Enzyme-passage free culture of mouse embryonic stem cells on thermo-responsive polymer surfaces

Cell based therapies offer potentially revolutionary treatments for a number of diseases, but are dependent on the culture and supply of defined cell types in appropriate numbers. In turn, this supply requires changes to current cell culture passaging methods, which commonly use enzymatic digestion. Here we demonstrate, for the first time, the use of thermoresponsive poly(methoxyethoxyethylmethacrylate-co-oligoethyleneglycolmethacrylate) (poly(MEO2MA-co-OEGMA)) as a responsive surface on which to culture embryonic stem cells (mouse; used as a model embryonic stem cell type). Thermo-responsive copolymer brushes composed of MEO2MA and OEGMA were grafted from hydroxyl plasma-polymer functionalised glass slides using atom transfer radical polymerisation (ATRP) and their thermo-responsive behaviour characterised. Feeder-free mouse embryonic stem cells (mESCs) were seeded on these surfaces following adsorption of fibronectin (to encourage cell attachment) and cultured for three days. Subsequent passaging experiments were performed over 10 passage cycles and the cells analysed for population doubling times and retention of the stem cell geno/phenotype. These thermoresponsive polymer/fibronectin surfaces were found to be suitable for mESC culture without promoting differentiation during the early stages of culture. This indicated that these materials have promise as a new generation of culture materials for enzyme free cell culture and passage suitable for generating cell populations for clinical applications.

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