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Method for preparing freeze-drying powder of human mesenchymal stem cells culture supernatant and prepared freeze-drying powder

A technology for stem cells and supernatant, applied in the field of human mesenchymal stem cells, can solve the problems of low stem cell activity, stem cell death, and inability to significantly increase the content of factors in the supernatant of stem cells, etc., and achieves simple preparation method, high induction efficiency, and yield. big effect

Active Publication Date: 2016-12-21
GUANGZHOU MACRO SOURCE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when conventionally cultured in a medium without fetal bovine serum, the activity of stem cells is low, the secreted factors are few, and a large number of stem cells die in a short period of time
Conventional methods, such as mechanical rubbing, UV irradiation, and starvation culture, did not significantly increase the content of factors in stem cell supernatants

Method used

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  • Method for preparing freeze-drying powder of human mesenchymal stem cells culture supernatant and prepared freeze-drying powder
  • Method for preparing freeze-drying powder of human mesenchymal stem cells culture supernatant and prepared freeze-drying powder

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] How to obtain human mesenchymal stem cells

[0027] A method for obtaining human mesenchymal stem cells, comprising the following steps:

[0028] (1) The cells used come from aseptic conditions. Take the umbilical cord of a full-term baby 5-10cm, wash it with normal saline until the tissue is bloodless, remove the epidermis, two arteries and one vein, cut the tissue into pieces, and use 0.1% Type I collagenase was digested at 37°C for 1 hour, centrifuged at 400g for 5 minutes, discarded the supernatant, mixed evenly and filtered through a 200-mesh screen, and the obtained cell suspension was centrifuged at 1200r / min for 8 minutes, and the cell pellet was added to DMEN containing 10% FBS / F12 medium (with double antibody), 37°C, saturated humidity, 5% CO 2 : After culturing for about one week, primary human umbilical cord mesenchymal stem cells can be obtained. When the cell confluency was 70%-80%, digest the cells with 0.25% trypsin (containing 0.02% EDTA) and subcu...

Embodiment 2

[0031] Preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder

[0032] The preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder comprises the following steps:

[0033] (1) Low temperature induces the secretion of stem cell factors: resuspend the low-generation cells collected in Example 1 in normal saline, and store them at 4°C, saturated humidity, and 5% CO 2 Under the same conditions, culture for 72 hours; during this period, when the human mesenchymal stem cells grow to 70-90% confluence, the culture supernatant is collected and mixed with an equal volume of DMEM serum-free medium, and continues to be used for the expansion of human mesenchymal stem cells Culture is repeated until the cells have been subcultured to 10-15 generations, and all cell culture supernatants are collected for subsequent preparation of freeze-dried powder.

[0034] (2) Preparation of freeze-dried powder: put the collected cul...

Embodiment 3

[0036] Different methods were used to induce and culture the stem cells of the fifth generation collected in Example 1, specifically as follows:

[0037] 1.1 blank control

[0038] Stem cells at passage 5 were taken, washed with physiological saline three times, added with DMEM serum-free medium to continue culturing for 72 hours, and the supernatant was collected. The stem cell culture supernatant was taken to determine its proliferative activity on human dermal fibroblasts.

[0039] 1.2 UV induction

[0040] Take the stem cells of passage 5, wash the cells three times with physiological saline, add DMEM serum-free medium, irradiate the cells with UVA for 2 hours (interval 22 hours, irradiate three times), culture for 72 hours, and collect the supernatant. The stem cell culture supernatant was taken to determine its proliferative activity on human dermal fibroblasts.

[0041] 1.3 Mechanical friction

[0042] Take the stem cells of passage 5, wash the cells three tim...

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Abstract

The invention discloses a method for producing stem cell factors by inducing human mesenchymal stem cells. The invention also discloses a method for preparing freeze-drying powder of a human mesenchymal stem cells culture supernatant and prepared freeze-drying powder. The method has the advantages of high induction efficiency and large output of stem cell factor, and can obtain a lot of stem cell factors in short time. The method of for preparing freeze-drying powder of the human mesenchymal stem cells culture supernatant is simple and convenient, compared with a low temperature induction method, the output is increased by 15%-20%; the method for preparing freeze-drying powder of the human mesenchymal stem cells culture supernatant preserves various cytokine mixtures having biological activity in the human mesenchymal stem cells culture supernatant, and establishes a base for application of the human mesenchymal stem cells culture supernatant on beauty treatment aspect.

Description

technical field [0001] The invention belongs to the field of human mesenchymal stem cells, and in particular relates to a method for preparing freeze-dried powder of human mesenchymal stem cell culture supernatant and the prepared freeze-dried powder. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) is an important pluripotent stem cells, mainly distributed in the mesoderm and ectoderm of early mating embryos. Among them, umbilical cord mesenchymal stem cells are a kind of multifunctional stem cells that exist in the umbilical cord tissue of newborns. Under specific induction conditions in vivo or in vitro, it can differentiate into various tissue cells such as bone, cartilage, tendon, ligament, nerve, liver, endothelium and cardiac muscle, and has broad clinical application prospects. Because of its low immunogenicity, high cell content, and excellent proliferation ability, it is widely used in cosmetic and plastic surgery and other field...

Claims

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Application Information

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IPC IPC(8): C12P21/02A01N1/02
CPCA01N1/0284C12P21/02
Inventor 张龙吴延恒
Owner GUANGZHOU MACRO SOURCE BIOTECH CO LTD
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