Low-temperature preservation method of multifunctional 3D recombinant skin model

A skin model and preservation method technology, applied in the field of biomedicine, can solve the problems of limiting factors of preservation time, prolongation of solid preservation time, and reduction of material exchange capacity, so as to extend the preservation time limit, prolong the preservation time, and improve the ability of material exchange. Effect

Active Publication Date: 2020-09-08
JINAN PANSHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been relevant reports on solid medium suitable for cryopreservation of transported skin models and tissue-engineered skin (such as Chinese patents 201410418456.X, 201510697694.3, 201510697887.9). limiting factors
[0004] Cytochalasin B has a variety of biological functions, especially the effect on the microfilament structure. As a cryoprotectant, it can increase the elasticity of the cytoskeleton, which is very important for our cryopreservation. At present, there is a Chinese patent 201410418456 .X Cytochalasin B is added to the preservation solution, but so far, the use of cytochalasin B in the reported literature all occurs in a liquid environment, and the difference in dosage addition and treatment time will have a negative impact on cells or tissues. make a large impact
Due to the poor fluidity of solid storage solutions compared with liquid storage solutions, the movement of many molecules is restricted, resulting in reduced material exchange capacity, which largely limits the extension of solid storage time.

Method used

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  • Low-temperature preservation method of multifunctional 3D recombinant skin model
  • Low-temperature preservation method of multifunctional 3D recombinant skin model
  • Low-temperature preservation method of multifunctional 3D recombinant skin model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Different applications of solid preservation and liquid preservation to skin models

[0036] 1. Experimental steps

[0037] 1. Preparation of liquid preservation solution

[0038] (1) The basal medium is prepared as DMEM, F12 or a mixed solution of DMEM and F12, and the volume ratio of DMEM and F12 in the mixed solution of DMEM and F12 is 4:1-12.

[0039] (2) Add the added factors according to the concentration in the basal culture medium, and the factor concentrations are as follows: the concentration of hydrocortisone is 1ug / ml, the concentration of adenine 20nM, ITT is 10.0ug / ml, the concentration of L-glutamine The concentration of EOP (ethanolamine and catalytic phosphatidylethanolamine) is 10nM, the concentration of human epidermal growth factor (hEGF) is 0.5ng / ml, the concentration of bovine pituitary extract (BPE) is 1ug / ml, and the concentration of selenium is 10 -2 nM, the volume fraction of fetal bovine serum is 5%. After the preparation is comp...

Embodiment 2

[0048] Example 2 Effect of the Concentration of Cytochalasin B on the Vitality of Skin Cells

[0049] 1. Experimental steps

[0050] During the experiment in step 1, cytochalasin B with a concentration of 20 mg / ml was diluted to the following test concentrations: 1 mg / ml, 10 ug / ml, 5 ug / ml, 1 ug / ml, 0.1 ug / ml.

[0051] Step 2 Take dermal fibroblasts and epidermal cells in good logarithmic growth phase, digest, centrifuge, and count, then resuspend with dermal medium and epidermal medium respectively, with 600,000 dermal cells per well and 800,000 epidermal cells per well. 10,000 inoculated in 96-well cell culture plates with a volume of 100ul per well.

[0052] Step 3 After the cells adhere to the wall, add cytochalasin B at different concentrations in step 1, the volume is 100ul, and each concentration is repeated three times. The dermal and epidermal control groups are respectively added dermal medium and epidermal medium with a volume of 100ul, and set At 37°C, 5% CO 2 c...

Embodiment 3

[0056] Embodiment 3 3D epidermis model cryopreservation

[0057] 1. Experimental operation

[0058] 1. Set up the experimental group

[0059] The 3D epidermis model is cultivated by cell culture technology. At the end of the 3D epidermis model culture, the cultured epidermis model is divided into the control group, the experimental group 1 and the experimental group 2. The treatment method of the control group is that no preservation is performed after the culture is completed. MTT detection was performed directly. Experimental groups 1 and 2 were directly subjected to traditional solid storage and solid storage using the three-step method of the present invention, and then the epidermal model was recovered, and the cell viability before and after recovery was compared.

[0060] 2. Treatment of experimental group 1

[0061] (1) The basal medium is prepared as DMEM, F12 or a mixed solution of DMEM and F12, and the volume ratio of DMEM and F12 in the mixed solution of DMEM and...

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Abstract

The invention discloses a low-temperature preservation method of a multifunctional 3D recombinant skin model. According to the invention, cytochalasin B is applied to solid preservation of a skin model for the first time, a three-step method is adopted for treatment, and the treatment method can maintain the cell activity of the model in a transportation process and reduce cell hypoxic injury, sothat the test accuracy and the cell activity of the 3D heavy skin group model are guaranteed, and the preservation time is prolonged to a great extent.

Description

technical field [0001] The invention relates to a low-temperature preservation method of a multifunctional 3D recombined skin model, belonging to the field of biomedicine. Background technique [0002] With the development of in vitro organ culture medium replacement technology, the 3D recombinant skin model based on 3D in vitro cell culture has shown a unique trend in the evaluation of the safety and effectiveness of irritation tests. The 3D epidermal model constructed in vitro is similar to the structure of human skin, and can be used for in vitro substitution tests for cosmetic testing; the 3D full-thickness skin constructed in vitro is tissue-engineered skin with living cells, with epidermis and dermis, and can participate in wounds and wounds It can be used for the repair of skin defects caused by various reasons, accelerate the healing of skin wounds, and form functional skin, so as to reduce the pain of patients and improve the quality of life of patients. [0003] H...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/071
CPCA01N1/0226A01N1/0221C12N5/0698C12N2501/30C12N2500/40C12N2501/39C12N2500/32C12N2500/46C12N2501/11C12N2500/84C12N2500/05
Inventor 李霄邢志青张甜甜王杰张平郑海阳
Owner JINAN PANSHENG BIOTECH
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