Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiation

Daryl E. Powers, Jeffrey R. Millman, Susan Bonner-Weir, Michael J. Rappel, Clark K. Colton

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO2gas). Because the cells are consuming oxygen supplied by diffusion, a difference between pO2gas and that experienced by the cells (pO2cell) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygenpermeable silicone rubber membranes can be used to control pO2cell during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene-propylene copolymer membranes. Cell attachment on silicone rubber was achieved by physical adsorption of fibronectin or Matrigel. We use these membranes for the differentiation of mouse embryonic stem cells to cardiomyocytes and compare the results with culture on polystyrene or on silicone rubber on top of polystyrene. The fraction of cells that are cardiomyocyte-like increases with decreasing pO2 only when using oxygen-permeable silicone membrane-based dishs, which contract on silicone rubber but not polystyrene. The high permeability of silicone rubber results in pO2cell being equal to pO2gas at the tissue-membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO2 distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO2cell, and these results suggest they are a valuable tool for investigating pO2 effects in many applications, such as stem cell differentiation.

Original languageEnglish
Pages (from-to)805-818
Number of pages14
JournalBiotechnology Progress
Volume26
Issue number3
DOIs
StatePublished - May 1 2010

Keywords

  • Differentiation
  • Embryonic stem cells
  • Hypoxia
  • Oxygen
  • Silicone rubber

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