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Method, culture medium and system for promoting iPSC to differentiate into peripheral neuronal cells

A neuron cell and peripheral nerve technology, applied in the field of cell biology, can solve problems such as moral disputes, consumption of embryonic tissue, low yield, etc., and achieve the effects of being conducive to industrial production, reducing quality fluctuations, and clear chemical composition

Pending Publication Date: 2021-09-21
HONG KONG REGEN MEDTECH LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, the acquisition of ESC itself requires the consumption of embryonic tissue, which has certain ethical controversies
In addition, the existing technology is mostly used for scientific research, and has not tried to explore the method of mass production of peripheral neuron cells, failing to meet the needs of industrial production and therapeutic applications
[0003] To sum up, at present, the access to peripheral neuron cells is limited, the preparation process steps are immature, the chemical composition is not completely clear, and the reagents and culture medium containing animal-derived components are used, and the quality of the obtained peripheral neuron cells varies greatly among batches. , high preparation cost and low yield limit its application, and there is an urgent need for a differentiation method and culture medium that simplifies the preparation process, shortens the preparation time, lowers the cost, lowers the risk of contamination, has high yield and purity, and is conducive to industrial production.

Method used

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  • Method, culture medium and system for promoting iPSC to differentiate into peripheral neuronal cells
  • Method, culture medium and system for promoting iPSC to differentiate into peripheral neuronal cells
  • Method, culture medium and system for promoting iPSC to differentiate into peripheral neuronal cells

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Experimental program
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Effect test

preparation example Construction

[0043] (3) Preparation of peripheral neuron cells (iPN)

[0044] Peripheral neural stem cells (NP) were treated with 0.5~3.0×10 4 piece / cm 2 Inoculate the cell culture vessel treated with recombinant laminin (Lm, animal-free, instead of mouse-derived Matrigel, etc.), using animal-free peripheral nerve induction medium-02 (pNiM-02AF) After culturing, the culture medium is replaced every 24-36 hours, and the peripheral neuron cells (iPN) are analyzed after 10-12 days of culture to detect their purity and yield.

[0045] (4) Preparation of iPN from iPSCs—exploring larger-scale preparations

[0046] Divide NP by 0.5×10 4 piece / cm 2 and 3.0×10 4 piece / cm 2 The density was inoculated on cell culture vessels with different surface areas treated with recombinant laminin (Lm), cultured with pNiM-02AF, and the culture medium was removed after 10-12 days to detect the purity and yield of iPN, and the surface area ( The ability of culture vessels with different sizes) to produce iP...

Embodiment 1

[0047] The cultivation of embodiment 1 iPSC

[0048] Dilute iPSCs at 1.0-2.5×10 4 piece / cm 2 The density (example inoculation density: 1.5 × 10 4 piece / cm 2 ) was inoculated on a cell culture dish (the embodiment culture dish: 6-well plate) processed through vitronectin (Vc), and cultured using E8 (the amount of the example: 2 mL per well of the 6-well plate), and the culture medium was replaced every 24 hours After 3-4 days, when the confluence of iPSCs reaches 60-80%, the iPSCs are treated with EDTA (0.5mM) (EDTA treatment time range: 3-8 minutes, the embodiment treatment time: 5 minutes), and the iPSCs are collected and passaged , also at 1.0~2.5×10 4 piece / cm 2 The density (example inoculation density: 1.5 × 10 4 piece / cm 2 ) density inoculated on Vc-treated cell culture dishes.

Embodiment 2

[0049] Example 2 NP differentiation of iPSCs

[0050] Divide iPSC at 1.0~4.0×10 4 piece / cm 2 The density is inoculated on the cell culture vessel (example optimum inoculation density: 2.0 × 10 4 piece / cm 2 ), cultured using animal-free peripheral nerve induction medium-01 (pNiM-01AF) (Example dosage: 2 mL per well of a 6-well plate), the formula of pNiM-01AF is shown in Table 1, in DMEM / Components numbered 2-23 were added to the F12 basal medium, and NPs obtained after iPSC differentiation were collected with trypsin substitute after 8-10 days of culture.

[0051] Table 1 Animal-derived Peripheral Nerve Induction Medium-01 (pNiM-01AF)

[0052]

[0053]

[0054] **: Y27632 was added during the first 36-48 hours of culture.

[0055] Among them, LDN193189, SB431542, CHIR99021, RO4902097, SU5402, and Y27632 are the code names of potential drug candidates.

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Abstract

The invention discloses a method, a culture medium and a system for promoting induced pluripotent stem cells to differentiate into peripheral neuronal cells, and the method comprises the following steps: S1, culturing the induced pluripotent stem cells: treating cell culture vessels with vitronectin; S2, inducing the pluripotent stem cells to differentiate into peripheral neural stem cells, using a cell culture vessel treated by recombinant laminin, and using a non-animal-origin peripheral nerve induction culture medium 01 at the same time; and S3, further differentiating the peripheral neural stem cells into peripheral neuronal cells, and culturing by using the cell culture vessel treated by recombinant laminin and a non-animal-origin peripheral nerve induction culture medium 02. Compared with an existing method, the technology is integrally optimized, high-purity peripheral neuronal cells can be prepared through two culture media, chemical components of the culture media are clear, the technology has obvious advantages in quality control of the culture media and quality control of final products, the whole technology can be free of animal origin, the pollution risk of animal-origin pathogenic factors is avoided, the method is suitable for the cell culture vessel with a large surface area, increases the yield of peripheral neuronal cells in each batch, and is beneficial to industrial production.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a method, culture medium and system for promoting iPSC differentiation into peripheral neuron cells. Background technique [0002] The neurons of the peripheral nervous system (Peripheral Nervous System Neurons, iPN) have great application potential in the fields of disease treatment, drug development, and scientific research. Among them, disease treatment and drug development require a large number and high purity of peripheral neuron cells. The existing method of obtaining peripheral neuron cells is mainly the stem cell differentiation method, in which embryonic stem cells (Embryonic Stem Cell, ESC) or induced pluripotent stem cells (induced Pluripotent Stem Cell, iPSC) are differentiated into precursors of the peripheral nervous system through in vitro differentiation. cells, which further differentiate into peripheral neurons. However, most of the existing in vitro dif...

Claims

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Application Information

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IPC IPC(8): C12N5/10
CPCC12N5/0623C12N5/0619C12N2506/45C12N2510/00C12N2533/52C12N2500/92C12N2500/32C12N2500/25C12N2500/46C12N2501/998C12N2501/71C12N2501/999C12N2500/30C12N2500/38C12N2500/36C12N2501/13C12N2500/40C12N2501/727
Inventor 徐轶冰施明耀
Owner HONG KONG REGEN MEDTECH LTD
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