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Method for enabling mononuclear cell of blood to perform reversion differentiation for producing human blood derived auto-retina stem cells as well as kit and application

A retina and kit technology, applied in the fields of biology and biomedicine, can solve the problems of low yield, long time and complicated process.

Active Publication Date: 2017-11-07
深圳百年干细胞技术研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to solve many problems in the current retinal stem cell preparation method, such as long time-consuming, immune rejection, unsuitable for retinal regeneration therapy, poor stability, high pollution rate, low yield, high risk of carcinogenicity and complicated process

Method used

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  • Method for enabling mononuclear cell of blood to perform reversion differentiation for producing human blood derived auto-retina stem cells as well as kit and application
  • Method for enabling mononuclear cell of blood to perform reversion differentiation for producing human blood derived auto-retina stem cells as well as kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 A group of culture fluids that make people's somatic cells reversely differentiate to produce autologous retinal stem cells

[0068] Including cell culture medium A1, cell culture medium A2 and cell culture medium A3.

[0069] It is operated in a safe operating bench with a cleanliness level of 10-100, and is prepared at a low temperature of 4-10°C.

[0070] Preparation of cell culture medium A1:

[0071] Add the following ingredients in 500mL DMEM culture solution (purchased from GICO company):

[0072] 10 μM RHO kinase inhibitor Y-27632 (purchased from Sigma Company), 10 ng / mL stem cell factor (purchased from R&D Company), 10 ng / mL interleukin-3 (purchased from R&D Company), 10 ng / mL interleukin Protein 6 (purchased from R&D Company), 10 ng / mL interleukin 11 (purchased from R&D Company), 10 ng / mL macrophage colony-stimulating factor (purchased from R&D Company), 10 ng / mL granulocyte colony-stimulating factor (purchased from R&D company), 10 μg / mL fucoidan...

Embodiment 2

[0079] Embodiment 2 is used to prepare the kit of autologous retinal stem cell

[0080] Including cell culture solution A1, cell culture solution A2, and cell culture solution A3 in Example 1; human somatic cells are also included.

Embodiment 3

[0081] Embodiment 3 adopts the method for preparing retinal stem cells from peripheral venous blood

[0082] 1. Source of blood samples: aseptically collected peripheral venous blood donated by scientific research staff. Before collecting peripheral venous blood, the consent of the blood donor or his immediate family members should be obtained, and the genetic and infectious disease history of the blood donor and his family, as well as the results of all relevant virus examinations in the hospital should be recorded.

[0083] 2. Separation of mononuclear cells: Blood samples are separated from mononuclear cells by Ficoll standard separation technology, and used as raw cells. The microscopic view of the raw cells is as follows: figure 1 -A shown.

[0084] 3. Preparation of retinal stem cells:

[0085] Cell culture solution A1, cell culture solution A2 and cell culture solution A3 prepared in Example 1 were adopted;

[0086] The obtained mononuclear cells were divided into 5x...

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Abstract

The invention belongs to the technical field of biology, in particular relates to the technical field of biomedicine and in particular relates to a cell culture method for enabling human somatic cells to perform reversion differentiation for producing human blood derived auto-retina stem cells as well as a kit and application. The preparation method comprises the following steps: taking somatic cells as raw cells, sequentially culturing the raw cells through a cell culture solution A1, a cell culture solution A2 and a cell culture solution A3, thereby obtaining the blood derived auto-retina stem cells. The human blood cells serve as the raw cells, and the raw cells are subjected to reversion differentiation so as to produce the human blood derived auto-retina stem cells. A cell culture solution formula composed of small molecular substances is used, and the human blood cells are rapidly subjected to reversion differentiation so as to produce the human blood derived auto-retina stem cells under the conditions that the human somatic chromosome DNA sequence is not changed and any foreign gene or DNA fragment is not inserted. The production speed, yield and purity in the invention are obviously superior to those in the prior art.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically relates to the field of biomedical technology, and more specifically relates to a cell culture method, kit and application for making human somatic cells reversely differentiate to generate autologous retinal stem cells. Background technique [0002] The retina is a specialized part of the central nervous system located in the fundus. It originates from the neuroectoderm and is a light-sensitive, fine membrane-like structure that forms the basis of various visual functions. Developed from the optic cup formed by the neuroectoderm during the embryonic period, the outer layer of the optic cup forms a single retinal pigment epithelium layer, and the inner layer of the optic cup differentiates into the retinal neurosensory layer. The neurosensory layers of the retina are, from outside to inside: cones, rods, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear l...

Claims

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Application Information

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IPC IPC(8): C12N5/0797A61L27/38
CPCA61L27/3804A61L27/3839A61L2430/16C12N5/0621C12N5/0623C12N2500/30C12N2500/34C12N2501/105C12N2501/115C12N2501/125C12N2501/195C12N2501/22C12N2501/2303C12N2501/2306C12N2501/2311C12N2501/727C12N2501/998
Inventor 林雄斌汤世坤任海英廖唤昭徐胜美黄卫红吴铭
Owner 深圳百年干细胞技术研究院有限公司
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