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Decellularized corneal limbus protective liquid and preparation method of decellularized corneal limbus

A corneal limbal and acellular technology, which is applied in the field of bioengineering medical materials, can solve the problem of rarely acellular limbal stroma, and achieves the effects of being conducive to proliferation and crawling, optimizing acellular conditions and having a wide range of sources.

Active Publication Date: 2017-05-10
健诺维(成都)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

With the extensive and in-depth research on biological tissue engineering, heterogeneous corneal stromal materials prepared by decellularization and virus inactivation have achieved preliminary therapeutic effects at the animal level and in clinical applications, but there are few acellular limbal stroma materials. Related reports

Method used

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  • Decellularized corneal limbus protective liquid and preparation method of decellularized corneal limbus
  • Decellularized corneal limbus protective liquid and preparation method of decellularized corneal limbus
  • Decellularized corneal limbus protective liquid and preparation method of decellularized corneal limbus

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preparation example Construction

[0031] The preparation method of the decellularized limbus of the present invention is to carry out the whole process of fresh porcine corneal limbal matrix containing Bowman's membrane in the decellularized limbus protection solution. The composition of the decellularized corneal limbal protective solution of the present invention is PBS containing 5-12g / L hyaluronic acid, 5-20g / L chondroitin sulfate, 3-10g / L low molecular weight dextran and 2.5-5mg / L tobramycin The buffer solution is preferably a PBS buffer solution containing 7-10 g / L hyaluronic acid, 10-15 g / L chondroitin sulfate, 5-8 g / L low molecular weight dextran and 3-4 mg / L tobramycin.

[0032] First, the fresh porcine limbal stroma containing the Bowman's membrane is sealed in the decellularized limbal protection solution, and the cells are broken by high static pressure treatment. The condition for high static pressure treatment of broken cells is 100-600 MPa, preferably 200-500 MPa, more preferably 200-400 MPa; th...

Embodiment 1

[0038] Cut out the fresh porcine limbal stroma (including 1mm sclera, 2mm peripheral cornea, about 0.2mm thick) containing Descemet's membrane.

[0039] The protection solution is used to protect the limbal tissue throughout the whole process. The composition of the protection solution is: 8g / L hyaluronic acid, 8g / L chondroitin sulfate, 5g / L low molecular dextran, 2.5mg / L Tobu Mycin, adjust the pH value to 7.3, and the osmotic pressure is 320mOsm;

[0040] Seal the cut fresh porcine limbal stroma containing Descemet's membrane in a plastic bag filled with protective solution; treat it twice under the condition of high static pressure of 200Mpa, each time is 2 minutes; after taking out the limbus , placed in a protective solution containing 0.3% sodium lauryl sulfate + 200 U / ml DNase, set the shaking table speed to 100 rpm, and the temperature to 20°C, and digested for 4 hours to remove the DNA components in the cornea; After the limbus was taken out, it was placed in a protec...

Embodiment 2

[0042] Cut out the fresh porcine limbal stroma (including 1mm sclera, 2mm peripheral cornea, about 0.2mm thick) containing Descemet's membrane.

[0043] The protection solution was used to protect the limbal tissue throughout the whole process. The composition of the protection solution was: PBS buffer solution with 5g / L hyaluronic acid, 17g / L chondroitin sulfate, 10g / L low molecular weight dextran, 3mg / L Tobramycin element, adjust the pH value to 7.4, and the osmotic pressure is 380mOsm;

[0044]Seal the cut fresh porcine limbal stroma containing Descemet's membrane in a plastic bag filled with protective solution; treat it 5 times under the condition of high static pressure of 400Mpa, each time is 1 minute; take out the limbus , placed in a protective solution containing 0.5% sodium lauryl sulfate + 2000U / ml DNase, set the shaking table speed to 120 rpm, and the temperature to 30°C, and digested for 2 hours to remove the DNA components in the cornea; After the limbus was ta...

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Abstract

The invention relates to the field of bioengineering medical materials, and discloses decellularized corneal limbus protective liquid and a preparation method of decellularized corneal limbus. The component of the decellularized corneal limbus protective liquid is a PBS buffer solution containing hyaluronic acid of 5 to 12 g / L, chondroitin sulfate of 5 to 20 g / L, low molecular dextran of 3 to 10 g / L and tobramycin of 2.5 to 5 mg / L. A fresh pig corneoscleral limbal matrix containing lamina elastica anterior is sealed in the decellularized corneal limbus protective liquid and cells are crushed through high hydrostatic pressure treatment; the corneal limbus of the crushed cells are put into the decellularized corneal limbus protective liquid added with a detergent and nuclease to perform oscillation decellularized treatment; and the decellularized corneal limbus is put into the decellularized corneal limbus protective liquid to be rinsed to finish preparation. The decellularized corneal limbus obtained by the method is complete in decellularization, minimally reduces damage to the corneal limbus structure, has high biocompatibility and is favorable for autologous cell proliferation and crawling.

Description

technical field [0001] The invention relates to the field of bioengineering medical materials, in particular to a decellularized limbal stroma. Background technique [0002] The corneal limbus is the transition zone between the cornea and the sclera, where the limbal stem cells (LSCs) are located, and plays an important role in the maintenance of corneal transparency and normal physiological functions. Severe ocular surface diseases can damage the limbus region, leading to the loss of LSCc and the destruction of the barrier structure, causing a series of ocular surface reactions and even blindness. For example, when the eye suffers severe chemical injury, thermal burn, etc., the limbus is extensively destroyed, and some or all limbal stem cells are lacking, resulting in difficulty in repairing the corneal epithelium, the growth of the conjunctival epithelium toward the cornea, the growth of new blood vessels, corneal conjunctiva and Scarring of the stroma eventually leads t...

Claims

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Application Information

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IPC IPC(8): A01N1/00A61L27/36
CPCA01N1/00A61L27/3687A61L27/3691A61L2430/16A61L2430/40
Inventor 史伟云周庆军董沐晨
Owner 健诺维(成都)生物科技有限公司
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