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Method for decellularization of human corneal stroma

A technology of corneal stroma and stromal cells, applied in medical science, prostheses, etc., can solve the problems of damaged corneal stroma tension, difficulty in clinical application, and retention, so as to avoid damage to tissue tension, protect biological characteristics, and maintain electrolyte levels. Effect

Inactive Publication Date: 2015-02-11
沈阳市第四人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many sources of corneal stroma material and decellularization methods, there are many problems: (1) corneal stroma material comes from human or animal eyes, and human corneal tissue compatibility is the best, but full-thickness corneal The sources are scarce, and the corneal stroma derived from animal eyes is difficult to avoid immune reactions and other problems; (2) In order to minimize the risk of immune reactions, the corneal stroma used for corneal transplantation needs to be decellularized in advance of the stromal cells on the corneal stroma
The development of the decellularization method from the initial freeze-thaw method to the more commonly used surfactant elution method has some unavoidable problems: for example, repeated freezing and thawing may cause collagen breakage and damage the tension of the corneal stroma; while surfactant All have relatively high toxicity, and it is difficult to apply clinically after treatment, and most of them stay in the laboratory stage at present.
[0005] There have been decades of research on corneal stroma decellularization methods. As mentioned earlier, many previous decellularization methods were difficult to promote clinically due to their limitations.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Use sterile micro-toothless forceps to put the corneal stromal flap with a smooth surface, 40-140 μm in thickness and 8±2 mm in diameter removed during laser refractive surgery into a flask containing 0.3 wt% penicillin and streptomycin from the operating table. normal saline in a 1ml eppendorf tube, and transferred to a secondary B2 type biological safety cabinet, inject physiological saline containing 0.3wt% penicillin and streptomycin into the petri dish, take out the corneal stroma flap, and wash it 3 times in the petri dish, every Then put the corneal stroma flap into a 5ml centrifuge tube, use a portable nitrogen bottle to fill the centrifuge tube with nitrogen gas, tighten the cap of the centrifuge tube and seal the tube with a sealing film, store at room temperature for 5 days; take out the corneal stroma flap after 5 days , transferred to the second-level B2 biological safety cabinet, washed 3 times in a petri dish with normal saline containing 0.3wt% penicillin...

Embodiment 2

[0024] Put the full-thickness corneal stroma from which other tissues have been removed with sterile microscopic forceps into a test tube containing normal saline containing 0.3 wt% penicillin and streptomycin, transfer it to a secondary B2 type biological safety cabinet, and put it in a petri dish Inject normal saline containing 0.3wt% penicillin and streptomycin in the medium, wash the corneal stroma in the culture dish 3 times, 2 minutes each time; take out the corneal stroma, put it into a 5ml centrifuge tube, and use a portable nitrogen bottle to fill the centrifuge tube with Nitrogen gas, screw the cap tightly and seal the tube with sealing film, store at room temperature for 7 days; take out the corneal stromal flap after 7 days, and wash it with normal saline containing 0.3wt% penicillin and streptomycin in a culture dish for 3 times, 2 minutes each time; Put the cleaned corneal stroma into a reagent bottle containing physiological saline containing 0.3wt% penicillin an...

Embodiment 3

[0027] Use sterile micro-toothless forceps to put the corneal stromal flap with a smooth surface, 40-140 μm in thickness and 8±2 mm in diameter removed during laser refractive surgery into a flask containing 0.3 wt% penicillin and streptomycin from the operating table. In a 1ml eppendorf tube of balanced salt solution, transfer it to a second-class B2 type biological safety cabinet, inject a balanced salt solution containing 0.3wt% penicillin and streptomycin into the petri dish, take out the corneal stroma flap, and wash it twice in the petri dish , 5 minutes each time; then put the corneal stromal flap into a 5ml centrifuge tube, use a portable nitrogen bottle to fill the centrifuge tube with nitrogen, tighten the cap of the centrifuge tube and seal the tube with sealing film, store at 4°C for 6 days; take it out after 6 days The corneal stroma flap was washed twice in a petri dish with balanced salt solution containing 0.3wt% penicillin and streptomycin for 5 minutes each ti...

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Abstract

The invention aims to solve the problem of donor shortage of corneal stroma and overcome defects of a traditional decellularization method, provides a method for decellularization of human corneal stroma, and belongs to the technical field of cornea in tissue engineering. The method comprises the following steps: collecting corneal stroma containing tissue cells to be put in a cleaning solution, and washing in the cleaning solution for 2-3 times and 2-5 minutes each time; sealing the corneal stroma in nitrogen, and storing for 5-7 days; taking out the corneal stroma from nitrogen, washing by the cleaning solution for 2-3 times and 2-5 minutes each time, shaking at constant temperature in the cleaning solution, changing the cleaning solution every 1-2 hours to obtain decellularized corneal stroma. The method can be used for completely decellularization, collagen of corneal stroma cannot be hurt, preparations applied in the whole process are non-toxic, the biological characteristics of the corneal stroma can be furthest protected, and the stroma cells can be effectively removed.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering cornea, and in particular relates to a decellularization method of human corneal stroma. Background technique [0002] About 3 to 5 million Chinese citizens are blinded by monocular and binocular corneal diseases, ranking second only to cataracts. The main diseases that lead to corneal blindness include various infectious corneal diseases, corneal degeneration, severe mechanical trauma, ocular surface chemical injury, severe dry eye, complex pterygium, ocular pemphigoid infection, etc., covering almost all corneal diseases kind. The only way for most of these patients to obtain useful vision is surgical treatment, and the main method of surgery is full-thickness or lamellar corneal transplantation. Due to the limitation of traditional concepts and eye bank conditions in my country, the number of corneal donors is very limited, the treatment cost is high, and some corneal donors cannot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/36
Inventor 齐飞李若溪
Owner 沈阳市第四人民医院
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