Abstract
Background:Dynamic cultures, characterized by continuous fluid reperfusion, elicit physiological responses from cultured cells. Mesenchymal stem cell-derived EVs (MSC-EVs) has been proposed as a novel approach in treating several renal diseases, including acute glomerular damage, by using traditional two-dimensional cell cultures and in vivo models. We here aimed to use a fluidic three-dimensional (3D) glomerular model to study the EV dynamics within the glomerular structure under perfusion.Methods:To this end, we set up a 3D glomerular model culturing human glomerular endothelial cells and podocytes inside a bioreactor on the opposite sides of a porous membrane coated with type IV collagen. The bioreactor was connected to a circuit that allowed fluid passage at the rate of 80 µl/min. To mimic glomerular damage, the system was subjected to doxorubicin administration in the presence of therapeutic MSC-EVs.Results:The integrity of the glomerular basal membrane in the 3D glomerulus was assessed by a permeability assay, demonstrating that the co-culture could limit the passage of albumin through the filtration barrier. In dynamic conditions, serum EVs engineered with cel-miR-39 passed through the glomerular barrier and transferred the exogenous microRNA to podocyte cell lines. Doxorubicin treatment increased podocyte apoptosis, whereas MSC-EV within the endothelial circuit protected podocytes from damage, decreasing cell death and albumin permeability.Conclusion:Using an innovative millifluidic model, able to mimic the human glomerular barrier, we were able to trace the EV passage and therapeutic effect in dynamic conditions.
Highlights
Federica Collino and Benedetta Bussolati have contributed to this work.Understanding the uptake and effect of therapeutic agents in dynamic conditions and possibly in three-dimensional models is relevant for their clinical application
We here aimed to use a fluidic three-dimensional (3D) glomerular model to study the EV dynamics within the glomerular structure under perfusion. To this end, we set up a 3D glomerular model culturing human glomerular endothelial cells and podocytes inside a bioreactor on the opposite sides of a porous membrane coated with type IV collagen
A standardized protocol for preparing glomerular co-cultures was obtained, starting from the coating of the porous membrane of a bioreactor with type IV collagen to improve endothelial cell adhesion and ensures the maintenance of the podocyte phenotype [25, 26]. h-GECs and h-PODs were seeded on opposite sides of the membrane, with a density of 100,000 cells/1.9 cm2, to allow the formation of uniform cell bilayers
Summary
Federica Collino and Benedetta Bussolati have contributed to this work.Understanding the uptake and effect of therapeutic agents in dynamic conditions and possibly in three-dimensional models is relevant for their clinical application. Three dimensional (3D) millifluidic systems, composed of living cells seeded in a bioreactor and a continuous fluid perfusion system [2], mimic the organ 3D architecture and offer an alternative solution to the animal experimentation studies [3]. We here aimed to use a fluidic three-dimensional (3D) glomerular model to study the EV dynamics within the glomerular structure under perfusion. The system was subjected to doxorubicin administration in the presence of therapeutic MSC-EVs. RESULTS: The integrity of the glomerular basal membrane in the 3D glomerulus was assessed by a permeability assay, demonstrating that the co-culture could limit the passage of albumin through the filtration barrier. CONCLUSION: Using an innovative millifluidic model, able to mimic the human glomerular barrier, we were able to trace the EV passage and therapeutic effect in dynamic conditions
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