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A hemopoietic stem cell medium

A technology of hematopoietic stem cells and culture medium, which is applied in the directions of cell culture active agents, blood/immune system cells, culture process, etc. The concentration is reduced too quickly, no toxic side effects, and the effect of increasing the amplification factor

Inactive Publication Date: 2017-08-29
DONGGUAN BOALAI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are two main in vitro proliferation pathways for hematopoietic stem cells. One is in vitro culture through matrix perfusion. However, the matrix contains various cells and various protein components. It is more difficult and costly, which greatly limits the application of in vitro expansion of hematopoietic stem cells; the second is to inoculate hematopoietic stem cells in hematopoietic stem cell medium for in vitro culture
However, most of the hematopoietic stem cells cultured in the existing hematopoietic stem cell medium have poor activity and proliferation ability, and the hematopoietic stem cells are easy to differentiate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] In this embodiment, a medium for hematopoietic stem cells includes DMEM medium and an additive added to the DMEM medium. In terms of final concentration, the additive includes the following components: FBS 150 mg / mL, sodium selenite 0.025μmol / mL, BCG polysaccharide nucleic acid 7mg / mL, fenugreek 70μmol / mL, trehalose 60ng / mL, vitamin C 50ng / mL, the first cytokine sustained-release microcapsules 2mg / mL, the second cytokine sustained-release Microcapsules 35mg / mL.

[0036] Preferably, the preparation method of the first cytokine sustained-release microcapsules comprises the following steps:

[0037] A. IL-3, IL-6, GM-CSF and polyethylene glycol are added to the aqueous solution pre-added with mannitol and zinc sulfate, and after stirring, mixture A is obtained. In the mixture A, IL-3, IL- 6. The concentration of GM-CSF is 1.5wt%, the concentration of polyethylene glycol is 8wt%; the concentration of mannitol is 3wt%, and the concentration of zinc carbonate is 2wt%;

[00...

Embodiment 2

[0054] Preferably, the hematopoietic stem cell culture medium includes DMEM medium and an additive added to the DMEM medium. In terms of final concentration, the additive includes the following components: FBS 100 mg / mL, sodium selenite 0.03 μmol / mL mL, BCG polysaccharide nucleic acid 5mg / mL, fenugreek 100μmol / mL, trehalose 50ng / mL, vitamin C 80ng / mL, the first cytokine slow-release microcapsules 1.5mg / mL, the second cytokine slow-release microcapsules 50 mg / mL.

[0055] Preferably, the preparation method of the first cytokine sustained-release microcapsules comprises the following steps:

[0056] A. IL-3, IL-6, GM-CSF and polyethylene glycol are added to the aqueous solution pre-added with mannitol and zinc sulfate, and after stirring, mixture A is obtained. In the mixture A, IL-3, IL- 6. The concentration of GM-CSF is 1 wt%, the concentration of polyethylene glycol is 10 wt%; the concentration of mannitol is 2 wt%, and the concentration of zinc carbonate is 3 wt%.

[0057]...

Embodiment 3

[0073] In this embodiment, a method for culturing hematopoietic stem cells includes the following steps:

[0074](1) Collect umbilical cord blood, add an equal volume of PBS to dilute to obtain umbilical cord blood diluent; add 0.3% methyl cellulose solution to the umbilical cord blood diluent, and the umbilical cord blood diluent and methyl cellulose solution are mixed. After mixing at a volume ratio of 1:0.15, sediment the red blood cells in the umbilical cord blood and aspirate the supernatant; after centrifuging the supernatant, discard the obtained supernatant, and add PBS to resuspend the cells to obtain a cell suspension; Add the lymphocyte separation solution to the suspension, let it stand, centrifuge, discard the supernatant, add the red blood cell lysate to the collected lower mononuclear cells, the volume of the red blood cell lysate and the collected lower mononuclear cells The ratio was 1:0.5, and the cells were lysed for 5 min at room temperature; PBS was added ...

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Abstract

The invention relates to the technical field of biology, and particularly relates to a hemopoietic stem cell medium. The medium includes a base medium and an additive added into the base medium. Based on final concentrations, the additive includes 100-200 mg / mL of FBS, 0.02-0.03 [mu]mol / mL of sodium selenite, 5-10 mg / mL of bacillus calmette-guerin polysaccharides and nucleic acids, 50-100 [mu]mol / mL of cepharanthine, 50-100 ng / mL of trehalose, 40-80 ng / mL of vitamin C, 1.5-2.5 mg / mL of a first cytokine slow-release microcapsule, and 25-50 mg / mL of a second cytokine slow-release microcapsule. The hemopoietic stem cell medium can significantly increase the propagation rate and cell activity of hemopoietic stem cells, allows the hemopoietic stem cells to be in a propagation undifferentiated state for long time, and maintains stem cell characteristics of the hemopoietic stem cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a hematopoietic stem cell culture medium. Background technique [0002] Hematopoietic stem cells are adult stem cells in the blood system. They are a heterogeneous group with long-term self-renewal ability and the potential to differentiate into various mature blood cells. It is a type of adult stem cells with the longest and most in-depth research history, and has important guiding significance for the study of various types of stem cells, including tumor stem cells. Because hematopoietic stem cells have the potential of multi-directional differentiation, transplantation of hematopoietic stem cells into the body is an effective method for the treatment of leukemia, lymphoma and other malignant tumors, metabolic diseases, autoimmune diseases and innate immune deficiencies that seriously endanger human health. to alleviate the suffering of patients. However, the number of hematopoie...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2500/12C12N2500/38C12N2501/125C12N2501/22C12N2501/2303C12N2501/2306C12N2501/727C12N2501/90C12N2501/999
Inventor 罗天恩
Owner DONGGUAN BOALAI BIOLOGICAL TECH CO LTD
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