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Method, culture medium and system for promoting differentiation of iPSC (induced pluripotent stem cells) into peripheral neural stem cells

A peripheral nerve and cell culture technology, applied in the field of cell biology, can solve the problems of incompletely clear chemical composition, no attempt to explore the method of mass production of NP, and immature preparation process, etc.

Pending Publication Date: 2021-09-14
HONG KONG REGEN MEDTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, the acquisition of ESC itself requires the consumption of embryonic tissue, and there are certain ethical disputes
In addition, the existing technology is mostly used for scientific research, and has not tried to explore the method of mass production of NP, failing to meet the needs of industrial production and therapeutic applications
[0003] To sum up, the access to NP is limited, the preparation process is immature, the chemical composition is not completely clear, and the reagents and culture medium containing animal-derived components are used. The quality of the obtained NP batches varies greatly, and the preparation cost is high and the yield is low. , which limits the application of NP, there is an urgent need for a differentiation method and culture medium that simplify the preparation process, shorten the preparation time, reduce the risk of contamination, high yield and purity, and facilitate industrial production.

Method used

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  • Method, culture medium and system for promoting differentiation of iPSC (induced pluripotent stem cells) into peripheral neural stem cells
  • Method, culture medium and system for promoting differentiation of iPSC (induced pluripotent stem cells) into peripheral neural stem cells
  • Method, culture medium and system for promoting differentiation of iPSC (induced pluripotent stem cells) into peripheral neural stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Culture of iPSC

[0042] 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

[0043] Example 2 NP differentiation of iPSCs

[0044] 24 hours before the start of differentiation, iPSCs were mixed with 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 , see Table 3 below for details), cultured without animal-derived peripheral nerve induction medium-01 (pNiM-01AF) (dosage in the example: 2 mL per well in a 6-well plate).

[0045] The final formulation of pNiM-01AF was determined through comparative experiments. Firstly, the "Animal-derived Peripheral Nerve Induction Medium-01 (pNiM-01AF)" was designed, and then compared with the medium containing animal-derived components in the existing process (Control) For comparison, see Tables 1-2 below, which are all components from No. 2 to the last row added to the DMEM / F12 basal medium No. 1.

[0046] Table 1 Existing Peripheral Nerve Induction Medium Containing Animal-derived Components (Control)

[0047]

[0048]

...

Embodiment 3

[0064] Example 3 NP differentiation of iPSCs—exploration of larger scale NP preparation

[0065] Convert iPSCs to 2.0 × 10 4 piece / cm 2 The density was inoculated on recombinant laminin (Lm)-treated cell culture dishes with different surface areas, and cultured with pNiM-01AF, see Table 4 below for details.

[0066] Table 4 NP differentiation results of culture vessels with different surface areas

[0067]

[0068] The conclusion is drawn from the data in Table 4, at 9.5, 25 and 75cm 2 For three surface areas, pNiM-01AF differentiated iPSCs yielded NPs with a purity of ≥90% ( image 3 ), while the size of the surface area had no significant effect on the yield of peripheral neural stem cells ( Figure 4 ). Both the inoculation density of iPSC and the feasible surface area of ​​the culture vessel are obtained through exploration in the present invention, which is beneficial to industrial production.

[0069] In summary, in the method of the present invention, the iPSC cu...

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Abstract

The invention discloses a method, culture medium and system for promoting differentiation of induced pluripotent stem cells (iPSC) into peripheral neural stem cells. A cell culture vessel treated by vitronectin is used in a culture process of the iPSC, a cell culture vessel treated by recombinant laminin is used during the differentiation of the iPSC into the peripheral neural stem cells, and meanwhile, a non-animal-origin peripheral nerve induction culture medium 01 is applied to culture. Compared with the existing methods, the method has the advantages that the whole process is optimized, chemical components of the culture medium are clear, the method has obvious advantages in quality control of the culture medium and quality control of a final product, the whole process can be free of animal origin, the risk of pollution caused by animal-origin pathogenic factors is avoided, the method is applicable to a cell culture vessel with a large surface area, the yield of the peripheral neural stem cells in each batch is increased, and thus, industrial production is facilitated.

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 the differentiation of induced pluripotent stem cells into peripheral neural stem cells. Background technique [0002] Neural stem cells (Peripheral Nervous System Neural Progenitor, NP) in the peripheral nervous system have great application potential in disease treatment, drug development, scientific research and other fields. Disease treatment and drug development require a large amount of high-purity NP. The existing methods for obtaining NP are mainly 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 NP by means of in vitro differentiation. However, most of the existing in vitro differentiation methods are immature in technology, use medium with incomplete chemical composition, and have...

Claims

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Application Information

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IPC IPC(8): C12N5/0797C12N5/074C12M3/00
CPCC12N5/0623C12M23/20C12N2533/52C12N2500/32C12N2500/25C12N2500/46C12N2501/998C12N2501/71C12N2501/999C12N2501/392C12N2500/38C12N2500/36C12N2501/15C12N2501/727C12N2501/155C12N2506/45
Inventor 徐轶冰施明耀
Owner HONG KONG REGEN MEDTECH LTD