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A composition and its use

A technology of culture system and concentration, used in microorganisms, tissue culture, animal cells, etc.

Active Publication Date: 2020-10-20
NEWISH TECH (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that MS275 can promote the generation of human induced pluripotent stem cells (iPSCs), but the maintenance and expansion of hematopoietic stem cells and the reprogramming of hematopoietic progenitor cells to hematopoietic stem cells have not been reported yet.

Method used

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  • A composition and its use

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Experimental program
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Effect test

preparation example Construction

[0128] The preparation of the umbilical cord blood hematopoietic stem cells includes: diluting the umbilical cord blood 2 to 3 times with normal saline, adding lymphocyte separation medium, centrifuging at 1500 to 2000 rpm / min for 20 minutes to obtain mononuclear cell layer (PBMC), washing with normal saline and resuspending Obtain PBMC cell aggregates; then separate CD34+ cells by magnetic beads method.

[0129]The composition or culture system provided by the present invention can be applied to the in vitro expansion of hematopoietic stem cells, and the hematopoietic stem cells can be derived from experimental animals (such as mice, etc.) or humans. Human hematopoietic stem cells can be derived from bone marrow, peripheral blood, umbilical cord blood and placental blood. In the embodiment of the present invention, umbilical cord blood hematopoietic stem cells are taken as an example, wherein the umbilical cord blood has been tested for hepatitis B, hepatitis C, syphilis, AIDS...

Embodiment 1

[0132] A large amount of umbilical cord blood hematopoietic stem cells are obtained from hematopoietic progenitor cells by using a special transformation medium for human umbilical cord blood hematopoietic stem cells, including the following steps:

[0133] 1. Obtain peripheral blood mononuclear cells (PBMC).

[0134] (1) Collect 80-120ml of umbilical cord blood with a disposable blood bag (containing anticoagulants such as heparin sodium), transfer the umbilical cord blood from the blood bag to a 500ml culture bottle, add normal saline to dilute 2-3 times, mix well and gradually Add dropwise to 0.4 times the volume of lymphocyte separation medium, be careful not to damage the interface.

[0135] (2) Centrifuge at 1500-2000rpm / min for 20 minutes. Due to different densities, the centrifuge tube is divided into four layers from top to bottom: the first layer is the plasma layer, the second layer is the ring-shaped milky white mononuclear cell layer (PBMC), the The third layer i...

Embodiment 2

[0153] The above-mentioned isolated human placental blood hematopoietic stem cells are subjected to phenotype identification, activity rate and purity detection and functional identification, including the following steps:

[0154] 1. Cell count

[0155] The cultured CD34+CD90+ and CD34+CD45RA- hematopoietic stem cells were counted respectively.

[0156] Table 2 Statistics on the number of cells after CD34+CD90- hematopoietic progenitor cells were cultured

[0157]

[0158] Table 3 Statistics of cell number after CD34+CD45RA+ hematopoietic progenitor cells were cultured

[0159]

[0160] 2. Cell Flow Cytometry Analysis

[0161] Using BD’s FACS Verse flow detection instrument, take 20 μl of the cell suspension and add 0.2 μl each of the following four antibodies dissolved in 0.5% BSA: FITC-labeled CD34, PE-labeled CD38, APC-Cy7-labeled CD45RA, APC Labeled CD90. After vortexing each tube, incubate at room temperature in the dark for 15 minutes, add an appropriate amoun...

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Abstract

Provided is a composition comprising MS275 and its use for promoting the transformation of human hematopoietic progenitor cells into human hematopoietic stem cells. During the expansion and culture of umbilical cord blood hematopoietic stem cells, adding cytokines and adding MS275 can not only increase the number of hematopoietic stem cells, but also improve the ability of hematopoietic stem cells to form CFU colonies, so that hematopoietic stem cells are in a state of proliferation and non-differentiation, which meets the needs of clinical transplantation .

Description

[0001] This application claims the priority of the Chinese patent application submitted to the China Patent Office on September 17, 2018, with the application number 201811084022.5, and the title of the invention is "A Method for Transforming Human Hematopoietic Progenitor Cells into Hematopoietic Stem Cells", the entire content of which is passed References are incorporated in this application. And claim the priority of the Chinese patent application with the application number 201811625923.0 and the title of the invention "Culture system, method and use for expanding hematopoietic stem cells" submitted to the China Patent Office on December 28, 2018, the entire contents of which are incorporated by reference in this application. technical field [0002] The invention relates to the technical field of hematopoietic stem and progenitor cells, in particular to a composition and its application. Background technique [0003] Hematopoietic stem cells are an extremely important...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2501/999C12N2501/125C12N2501/145
Inventor 孙忠杰陈立功刘德芳肖雄郭潇严小娥齐海龙
Owner NEWISH TECH (BEIJING) CO LTD
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