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Three-dimensional culture method of umbilical cord mesenchymal stem cells

A technology of stem cells and three-dimensional culture, applied in the biological field, can solve the problems of limiting the expansion multiple of stem cells, unable to meet the demand of exosomes, and low yield.

Active Publication Date: 2022-04-12
深圳市旷逸生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional culture of umbilical cord mesenchymal stem cells adopts a two-dimensional culture method (planar method), the area of ​​the culture surface limits the expansion multiple of stem cells, and the output is low, which cannot meet the market demand for exosomes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The mesenchymal stem cells in Example 1 are all human umbilical cord-derived mesenchymal stem cells. The primary cells were collected from umbilical cord tissue Wahrenheit jelly. The obtained cells) were cryopreserved, and the cell content was 2 million cells per cryopreservation tube, with a capacity of 1 mL. The stem cell medium used in Example 1 is an animal-derived substance-free stem cell medium (LONZA, 12-725F) with Ultroser G as a serum substitute, wherein the content of Ultroser G is 2%.

[0036] The steps of the three-dimensional culture method of the umbilical cord mesenchymal stem cells of embodiment 1 are:

[0037] (1) Take out the stem cell cryopreservation tube frozen in the liquid nitrogen tank, and immediately put it into the preheated 37°C alcohol for rapid thawing.

[0038] (2) The thawed stem cells are transferred to a T175 culture flask containing stem cell culture medium, and placed in a constant temperature incubator at 37°C and 5% CO 2 After cul...

Embodiment 2

[0045] The umbilical cord mesenchymal stem cells, medium and other reagents used in Example 2 are the same as those in Example 1, except that the 96-well plate used in Example 2 is a flat-bottomed well plate. Specifically, the steps of the three-dimensional culture method of umbilical cord mesenchymal stem cells in Example 2 are:

[0046] (1) Take out the cryopreservation tube of umbilical cord mesenchymal stem cells frozen in the liquid nitrogen tank, and immediately put it into preheated 37°C alcohol for rapid thawing.

[0047] (2) The thawed stem cells are transferred to the T175 culture flask containing the stem cell medium, and placed in a constant temperature incubator at 37°C and 5% CO 2 After culturing for 3 days, when the fusion rate reached 80%, the stem cells were digested with 0.25% trypsin, and the stem cells were passaged to the P4 generation by successive methods.

[0048] (3) P4 mesenchymal stem cells were seeded in 96-well plates (Thermo Scientific TM Nunc ...

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Abstract

The invention relates to a three-dimensional culture method of umbilical cord mesenchymal stem cells. The three-dimensional culture method of umbilical cord mesenchymal stem cells comprises the following steps: seeding umbilical cord mesenchymal stem cells in a cell culture device, and then rotating and culturing the umbilical cord mesenchymal stem cells at a horizontal rotation speed of 20 rpm to 40 rpm, wherein the cell culture device The inner surface of the bottom is curved, and the inner surface of the bottom is treated with anti-cell attachment. The above three-dimensional culture method of umbilical cord mesenchymal stem cells can increase the yield of exosomes secreted by umbilical cord mesenchymal stem cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a three-dimensional culture method for umbilical cord mesenchymal stem cells. Background technique [0002] Exosomes are subcellular double-layer membrane vesicles with a uniform size and a molecular diameter of 40nm to 100nm, which are formed by cells through a series of regulatory processes such as "endocytosis-fusion-efflux", and are actively secreted to the outside of the cell. Contains proteins, miRNA and mRNA related to cell origin. Exosomes can not only directly activate recipient cells through plasma membrane receptors, but also transport proteins, miRNA, mRNA and even organelles into recipient cells, and can also carry specific substances contained in cells in different case states. It plays an important role in both physiology and pathology. [0003] Umbilical cord mesenchymal stem cells (UC-MSCs) have the potential of self-renewal and multi-lineage differentiation, and c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2513/00C12N2509/00
Inventor 胡士庶宋芸娟刘露林沈洁沈艳
Owner 深圳市旷逸生物科技有限公司
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