Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Control of ES cell self renewal and lineage specification, and medium therefor

A culture medium and cell technology, applied in the direction of embryonic cells, cell culture active agents, culture process, etc., can solve the problems of pollution, blocking the development of ES cells and their progeny, and achieve the effect of reducing differentiation

Inactive Publication Date: 2005-08-24
THE UNIV OF EDINBURGH
View PDF7 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] As a result, ES cells and any differentiated progeny obtained by culturing in such complex media are at risk of contamination by the media and or cells (e.g. feeder cells required to maintain ES cells)
These factors hinder the development of superior manufacturing scales for ES cells and their progeny for therapeutic and other applications

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
  • Control of ES cell self renewal and lineage specification, and medium therefor
  • Control of ES cell self renewal and lineage specification, and medium therefor
  • Control of ES cell self renewal and lineage specification, and medium therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Method of Culturing ES Cells in a Serum-Free and Feeder-Free Defined Medium

[0104] N2B27 (DMEM / F12 medium supplemented with modified N2 (25 μg / ml insulin, 100 μg / ml apotransferrin, 6 ng / ml luteinizing hormone, 16 μg / ml putrescine, 30 nM sodium selenite and 50 μg / ml bovine serum albumin) and a 1:1 mixture of B27-supplemented Neurobasal Medium)

[0105] ES cells were cultured in N2B27 medium supplemented with LIF (100 U / ml) and BMP4 (100 ng / ml) in a culture dish coated with 0.1% gelatin. For passaging, detach cells with standard protein-free cell dissociation buffer.

[0106] Cells were seeded at a density of approximately 1-5 x 10 4 / cm 2 .

[0107] At the beginning of culture, SU5402 (5 μM) was supplemented in the medium to inhibit differentiation. Cells were transferred to SU5402-free medium after 2 passages.

[0108] ES cells were maintained under these serum-free conditions for 20 passages over 3 months. Cells are typically passaged every 2-4 days,...

Embodiment 2

[0110] Example 2 maintains Oct4-GFP expression in ES cells cultured under serum-free conditions

[0111] Oct4GFP (clone C1 ) ES cells were cultured in N2B27 (see Example 1) supplemented with LIF and SU5402 in 0.1% gelatin coated plates. After 2 passages, cells were cultured in N2B27 medium supplemented with LIF and BMP4. After another 2 passages, light microscope images of the cells were taken under phase contrast conditions showing morphology and UV fluorescence to reveal GFP expression. Figure 1 shows the microscope images.

[0112] What is clear is the morphology and expression of GFP, indicating that ES cells maintain their pluripotent phenotype after 4 passages in serum-free medium.

Embodiment 3

[0113] Example 3 Stable transfection of ES cells

[0114] E14TG2A ES cells were cultured in DMEM F12+ neural basal medium supplemented with N2-B27 supplement and LIF, BMP4. Cells were propagated in 0.1% gelatin-coated plates, harvested and transfected with pPCAG-tauGFP-IP electroporation. transfected cells at 10 5 -10 6 The density per dish was reseeded on 10 cm diameter Petri dishes. After 24 hours, 0.5 μg / ml puromycin was added to select positive clones.

[0115] Between 8-10 days thereafter, single GFP-positive clones were picked into individual wells of 96-well plates, and cells were cultured in the same medium as described above.

[0116] GPF green fluorescence indicates stable transfection of ES cells, and expansion of morphologically undifferentiated ES cells is observed, as shown in figure 2 shown.

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
Inoculation densityaaaaaaaaaa
Login to View More

Abstract

Self renewal of pluripotent cells in culture is promoted using a combination of an activator of a signalling pathway downstream of a receptor of the TGF-beta superfamily and an activator of a gp130 downstream signalling pathway.

Description

technical field [0001] The invention relates to culture conditions and methods for cultivating pluripotent stem cells to promote stem cell self-renewal and prevent or control stem cell differentiation. The invention also provides methods of isolating and maintaining homogeneous preparations of pluripotent stem cells. The provided methods and compositions are suitable for culturing and isolating pluripotent stem cells, such as embryonic stem cells (ES cells). Background technique [0002] The establishment and maintenance of in vitro pluripotent stem cell cultures in media containing serum and leukemia inhibitory factor (LIF) is well known (Smith et al. (1988) Nature 336:688-90). These methods are used to maintain pluripotent embryonic stem cells (ES cells) in permissive mouse strains for many generations. The maintenance and self-renewal of pluripotent stem cell cultures is further supported when the stem cells are cultured in the presence of feeder cells or their extracts...

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): C12N15/09C12N5/00C12N5/02C12N5/07C12N5/0735
CPCC12N2501/155C12N2501/15C12N2500/99C12N2501/235C12N5/0606C12N2500/90
Inventor A·G·史密斯应其龙
Owner THE UNIV OF EDINBURGH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com