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Application of cepharanthine and culture medium and method for amplifying hematopoietic stem cells

A technology of hematopoietic stem cells and stephenine, which is applied in the direction of cell culture active agents, blood/immune system cells, biochemical equipment and methods, etc., can solve the problems of lack of proliferation of the number and content of hematopoietic stem cells, and achieve strong stemness, High activity, the effect of improving the amplification effect

Inactive Publication Date: 2017-02-22
GUANGZHOU KOHEALA BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after research, it is found that the above-mentioned medium is still insufficient for the proliferation of the number and content of hematopoietic stem cells, and needs to be further improved.

Method used

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  • Application of cepharanthine and culture medium and method for amplifying hematopoietic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Extraction and separation of umbilical cord blood hematopoietic stem cells

[0034] Obtain the consent of the obstetrics and gynecology department of Huaqiao Hospital in advance, and collect cord blood through the formal collection method of Huaqiao Hospital in the form of voluntary donation.

[0035] Mix umbilical cord blood with PBS at a volume ratio of 1:1, then add 6% hydroxyethyl starch solution at a volume ratio of 4:1 and mix evenly, and let stand at room temperature for 20-30 minutes until the red blood cells naturally settle to a clear boundary , suck out the upper layer solution into a 50ml centrifuge tube;

[0036] Centrifuge at 1500rpm for 5min. After centrifugation, take out the centrifuge tube, collect the mononuclear cells in the lower layer, wash with 3-5ml of PBS, and resuspend the cells for later use;

[0037] Cells were counted with countstar and adjusted to a density of 2×10 8 / ml cell suspension, add 100 μl CD34 antibody to the mixture ...

Embodiment 2

[0039] Example 2: Safety test of stephenine on umbilical cord blood hematopoietic stem cells

[0040] Stephanine was diluted with DMEM / F12 basal medium to drug concentrations of 0, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μmol / ml.

[0041] The cells isolated in Example 1 were mixed with 1×10 4 Seed in 96-well plates at a density of 1 / mL, add different concentrations of stephine, 100ul per well, take the basal medium of umbilical cord blood hematopoietic stem cells without stephine as the normal cell group, place at 37°C, 5% CO 2 Cultivate under the condition for 48h and 72h.

[0042] According to the instructions of the MTT kit, the OD value was detected by a microplate reader, and the survival rate of the cells was calculated. The results are shown in figure 1 .

[0043] Depend on figure 1 It can be seen that whether it is cultured for 48h or 72h, when the concentration of stephine is 50-100 μmol / ml, the survival rate of hematopoietic stem cells is the highest, so 50-100...

Embodiment 3

[0044] Embodiment 3: the comparative test of the amplification effect of different expansion medium

[0045] 1. Medium

[0046] Control medium 1: DMEM / F12+10% FBS;

[0047] Control medium 2: expansion medium of CN105112374A Example 3;

[0048] Positive control medium: DMEM / F12+10%FBS+15ng / ml thrombopoietin+100ng / ml stem cell growth factor+33ng / ml human FMS-like tyrosine kinase 1 ligand+0.2ng / ml interleukin-1+0.2 ng / ml interleukin-6+1uMSR-1 (Note: SR-1 is a puromycin derivative that has been obtained through screening technology, and it has been confirmed so far that it can promote the massive expansion and self-renewal of human CD34+ hematopoietic stem cells Small molecules can generally increase the number of cells by 50 times, so they can be used as positive control drugs, but their cost is relatively high and they are not easy to purchase);

[0049] Culture medium 1 of the present invention: DMEM / F12 medium+8%FBS+20ng / ml thrombopoietin+150ng / ml stem cell growth factor+45...

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Abstract

The invention relates to the technical field of biology, and discloses the application of cepharanthine in the preparation of in-vitro amplification culture medium of hematopoietic stem cells and / or in-vitro amplification of the hematopoietic stem cells, a cepharanthine-containing culture medium for amplifying umbilical cord blood stem cells and a method. According to the culture medium provided by the invention, the in-vitro amplification culture medium is composed of multiple appropriate components and the cepharanthine, and the amplification effect can be obviously improved during in-vitro amplification of the hematopoietic stem cells, so that the culture medium has the characteristics of high dryness and high activity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of stephine and a culture medium and method for expanding hematopoietic stem cells. Background technique [0002] Hematopoietic stem cells are a group of primitive hematopoietic cells that exist in hematopoietic tissues. They are adult stem cells in the blood system. They have the ability of long-term self-renewal and the potential to differentiate into various mature blood cells. It is a type of adult stem cell with the longest and most in-depth research history, and it has important guiding significance for the study of various stem cells, including tumor stem cells. Hematopoietic stem cells are an effective method for the treatment of leukemia, lymphoma and other malignant tumors, autoimmune diseases and congenital immune defects and other diseases that seriously endanger human health, and can effectively alleviate the suffering of patients. [0003] Since Gluckma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2500/30C12N2501/125C12N2501/145C12N2501/2301C12N2501/2306C12N2501/727
Inventor 王一飞陈海佳葛啸虎马岩岩王小燕
Owner GUANGZHOU KOHEALA BIOTECHNOLOGY CO LTD
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