Retinal pigment epithelial cell induction medium and application thereof

A retinal pigment and induction medium technology, applied in the field of retinal pigment epithelial cell induction medium, can solve the problems of fetal tissue ethics, low dopamine production, no proven therapeutic effect, etc., and achieve short differentiation culture time and high safety , high yield effect

Active Publication Date: 2021-10-08
BEIJING TAIDONG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a randomized double-blind study using the same RPE cells did not demonstrate the same therapeutic effect
In addition, ethical issues remain regarding the clinical use of fetal tissue
[0004] The inventors of the present invention have previously conducted research on inducing the differentiation of retinal pigment epithelial cells into dopaminergic neuron-like cells, but the induction culture method used still has the problem of low dopamine production

Method used

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  • Retinal pigment epithelial cell induction medium and application thereof
  • Retinal pigment epithelial cell induction medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] A method for inducing and culturing retinal pigment epithelial cells.

[0067] Retinal pigment epithelial cells were divided into 2 × 10 per well 4 The density of cells was spread on a 6-well plate, and the neural differentiation medium contained B27 supplement (1:50), L-glutamine 2mM, sodium pyruvate 1mM, β-mercaptoethanol 0.1mM, L-ascorbic acid Neurobasal medium with 50 μM, cAMP 1.5 mM, LDN193189 100 nM, CHIR9021 10 μM, ALK inhibitor SB431542 15 μM, SHH35ng / ml. Thereafter, cells were passaged 3 days later. Cells were cultured in neuron induction medium until day 7.

Embodiment 2

[0069] A method for inducing and culturing retinal pigment epithelial cells.

[0070] Retinal pigment epithelial cells were divided into 2 × 10 per well 4The density of cells was laid on a 6-well plate, and the neural differentiation medium contained B27 supplement (1:60), L-glutamine 5mM, sodium pyruvate 0.5mM, β-mercaptoethanol 0.15mM, L- Ascorbic acid 30 μM, cAMP 3 mM, LDN193189 120 nM, CHIR9021 5 μM, ALK inhibitor SB431542 5 μM, SHH 60ng / ml Neurobasal neural base medium. Thereafter, cells were passaged 3 days later. Cells were cultured in neuron induction medium until day 7.

Embodiment 3

[0072] A method for inducing and culturing retinal pigment epithelial cells.

[0073] Retinal pigment epithelial cells were divided into 2 × 10 per well 4 The density of cells was spread on a 6-well plate, and the neural differentiation medium contained B27 supplement (1:40), L-glutamine 1mM, sodium pyruvate 3mM, β-mercaptoethanol 0.05mM, L-ascorbic acid Neurobasal medium with 120 μM, cAMP 0.5 mM, LDN193189 30 nM, CHIR9021 20 μM, ALK inhibitor SB431542 25 μM, SHH 15ng / ml. Thereafter, cells were passaged 3 days later. Cells were cultured in neuron induction medium until day 7.

[0074] Immunofluorescence:

[0075] Immunofluorescent staining was performed on Example 2. Cells on coverslips were fixed in 4% PFA pre-cooled on ice for 15 min. After washing three times in phosphate buffered saline, the cells were blocked with phosphate buffered saline containing 5% bovine serum albumin for 30 min, and then incubated with appropriate primary antibodies at 4°C for 12 h.

[0076] ...

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Abstract

The invention relates to a retinal pigment epithelial cell induction medium and application thereof. The induction medium is a Neurobasic medium containing the following components: a B27 supplement, 1 mM to 5 mM of L-glutamine, 0.5 mM to 3 mM of sodium pyruvate, 0.05 mM to 0.15 mM of beta-mercaptoethanol, 30 mM to 120 [mu] M of L-ascorbic acid, 0.5 mM to 3 mM of cAMP, 30 nM to 120 nM of a BMP inhibitor, 5 [mu] M to 20 [mu] M of a GSK-3beta inhibitor, 5 [mu] M to 25 [mu] M of an ALK inhibitor and 15 ng/ml to 60 ng/ml of SHH. The medium can be subjected to directional differentiation culture by taking human retinal pigment epithelial cells as raw materials to obtain dopaminergic neuron-like cells capable of generating dopamine.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a medium for inducing retinal pigment epithelial cells and its application. Background technique [0002] Parkinson's disease is one of the most common neurodegenerative diseases in the elderly population. People with Parkinson's disease often suffer from resting tremor, stiffness, hypokinesia, gait, balance and autonomic dysfunction, depression and dementia. From an etiological point of view, Parkinson's disease is caused by the loss of dopamine-producing neurons in the substantia nigra pars compacta and the nigro-striatal neuronal projections are responsible for motor symptoms during the disease. Starting with changes in midbrain dopamine neurons, the disease eventually affects forebrain neurons, such as cortical neurons. Over the past decade, several predisposing factors have been identified. This study suggests that protein misfolding, abnormally increased oxidative s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793C12N5/079
CPCC12N5/0621C12N5/0619C12N2500/32C12N2500/44C12N2500/30C12N2501/999C12N2501/01C12N2500/38
Inventor 张晗
Owner BEIJING TAIDONG BIOTECH CO LTD
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