Preparation of exosomes from dental epithelial cells, preparation and application of exosome implants

A technology of epithelial cells and exosomes, applied in the field of biomedical engineering, to achieve the effects of overcoming immunogenicity, avoiding immunogenic problems, and being easy to store and transport

Active Publication Date: 2021-02-23
成都世联康健生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are also many difficulties in the application of engineered dental epithelial exosomes, such as the screening of exosome scaffold materials. An ideal scaffold material must first be safe and harmless, and be able to adhere to and release exosomes slowly. It can be gradually absorbed in the body without affecting the microenvironment of the host in vivo; secondly, because there will be a series of physical and chemical operations in the extraction process of exosomes, it is difficult to ensure their safety and harmlessness

Method used

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  • Preparation of exosomes from dental epithelial cells, preparation and application of exosome implants
  • Preparation of exosomes from dental epithelial cells, preparation and application of exosome implants
  • Preparation of exosomes from dental epithelial cells, preparation and application of exosome implants

Examples

Experimental program
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Effect test

Embodiment 1

[0046] A preparation of exosomes from dental epithelial cells,

[0047] (1) Treatment of engineered dental epithelial cells

[0048] The engineered dental epithelial cell line (HERS-H1) preserved in liquid nitrogen was revived with warm water at 45°C, inoculated in a 10cm culture dish, added epithelial cell medium medium containing 2% fetal bovine serum, and stored at 37°C. 5%CO 2 After 28 hours, the medium was changed, and the cells were subcultured with trypsin after they were fused to 90%. When the cells in each dish expand to 90-95% of its volume, the extraction of the engineered dental epithelial cell line extract is carried out.

[0049] (2), acquisition of engineered dental epithelial cell extract

[0050] After sufficient cells have been amplified in step (1), inoculate the epithelial cell line obtained in step (1) into a T75 culture flask, and when it reaches 70% confluence, discard the medium, and wash the cells twice with PBS , add epithelial cell medium medium ...

Embodiment 2

[0054] This embodiment is on the basis of embodiment 1,

[0055] Identification of engineered dental epithelial cell-derived exosomes:

[0056] Step 1. Morphological Identification by Transmission Electron Microscopy

[0057] Exosomes were fixed with 1% glutaraldehyde, overnight at 4°C. After washing off the fixative, the mixed exosomes were dropped onto the sample-loading copper grid, and then water phosphotungstic acid was added dropwise to negatively stain the exosomes for 1 min. Transmission electron microscopy (Hitachi H-7650) imaged and photographed, a circular vesicle-like structure with a diameter of 50-150nm can be seen ( figure 1 ).

[0058] Step 2. Particle size distribution identification

[0059] Take 50 microliters of sample and inject it into a special channel to measure the particle size distribution of exosomes. It can be seen that the peak of the particle size distribution is around 110nm, and the particle size is mainly distributed between 60-150nm ( fi...

Embodiment 3

[0061] A collagen gel composite engineered dental epithelial cell-derived exosome implant was prepared. The standard type of collagen gel was placed on ice, and the required tip was pre-cooled at the same time. Add collagen gel, 10×basic medium and buffer to pre-cooled glass centrifuge tubes at a ratio of 8:1:1. The buffer composition was 2.2% sodium bicarbonate, 0.05N sodium hydroxide and 200M hydroxyethylpiperazine ethanesulfonic acid. Take 800 micrograms of exosome pellets and resuspend them with 400 microliters of liquid collagen gel mixture (the final concentration of exosomes is 2 mg / ml), and transfer the obtained liquid collagen gel mixed with engineered dental epithelial cell-derived exosomes to Put it into a pre-cooled centrifuge tube, and put it on ice for later use, to ensure that the solvent is in a liquid state and the time is cut off before the in vivo experimental model is stacked, within 0.5h. The standard type of collagen gel refers to the basic type of colla...

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Abstract

The invention discloses a preparation of exosomes of dental epithelial cells, comprising the following steps: (1) processing of engineered dental epithelial cell lines; (2) obtaining supernatant of engineered dental epithelial cell lines; (3) engineering Extraction of dental epithelial cell lines; (4) acquisition of engineered dental epithelial cell exosomes. The invention also discloses a preparation method of a collagen gel compound engineered dental epithelial cell exosome implant. The invention also discloses the application of an engineered dental epithelial cell exosome in promoting periodontal and dental pulp regeneration in vivo. The immortalized Hertwig epithelial cell line (HERS‑H1) was used for long-term stable expansion and large-scale preparation of exosomes derived from dental epithelium. And because exosomes do not contain major histocompatibility complex, the immune rejection problem faced in stem cell-based regeneration is avoided, and exosomes are easy to store and transport.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to the preparation of exosomes from dental epithelial cells, the preparation and application of exosome implants. Background technique [0002] Periodontitis is one of the common oral diseases of middle-aged and elderly people, which can lead to the absorption of alveolar bone and loosening of teeth, eventually leading to tooth loss. At present, conventional cleaning and curettage is the main method clinically, which can only delay the bone resorption process, and there is no radical treatment method yet. Pulp disease caused by caries, various traumas, etc. is one of the most common oral infectious diseases in the world, which can lead to pulp inflammation and even necrosis. However, root canal therapy, as the most effective means of pulp treatment at present, is The replacement of active dental pulp with artificial materials results in the loss of dental pulp, which...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10A61K35/36A61K9/06A61K47/42A61P1/02A61L27/38A61L27/24A61L27/52A61L27/54
CPCA61K9/06A61K35/36A61K47/42A61L27/24A61L27/3813A61L27/3865A61L27/3895A61L27/52A61L27/54A61L2300/30A61L2300/412A61P1/02C12N5/0625C12N2509/00C12N2509/10C08L89/00
Inventor 乔鞠陈国庆张思诚
Owner 成都世联康健生物科技有限公司
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