Method for efficiently acquiring inductive pluripotent stem cells (iPSCs)

A pluripotent stem cell, inducible technology, applied in the field of cells, can solve the problems of low efficiency and long cycle of pig iPSCs, and achieve the effect of improving efficiency, shortening cycle and improving quality

Active Publication Date: 2014-03-26
ANHUI AGRICULTURAL UNIVERSITY
View PDF4 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In order to overcome the above-mentioned problems of low efficiency and long cycle of inducing porcine iPSCs, the object of the present invention is to provide a technical method for efficiently obtaining porcine iPSCs. The method is to induce reprog

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for efficiently acquiring inductive pluripotent stem cells (iPSCs)
  • Method for efficiently acquiring inductive pluripotent stem cells (iPSCs)
  • Method for efficiently acquiring inductive pluripotent stem cells (iPSCs)

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0046] Example 1. Preparation and culture of cells

[0047] 1.1 Cultivation of Porcine Adipose Stem Cells (ADSCs)

[0048] Separate adipose tissue from pig back and abdomen subcutaneously, remove blood vessel and muscle residue, wash thoroughly with DPBS buffer (Gibco), then use surgical scissors to fully cut the adipose tissue until there is almost no shear resistance, and transfer to centrifugation In the tube, add 0.09% type I collagenase digestion solution (Sigma) equivalent to 2 to 3 times the total volume of adipose tissue, and place it in a 37°C water bath for shaking and digestion; after the tissue suspension is gelatinized, use Pasteur Pipette repeatedly to disperse tissue debris, centrifuge at 1,200 rpm for 5 minutes at room temperature, discard the mature adipose tissue in the upper layer of the centrifuge tube, and the digestive fluid in the middle layer, and then use DMEM / F-12 basal medium containing 10% fetal bovine serum (HyClone) ) Fully resuspend the cells at the ...

Example Embodiment

[0053] Example 2. Viral vector infects pig ADSCs

[0054] According to the method described in Example 1, inoculate low-passage pig ADSCs in cell culture flasks at 37°C, 5% CO 2 After culturing to a confluence of 80-90% under conventional culture conditions, use 0.25% trypsin-EDTA (Gibco) to digest into single cell suspension at 37°C, and then infect 1×10 with the collected viral supernatant 5 ADSCs with a multiplicity of infection (MOI) of 3 (the titer of the virus used is 5-10×10 6 IU / mL, the four viruses carrying pluripotency factor cDNA are mixed at 1:1:1:1), and then inoculated into a 6-well culture plate, and then kept at 37℃, 5% CO 2 Cultivate under the conventional culture conditions. The virus supernatant was transfected into 293T cells (Fugene HD) with a drug-inducible (RevTet-On) lentiviral expression vector (SiDanSai company) containing human Oct4, Sox2, Klf4 and c-Myc cDNA according to conventional methods. , Roche) (see "Molecular Cloning Experiment Guide", edited by...

Example Embodiment

[0055] Example 3. Continued culture and clone screening of infected cells

[0056] On the second day after infection, wash the culture well twice with DPBS buffer, and then digest the infected ADSCs with 0.25% trypsin-EDTA (Gibco) at 37°C into a single cell suspension, and press 2,500 cells / cm 2 Inoculate into a new 6-well culture plate with a density of 5%, and a total of 4 culture wells are inoculated. The surface of the culture plate is pre-coated with Matrigel (BD Pharmingen). Continue to use ADSCs complete medium. Among the parallel samples of 4 wells, one well was used for alkaline phosphatase (AP) staining (SiDanSai) to count the number of positive clones, and the other 3 wells were used for clone selection. The experiment was repeated three times. On the third day after infection, the ADSCs complete medium was replaced with the porcine iPSCs serum-free complete medium described in Example 1, and the temperature was continued at 37°C, 5% CO 2 Cultivate under the conventio...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Titeraaaaaaaaaa
Login to view more

Abstract

The invention discloses a method for efficiently acquiring inductive pluripotent stem cells (iPSCs). Adipose-derived stem cells (ADSCs) are induced and reprogrammed to form the iPSCs by using the method. The method concretely comprises the following steps: (1) inducing cDNA (Complementary Desoxvribose Nucleic Acid) of a multifunctional factor to the ADSCs; (2) culturing the ADSCs by using a serum-free culture medium in a feed-layer-free system, wherein the ADSCs is obtained in the step (1); (3) after cloning sample cells of embryonic stem cells (ESCs), further culturing by using a culture medium with an MEK (Methyl Ethyl Ketone) and GSK3 (Glaxo Smith Klein) signal channel inhibitor, selecting cells to clone, and enlarging culture; and (4) authenticating the cell cloning pluripotency. In the method, the ADSCs are low in cost and easily available on a large scale, and the reprogramming speed and efficiency are very high; the feed-layer-free system can be used for increasing the cloning purity of positive iPSCs; the serum-free culture medium can be used for avoiding unstable factors brought by undefined components in serum and batch difference; the signal channel inhibitor can be used for promoting cells to be up to the sufficient reprogramming state and improving the cloning quality of the positive iPSCs.

Description

technical field [0001] The invention relates to the field of cells, in particular to a method for efficiently obtaining porcine induced pluripotent stem cells. Background technique [0002] Stem cells are the initial source of the human body and its various tissue cells. Their most notable biological characteristics are the ability of self-renewal and continuous proliferation, as well as the potential of multidirectional differentiation. Stem cells are divided into adult stem cells (Adult stem cells) and embryonic stem cells (Embryonic stem cells, ESCs) according to different sources. Adult stem cells include bone marrow mesenchymal stem cells, pancreatic stem cells, neural stem cells, adipose stem cells, etc., which exist in adult tissues. [0003] In 1981, the isolation and culture of ESCs was first achieved in mice, and it is the most widely studied and mature stem cell system so far. The generation of human stem cells began in 1998. American scientist James A. Thomson ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/867C12N5/10
Inventor 张运海张宇魏超章孝荣
Owner ANHUI AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap