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Method for producing induced pluripotent stem cells without using hydrogel

A pluripotent stem cell and inducible technology, applied in the field of induced pluripotent stem cell production, can solve the problems that the base glue hinders the development of the technical field and the base glue is expensive

Pending Publication Date: 2022-04-29
NEXT&BIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, base glue is also expensive
Therefore, although base gel is a material that contributes to the development of the field of cell culture technology, there is also the fact that base gel hinders the development of this technical field

Method used

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  • Method for producing induced pluripotent stem cells without using hydrogel
  • Method for producing induced pluripotent stem cells without using hydrogel
  • Method for producing induced pluripotent stem cells without using hydrogel

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1. Experimental method

[0100] 1-1: Culture of fibroblasts and production of induced pluripotent stem cells

[0101] German authorities / RKI: AZ 1710-79-1-4-41E01 (F134) is a human fibroblast cell line, which contains 10% FBS (fetal bovine serum, Invitrogen, USA) and 1mM L- glutamine (Invitrogen, USA) in a 35mm or 100mm Petri dish. The cultured fibroblasts were reprogrammed by electroporation with free iPSC reprogramming vector (EP5 TM kit: Cat.No.A16960.Invitrogen, Carlsbad, CA, USA) (NeonTM transfection system). Electroporation was performed at 1,650V, 10ms and 3 pulses.

[0102] such as Figure 4 A, transfected fibroblasts were inoculated into the 3D cell culture plate of the present invention (without base glue, Example), 2D 12 well plate (coated with base glue, Comparative Example 1) and commercial product Addgene (Comparative Example 2, coated with base glue, Figure 4 Not shown in A) and cultured in N2B27 medium (containing bFGF). After 15 days of fibroblast cu...

Embodiment 2

[0114] Example 2 Confirmation of Reprogramming Efficiency

[0115] refer to Figure 4 B, it can be seen that in the case of 2D culture, only a few colonies began to form on D15. After inducing iPSC to be reprogrammed to D15, 3D iPSC was laid on 2D board, and the number of colonies in Comparative Example 1 and Example 1 was compared by laying 3D iPSC on 2D board. As a result, the number of colonies formed was quite different. It can be seen that the iPSC reprogramming yield of Example is high, because cells well differentiated into iPSC form colonies. refer to Figure 5 and Figure 7 B, it can be seen that the difference of colony number between 2D culture (Comparative Example 1) and 3D culture (Example) is very large. refer to Figure 6 It can be seen that as a result of alkaline phosphatase (AP) staining, the reprogramming efficiency in 3D iPSC spheroid (Example, 3D sph-iPCS) is very high. Further, refer to Figure 6 and Figure 7 A, when the 2D base glue (Comparative Example 1) and 3D...

Embodiment 3

[0118] Example 3 Characteristic Analysis of Induced Pluripotent Stem Cells

[0119] refer to Figure 9 It can be seen that the iPSC produced according to the present invention has very high expression of pluripotent markers.

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Abstract

Provided is a method for producing induced pluripotent stem cells without using a hydrogel.

Description

Technical field [0001] The invention relates to a method for producing induced pluripotent stem cells without using hydrogel. More specifically, the present invention relates to a method for producing induced pluripotent stem cells from somatic cells using a 3D cell culture plate without using hydrogel. technical background [0002] The process of restoring differentiated somatic cells to undifferentiated cells (such as stem cells) is called reprogramming. Induced pluripotent stem cells (iPSC) are also called reverse differentiated stem cells and reverse differentiated pluripotent stem cells. Reprogramming is to use reprogramming factors (Oct4, Klf4, Sox2, c-Myc, etc.) to transform somatic cells into stem cells (non-patent documents 1 and 2). [0003] Induced pluripotent stem cells are widely used in fields such as cell therapy, biological tissue engineering, new drug development, toxicology and precision medicine. These applications need to cultivate a large number of high-effic...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/077C12M3/00
CPCC12N5/0696C12M23/12C12N2513/00C12N2506/1307C12N2535/00C12M25/04C12N15/85C12N2800/108
Inventor 郑锡梁智勋田艺瑟罗奎唤
Owner NEXT&BIO INC