3D model for culturing hematopoietic stem cells by simulating bone marrow environment as well as preparation method and application of 3D model

A hematopoietic stem cell and environment technology, which is applied to the 3D model of culturing hematopoietic stem cells in a simulated bone marrow environment and its preparation, and the field of cultivating hematopoietic stem cells by simulating a complex environment in the body, which can solve the problems of difficulty in mass production and high culture cost, and achieve good application prospects. Effect

Pending Publication Date: 2021-03-26
上海东方星际干细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing 3D models have certain limitations, such as high training costs and difficulties in mass production.

Method used

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  • 3D model for culturing hematopoietic stem cells by simulating bone marrow environment as well as preparation method and application of 3D model
  • 3D model for culturing hematopoietic stem cells by simulating bone marrow environment as well as preparation method and application of 3D model
  • 3D model for culturing hematopoietic stem cells by simulating bone marrow environment as well as preparation method and application of 3D model

Examples

Experimental program
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Effect test

Embodiment 1

[0021]3D model of simulating bone marrow environment culture hematopoietic stem cells, including cell culture medium and collagen solution; wherein cell culture medium comprises: V / V is 1% penicillin / streptomycin-free serum cell base medium, 100 ng / ml recombinant human stem cytometry, 50 ng / ml recombinant human platelet genetin, 50 ng / ml recombinant human FMS sample tyrosine kinase 3 and 50 ng / ml interpolarin-3.

[0022]The formulation of collagen solution was 100 μl of 10 × PBS, 10 μL of 1 mol / l NaOH, 250 μl of water and 640 μl of 3.2 mg / ml collagen.

[0023]The 3D model of the simulated bone marrow environment was cultured in the mandatory bone marrow environment, which first inoculated hematopoietic stem cells to be cultured in collagen solution, and then incubated with collagen solution containing hematopoietic stem cells at 37 ° C. The cell culture medium was added to the primary protein polymerization to prepare a 3D model obtained by simulated bone marrow environmen...

Embodiment 2

[0026]3D models of hematopoietic stem cells were used to detect stem cell activity and content by CD34 using Example 1.+Cell is an example:

[0027]Umbilical cord blood collection and CD34+Cell isolation purification: collect fresh umbilical cord blood, dilute undated umbilical cord blood cells with hydroxyethyl starch solution, using CD34+Magnetic beads CD34+cell.

[0028]Take 105CD34+The cells are inoculated into the collagen solution, and 24-well plates are preheated, and then the collagen solution containing hematopoietic stem cells is added to the 24-well plate, and is placed in a 37 ° C incubator for incubation, and the cell culture medium is added after the collagen polymerization Replace the medium daily.

[0029]And use 2D cell culture model as a control: 105The blood stem cells were directly added to a 24-well plate containing the above medium, and the medium was placed in a 37 ° C incubator and replaced daily.

[0030]Cell activity and CD34+Cell content detection: After 7 days, the c...

Embodiment 3

[0032]The 3D model of Example 2 was observed by immunodeficiency NOD-SCID mice, the hematopoietic ability of the 3D model culture of Example 2 was observed: 10 days after culture, two groups of cells were transferred into NOD-SCID mice, and 600 per mouse were transplanted. Cells, 4 weeks after detection of mouse peripheral blood human CD45+The amount of cells. Such asFigure 4As shown, the 3D model cultured cells have detected a number of more than 2d cultured cells.

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Abstract

The invention discloses a 3D model for culturing hematopoietic stem cells by simulating the bone marrow environment as well as a preparation method and application of the 3D model. The 3D model comprises a cell culture medium and a collagen solution, wherein the cell culture medium comprises 1% (v / v) of a penicillin / streptomycin serum-free stem cell basal culture medium, 100 ng / mL of a recombinanthuman stem cell factor, 50 ng / mL of recombinant human thrombopoietin, 50 ng / mL of recombinant human FMS-like tyrosine kinase 3 and 50 ng / mL of interleukin-3. Compared with the prior art, the 3D modelhas the following advantages that (1) the 3D model firstly adds cells into collagen to promote cell polymerization, thereby simulating the environment of hematopoietic stem cells in extracellular matrix to the greatest extent; (2) the cell culture medium is subsequently added into the collagen containing stem cells by the 3D model, so that the collagen can well permeate to provide nutrition for cell proliferation; and (3) the 3D model reserves the multidirectional differentiation capability of the hematopoietic stem cells, so that a good application prospect is provided for in-vitro expansionof the hematopoietic stem cells.

Description

Technical field[0001]The present invention belongs to the field of cell in vitro culture technology, and is related to a method for culturing hematopoietic stem cells in an analog, specifically a 3D model and preparation method and application thereof of simulated bone marrow environment culture hematopoietic stem cells.Background technique[0002]In the bone marrow, hematopoietic stem cells have an unlimited self-renewal, producing the same progeny as the original cells. They are usually in the G0 period of the cell cycle, and can differentiate into cells having a specific function. Because of its ability to self-renew and multi-differentiation, hematopoietic stem cells have widespread use in the treatment of blood system malignant and non-malignant disease, and research and development of an efficient in vitro amplification technology is more important.[0003]Conventional 2D cell culture technology is to cultivate hematopoietic stem cells into the culture bottle or petriper, where se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2513/00C12N2533/54C12N2500/90C12N2501/125C12N2501/145C12N2501/2303C12N2501/727
Inventor 刘中民乐文俊陈炳地胡益辉梅天笑
Owner 上海东方星际干细胞科技有限公司
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