Ovarian Cancer (OC) research is limited by the lack of an appropriate in vitro model of the tumor microenvironment. We utilized a perfusion bioreactor, suitable for the culture of up to 40 cell-seeded scaffolds (1), at velocities mimicking the cellular conditions in the extracellular fluid flow (2), to construct a new OC model. Our hypothesis was that operating the reactor at low volumetric velocities, with a vertical flow model will allow better homogenous cultivation, compared to a horizontal fluid, highly affected by gravitation forces.
Velocities and shear stresses throughout the reactor body were simulated by ANSYS Fluent, at different velocities, of vertical or horizontal flow. 433 and ES2 OC cells were seeded into alginate macroporous scaffolds and cultured in the perfusion bioreactor for 3 days under the simulated conditions. Cell viability was examined by Presto Blue assay. RT-PCR analysis was conducted for Sphingosine-1-Phosphate receptors (S1PRs), associated with OC. The mRNA expression levels were compared to other culture methods (monolayer, static seeded scaffolds, spheroids) and to OC samples of primary tumor and effusions.
ANSYS Fluent simulation indicated higher homogeneity at 50mL/h vertical flow, compared to horizontal flow conditions. ES-2 seeded scaffolds culture at 50 mL/h vertical flow, resulted in more homogenous cellular viability, compared to horizontal flow; supporting the simulation results. mRNA expression levels of S1PR1 and S1PR2 were shown to be significantly lower when 500 mL/h volumetric velocities were applied, compared to 50 mL/h and static conditions (p<0.05); emphasizing the necessity of specific velocity for the OC in vitro model. 433-seeded scaffolds, cultured at 50 mL/h resulted in S1P receptor mRNA expression levels, similar to those of the primary OC samples, compared to monolayer, static scaffold, and spheroid cultures.
DISCUSSION & CONCLUSIONS:
Vertical perfusion flow at relatively low velocity was found to be efficient for constructing novel in vitro models of OC Primary and effusion.
ACKNOWLEDGEMENTS: Azrieli College of Engineering, Jerusalem (AB).
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