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Conditioned medium, freeze-dried powder, preparation method and application

A technology of conditioned medium and freeze-dried powder, which is applied in the field of stem cells to achieve high safety, improve adhesion performance and proliferation rate, and solve the effects of cytotoxic side effects

Pending Publication Date: 2021-04-23
苏州京赛诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is believed in medicine that aging (cell aging) is an irreversible process. The conventional drug treatment on the market can only achieve short-term short-term improvement of the appearance of cell aging, and cannot fundamentally solve the problem of cell aging and apoptosis.

Method used

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  • Conditioned medium, freeze-dried powder, preparation method and application
  • Conditioned medium, freeze-dried powder, preparation method and application
  • Conditioned medium, freeze-dried powder, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Preparation and subculture of human adipose-derived mesenchymal stem cells

[0062] (1) Soak the removed human fresh adipose tissue in PBS containing penicillin and streptomycin (100U / mL), sterilize the sample bag with 75% ethanol, and then transfer it to the ultra-clean bench;

[0063] (2) Take out the adipose tissue and place it in a 10cm petri dish, wash it three times with PBS containing penicillin and streptomycin (100U / mL);

[0064] (3) Cut the adipose tissue into small pieces with scissors and tweezers, and remove blood vessels and connective tissue under a dissecting microscope;

[0065] (4) Transfer the separated adipose tissue block into a 50 ml centrifuge tube, add collagenase, shake and digest at 37°C for 30 minutes (the shaking speed should not allow the tissue block to sink to the bottom);

[0066] (5) Add an equal volume of PBS to stop the digestion, centrifuge at 500×g for 10 min,

[0067] (6) The suspension after centrifugation is divided i...

Embodiment 2

[0072] Embodiment 2, preparation and subculture of human dermal fibroblasts

[0073] (1) Soak the surgically excised human fresh foreskin in PBS containing penicillin and streptomycin (100 U / mL), sterilize the sample bag with 75% ethanol and move it into the ultra-clean bench;

[0074] (2) Take out the skin piece and place it in a 10cm petri dish, rinse twice with PBS containing penicillin and streptomycin (100U / mL), after washing away the residual impurities and blood stains, replace it with a fresh medium containing 1% double antibody + PBS to continue Soak for 30min;

[0075] (3) After removing subcutaneous fat and connective tissue with scissors and tweezers, cut the skin slices into 1cm×1cm size, separate them upwards and stick them on the bottom of 10cm petri dish;

[0076] (4) Add an appropriate amount of dispase II enzyme solution with a final concentration of 2mg / mL, and digest overnight in a refrigerator at 4°C

[0077] (5) Separate the epidermis and dermis in an u...

Embodiment 3

[0082] Example 3, Preparation of Human Adipose-derived Mesenchymal Stem Cell Conditioned Medium

[0083] (1) The human adipose-derived mesenchymal stem cells that have been subcultured to the 3rd to 6th passages are inoculated in a 10cm culture dish, and cultured in DMEM / F12 medium containing 10% FBS until the cell growth confluence reaches 70%;

[0084] (2) Discard the original serum-containing medium, replace it with Lonza UltraCULTURE serum-free medium, and culture it in a cell culture incubator at 37°C containing 5% CO2;

[0085] (2) After continuing to culture for 48 hours, collect the cell supernatant into a 50 mL centrifuge tube.

[0086] (3) Centrifuge at 4°C and 200×g for 15 minutes, and discard the precipitate.

[0087] (4) Take the supernatant and filter it with a 0.22 μm filter membrane, and the obtained filtrate is the conditioned medium of the adipose-derived mesenchymal stem cells.

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Abstract

The invention relates to a conditioned medium based on human adipose tissue-derived stromal cells, freeze-dried powder, a preparation method and application. Specifically, the invention provides a preparation and subculture method of human adipose-derived mesenchymal stem cells and human dermal fibroblasts, and screens out the optimal variety and proportion of the freeze-dried powder protective additive and the optimal dilution proportion of the conditioned medium. According to the culture medium provided by the invention, the adherence performance and the proliferation rate of the mesenchymal stem cells can be remarkably improved; ad the conditioned medium freeze-dried powder provided by the invention has a remarkable anti-aging effect.

Description

technical field [0001] The application belongs to the technical field of stem cells, and in particular relates to a freeze-dried powder based on human adipose-derived mesenchymal stem cell conditioned medium and its anti-oxidative and aging effects. Background technique [0002] Aging is accompanied by the gradual loss of structure and function of multiple organs. The skin is the largest organ of the human body. Skin aging is the most intuitive manifestation of body aging. Its main features are dry and rough skin, weakened elasticity, sagging, and deepened wrinkles. With the continuous improvement of living standards, people pay more attention to skin and body maintenance than before. Therefore, the development of new anti-aging treatments has important research significance and good clinical application prospects. [0003] Skin aging is a complex process, including endogenous aging and exogenous aging. Endogenous aging refers to natural aging caused by factors such as agi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61K9/19A61P17/18A61P17/00A61K8/99A61K8/98A61K8/02A61Q19/00A61Q19/08
CPCC12N5/0667A61K35/28A61K9/19A61P17/18A61P17/00A61K8/99A61K8/981A61K8/022A61Q19/08A61Q19/004C12N2509/00
Inventor 张京钟余爽汪婧雯
Owner 苏州京赛诺生物科技有限公司
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