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Gingival epithelium model and in-vitro building method thereof

A construction method and gingival technology, applied in the field of tissue engineering biomaterials, can solve the problems of long construction time, poor model stratification, and reduced model application, and achieve the effects of reducing production cost, shortening construction time, and improving barrier function.

Inactive Publication Date: 2017-11-03
GUANGDONG BOXI BIO TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Lauer et al. used gingival epithelium, using 3T3 cells as a trophoblast, and cultured in vitro for 3 to 4 weeks to form a complete epithelial membrane containing 3 to 6 layers of cells. The test results found that the cultured epithelium was well differentiated, similar to normal tissue, However, the application of heterogeneous trophoblast cells has a certain impact on the accuracy of the test results
[0004] From the above analysis, it can be seen that although relevant gingival epithelial models have been developed, these models have a series of defects: First, the model scaffolds often use exogenous cells, which have certain limitations in later applications; 2. The construction time is long. It takes up to one month to construct a batch of models in vitro. Too long construction time will increase the uncontrollable factors in the process and affect the stability of the model; 3. The single culture system cannot adapt to the nutrition of cells at different stages demand, resulting in poor model stratification, inhomogeneous and complete structure, weakened barrier function, and seriously reducing the application of the model

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  • Gingival epithelium model and in-vitro building method thereof

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Embodiment 1

[0026] Embodiment 1: This embodiment emphatically introduces the in vitro construction method steps of a kind of gingival epithelial model:

[0027] Step 1. Primary culture and expansion of human gingival epithelial cells:

[0028] Normal human gingival tissue was washed with 0.01M PBS containing 100IU / mL penicillin and 100μg / mL streptomycin under the condition of pH=7.4. Trim the tissue block, remove the subcutaneous connective tissue, and cut the tissue block into about 10mm 3 Wash with 0.01M PBS containing 100IU / mL penicillin and 100μg / mL streptomycin at pH=7.4, and digest with Dispase solution at 4°C for 16h. The epithelial layer and subcutaneous connective tissue layer were separated, and the separated epithelial layer was transferred to a centrifuge tube pre-prepared with 1.5 mL of 0.25% trypsin, incubated at 37°C for 5 min, and digested to obtain a single-cell suspension. Add keratinocyte serum-free medium to resuspend the cells, transfer them to culture flasks, chang...

Embodiment 2

[0040] This embodiment focuses on the in vitro construction method steps of a gingival epithelial model:

[0041] Step 1. Primary culture and expansion of human gingival epithelial cells:

[0042] Normal human gingival tissue was washed with PBS 0.01M containing 100IU / mL penicillin and 100μg / mL streptomycin at pH=7.4. Trim the tissue block, remove the subcutaneous connective tissue, and cut the tissue block into about 10mm 3 Wash with PBS 0.01M containing 100IU / mL penicillin and 100μg / mL streptomycin at pH=7.4, and digest with Dispase solution at 4°C for 16h. The epithelial layer and subcutaneous connective tissue layer were separated, and the separated epithelial layer was transferred to a centrifuge tube pre-prepared with 1.5 mL of 0.25% trypsin, incubated at 37°C for 5 min, and digested to obtain a single-cell suspension. Add keratinocyte serum-free medium to resuspend the cells, transfer them to culture flasks, change the medium after 12 hours, and change the medium ever...

Embodiment 3

[0054] This embodiment focuses on the in vitro construction method steps of a gingival epithelial model:

[0055] Step 1. Primary culture and expansion of human gingival epithelial cells:

[0056] Normal human gingival tissue was washed with PBS 0.01M containing 100IU / mL penicillin and 100μg / mL streptomycin at pH=7.4. Trim the tissue block, remove the subcutaneous connective tissue, and cut the tissue block into about 10mm 3 Wash with PBS 0.01M containing 100IU / mL penicillin and 100μg / mL streptomycin at pH=7.4, and digest with Dispase solution at 4°C for 16h. The epithelial layer and subcutaneous connective tissue layer were separated, and the separated epithelial layer was transferred to a centrifuge tube pre-prepared with 1.5 mL of 0.25% trypsin, incubated at 37°C for 5 min, and digested to obtain a single-cell suspension. Add keratinocyte serum-free medium to resuspend the cells, transfer them to culture flasks, change the medium after 12 hours, and change the medium ever...

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Abstract

The invention discloses a gingival epithelium model and an in-vitro building method thereof. The method is characterized by including following steps: step 1, primary culture and amplification of gingival epithelium cells; step 2, inoculation culture of gingival epithelium cell sap; step 3, proliferation and differentiation culture of a gingival epithelium model air-liquid face. Human-derived gingival epithelium cells are used, so that model structure and function are closer to normal human gingival epithelium tissue, and detection indexes can be reflected more really; in-vitro building is high in repeatability, and industrial preparation can be realized; an air-liquid face staged culture method ensures different nutritional needs of cells in different stages, shortens building time and lowers production cost; by adding factors into a culture medium, normal stratifying of the model is ensured, influence of this own structure and extracellular microenvironment can further improve a barrier function of the model, and a finally-built in-vitro gingival epithelium model is highly similar to human gingival epithelium tissue in structure and function.

Description

technical field [0001] The invention relates to the technical field of tissue engineering biomaterials, in particular to an in vitro replacement of gingival epithelium for dental care products, medical devices, dental materials, chemicals, small molecular compounds, active proteins and other products for safety and efficacy testing and A gingival epithelial model and its construction method for evaluating the safety and efficacy of other materials in contact with the gingival epithelium. Background technique [0002] At present, the gingival epithelium has received more and more attention in the safety and efficacy testing of dental care products, the carcinogenicity testing of tobacco components, and the pathogenic mechanism of oral pathogens. However, the source of gingival epithelium in the current research is mainly animal gingival epithelium, and the gingival epithelium tissue of patients is surgically removed. Both have certain application drawbacks. The gingival epit...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0625C12N2500/12C12N2500/14C12N2500/24C12N2500/32C12N2500/34C12N2500/36C12N2500/38C12N2500/40C12N2500/84C12N2501/11C12N2501/33
Inventor 何欣李潇卢永波
Owner GUANGDONG BOXI BIO TECH CO LTD
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