Speaker
Description
Introduction: Customized bioreactors can replicate diverse physiological conditions, such as shear stress, pulsatile pressure, and strain, while enhancing diffusion and nutrient exchange. These conditions stimulate cellular processes including proliferation, differentiation, gene expression, and substrate remodeling. However, in two-dimensional cultures, these stimuli promote adipose-derived stromal cells (AdSC) and endothelial co-culture into smooth muscle-like cells, improving cell-substrate and cell-cell interactions, and promoting ECM production. Despite these advancements, the result is still only a few layers of cells rather than fully developed tissue. Introducing a third dimension with collagen bioprinted scaffolding enables time- and stimuli-dependent remodeling, leading to structures that more closely resemble real tissue [1].
Methods: In our studies, we used porcine-based collagen bioink at concentrations of 20–50 mg/ml, incorporating porcine AdSCs at densities of 10–20 million cells/ml. The collagen was mixed using a custom-built mixing system. Substrates were printed with a custom bioprinter utilizing syringe-based extrusion. Gelling was achieved through pH neutralization and temperature change [2]. Samples were printed onto glass or PLCL nanofibers with defined rectangular dimensions (up to 25 x 75 mm) and varying thicknesses ranging from 0.5 to 2 mm. Printed samples were mounted either as fixed or freely floating in custom cultivation bioreactor chambers connected to a customized programmable pulsatile pressure flow generator. We focused on varying multiple conditions, including generic perfusion for nutrient replacement, increased partial pressures to promote diffusion, pulsatile pressures to enhance diffusion and provide mechanical stimulation, and defined shear stress. These conditions were applied continuously or with intermittent resting pauses. Culture media were programmatically altered to promote cellular proliferation, differentiation, and ECM remodeling.
Discussion: Perfusion with altered partial pressures of CO2 and O2 improved cell viability. Depending on cell densities and cultivation time, the initial collagen was remodeled into compressed, more stable, and stiffer structure, resulting in an increased compression modulus [2]. Differentiation media with pulsatile stress promoted smooth muscle cell (SMC) differentiation and new extracellular matrix (ECM) formation. Controlled shear stress oriented the cells, and with the addition of arterial endothelial cells (ECs), vessel-like formations were created. The initial 3D bioprinting provided a shape that was remodeled over time. By controlling these parameters during cultivation, it is possible to create functionalized tissue constructs for various applications, such as vessel patching and grafting, with defined microstructure and biomechanical properties, unlike static culture [3].
Fundings:
This research was funded by the Ministry of Health of the Czech Republic grant No. NW24-08-00064 and NW24J-02-00061 and by the Grant Agency of the Czech Technical University in Prague (grant No. SGS25/183/OHK4/3T/17).
References:
[1] Kanokova, D.; Matejka, R.; Zaloudkova, M.; Zigmond, J.; Supova, M.; Matejkova, J. Active Media Perfusion in Bioprinted Highly Concentrated Collagen Bioink Enhances the Viability of Cell Culture and Substrate Remodeling. Gels 2024, 10, 316.
[2] Matejkova, J.; Kanokova, D.; Supova, M.; Matejka, R. A New Method for the Production of High-Concentration Collagen Bioinks with Semiautonomic Preparation. Gels 2024, 10, 66.
[3] Matějková, J.; Kaňoková, D.; Matějka, R. Current Status of Bioprinting Using Polymer Hydrogels for the Production of Vascular Grafts. Gels 2025, 11, 4
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