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Preparation method of functionalized retinal pigment epithelial cell graft

A technology of retinal pigment and epithelial cells, applied in the field of preparation of functionalized retinal pigment epithelial cell transplants, can solve the problems of poor mechanical properties, difficult to meet clinical application, long time, etc., to promote the formation of microstructure and morphology , avoid RPE cell death, good cell affinity effect

Active Publication Date: 2015-04-15
WENZHOU MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although artificial synthetic materials have good plasticity and mechanical properties, they have insufficient cell affinity, while natural biomaterials are rich in cell recognition signal molecules, but their mechanical properties are poor, and they are difficult to meet clinical applications.
In addition, it takes a long time to form functionalized RPE cell grafts. However, whether these membranes can support the formation of RPE cell functions for a long time and whether they will cause the expression of inflammation-related genes have been rarely studied in the existing studies. involve

Method used

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  • Preparation method of functionalized retinal pigment epithelial cell graft
  • Preparation method of functionalized retinal pigment epithelial cell graft
  • Preparation method of functionalized retinal pigment epithelial cell graft

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 prepares ultra-thin porous tussah silk fibroin / polycaprolactone / gelatin composite nanofiber bionic Bruch's membrane preparation method comprises the following steps:

[0038] 1. Tussah silk treatment: degumming tussah silk, then dissolving, dialysis, purification and freeze-drying to obtain pure tussah silk fibroin;

[0039] 2. Preparation of electrospinning solution: Weigh 0.125g of tussah silk fibroin, 2.125g of polycaprolactone, and 0.25g of gelatin into a 50mL conical flask, add 10mL of 98% formic acid solution, and stir overnight at room temperature to obtain static electricity. spinning solution;

[0040] 3. Electrospinning process: put the electrospinning solution into the syringe, and the syringe is equipped with a blunt needle with a diameter of 0.6mm. Electrospinning is performed under the conditions of voltage 12-20kv, collection distance 10-20cm, and extrusion speed 1-5mL / h ;The preferred voltage is 18kv, the collection distance is 15cm, and th...

Embodiment 2

[0045] Example 2 Nano-bionic Bruch's membrane pretreatment of the present invention; cell planting and cultivation; and preparation of functionalized retinal pigment epithelial cell transplant sheet of the present invention

[0046] The nano-bionic Bruch's membrane prepared in Example 1 was further studied as a carrier for RPE cell transplantation. Pretreatment such as disinfection must be carried out before co-culture with cells. In addition, factors such as cell planting density, method, and cell culture conditions were also optimized.

[0047] 1. Pretreatment before use such as disinfection of the ultra-thin porous nano-bionic Bruch’s membrane prepared in the above-mentioned embodiment 1: cutting the ultra-thin porous nano-bionic Bruch’s membrane into 2×2cm 2 sized diaphragm and place it in a Petri dish. After soaking in 75% ethanol for 30-120min, suck out the ethanol, wait for the ethanol to completely volatilize on the ultra-clean bench, add PBS or HBSS buffer and soak ...

Embodiment 3

[0051] Adhesion evaluation of RPE cells on each nano-bionic Bruch's membrane in embodiment 3 of the present invention

[0052] According to the following steps to study the influence of each nano-bionic Bruch's membrane on the cell adhesion ability, such as figure 1 shown.

[0053] 1. according to the step 2 in embodiment 2,3,4 operation, RPE cell is inoculated to each nano-bionic Bruch's film (respectively polycaprolactone nano-bionic Bruch's film, tussah silk fibroin / polycaprolactone nano-bionic Bruch's film) membrane, tussah silk fibroin / polycaprolactone / gelatin nano-bionic Bruch's membrane) and control TCP, remove the culture medium after 24 hours, and wash 4 times with PBS or HBSS to remove non-adherent cells;

[0054] 2. Observe cell adhesion under an inverted microscope, such as figure 1 As shown, among them, A indicates that RPE cells are planted on TCP, B indicates that RPE cells are planted on polycaprolactone nano-bionic Bruch's membrane, and C indicates that RPE ...

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Abstract

The invention provides a preparation method of a functionalized retinal pigment epithelial cell graft. The preparation method comprises the following steps: taking a nano bionic Bruch's membrane as a retinal pigment epithelial cell carrier, inoculating retinal pigment epithelial cells onto the nano bionic Bruch's membrane, and performing co-culture under low-serum and low-sugar culture conditions to obtain the functionalized retinal pigment epithelial cell graft, wherein the nano bionic Bruch's membrane is an ultrathin porous antheraea pernyi silk fibroin / polycaprolactone / gelatin compound nanofiber membrane prepared by dissolving the antheraea pernyi silk fibroin, polycaprolactone and the gelatin in an organic solvent to form an electrostatic spinning solution and performing electrostatic spinning on the electrostatic spinning solution by an electrostatic spinning method. The functionalized retinal pigment epithelial (RPE) cell graft prepared by the method is capable of effectively avoiding the problems of high possibility of the RPE cells, cell polarity disorder and the like after transplantation in a conventional method, and can be widely applied to cell transplantation.

Description

technical field [0001] The invention relates to the field of tissue engineering of regenerative medicine, in particular to a preparation method of a functionalized retinal pigment epithelial cell graft. Background technique [0002] Age-related macular degeneration (AMD) is a common cause of blindness worldwide over the age of 50. The main cause is the abnormal structure or function of the retinal pigment epithelium (RPE), which eventually causes the apoptosis of retinal photoreceptor cells and leads to irreversible degeneration of the neural retina. With the increase of the aging degree in our country, the incurable blindness caused by such diseases is becoming a major burden on the current national economic construction and social development. However, most of the current treatments for this disease (anti-VEGF drugs, etc.) are measures aimed at complications, and there is no intervention method for patients with advanced disease and atrophic (dry) AMD. [0003] RPE trans...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/26A61L27/24A61L27/22A61L27/18A61L27/56A61L27/50D04H1/4382D04H1/728
Inventor 金子兵向萍李敏项略
Owner WENZHOU MEDICAL UNIV
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