Tissue scaffold based sodium ion channel detection method of three dimensional optic cup originated neuron-like cells

A sodium ion channel and tissue scaffold technology is applied in the field of sodium ion channel detection of three-dimensional optic cup-derived nerve-like cells, which can solve the problems of incomplete growth and differentiation of transplanted cells, low cell survival rate, and difficulty in precise control of cell growth sites.

Active Publication Date: 2016-03-30
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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Problems solved by technology

However, there are many complex and critical problems in stem cell transplantation: for example, it is difficult to precisely control the cell growth site; the transplanted cells cannot fully gro

Method used

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  • Tissue scaffold based sodium ion channel detection method of three dimensional optic cup originated neuron-like cells
  • Tissue scaffold based sodium ion channel detection method of three dimensional optic cup originated neuron-like cells
  • Tissue scaffold based sodium ion channel detection method of three dimensional optic cup originated neuron-like cells

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Embodiment 1

[0018] 1. Human iPSc-derived 3D retinal isolation and digestion:

[0019] 1. Using the method of mechanical separation, put the nerve fiber layer of human iPSc-derived 3D retina cultured for 50-60 days (specific preparation method reference: XiufengZhong, et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs, NatCommun.2014Jun10; 5:4047) Put it into D-Hanks solution, use a 0.45mm needle to separate the tissue under a 50 times upright microscope, discard the pigmented part (pigment layer) in the retinal tissue, and keep the golden yellow tissue part (nerve fiber layer); the separated nerve fiber Use a pipette to transfer the layered tissue to a 3.5cm culture dish, rinse with PBS for 10 minutes; absorb the PBS, digest the cells with accutase cell digestion solution, and place them in a 37°C incubator for 30 minutes; at the end of the process, the cells can be sucked into the centrifuge tube and blown. Under a 10x upright microscope...

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Abstract

The invention discloses a tissue scaffold based sodium ion channel detection method of three dimensional optic cup originated neuron-like cells. According to the method, the nerve fiber layer of human iPSc source 3D retina is digested into single cells; then the single cells are taken as the seed cells, after induced differentiation and culture medium re-suspension, the seed cells are inoculated to a matrix gel embedded polylactic acid-hydroxyacetic acid polymer scaffold, then the seed cells are cultured in an induced differentiation culture medium, which is composed of a neurotrophic factor containing retina differentiation nutrient liquid with a constant concentration; and after the culture lasts for a while, the neuron-like cells on the polylactic acid-hydroxyacetic acid polymer scaffold are subjected to sodium ion channel detection to detect the neurological basic functions of the neuron-like cells. The nerve cells cultured on the PLGA membrane is subjected to sodium ion channel immuno-fluorescent staining; the state of cells on the tissue membrane can be represented more realistically; and reference and basis are provided for the membrane clamp detection on functional potential of cells on the membrane.

Description

Technical field: [0001] The invention belongs to the field of stem cell tissue engineering and the field of neurobiology, in particular to a method for detecting sodium ion channels of three-dimensional optic cup-derived nerve-like cells based on tissue scaffolds. Background technique: [0002] Glaucoma is a group of clinical syndromes or eye diseases that threaten and damage the optic nerve and its visual pathways, eventually leading to impairment of visual function, mainly related to pathological elevated intraocular pressure. Its optic neuropathy is characterized by progressive loss of retinal ganglion cells (RGCs) and damage to their axonal connections to the central brain. At present, the main treatment for lowering intraocular pressure clinically can slow down the progression of the disease, but it cannot completely prevent the further development of the disease, especially for patients with advanced glaucoma. Therefore, it is necessary to find a new therapeutic strat...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68G01N2333/705
Inventor 葛坚顾怀宇钟秀风李康寯杨斯婧
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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