Method of separating multipotent adult progenitor cells from umbilical cord blood

A technology for umbilical cord blood and progenitor cells, which can be applied to blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of high cost and complicated steps, and achieve the effect of reducing the impact

Inactive Publication Date: 2009-01-21
INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Domestic reports are similar (29.17%, Zhu Meiling, Hu Yanfen, Wen Guanmei, etc., Isolation and Culture of Human Placental Blood-Mesenchymal Stem Cells, Chinese Journal of Pathophysiology 2004, 20(9): 1743-1744), and the analysis using immunomagnetic beads The selection has complex steps and high cost

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Isolation, purification and expansion of umbilical cord blood multipotent adult progenitor cells

[0034] After the umbilical cord blood was subjected to density gradient centrifugation in lymphocyte separation medium, mononuclear cells were collected, washed twice with DMEM / F12 medium, and the cells were suspended in DMEM / F12 medium.

[0035] The isolated mononuclear cells were divided into 1×10 6 / cm 2 The inoculation density was inoculated into DMEM / F12 medium containing 10% fetal bovine serum, and each T-25 culture bottle had 7ml of medium. Placed at 37°C, 5% CO 2 , 100% humidity incubator, cultured for 72 hours. Discard non-adherent cells, replace the cell culture medium with 40% MCDB-201, 2% fetal bovine serum, 10ng / ml bFGF, 10ng / ml EGF, 10ng / ml PDGF-BB, 10 -4 M ascorbic acid, 10 -8 M dexamethasone, 1 × ITS DMEM / F12 culture medium continued. Change the medium 2-3 times a week. Adherent cells were seen in 70% of the cord blood samples after cultur...

Embodiment 2

[0036] Example 2. Surface antigen characteristics and cell cycle detection of umbilical cord blood multipotent adult progenitor cells

[0037] The umbilical cord blood multipotent adult progenitor cells expanded for three generations were taken, and the culture medium was removed. The cells were washed twice with PBS, treated with trypsin (0.25%), and the cells were recovered and counted. Make 5 x 10 in PBS 5 / ml single cell suspension, add 500μl to each tube, add 10μl anti-human CD3, CD34, CD11a, CD29, CD105, CD45, CD44, CD73, CD49d, CD49e, CD31, CD62L, CD14, HLA-DR, CD144 , vWF and anti-mouse IgG 1 Isotype control fluorescent antibody. Incubate at 4°C in the dark for 30 minutes, add 1ml PBS to wash, discard the supernatant, add 300μl PBS containing 1% paraformaldehyde, and perform flow cytometric detection. CD29, CD44, CD105, CD49d, and CD73 were positively expressed, and CD34, CD45, CD11a, CD14, CD31, HLA-DR, CD144, vWF, and CD62L were negatively expressed.

[0038] In...

Embodiment 3

[0039] Example 3. Induced differentiation characteristics of umbilical cord blood multipotent adult progenitor cells

[0040] The 3rd generation multipotent adult progenitor cells expanded in vitro were digested, and 5×10 3 / cm 2 seeded in 6-well plates pre-placed with coverslips. When the cells reached 60%-70% confluency, each well was replaced with 10 -7 mol / L dexamethasone, 10mmol / L β-glycerol phosphate, 0.05mol / L vitamin C and 10% FCS of DMEM / F-12 osteogenic differentiation induction solution 2ml for induction culture for 3 weeks, change the medium 2 times a week The second time, the slides were taken out on the 14th and 21st days of induction, fixed, observed the morphological changes and carried out histological staining.

[0041] The 3rd generation multipotent adult progenitor cells expanded in vitro were digested, and 5×10 3 / cm 2 seeded in 6-well plates pre-placed with coverslips. When the cells reached 60%-70% confluency, each well was replaced with 10 -8 mol / ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
densityaaaaaaaaaa
Login to view more

Abstract

The invention discloses the method for separating pluripotent ancestral cell from umbilical cord blood, comprising the following steps: using lymphoblast to separate umbilical cord blood monocyte, culturing for 3 days at culture medium with 8-15% bovine serum, throwing away cell which is not sticked on wall, replacing the culture medium with 2-5% bovine serum, bFGF, EGF, PDGF-BB, MCDB-201, ascorbic acid, anaflogistico and ITS addition agent, culturing, passage purifying, and separating. The method has the advantages of high separation rate. The cell phenotype, cell periodic time and evoked differentiation ability are the same.

Description

technical field [0001] The invention relates to a method for isolating multipotent adult progenitor cells (multipotent adult progenitor cells, MAPC) from umbilical cord blood, in particular to a method for isolating multipotent adult progenitor cells from umbilical cord blood in vitro. Background technique [0002] As the basic research of tissue engineering, stem cell engineering has an extremely important role and far-reaching impact on future tissue and organ repair and replacement. The key is to obtain stem cells that can differentiate into desired tissues. [0003] Stem cells can be divided into embryonic stem cells and adult stem cells according to the order in which they appear during individual development. Due to the lack of knowledge about the regulatory mechanism of embryonic stem cell differentiation and development, the limited source of embryonic stem cells and the risk of inducing tumors after transplantation, and the research and application of embryonic stem...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08C12N5/0789
Inventor 邱录贵李云涛徐燕孟恒星于珍韩俊领
Owner INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap