Separation method of buffering stem cell in human placenta

A technology of mesenchymal stem cells and human placenta, applied in the field of separation of human placental mesenchymal stem cells, can solve the problems of cumbersome separation methods

Inactive Publication Date: 2004-11-24
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
View PDF0 Cites 66 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the separation methods descr

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

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Separation method of buffering stem cell in human placenta
  • Separation method of buffering stem cell in human placenta
  • Separation method of buffering stem cell in human placenta

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 Placental MSC isolation and culture method

[0039] According to the anatomical structure of the placenta, under sterile conditions, a polyethylene catheter was inserted from the umbilical vessels (two umbilical arteries and one umbilical vein) to form a circulating fluid perfusion system, and heparin-containing DMEM or IMDM medium was used as the perfusion fluid. First use perfusate to wash out the residual blood within 0.5-2.5 hours postpartum through arteriovenous circulation, and then incubate with 100-250ml perfusate at 20-25°C for 12-24 hours. Mononuclear cells were isolated from the perfusate by density gradient centrifugation, and the cells were suspended in MSC medium after washing, at 37°C, 5% CO 2 After culturing for 24-48 hours under full-humidity conditions, the medium was changed to remove unattached suspended cells, and the culture was continued, changing the medium every 3-4 days. After the early scattered cells form clones, pi...

Embodiment 2

[0040] Example 2 Identification of biological characteristics of placental MSCs

[0041] 1. Cell growth characteristics and morphological characteristics

[0042] It takes about 3 days to cultivate by the method of Example 1. Scattered spindle-shaped adherent cells can be seen under the microscope, and radial clones are formed in about 7-10 days. Each clone is picked out and cultured separately in a 24-well plate. After the cells were purified to passage 3, they were used for the detection of cell biological characteristics. During the culture process, it was found that the cell shape was relatively uniform, the growth rate was fast, the adherence speed was fast, and it was easily digested by trypsin. After passage to more than 15 generations, its shape and growth characteristics did not change significantly. See figure 1 .

[0043] 2. Identification of MSC surface markers by flow cytometry

[0044] The 3rd, 6th, 9th, 12th, and 15th passage cells were collec...

Embodiment 3

[0052] Example 3 Identification of multilineage differentiation potential of placental MSCs

[0053] 1. Adipogenic induction

[0054] More than 3 generations of MSC, press 1×10 5 Inoculate each well in a six-well plate, and after culturing the standard medium for 24 hours, replace it with high-sugar DMEM containing 10% screened FBS, and add 1 μM of dexamethasone, 200 μM of indomethacin, 0.5 mM of IBMX, and 10 μg / ml of insulin, half the amount every 3 days The medium was changed, induced for 2w, and oil red staining was used to identify lipid droplet formation.

[0055] In DMEM-HG containing 10% filtered FBS, add dexamethasone 1μM, indomethacin 200μM, IBMX 0.5mM, insulin 10μg / ml and culture for 3 days, the cells will undergo morphological changes, gradually shrinking from spindle-shaped fibroblasts to Short, more than 90% of the cells become cubic or polygonal; continuous culture for 7 days, microscopic lipid droplets appear in the cells, as the culture time prolon...

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

No PUM Login to view more

Abstract

The present invention discloses the separation method of mesenchymal stem cell in human placenta. On the basis of past separation of tissue cell, the present inventor separates from placenta mesenchymal stem cell with high purity via perfusion process based on the special anatomical structure of placenta. Identification result shows that the mesenchymal stem cell separated from placenta has the biological characteristic and polydirectional differentiation capacity as the reported mesenchymal stem cell of marrow. Owing to the infantile cell component and wide source of placenta, like cord blood, the present invention will have wide clinical application foreground.

Description

technical field [0001] The invention relates to a method for separating cells, in particular to a method for separating human placenta mesenchymal stem cells. Background technique [0002] Human mesenchymal stem cells (MSCs) are the first type of tissue stem cells isolated from bone marrow with multi-directional differentiation potential and self-renewal ability. They are derived from the mesoderm during embryogenesis. Can differentiate into osteoblasts, chondrocytes, adipocytes, muscle cells, tenocytes, hepatocytes and even glial cells (1. Caplan AI. Mesenchymalstem cells. J Orthop Res. 1991, 9: 641-650.2. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284: 143-147.). MSCs are easy to isolate and purify from bone marrow, and can be effectively expanded in vitro. The research results on their biological effects show that MSCs can improve the bone marrow microenvironment and promote hematopoietic reconstr...

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
IPC IPC(8): C12N5/0775C12Q1/02
Inventor 张毅李长东江小霞何津刘元林张双喜吴英毛宁
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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