Large-Scale Expansion of Mammary Epithelial Stem Cell Aggregates in Suspension Bioreactors

Abstract

Mutations in the pathways regulating mammary epithelial stem cell (MESC) selfrenewal and differentiation are currently hypothesized to result in uncontrolled cell division and, in turn, breast tumor formation. Although research is aggressively being pursued to understand how such pathways result in breast cancer formation, current studies have been greatly limited by MESC scarcity. To address this issue, this study has successfully developed large-scale expansion protocols for MESC through the subculture of murine mammary epithelial tissue aggregates, called mammospheres, in suspension bioreactors. Growth kinetics of mammospheres cultured in 125 mL suspension bioreactors and T-flasks were found to be comparable, achieving cell densities of 3.10 × 10 5 and 2.75 × 10 5 cells/mL, respectively. This corresponded to a 4-fold expansion over 8 days. Yields were also found to be strongly affected by liquid shear forces, where high agitation rates reduced overall cell numbers. Bioreactor cultures were scaled up to 1000 mL operating volumes, resulting in the production of 4.21 × 10 8 total cells (5.6-fold expansion) from a single passage. Furthermore, intermittent replacement of culture medium with fresh medium dramatically improved maximum cell densities, resulting in an 11-fold expansion, thereby enabling the generation of stem cells in quantities sufficient for standard biochemical and genetic analyses. After being cultured in suspension bioreactors for several passages, analysis by flow cytometry of Ki-67 revealed that 85% of the population was composed of proliferating cells. The successful development of expansion protocols for MESC aggregates in suspension bioreactors makes available experimental avenues that were not previously accessible for breast cancer research, thereby facilitating future investigations into elucidating the role of MESCs in breast cancer tumorigenesis.

Gamma-secretase inhibitors target tumor-initiating cells in a mouse model of ERBB2 breast cancer

“…Mammospheres or tumorspheres were established by plating primary cells in stem cell medium (Reynolds and Weiss, 1992), which comprises Dulbecco's modified Eagle's medium:Ham's F-12 (3:1), 4 mg/ml of B-27, 20 ng/ml EGF, 40 ng/ml FGF-2 and 4 ng/ml Heparin (all from Invitrogen, Carlsbad, CA, USA) (Youn et al, 2005(Youn et al, , 2006Kurpios et al, 2009). Spheres arising from primary cells were passaged every 4 days by mechanical dissociation followed by plating the dispersed cells at a density of B30 000 cells per ml in stem cell medium.…”

Expansion of Human Neural Precursor Cells in Large‐Scale Bioreactors for the Treatment of Neurodegenerative Disorders

“…(a) Experimental setup for the 500-mL suspension bioreactor showing: (1) 500-mL suspension bioreactor, (2) heating pad, (3) stirrer plate, (4) gas inlets to the bioreactor connected to O 2 , CO 2 , N 2 , and air gas cylinders via Wheaton pump, (5) Wheaton pump, (6) the control tower (Wheaton) connected to the dissolved oxygen (DO), pH, and temperature probes to monitor the level of each parameter, (7) DO, pH, and temperature probes, and (8) laboratory involving the expansion of mNPCs, 2,28-33 mammary epithelial stem cells (MESCs), 34 and breast cancer stem cells (BrCSCs), 35 the oxygen demand of the cells in the large-scale bioreactor was satisfied through headspace aeration using a gas mixture containing O 2 , CO 2 , and N 2 . Standard process control techniques were used to control the temperature at 37 C, pH at 7.2 ,and dissolved oxygen concentration at 70% air saturation and monitored using a computer.…”

Bioreactor development for stem cell expansion and controlled differentiation

“…Mouse neural stem cell culture was scaled up to 500 mL in an instrumented and controlled bioreactor [11••]. Mouse mammary epithelial stem cells were expanded as aggregates (or mammospheres) in spinner flasks [12]. Mouse breast cancer stem cells expanded as aggregates in an instrumented and controlled stirred bioreactor retained the potential for clonal growth, including secondary colony formation [13••].…”

“…Different stem cell types have different optimal shear stress values. 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].…”

“…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]. Under conditions for which oxygen was not limiting, Zhao et al reported greater human MSC expansion at a lower perfusion rate [7].…”