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Biomanufacturing

Biomanufacturing - Poster Session B

Poster B2 - Characterization of Decellularized Plant Scaffolds Behavior in Fluidized Bed Bioreactor

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The widespread adoption of cultured meat is limited due to the low productivity of industrial cell cultures. Factors reducing productivity include accumulation of biological waste, depletion of nutrients, and batch processes. Fluidized bed bioreactors (FBBs) operate with continuous flow of fluid and suspension of the particles. This mode of production allows for reduced water usage, and repletion of nutrients and continuous operation. FBBs combined with edible decellularized plant scaffolds present a sustainable and more productive alternative for cultured meat at industrial scale. In this work, we characterize the behavior of decellularized plant scaffolds (broccoli florets) in a model FBB system.

Materials and

Methods:  Decellularization Process
Fresh broccoli florets were placed in a 50.0g/L sodium dodecyl sulfate (SDS) solution on a rotating platform for two days, then 50.0 g/L SDS with 5% (v/v) bleach for one day.

Fluidized Reactor Characterization
The fluidized reactor uses a 15mm Omnifit Labware column with total volume of 22.53 mL attached to a RP-1 Rainin Dynamax peristaltic pump with flow rates of 7.5, 10.0, and 15.0 mL/min. An air trap was introduced between the pump and column with a modified 50 mL conical tube to allow dye injections into the closed system. The total media volume in the system was 52.0 mL. Methylene blue dye (0.5 mL) was injected using a 2 mL syringe. Samples of the DMEM/F-12 media were every 45 seconds over 10 minutes to determine the time necessary to reach system equilibrium via absorbance at wavelength 666 with a BioTek Cytation1 Reader (three replicates). Packing effect was measured by volume displacement. Decellularized florets (1.440 g, n = 10) were added to the bioreactor column with DMEM/F-12 media. The packed volumes were measured at static state and each of the three standard flow rates after five minutes.

Imaging and Image processing
Descension and packing videos were recorded using a Fastec HiSpec4 4G Mono camera at 10 FPS. Videos were uploaded to Fiji (ImageJ) software for processing where individual floret displacements were measured in terms of movement and angular change during descent.

Results, Conclusions, and Discussions: Results

Descent Speed
Scaffold velocity, position, angle, average acceleration, angular velocity, and angular acceleration were all recorded for each individual floret that was dropped via forceps into the static system. A total of 10 florets were recorded, 1 was excluded due to air bubbles creating buoyancy, 1 was determined to be an outlier. Average linear acceleration was 0.03 +/- 0.01 mm/s2. Average angular acceleration was 0.08 +/- 0.05 degrees/s2.

Mixing Rate
Mixing rates were analyzed by tracking the change in absorbance after the injection of the blue dye. Peak absorbance was reached after 135 seconds with 7.5 mL/min, 90 seconds with 10 mL/min and 15.0 mL/min. Complete mixing was achieved when absorbance change between timepoints was ≤0.001/second. Average mixing time was 315 seconds for 7.5 mL/min, 270 seconds for 10.0 mL/min and 225 for 15.0 mL/min.
Packing
The expansion coefficient was measured by comparing the height of the florets in the column at static state to the height during dynamic state. The expansion coefficient was 1.197 for 7.5 mL/min, 1.221 for 10 mL/min and 1.554 for 15.0 mL/min. The apparent porosity coefficient was measured as the volume of added volume as the void space divided by the total volume in the column of the packed florets under flow. The porosity coefficient was 16.5% for 7.5 mL/min, 18.1% for 10.0 mL/min, and 35.7% for 15.0 mL/min.

Conclusions
The working parameters for the FBBs with decellularized plant scaffolds were determined and the minimum and maximum experimental conditions were identified. Higher flow rates led to faster mixing and higher porosity coefficients. The impact of such flow rates on cell growth and viability must be investigated. A model will be developed to describe scaffold and cell behaviors within these reactors to further develop FBBs and scale-up cultured meat production.

Acknowledgements (Optional):