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Preparation method and application of a high-concentration glucose solution-activated mesenchymal stem cell conditioned medium

A technology of glucose solution and conditioned medium, which is applied in the field of preparation of mesenchymal stem cell conditioned medium anti-aging solution, can solve the problems of poor anti-aging effect, achieve easy mass production and storage, reduce ROS production, anti-aging effect

Active Publication Date: 2021-04-13
北京恒峰铭成生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problem that the existing MSC-CM anti-aging effect is not very good, the present invention provides a preparation method and application of a conditioned medium for mesenchymal stem cells activated by a high-concentration glucose solution

Method used

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  • Preparation method and application of a high-concentration glucose solution-activated mesenchymal stem cell conditioned medium
  • Preparation method and application of a high-concentration glucose solution-activated mesenchymal stem cell conditioned medium
  • Preparation method and application of a high-concentration glucose solution-activated mesenchymal stem cell conditioned medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Preparation of mesenchymal stem cells

[0036] Preparation of umbilical cord mesenchymal stem cells:

[0037] Operate in an ultra-clean workbench, take fresh full-term healthy fetal umbilical cord, wash it with normal saline containing 2 times 100U / mL penicillin and streptomycin to remove blood stains, peel off Walton’s jelly, and cut it into pieces less than 1 cubic millimeter Shape, spread evenly, slowly add 10mL serum-free medium, and culture in a 37°C, 5% carbon dioxide, saturated humidity incubator. After the primary cells were cultured for 7 days, the serum-free medium was replaced and cultured until the cells reached 70% confluence, the serum-free medium was removed, and trypsin-sodium edetate ( EDTA) for 1 min, the umbilical cord MSCs shrank and detached from the bottle wall. At this time, the previously removed culture supernatant was added to neutralize the trypsin-EDTA solution, and gently blown with a pipette, and the umbilical cord MSC suspensio...

Embodiment 2

[0038] Example 2. Preparation of anti-aging solution, i.e. high glucose-activated mesenchymal stem cell conditioned medium

[0039] When the third-generation cells of the umbilical cord MSCs prepared in Example 1 were cultured in DMEM / F12 containing 10% FBS to 40-50% monolayer cells, glucose solution was added, and the final concentration of glucose was 20-50 mmol / L, and then continued Culture was continued for 48±2 hours in DMEM / F12 containing 10% FBS. Centrifuge at 2000rpm for 5 minutes to collect the culture supernatant, i.e. the conditioned medium, as umbilical cord MSC-CM (UC-MSC-CM), use an ultrafiltration membrane with a molecular weight cut-off of 5kDa to further concentrate the conditioned medium 10 times to obtain high glucose Activated mesenchymal stem cell conditioned medium, namely anti-aging solution.

[0040] The concentrated anti-aging solution was filtered through a 0.2 μm filter and stored at -80°C until use.

Embodiment 3

[0041] Example 3. Culture of fibroblasts

[0042] Foreskin (FF) samples were collected by routine circumcision. Collected foreskin samples were washed with phosphate buffered saline (PBS) supplemented with 1% penicillin-streptomycin solution (PS). All samples were then cut into small pieces and incubated in type I collagenase (Sigma) at 37°C for 1 hour. Digestion was terminated and the supernatant collected for centrifugation. The cell pellet was gently resuspended in DMEM containing 10% fetal bovine serum (FBS), and the medium was released every two days.

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Abstract

The invention provides a preparation method and application of a mesenchymal stem cell conditioned medium activated by a high-concentration glucose solution, belonging to the field of stem cell anti-aging. The conditioned medium for mesenchymal stem cells activated by a high-concentration glucose solution is characterized in that the mesenchymal stem cells are pretreated in a glucose medium with a final concentration of 20-50 mmol / L for 48 ± 2 hours, and the mesenchymal stem cells are collected Stem cell conditioned medium, which is concentrated and purified. The high-concentration glucose solution activated mesenchymal stem cell conditioned medium of the present invention can be used for treating cell aging and anti-oxidation. The conditioned medium of mesenchymal stem cells activated by the high-concentration glucose solution can be made into freeze-dried powder, which is easier to store.

Description

technical field [0001] The invention belongs to the field of stem cell anti-aging, and relates to a preparation method and application of a mesenchymal stem cell conditioned medium anti-aging liquid. Background technique [0002] With the increasingly prominent problem of population aging, the field of anti-aging has become the focus of global attention. The skin is the largest organ of the human body, responsible for many aspects such as protection, sensation, temperature regulation, secretion, excretion and immunity. Regression, shrinkage, wrinkling. The appearance of clinical symptoms such as wrinkles and senile plaques makes people realize that they are beginning to age, which has complex psychological and physical effects on people's life and work. It can cause a series of psychological problems such as anxiety, depression, and low self-esteem. Therefore, anti-aging treatment, especially anti-aging treatment for skin has become one of the focuses of research, and peop...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775A61K35/28A61K8/99A61K8/60A61Q19/08A61P17/18A61P17/00A61P39/06A61K31/7004
CPCA61K8/60A61K8/99A61K31/7004A61K35/28A61Q19/08C12N5/0668C12N2500/34A61K2300/00
Inventor 郝好杰李梓源易军周严恒刘刚陈惠华
Owner 北京恒峰铭成生物科技有限公司
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