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In-vitro culture method of umbilical cord blood megakaryocyte progenitor cells

A megakaryocyte progenitor cell, in vitro culture technology, applied in cell culture medium, cell culture active agent, blood/immune system cells, etc. Differentiation and other issues, to achieve the effect of improving the ability to differentiate into platelets, improving the characteristics of progenitor cells, and reducing dosage

Inactive Publication Date: 2017-03-08
浙江绿蔻生物技术有限公司
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Problems solved by technology

Although the existing technology has achieved certain results in the in vitro expansion of megakaryotic progenitor cells, the progenitor cell characteristics of megakaryotic progenitor cells cannot be well maintained during the culture process, thereby reducing the ability of megakaryotic progenitor cells to differentiate into platelets in the long term

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  • In-vitro culture method of umbilical cord blood megakaryocyte progenitor cells
  • In-vitro culture method of umbilical cord blood megakaryocyte progenitor cells
  • In-vitro culture method of umbilical cord blood megakaryocyte progenitor cells

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Embodiment 1

[0049]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. Proceed as follows to expand megakaryotic progenitor cells:

[0050] (1) Preparation of hematopoietic stem cell culture supernatant

[0051] Obtain mononuclear cells from fresh healthy human cord blood within 24 hours, and then obtain CD34 by immunomagnetic bead sorting + The sorted hematopoietic stem cells were added to Stem Span medium for culture and expansion. At the same time, five cytokines including 20ng / mL IL-6, 20ng / mL IL-3, 50ng / mL SCF, 50ng / mL Flt-3L and 20ng / mL TPO need to be added. After culturing for about 3 days, the culture supernatant of hematopoietic stem cells was collected. Th...

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Abstract

The invention discloses an in-vitro culture method of umbilical cord blood megakaryocyte progenitor cells. According to the method, a specific megakaryocyte progenitor cell medium is inoculated with umbilical blood cells for culture. The specific megakaryocyte progenitor cell medium is a serum-free medium containing 40vol%-50vol% of hematopoietic stem cell culture supernatant, 50+ / -10 ng / mL SCF and 50+ / -10 ng / mL TPO, wherein the hematopoietic stem cell culture supernatant is obtained by culturing umbilical cord blood hematopoietic stem cells in a serum-free medium containing IL-6, IL-3, SCF, Flt-3L and TPO for 3-4 days. With the adoption of the method, the progenitor cell characteristics of umbilical cord blood megakaryocyte progenitor cells realizing in-vitro amplification are effectively improved, the megakaryocyte progenitor cell obtaining efficiency is improved, and accordingly, the capacity of the megakaryocyte progenitor cells differentiating into blood platelet is improved. In the amplification stage of the megakaryocyte progenitor cells, the consumption of added cell factors is reduced, accordingly, material consumption is reduced, and the cost is reduced.

Description

technical field [0001] The invention belongs to the field of cell technology, and in particular relates to a method for culturing 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] Before hematopoietic stem cell transplantation, the recipient usually needs to undergo high-dose radiotherapy / chemotherapy to achieve myeloablative effect, and radiotherapy / chemotherapy will seriously damage the recipient's hematopoietic system, resulting in a severe lack of platelets for a period of time, which is prone to bleeding and even causes Death, so a large n...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N5/0037C12N2501/125C12N2501/145
Inventor 嵐山芮魏伟许超
Owner 浙江绿蔻生物技术有限公司
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