all the cells can be administered at once, or a part of them can be preserved and additionally administered as necessary.

  The term “subject” in the present invention means any living individual, preferably an animal, more preferably a mammal, more preferably a human individual. In the present invention, the subject typically has some damage site.

  The present invention can also be used in an evaluation method for evaluating whether or not cells cultured in vitro are cells that can be used for repair and regeneration of living tissue. In one embodiment, the evaluation method according to the present invention includes a step of measuring the expression level of a specific gene in a cell to be evaluated. In one embodiment, the evaluation method according to the present invention may include a step of measuring the expression level of at least one of the genes described in Tables 1 to 4 in the cell to be evaluated. The evaluation method according to the present embodiment may further include a step of comparing at least one of the genes listed in Tables 1 to 4 with the expression level of the gene in a control cell.

  In the present invention, the control cell is a cell prepared from the same source as the cell to be evaluated and cultured with heterologous serum. The expression level of the target gene in the control cell may be measured simultaneously when measuring the expression level of the target gene in the cell to be evaluated, or may be acquired in advance. That is, the evaluation method according to the present invention may further include a step of measuring the expression level of the target gene in the control cell.

  According to the present invention, when the target gene is the gene described in B of Tables 2 to 4, when the expression level of the target gene in the cell to be evaluated is lower than the expression level of the gene in the control cell, It is evaluated as a cell that can be used for the repair and regeneration of living tissue. In addition, when the target gene is the gene described in A of Tables 2 to 4, when the expression level of the target gene in the cell to be evaluated is higher than the expression level of the gene in the control cell, the repair of living tissue And evaluated as a cell available for regeneration.

  In the present invention, the gene expression level may be measured based on the mRNA level or the protein level. As described above, the “genes described in the table” is intended to include genes (including mutants that retain the function of the gene) specified by the “Probe Set ID” described in the table. If present, it is easily understood based on the “Gene Title” in the table, so that those skilled in the art can easily construct a sequence tool (for example, primer or probe) necessary for measuring the amount of mRNA and protein The antibodies necessary for measuring the amount can be easily obtained. In addition, the evaluation method according to the present invention may be performed using as an index that the ratio of gene expression levels is 4 times or more in the system described in this example.

  The evaluation method according to the present invention may include a step of comparing the growth rate of cells to be evaluated in the presence of allogeneic serum with the growth rate in the presence of heterologous serum. The present invention is preferably applied to human-derived cells. In this case, the heterologous serum is preferably FBS. So far, human serum is known to have a significantly inferior growth-promoting activity compared to FBS, and human serum alone is known not to exhibit sufficient cell growth-promoting activity. Cells when using human serum (particularly autoserum) are known. It was unexpected that it could be evaluated whether it can be used for the repair and regeneration of living tissue by comparing the growth rate with the cell growth rate when using FBS.

  The following examples further illustrate the proliferation of cells according to the method of the present invention and the medicament for the repair / regeneration of tissues containing cells grown by the method of the present invention. It is not intended to limit the range. Those having ordinary knowledge and skills in the field of cell culture and / or cell therapy can make various modifications without departing from the spirit of the present invention.

Example 1
Using a blood collection tube whose inner wall was previously wetted with a small amount of heparin (0.1 U per mL of bone marrow fluid), 60 mL of bone marrow fluid was collected from a patient with brain disease and added to 210 mL of medium to make a total amount of 270 mL. This bone marrow fluid-containing culture broth was divided into 18 equal parts and seeded in 15 ml portions in a 150 mm diameter dish (Tissue culture dish # 3030-150 manufactured by IWAKI), and 5 ml of the medium was added to make a total volume of 20 ml per dish. After sowing, each of the 9 dishes was left in a separate incubator and cultured at 37 ± 0.5 ° C. in a 5% carbon dioxide atmosphere. Bone marrow fluid was confirmed in advance by peripheral blood examination to be free of HIV, ATL, HB, HC, syphilis, human parvovirus B19, and the like.

  The medium is 500 mL of Dulbecco's modified Eagle medium, 56.8 mL of serum derived from autologous peripheral blood, 5.7 mL of antibiotics (consisting of penicillin 10,000 U / mL, streptomycin 10 mg / mL), and 5.7 mL of glutamine (292.3 mg). / L) was added, sterilized by filtration, and divided into small portions and stored in a 4 ° C. cool box, and kept at 37 ° C. in advance.

  In washing the mesenchymal stem cells adhering to the culture vessel on the 4th day, the medium and blood cell components floating in the medium are aspirated, separated and removed, and then 5 ml of phosphate buffered saline is used as a washing solution. The adhering mesenchymal stem cell surface was washed 6 times.

  For the first passage on the 8th day, after washing with 5 mL of phosphate buffered saline, 4 mL of the separation solution (0.25% trypsin-2.21 mM EDTA) was dished in order to peel off the attached stem cells. And incubated at 37 ° C. for 3 minutes to confirm peeling. The same amount of medium was added to the separated and separated solution, and the whole amount was collected by the gradient method. Cells for nine dishes were transferred to nine centrifuge tubes and centrifuged at 800 rpm for 5 minutes using a centrifuge. After separation, the supernatant of each centrifuge tube was removed, and DMEM was added to collect cells. The collected cell solution was centrifuged again at 800 rpm for 5 minutes. After centrifugation, the supernatant of each centrifuge tube was removed, and 300 mL of medium was added to collect cells. This cell solution was subdivided into 15 dishes, and the cells were subcultured and incubated at 37 ± 0.5 ° C. and a carbon dioxide gas concentration of 5% as in the primary culture. The same passage operation was performed for the remaining nine dishes.

On the 13th day, washing, detachment, and centrifugation were carried out in the same manner as described above, and when the number of cells was measured with a hemocytometer using a blood cell counter, it reached 1.1 × 10 ⁶ cells. It was. The culture was continued and the number of cells was counted on the 20th day. As a result, the total number of cells reached 1.0 × 10 ⁸ , and washing, detachment, and centrifugation were performed, and a cryopreservation solution (normal filtration sterilization) The suspension was suspended in 20.5 mL of RPMI and 20.5 mL of autoserum collected from the patient, 5 mL of dextran, and 5 mL of DMSO) and frozen at -150 ° C. The proportion of mesenchymal stem cells in the cells was 98% or more (CD105 positive (positive rate = 99.9%), CD34 negative (negative rate = 98.8%), CD45 negative (negative rate = 98.5%). .

Comparative Example 1
The culture was performed under the same conditions as in Example 1 except that the content of heparin in the sample was changed to 2 mL (267 U / mL). The results are shown in FIG. 1 and FIG. When only a very small amount of heparin (0.1 U / mL) is added, 8 × 10 ⁵ growths are obtained 4 days after the start of the culture, whereas when 2 mL of heparin is added, 2 × 10 ⁴ growths are obtained. When the amount of heparin was very small, a growth rate of about 40 times was obtained (FIGS. 1 and 2). When the culture was continued, the number of cells 12 days after the start of the culture increased to 1.4 × 10 ⁷ cells added with a very small amount of heparin, whereas cells added with 2 mL of heparin increased to 2.9 ×. 10 ⁶ (FIG. 1). These results indicate that the addition of heparin has an inhibitory effect on cell proliferation, and the cells of the present invention can be produced by the method of the present invention even when the anticoagulant is added in a very small amount or not substantially added. It is confirmed that it is possible to rapidly increase the amount of heparin, and further, by making the amount of heparin in the sample extremely small, a significant improvement in the growth rate is obtained.

Comparative Example 2
The culture was performed for 8 days under the same conditions as in Example 1 except that FBS was used instead of serum derived from human peripheral blood. A comparison of the proliferation rate of mesenchymal stem cells by counting the number of cells is shown in FIG. In the case of culturing human mesenchymal stem cells under the conditions of the present invention, when using human adult serum, it is about 2.6 times after 5 days from the start of culture and about 8 days later when compared with the case of using FBS. A 6-fold growth rate was obtained. This result shows that in human bone marrow cell culture, the growth rate higher than that of FBS can be obtained using human adult serum by making the amount of heparin in the sample extremely small.

Comparative Example 3
Experiments were performed using rat mesenchymal stem cells. Bone marrow cells collected from two rat femurs were cultured in the same medium as in Example 1 with heparin added at 1 U / mL, 10 U / mL, 100 U, or 1000 U / mL, respectively. The culture was performed under the same conditions as in Example 1 except that FBS was used instead of serum derived from human peripheral blood. The results are shown in FIG. It was shown that the higher the concentration of heparin in the medium, the higher the inhibitory effect on growth.

Comparative Example 4
Experiments were performed using rat mesenchymal cells as follows. Group 1: Bone marrow fluid was pushed out from one rat femur using 4 mL of DMEM to obtain a total of slightly more than 4 mL of samples. This sample was added to 36 mL of DMEM to which 160 U of heparin was added, and culture was started. The heparin concentration in the medium was 4 U / mL. Group 2: From one rat femur, bone marrow fluid was pushed out using 4 mL of DMEM to which 160 U of heparin was added, and a total of 4 mL samples were obtained. This sample was left exposed to high concentration heparin as it was for 5 minutes at room temperature, and then added to 36 mL of DMEM to start culture. The heparin concentration in the medium was 4 U / mL. Changes in the number of cells in these two groups are shown in FIG. In group 1, a significant reduction in proliferation rate was observed compared to group 2. This result shows that when heparin is added to a cell sample at the time of sampling (or before shifting to culture), the proliferation efficiency of the cell is lower than when heparin is added to the culture medium during culture. I suggest that.

Example 2
The culture was performed under the same conditions as in Example 1 except that αMEM medium was used instead of DMEM as the standard medium. The results are shown in FIG. Even in the case of using the αMEM medium, it was confirmed that by suppressing the amount of heparin, it was possible to obtain faster growth than FBS using human serum.

Comparative Example 5
All were cultured for 7 days in the same manner as in Example 1 except that the medium did not contain glutamine. FIG. 7 shows a comparison of the proliferation rate of mesenchymal stem cells by counting the number of cells. One week after the start of the culture, a growth rate of about 1.6 times was observed when glutamine was used. This result indicates that addition of glutamine is necessary for rapid proliferation of mesenchymal stem cells.

Example 3
〔Method〕
Three rat femurs were collected, bone marrow cells were washed with DMEM (5 ml), and the following were added to prepare three groups of samples.
(i) DMEM (5ml)
(ii) DMEM (5 ml) + heparin 2 μl
(iii) DMEM (5 ml) + 2 μl heparin + 2 μl protamine
Each sample was washed with DMEM, placed in a 10 ml culture solution (DMEM + 10% FBS + 1% penicillin / streptomycin + 2 mM L-Glutamine), seeded in a 10 cm dish, and cultured for 14 days. Passaging was performed when the number exceeded 5,500 cells / cm ² .

〔result〕
As shown in FIG. 8, the cells that did not undergo heparin treatment had a larger initial amount than the cells that received heparin treatment, but also had a high proliferation rate, which was prominent in 8-11 days of culture. In addition, cells added with protamine, which is a substance that inhibits the activity of heparin, had a low initial amount and a high growth rate, similar to cells that had undergone heparin treatment.

Example 4
The mesenchymal stem cells cultured in autoserum and the mesenchymal stem cells cultured in FBS were compared in a state where the cells were not substantially in contact with heparin, that is, under conditions of low concentration heparin.

[Synthesis and purification of cDNA]
The mesenchymal stem cells cultured in the autoserum-containing medium according to the method of Example 1 and the mesenchymal stem cells cultured in the FBS-containing medium according to the method of Comparative Example 2 were separately adjusted to 1 × 10 ⁷ cells / sample. From each, total RNA was extracted using RNeasy Protect Min Kit (QIGEN, Cat. No. 74124). QIA shredder (QIAGEN, Cat. No. 79654) was used for cell destruction. The resulting RNA solution was adjusted to 0.5 μg / μl and RNA quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies).

GeneChip Eukaryotic Poly-A RNA Control Kit (AFFYMETRIX, P / N900433) and MessageAmpII-Biotin Enhanced Kit (Ambion, Catalog # 1791) ^was used to synthesize 1 st strand cDNA. Specifically, reaction solution (II) was added to reaction solution (I) reacted at 70 ° C. for 10 minutes, and reacted at 42 ° C. for 2 hours in a 20 μl reaction system.

Subsequently, MessageAmpII-Biotin Enhanced Kit (Ambion , Catalog # 1791) ^was used to synthesize and purify the 2 nd strand cDNA. Specifically, the reaction solution (III) is added to the reaction solution (II) after the reaction and reacted in a 100 μl reaction system at 16 ° C. for 2 hours, and finally, using the cDNA Filter Cartridge attached to the kit. The cDNA was purified by adding 24 μl of Nuclease-free Water to the Filter in two portions and eluting.

[IVT reaction]
IVT reaction and aRNA purification were performed using MessageAmpII-Biotin Enhanced Kit (Ambion, Catalog # 1791). Specifically, after reacting the reaction solution (IV) at 37 ° C. for 14 hours, 60 μl of Nuclease-free Water was added to stop the reaction, and finally using the aRNA Filter Cartridge attached to the kit, The aRNA was purified by adding 100 μl of Nuclease-free Water to the Filter and eluting. The resulting RNA solution was adjusted to 20 μg / 32 μl and RNA quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies).

(Preparation of Hybridization Cocktail)
Using a MessageAmpII-Biotin Enhanced Kit (Ambion, Catalog # 1791), aRNA was fragmented to prepare Hybridization Cocktail. Specifically, the reaction solution (V) was reacted at 94 ° C. for 35 minutes, and then a hybridization cocktail was prepared using GeneChip Expression 3′-Amplification Reagents Hybridization control kit (AFFYMETRIX, P / N900454). And then reacted at 45 ° C. for 5 minutes.

[Hybridase]
GeneChip Human Genome U133 Plus 2.0 Array (AFFYMETRIX, P / N900466) was filled with 1 × Hybridization buffer and prehybridization was performed at 60 rpm and 45 ° C. for 10 minutes. Then, 1x Hybridization buffer was removed, filled with the prepared Hybridization Cocktail, and hybridization was performed overnight at 60 rpm and 45 ° C.

[Washing and dyeing]
Set Wash Buffer A (6 × SSPE, 0.01% Tween-20), Wash Buffer B (100 mM MES, 0.1 M [Na +], 0.05% Tween-20) and Water in Fluidics Station, and use GCOS (GeneChip Operating Software) Priming was performed from the program. Next, except Hybridization Cocktail, the array filled with Wash Buffer A and SAPE Solution Mix (1 × Stain buffer, 2 mg / ml BSA, 10 μg / ml SAPE) and Antibody Solution Mix (1 × Stain buffer, 2 mg / ml BSA, 0.1 mg / ml Goat IgG Stock, 3 μg / ml biotinylated antibody) was set in Fluidics Station. Thereafter, washing and staining were performed by a GCOS program.

〔scan〕
Array was set in Gene Array Scanner, and scanning and analysis were performed. The analysis results are shown in Tables 1-5.

〔result〕
Table 1 shows cell surface antigens that were increased or decreased in common in five cases in FBS cultured cells and autologous serum cultured cells. Table 2 lists growth factor-related factors whose expression was increased or decreased in common in five cases. Table 3 shows cytokines whose expression was increased or decreased in common in the five cases. Further, Table 4 shows gene groups that have a difference of two times or more in expression level in common in five cases.

  As shown in Table 1, the differentiation marker CD24 expressed in the FBS cultured cells is not expressed in the autoserum cultured cells, indicating that the undifferentiated state is maintained. In addition, as shown in Tables 2 to 4, autologous serum cultured cells expressed a series of growth factors, and some growth factors showed increased expression compared to FBS cultured cells. Furthermore, as a result of this analysis, it was confirmed that a series of cancer-related genes were low and safety was high in cultured autoserum cells.

  From the above, it was confirmed that in the autoserum culture method of the present invention, cells can be cultured with high proliferation efficiency without using FBS, and cells that are safer and have higher differentiation ability can be obtained.

Example 5
The patient (52 years old, male) had ischemic neurological disease (cerebral infarction: right internal carotid artery occlusion) and developed left hemiplegia on February 4, 2007. On February 19, the same year, Sapporo Medical University Hospital I was transferred to the hospital. Symptoms before treatment: left hemiplegia, especially strong paralysis in the upper limbs; cannot open or hold hands at all; cannot hold or release objects (such as blocks); shoulder arms I couldn't raise it above the position; I couldn't bend or stretch my wrist. From this patient, mesenchymal stem cells were collected and proliferated as described in Example 1, and the total amount thereof was frozen storage solution (20.5 mL of normal filter-sterilized RPMI and 20.5 mL of autologous serum collected from the patient). Dextran 5 mL, DMSO 5 mL) was added to produce the therapeutic agent. In addition, it was confirmed in advance that the bone marrow fluid was not infected with HIV, ATL, HB, HC, syphilis, human parvovirus B19, etc. by examination of peripheral blood. This therapeutic agent was administered intravenously over 30 minutes to the patient on March 19th. No side effects were observed.

Results This patient had a motor dysfunction of all 5 fingers before cell therapy, but the left finger that did not move at all moves the next morning after cell administration so that it can be grasped and opened. became. One week later, motor function improved, and stake transportation was possible. Two weeks later, it was confirmed by MRI that the cerebral infarction was significantly reduced (FIG. 10). In addition, you can now hold and open your hands faster, and you can pinch and release blocks. The arm can be raised higher than the shoulder position and can be “banzai”. Elbows and wrists can now be bent and stretched. Changes in cerebral infarction level of this patient before and after cell therapy are well-known scales for evaluation of cerebral infarction (NIHSS: National Institutes of Health Stroke Scale, JSS: Stroke Scale of the Japan Stroke Society, MRS: Modified Rankine Scale) It shows in FIG.

  FIG. 10 is a brain MRI image of this patient. Reduction of the site (white part) damaged by cerebral infarction in the right cerebrum is observed. FIG. 12 is an image of cerebral blood flow of the same patient, but recovery of cerebral blood flow at the damaged site is observed one week after treatment. These results, together with the above-described recovery of motor function, demonstrated that the administration of this administration agent shows a marked improvement effect after the subacute phase. Together with the above recovery of motor function, these results demonstrated that the administration of the medicament of the present invention has a marked improvement effect on cerebral infarction after the subacute phase.

Example 6
The patient (fifties, female) has been suffering from Willis arterial occlusion (moyamoya disease) for a long time, thereby developing left hemiparesis. From this patient, mesenchymal stem cells were collected and cultured in the same manner as in Example 1, and intravenously administered to the patient about 2 months after the onset. Improvement in cerebral blood flow was confirmed by MRI (PWI) examination performed after administration.

Example 7
A patient (60s, male) developed left hemiplegia due to atherothrombosis. From this patient, mesenchymal stem cells were collected and cultured in the same manner as in Example 1, and intravenously administered to the patient 4 months after the onset. As a result, relaxation of stiffness and expansion of the range of motion of the joint were observed from the day after the administration, and muscle strength was remarkably recovered even after that, and grip strength could be measured (4 kg). Furthermore, about two and a half months after administration, he became able to walk alone, and his left hand recovered to a level where it could be used practically.

Example 8
The patient (50s, male) developed left hemiplegia due to atherothrombosis. From this patient, mesenchymal stem cells were collected and cultured in the same manner as in Example 1, and intravenously administered to the patient 6 weeks after the onset. As a result, finger movement improved from the next day of administration, muscle strength was remarkably recovered even after that, and grip strength could be measured (8 kg). In addition, more detailed work can be performed more quickly, and it becomes possible to split disposable chopsticks, and the power of the fingertips has been restored, enabling operations that are useful in daily life.

Example 9
A patient (60s, male) developed left hemiplegia due to atherothrombosis. From this patient, mesenchymal stem cells were collected and cultured in the same manner as in Example 1, and intravenously administered to the patient 5 weeks after the onset. As a result, the improvement of lower limb movement was noticed mainly from the night of the administration day, and the improvement of hand movement was also noticed the day after administration. Since then, motor function has continued to improve.

Example 10
The patient (70s, male) developed left hemiplegia and grammatical disorder due to lacunar infarction. From this patient, mesenchymal stem cells were collected and cultured in the same manner as in Example 1, and intravenously administered to the patient 8 weeks after the onset. As a result, the toes began to move from the morning after the administration, and improvements in shoulder and elbow movements were also observed. And 3 days after the administration, it recovered enough to move the finger of the hand.

Example 11
In the same manner as in Example 1, mesenchymal stem cells were collected from a chronic diabetic patient, cultured, and intravenously administered to the patient. As a result, the blood glucose level, which was about 190 mg / dl before administration, was improved to a normal level about 1 month after administration, and other diabetic indicators were also markedly improved (FIG. 13).

Example 12
In the same manner as in Example 1, mesenchymal stem cells were collected from a patient with prostatic hypertrophy, cultured, and intravenously administered to the patient. As a result, the PSA value, which is an index of prostate enlargement, was significantly improved after administration as compared to before administration (FIG. 14).

Example 13
In the same manner as in Example 1, mesenchymal stem cells were collected from a hepatic disorder patient, cultured, and intravenously administered to the patient. As a result, the γ-GTP value, GOT value, and GPT value, which are indicators of liver damage, were significantly improved after administration compared to before administration (FIG. 15).

Example 14
In the same manner as in Example 1, mesenchymal stem cells were collected from a renal disorder patient, cultured, and intravenously administered to the patient. As a result, the β _2- microglobulin level, which is an indicator of renal damage, was significantly improved after administration compared to before administration, and other renal injury indices (BUN value and / or creatinine level) were also observed. A significant improvement was seen (FIG. 16).

Example 15
Mesenchymal stem cells were collected and cultured from a hyperlipidemic patient in the same manner as in Example 1 and intravenously administered to the patient. As a result, the triglyceride level, which is an index of hyperlipidemia, markedly improved after administration compared to before administration, and other hyperlipidemia indicators also showed marked improvement. (FIG. 17).

Example 16
Mesenchymal stem cells were collected and cultured from patients with higher brain dysfunction / aphasia in the same manner as in Example 1 and intravenously administered to the patients. Before and after administration, standard aphasia test (SLTA) and Wexler adult intelligence test (WAIS-R) were performed. As a result, the SLTA and WAIS-R values were significantly improved compared with those before administration (FIG. 18).

Example 17
Mesenchymal stem cells were collected and cultured from healthy human subjects in the same manner as in Example 1. The aforementioned human mesenchymal stem cells were prepared for a rat cerebral infarction model (middle cerebral artery occlusion model). Next, (i) non-transplanted group, (ii) cells (1.0 × 10 ⁶ ) intravenous group, (iii) Angiopoietin gene-introduced cells (1.0 × 10 ⁶ ) intravenous group, (iv) VEGF gene-introduced cells ( The treatment was divided into 1.0 × 10 ⁶ ) intravenous injection group, (v) angiopoietin and VEGF gene-introduced cells (1.0 × 10 ⁶ ) intravenous injection group, and the therapeutic effect was compared. As a result of evaluating the therapeutic effect by MRI (FIG. 19A), (ii), (iii), and (v), a therapeutic effect was observed. The strength of the therapeutic effect was (v)> (iii) > (Ii).

In addition, as a result of analyzing the therapeutic effect from the aspect of angiogenesis (FIG. 19B), (ii), (iii), (iv), and (v) showed a therapeutic effect, but the strength of the therapeutic effect (V)>(iii)>(iv)> (ii).
In addition, as a result of analyzing the therapeutic effect from the behavioral side using the Treadmill stress test (FIG. 20), (ii), (iii), (v), the therapeutic effect was seen. The strength was (v)>(iii)> (ii).

Example 18
Mesenchymal stem cells were collected and cultured from healthy human subjects in the same manner as in Example 1. The above-mentioned human mesenchymal stem cells (1.0 × 10 ⁶ ) were intravenously administered to a rat cardiopulmonary arrest model to determine the therapeutic effect. Apoptosis was suppressed by treatment (the number of Tunnel-positive cells decreased) (FIG. 21), and the number of surviving neurons was also large (FIG. 22). Moreover, as a result of evaluating the higher brain function by the Morris water maze test, the improvement was seen in the treatment group.

Example 19
Mesenchymal stem cells were collected and cultured from healthy human subjects in the same manner as in Example 1. A spinal cord injury model was created using NYU imactor on rat spinal cord (Th 11) after Laminectomy, and the above-mentioned human mesenchymal stem cells (1.0 x 10 ⁶ ) after 6 hours, 1 day, 3 days, 7 days, and 14 days later Were transplanted from the femoral vein, and the movement was evaluated over time by a treadmill test. In other words, running a rat for 20 minutes a day for 2 days a week on a treadmill that moves at a speed of 20 m / min that is designed to receive an electric shock when you stop running in advance of the creation of a cerebral infarction. After training, the degree of recovery over time in each group was graphed. The horizontal axis is time, and the vertical axis is maximum speed. A marked therapeutic effect was seen in the groups transplanted 6 hours and 1 day after spinal cord injury (FIG. 23).

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

  The present invention enables rapid provision of a therapeutic agent having a remarkable effect on tissue repair / regeneration. By speeding up the provision of the therapeutic agent, a disease in which early cell therapy is particularly effective, such as cranial nerves, can be provided. It has a tremendous effect on the regeneration of cranial nerves of patients who have been damaged, and further reduces the shortage and physical burden of cell donors. The pharmaceutical produced by the method of the present invention is not only effective as a therapeutic agent, but is rapidly provided to amplify the therapeutic effect, significantly improve the patient's QOL, and reduce the burden on the caregiver and the cost of care. By reducing it, it will lead to a reduction in social burden, and will shed light on an aging society.

Claims (16)

  A cell characterized in that at least 90% of the positive CD antigen group described in Table 1 is expressed and at least 90% of the negative CD antigen group is not expressed.   The amount of anticoagulant added to the collected sample is less than 0.2 U / mL with respect to the volume of the sample, so that cells are collected from bone marrow or blood without substantial contact with the anticoagulant. The cell according to claim 1, which is obtained by culturing and growing the cultured cell with the amount of the anticoagulant present in the medium being less than 0.02 U / mL relative to the volume of the medium.   The cell according to claim 2, which is cultured in a medium containing allogeneic serum.   The cell according to any one of claims 2 and 3, wherein an anticoagulant is not added to the collected sample.   The cell according to any one of claims 3 and 4, wherein the allogeneic serum is autologous serum of a subject from whom the cells were collected.   The cell according to any one of claims 1 to 5, wherein the cell is a stem cell.   The cell according to any one of claims 1 to 6, wherein the stem cell is a mesenchymal stem cell.   The cell according to any one of claims 1 to 7, wherein the cell is a human cell.   The cell according to any one of claims 2 to 8, wherein the cell is grown in an undifferentiated state.   EWI-FLI-1, FUS-CHOP, EWS-ATF1, SYT-SSX1, PDGFA, FLI-1, FEV, ATF-1, WT1, NR4A3, CHOP / DDIT3, FUS / TLS, BBF2H7, CHOP, MDM2, CDK4, HGFR, c-met, PDGFα, HGF, GFRA1, FASN, HMGCR, RGS2, PPARγ, YAP, BIRC2, lumican, caldesmon, ALCAM, Jam-2, Jam-3, cadherin II, DKK1, Wnt, Nucleostemin, Neurofibromin, RB , CDK4, p16, MYCN, telomere, hTERT, ALT, Ras, TK-R, CD90, CD105, CD133, VEGFR2, CD99, ets, ERG, ETV1, FEV, ETV4, MYC, EAT-2, MMP-3, FRINGE , ID2, CCND1, TGFBR2, CDKNIA, p57, p19, p16, p53, IGF-1, c-myc, p21, cyclin D1, p21 The cell according to any one of claims 1 to 9.   A medicine for tissue repair / regeneration comprising the cell according to any one of claims 1 to 10.   Aging that is mesenchymal stem cells and is administered intravenously, with lumbar puncture, intracerebral, intracerebroventricular, topical, or intraarterial, to aid repair of the damaged site, or due to aging 12. A medicament according to claim 11 for assisting in the repair of a damaged site.   13. The medicine according to claim 12, wherein the damaged site or an age-aged site is a nervous system tissue, and is used for intravenous administration.   The medicine according to claim 13, wherein the nervous system tissue is a brain.   The medicament according to any one of claims 11 to 14, which is administered after the subacute phase of a disease or disorder.   The medicine according to any one of claims 12 to 15, which assists repair of an injured site by cytokine secretion, angiogenesis and / or nerve regeneration.

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