CN102641293B - Preparation being used for the treatment of ischemic cerebrovascular and preparation method thereof - Google Patents
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Abstract
The present invention relates to a kind of preparation for the treatment of ischemic cerebrovascular and preparation method thereof, the preparation method of said preparation obtains mescenchymal stem cell (Mesenchymal by induced monocyte? stem? cells, MSC), and then cultivate above-mentioned MSC cell at low serum condition, finally be separated MSC cell and obtain cell-free medium, and corresponding post processing is carried out to this cell-free medium, described preparation can be obtained.In vitro and in vivo experiment shows, the preparation for the treatment of ischemic cerebrovascular of the present invention can promote the recovery of the corresponding function of the cerebral tissue of ischemic necrosis significantly.
Description
Technical Field
The invention relates to a preparation for treating ischemic cerebrovascular diseases and a preparation method thereof.
Background
Cardiovascular and cerebrovascular diseases are the diseases with the highest fatality rate and morbidity in the world. According to the statistical report of the heart society of the united states in 2010, about 1750 thousands of people die of various cardiovascular and cerebrovascular diseases in 2005 worldwide, accounting for about 30% of the total number of deaths. The number of deaths due to cardiovascular and cerebrovascular diseases is estimated to reach 2500 million worldwide by 2020. In 2004, 430 million people died from various cardiovascular and cerebrovascular diseases, up to 48% of the total death. Among the various cardiovascular and cerebrovascular diseases, thrombosis and vascular blockage caused by atherosclerosis are the most common and important pathogenesis. In the early stages of the disease, modification of low density lipoproteins by reactive oxygen species leads to progressive vascular endothelial dysfunction. The body's immune system produces chronic inflammation and continues to secrete leukocyte populations rich in T cells, macrophages, mast cells, and forms fatty streaks, fibrous plaques, and atheromatous plaques, narrowing the lumen of the blood vessel and resulting in reduced blood flow. When the final atheromatous plaque is broken, the dislodged plaque fragments can lead to platelet aggregation and the development of massive occlusive thrombosis and other secondary changes, including ischemia or necrosis of the tissues or organs served by the artery. The ischemic cardiovascular and cerebrovascular diseases caused by atherosclerosis mainly include coronary heart disease, ischemic stroke and peripheral occlusive arterial diseases. Ischemic stroke is the lack of blood supply to the brain caused by a thrombus (the brain forms an occluding clot), an embolism (the embolism forms from elsewhere, see below), systemic insufficiency (general systemic insufficiency, such as shock) or venous thrombosis, resulting in brain tissue dysfunction and necrosis. Worldwide, stroke patients are 1500 million people per year and result in 500 million lifelong disabilities and 570 million deaths, of which 87% are attributed to ischemic stroke. In the united states, on average, one person per 40 seconds can develop a stroke, and on average, one patient dies every 4 minutes. Stroke is also the 2 nd leading cause of death worldwide. Despite the traditional therapeutic approaches of conservative exercise, anticoagulant drugs, and surgery (carotid endarterectomy or carotid angioplasty), most patients with ischemic stroke caused by atherosclerosis still cannot be treated effectively, mainly because of the side effects of anticoagulant drugs and physical or disease conditions, and the like.
Stem cell therapy is a new direction that has been expected most recently in the field of treatment of cancer, cardiovascular and cerebrovascular diseases, and the like. Theoretically, stem cells can be used for the treatment of various diseases, but the most suitable diseases are mainly tissue necrosis diseases such as myocardial necrosis caused by ischemia, degenerative diseases such as parkinsonism, autoimmune diseases such as insulin-dependent diabetes mellitus, and the like. Especially has the special effects of promoting the regeneration of blood vessels and recovering the functions of organs, and has great curative effect in the treatment of cardiovascular and cerebrovascular diseases.
Mesenchymal Stem Cells (MSCs) are an adult stem cell with multipotent differentiation potential derived from early mesoderm. MSCs can theoretically differentiate into all mesoderm-derived cells such as endothelial cells, osteoblasts, adipocytes, chondrocytes, cardiomyocytes, nerve cells, etc., and have low immunological activity, and allograft transplantation does not generate immunological rejection. MSC is widely present in connective tissues and organs of the whole body, is most abundant in bone marrow tissues, can be separated from fetal umbilical cord blood, and is also present in placenta, amniotic fluid, umbilical vein subendothelial layer, peripheral blood, liver, fat, muscle, skin and other tissues. Under the appropriate condition, MSC can gather in the ischemic brain tissue of the rat with focal cerebral ischemia, and has the tendency of promoting the formation of new blood vessels in the ischemic brain region and improving the nerve function, the formation of the new blood vessels and the reconstruction of blood circulation are beneficial to the repair of the damaged and non-dead neurons in the ischemic penumbra region of the brain, and the MSC can migrate and differentiate the autologous nerve stem cells to the ischemic brain region and has important significance for the repair and reconstruction of the damaged nerve tissue.
The reason why MSCs promote angiogenesis in the ischemic region is that endothelial progenitor cells derived from MSCs differentiate into vascular endothelial cells and increase the expression of growth factors and cytokines (e.g., VEGF) in the ischemic region, thereby promoting angiogenesis in the ischemic region. In fact, the growth factors and cytokines secreted by the transplanted MSC involved in the repair of ischemic brain tissue can be obtained by the MSC in the conditioned medium secreted in the specific environment in vitro, and it is fully possible to apply the MSC in the repair and regeneration of damaged blood vessels and peripheral tissues, thereby restoring the corresponding functions of ischemic necrotic brain tissue. Therefore, the growth factors and cytokines secreted by the MSC in the in vitro artificial environment for promoting the repair or regeneration of blood vessels and peripheral tissues have great potential in clinic, and can be used as an effective substitute or an auxiliary drug of a stem cell therapy to be applied to the treatment of ischemic cerebrovascular diseases.
Disclosure of Invention
The invention aims to provide a preparation capable of effectively treating ischemic cerebrovascular diseases, which is used for overcoming the defects of the existing medicines and providing a new treatment medicine for clinically treating the ischemic cerebrovascular diseases.
The ischemic cerebrovascular disease mainly refers to diseases of vessel blockage caused by atherosclerosis, and specifically comprises the following steps: apoplexy, cerebral ischemia, cerebral infarction, brain atrophy and infarction type dementia caused by cerebral arteriosclerosis.
The present invention also aims to provide a method for preparing a preparation effective for treating ischemic cerebrovascular diseases, which comprises the steps of:
1) obtaining peripheral blood mononuclear cells from blood of a healthy person by leukocyte replacement (leukapheresis), or obtaining bone marrow mononuclear cells from bone marrow (by means of density gradient centrifugation, optional steps), or directly extracting MSCs from a human liposuction (lipoaspirate);
2) screening and culturing peripheral blood or bone marrow mononuclear cells to obtain MSC cells;
3) culturing MSC cells under specific conditions to secrete growth factors and cytokines for promoting tissue repair and regeneration, separating the MSC cells from a culture medium to obtain a cell-free culture medium rich in angiogenesis promoting factors and cytokines;
4) post-treating the cell-free medium obtained in step 3), the post-treating step comprising: filtering cell debris, purifying the cell-free culture medium, detecting the components and the content of each effective growth factor, and carrying out freeze-drying treatment or cryopreservation on the culture medium to obtain the preparation for treating the ischemic cerebrovascular disease.
In the method for preparing the preparation capable of effectively treating ischemic cerebrovascular diseases, the source of the healthy human blood or bone marrow in the step 1) can be autologous or allogeneic, and the obtaining way can be direct bone marrow extraction or peripheral blood extraction, or a healthy human peripheral blood leukocyte suspension sample directly purchased from a blood bank, or clinical leukocyte replacement. The step of gradient centrifugation to obtain monocytes as described in the optional step is: subjecting the bone marrow or peripheral blood obtained to gradient centrifugation in a density gradient medium, such as Ficoll-Paque (GEhealthcare), Histopaque-1077(Sigma), or the like, preferably Histopaque-1077; the temperature range suitable for use is from 15 to 25 deg.C, preferably 25 deg.C. The specific operation is as follows: centrifuging container containing bone marrow or peripheral blood and gradient agent at 200-500 g for 20-40 min, layering, sucking the opaque layer with sterile disposable needle tube to obtain suspension rich in mononuclear cells, which is identified to be derived from multiple cell subsets contained in marrow stem cells and present polymorphism.
In the method for preparing the preparation capable of effectively treating ischemic cerebrovascular diseases, when culturing the mononuclear cells in the step 2) to obtain the MSC cells, the used culture medium can be one of M119, DMEM, F12 and RPMI-1640, and heparin (0-100U/ml) can be added. The culture medium can also contain 5 to 20 mass percent of fetal bovine serum or human serum albumin or autologous serum. Culturing in a cell culture box with the preset conditions of the culture temperature of 37 ℃ and the carbon dioxide concentration of 5-7%.
In the method for preparing the preparation capable of effectively promoting the treatment of ischemic cerebrovascular diseases, the step of culturing the mononuclear cells to obtain the MSC cells in the step 2) is one of the following methods (i) to (iv):
method ① monocyte is cultured at 5 × 10 per square centimeter5To 2 × 106Culturing for 1-2 days, removing suspension cells, and culturing adherent cells for 7-21 days. The obtained type I MSC is in a spindle shape,has typical MSC characteristics, namely expresses a large amount of cell surface receptors CD90, CD105 and CD73, and does not contain hematopoietic stem cells such as CD14, CD34, CD45 and the like and monocyte receptors.
Method ② includes culturing monocytes in a culture medium with 1000mg of fibrin microspheres (50-250 μm in diameter, commercially available, such as Forticell bioscience) for 1-2 days to adsorb 1 × 106To 1 × 108Density of individual cells, suspension cells were removed, and cells attached to the fibrin microspheres were cultured for 7-21 days. The obtained type I MSC is spindle-shaped, has typical MSC characteristics, namely expresses a large amount of cell surface receptors CD90, CD105 and CD73, and does not contain hematopoietic stem cells such as CD14, CD34, CD45 and the like and monocyte receptors.
Method ③ magnetic bead sorting of monocytes by CD14, or CD45 specific antibodies (Manufactured by miltenyi biotec, germany), a subpopulation of monocytes not containing CD14 or CD45 was selected and the CD14 was isolated-Or CD45-Monocyte subpopulation at 5 × 10 per square centimeter5To 2 × 106The density of individual cells is cultured for 1-2 days (or cultured by ②), suspension cells are removed, and adherent cells (or attached to fibrin microspheres) are cultured for 7-21 days, the obtained type II MSCs are spindle-shaped, have typical MSC characteristics, i.e., express a large amount of cell surface receptors CD90, CD105 and CD73, and do not contain hematopoietic stem cells such as CD14, CD34, CD45 and monocyte receptors.
Method ④ comprises washing human liposuction extract (about 50-100ml) in Phosphate Buffered Saline (PBS) with pH 7.4 or 0.9% physiological saline, digesting with collagenase type I and type II (0.05% -0.1%), shaking at 37 deg.C for 30-60 min, and dispersing the digested mononuclear cells at a concentration of 5 × 10/sq cm5To 2 × 106Culturing the cells at a density of 1-2 days (or using ②), and culturing the adherent cells (or attached to the fibrin microspheres) for 7-21 days. The obtained type III MSC is spindle-shaped, has typical MSC characteristics, namely expresses a large amount of cell surface receptors CD90, CD105 and CD73, and does not contain hematopoietic stem cells such as CD14, CD34, CD45 and the like and monocyte receptors.
In the method for preparing the preparation capable of effectively treating ischemic cerebrovascular diseases, the method for culturing the MSC cells under the specific conditions in the step 3) is one of the following methods (i) or (ii):
the method comprises the following steps: culturing the type I, II, III MSCs obtained in step 2) in a medium without any growth factor in an atmosphere of 0.5% to 2% oxygen at 37 ℃ for 1 to 3 days in the presence of M119, DMEM, RPMI-1640, F12, phosphate buffer (pH 7.4), or 0.9% physiological saline for medical use, and optionally adding 1% human serum albumin or autologous serum for medical use.
The method II comprises the following steps: culturing the type I, II, III MSC obtained in step 2) in a vascular endothelial cell growth promoting medium (M119, DMEM, F12, RPMI-1640, EBM-2, optionally supplemented with 0.5-1% of one of growth factor additives EGM, EGM-MV, EGM-2 or EGM2-MV (commercially available from Lonza of Switzerland, wherein the growth factor comprises vascular endothelial growth factor VEGF-1, basic fibroblast growth factor FGF-2, epidermal growth factor EGF, and insulin-like growth factor IGF-1) for 1-2 days; or adding ECGF (10-100 μ g/ml); and optionally heparin (0-100U/ml); the culture medium can also contain 5 to 20 mass percent of fetal bovine serum or human serum albumin or autologous serum. After 1 to 2 days, MSCs were transferred to a medium without any growth factor, and cultured in an atmosphere of 0.5% to 2% oxygen at 37 ℃ for 1 to 3 days in the presence of M119, DMEM, RPMI-1640, F12, phosphate buffer (pH 7.4) or 0.9% physiological saline for medical use, and 1% human serum albumin or autologous serum for medical use was added.
After the culture is completed according to the above-mentioned method (i) or (ii), the conditioned medium rich in cell growth factors is collected, and the MSC cells are discarded.
In the method for preparing the preparation capable of effectively treating ischemic cerebrovascular diseases, the post-treatment step of the step 4) comprises the following steps:
filtering the cell-free culture medium collected in the step 3), and removing cell impurities and debris by high-speed centrifugation or a filter.
② identifying the components of the filtered cell-free medium and the content of representative pro-vascular and peripheral tissue growth factors and cytokines such as MCP-1, EGF, MMP-9, MMP-2, PDGF, SDF-1, FGF, VEGF contained therein, by proteomics, cytokine arrays, enzyme linked immunosorbent assay (ELISA), or Bio-PlexTMAnd (4) testing the cell factors.
Freeze drying or packing and freeze preserving the filtered cell-free culture medium for long term preservation of the activity of the growth factor and the cell activity component.
Drawings
FIG. 1: the preparation for treating ischemic cerebrovascular diseases has the influence on the proliferation of the brain microvascular endothelial cells of rats.
FIG. 2: the preparation for treating ischemic cerebrovascular diseases has the effects of activating rat brain microvascular endothelial cells, promoting migration and repairing injury.
Detailed Description
The present invention is further illustrated by the following specific examples, which include, but are not limited to, the following steps and contents.
Example 1 preparation of monocytes.
The extracted bone marrow or a suspension of leukocytes from healthy human peripheral blood directly purchased from a blood bank was added to a density gradient reagent Histopaque-1077(Sigma) in an amount of 30mL per 15mL of the suspension of bone marrow or peripheral blood leukocytes. The bone marrow extract or the peripheral blood leukocyte suspension is centrifuged at the speed of 400G for 30 minutes at normal temperature in the presence of a gradient agent. After layering, the opaque layer is absorbed by a sterile disposable needle tube, namely the suspension liquid rich in the marrow mononuclear cells.
Example 2 acquisition of MSC cells.
The method for obtaining class I MSC cells comprises adding bone marrow or peripheral blood mononuclear cells into DMEM culture solution containing 10% human serum albumin or autologous serum at a ratio of 1 × 10 per square centimeter6Density culture of individual cells for 2 days, removal of suspension cells, and continuous culture of adherent cells for 21 days. The obtained type I MSC is spindle-shaped, has typical MSC characteristics, namely expresses a large amount of cell surface receptors CD90, CD105 and CD73, and does not contain hematopoietic stem cells such as CD14, CD34, CD45 and the like and monocyte receptors.
The method for obtaining class II MSC cells comprises adding bone marrow or peripheral blood mononuclear cells into DMEM culture solution containing 10% human serum albumin or autologous serum at a ratio of 1 × 10 per square centimeter6Density culture of individual cells for 2 days, magnetic bead sorting by CD14, or CD45 specific antibodies (Manufactured by miltenyi biotec, germany), a subpopulation of monocytes not containing CD14 or CD45 was selected and the CD14 was isolated-Or CD45-Monocyte subpopulation in DMEM supplemented with 10% human serum albumin or autologous serum at 1 × 10 per square centimeter6Density culture of individual cells for 2 days, removal of suspension cells, and continuous culture of adherent cells for 21 days. The obtained type II MSCs are spindle-shaped, have typical MSC characteristics, namely express a large amount of cell surface receptors CD90, CD105 and CD73, and do not contain CD14, CD34 and CD45 hematopoietic stem cell and monocyte receptor.
The method for obtaining class III MSC cells comprises washing human liposuction (about 50-100ml) in medical normal saline with pH 7.4 and 0.9%, digesting with collagenase type II (mass concentration 0.075%), shaking at 37 deg.C for 30 min, and adding the digested mononuclear cells into DMEM culture solution containing 10% human serum albumin or autologous serum at a concentration of 1 × 10/cm6The density of individual cells was cultured for 2 days, and adherent cells were further cultured for 21 days. The obtained type III MSC is spindle-shaped, has typical MSC characteristics, expresses a large amount of cell surface receptors CD90, CD105 and CD73, and does not contain hematopoietic stem cells such as CD14, CD34, CD45 and the like and monocyte receptors.
Example 3 acquisition of an agent for treating ischemic cerebrovascular disease.
Each type of MSC obtained in example 2 was cultured in 0.9% physiological saline for medical use in an environment of 0.5% oxygen concentration for 2 days (2 × 10 per square centimeter) in a medium without any growth factor5Individual cells) and 1% of human serum albumin or autologous serum for medical use may be added. And collecting a cell-free culture medium rich in cell growth factors after the culture is finished, and discarding adherent MSC cells. Removing cell impurities and debris from the collected cell-free culture medium by a filter with a pore size of 0.2 micrometer, and subpackaging in a refrigeration house at-80 deg.C for cold storage. As the case may be, every 1x1062-5ml of the preparation for promoting the repair or regeneration of blood vessels and peripheral tissues can be prepared by each MSC cell.
Example 4 identification of growth factors and cytokines in formulations for treating ischemic cerebrovascular disorders.
The growth factors and cytokine components contained in the preparation for treating ischemic cerebrovascular diseases prepared in example 3 were identified by cytokine array (purchased from R & DSystems), and the effective components of the preparation for treating ischemic cerebrovascular diseases include but are not limited to the following growth factors: MCP-1, EGF, IL-6, IL-8, MMP-9, SDF-1, HGF, VEGF, and PDGF. Measured by enzyme-linked immunosorbent assay (ELISA), wherein the content of active ingredients is, MCP-1: 5-50 ng/ml; IL-8: 1-5 mug/ml; SDF-1: 0.5-5 ng/ml; IL-6: 5-20 ng/ml; PDGF-BB: 0.1-10 ng/ml; VEGF: 1-20 ng/ml. The other composition is shown in Table 1.
TABLE 1 ingredient list of the preparation for treating ischemic cerebrovascular disease according to the present invention (including but not limited to the following ingredients)
| ANG-1 | IGF-II | MCP-4 | SDF-1 |
| ANG-2 | IL-1 | M-CSF | Sfrp |
| bFGF | IL-11 | MMP-13 | TB4 |
| b-NGF | IL-12 | MMP-2 | TGFbeta |
| EGF | IL-6 | MMP-9 | TIMP-1 |
| FGF-7 | IL-7 | PA | TNFalpha |
| G-CSF | IL-8 | PDGF | TSP-1 |
| GM-CSF | LIF | PIGF | TSP-2 |
| HGF | MCP-1 | RANTES | VEGF |
| IGF-I | MCP-2 | SCF | VEGF-D |
Example 5 in vitro assay of the effect of an agent for the treatment of ischemic cerebrovascular disease on the proliferation of brain microvascular endothelial cells in rats.
Rat brain microvascular endothelial cells (rBCEC), commercially available, e.g., CellAplicationInc., were prepared at 5 × 10 per well3The density of each cell was inoculated into a 96-well plate, cultured for 18 hours in EBM-2 medium containing 10% human serum albumin and added with 1% growth factor additive EGM (purchased from Lonza, Switzerland, containing vascular endothelial growth factor VEGF-1, basic fibroblast growth factor FGF-2, epidermal growth factor EGF, and insulin-like growth factor IGF-1), discarded, placed again in the agent for treating ischemic cardiovascular diseases or negative control medium (PBS containing 1% human serum albumin alone, pH 7.4) described in the present invention, cultured for 24 hours in a cell culture chamber at 37 ℃ and 5% carbon dioxide concentration, and then cultured in a cell culture chamber at 5% carbon dioxide concentrationThe NF cell proliferation kit (purchased from Invitrogen) measures the viable cell content (see fig. 1 for results). The result shows that the preparation for treating ischemic cardiovascular diseases can obviously increase the survival and proliferation rate of rBCEC cells in a low serum environment, and the number of the rBCEC cells cultured in the preparation for treating ischemic cardiovascular diseases is 2.60 +/-0.75 times (p is less than 0.05) of that of a negative control group.
Example 6 in vitro testing of the activation and migration promoting and injury repairing effects of the preparation for treating ischemic cerebrovascular diseases on rat brain microvascular endothelial cells.
Rat brain microvascular endothelial cells were plated at 5 × 10 per well4The density of individual cells was inoculated into 24-well plates, cultured for 24 hours in EBM-2 medium containing 10% human serum albumin and growth factor additive EGM (produced by Lonza, Switzerland, containing 0.5mL of vascular endothelial growth factor VEGF-1, 2mL of basic fibroblast growth factor FGF-2, 0.5mL of epidermal growth factor EGF and 0.5mL of insulin-like growth factor IGF-1), the original medium was discarded and a cell blank area (i.e., damaged area) was drawn on the cell layer of the plate bottom with a pipette tip, and the cells were replaced with the agent for treating ischemic cerebrovascular disease or the negative control medium (i.e., PBS containing only 1% human serum albumin, pH 7.4) described in the present invention, and the degree of healing (i.e., damaged area ratio; results are shown in FIG. 2) of the scratch healing was measured after culturing for 10 hours. The result shows that the preparation for treating ischemic cerebrovascular diseases can remarkably activate the repair and migration speed of rat brain microvascular endothelial cells to the damaged area in a low serum environment, and compared with the scratch area (27.1 +/-1.39%) reduced by a negative control group, the preparation for treating ischemic cerebrovascular diseases reduces the scratch area (p < 0.05) by 86.3 +/-4.7%.
Claims (5)
1. A preparation method of a preparation for treating ischemic cerebrovascular diseases is characterized by comprising the following steps:
1) obtaining peripheral blood mononuclear cells from blood of healthy people through leukocyte replacement, or obtaining bone marrow mononuclear cells from bone marrow through a density gradient centrifugation method, or directly extracting MSC from human fat aspirate;
2) screening and culturing peripheral blood or bone marrow mononuclear cells to obtain MSC cells;
3) culturing MSC cells under specific conditions to secrete growth factors and cytokines for promoting tissue repair and regeneration, separating the MSC cells from a culture medium to obtain a cell-free culture medium rich in angiogenesis promoting factors and cytokines;
4) post-treating the cell-free medium obtained in step 3), the post-treating step comprising: filtering cell debris, purifying the cell-free culture medium, detecting the components and the content of each effective growth factor, and carrying out freeze-drying treatment or cryopreservation on the culture medium to obtain the preparation for treating the ischemic cerebrovascular disease;
wherein,
the source of the healthy human blood or bone marrow in the step 1) is autologous source or allogeneic source, and the obtaining way is direct bone marrow extraction or peripheral blood extraction, or a healthy human peripheral blood leukocyte suspension sample directly purchased from a blood bank, or clinical leukocyte replacement;
the method for obtaining the peripheral blood single cells from the blood or the bone marrow through density gradient centrifugation in the step 1) comprises the following steps: using a gradient agent of Ficoll-Paque, Histopaque-1077 or other similar products, controlling the temperature at 15-25 ℃, the centrifugal force at 200-500 g for 20-40 minutes, and sucking an opaque layer after the centrifugation is finished to obtain the monocyte suspension;
when culturing the mononuclear cells in the step 2) to obtain MSC cells, adding 0-100U/ml heparin into one of M119, DMEM, F12 and RPMI-1640 as a culture medium, adding 5-20% by mass of fetal bovine serum or human serum albumin or autologous serum into the culture medium, and culturing in a cell culture box at a preset condition of a culture temperature of 37 ℃ and a carbon dioxide concentration of 5-7%;
the method for culturing the mononuclear cells to obtain the MSC cells in the step 2) is one of the following methods (i), (ii), (iii) or (iv):
method ① monocyte is cultured at 5 × 10 per square centimeter5To 2 × 106Performing density culture on each cell for 1-2 days, removing suspended cells, and continuously culturing adherent cells for 7-21 days to obtain type I MSC;
method ② includes culturing monocyte and fibrin microsphere for 1-2 days, wherein 1000mg of the microsphere can adsorb 1 × 106To 1 × 108Removing suspended cells according to the cell density, and continuously culturing the cells attached to the fibrin microspheres for 7-21 days to obtain I-type MSC;
③ method comprises selecting monocyte by magnetic bead sorting with CD14 or CD45 specific antibody, screening monocyte subpopulation without CD14 or CD45, and screening the subpopulation with CD14-Or CD45-Monocyte subpopulation at 5 × 10 per square centimeter5To 2 × 106Performing density culture for 1-2 days or using ② method, removing suspension cells, and continuously culturing cells adherent to or attached to the fibrin microspheres for 7-21 days to obtain type II MSC;
method ④ comprises washing 50-100ml of human fat extract in phosphate buffer solution with pH of 7.4 or 0.9% medical normal saline, digesting with collagenase type I and type II with concentration of 0.05% -0.1%, shaking at 37 deg.C for 30-60 min, and dispersing the digested mononuclear cells at 5 × 10/sq cm5To 2 × 106Culturing the cells at a density for 1-2 days or using a method ②, and continuously culturing adherent or attached fibrin microballon cells for 7-21 days to obtain type III MSC;
the method for culturing the MSC cells under the specific conditions described in the step 3) is one of the following methods (i) or (ii):
the method comprises the following steps: placing the type I, II, III MSCs obtained in step 2) in a medium without any growth factor, culturing for 1 to 3 days in an environment with an oxygen concentration of 0.5% to 2% and a temperature of 37 ℃, using M119, DMEM, RPMI-1640, F12, a phosphate buffer solution with a pH of 7.4 or 0.9% of a physiological saline solution for medical use, and adding 1% of human serum albumin for medical use or autologous serum;
the method II comprises the following steps: culturing the type I, II and III MSCs obtained in the step 2) in a culture medium for promoting the growth of vascular endothelial cells for 1-2 days, wherein the culture medium is one of M119, DMEM, F12, RPMI-1640, EBM and EBM-2, and is added with 0.5-1% of one of growth factor additives EGM, EGM-MV, EGM-2 or EGM 2-MV; or adding 10-100 μ g/ml ECGF; 0-100U/ml heparin is added; the culture medium also contains 5-20% by mass of fetal bovine serum or human serum albumin or autologous serum, 1-2 days later, MSC is transferred into a culture medium without any growth factor, and is cultured for 1-3 days in an environment with oxygen concentration of 0.5-2% and temperature of 37 ℃, the used culture medium is M119, DMEM, RPMI-1640, F12, phosphate buffer solution pH 7.4 or 0.9% medical physiological saline, and 1% medical human serum albumin or autologous serum is added.
2. The method according to claim 1, wherein 1% of human serum albumin or autologous serum for medical use is not added.
3. The method according to claim 1, wherein the MSC cells are type I MSC cells.
4. A formulation obtainable by the process of any one of claims 1 to 3.
5. Use of the formulation of claim 4 for the manufacture of a medicament for the treatment of ischemic cerebrovascular disease.
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