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Method for preparing tissue engineered cornea and device of method

A technology of tissue engineering and cornea, which is applied in the field of tissue engineering cornea preparation for clinical transplantation, can solve the problems of large corneal epithelial defect, increased rejection risk, immune rejection, etc. The effect of layers

Active Publication Date: 2014-06-18
SHAANXI RUISHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Chinese patent application 03140113.9 discloses a biological cornea of ​​artificial tissue engineering. Although epithelial and endothelial cells have been compounded, the carrier used is fresh cornea containing stromal cells or cornea without stromal cells after preservation. Infected cells or cell residual components can cause immune rejection, especially in high-risk transplants with loss of immune pardon.
[0007] In addition, there are also literature and patent reports, there are deficiencies in using autologous cells to expand and culture in vitro or planting stem cells on scaffold materials such as amnion, fibrin glue, collagen, etc.
[0008] In Chinese patent application 03150375.6, autologous limbal cells are used as seed cells, which are only suitable for patients with unilateral limbal damage. When both eyes are damaged, materials cannot be obtained, and there is a risk of damage to healthy eyes; the important thing is that it cannot be realized. can not meet the urgent needs of patients
[0009] Chinese patent application 201110039014.0 discloses a method for constructing a human corneal epithelial cell line. The source of the seed cells has immunogenicity problems. In high-risk corneal transplantation, the status of immune pardon is broken, which increases the risk of rejection; Bovine serum, introducing a risk of transmission of bovine-derived viruses
The formed corneal epithelium has poor mechanical strength and stability. When applied to high-risk transplantation, the complex inflammatory environment of the implant bed and a large amount of matrix metalloproteinases can cause the degradation of the graft and increase the failure rate of the graft; and the collagen gel is not With the microstructure of the natural cornea, it is more difficult to integrate with the implant bed after transplantation compared with allografts
[0012] Chinese patent application 200710129136.2 discloses a preparation method of tissue-engineered corneal epithelial transplantation membrane, which has the problems of allogeneic cell immunogenicity and the risk of using fetal bovine serum, and only considers the maintenance of stem cells, not the corneal epithelium The natural composition structure and the combined action of centripetal and superficial cell renewal means that there is a greater risk of corneal epithelial defect after transplantation

Method used

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  • Method for preparing tissue engineered cornea and device of method
  • Method for preparing tissue engineered cornea and device of method
  • Method for preparing tissue engineered cornea and device of method

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Step 1. Inoculation of amnion epithelial cells: place the decellularized porcine corneal stroma in a 12-well cell culture plate, and inoculate the second-generation amnion epithelial cells cultured in culture solution A at 1×10 5 / cm 2 Density seeding on the Bowman's membrane surface of the decellularized corneal stroma; add 2ml of culture medium A, and cultivate under the liquid surface; the culture medium A is based on the MCDB153 medium aqueous solution with a concentration of 17.6g / L, and contains: EGF 2μg / L, human insulin 10mg / L, L-glutamine 2mM, non-essential amino acid 10ml / L, sodium pyruvate 1.18g / L, arachidonic acid 2mg / L, adenine 10mg / L, triiodine Thyrosine 20ng / L, sodium selenite 5mg / L, transferrin 100mg / L; the condition of cell incubator is, in the volume concentration respectively is 90% N 2 , 5% CO 2 , 5% O 2 In a mixed gas environment, culture at 37°C for 7 days; replace the culture medium every two days, and when the amniotic epithelial cells grow and...

Embodiment 2

[0041] Step 1. Inoculation of amnion epithelial cells: place decellularized bovine corneal stroma in a 12-well cell culture plate, press 8×10 5 / cm 2 Density inoculation of primary amniotic epithelial cells on the surface of the Bowman's membrane of the decellularized corneal stroma; Add 4ml of culture solution A and culture under the liquid surface; the culture solution A is the same as in Example 1; the conditions of the cell culture box are, in volume Concentrations were 93% N 2 , 5% CO 2 , 2% O 2 In a mixed gas environment, culture at 37°C for 5 days; change the medium every two days, and when the amniotic epithelial cells grow and confluence on the surface of the descemet layer of the decellularized corneal stroma, the cells grow to 1-2 layers. A construct in which amniotic epithelial cells were complexed on the surface of the decellularized corneal stroma was obtained.

[0042] Step 2, air-liquid alternate culture of epithelial cell membrane: same as Example 1, use t...

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Abstract

The invention discloses a method for preparing tissue engineered cornea and a device of the method. According to the method, amniotic membrane epithelial cells are inoculated on acellular corneal stroma, construction bodies of composite amniotic membrane epithelial cells are obtained through culturing, and the tissue engineered cornea is prepared by a double-face culturing mode through alternative culturing of gas-liquid alternating illumination culturing and illumination-free culturing in liquid. The prepared tissue engineered cornea is an excellent cornea substitute, and has the heat stability and the transparency which are close to those of natural cornea; under the wavelength of 680nm, the light transmittance of the tissue engineered cornea is 90 to 95 percent; due to the adoption of a double-face culturing device, the surfaces of the construction bodies can fully contact with gas, the number of laminated layers of epithelial cell sheets can be increased, 5 to 7 layers of cells can be accumulated, the survival rate is more than 90 percent, and good stem cell performance can be kept; and the tissue engineered cornea can be used for various lamellar cornea transplantation in keratohelcosis, leukoma, corneal dystrophy and the like and can show a very good clinical effect in high-risk cornea transplantation with persistent corneal epithelial defects and cornea limbal stem cell deficiency.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering medical biomaterials, and in particular relates to a method for preparing a tissue engineering cornea for clinical transplantation and a device thereof. Background technique [0002] The cornea is a transparent, avascular tissue structure that is divided from outside to inside into an epithelial layer, a Bowman's membrane, a stroma, a Bowman's membrane, and an endothelial layer. The epithelial cell layer is composed of 5 to 7 layers of epithelial cells, which are the superficial layer, the upper basal layer and the basal layer from the outside to the inside; there is a basement membrane structure formed by the deposition of extracellular matrix (ECM) between the basal layer and the Bowman's membrane. The bottom layer is composed of a single layer of columnar basal cells, containing a large number of stem cells, which divide upwards and continuously produce offspring cells; the offspring...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12M3/00A61L27/38A61L27/40
Inventor 卢永波
Owner SHAANXI RUISHENG BIOTECH
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