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Updated: Aug 4, 2026
Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells
Published on: June 21, 2012
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.
Abstract:
A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm2, above the recommended value of 20,000 cells/cm2, appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm2), the ES cells appear to stack up to form a "bulge." This was not observed under the recommended feeder cell density of 20,000 cells/cm2. By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm2. This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.
Insights
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.
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:
- 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.
