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TA culture solution for constructing barrier-reinforced in-vitro recombinant cuticle model

A technology of culture medium and reinforcing body, applied in the field of TA culture medium, can solve the problems of imperfect barrier function of the reconstituted epidermis model, affecting the accuracy of the model to chemical substances, incomplete intercellular connection structure, etc., to meet the needs of skin absorption and penetration. Test requirements, enhancement of tight junction permeability barrier, effect of strong proliferation and differentiation ability

Inactive Publication Date: 2020-11-10
GUANGDONG BOXI BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to abnormalities in lipid composition and lamellar structure, the factors causing barrier function defects in the model may also include incomplete intercellular connection structures, damage to the normal exfoliation process of the stratum corneum, and the existence of non-keratinized microscopic lesions. The defects of the existing recombinant epidermal models lead to imperfect barrier function, which limits the application of these skin models in other aspects such as skin absorption and penetration experiments
[0005] Although the full-thickness skin model with fibroblast dermis and epidermis is more similar to human natural skin in tissue structure and can reduce permeability to a certain extent, the addition of dermis will introduce other effects, such as in the model During the preparation process, the source, quantity and state of fibroblasts in the lower layer have a great impact on the proliferation and differentiation of epidermal cells, resulting in poor stability of the model and affecting the accuracy of the model in judging chemical substances
In addition, during the process of in vitro differentiation, fibroblasts will secrete collagen themselves to shape the external collagen scaffold or other biomaterial scaffolds, which will easily cause the dermis and even the model to shrink, causing the test object to leak from the edge of the model. Interfering with the evaluation of test results; finally, the existence of fibroblasts also requires that the culture medium for constructing the system must contain serum, which introduces unpredictable safety risks
Moreover, the model construction process is cumbersome, the preparation cycle is long, and the structure is complex

Method used

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  • TA culture solution for constructing barrier-reinforced in-vitro recombinant cuticle model
  • TA culture solution for constructing barrier-reinforced in-vitro recombinant cuticle model
  • TA culture solution for constructing barrier-reinforced in-vitro recombinant cuticle model

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The embodiment of the present invention provides a method for constructing an in vitro recombinant epidermal model with enhanced barrier function, which specifically includes the following steps:

[0022] 1. Preparation of epidermal model construction medium:

[0023] Epidermal cell culture solution preparation: KC-growth culture solution is used as the base solution, and L-glutamine 1.0-1.5mM and bovine pituitary extract 12-25mg are added to every 500ml.

[0024] Epidermal cell TU culture medium: on the basis of (1), add 0.5-5 μg of epidermal growth factor, 2-50 μg of hydrocortisone, 1-10 mg of insulin, 5-25 mg of adenine, 2.5-6 μg of transferrin, calcium chloride 0.03~0.2mM.

[0025] Epidermal cell TA culture medium: on the basis of (2), add lipid mixture (L-serine 0.25~5mM, palmitic acid 0.4~5μM, linoleic acid 0.01~0.05mM, argininosuccinic acid 0.025~0.05mM , bovine serum albumin 0.01-0.05mM), macrophage-activating lipopeptide MALP-2 (Macrophage-activating Lipopept...

Embodiment 2

[0031] The embodiment of the present invention provides a method for constructing an in vitro recombinant epidermal model with enhanced barrier function, which specifically includes the following steps:

[0032] 1. Preparation of special medium for epidermal model construction:

[0033] Epidermal cell culture solution preparation: KC-growth culture solution is used as the base solution, and L-glutamine 1.0-1.5mM and bovine pituitary extract 12-25mg are added to every 500ml.

[0034] Epidermal cell TU culture medium: on the basis of (1), add 0.5-5 μg of epidermal growth factor, 2-50 μg of hydrocortisone, 1-10 mg of insulin, 5-25 mg of adenine, 2.5-6 μg of transferrin, calcium chloride 0.03~0.2mM.

[0035] Epidermal cell TA culture medium: on the basis of (2), add lipid mixture (L-serine 0.25~5mM, palmitic acid 0.4~5μM, linoleic acid 0.01~0.05mM, argininosuccinic acid 0.025~0.05mM , bovine serum albumin 0.01-0.05mM), macrophage-activating lipopeptide MALP-2 (Macrophage-activat...

Embodiment 3

[0041] The application of the in vitro reconstituted epidermal model in the chemical skin absorption and penetration experiment is as follows:

[0042] 1. Skin pretreatment

[0043] Select healthy human abdominal skin, carefully remove the subcutaneous mucosa and fat tissue, rinse it with normal saline, absorb the normal saline on the surface of the skin with filter paper, and select undamaged skin of appropriate size under a dissecting microscope for future use. The in vitro reconstituted epidermis model (stimulation model) and the barrier-enhanced epidermis model (permeation model) were cultured on the air-liquid surface for 10 or 14 days, respectively, for the permeation test. Check the integrity of the human skin suit and epidermal model before the experiment to ensure that there is no damage to the barrier.

[0044] 2. Percutaneous penetration test

[0045] (1) Franz diffusion cell was used for transdermal test (the effective penetration surface S was 0.196cm 2 , the v...

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Abstract

The invention provides a TA culture solution for constructing a barrier-reinforced in-vitro recombinant cuticle model, and relates to the technical field of tissue engineering. The barrier function ofthe tissue engineering reconstructed cuticle model can be strengthened from two aspects of chemical composition and physical structure, the requirements of skin absorption and penetration tests can be met, and the application range of products can be enlarged.

Description

technical field [0001] The invention relates to the technical field of tissue engineering, in particular to a TA culture solution for constructing a barrier-strengthening in vitro reconstituted epidermis model. Background technique [0002] The skin is the barrier between the human body and the external environment, protecting various tissues and organs in the body from physical, mechanical, chemical and pathogenic microorganisms. Human skin is mainly composed of three parts, namely the epidermis, dermis and subcutaneous tissue. The epidermis is the outermost layer of the skin and consists of the basal layer, acanthocyte layer, granule cell layer, and stratum corneum. The stratum corneum is the final product of epidermal cell differentiation. It is composed of keratinocytes and their intercellular lipid matrix, with a thickness of about 10 μm. It is the main structure of the skin barrier function. Therefore, the barrier function of the stratum corneum has been the main rese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0625C12N2501/11C12N2500/14C12N2500/25C12N2500/32C12N2500/36C12N2500/40C12N2500/38C12N2500/84C12N2533/30C12N2501/998
Inventor 李润芝李潇卢永波
Owner GUANGDONG BOXI BIO TECH CO LTD