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In vitro amplification culture system and method of hematopoietic stem cells

A technology for hematopoietic stem cells and a culture system is applied in the field of efficient expansion and cultivation of hematopoietic stem cells and a culture system for in vitro expansion of hematopoietic stem cells, which can solve the problems of insufficient application of hematopoietic stem cells and the like.

Pending Publication Date: 2020-04-03
武汉济源高科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Through the system and method provided by the present invention, because it does not contain animal serum and feeder layer components, the purity of CD34 hematopoietic stem cells is high, and there are few other types of blood cells differentiated from hematopoietic stem cells. The method is simple to operate, and a large number of clinical hematopoietic stem cells can be cultivated. Solve the problem of insufficient application of clinical hematopoietic stem cells

Method used

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  • In vitro amplification culture system and method of hematopoietic stem cells
  • In vitro amplification culture system and method of hematopoietic stem cells
  • In vitro amplification culture system and method of hematopoietic stem cells

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Embodiment 1

[0016] A hematopoietic stem cell culture system in this embodiment uses F12 medium as the basal medium and configures hematopoietic stem cell serum-free medium, specifically as follows: when configuring F12 medium, add the following components and their contents (final concentrations) as follows: Polyvinyl alcohol 80mg / ml, dexamethasone 0.2nM, nicotinamide 4ug / ml, epinephrine 50ng / ml; and cytokine TPO 100ng / ml, SCF factor 50ng / ml.

Embodiment 2

[0018] A serum-free medium for hematopoietic stem cells obtained in Example 1 was used to culture peripheral blood hematopoietic stem cells in vitro, and the culture method was as follows.

[0019] Coating: Aseptically prepare laminin coating solution with a final concentration of 5ug / ml, use a pipette to draw 12ml of coating solution and add it to T75cm 2 For the cell culture bottle, slowly shake the bottom of the bottle horizontally to make the coating solution evenly coated, then seal the coated bottle with a parafilm, and slowly place it horizontally in a 4°C refrigerator overnight or in a 37°C constant temperature and humidity incubator to avoid light 0.5 to 1 hour.

[0020] Separation and cultivation: Collect 50ml of peripheral blood from healthy volunteers, enrich the peripheral blood hematopoietic stem cells by magnetic beads or other methods, wash the cells with normal saline, centrifuge at 400 g for 10 min, and add a hematopoietic stem cell prepared in Example 1 Res...

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Abstract

The invention discloses in vitro amplification culture system and method of hematopoietic stem cells. According to the in vitro amplification culture system and method of the hematopoietic stem cells,coating is carried out in advance by adopting a combination of one or more selected from gelatin, collagen, laminin and fibronectin; a serum-free medium is used as a basal medium; and polyvinyl alcohol, dexamethasone, nicotinamide, metanephrines as well as a combination of a cytokine thrombopopoietin (TPO) and a stem cell factor (SCF) are added. Compared with conventional culture systems and methods, the in vitro amplification culture system and method of the hematopoietic stem cells disclosed by the invention are capable of efficiently expanding number of CD34 + cells that the number of theCD34 + cells can be expanded by 200-1000 times after about 4 weeks of culturing in vitro; moreover, the active hematopoietic stem cells can be retained for a long term. The in vitro amplification culture system and method of the hematopoietic stem cells is capable of providing a large number of hematopoietic stem cells for clinical use, so that the problem of insufficient application of clinical hematopoietic stem cells is effectively solved.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a composition for expanding stem cells in vitro, in particular to a culture system for expanding hematopoietic stem cells in vitro, and a method for efficiently expanding and culturing hematopoietic stem cells. Background technique [0002] Hematopoietic stem cells (hematopoietic stem cells, HSCs) have the ability of self-renewal cell proliferation and differentiation into various blood tissue cells. Hematopoietic stem cells play a vital role in the development, maintenance and regeneration of the blood system. At present, hematopoietic stem cell transplantation is widely used in the treatment of hematological malignancies, some malignant tumors, some genetic diseases, and immunodeficiency diseases. [0003] At present, the biggest obstacle restricting its clinical application at home and abroad is the insufficient number of hematopoietic stem cell transplantation. The in ...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2500/50C12N2501/30C12N2500/38C12N2501/125C12N2501/20C12N2533/54C12N2533/52
Inventor 陈杰孔德照方松刚
Owner 武汉济源高科技有限公司
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