Method for preparing retinal pigment epithelial cell slice by using autologous immunocyte

A technology of retinal pigment and autoimmunity, applied in the field of cell sheet preparation, can solve problems such as limited quantity, difficult source of RPE cells, unresolved social and ethical issues, etc.

Active Publication Date: 2018-10-12
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the source of autologous RPE cells is difficult and the number is limited. How to obtain sufficient low immune rejection and tumorigenic RPE cells is also an urgent problem to be solved.
[0004] In 2015, Schwartz et al. transplanted hESC-RPE cell suspensions into patients with atrophic AMD and Stargardt's disease, but the extent of cell survival and vision recovery is unclear
However,

Method used

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  • Method for preparing retinal pigment epithelial cell slice by using autologous immunocyte
  • Method for preparing retinal pigment epithelial cell slice by using autologous immunocyte
  • Method for preparing retinal pigment epithelial cell slice by using autologous immunocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Obtaining autologous immune cells from the patient's own peripheral blood

[0093] Obtaining autologous immune cells from the patient's own peripheral blood includes the following steps:

[0094] Firstly, the patient's own peripheral blood is diluted with normal saline, then added to the lymphocyte separation medium, centrifuged horizontally at room temperature, and stratified; Discard the supernatant and collect the autologous immune cells.

[0095] The detailed steps of this embodiment are:

[0096] Collect 50ml of the patient's own peripheral blood, and use TBD sample density separation medium (purchased from Tianjin Haoyang Huake Biology) to obtain autoimmune cells:

[0097] 1) Dilute 50ml of peripheral blood with normal saline at a ratio of 1:1. Carefully add the diluted blood to the same volume of lymphocyte separation medium to form obvious layers, and centrifuge horizontally at room temperature at 1200rpm / min for 20min. At this time, four layers ar...

Embodiment 2

[0099] Example 2 Dedifferentiation of autologous immune cells into pluripotent induced stem cells in vitro

[0100] The in vitro dedifferentiation of autologous immune cells into pluripotent induced stem cells includes the following steps:

[0101] After the immune cells obtained from RPMI (gibco) medium were cultured overnight, the cells were transfected with four kinds of lentiviruses respectively carrying transformation factors OCT4, SOX2, KLF4 and C-MYC, and the cells were constructed with transformation factors OCT4, The four plasmids of SOX2, KLF4 and C-MYC are ready for use after being sequenced correctly;

[0102] Using the lentiviral packaging kit, inoculate the lentiviral packaging cell line 293T in a culture dish, culture it, and use the obtained four plasmids with transformation factors OCT4, SOX2, KLF4 and C-MYC to transfect the lentiviral packaging cells respectively Line 293T, obtained recombinant pLent-OCT4 lentivirus, recombinant pLent-SOX2 lentivirus, recomb...

Embodiment 3I

[0131] Identification of Example 3IPS (Induced Multi-Pluripotent Stem Cells)

[0132] The IPS cells obtained in Example 2 were subcultured to the 4th generation and then detected by fluorescent quantitative PCR.

[0133] In this example, the specific method: centrifuge to collect IPS cells into EP tubes, and use RNAisoTM Plus to lyse the cells to extract total RNA. Put the above-mentioned EP tube into a centrifuge at 4°C and centrifuge at 12000rpm for 10min, transfer the supernatant to a new EP tube, and discard the precipitate. An equal volume of chloroform solution was added to the supernatant, vortexed, and allowed to stand at room temperature for 7 minutes. Centrifuge at 4°C and 12000rpm for 15 minutes. At this time, the liquid in the EP tube is divided into three layers, and the white layer in the middle is the protein layer. Take the upper aqueous phase and transfer it to a new EP tube (RNA exists in the aqueous phase); add, etc. Add a volume of isopropanol to the tran...

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Abstract

The invention relates to a method for preparing a retinal pigment epithelial cell slice by using an autologous immunocyte, which comprises the following steps of: S1, obtaining an autologous immunocyte from the peripheral blood of a patient, and differentiating the immunocyte, in vitro, to a multifunctional induction stem cell; S2, directionally inducing the multifunctional induction stem cell obtained in S1 to generate a retinal pigment epithelial cell, so as to obtain a retinal pigment epithelial cell slice. The used RPE is derived from the autologous immunocyte of the patient and then the PRE is transplanted to the same patient, thus reducing the immune rejection of the patient. Besides, no problems of organization matching and ethic exist; therefore, the ethical and post-operative results of the test are not obstacles to carrying out such clinical trials. Moreover, the method has a good prospect on the treatment of macular degeneration.

Description

technical field [0001] The invention relates to the technical field of cell sheet preparation, in particular to a method for preparing retinal pigment epithelial cell sheet by using autologous immune cells. Background technique [0002] Age-related macular degeneration (AMD) is the most common blinding eye disease in the aging population. It belongs to the category of retinal degenerative diseases. The pathological basis of such diseases is the irreversible damage of retinal neuron cells. . Currently commonly used treatment methods: There are currently two types of treatment for exudative AMD: anti-neovascular treatment and retinal transposition surgery. However, the former requires long-term use to suppress the disease; although the latter repairs the macula, it does not prevent disease recurrence. There is currently no effective treatment for atrophic AMD. [0003] Currently, retinal pigment epithelial (retinal pigment epithelium, RPE) cell transplantation is a new hope...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12N15/867C12N15/66
Inventor 刘明录郭爱萍韩国英金海锋刘玉强邦明冯建海张传鹏
Owner SHANDONG XINRUI BIOTECH CO LTD
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