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

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 oxygen‐permeable 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. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010

[1]  Adam J Engler,et al.  Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating , 2008, Journal of Cell Science.

[2]  Ryan B. Huang,et al.  Effects of oxygen on mouse embryonic stem cell growth, phenotype retention, and cellular energetics. , 2008, Biotechnology and bioengineering.

[3]  J. Stuart,et al.  Metabolic rate does not scale with body mass in cultured mammalian cells. , 2007, American journal of physiology. Regulatory, integrative and comparative physiology.

[4]  R. M. Barrer,et al.  Solution and diffusion of gases and vapors in silicone rubber membranes , 2007 .

[5]  D. Mills,et al.  Gelatin-glutaraldehyde cross-linking on silicone rubber to increase endothelial cell adhesion and growth , 2002, In Vitro Cellular & Developmental Biology - Animal.

[6]  Daryl E. Powers.  Effects of oxygen on embryonic stem proliferation, energetics, and differentiation into cardiomyocytes , 2007 .

[7]  G. Garin,et al.  Flow-mediated signaling modulates endothelial cell phenotype. , 2006, Endothelium : journal of endothelial cell research.

[8]  H. Callesen,et al.  Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system. , 2005, Reproduction.

[9]  G. Weir,et al.  High-density culture of human islets on top of silicone rubber membranes. , 2005, Transplantation proceedings.

[10]  E. Pettersen,et al.  Pericellular oxygen depletion during ordinary tissue culturing, measured with oxygen microsensors , 2005, Cell proliferation.

[11]  D. Wilson,et al.  Stability of plasma-treated silicone rubber and its influence on the interfacial aspects of blood compatibility. , 2004, Biomaterials.

[12]  J. B. Pitner,et al.  Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration , 2004, Biotechnology and bioengineering.

[13]  B. Glennon,et al.  Mass transfer analysis of a membrane aerated reactor , 2004 .

[14]  N. Cruz,et al.  Investigations on the Stability of Plasma Modified Silicone Surfaces , 2004 .

[15]  P. Hammond,et al.  Controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[16]  B. Glennon,et al.  Characteristics of a Methanotrophic Culture in a Membrane‐Aerated Biofilm Reactor , 2004, Biotechnology progress.

[17]  Izumi Ichinose,et al.  Biocompatibility of layer-by-layer self-assembled nanofilm on silicone rubber for neurons , 2003, Journal of Neuroscience Methods.

[18]  C. Otto,et al.  In vitro intermittent hypoxia: challenges for creating hypoxia in cell culture , 2003, Respiratory Physiology & Neurobiology.

[19]  M. Rubner,et al.  New class of ultrathin, highly cell-adhesion-resistant polyelectrolyte multilayers with micropatterning capabilities. , 2003, Biomacromolecules.

[20]  A. Mas,et al.  Improvement of silicone endothelialization by treatment with allylamine and/or acrylic acid low‐pressure plasma , 2003 .

[21]  Allon I Hochbaum,et al.  Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films. , 2003, Biomacromolecules.

[22]  J. Hescheler,et al.  Hypoxia influences generation and propagation of electrical activity in embryonic cardiomyocyte clusters. , 2002, Comparative biochemistry and physiology. Part A, Molecular & integrative physiology.

[23]  J. Linderman,et al.  Quantification of fibronectin adsorption to silicone-rubber cell culture substrates. , 2002, BioTechniques.

[24]  W. Deen,et al.  Analysis of the effects of cell spacing and liquid depth on nitric oxide and its oxidation products in cell cultures. , 2001, Chemical research in toxicology.

[25]  V. Falanga,et al.  The levels and kinetics of oxygen tension detectable at the surface of human dermal fibroblast cultures , 2000, Journal of cellular physiology.

[26]  Jeffrey A. Hubbell,et al.  Thin Polymer Layers Formed by Polyelectrolyte Multilayer Techniques on Biological Surfaces , 1999 .

[27]  F W Falkenberg,et al.  Production of monoclonal antibodies in the miniPERM bioreactor: comparison with other hybridoma culture methods. , 1998, Research in immunology.

[28]  K. Toda,et al.  Cellulose production by Acetobacter pasteurianus on silicone membrane , 1996 .

[29]  W. Jelkmann,et al.  Pericellular PO2 and O2 consumption in monolayer cell cultures. , 1995, Respiration physiology.

[30]  H. Busscher,et al.  Hydrophobic recovery of repeatedly plasma-treated silicone rubber. Part 2. A comparison of the hydrophobic recovery in air, water, or liquid nitrogen , 1995 .

[31]  Y. Ikada,et al.  Surface modification of silicone for percutaneous implantation. , 1995, Journal of biomaterials science. Polymer edition.

[32]  W. Jelkmann,et al.  Microelectrode measurements of pericellular PO2 in erythropoietin-producing human hepatoma cell cultures. , 1993, The American journal of physiology.

[33]  H. J. Henzler,et al.  Oxygenation of cell cultures , 1993 .

[34]  C. C. Wang,et al.  ppHEMA-modified silicone rubber film towards improving rabbit corneal epithelial cell attachment and growth. , 1993, Biomaterials.

[35]  R. Tompkins,et al.  Hepatic Tissue Engineering: Development of Critical Technologies , 1992, Annals of the New York Academy of Sciences.

[36]  J. Feijen,et al.  The influence of protein adsorption on interactions of cultured human endothelial cells with polymers. , 1987, Journal of biomedical materials research.

[37]  I. Silver,et al.  The oxygen dependence of cellular energy metabolism. , 1979, Archives of biochemistry and biophysics.

[38]  D. Wallach,et al.  Diffusion in tissue cultures on gas-permeable and impermeable supports. , 1976, Journal of theoretical biology.

[39]  G. Moore,et al.  Kinetics of gas diffusion in mammalian cell culture systems. I. Experimental , 1968 .

[40]  W. L. Robb,et al.  THIN SILICONE MEMBRANES‐THEIR PERMEATION PROPERTIES AND SOME APPLICATIONS , 1968, Annals of the New York Academy of Sciences.

[41]  K. Stevens,et al.  Oxygen Requirements for Liver Cells in vitro , 1965, Nature.

[42]  L. Rosenhead.  Conduction of Heat in Solids , 1947, Nature.