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Method for inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells, and applications thereof

A technology of retinal pigment and epithelial cells, applied in the application field of the treatment of retinal degenerative diseases, can solve the problems of limited source of embryonic stem cells and complicated culture technology

Active Publication Date: 2019-09-06
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, embryonic stem cells have limited sources and ethical issues, while induced pluripotent stem cells (iPS) have problems such as complex culture techniques

Method used

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  • Method for inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells, and applications thereof
  • Method for inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells, and applications thereof
  • Method for inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The preparation of embodiment 1 human amnion cell test solution

[0052] Step 1: Preparation of amnion epithelial cell culture medium: add 95ml KSR to 500ml DMEM / F12 1:1 (1X); 6.5ml 100mM L-Glutamine; 6.5ml 100mM Sodium Pyruvate; 6.5ml 100mM MEM NEAA; 2000× EGF Add before use with 100× double antibody (Penicillin-Streptomycin);

[0053] Step 2: Preparation of 2000×EGF: Add 1ml of sterile ddH2O to the 100ug EGF packaging tube, let it stand for 5-10min to dissolve, then add 4ml of diluent (PBS containing 5% trehalose), mix well and dispense into 1.5ml EP tubes, and dispense 100μl per tube;

[0054] Step 3: Preparation of digestion stop solution: DMEM / F12 1:1(1X)+10%FBS;

[0055] Step 4: Preparation of cryopreservation solution: 40% FBS + 50% culture medium + 10% DMSO.

Embodiment 2

[0056] Example 2 Separation of human amniotic epithelial cells

[0057] 1. The source of human amniotic membrane

[0058] After the mother’s authorization and consent, the placental tissue of the healthy mother (HIV, syphilis, hepatitis A, hepatitis B, hepatitis C and other serological reactions were all negative) after caesarean section was taken, the placenta was cut with a cross knife, and the whole amniotic membrane was obtained by mechanical separation.

[0059] 2. Isolation of human amniotic epithelial cells

[0060] Step 1: Wash the amniotic membrane three times with sterile PBS solution added with double antibody (P / S), wash away blood and other impurities, and transfer the amniotic membrane to a 50ml centrifuge tube.

[0061] Step 2: Add 10ml of 0.25% trypsin (bathed at 37°C in advance) to digest for 30s, invert 20 times, and transfer the amnion into another 50ml centrifuge tube.

[0062] Step 3: Add 15ml of 0.25% trypsin to the centrifuge tube (bathed at 37°C in ad...

Embodiment 3

[0066] Embodiment 3 Inoculation culture and cryopreservation of human amniotic membrane epithelial cells

[0067] 1. Cell counting culture: 1×10 7 Cells were seeded into a 15 cm Petri dish. The culture medium was changed after the cells adhered to the wall, and the culture medium was changed every three days thereafter.

[0068] 2. Cell cryopreservation: Digest the cells after the cells cover the plate for cryopreservation: add 5ml trypsin to a 15cm dish, observe under the microscope after 10 minutes, and add when the cells become round and all the cells become suspended when the plate is shaken on the plane. Amount of digestion stop solution to stop digestion. Use a micropipette to blow down the cells on the culture dish in the same direction, transfer to a 15ml centrifuge tube, centrifuge at 800g for 3min, collect the cells and then count the cells. Add cryopreservation solution into the cryopreservation tube, mark the date of freezing, batch and cell number, put the cell...

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Abstract

The invention relates to a method for inducing the differentiation of human amniotic epithelial cells into retinal pigment epithelial cells, and applications thereof in treating retinal degenerative diseases. The method includes adding an inducing composition including 10-100 mM of nicotinamide and 1-10 [mu]M of trichostatin A into human amniotic epithelial cells; and inducing the generated cellsto highly express the typical genes of retinal pigment epithelial cells such as MITF, PMEL17, RPE65 and Bestrophin. Cells induced and generated through the method can be injected into an RCS rat subretinal cavity, the electrophysiological signal of the eyes of a rat can be obviously enhanced through ERG and fundus detection, and fundus structures can be obviously improved, and the thickness of retina can be obviously increased through the displaying of eyeball slice HE staining; the cells induced and differentiated through the above schemes can recover the eyesight of the rat to a certain extent after injection; and the inducing composition can be used for the differentiation of the human amniotic epithelial cells into the retinal pigment epithelial cells and the treatment of retinal damage related eye diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for inducing differentiation of human amniotic epithelial cells into retinal pigment epithelial cells and its application in treating retinal degenerative diseases. [0002] technical background [0003] Retinal neurodegenerative diseases have become the main cause of irreversible blindness worldwide, affecting the vision health of tens of millions of people, such as retinitis pigmentosa (Retinitis Pigmentosa, RP), age-related macular degeneration (Age-related macular degeneration) Degeneration, AMD) and glaucoma. The main manifestations are the progressive damage and loss of retinal pigment epithelial (RPE) cells and photoreceptor cells (Photoreceptor cells), which cause retinal dysfunction, and eventually lead to progressive irreversible visual function decline or loss in patients. There is no effective treatment, and there is currently no effective treatment f...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N5/071C12N5/079A61K35/30A61P27/02A61P27/06
CPCA61K35/30A61P27/02A61P27/06C12N5/0605C12N5/0621C12N5/0625C12N2500/38C12N2501/065C12N2506/025C12N2509/00
Inventor 余路阳张传宇李金英袁惟芯邱晨郭礼和邵小燕刘佳
Owner ZHEJIANG UNIV
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