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Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium

A solid medium, low temperature preservation technology, applied in the field of biomedicine, can solve the problems of reduced vitality and structural damage, and achieve the effects of increasing stability, preventing deterioration and facilitating long-distance transportation.

Active Publication Date: 2014-12-24
GUANGDONG BOXI BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In view of the defects existing in the prior art, the object of the present invention is to provide a solid cryopreservation transportation medium and its use method for the problems of tissue engineered skin such as reduced vitality and structural damage during cryopreservation and transportation.

Method used

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  • Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium
  • Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium
  • Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Tissue engineered epidermal skin cryopreservation

[0040] Preparation of cryopreservation medium: The cryopreservation medium is based on epidermal cell culture medium MCDB153, take 100ml MCDB153 medium, add to it according to the final concentration shown: HEPES 100mmol / L, trehalose 0.1mmol / L, sucrose 30mmol / L , glycine 50mmol / L, alanine 0.5mmol / L, adenosine 5mmol / L, α-tocopherol 1mmol / L, vitamin C 0.01mmol / L, deferoxamine (DFO) 10mmol / L, 1,6-di Fructose phosphate (FDP) 0.5mmol / L; after dissolving, it was mixed with 0.5% agarose solution which was pre-autoclaved in accordance with 1:1 and then prepared into a solid medium with a concentration of 0.25% agarose.

[0041] The preparation method is as follows:

[0042] Liquid A: In the cell culture medium, add HEPES, agarose, trehalose, sucrose, glycine, alanine, adenosine, α-tocopherol, vitamin C, deferoxamine (DFO), 1,6-di Fructose phosphate (FDP), added in no order, after fully dissolved, filter to steriliz...

Embodiment 2

[0048] Example 2 Low Temperature Preservation of Tissue Engineering Skin Containing Melanocytes

[0049] Preparation of cryopreservation medium: take 50ml of F12 / DMEM (1:1) medium, add subsequent protective substances to it, so that the final concentrations are: HEPES 1mmol / L, trehalose 10mmol / L, sucrose 0.1mmol / L, Glycine 100mmol / L, Alanine 50mmol / L, Adenosine 1mmol / L, α-tocopherol 10mmol / L, Vitamin C 10mmol / L, Desferoxamine (DFO) 0.01mmol / L, Fructose 1,6-diphosphate (FDP) 0.1mmol / L; After dissolving, it is mixed with 2% agarose solution which has been pre-autoclaved in accordance with 1:1, and then prepared into a solid medium with a concentration of 1% agarose.

[0050] The successfully constructed tissue-engineered skin containing melanocytes together with the culture utensils was embedded on the surface of the solid medium, sealed under sterile conditions, and stored at 4°C for 24 hours.

[0051] After storage, the tissue engineering model was rewarmed with 37°C pre-warm...

Embodiment 3

[0052] Example 3 Full-thickness tissue engineered skin cryopreservation

[0053] Preparation of cryopreservation medium: the cryopreservation medium is epidermal cell culture medium F12 / DMEM (1:3), take 50ml of F12 / DMEM (1:3) medium, add to it according to the final concentration shown: HEPES 50mmol / L, trehalose 5mmol / L, sucrose 10mmol / L, glycine 1mmol / L, alanine 10mmol / L, adenosine 50mmol / L, α-tocopherol 0.01mmol / L, vitamin C 5mmol / L, deferoxamine (DFO) 2.5mmol / L, 1,6-diphosphofructose (FDP) 10mmol / L; after dissolving, mix with 3% agarose solution which has been pre-autoclaved in accordance with 1:1 to prepare 1.5% agarose concentration solid medium.

[0054] The preparation method is as follows:

[0055] Liquid A: In the cell culture medium, add HEPES, agarose, trehalose, sucrose, glycine, alanine, adenosine, α-tocopherol, vitamin C, deferoxamine (DFO), 1,6-di Fructose phosphate (FDP), added in no order, after fully dissolved, filter to sterilize;

[0056] Solution B: D...

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Abstract

The invention discloses a low-temperature preserved solid culture medium used for a tissue engineering skin model and a preservation method of the low-temperature preserved solid culture medium. The low-temperature preserved solid culture medium is prepared by the following steps: adding a low-temperature protection agent into a common culture medium for epidermis cells to prepare a basic culture solution; and mixing the basic culture solution with agarose gel and condensing to form the solid culture medium suitable for the skin model. The tissue engineering skin model is embedded into the culture medium, is preserved at 4-8 DEG C for 24-72 hours, and then is resuscitated by a common epidermis cell culture solution. The solid culture medium can be used for reducing tissue activity reduction and structure damages of cells and skin tissues, caused by a low-temperature preservation condition, to the greatest extent.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a solid medium for skin model cryopreservation and transportation and a preservation method thereof. Background technique [0002] With the rapid development of in vitro alternative science, skin models, as a substitute for skin safety testing, have been widely used in the testing of pharmaceuticals and cosmetics. The European Council of Alternative Methods (ECVAM) has passed the verification of EpiDerm and EpiSkin skin model products and the corresponding test laws. [0003] Due to the characteristics of the skin model's own performance, it is required to maintain its structure and activity in vitro, so as to ensure the accuracy of the test results. Therefore, the scope of promotion and application of the model is restricted by its storage and transportation conditions, and foreign chemicals should be excluded to the greatest extent. Impact. Therefore, it is of great significance to d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 支旭勃徐佳洁罗海浪卢永波李潇
Owner GUANGDONG BOXI BIO TECH CO LTD
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