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 (pO(2gas)). Because the cells are consuming oxygen supplied by diffusion, a difference between pO(2gas) and that experienced by the cells (pO(2cell)) 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 pO(2cell) 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 pO(2) 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 pO(2cell) being equal to pO(2gas) at the tissue-membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO(2) distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO(2cell), and these results suggest they are a valuable tool for investigating pO(2) effects in many applications, such as stem cell differentiation.
Powers
Daryl E
DE
Dept. of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Millman
Jeffrey R
JR
Bonner-Weir
Susan
S
Rappel
Michael J
MJ
Colton
Clark K
CK
D000818:Animals; D002448:Cell Adhesion; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D003094:Collagen / Q000378:metabolism; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology*; D005353:Fibronectins / Q000378:metabolism; D020342:Finite Element Analysis; D007700:Kinetics; D007797:Laminin / Q000378:metabolism; D016014:Linear Models; D051379:Mice; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000166:cytology; D010100:Oxygen / Q000378:metabolism*; D010313:Partial Pressure; D011137:Polystyrenes; D011509:Proteoglycans / Q000378:metabolism; D012825:Silicon / Q000737:chemistry*
D004338:Drug Combinations; D005353:Fibronectins; D007797:Laminin; D011137:Polystyrenes; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D010100:Oxygen; D012825:Silicon