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Related Experiment Videos

Feeder cell density--a key parameter in human embryonic stem cell culture.

Boon Chin Heng1, Hua Liu, Tong Cao

  • 1Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, 119074 Singapore.

In Vitro Cellular & Developmental Biology. Animal
|February 23, 2005
PubMed
Summary

Murine embryonic fibroblast (MEF) feeder cell density significantly impacts human embryonic stem (ES) cell culture. Suboptimal densities hinder ES cell maintenance, while excessive densities impede ES colony attachment and growth.

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Area of Science:

  • Stem Cell Biology
  • Developmental Biology
  • Cell Culture Technology

Background:

  • Human embryonic stem (ES) cell culture relies on murine embryonic fibroblast (MEF) feeder cells for support.
  • Variability in MEF feeder cell density across studies is a critical, overlooked factor.
  • Feeder cell density influences secreted factors, extracellular matrix, and cell-cell contacts essential for pluripotency maintenance.

Purpose of the Study:

  • To investigate the impact of varying murine embryonic fibroblast (MEF) feeder cell densities on human embryonic stem (ES) cell culture.
  • To determine the optimal MEF feeder cell density for robust human ES cell attachment and proliferation.
  • To analyze the morphological changes in human ES cell colonies under different feeder densities.

Main Methods:

Related Experiment Videos

  • Human ES cell line H9 cultured on MEF feeder layers at densities of 20,000 cells/cm² and 32,000 cells/cm².
  • Serial passaging of ES cell colonies to assess attachment and growth.
  • Microscopic observation to document colony morphology and identify any abnormalities.

    • Main Results:

    • An elevated MEF density of 32,000 cells/cm² was detrimental to the attachment and growth of H9 human ES cell colonies.
    • Human ES cells exhibited abnormal stacking and "bulge" formation at higher MEF densities.
    • The recommended MEF density of 20,000 cells/cm² supported better ES colony attachment and morphology.

    Conclusions:

    • MEF feeder cell density is a critical parameter that requires careful optimization for consistent human ES cell culture.
    • Excessive MEF density can negatively affect human ES cell behavior, potentially hindering pluripotency maintenance.
    • Further research is needed to establish optimal feeder densities for various human ES cell lines and culture conditions.