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Stem cell preparation for treating aplastic anemia and preparation method thereof

A technology for aplastic and stem cell preparations, applied in animal cells, extracellular fluid diseases, vertebrate cells, etc., can solve the problem of low immunosuppressive ability, achieve strong anti-radiation response ability, strong cell activity, and enhance immunosuppression The effect of the function

Inactive Publication Date: 2015-12-30
奥思达干细胞有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to address the deficiencies in the prior art, to solve the specific problems in the prior art of the low immunosuppressive ability of mesenchymal stem cells for the treatment of aplastic anemia patients, and the defects of stem cell preparations for the treatment of aplastic anemia and their preparation methods. Infusion dosage, the specific influence of mesenchymal stem cell secretion ability and differentiation potential on hematopoietic stem cells, etc., the present invention provides a preparation method of stem cell preparation for treating aplastic anemia, which can make up for the deficiencies of the prior art and reduce the risk of graft-versus-host The incidence of disease, more mesenchymal stem cells with strong immunosuppressive function can be obtained in a short period of time, which can meet the clinical needs, provide a new idea for the transplantation effect of mesenchymal stem cells, and play an important practical application value

Method used

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  • Stem cell preparation for treating aplastic anemia and preparation method thereof
  • Stem cell preparation for treating aplastic anemia and preparation method thereof
  • Stem cell preparation for treating aplastic anemia and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] The preparation of embodiment 1 culture medium

[0045] 1. Preparation of medium A: prepare DMEM / F12 medium containing 10% fetal bovine serum and 1× double antibody;

[0046] 2. Preparation of medium B: preparation containing 75 μg / L gamma interferon (INF-γ), 75 μg / L tumor necrosis factor-α (TNF-α), 15 mg / L basic fibroblast growth factor (bFGF), 15 mg / L L epidermal growth factor (EGF), 5g / L bovine serum albumin (BSA), 1.5mg / L reduced glutathione, 1× insulin-transferrin-selenium-ethanolamine additive, 0.05mmol / L β-mercaptoethanol , 10mg / L hydrocortisone, 100nmol / L dexamethasone and 1% non-essential amino acid serum-free DMEM / F12 medium;

[0047] 3. Preparation of medium C: prepare human serum albumin with a mass volume ratio of 5%, low molecular weight heparin calcium with a mass volume ratio of 0.5%, and compound amino acids with a mass volume ratio of 10% with a mass volume ratio of 0.5% vitamin C, 20ng / ml interleukin 3 (IL-3), 20ng / ml recombinant human granulocyte c...

Embodiment 2

[0048] Preparation of embodiment 2 stem cells

[0049] The specific steps for the preparation of stem cells are as follows:

[0050] (1) Take P3-P6 mesenchymal stem cells derived from bone marrow, umbilical cord or placenta, and first conduct flow cytometric identification, in which markers CD29, CD44, CD90, CD105, and CD166 are expressed in more than 90%; CD34 is not expressed , CD45, below 5%;

[0051] (2) Put it in 2×10 4 The concentration of each / ml was mixed in medium A, and placed at 37°C, 5% CO 2 , Adhesive culture in a cell incubator with saturated humidity, change the medium on the second day of cultivation, and change the medium once every 2 days thereafter;

[0052] (3) When the above-mentioned cell culture confluence reaches 80%-90%, digest with 0.025% trypsin-EDTA at 37°C for 3-4 minutes, prepare a cell suspension, and dilute 2×10 4 The concentration of each / ml was mixed in medium B, placed at 37°C, 5% CO 2 , Adhesive culture in a cell incubator with saturate...

Embodiment 3

[0056] Example 3 Stem cells inhibit lymphocyte proliferation experiment

[0057] 1. Preparation of lymphocytes: use lymphocyte separation medium to separate peripheral blood, and obtain mononuclear cells 1×10 8 , the cells were resuspended in 50ml of DMEM / F12 culture medium containing 10% fetal bovine serum and 2mmol / L glutamine, adding anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody 100ng / ml each, interleukin 210ng / ml ml at 37°C, 5% CO 2 After culturing in the incubator for 5 days, the lymphocytes formed a large number of suspension colonies.

[0058] 2. Take the stem cell pellet cultured in the above-mentioned embodiment 2 and resuspend with DMEM / F12 culture medium containing 10% fetal bovine serum and 2mmol / L glutamine; adjust the concentration of the stem cell suspension to 2×10 5 / ml, placed in a 96-well cell culture plate at 100 μl / well, as the experimental group; meanwhile, the stem cells cultured without the steps described in Example 2 were taken as the ...

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Abstract

The invention provides a stem cell preparation for treating aplastic anemia and a preparation method thereof. The defects in the prior art are overcome, the occurrence rate of the graft versus host disease is decreased, more mesenchyme stem cells with the high immunosuppression function are obtained within a short period of time, the clinical requirements are met, a new thought is provided for the transplantation effect of stem cells, and important practical application value is brought into play. The invention further provides the preparation method of the stem cell preparation for treating aplastic anemia. According to the method, the mesenchymal stem cells are induced and cultivated through a special method, the immunosuppression capacity of the mesenchymal stem cells can be effectively improved and enhanced, and the mesenchymal stem cells can be further prepared into the stem cell preparation for treating aplastic anemia.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a stem cell preparation for treating aplastic anemia and a preparation method thereof. Background technique [0002] Aplastic anemia (AA, referred to as aplastic anemia) is a common autoimmune blood system disease. The mechanism involves abnormal hematopoietic microenvironment, hematopoietic stem / progenitor cell defect and immune dysfunction. The specific manifestations are peripheral blood pancytopenia, and bone marrow aspiration examination also shows three lineages of hematopoietic cells: neutrophils <0.5×10 9 / L, platelets <20×10 9 / L, reticulocytes <1%, non-hematopoietic cells increase, and the cytokines with increased expression include IFN-γ, IL-2, TNF-α, etc. AA is a severe disease with great harm, poor curative effect and high mortality. It is a refractory disease. [0003] At the same time, researchers generally believe that aplastic anemia is a kind of acquire...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/28A61K38/38C12N5/0775A61P7/06A61K31/375A61K31/727
Inventor 周萱王云娟
Owner 奥思达干细胞有限公司
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