A method for constructing tissue engineered corneal limbus

A tissue engineering and limbal technology, applied in the field of bioengineering medical materials, can solve the problem of few decellularized limbal stroma, and achieve the effect of strong in vitro proliferation ability, wide source, and guaranteed stability

Active Publication Date: 2019-11-19
SHANDONG EYE INST
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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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Cut out the fresh porcine limbal stroma containing Descemet's membrane, including 1mm sclera, 2mm peripheral cornea, and a thickness of about 0.2mm.

[0035] 1. Preparation of carrier

[0036] The limbal tissue is protected with a protective solution throughout the process. The components of the protective solution are: PBS buffer solution with 6g / L hyaluronic acid, 7g / L chondroitin sulfate, 6g / L dextran and 2.5mg / L tobramycin , adjust the pH value to 7.2, and the osmotic pressure to 310mOsm;

[0037] Put the corneal limbus in the protective solution containing 0.2% Triton+200U / ml DNase, set the shaker speed to 100 rpm, and the temperature to 25°C, and digest for 3 hours to remove the DNA components in the cornea; the decellularized The corneal limbus was placed in the protective solution and rinsed for 4 hours to obtain the decellularized limbal carrier.

[0038] 2. Construction of tissue engineered limbus

[0039] Limbal cells or stem cells were divided into 2 × 10...

Embodiment 2

[0041] Cut out the fresh porcine limbal stroma containing Descemet's membrane, including 1mm sclera, 2mm peripheral cornea, and a thickness of about 0.2mm.

[0042] 1. Preparation of carrier

[0043] The limbal tissue was protected with protective solution throughout the whole process. The components of the protective solution were: PBS buffer solution with 8g / L hyaluronic acid, 10g / L chondroitin sulfate, 5g / L dextran and 3mg / L tobramycin, Adjust the pH value to 7.3 and the osmotic pressure to 320mOsm;

[0044] Put the limbus in a protective solution containing 0.3% sodium lauryl sulfate + 1000U / ml DNase, set the shaking table speed to 150 rpm, and the temperature to 20°C, digest for 4 hours to remove the DNA components in the cornea ; Rinse the decellularized limbus in protective solution for 3 hours to obtain the decellularized limbal carrier.

[0045] 2. Construction of tissue engineered limbus

[0046] Limbal cells or stem cells were divided into 2 × 10 4 / cm 2 The de...

Embodiment 3

[0048] Cut out the fresh porcine limbal stroma containing Descemet's membrane, including 1mm sclera, 2mm peripheral cornea, and a thickness of about 0.2mm.

[0049] 1. Preparation of carrier

[0050] The limbal tissue was protected with a protective solution throughout the process. The components of the protective solution were: 5g / L hyaluronic acid, 18g / L chondroitin sulfate, 10g / L dextran and 4mg / L tobramycin were added to the PBS buffer. Adjust the pH value to 7.4 and the osmotic pressure to 340mOsm;

[0051] Put the corneal limbus in a protective solution containing 0.5% sodium dodecylsulfonate + 500U / ml DNase, set the shaking table speed to 120 rpm, and the temperature to 30°C, digest for 2 hours to remove the DNA in the cornea Composition: the decellularized corneal limbus is placed in the protective solution and rinsed for 5 hours to obtain the decellularized limbal carrier.

[0052] 2. Construction of tissue engineered limbus

[0053] Oral mucosal epithelial cells o...

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Abstract

The invention relates to the field of bioengineering medical materials and discloses a method for building tissue engineering limbus corneae. The method comprises the following steps of putting fresh pig corneoscleral limbus matrix containing a lamina elastica anterior into a decellularized limbus corneae protecting liquid added with a detergent and nuclease for oscillating acellular treatment; putting decellularized limbus corneae into the decellularized limbus corneae protecting liquid for rinsing to obtain the decellularized limbus corneae; inoculating seed cells into the decellularized limbus corneae for amplification in vitro; obtaining the tissue engineering limbus corneae when the seed cells are amplified to 3-5 layers on the decellularized limbus corneae, wherein the decellularized limbus corneae protecting liquid is a PBS buffer solution containing 5-12g / L hyaluronic acid, 5-20g / L chondroitin sulfate, 3-10g / L dextran and 2.5-5mg / L L-tobramycin. Human limbus corneae can be replaced with the tissue engineering limbus corneae material.

Description

technical field [0001] The invention relates to the field of bioengineering medical materials, in particular to a preparation method of tissue engineering corneal limbus. Background technique [0002] Limbal stem cells (LSCs) are located at the junction of cornea and sclera, which is the basis for self-renewal, repair and regeneration of corneal epithelial cells, and is also an important barrier structure between cornea and conjunctiva. Functionality plays an extremely important role. Severe ocular surface diseases, such as inflammation, ocular surface thermal and chemical injury, and ocular surface immune 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. Currently LSCs transplantation is the main treatment. However, the severe shortage of LSCs donors and the difficulty of in vitro culture and expansion make many patients lose the chance of treat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/36A61L27/38A61L27/50
CPCA61L27/3604A61L27/3687A61L27/3813A61L27/3834A61L27/3895A61L27/50A61L2430/16
Inventor 周庆军董沐晨史伟云杨玲玲段豪云
Owner SHANDONG EYE INST
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