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Method of simulating bone marrow microenvironment to culture hematopoietic stem cells

A technology of hematopoietic stem cells and microenvironment, applied in the field of cell engineering, can solve the problems of poor cell activity, low proliferation rate, and easy loss of stem cell characteristics of hematopoietic stem cells, and achieve the effect of promoting growth and proliferation and increasing the contact area.

Inactive Publication Date: 2016-02-10
深圳华毓造血干细胞研究有限公司
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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a method that can improve hematopoietic stem cells in view of the disadvantages of poor cell activity, low proliferation rate, and easy loss of stem cell characteristics of hematopoietic stem cells in the prior art using a two-dimensional induction culture system to induce hematopoietic stem cells. Method for cultivating hematopoietic stem cells in a simulated bone marrow microenvironment capable of maintaining activity and proliferation rate and maintaining stem cell characteristics

Method used

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Embodiment 1

[0059] The method for culturing hematopoietic stem cells in a simulated bone marrow microenvironment provided by the present invention specifically includes the following steps:

[0060] S1a, obtaining bone marrow mesenchymal stem cells;

[0061] Bone marrow samples were collected and anticoagulated with heparin. Each sample was added to percoll separation solution with a density of 1.073g / ml at a ratio of 1:2, centrifuged at 2300rpm for 30min, the middle layer of mononuclear cells was taken, added with PBS buffer, centrifuged at 1000rpm for 10min, washed 3 times, and discarded. The serum is obtained to obtain bone marrow mesenchymal stem cells.

[0062] S2a, preparation of biologically derived bone;

[0063] The cancellous bone of the metaphysis of the animal long bones was taken and processed into 1cm×0.5cm×0.5cm bone blocks, and the mass fraction of the bone blocks was about 30%H. 2 O 2 The solution was soaked at 38°C for 72h, changed every 24h, soaked in double-distill...

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Abstract

The invention relates to a method of simulating bone marrow microenvironment to culture hematopoietic stem cells. The method includes following steps: acquiring hematopoietic stem cells, and inoculating the hematopoietic stem cells onto bio-derived bone containing osteoblasts for in-vitro amplification culture. The bio-derived bone containing the osteoblasts is adopted as a three-dimensional culture scaffold, and the hematopoietic stem cells and the osteoblasts are co-cultured in the bio-derived bone, so that contact area between the hematopoietic stem cells and a culture medium as well as osteoblast juxtacrine is increased, and growing and proliferating of the hematopoietic stem cells are facilitated; the osteoblasts can secrete factors conducive to the hematopoietic stem cells to maintaining characteristics, and the hematopoietic stem cells after amplification can still have potential of multidirectional differentiation, so that the problems of low cell activity and low proliferation rate when a two-dimensional culture system is adopted to culture the hematopoietic stem cells and the problem that the hematopoietic stem cells are prone to losing the stem cell characteristics in the prior art are solved.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a method for culturing hematopoietic stem cells by simulating a bone marrow microenvironment. Background technique [0002] At present, hematopoietic stem cells usually come from three sources: bone marrow, peripheral blood and umbilical cord blood. Hematopoietic stem cells have strong self-proliferation ability, and umbilical cord blood is rich in earlier hematopoietic stem cells, which is convenient to collect. There are few antibodies produced by the derived antigen, no strict matching is required during transplantation, less mature T cells, easy collection and storage, no tumor cell contamination, no damage and side effects to the donor, CD34+CD38- and CD34+CD33 -The ratio of umbilical cord blood hematopoietic stem cells is of great clinical application value. However, the content of hematopoietic stem cells in a single cord blood is low, which makes it difficult to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
Inventor 曾宪卓鲁菲
Owner 深圳华毓造血干细胞研究有限公司
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