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Method for extracting and separating umbilical cord-derived skeletal stem cells

A separation method and stem cell technology, applied in the field of extraction and separation of umbilical cord-derived skeletal stem cells, can solve the problems of difficulty in obtaining skeletal stem cells, large damage to the donor, and large secondary damage to the donor, etc., to shorten the primary culture time and increase the number and quality, less controversial effects of ethics

Pending Publication Date: 2022-05-13
深圳市奥思摩尔生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The traditional method of isolating skeletal stem cells (SSC) is to obtain human skeletal stem cells from bone marrow, cartilage, and fetal tissue, and then sort them. It is difficult to obtain skeletal stem cells from the above tissues, and it faces great harm to the donor, and the number of cells that can be obtained is low. , and ethical issues
For example, to obtain SSC from bone marrow in clinical practice, bone marrow needs to be obtained during surgery. At present, most of the bone marrow that can be obtained clinically are orthopedic surgery patients aged around 70. Due to the age of patients, the viability of related cells can be obtained from this source. The amount of bone marrow available during surgery is scarce, so the quantity and quality of available SSCs are difficult to meet the needs of clinical research or therapeutic applications
It is more difficult to obtain SSC from cartilage clinically, and there are few cartilage tissues in the human body. At present, the vast majority of cartilage that can be obtained clinically is obtained from total joint replacement surgery for osteoarthritis. The patient's own condition significantly affects the quantity and quality of SSC obtained. Normal people Cartilage is difficult to obtain, and it will cause secondary damage to the donor, and it is difficult to determine the quantity and quality of the obtained SSC

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  • Method for extracting and separating umbilical cord-derived skeletal stem cells
  • Method for extracting and separating umbilical cord-derived skeletal stem cells
  • Method for extracting and separating umbilical cord-derived skeletal stem cells

Examples

Experimental program
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Embodiment 1

[0058] A method for extracting umbilical cord derived bone stem cells, comprising the steps of:

[0059] S1. Primary isolation of human umbilical cord-derived stem cells (hUC-MSCs)

[0060] (1) After the human umbilical cord has undergone strict testing procedures (ABO / Rh blood type testing, HLA typing testing, syphilis antibody testing, HIV immune testing, CMV antibody testing, hepatitis B antigen antibody testing, etc.) to determine the safety, take a 20cm umbilical cord Put it into a collection bottle of PBS buffer containing double antibodies (final concentration of penicillin is 100U / mL and final concentration of streptomycin is 0.1mg / mL), seal and send to a sterile ultra-clean bench for processing;

[0061] (2) Disinfect the outer wall of the collection bottle with 75% alcohol, take out the umbilical cord tissue with sterile tweezers (big long tweezers) and transfer it to a petri dish, and wash it with double antibodies (final concentration of penicillin is 100U / mL and s...

Embodiment 2

[0095] Example 2 Cell culture of umbilical cord-derived skeletal stem cells (U-hSSCs) and bone marrow-derived skeletal stem cells (B-hSSCs)

[0096] The U-hSSCs prepared in Example 1 and the B-hSSCs prepared in Comparative Example 1 were respectively inoculated into T25 cell culture flasks containing complete medium, and placed in CO 2 Culture in an incubator with a concentration of 5%, a humidity of 95%, and a temperature of 37°C; replace the medium every 48 hours, and wash the cells twice with PBS when the cell confluence reaches 80% to 90%; discard the PBS , add 1mL of digestion solution (trypsin-EDTA digestion solution (0.25%) containing phenol red, Solarbio, Cat#T1320), and put it in the incubator for digestion for 1min; discard the digestion solution, and stop the digestion with complete medium immediately; Transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 800rpm for 5min; discard the supernatant, resuspend the cells with complete culture, and inoc...

Embodiment 3

[0098] Cartilage differentiation of hSSCs derived from the umbilical cord of embodiment 3

[0099] Umbilical cord-derived skeletal stem cells (U-hSSCs), human umbilical cord stem cells (hUC-MSCs) (produced in Example 1), human bone marrow stromal stem cells (hBMSCs) and human bone marrow-derived skeletal stem cells (B- hSSCs) (prepared in Comparative Example 1) were inoculated in 12-well plates, with 2 wells for each type of cell, about 100,000 cells in each well, added 2 mL of complete medium, and placed at 37 ° C, 5% CO 2 Culture in the incubator until it was overgrown; the medium in the 12-well plate was aspirated and discarded, and 1 mL of chondrogenic differentiation medium (50% StemPro chondrogenic differentiation reagent (Thermo Fisher Scientific) was added to one well of each cell. TM , Cat#A1007101, 50% MEM Alpha (1×) medium)) for chondrogenic induction and differentiation, another well was added with the same volume of complete medium as a blank control; after the 4t...

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Abstract

The invention belongs to the technical field of cell biology, and discloses an extraction and separation method of umbilical cord-derived skeletal stem cells, and the separation method comprises the following steps: (1) cutting umbilical cord tissues into small blocks, standing for 25-40 minutes, adding a culture medium, and culturing; (2) after cells climb out of the edge of the umbilical cord tissue block, stripping the umbilical cord tissue block, and collecting the climbed cells; and (3) sorting the cells collected in the step (2) to obtain the umbilical cord-derived skeletal stem cells. According to the method provided by the invention, the skeletal stem cells can be quickly separated from the umbilical cord, the primary culture time for obtaining the skeletal stem cells is greatly shortened, and more cells climb out, are uniformly distributed and grow quickly.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a method for extracting and separating umbilical cord-derived bone stem cells. Background technique [0002] Skeletal stem cells (Skeletal stem cells, SSCs) are a subtype of stem cells discovered in chondrocytes in recent years, and have been reported in Nature, Cell and other well-known journals. This type of stem cells has the ability to differentiate into cartilage and bone. SSC has the ability to self-renew in vitro. Experiments have shown that transplanted under the mouse kidney, it can linearly differentiate into multilineage ossicles containing bone, cartilage and matrix: first differentiate the progenitor of early bone, cartilage and matrix Cells (human bone, cartilage and stromal progenitor, hBCSP), followed by osteoprogenitor cells (human osteoprogenitor, hOP) and cartilage progenitor cells (human chondroprogenitor, hCP), and finally differentiated into...

Claims

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Application Information

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IPC IPC(8): C12N5/074A61K35/51A61P19/02A61P19/04A61P19/08
CPCC12N5/0662A61K35/51A61P19/08A61P19/02A61P19/04C12N2509/00
Inventor 刘衍志高翔
Owner 深圳市奥思摩尔生物科技有限公司
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