PhD student Syracuse University Syracuse, New York, United States
Introduction: Recreating the 3D spatial organization of single-cell networks will help elucidate underlying mechanisms related to their emergent functional properties exhibited at the tissue level. Although technological advances have led to an array of methods to arrange cells in 2D and 3D to mimic tissue specific architectures, single-cell resolution with control over cell-to-cell connectivity remains challenging. Methods based on 3D photo-patterning of adhesive peptides within specialized semi-synthetic hydrogels have emerged, but they have failed to realize 3D single cell networks. At present, only methods based on multi-photon absorption (MPA) can pattern features at single-cell resolutions although limitations related to specialized photosensitive materials, water soluble low-toxicity photoinitiators, cell viability during laser scanning in the presence of cells, and low scalability, have limited its use in the field. Thus, the field continues to rely on self-assembly based methods, that involve mixing relevant cells in natural ECM like collagen or fibrin. This however results in randomly organized 3D cell networks without precise control over network density, connectivity, and architecture, making systematic mechanistic study on networks’ properties challenging. Bioprinting methods can provide spatial control over cell placements within 3D ECMs, however single cell resolution is not possible. Microfluidic devices integrated with acoustic, dielectric, and magnetic field stimulation have also been used to directly manipulate cells in a contactless manner. However, these methods cannot achieve single-cell resolution or user-defined multi-layer 3D patterns, and its control over intercellular connectivity remains poor.
Materials and
Methods: First, Digital Light Projection (DLP) was used to rapidly design and print master molds which were used to generate custom three-chambered PDMS devices. Second, ECM of interest (type I collagen) was perfused into central chambers and thermally crosslinked to generate a barrier between chambers 1 and 3. Third, Two Photon Ablation (TPA) was used to ablate 3D microchannel network within collagen. Model cells, seeded within the device, self-assemble within ablated network, and generate an interconnected, 3D, functional circuits coined as CELLNETs. We show that CELLNETs are compatible with standard imaging methods (brightfield, immunostaining, time-lapse microscopy), co-culturing cells and in situ manipulation such as application of fluid flow and/or biochemical stimuli, or injury to target cells to generate user-defined disrupted networks.
Results, Conclusions, and Discussions: Here, we report a novel technology, coined as CELLNET, to generate normal and disrupted 3D single-cell networks within type I collagen matrix in user-defined configurations and study the real-time signaling of single cells and signal propagation across the entire network. We show that this template-based strategy works with many cell types, is highly reproducible and provides user control over cell-cell connectivity and cell-network layout. Use of DLP allows rapid and inexpensive iteration of master molds enabling rapid production of custom internal microchannel designs within standard microfluidic devices. We show that real-time Ca signaling of individual cells and signal propagation within CELLNETs can be monitored when subjected to biophysical and biochemical stimuli. Moreover, femtosecond laser irradiation can ablate target cells within CELLNETs at defined locations and time-points to design custom disrupted networks and study real-time changes in their signaling dynamics. This allows entire CELLNETs or individual cells within the network to be manipulated in a noninvasively, contactless, and sterile manner during active culture. To test the capability of studying real-time signaling within CELLNETs, we choose osteocytes as our model cells, due to our groups’ prior experience with bone tissue engineering. With CELLNET, a user-defined templated in collagen is generated before cells are seeded/pipetted in target microfluidic chambers. This decoupling provides flexibility to generate 3D cell networks in any bioink including native and unmodified ECM like collagen which in turn results in close to 100% cell viability as laser scanning is not performed in the presence of cells. We envision CELLNET to be an ECM- and cell-agnostic technology that can be broadly used to design tissue-specific CELLNETs for a range of applications. We envision that CELLNET will transform the study of tissue-scale system biology by linking individual cells and tissue networks’ function thereby elucidating higher-level emergent property.
Acknowledgements (Optional): National Institutes of Health, R21GM141573, R21AR076642 (PS).
