Merife, A.-B. | Search bioRxiv

bioRxiv2025

3D osteocyte networks under Pulsatile Unidirectional Fluid Flow Stimuli (PUFFS)

Although osteocytes are known to play a key role in skeletal mechano-adaptation, few in vitro models have investigated how pulsatile mechanical stimuli influence the properties of 3D osteocyte networks. Here we design and develop a microfluidic based in vitro model to study 3D osteocyte networks cultured under Pulsatile Unidirectional Fluid Flow Stimuli (PUFFS). Digital light projection stereolithography was used to design and fabricate a three-chambered PDMS microfluidic chip. Model osteocytes (murine MLO-Y4) were encapsulated in collagen matrix within the chip to form self-assembled three-dimensional (3D) cell networks. Daily stimulus in the form of PUFFS was then applied for upto 21 days. A combination of experiments, computational simulation and analytical modeling was used to characterize the mechanical environment experienced by embedded cells during PUFFS. Viability, morphology, cell-connectivity, expression of key proteins, and gene expression, and real-time calcium signaling within 3D osteocyte networks were characterized at select time-points and compared to static conditions. Results show that PUFFS stimulation at 0.33 and 1.66 Hz can initiate mechanotransduction via calcium signals that are propagated across the network of collagen encapsulated osteocytes via Cx43 junctions. Furthermore, osteocytes cultured in these devices maintain expression of several key osteocyte genes for up to 21d. Taken together, this model can potentially serve as a testbed to study how 3D osteocyte networks respond to dynamic mechanical stimulation relevant to skeletal tissues.

bioengineering↗

bioRxiv2024

CELLNET technology: Spatially organized, functional 3D networks at single cell resolution

Cells possess the remarkable ability to generate tissue-specific 3D interconnected networks and respond to a wide range of stimuli. Understanding the link between the spatial arrangement of individual cells and their networks emergent properties is necessary for the discovery of both fundamental biology as well as applied therapeutics. However, current methods spanning from lithography to 3D photo-patterning to acoustofluidic devices are unable to generate interconnected and organized single cell 3D networks within native extracellular matrix (ECM). To address this challenge, we report a novel technology coined as CELLNET. This involves the generation of crosslinked collagen within multi-chambered microfluidic devices followed by femtosecond laser ablation of 3D microchannel networks and cell seeding. Using model cells, we show that cell migrate within ablated networks within hours, self-organize and form viable, interconnected, 3D networks in custom architectures such as square grid, concentric circle, parallel lines, and spiral patterns. Heterotypic CELLNETs can also be generated by seeding multiple cell types in side-chambers of the devices. The functionality of cell networks can be studied by monitoring the real-time calcium signaling response of individual cells and signal propagation within CELLNETs when subjected to flow stimulus alone or a sequential combination of flow and biochemical stimuli. Furthermore, user-defined disrupted CELLNETs can be generated by lethally injuring target cells within the 3D network and analyzing the changes in their signaling dynamics. As compared to the current self-assembly based methods that exhibit high variability and poor reproducibility, CELLNETs can generate organized 3D single-cell networks and their real-time signaling responses to a range of stimuli can be accurately captured using simple cell seeding and easy-to-handle microfluidic devices. CELLNET, a new technology agnostic of cell types, ECM formulations, 3D cell-connectivity designs, or location and timing of network disruptions, could pave the way to address a range of fundamental and applied bioscience applications. TeaserNew technology to generate 3D single cell interconnected and disrupted networks within natural extracellular matrix in custom configurations.

bioengineering↗