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Method of prodcing megacaryocyle using umbilical blood CD 344+cell in vitro induction method

A technology for megakaryocytes and cell bodies, which is applied in the field of in vitro induction of megakaryocytes, can solve the problems of affecting the induction efficiency of megakaryocytes, limited number of megakaryocytes, and limited content.

Inactive Publication Date: 2004-12-22
上海伯瑞生物技术发展有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with bone marrow and mobilized peripheral blood, CD34 contained in cord blood + Cells are suitable starting cells for inducing megakaryocytes in vitro (Helen Tao, Leonie Gaudry, Alison Rice, et al. Cord blood is better than bonemarrow for generating megakaryocytic progenitor cells[J]. Experimental Hematology, 1999, 27: 293- 301.) These methods are directly from freshly isolated cord blood CD34 + cells set off to induce megakaryocyte production, as CD34 in cord blood + The content of cells is limited, and the absolute number is small. As the starting cells for inducing megakaryocytes, the number directly affects the induction efficiency of megakaryocytes, resulting in a limited number of induced megakaryocytes that cannot meet clinical requirements. Therefore, it is urgent to find A method to improve the cell effect of megakaryocyte expansion in vitro

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] After the umbilical cord blood was subjected to density gradient centrifugation with lymphocyte separation medium (Ficoll), mononuclear cells were collected, washed twice with IMDM medium, and the cells were suspended in IMDM medium at a concentration of 1×10 8 cells / 300uL, add 50uL magnetic bead-coupled mouse anti-human CD34 antibody, react at 4°C for 30 minutes, wash with PBS containing 2mM EDTA, 0.5% human serum albumin, and remove non-CD34 on the separation column in a magnetic field + After the cells were washed, CD34 cells were separated from the magnetic field with PBS containing 2mM EDTA and 0.5% human serum albumin. + Cells were eluted and resuspended in IMDM medium.

[0033] The isolated CD34 + Cells at 5 x 10 4 The inoculation density of cells / mL was inoculated into IMDM medium containing 10% fetal bovine serum, and 50 ng / mL of human stem cell factor (SCF), human thrombopoietin (TPO), and flt-3 ligand were added to the medium. At 37°C, 5% CO 2, in an incu...

Embodiment 2

[0035] After the umbilical cord blood was subjected to density gradient centrifugation with lymphocyte separation medium (Ficoll), mononuclear cells were collected, washed twice with IMDM medium, and the cells were suspended in IMDM medium at a concentration of 1.5×10 8 cells / 300ul, add 100ul magnetic bead-coupled mouse anti-human CD34 antibody, react at 4°C for 30 minutes, wash with PBS containing 2mM EDTA, 0.5% human serum albumin, and remove non-CD34 on the separation column in a magnetic field + After the cells were washed, CD34 cells were separated from the magnetic field with PBS containing 2mM EDTA and 0.5% human serum albumin. + Cells were eluted and resuspended in IMDM medium.

[0036] The isolated CD34 + Cells were inoculated into IMDM medium containing 10% fetal bovine serum, and human stem cell factor (SCF) 100 ng / mL, human thrombopoietin (TPO) 50 ng / mL, and flt-3 ligand 25 ng / mL were added to the medium. At 37°C, 5% CO 2 , in an incubator with 100% humidity, co...

Embodiment 3

[0038] After the umbilical cord blood was subjected to density gradient centrifugation with lymphocyte separation medium (Ficoll), mononuclear cells were collected, washed twice with IMDM medium, and the cells were suspended in IMDM medium at a concentration of 6×10 7 cells / 300ul, add 20uL magnetic bead-coupled mouse anti-human CD34 antibody, react at 4°C for 40 minutes, wash with PBS containing 2mM EDTA, 0.5% human serum albumin, and remove non-CD34 on the separation column in a magnetic field + After the cells were washed, CD34 cells were separated from the magnetic field with PBS containing 2mM EDTA and 0.5% human serum albumin. + Cells were eluted and resuspended in IMDM medium.

[0039] The isolated CD34 + Cells were inoculated into IMDM medium containing 10% fetal bovine serum, human stem cell factor (SCF) 100ng / mL, human thrombopoietin (TPO) 50ng / mL were added to the medium, placed at 37°C, 5% CO2, 100 % humidity of the incubator, after 7 days of culture, the cells we...

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Abstract

A process for generating megacaryocyte by in vitro induction to umbilical blood cell CD34+ includes in vitro amplifying the CD34+ in the culture medium containing fetal calf serum, human SCF, human TPO and / or ligand flt-3 and inducing the generation of megacaryocytes in the non-serum culture medium containing human TPO, human interleukin 3(IL-3) and / or human GM-CSF. Its advantage is high inducing efficiency.

Description

technical field [0001] The invention relates to a method for inducing megakaryocytes in vitro, in particular to a method for generating megakaryocytes from umbilical cord blood CD34 + A method for inducing megakaryocytes from cells in vitro. Background technique [0002] Megakaryotic hematopoiesis is a complex biological process initiated by pluripotent hematopoietic stem cells, through the formation of mature megakaryocytes, and finally platelets. [0003] The generation of megakaryocytes is generally divided into three developmental stages: progenitor cells, immature megakaryocytes (megakaryotic preblasts) and mature megakaryocytes. Some mitotic signals, such as cytokines and extracellular matrix, can promote the proliferation of megakaryotic progenitor cells. Megakaryotic preblasts are transient cells that act as a bridge between progenitor, mature, and postdividing cells. [0004] Megakaryopoiesis is a unique hematopoietic process in which the development of megakaryo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/08
Inventor 谭文松蔡海波顾小华
Owner 上海伯瑞生物技术发展有限公司
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