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Blood retinal outer barrier model, construction method thereof and culture medium combination adopted for constructing model

A construction method and medium technology, applied in the field of medium combination, blood-retinal barrier model and its construction, can solve the problems of inability to accurately construct oBRB model derived from patients

Active Publication Date: 2021-01-05
AIER EYE HOSPITAL GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The source of the two cells is different, and it is impossible to accurately construct a patient-derived oBRB model

Method used

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  • Blood retinal outer barrier model, construction method thereof and culture medium combination adopted for constructing model
  • Blood retinal outer barrier model, construction method thereof and culture medium combination adopted for constructing model
  • Blood retinal outer barrier model, construction method thereof and culture medium combination adopted for constructing model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 iPSC Induced Differentiation Vascular Network

[0047] Digest iPSCs with a confluence of 70% to 80% into single cells, inoculate them into low-adhesion 6-well plates for culture, and inoculate at a density of 2×10 5 , 37°C, 5% O 2 , 5% CO 2 to cultivate. There are 3 types of media involved, which are replaced and used in chronological order, including medium 1 (mTeSR1+25μM Y2); medium 2: (DMEM+6μM CHIR99021); medium 3 (DMEM+20ng ml - 1 BMP4+60ng ml -1 VEGF-A+50ngml -1 bFGF).

[0048] After 7 days, transfer the cultured single-well cell spheres to a 2ml centrifuge tube with a pipette and discard the supernatant. Add 1.5ml of frozen collagen I to the centrifuge tube and mix well, quickly transfer the mixture to a new 6-well plate with a pipette, shake to cover the bottom of the well and put it in a 37°C incubator for 20 minutes to make it solidify. Then take it out from the incubator, buckle it upside down on the nylon membrane, and cover the upper layer...

Embodiment 2

[0049] Example 2 iPSC induced differentiation of vascular network

[0050] Digest iPSCs with a confluence of 70% to 80% into single cells, inoculate them into low-adhesion 6-well plates for culture, and inoculate at a density of 5×10 5 , 37°C, 5% O 2 , 5% CO 2 to cultivate. There are 3 kinds of media involved, which are replaced and used in chronological order, including medium 1 (mTeSR1+50μM Y2); medium 2: (DMEM+6μM CHIR99021); medium 3 (DMEM+10ng ml - 1 BMP4+100ng ml -1 VEGF-A+10ngml -1 bFGF).

[0051] After 7 days, transfer the cultured single-well cell spheres to a 2ml centrifuge tube with a pipette and discard the supernatant. Add 2ml of frozen collagen I to the centrifuge tube and mix well, quickly transfer the mixture to a new 6-well plate with a pipette, shake to cover the bottom of the well and put it in a 37°C incubator for 40 minutes to make it solidify. Then take it out from the incubator, buckle it upside down on the nylon membrane, and cover the upper lay...

Embodiment 3

[0052] Example 3 iPSC induced differentiation of vascular network

[0053] Digest iPSCs with a confluence of 70% to 80% into single cells, inoculate them into low-adhesion 6-well plates for culture, and inoculate at a density of 3×10 5 , 37°C, 5% O 2 , 5% CO 2 to cultivate. There are 3 types of media involved, which are replaced and used in chronological order, including medium 1 (mTeSR1+35μM Y2); medium 2: (DMEM+6μM CHIR99021); medium 3 (DMEM+30ng ml - 1 BMP4+30ng ml -1 VEGF-A+100ng ml -1 bFGF).

[0054] After 7 days, transfer the cultured single-well cell spheres to a 2ml centrifuge tube with a pipette and discard the supernatant. Add 1ml of frozen collagen I to the centrifuge tube and mix well, quickly transfer the mixture to a new 6-well plate with a pipette, shake to cover the bottom of the well and put it in a 37°C incubator for 30 minutes to make it solidify. Then take it out from the incubator, buckle it upside down on the nylon membrane, and cover the upper la...

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Abstract

The invention relates to the field of biological medicine, in particular to a blood retinal outer barrier model, a construction method thereof and a culture medium combination adopted for constructingthe model. The invention provides a construction method of a scaffold-free material integrated oBRB model differentiated from human iPSC. The preparation method comprises the following main steps offirstly, inducing embryoid mesoderm cell spheres from iPSC in a 3D culture manner, then embedding the cell spheres into collagen I, and inducing the collagen I to form a vascular network sheet. And inoculating the RPE cells from the iPSC to the vascular network sheet, and co-culturing for a period of time to form an integral structure. The iPSC differentiation technology and the tissue engineeringtechnology are combined, the blood-retinal outer barrier structure is better simulated, a new research way for obtaining the oBRB model is provided, and a new replacement way is provided for stem cell treatment of AMD and other diseases.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a blood-retinal outer barrier model and a construction method thereof, and a culture medium combination used to construct the model. Background technique [0002] The blood-retinal barrier (BRB), consists of an inner barrier and an outer barrier. The retinal capillary endothelium and its connections form the inner blood retinal barrier (iBRB); the choriocapillary layer and retinal pigment epithelium (RPE) form the outer blood retinal barrier (oBRB). Its main function is through the formation of tight junctions between RPE cells. The RPE is a monolayer of hexagonal cells with folds in the base membrane for transport and constitutes the inner layer of Bruch's membrane. Part of Bruch's membrane is composed of RPE and the other part is synthesized by choroidal cells. In addition to the RPE as a selective permeability barrier to the retina, Bruch's membrane also forms a barrier to certai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/071C12N5/074C12Q1/02
CPCC12N5/0621C12N5/0625C12N5/0691G01N33/5082C12N2506/45C12N2533/54C12N2500/44C12N2500/38C12N2501/16C12N2501/405C12N2513/00C12N2501/155C12N2501/165C12N2501/115C12N2503/04G01N2500/10
Inventor 顾佳宁陈建苏王译妮唐仕波
Owner AIER EYE HOSPITAL GRP CO LTD
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