Fig 1.
Dynamic suspension bioreactor.
(A) Schematic draw of the bioreactor showing its internal components and its axial symmetry (red lines). (B) Picture of the bioreactor. (C) Schematic representation of the set-up of the bioreactor connected to the closed loop recirculation circuit and positioned within the incubator.
Fig 2.
Flow field within the bioreactor.
Flow field visualization of the mutual interaction between the medium (primary phase) and the cells/constructs (dispersed phase) within the culture chamber for ultralow (A and A1) and low-to-moderate (B and B1) shear stress conditions. Flow field is depicted using both linear integral convolution lines (A and B), and a classical streamline representation (A1 and B1). Yellow arrows indicate the flow inlet and outlet. Blue arrows indicate the primary buoyant vortices. Red arrows indicate the secondary vortices.
Fig 3.
Temporal evolution of the volume fraction occupied by suspended cells inside the bioreactor culture chamber.
Contour plots of the temporal evolution of the VF occupied by the suspended cells inside the bioreactor culture chamber from 0 to 60 min of simulated time, with an imposed flow rate of 5 mL/min (ultralow shear stress condition, similarly to the experimental in vitro test) and 9x106 inoculated cells (initial VF = 0.48%). After a transient of about 5 min, the 95.3% of the inoculated cells are suspended at an average VF value of approximately 0.33%, very close to the initial VF value. At the bottom of the culture chamber, a small volume of about 194 μL is characterized by a VF value around 6%, more than three times lower than the set threshold value of sedimentation (20%), which dynamically involves only the 2% of the inoculated cells.
Fig 4.
Probability density functions of volume fraction and shear stresses.
Probability density functions (PDF) of cell VF (A) and shear stresses (B) values experienced by the cellular phase within the bioreactor culture chamber after 60 min, with an imposed flow rate of 5 mL/min and 9x106 inoculated cells.
Fig 5.
Morphological comparison by phase contrast microscopy.
After 5 days of suspension culture, (A) Calu-3 cells cultured in static suspension show individual cells or very small clusters, (B) Calu-3 cells cultured under dynamic suspension show the formation of spheroids. Scale bars 200 μm.
Fig 6.
Ultrastructural comparison by TEM.
The TEM images show (A) a small cluster (3 cells) of Calu-3 cells grown in static suspension, and (B) a larger spheroid (9 cells) of Calu-3 cells cultured within the bioreactor, harvested both after 5 days of suspension culture. Prominent nucleoli (N: nuclei), cytoplasmic structures and longitudinally and transversally oriented microvilli are characteristic features of NSCLC cell line Calu-3. High magnification views of areas included in black rectangles in panels A and B shown, respectively, (A1) a single tiny adherence junction (arrowhead) among cells cultured under static suspension, and (B1) several well-developed adherence junctions (arrowheads) developed by Calu-3 cultured within the bioreactor. Scale bars: A and B = 5 μm; A1 and B1 = 1 μm.
Fig 7.
Quantitative comparison of cycling cells and double DNA strand breaks.
(A) Bar graph of the measurement of Ki67 positive cells, showing the fraction of cycling Calu-3 cells after static and dynamic suspension culture (*: p<0.05 vs static suspension). (B) Bar graph of the measurement of γH2AX positive cells, quantifying the double DNA strand breaks in Calu-3 cells harvested from static and dynamic suspension culture.