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

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 (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.

Oxygen Transport and Stem Cell Aggregation in Stirred-Suspension Bioreactor Cultures

“…This facilitates the comparison with findings from previous studies and eliminates potential discrepancies due to differences in culture media utilized in those reports. Even though we cannot rule out potential changes in parameter values if the measurements were performed in culture medium, these changes are expected to be minimal as shown for other systems [36], [37], [40].…”

Rat islet cell aggregates are superior to islets for transplantation in microcapsules

“…Encapsulated islet-cell aggregates are superior to intact islets in their ability to survive and function in a low-oxygen environment in vitro and following transplantation. In this study, we cultured encapsulated islet tissue on silicone rubber membranes so as to make the p O 2 to which the capsules were exposed about the same as in the gas phase [28]. The low oxygen concentration culture conditions were chosen to approximate the levels of hypoxia expected to be present in encapsulated non-vascularised islets or islet cell aggregates.…”

Quantitative Assessment of Islets of Langerhans Encapsulated in Alginate

“…In these plates, holes were drilled in the polystyrene bottom, and inserts with a silicone rubber bottom were placed on top of spacers in each well (PR-AY-7-0004; Wilson Wolf) to yield culture wells having a silicone rubber bottom wherein the pO 2 at the membranemedium interface was the same as in the gas phase. 53 …”