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Method for in vitro amplification of hemopoietic stem cells and precursor cells

A precursor cell and in vitro expansion technology, applied to animal cells, etc., can solve problems that hinder large-scale therapeutic applications, complex cell microenvironment, and limit the application of stem cells and precursor cells

Active Publication Date: 2014-04-30
杭州中赢生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This complex microenvironment also greatly hinders the definition of elements that affect the developmental stages of hematopoietic stem cells
Although first-generation stromal co-culture systems were able to sustain long-term production of stem cells, these stem cells are currently difficult to use for therapeutic purposes
First, the cellular microenvironment in this system is very complex and difficult to substantially improve
Second, the matrix that needs to be established in this system often takes several weeks, hindering the application of large-scale treatments
However, these perfusion bioreactor systems are simple modifications to liquid culture systems and are still unable to expand stem and precursor cells on a large scale
This also limits the use of stem and precursor cells in bone marrow transplantation and gene therapy

Method used

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  • Method for in vitro amplification of hemopoietic stem cells and precursor cells
  • Method for in vitro amplification of hemopoietic stem cells and precursor cells
  • Method for in vitro amplification of hemopoietic stem cells and precursor cells

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Stem cell expansion

[0039] Human bone marrow cells and human peripheral blood cells come from healthy donors. The method of extracting CD34-positive stem cells and precursor cells is well-known to those with basic skills in the industry. First, the method of density gradient centrifugation is used to separate human bone marrow cells and lymphocytes from human peripheral blood cells. CD34 positive bone marrow stem cells and precursor cells were further purified using antibody magnetic beads method.

[0040] After the stem cells are purified through this process, monitoring by flow cytometry shows that their purity exceeds 99%. The isolated and purified CD34-positive stem cells and precursor cells can be frozen and stored in liquid nitrogen for a long time (1 million-5 million cells / ml). Frozen cells can be quickly thawed in a 37°C water bath and washed twice with culture medium. Trypan blue staining shows that 99% of the cells are still alive.

[0041] The methy...

Embodiment 2

[0047] Example 2 Mice transplantation

[0048] Female mice aged 12-14 weeks (C57BL / 6) were purchased from Jackson Laboratory, USA. The animals are maintained on a 12-hour light / dark cycle. HUVEC cells were cultured for 7 days after adding IMDM. 10% inactivated fetal bovine serum (FBS) and 1% penicillin were added to IMDM.

[0049] Bone marrow cell expansion in vitro

[0050] Mouse stem cells are isolated from femoral bone marrow cells. The separated stem cells were seeded into a Petri dish full of HUVEC. After adding GM-CSF (5ng / ml), IL-3 (5ng / ml) at 37°C and 5% carbon dioxide for 7 days, the non-adherent cells were gently removed from the HUVEC layer. Wash with PBS containing 1% FBS and 1% penicillin, and concentrate.

[0051] The mice were sacrificed by cervical dislocation, and the femur and spleen were taken out. The cells in the femur were washed with 5 ml of PBS containing 10% heat-inactivated fetal bovine serum. Use a 25-gauge needle to break up the spleen tissue until t...

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Abstract

The invention discloses a method for in vitro amplification of stem cells, in particular a method for amplifying the hemopoietic stem cells and the precursor cells by co-culturing hemopoietic stem cells and precursor cells, which are separated from human peripheral blood or marrow, and endothelial cells. The method comprises the following steps of: separating the CD34 positive marrow stem cells and precursor cells; making the stem cells and the precursor cells directly contacted with the endothelial cells for culture; adding at least one type of cytokine to amplify the stem cells and the precursor cells; and recognizing the stem cells and the precursor cells by the cell surface antigens, and selecting the stem cells and the precursor cells, which have positive CD34 negative CD38, negative HLA-DR, negative CD15, negative Lin, positive c-kit, and no less than 85 percent purity. The method has the advantages of having good cell amplification effect, obviously reducing the drawing amount of the marrow and peripheral blood of a patient, lowering the necessity of general anaesthesia, enforcing quick recovery of hemopoiesis, and shortening the time when the patient stays in the hospital. The method has a wide application prospect in disease treatment.

Description

Technical field [0001] The present invention relates to a method for in vitro expansion of stem cells, in particular to a method for co-cultivating hematopoietic stem cells and precursor cells isolated from human peripheral blood or bone marrow with endothelial cells to expand hematopoietic stem cells and precursor cells. technical background [0002] The formation of mature blood cells is a very complicated process. The maturation of blood cells mainly occurs in the bone marrow. Hematopoietic stem cells proliferate and differentiate to produce different types of mature blood cells. Hematopoietic stem cells and precursor cells have extensive and long-term self-renewal capabilities and the ability to differentiate into all lymphocytes. CD34-labeled hematopoietic cell surface antigen is an important marker for hematopoietic stem cells and precursor cells. 1-5% of cells in bone marrow express CD34 antigen, and 0.1-0.5% of cells in peripheral blood express CD34 antigen. CD34 antig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07
Inventor 徐以兵吴忠福董升炬
Owner 杭州中赢生物医疗科技有限公司
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