Introduction: Meter-long alginate hydrogel microfibers or microstrands have shown great potential for in vitro 3D culture and in vivo cell delivery, providing an alternative hydrogel scaffold system for proper cell distribution, maintenance and expansion in tissue regeneration applications. These microfibers provide several advantages, including uniform cell distribution, free access to gases and nutrients by diffusion, and ease of handling. Current state-of-the-art microfiber fabrication technologies include coaxial or multi-axial microfluidics, spinning, extrusion, and bioprinting. However, most of these methods face challenges such as initial accumulation of a large drop of alginate or gel at the nozzle tip or printer head, clogging of the channels or nozzles, and mismatches of flows.
Materials and
Methods: To address these challenges, we invented a vacuum-driven approach to fabricating meter-long cell-laden alginate hydrogel microstrands. We developed non-invasive methods to characterize cell-laden alginate hydrogel microstrands using Raman spectroscopy and monitor 3D cell growth using alamarBlue assay. We further established the indentation testing method to determine the elasticity and viscoelasticity of alginate hydrogel microstrands in the absence and presence of NIH 3T3 fibroblasts at different cell seeding density (1 or 5 × 10E6 cells/mL) with or without CaCl2 supplement on days 1, 3, 7 and 14. Additionally, we encapsulated and cultured different cell types in alginate hydrogel microstrands, ranging from stem cells such as embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), to stromal cells (such as NIH 3T3 fibroblasts), to epithelial cells (such as salivary gland ductal epithelial SIMS cells). Cell growth in alginate hydrogel microstrands was monitored by optical microscopy and cell viability was evaluated by LIVE/DEAD assay.
Results, Conclusions, and Discussions: We were able to reproducibly fabricate alginate hydrogel microstrands with diameter as small as 157 µm, which can be 51.5-meter in length and 0.0254 m2 in surface area for 1 mL alginate hydrogel. The alginate hydrogel microstrand exhibited characteristic Raman peaks of alginate and the cell-laden microstrand showed distinct Raman peaks different from those of alginate. These alginate hydrogel microstrands supported high density viable cell growth. The elasticity of alginate hydrogel microstrands, which was measured by the indentation modulus, increased in the presence of CaCl2 with time while the viscoelasticity did not show dramatical changes. Additionally, using cell types other than NIH 3T3 fibroblasts, such as mouse ESCs, MSC-like primary embryonic day 16 (E16) mesenchyme cells, and salivary gland ductal epithelial SIMS cells, we demonstrated the capability of our vacuum-driven approach to high-density cell encapsulation and large-scale production of cell-laden hydrogel microstrands. 3D culture of these cells showed high cell viability for 7-14 days. The feasibility of using cell-laden alginate hydrogel microstrands was further demonstrated. Thus, vacuum-driven fabrication of cell-laden hydrogels is a promising alternative to macroscopic scaffolds for their use in cell culture and tissue regeneration.
Acknowledgements (Optional): This work is supported by NIH National Institute of Dental & Craniofacial Research (NIDCR) under the grant number 1R01DE02795301. The authors thank Dr. Melinda Larsen for providing primary E16 mesenchyme and SIMS cells, Dr. Alexander Khmaladze and Monireh Pourrahimi for Raman microscopy, and Dr. Kristen L. Mills and Subbir M Parvej for advising on indentation testing.
