Extracellular matrix freeze-drying protection liquid and application method thereof

A technology of exogenous matrix and protective solution, applied in the preparation method of peptides, chemical instruments and methods, applications, etc., can solve the problems of decreased activity, changes in the physical and chemical properties of the matrix, and weak electronegativity, and achieve low cytotoxicity and stable protection Sex, good freeze-drying effect

Active Publication Date: 2012-10-24
西安博和医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the extracellular matrix is ​​insoluble in water and cannot form a eutectic point with the lyoprotectant, so the protective agent and the extracellular matrix cannot form a stable co-state during the freezing process. Therefore, the lyoprotectant for soluble small molecular proteins is used in When the extracellular matrix is ​​removed, the generated ice crystals will damage the three-dimensional structure of the matrix, resulting in changes in the physical and chemical properties of the matrix
In addition, the surface of collagen molecules has a large number of positive charges, while the electronegativity of carbohydrates is weak, so the adhesion to collagen is weak, and the ability to compete with water molecules to replace hydrogen bonds is also weak, and the protection of collagen molecules is insufficient. , easy to collapse the collagen structure and decrease its activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0020] Example 1. Freeze-drying protection of collagen (collagen used was purchased from Sigma, USA).

[0021] Use normal saline as a solvent to prepare freeze-dried protection solution, which contains chondroitin sulfate 60mg / ml, vitamin C 2mg / ml, polyethylene glycol 130mg / ml, trehalose 20mg / ml, dextran 20mg / ml, serine 3μg / ml ml, threonine 3 μg / ml.

[0022] Example of use:

[0023] Because collagen can dissolve weak acid without denaturation. Therefore, collagen is first dissolved in an acetic acid solution with a volume concentration of 3%, and then the pH is adjusted to 7.2 to form a collagen solution with a final concentration of 20 mg / ml; then the collagen solution is mixed with 3 times the volume of lyoprotectant solution, and the Shake at room temperature for 5 minutes, then transfer to 4°C water bath; then cool down to -65°C at a rate of 1°C / min, and start vacuuming after maintaining for 4 hours. The speed is raised to -35°C and maintained for 24 hours; finally, the...

example 2

[0025] Example 2. Freeze-drying protection of decellularized small intestinal mucosa (the decellularized small intestinal mucosa used was prepared according to the method in Chinese patent application 200810150792.5).

[0026]Prepare a freeze-dried protection solution with physiological saline as a solvent, containing chondroitin sulfate 120mg / ml, vitamin C 4mg / ml, polyethylene glycol 250mg / ml, trehalose 40mg / ml, dextran 30mg / ml, serine 8μg / ml, threonine 8 μg / ml.

[0027] Example of use: Soak the decellularized small intestinal mucosa in 8 times the volume of freeze-dried protection solution, shake at room temperature for 20 minutes, then transfer to a 4°C water bath; then cool down to -65°C at a rate of 2°C / min, and maintain for 5 hours Then start vacuuming, make the absolute vacuum pressure reach 10Pa within 30 minutes, then raise the temperature to -35°C at a rate of 3°C / min and then maintain it for 15 hours; finally carry out analytical temperature rise, and raise the tem...

example 3

[0029] Example 3. Freeze-drying protection of decellularized cowhide matrix (the decellularized cowhide matrix used was purchased from Yantai Zhenghai Company).

[0030] Use normal saline as a solvent to prepare freeze-dried protection solution, which contains chondroitin sulfate 80mg / ml, vitamin C 4mg / ml, polyethylene glycol 350mg / ml, trehalose 40mg / ml, dextran 15mg / ml, serine 8μg / ml, threonine 8 μg / ml.

[0031] Example of use: Soak the decellularized cowhide matrix in 20 times the volume of freeze-dried protection solution, shake at room temperature for 20 minutes, then transfer to a 4°C water bath; then cool down to -65°C at a rate of 2°C / min, and maintain for 5 hours Then start vacuuming, and make the absolute vacuum pressure reach 10Pa within 30 minutes, then raise the temperature to -35°C at a rate of 3°C / min, and then maintain it for 15 hours; finally carry out analytical heating, and raise the temperature to 25°C at a rate of 10°C / min After maintaining for 6 hours, t...

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PUM

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Abstract

The invention relates to an extracellular matrix freeze-drying protection liquid. The extracellular matrix freeze-drying protection liquid takes normal saline as a solvent and comprises the following components: chondroitin sulfate, vitamin C, polyethylene glycol, trehalose, glucan, serine and threonine. The invention has the following advantages: the protection liquid has high penetrability, can be vitrified under a low-temperature condition, is rich in negative polar groups and hydroxyl groups, and can be efficiently bonded with collagen molecules; the stable and uniform extracellular matrix freeze-drying effect can be ensured; the protection liquid can be formed into the vitreous state at low temperature, thereby preventing ice crystals from damaging the protein structure; and a protection film is formed at the outer layer of the matrix in stead of hydrogen bonds formed by water molecules and collagen, thereby protecting the collagen function and the stability of the three-dimensional structure. The extracellular matrix freeze-drying protection liquid has low cytotoxicity, and can be used immediately after being rinsed with normal saline or sterilized water; the freeze-dried extracellular matrix can be preserved at normal temperature for more than three years when being packaged in vacuum; and the extracellular matrix freeze-drying protection liquid is suitable for the preservation of purified extracellular matrix such as collagen, elastin, glucoprotein and the like, and is also suitable for various biologic materials constructed by extracellular matrix materials.

Description

technical field [0001] The invention belongs to the technical field of medical biological material protection, and in particular relates to a protective solution for freeze-drying of extracellular matrix (abbreviated as freeze-drying) and a use method thereof. Background technique [0002] Extracellular matrix is ​​secreted by cells and can initiate intracellular signaling pathways through specific surface receptors to regulate cell differentiation, proliferation, migration, adhesion and other functions. It is derived from natural tissues and has good biocompatibility. The extracellular matrix is ​​mainly composed of collagen, laminin, fibronectin and other components; it is obtained by removing cellular components in biomaterials by physical and chemical means. This type of biomaterials is used in clinical repair and tissue engineering tissue construction. It has obvious curative effects and advantages; currently there are dozens of products on the market based on extracel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/00A01N1/02
Inventor 罗海浪
Owner 西安博和医疗科技有限公司
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