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Method for decellularizing mammalian cornea

A mammalian and decellularized technology, applied in the field of tissue engineering, can solve the problems of unincubated cornea softening, cornea easily swelled, and corneal stroma structure destructive, etc., and achieve the effect of shortening the contact time and maintaining the basic structure.

Active Publication Date: 2020-02-07
SHENZHEN AINEAR CORNEA ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both of the above two patented technologies use a hypotonic solution, which causes the cornea to swell easily and is more destructive to the corneal stroma structure, and does not incubate and soften the decellularized cornea

Method used

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  • Method for decellularizing mammalian cornea
  • Method for decellularizing mammalian cornea
  • Method for decellularizing mammalian cornea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This embodiment exemplifies the decellularization method of mammalian porcine cornea, including a pretreatment step and a decellularization step. The pretreatment step includes cleaning, scraping and cutting; the decellularization step includes freezing, thawing and removal; in:

[0041] Cleaning refers to that the obtained mammalian eyeball is cleaned with a cleaning solution, and the cleaning solution is a sterile PBS solution containing antibiotics, and the antibiotics are antibiotics known in the field of medicine; scraping refers to scraping off the corneal epithelium to make Bowman's membrane is exposed to remove epithelial cells; cutting refers to cutting the cleaned and scraped cornea to obtain a lamellar cornea with a thickness of 200-500 μm.

[0042] Freezing refers to sealing the cornea and placing it in liquid nitrogen for 5-60 minutes; warming and thawing refers to placing the frozen cornea in a water bath to thaw it; thawing refers to the process of thawin...

Embodiment 2

[0045] This example exemplifies the decellularization method of mammalian cornea. On the basis of implementation 1, a sterile PBS solution containing 300,000 units / L of fosfomycin was selected as the cleaning solution, and the cornea with a thickness of 250 μm was obtained after scraping and cutting. Lamellar cornea. Seal the cornea and place it in liquid nitrogen for 60 minutes; take it out and place it in a water bath at 20°C for 30 minutes, then place it in an aqueous solution containing 1.0% neutral protease and 0.01% EDTA at 4°C for 120 minutes, and then place it in normal saline Ultrasound treatment was performed for 8 minutes, and the ultrasonic frequency was 10 kHz to obtain acellular corneal stroma with good transparency, compact structure, and close to the natural characteristics of human cornea.

Embodiment 3

[0047] This example exemplifies the decellularization method of the mammalian cornea. On the basis of implementation 1, the cleaning solution is a sterile PBS solution containing 2 million units / L of streptomycin, and a 300 μm thick cornea is obtained after scraping and cutting. Lamellar cornea. Seal the cornea and place it in liquid nitrogen for 5 minutes; after taking it out, put it in a water bath at 37°C for 10 minutes, then place it in an aqueous solution containing 0.2% neutral protease and 0.20% EDTA at 45°C for 10 minutes, and then place it in PBS buffer Ultrasonic treatment for 15 minutes at an ultrasonic frequency of 100 kHz yields a decellularized corneal stroma with good transparency, compact structure, and close to the natural characteristics of the human cornea.

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Abstract

The invention discloses a method for decellularizing mammalian cornea. The method comprises a pretreatment step and a decellularization step, wherein the pretreatment step comprises cleaning, scrapingand cutting to obtain lamellar corneas with a thickness of 200-500 [mu]m, and the decellularization step comprises freezing, warm melting and removing to remove cells so as to obtain the decellularized corneal stroma which is good in transparency, compact in structure and close to the natural characteristics of human corneas. According to the method, mild neutral protease and EDTA are used as decellularization reagents, so that the microstructure of the cornea can be kept. According to the method, the cornea is soaked in the decellularization liquid for oscillation treatment, and then ultrasonic treatment is performed in the isotonic liquid, so that the contact time of the cornea and enzyme is effectively shortened, and the basic structure of the cornea can be maintained while the purposeof decellularization is achieved.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering, and in particular relates to a method for preparing a decellularized cornea from a mammalian cornea through steps such as pretreatment and decellularization. Background technique [0002] Corneal disease is the second leading cause of corneal blindness. Among them, 80% of patients with corneal disease can avoid blindness through corneal transplantation. However, under the influence of traditional Chinese culture, cornea donations are less, resulting in the shortage of allogeneic corneal donors. There are only more than 3,000 corneal transplants in the country every year, and the emergence of artificial corneas has brought hope to patients with corneal diseases. . The initial artificial corneas were corneal substitutes processed from inorganic or organic materials. However, these materials cannot simulate the microstructure of the natural cornea, and their biocompatibility is poor, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/36
CPCA61L27/3633A61L27/3641A61L2430/40A61L2430/16
Inventor 王海利张晋南宇咏梅唐小琪马百双甘良波
Owner SHENZHEN AINEAR CORNEA ENG