Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for preparing adipose tissue-derived stem cells by using serum-free culture medium

A kind of stem cell and culture medium technology, applied in cell dissociation method, biochemical equipment and method, culture process, etc., can solve the problem of unfavorable survival of stem cells and achieve excellent technical effects

Active Publication Date: 2021-02-02
BOYALIFE
View PDF7 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this literature method, mesenchymal stem cells are targeted at the same time when the cells are lysed, which is unfavorable for the survival of stem cells

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
  • Method for preparing adipose tissue-derived stem cells by using serum-free culture medium
  • Method for preparing adipose tissue-derived stem cells by using serum-free culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Isolation and culture of primary adipose-derived mesenchymal stem cells

[0076] (1) The fat donated by volunteers (sample A) was processed in a biological safety cabinet and transported to the laboratory through a cold chain at 2-8°C;

[0077] (2) Centrifuge at 100xg for 5 minutes, remove the upper layer of adipose tissue, wash it again with D-Hanks solution, centrifuge at 100xg for 5 minutes, remove the adipose tissue, add 2 times the volume of 1% type Ⅱ collagenase, shake and digest for 30 minutes;

[0078] (3) After digestion, add double volume of D-hanks solution to dilute, centrifuge at 100xg for 5min, and keep the bottom cell pellet for the next operation;

[0079] (4) Take the cell pellet obtained in step (3), add the primary complete medium to resuspend, take a sample and count, according to 2×10 4 / cm 2 Inoculate into a T75 bottle; place in CO 2 Incubator (5% CO 2 , 37°C, saturated humidity);

[0080] (5) After culturing for 3 days, the cultu...

Embodiment 1a

[0082] Example 1a: Isolation and culture of primary adipose-derived mesenchymal stem cells

[0083] The operation and material of step (1) to step (3) are continued in embodiment 1;

[0084] (4) Take the cell pellet obtained in step (3) of Example 1, add the primary supplementary medium to resuspend, take a sample and count, according to 2×10 4 / cm 2 Inoculate into a T75 bottle; place in CO 2 Incubator (5% CO 2 , 37°C, saturated humidity);

[0085] (5) After culturing for 3 days, the culture medium was completely changed once, until (about 5 days) the cell confluence reached over 80%, the old medium was removed, the cells were washed with D-hanks solution, and 2ml of recombinant trypsin solution was added to each bottle to digest the cells The cells were detached for 2 minutes, diluted by adding 10ml of D-hanks solution to each bottle, centrifuged at 100xg for 10 minutes, and the cell pellet was resuspended in the primary supplemented medium to obtain primary adipose-der...

Embodiment 2

[0093] Example 2: Isolation and culture of primary adipose-derived mesenchymal stem cells

[0094] (1) The fat donated by volunteers (sample B) was processed in a biological safety cabinet and transported to the laboratory through a 2-8°C cold chain;

[0095] (2) Centrifuge at 100xg for 5 minutes, remove the upper layer of adipose tissue, wash it again with D-Hanks solution, centrifuge at 100xg for 5 minutes, remove the adipose tissue, add 2 times the volume of 1% type Ⅱ collagenase, shake and digest for 30 minutes;

[0096] (3) After digestion, add double volume of D-hanks solution to dilute, centrifuge at 100xg for 5min, and keep the bottom cell pellet for the next operation;

[0097] (4) Take the cell pellet obtained in step (3), add the primary complete medium to resuspend, take a sample and count, according to 2×10 4 / cm 2 Inoculate into a T75 bottle; place in CO 2 Incubator (5% CO 2 , 37°C, saturated humidity);

[0098] (5) After culturing for 3 days, the culture ...

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 invention relates to a method for preparing adipose tissue-derived stem cells by using a serum-free culture medium, in particular to a method for serum-free isolated culture of the primary adiposetissue-derived stem cells. The method comprises the following steps: treating an adipose sample; centrifuging to obtain upper-layer adipose tissue, washing with a D-Hanks solution, centrifuging to obtain adipose tissue, and digesting by using type II collagenase; diluting with the D-hanks solution, centrifuging, taking a cell precipitate, adding a primary complete culture medium, resuspending, sampling, counting, and inoculating into a culture bottle according to a specified cell amount; culturing in a CO2 culture box; and after the cells are cultured until the cell fusion degree reaches 80%or above, removing the old culture medium, cleaning the cells by using the D-hanks solution, adding a recombinant pancreatin solution to digest the cells to enable the cells to fall off, adding the D-hanks solution to dilute, centrifuging, and resuspending the cell precipitate by using a primary complete culture medium to obtain the primary adipose tissue-derived stem cells. The method disclosed by the invention has excellent technical effects as shown in the specification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for separating fat mesenchymal stem cells from fat. The present invention also relates to the culture medium and related test solution used in the above-mentioned culture of adipose-derived mesenchymal stem cells. When the method of the present invention is used to isolate and culture the adipose-derived mesenchymal stem cells, the excellent technical effect of the present invention can be presented. In particular, the present invention relates to a method for isolating and culturing mesenchymal stem cells from fat using a serum-free medium. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) such as human mesenchymal stem cells were first isolated from bone marrow, a type of tissue stem cells derived from mesoderm with multi-lineage differentiation potential and self-renewal ability, in vivo and Under specific conditions in vitro, it has the ...

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): C12N5/0775
CPCC12N5/0667C12N2509/00C12N2500/84C12N2501/998C12N2501/33C12N2501/11C12N2501/115C12N2500/34C12N2500/35
Inventor 王正肖海蓉
Owner BOYALIFE
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com