Expansion of Undifferentiated Murine Embryonic Stem Cells as Aggregates in Suspension Culture Bioreactors

Abstract

Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for regenerating tissues lost to injuries and degenerative diseases. For clinical implementation of this technology, a quality controlled, reproducible culture system is necessary for the expansion and differentiation of the cells. Used in many bioprocess applications, suspension bioreactors have gained considerable attention for the regulated large-scale expansion of cells. The current study presents a bioreactor process for the large-scale expansion of undifferentiated murine ESCs as aggregates. In this system, the level of ESC aggregation and differentiation was effectively controlled by adjusting shear forces and inoculation density, achieving a 31-fold expansion in 5 days. Pluripotency markers Oct-4, Nanog, SSEA-1, ALP, and rex-1 were assessed using flow cytometry analysis and gene expression profiles and showed that the undifferentiated nature of the cells within the ESC aggregates was maintained. Colony-forming efficiencies and embryoid body formation tests of the expanded cultures demonstrated that characteristic functional attributes of undifferentiated cells were not lost. Overcoming a major impediment in the area of ESC expansion, this study describes a successful process for the controlled and reproducible largescale expansion of ESCs using suspension culture bioreactors.

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

“…Spinner flasks are one of the oldest and most widely used reactor systems and have been used extensively for the expansion of mouse as well as human ESC. Both mouse and human ESC have been expanded as aggregates in spinner flasks (Fok and Zandstra 2005;Cormier et al 2006;Abbasalizadeh et al 2012;Krawetz et al 2010;Steiner et al 2010). Extensive study in the field of large scale expansion of ES cells has given rise to novel and better systems for these cells but the field of dynamic culture of iPS cells has just started.…”

Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner

“…Due to their capacity of unlimited proliferation and ability to differentiate into cell types of all three germ layers, pluripotent stem cells are considered as highly interesting for both cell based therapies and the use in the pharmaceutical industry for drug screening and drug target identification. In order to facilitate the investigations of these cells for the mentioned applications, controlled, reproducible and scalable culture systems are needed to generate high amounts of undifferentiated cells [10], [11].…”

Bioreactor development for stem cell expansion and controlled differentiation

“…For example, the optimal shear stress for mammary epithelial stem cell aggregate culture was 0.21 Pa [12]. In contrast, mESC aggregate cultures had an optimal shear stress of 0.61 Pa, and exhibited extensive clumping at a shear stress of 0.45 Pa [15]. Encapsulation of mESC aggregates in agarose allowed for embryoid body formation and expansion at high agitation rates [21••].…”

“…Perfusion or frequent feeding also enhances culture performance by replacing depleted nutrients and/or removing inhibitory metabolic byproducts. For example, Cormier et al attributed the decline in viability of mESC aggregates after 6 days in batch culture to the associated build-up of ammonia (to 2.5 mM) and decrease in pH (from 7.6 to 6.6) [15]. Perfusion, frequent feeding, and/or the use of gas-permeable culture surfaces increased the expansion of MSCs [4,5,6•], ESCs [32,33] and mammary epithelial stem cells [12], while retaining stem cell potential [5,9,32,33].…”