Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures

Abstract

The development of efficient and reproducible culture systems for embryonic stem (ES) cells is an essential pre-requisite for regenerative medicine. Culture scale-up ensuring maintenance of cell pluripotency is a central issue, because large amounts of pluripotent cells must be generated to warrant that differentiated cells deriving thereof are transplanted in great amounts and survive the procedure. This study aimed to develop a robust scalable cell expansion system, using a murine embryonic stem cell line that is feeder-dependent and adapted to serum-free medium, thus representing a more realistic model for human ES cells. We showed that high concentrations of murine ES cells can be obtained in stirred microcarrier-based spinner cultures, with a 10-fold concentration of cells per volume of medium and a 5-fold greater cell concentration per surface area, as compared to static cultures. No differences in terms of pluripotency and differentiation capability were observed between cells grown in traditional static systems and cells that were replated onto the traditional system after being expanded on microcarriers in the stirred system. This was verified by morphological analyses, quantification of cells expressing important pluripotency markers (Oct-4, SSEA-1, and SOX2), karyotype profile, and the ability to form embryoid bodies with similar sizes, and maintaining their intrinsic ability to differentiate into all three germ layers.

Agathisflavone Enhances Retinoic Acid-Induced Neurogenesis and Its Receptors α and β in Pluripotent Stem Cells

“…mES cells and miPS cells were grown in ES medium as previously described [13,14]. Briefly, cell colonies were grown on mouse embryonic fibroblast (MEF), mitotic inactivated with mitomycin C (Sigma), in 15% knockout serum replacement (KSR; Gibco)-DMEM/F12 (Gibco) supplemented with 1% nonessential amino acid solution (Gibco), 2 mM l-glutamine (Gibco), 0.1 mM b-mercaptoethanol (Gibco), 40 mg/mL gentamicin sulfate (Scheing-Plough), and 0.2% of conditioned medium of CHO (Chinese hamster ovary) cells producing leukemia inhibitory factor (LIF).…”

Synchrotron Radiation X-Ray Microfluorescence Reveals Polarized Distribution of Atomic Elements during Differentiation of Pluripotent Stem Cells

“…EBs were collected from plates and immediately fixed in 4% paraformaldehyde, and then the material was processed for obtaining 10 µm cryosections (Figure S1) as previously described [23].…”

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells

“…Commercially available microcarriers-such as Cultisphere S, Cytodex 3, Solohill carriers and Hillex II have been widely used for the expansion of ESC, with Cytodex 3 being the most common. The speed used for mouse ESC, varies between 40 and 70 RPM (Fok and Zandstra 2005;Abranches et al 2007;Alfred et al 2011;Fernandes et al 2007;Marinho et al 2010;Storm et al 2010;Tielens et al 2007), while the range for human ESC lies within 24-80 RPM (Storm et al 2010;Chen et al 2011;Fernandes et al 2009;Kehoe et al 2010;Leung et al 2011;Lock and Tzanakakis 2009;Marinho et al 2013;Nie et al 2009;Oh et al 2009;Phillips et al 2008;Serra et al 2010). iPSC are by nature, delicate, making their large scale culture in spinner flasks using microcarriers a much more difficult proposition.…”