Fig 1.
Two types of bioreactors and experimental setup for fluidization.
(A) Schematic of the diversion-type microcapsule suspension fluidized bed bioreactor (DMFBB). (1) inlet; (2) bottom cap; (3) pool of incoming buffer; (4) turbine guide vanes; (5) 300 mesh membrane filters; 6) cylindrical bioreactor containing the microcapsules; (7) end cap; and (8) outlet. The dimensions associated with the turbine guide vanes were: height of the turbine = 15 mm; thickness of each turbine blade = 10 mm, blade inlet angle = 90°, blade outlet (outer edge) angle = 26°, blade outlet (inner edge) = 71°, screw pitch = 70 mm; number turbine vane rotations = 0.207, and diameter of fixed middle axis = 7 mm. (B) Schematic of the traditional fluidized bed bioreactor (FBB). (1) inlet; (2) bottom top; (3) pool of incoming buffer; (4) 300 mesh membrane filters; (5) cylindrical bioreactor containing the microcapsules; (6) end cap; and (7) outlet. (C) The experimental set up for dynamic culture of C3A cells within the fluidized bed bioreactor. C3A cells were encapsulated in alginate/chitosan microspheres with a diameter of 800 μm.
Table 1.
Primers Used in the Real-time Quantitative PCR Analyses.
Fig 2.
Fluidization performance of two bioreactors.
(A) Fluidization performance of the FBB at 90 ml/min. (B) Fluidization performance of the FBB at 150 ml/min. (C) Fluidization performance of the DMFBB at 90 ml/min. (D) Fluidization performance of the DMFBB at 150 ml/min. At the beginning, the red indicates fixed microcapsules at the bottom of the reactor, and the blue indicates fluidized flow (DMEM). As the flow rate was increased, microcapsules in the center at the bottom of the FBB were forced upwards to the top of the bioreactor, leading to weak fluidization. Conversely, the microcapsules in the DMFBB were gradually mixed with the flowing medium, and eventually, dynamic and balanced fluidization was established. In the images, the colors from red to yellow or green represented different microcapsule densities under different conditions. (E) Fluidization in DMFBB and FBB in terms of bed expansion (h/h0) as the perfusion flow rate was increased from 0 to150 ml/min.
Fig 3.
Effects of fluidization on empty microcapsule integrity within the bioreactors.
(A) Microcapsule retention rates of the DMFBB and FBB operated at 90 and 150 ml/min. (B) Swelling rates (%) of microcapsules in the DMFBB and FBB operated at 90 and 150ml/min. (C) Percentages of broken microcapsules in the DMFBB and FBB operated at 90 and 150 ml/min. The following results were obtained: (A): When the DMFBB was operated at 90 ml/min, the rate of microcapsule retention was 99.8% compared to 91.74% in the FBB at day 1 (p = 0.0025), 98.08% compared to 90.3% at day 2 (p = 0.0022), and 96.55% compared to 87.68% at day 3 (p = 0.0024).When the DMFBB was operated at 150 ml/min, the rate of microcapsule retention was 99.78% compared to 92.14% in the FBB at day 1 (p = 0.0051), 97.49% compared to 87.98% at day 2 (p = 0.0014), and 94.71% compared to 84.95% at day 3 (p = 0.0008). (B): When the DMFBB was operated at 90 ml/min, the swelling rate (%) of microcapsules was 7.7% compared to 15.81% in the FBB at day 1 (P = 0.0176), 11.59% compared to 19.64% at day 2 (p = 0.0027), and 12.8% compared to 34.81% at day 3 (p = 0.0005). When the DMFBB was operated at 150 ml/min, the swelling rate of microcapsules was 5.49% compared to 23.49% in the FBB at day 1 (p = 0.0328), 14.35% compared to 27.59% at day 2 (p = 0.0241), and 30.11% compared to 36.66% at day 3 (p = 0.3258). (C): When the DMFBB was operated at 90 ml/min, the percentage of broken microcapsules in the DMFBB was 1.6% compared to 3.2% in the FBB at day 1 (p = 0.0095), 4.8% compared to 7.4% at day 2 (p = 0.0117), and 6.4% compared to 11.6% at day 3 (p = 0.0017). When DMFBB was operated at 150 ml/min, the percentage of broken microcapsules in the DMFBB was 1.9% compared to 4.2% in the FBB at day 1 (p = 0.0042), 5.6% compared to 9.8% at day 2 (p = 0.0114), and 7.4% compared to 11.8% at day 3 (p = 0.0031).
Fig 4.
C3A cell viability in fluidized and static culture conditions.
(A) Cell viability according to MTT assay results; *p<0.05. Cell viability in the DMFBB was significantly improved compared to that in the FBB on each day of the 3-day experiment (p = 0.0155, 0.0098, and 0.0112). The differences between the FBB and static group were not significant over the 3-day time course (p = 0.0987, 0.0512, and 0.3086). (B) Images of live (CM-FDA stained) and dead (propidium iodide stained) C3A cells in microspheres. Scale bar, 500 μm.
Fig 5.
Hepatocyte-related functions of C3A cells in fluidized and static culture conditions.
(A and B) Activities of phase I enzymes CYP 1A2 (A) and CYP3A4 (B) measured via fluorometric substrates in different fluidized and static culture conditions. (C and D) Rates of albumin secretion (C) and urea synthesis (D) of C3A cells in different fluidized and static culture conditions. Columns labeled with the same letter indicate the results were not statistically different; for all other comparisons, p<0.05.
Fig 6.
Normalized gene expression of CYP450 and phase II enzymes in C3A cells after 72 h of culture in fluidized and static conditions.
All data were normalized to the activity of C3A cells cultured in static conditions.