Assessment of the interplay between scaffold geometry, induced shear stresses, and cell proliferation within a packed bed perfusion bioreactor

Abstract

By favoring cell proliferation and differentiation, perfusion bioreactors proved efficient at optimizing cell culture. The aim of this study was to quantify cell proliferation within a perfusion bioreactor and correlate it to the wall shear stress (WSS) distribution by combining 3‐D imaging and computational fluid dynamics simulations.NIH‐3T3 fibroblasts were cultured onto a scaffold model made of impermeable polyacetal spheres or Polydimethylsiloxane cubes. After 1, 2, and 3 weeks of culture, constructs were analyzed by micro‐computed tomography (μCT) and quantification of cell proliferation was assessed. After 3 weeks, the volume of cells was found four times higher in the stacking of spheres than in the stacking of cube.3D‐μCT reconstruction of bioreactors was used as input for the numerical simulations. Using a lattice‐Boltzmann method, we simulated the fluid flow within the bioreactors. We retrieved the WSS distribution (PDF) on the scaffolds surface at the beginning of cultivation and correlated this distribution to the local presence of cells after 3 weeks of cultivation. We found that the WSS distributions strongly differ between spheres and cubes even if the porosity and the specific wetted area of the stackings were very similar. The PDF is narrower and the mean WSS is lower for cubes (11 mPa) than for spheres (20 mPa). For the stacking of spheres, the relative occupancy of the surface sites by cells is maximal when WSS is greater than 20 mPa. For cubes, the relative occupancy is maximal when the WSS is lower than 10 mPa. The discrepancies between spheres and cubes are attributed to the more numerous sites in stacking of spheres that may induce 3‐D (multi‐layered) proliferation.

Viscous Microcapsules as Microbioreactors to Study Mesenchymal Stem/Stromal Cells Osteolineage Commitment

“…[ 48 ] In another study, it has been reported that shearing flow did not have a significant effect on MSCs proliferation, using a parallel plate flow chamber. [ 49 ] In contrary, other studies reported enhanced proliferation of MSCs and NIH‐3T3 fibroblasts [ 50,51 ] in dynamic cultures using scaffolds. Such contradictory findings may be due to the use of different shear stress magnitudes and modes.…”

Channeling Effect and Tissue Morphology in a Perfusion Bioreactor Imaged by X-Ray Microtomography

“…The samples were scanned by X-ray micro-tomography and the tridimensional arrangements of the beads and the tissue were obtained after image reconstruction. Preliminary experiments showed that tissue proliferation on glass and polyacetal bead stacks in static conditions (without flow) was negligible compared to dynamic conditions [39,40], producing too little tissue volume to be resolved by X-ray microtomography. Additionally, mass transport estimates in similar culture conditions and close flow rate indicate that oxygen and nutrient supply is in excess during most of the three weeks of culture [39].…”

“…The cylindrical growth chamber (13.8 mm diameter, 5 cm high, made of polycarbonate) is randomly filled by mono disperse polyacetal beads (2 mm diameter). Let us note that bead-based scaffolds have been identified as good candidates for tissue engineering and therefore have been investigated multiple times [4,32,39,40,[44][45][46][47]. The beads are immobilized by two polycarbonate grids (0.5 cm high), inserted in the growth chamber at the bioreactor inlet and outlet.…”

“…The following procedure has been followed in order to seed the cells on the beads before their introduction into the bioreactor [40,48]. First, a suspension of pre-confluent NIH-3T3 cells (10 6 cells/ml) in culture medium was gently poured onto the polyacetal beads previously coated with fibronectin (10 µg/ml, 1 hour at 37 • C), then left overnight in a CO 2 incubator to let the cells adhere to the beads.…”

“…2 Materials and Methods Following previous tissue engineering studies using murine fibroblasts [38][39][40], these cells have been chosen for our model system because they belong to the family of connective tissue cells (like chondrocytes, osteoblasts, ...) [41]. Additionally, they respond to mechanical stimuli [42], which is relevant to our perfusion bioreactor model and have a rapid proliferation rate.…”