Method for culturing hematopoietic stem/progenitor cells

A technology of hematopoietic stem cells and a culturing method is applied in the field of in vitro expansion of human cord blood hematopoietic stem and progenitor cells by using an alginate three-dimensional culture system, which can solve the problems of expanding human hematopoietic stem cells, and achieves simple and fast operation process and enhanced interaction. , the effect of expanding human cord blood hematopoietic stem progenitor cells

Inactive Publication Date: 2009-12-09
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Application Information

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Problems solved by technology

The alginate three-dimensional system has been used in the culture of other kinds of cells before, but it mainly focuses on cells with adherent growth mode, and there is no report on the expansion of human hematopoietic stem cells

Method used

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  • Method for culturing hematopoietic stem/progenitor cells
  • Method for culturing hematopoietic stem/progenitor cells
  • Method for culturing hematopoietic stem/progenitor cells

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Experimental program
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Embodiment 1

[0030] ①Experimental materials

[0031] (1) Isolation of human umbilical cord blood mononuclear cells

[0032] Mononuclear cell isolation medium: Ficoll-Paque PLUS, Amersham Biosciences AB, Sweden

[0033] PBS: Gibco, Invitrogen

[0034] 0.6% ACD(A)-PBS: Dissolve 2g of sodium citrate, 0.8g of citric acid, and 2.23g of glucose in 1L of PBS, adjust the pH to 7.2, and filter to sterilize at 0.22μm.

[0035] Disposable blood collection bag: Shanghai Blood Transfusion Technology Co., Ltd.

[0036] (2) Alginate three-dimensional system packaging and cell recycling

[0037] Sodium alginate: Sigma-Aldrich, USA

[0038] Gelatin: Sigma-Aldrich, USA

[0039] Dissolution buffer: 50 mM sodium citrate, 0.45% NaCl, 10 mM HEPES, pH adjusted to 7.2, sterilized by 0.22 μm filter.

[0040] (3) Cell culture

[0041] Culture medium: IMDM (Iscove’s Modified Dulbecco’s Medium), Gibco Invitrogen, Fetal bovine serum (FBS): Gibco Invitrogen

[0042] Cytokines:

[0043] SCF (stem cell factor): ...

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Abstract

The invention belongs to the field of biotechnology, and relates to a method for expanding the hematopoietic stem / progenitor cells from umbilical cord blood in vitro by an alginate three-dimensional culture system. The method combines SCF, TPO and FL cytokines to support the expansion of the hematopoietic stem / progenitor cells in a single karyocyte of human umbilical cord blood, does not need adding other cytokines, and cultures the single karyocyte of human umbilical cord blood packaged in an alginate microsphere in a three-dimensional stationary culture system and a three-dimensional rotary culture system to observe the expansion effect of the single karyocyte of human umbilical cord blood packaged in the alginate microsphere in the rotary culture system. Compared with the conventional two-dimensional system, the three-dimensional culture system improves the total cells and obviously increases CD34+ cells under the condition of culturing low-concentration cytokines; and colony forming unit-granulocytes and macrophages of cells expanded in the three-dimensional culture system are cloned to form obviously increased cells, and the single karyocyte of human umbilical cord blood in a transplanted NOD / SCID mouse has better engraftment and reconstructs hemopoiesis.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for culturing hematopoietic stem and progenitor cells, in particular to a method for expanding human umbilical cord blood hematopoietic stem and progenitor cells in vitro using an alginate three-dimensional culture system. Background technique [0002] The current conventional culture system for expanding human hematopoietic stem and progenitor cells in vitro is to culture mononuclear cells or purified CD34+ cells from bone marrow, peripheral blood or umbilical cord blood in the form of single cell suspension culture in culture dishes or culture flasks. Hematopoietic stem cells are usually in a relatively dormant and inert state. A large dose of a combination of various cytokines needs to be added to the cell culture medium of conventional culture systems to stimulate hematopoietic stem cells to enter the proliferation cycle and induce cell division and expansion. Stud...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08
Inventor 袁燕谢毅
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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