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Cultivation of primate embryonic stem cells

a technology of embryonic stem cells and primate embryos, which is applied in the field of primate embryonic stem cell culture to achieve the effects of avoiding the variability of results due to differences in animal serum batches, avoiding the use of animal serum, and increasing the efficiency of cloning

Inactive Publication Date: 2005-07-07
WISCONSIN ALUMNI RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0020] The culturing permits the embryonic stem cells to stably proliferate in culture for over one month (preferably over six months; even more preferably over twelve months) while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells.
[0024] Variability in results due to differences in batches of animal serum is thereby avoided. Further, it has been discovered that avoiding use of animal serum while using fibroblast growth factor can increase the efficiency of cloning.

Problems solved by technology

However, when one or more well defined purified component(s) of such serum is added, they do not.

Method used

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Examples

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examples

[0031] In the first experiments described here human ES cells were plated on irradiated (35 gray gamma irradiation) mouse embryonic fibroblasts. Culture medium for the present work consisted of 80% “KnockOut” Dulbeco's modified Eagle's medium (DMEM) (Gibco BRL, Rockville, Md.), 1 mM L-Glutamine, 0.1 mM β-mercaptoethanol, and 1% nonessential amino acids stock (Gibco BRL, Rockville, Md.), supplemented with either 20% fetal bovine serum (HyClone, Logan, Utah) or 20% KnockOut SR, a serum-free replacement originally optimized for mouse ES cells (Gibco BRL, Rockville, Md.). The components of KnockOut SR are those described for serum replacements in WO 98 / 30679.

[0032] In alternative experiments medium was supplemented with either serum or the aforesaid serum replacer KnockOut SR, and either with or without human recombinant basic fibroblast growth factor (bFGF, 4 ng / ml). The preferred concentration range of bFGF in the culture was between 0.1 ng / ml to 500 ng / ml.

[0033] To determine clonin...

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Abstract

The invention relates to methods for culturing human embryonic stem cells by culturing the stem cells in an environment essentially free of mammalian fetal serum and in a stem cell culture medium including amino acids, vitamins, salts, minerals, transferring, insulin, albumin, and a fibroblast growth factor that is supplied from a source other than just a feeder layer the medium. Also disclosed are compositions capable of supporting the culture and proliferation of human embryonic stem cells without the need for feeder cells or for exposure of the medium to feeder cells.

Description

CROSS REFERENCES TO RELATED APPLICATIONS [0001] This application is a continuation-in-part application of U.S. patent application Ser. No. [0002]09 / 522,030 filed Mar. 9, 2000.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0003] To be determined. BACKGROUND OF THE INVENTION [0004] The present invention relates to methods for culturing primate embryonic stem cell cultures and culture media useful therewith. [0005] Primate (e.g. monkey and human) pluripotent embryonic stem cells have been derived from preimplantation embryos. See for example, U.S. Pat. No. 5,843,780 and J. Thomson et al., 282 Science 1145-1147 (1998). The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein. Notwithstanding prolonged culture, these cells stably maintain a developmental potential to form advanced derivatives of all three embryonic germ layers. [0006] Primate (particularly human) ES cell lines have widespread utili...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/22C12N5/07A61K31/495C12NC12N5/00C12N5/02C12N5/0735C12R1/91
CPCC12N5/0606C12N2501/115C12N2500/99C12N2500/90C12N5/06
Inventor THOMSON, JAMES A.LEVENSTEIN, MARK
Owner WISCONSIN ALUMNI RES FOUND
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