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

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Problems solved by technology

However, embryonic stem cells have limited sources and ethical issues, while indu

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
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Example Embodiment

[0051] Example 1 Preparation of human amniotic membrane cell experimental solution

[0052] Step 1: Preparation of amniotic membrane epithelial cell culture solution: 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 a 100ug EGF packaging tube, let stand for 5-10min to dissolve, and then add 4ml of diluent (PBS containing 5% trehalose), mix well and dispense To 1.5ml EP tube, aliquot 100μl per tube;

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

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

Example Embodiment

[0056] Example 2 Isolation of human amniotic membrane epithelial cells

[0057] 1. The source of human amniotic membrane

[0058] With the authorization and consent of the parturient, the placenta tissue of the healthy parturient (HIV, syphilis, hepatitis A, hepatitis B, hepatitis C, etc. are all negative) after caesarean section, the placenta is cut with a cross knife, and the whole amniotic membrane is 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) to wash away blood and other impurities, and transfer the amniotic membrane into a 50ml centrifuge tube.

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

[0062] Step 3: Add 15ml of 0.25% pancreatin to the centrifuge tube (previously bathed at 37°C). Afte...

Example Embodiment

[0066] Example 3 Inoculation, culture and cryopreservation of human amniotic epithelial cells

[0067] 1. Cell counting culture: 1×10 7 Each cell was seeded into a 15cm petri dish. Change the culture medium after the cells adhere to the wall, and then change the culture medium once every three days.

[0068] 2. Cell cryopreservation: After the cells are overgrown on the plate, the cells are digested and cryopreserved: add 5ml trypsin to a 15cm dish, observe under a microscope after 10 minutes, and wait when the cells become rounded and the plate shakes the plate when the cells become suspended Amount of digestion stop solution to terminate the digestion. Use a micropipette to blow down the cells on the petri dish in the same direction, move them into a 15ml centrifuge tube, centrifuge at 800g for 3 minutes, collect the cells and then count the cells. Add the cryopreservation solution to the cryopreservation tube, mark the date, batch and number of cells, put the cells in the cryo...

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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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