Feeder Layer- and Serum-Free Culture of Human Embryonic Stem Cells1

TLDR

Human embryonic stem cells are valuable for developmental research and cell therapies but are traditionally grown on mouse feeder layers, which pose risks of animal virus contamination. The study aims to provide a feeder‑layer‑free, serum‑free culture system that facilitates research and offers a safer alternative for clinical use of hES cells. The system uses a defined medium containing 15 % serum replacement plus TGFβ1, leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix to support hES cell growth. Under these conditions, hES cells retain pluripotency, normal karyotype, and unlimited proliferation, while the system offers a well‑defined, pathogen‑reduced culture platform.

Abstract

In addition to their contribution to the research on early human development, human embryonic stem (hES) cells may also be used for cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast feeder layers, which allow their continuous growth in an undifferentiated state. However, the use of hES cells in human therapy requires an animal-free culture system, in which exposure to mouse retroviruses is avoided. In this study we present a novel feeder layer-free culture system for hES cells, based on medium supplemented with 15% serum replacement, a combination of growth factors including transforming growth factor β1 (TGFβ1), leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix. Human ES cells grown in these conditions maintain all ES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. The culture system presented here has two major advantages: 1) application of a well-defined culture system for hES cells and 2) reduced exposure of hES cells to animal pathogens. The feeder layer-free culture system reported here aims at facilitating research practices and providing a safer alternative for future clinical applications of hES cells.