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A method for inducing the differentiation of human amniotic epithelial cells into retinal photoreceptor cells and its application

An epithelial cell and photoreceptor cell technology, applied in epidermal cells/skin cells, medical preparations containing active ingredients, animal cells, etc., can solve the problems of complex culture technology and limited source of embryonic stem cells.

Active Publication Date: 2021-04-13
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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  • A method for inducing the differentiation of human amniotic epithelial cells into retinal photoreceptor cells and its application
  • A method for inducing the differentiation of human amniotic epithelial cells into retinal photoreceptor cells and its application
  • A method for inducing the differentiation of human amniotic epithelial cells into retinal photoreceptor cells and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The preparation of embodiment 1 amnion cell test solution

[0051] 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);

[0052] Step 2: Preparation of 2000×EGF: add 1ml sterile ddH to 100ug EGF packaging tube 2 O, let it stand for 5-10min to dissolve, then add 4ml diluent (PBS containing 5% trehalose), mix well and then dispense into 1.5ml EP tubes, 100μl per tube;

[0053] Step 3: Preparation of digestion stop solution: DMEM / F12 1:1(1X)+10%FBS;

[0054] Step 4: Preparation of cryopreservation solution: 40% FBS + 50% culture medium + 10% DMSO.

Embodiment 2

[0055] Example 2 Separation of human amniotic epithelial cells

[0056] 1. The source of human amniotic membrane

[0057] 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.

[0058] 2. Isolation of human amniotic epithelial cells

[0059] 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.

[0060] 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.

[0061] Step 3: Add 15ml of 0.25% trypsin to the centrifuge tube (bathed at 37°C in ad...

Embodiment 3

[0065] Embodiment 3 Inoculation culture and cryopreservation of human amniotic membrane epithelial cells

[0066] 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.

[0067] 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 photoreceptor cells and its application in treating retinal degenerative diseases. The invention provides a method for inducing the differentiation of human amniotic epithelial cells into retinal photoreceptor cells, culturing human amniotic epithelial cells under suitable conditions, adding an inducing composition to induce the differentiation of human amniotic epithelial cells into retinal photoreceptor cells, and inducing the cells produced High expression of typical genes of retinal photoreceptor cells such as Chx10, Rx, Crx, NRL. After the induced differentiation cells were collected and injected into the subretinal space, the electrophysiological signals of the eye were significantly enhanced, the fundus structure was significantly improved, and the retinal thickness was significantly increased through ERG and OCT detection, which showed that it could treat and / or improve the progress of retinal degenerative diseases. The clinical application of ophthalmic diseases will have broad prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for inducing the differentiation of human amniotic epithelial cells into retinal photoreceptor cells and its application in treating retinal degenerative diseases. Background technique [0002] 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 pathological basis of visual function damage caused by such diseases is the irreversible damage of retinal neuron cells, and there is currently no effective treatment for this in clinical practice. However, with the development of technology in recent years, methods such as gene therapy, stem cell therapy, and artificial vision have provided the possibility ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0793A61K35/30A61P9/10
CPCA61K35/30A61P9/10C12N5/062C12N2506/09
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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