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A method for improving the differentiation efficiency of umbilical cord blood megakaryocyte progenitor cells in vitro

A technique for inducing differentiation of megakaryotic progenitor cells, which is applied in the field of improving the efficiency of in vitro induction and differentiation of umbilical cord blood megakaryotic progenitor cells, which can solve problems such as pollution of operation steps, adverse effects on the efficiency of expansion and differentiation of hematopoietic stem cells, and increased costs, so as to reduce toxicity Function, low cost, and effect of improving efficiency

Active Publication Date: 2018-08-28
广州市天河诺亚生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Total nucleated cells contain a large number of cells of various types, and the complex composition adversely affects the efficiency of in vitro expansion and differentiation of hematopoietic stem cells
Although purer hematopoietic stem cells can be obtained by magnetic bead sorting to optimize the results of in vitro expansion and differentiation, the substantial increase in cost greatly restricts the need for large-scale expansion, and more operating steps also bring greater risk of contamination

Method used

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  • A method for improving the differentiation efficiency of umbilical cord blood megakaryocyte progenitor cells in vitro
  • A method for improving the differentiation efficiency of umbilical cord blood megakaryocyte progenitor cells in vitro
  • A method for improving the differentiation efficiency of umbilical cord blood megakaryocyte progenitor cells in vitro

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Experimental program
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Effect test

Embodiment 1

[0057] Umbilical cord blood was collected from healthy pregnant women and infants. The tests for hepatitis B, hepatitis C, syphilis, AIDS, cytomegalovirus, mycoplasma, chlamydia, G-DPD and thalassemia were all negative. The transportation temperature of the specimens from collection to the blood bank is kept at 4-8°C, and they are transported to the cord blood bank within 24 hours. Perform directed differentiation of megakaryotic progenitor cells as follows:

[0058] (1) Preparation of resveratrol solution and specific medium

[0059] Resveratrol (≥99%, GC), dissolved in the cell protection agent, is prepared into a concentration of 10mmol / L resveratrol storage solution; then the resveratrol storage solution is added to the stem cell culture medium to obtain Type I stem cell medium of resveratrol; the final concentration of resveratrol in the stem cell medium is 50 μmol / L; another stem cell medium (type II stem cell medium) without any resveratrol component is set as Control...

Embodiment 2

[0082] Directed expansion of umbilical cord blood hematopoietic stem cells to megakaryotic progenitor cells with reference to the method of Example 1:

[0083] (1) On the first day after inoculation:

[0084] The initial cell mass (microscopic cell count) of the Control group and the treatment group (50 μmol / L resveratrol cultured for 72 hours) were respectively: 4.26 × 10 6 pcs / mL, 4.28×10 6 individual / mL.

[0085] (2) On the 6th day after inoculation:

[0086] Microscopic cell counting results: the amount of cells in the Control group and the treatment group (50 μmol / L resveratrol cultured for 72 hours) were respectively: 3.34 × 10 6 pcs / mL, 2.61×10 6 a / mL;

[0087] Flow cytometry results: in CD41 + In terms of cell ratio, the values ​​of the Control group and the treatment group (50 μmol / L resveratrol cultured for 72 hours) were 6.35% and 10.38%, respectively.

[0088] (3) On the 12th day after inoculation:

[0089] Microscopic cell counting results: the cell amount...

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Abstract

The invention belongs to the field of cell technology, and in particular relates to a method for improving the differentiation efficiency of umbilical cord blood megakaryocyte progenitor cells in vitro. In the present invention, the type I stem cell culture medium containing 50 μmol / L resveratrol is added to the total nucleated cells of the umbilical cord blood for 72 hours, and the type II stem cell culture medium without resveratrol is replaced to continue the culture, and finally the High differentiation efficiency of megakaryotic progenitor cells. The present invention discloses that after culturing and treatment with Type I stem cell medium containing 50 μmol / L resveratrol, and then replacing Type II stem cell medium without any resveratrol component to continue culturing, through successive cultivation of these two mediums Sequential combination can significantly improve the efficiency of directional differentiation of umbilical cord blood hematopoietic stem cells to megakaryotic progenitor cells, and can prepare megakaryotic progenitor cells with higher purity. The process of the method is simple, reliable and repeatable, the possibility of pollution is low, and the economy of the preparation cost and the operation safety are taken into consideration.

Description

technical field [0001] The invention belongs to the field of cell technology, and in particular relates to a method for improving the differentiation efficiency of umbilical cord blood megakaryocyte progenitor cells in vitro. Background technique [0002] After the world's first successful transplantation of umbilical cord blood hematopoietic stem cells in 1989, the clinical application of umbilical cord blood hematopoietic stem cells has become more and more extensive. After more than 20 years of development, umbilical cord blood has become hematopoietic stem cells together with bone marrow and peripheral blood. of the three sources. [0003] Compared with bone marrow, the recovery rate of platelets after cord blood transplantation is slightly slower. It is generally believed that the reason is that the number of megakaryotic progenitor cells in cord blood is insufficient and their differentiation and maturation are slow. Before hematopoietic stem cell transplantation, the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789
Inventor 嵐山芮魏伟袁嘉恩
Owner 广州市天河诺亚生物工程有限公司
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