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Cryopreservation solution of tissue engineering products and application method thereof

A cryopreservation and tissue engineering technology, applied in the field of tissue engineering of biomedical materials, can solve the problems of low cryopreservation efficiency, unstable structure, and decline in the effect of tissue engineering products, and achieve long storage time, low toxicity, and wide application range Effect

Inactive Publication Date: 2013-10-30
FOURTH MILITARY MEDICAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficiency of such cryopreservation is still low, and its effect will be further reduced when applied to tissue engineering products with relatively unstable structures.

Method used

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  • Cryopreservation solution of tissue engineering products and application method thereof
  • Cryopreservation solution of tissue engineering products and application method thereof
  • Cryopreservation solution of tissue engineering products and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0017] Example 1. Cryopreservation of tissue-engineered skin

[0018]1. Preparation and pretreatment of cryopreservation solution: The cryopreservation solution uses DMEM culture medium as a solvent, which contains: vitamin B 6 μg / ml, vitamin C 6 μg / ml, chondroitin sulfate 3% w / v, β-integration Chlorophyllin 0.4% w / v, Sodium Cromolyn 4μg / ml, Cytochalasin B 3μg / ml, L-Glutamine 4mM, Bovine Serum Albumin 2%w / v, Fetal Bovine Serum 10%w / v, Na 2 CO 3 0.3% w / v, trehalose 3% w / v, polysucrose-70 2% w / v;

[0019] Put the tissue-engineered skin into a freeze-resistant container (such as a polyethylene or polypropylene plastic container), add a preservation solution to immerse the skin, and vibrate at 100 rpm for 50 minutes at room temperature; add dimethyl sulfoxide to the preservation solution to make When the concentration reaches 5%, let it stand for 10 minutes after mixing; cool down the preservation solution containing the skin to 4°C, add dimethyl sulfoxide to the preservation s...

example 2

[0021] Example 2. Cryopreservation of tissue engineered blood vessels

[0022] 1. Preparation and pretreatment of cryopreservation solution: The cryopreservation solution uses DMEM culture medium as a solvent, which contains: vitamin B 15 μg / ml, vitamin C 15 μg / ml, chondroitin sulfate 5% w / v, β-integration Chlorophyllin 0.4% w / v, Sodium Cromolyn 2μg / ml, Cytochalasin B 1.5μg / ml, L-Glutamine 4mM, Bovine Serum Albumin 3%w / v, Fetal Calf Serum 10%w / v, Na 2 CO 3 0.2% w / v, trehalose 3% w / v, polysucrose-70 2% w / v.

[0023] Put the blood vessel into a freeze-resistant container, add protection solution to submerge the blood vessel, shake at 80 rpm for 30 minutes at room temperature; add dimethyl sulfoxide to the protection solution to make the concentration reach 3%, mix well and let stand 10 minutes; cool down to 4°C, then add dimethyl sulfoxide to the protection solution to make the concentration reach 6%, mix well and let stand for 10 minutes; cool down the protection solution to...

example 3

[0025] Example 3. Cryopreservation of tissue-engineered corneas

[0026] 1. Preparation and pretreatment of cryopreservation solution: The cryopreservation solution uses DMEM culture medium as a solvent, which contains: vitamin B 10 μg / ml, vitamin C 10 μg / ml, chondroitin sulfate 5% w / v, β-integration Chlorophyllin 0.4% w / v, Sodium Cromolyn 3μg / ml, Cytochalasin B 5μg / ml, L-Glutamine 4mM, Bovine Serum Albumin 3%w / v, Fetal Bovine Serum 10%w / v, Na 2 CO 3 0.2% w / v, trehalose 6% w / v, polysucrose-70 3% w / v;

[0027] Put the tissue-engineered cornea into a freeze-resistant container, add protection solution to immerse the cornea, shake at 60 rpm for 40 minutes at room temperature; then add dimethyl sulfoxide to the protection solution to make the concentration reach 2%, mix well Then let it stand for 20 minutes; cool down to 4°C, add dimethyl sulfoxide again to make the concentration reach 4%, mix well and let stand for 10 minutes; then cool down to 0°C, add dimethyl sulfoxide to m...

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Abstract

A cryopreservation solution of tissue engineering products uses DMEM culture solution as a basic solvent which is added with vitamin B, vitamin C, chondroitin sulfate, beta-integrin, cromolyn sodium, cytochalasin B, L-glutamine, bovine serum albumin, fetal bovine serum, trehalose, sodium carbonate, polysucrose-70, and dimethyl sulfoxide added during freezing storage. The cryopreservation solution provided by the invention has little toxicity to cells and long storage time, and can be stored for six months under 80 DEG C below zero, and 12 to 18 months in liquid nitrogen; and the cell viability of the resuscitated cells can be over 60%. The stored tissues can be simply and conveniently used after being resuscitated for only three to five min in water bath under the temperature of 37 DEG C and being washed by sterilized saline water. The invention can be widely used, and is suitable for tissue engineering skin, tissue engineering cornea, tissue engineering blood vessel, tissue engineering nerve and the like, and also is applicable to the cryopreservation of normal tissues.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering of biomedical materials, and in particular relates to a low-temperature preservation method of tissue engineering products. Background technique [0002] Tissue engineering products are artificial tissues or organs constructed by cells and three-dimensional scaffolds. For example, tissue engineered skin constructed in vitro is a functional skin substitute constructed by combining seed cells and scaffold materials by applying tissue engineering methods, and then planted on On the wound surface, in the process of gradual degradation and absorption of scaffolding materials, cells can grow normally, differentiate and form tissues with certain functions, so as to achieve the purpose of replacing, repairing and regenerating damaged tissues or organs. The ultimate goal of tissue engineering is to achieve large-scale industrialization, so long-term preservation of tissue engineering products is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
Inventor 金岩罗海浪张勇杰
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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