Preparation method of acellular human corneal stroma

A corneal stroma, decellularization technology, applied in medical science, tissue regeneration, prosthesis, etc., can solve the problems of affecting the optical and mechanical properties of the corneal stroma, damaging the tension of the corneal stroma, and destroying the microstructure of the cornea, so as to maintain the original microstructure Effect of structure and transparency, prevention of pollution, avoidance of toxic effects

Inactive Publication Date: 2020-10-30
镇江雷音再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Repeated freezing and thawing or ultrasonic disruption of cells during the decellularization process can easily damage the microstructure of the cornea, cause collagen breakage, damage the tension of the corneal stroma, and affect the optical and mechanical properties of the corneal stroma, resulting in poor recovery of transparency after transplantation. Affect the patient's vision; and some chemical reagents such as surfactants are added to some decellularization methods, and if the post-treatment is not good, there will be a large toxic residue
These factors all determine whether the corneal material can be successfully transplanted clinically and affect the surgical effect.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] This embodiment is to prepare decellularized human corneal stroma, including the following steps:

[0027] 1) Pre-treatment of decellularization: the collected human corneal stroma material derived from the full femtosecond laser refractive surgery was washed twice in the prepared washing solution; each washing time was 3 minutes; the washing solution contained 1% ((V / V)) penicillin-streptomycin-amphotericin B mixed solution (100× third antibody) in PBS buffer, which can wash away the host cells on the surface of corneal stromal materials derived from femtosecond laser surgery, At the same time, it can inhibit the growth of bacteria and fungi, and avoid the chance of contamination during the treatment process.

[0028] 2) Cell crushing treatment: the cell membrane and nuclear membrane of the remaining cells on the human corneal stroma material are ruptured by the osmotic pressure shock method of hypertonic fluid and low osmotic fluid, and the DNA and RNA components cont...

Embodiment 2

[0033] This embodiment is also to prepare decellularized human corneal stroma, the operation is as follows:

[0034]Put the collected human corneal stromal material from femtosecond laser refractive surgery into the prepared washing solution for pre-decellularization; the washing solution contains 1% (V / V) penicillin-streptomycin- Amphotericin B mixed solution (100×third antibody) in PBS buffer, the number of washes is 3 times; each wash time is 2min.

[0035] Then, the osmotic pressure shock method is used to crush the cells of the human corneal stroma material, so that the cell membrane and nuclear membrane of the remaining cells on it are broken, and the DNA and RNA components contained in it are released; the human corneal stroma material is first placed in the hypertonic fluid medium, soak for 5 hours, and after the internal and external osmotic pressure of the cells reaches equilibrium, move them into the hypotonic solution and soak for 5 hours, so that the cells will sw...

Embodiment 3

[0039] Place the collected human corneal stromal material derived from femtosecond laser refractive surgery into HBSS buffer containing 1% (V / V) penicillin-streptomycin-amphotericin B mixed solution (100×three antibodies) , wash 3 times; wash 2min each time. Then put the human corneal stromal material in 1.5mol / L NaCl hypertonic solution and soak for 2 hours. After the osmotic pressure inside and outside the cells reaches equilibrium, move it into the triple distilled water and soak in the hypotonic solution for 3 hours, so that the remaining cells on the human corneal stromal material The cell and nuclear membranes rupture, releasing their DNA and RNA components. Then, the human corneal stromal material was digested with PBS buffer solution containing 1000U / ml DNase and 500U / ml RNase at a temperature of 25°C for 1 hour to remove DNA and RNA components released by cell rupture. Then put the human corneal stroma material into the repair solution for rinsing and repairing, the ...

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Abstract

The invention discloses a preparation method of an acellular human corneal stroma, comprising the following steps of: 1) cell pretreatment: flushing with a buffer solution containing 1% (V / V) penicillin-streptomycin-amphotericin B (100 * three antibodies) mixed solution to effectively kill bacteria and fungi and prevent pollution; (2) cell disruption treatment: disrupting cells by adopting an osmotic pressure impact method, and well maintaining the tissue physiological structure of corneal stroma and the form of collagenous fibers while swelling and disrupting the cells; 3) enzyme digestion treatment: adopting nuclease treatment to effectively remove DNA and RNA components of cells, wherein the decellularization treatment is safe and thorough; 4) rinsing and repairing: cleaning corneal cell residues, protecting corneal collagen fibers and promoting self-repairing of corneal wounds, and 5) sterilizing The preparation method has the advantages of complete decellularization, no damage tocorneal stroma collagen, maximum protection of the biological characteristics of corneal stroma, and effective removal of stroma cells.

Description

technical field [0001] The invention relates to a preparation method of decellularized human corneal stroma. Background technique [0002] There are about 5 million people blinded by corneal disease in China, second only to cataract, ranking second in ophthalmology blinding diseases. About 80% of patients can avoid blindness through corneal transplantation. However, due to the limitation of traditional concepts and eye bank conditions in our country, the number of corneal donors is very limited, and the treatment cost is expensive. The rise and development of modern tissue-engineered cornea research has opened up another direction for the search for corneal replacement materials, and then corneal substitutes of synthetic materials and natural biomaterials have also appeared. Although these materials have good biocompatibility, they can be maintained after transplantation. The integrity of the eyeball can restore the transparency of the cornea to a certain extent, but due to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/36
CPCA61L27/3633A61L27/3641A61L27/3687A61L27/3691A61L2430/16A61L2430/40
Inventor 郭文广蔡枫苏萍肖丹许伟吴佳宇
Owner 镇江雷音再生医学科技有限公司
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