Layer by Layer Three-dimensional Tissue Epitaxy by Cell-Laden Hydrogel Droplets

TLDR

3D tissue engineering holds promise for treating diseases, yet existing methods struggle to replicate native microvasculature and suffer from cell viability loss and clogging. The authors aim to develop a bioprinter that uses mechanical valves to print high‑viscosity hydrogel precursors containing cells, enabling 3‑mm‑scale smooth‑muscle‑cell patches. Their system employs valve‑controlled droplet generation to deposit collagen‑encapsulated smooth‑muscle cells in precise multilayered patterns. The platform produces 16‑µm‑thick layers with 18‑µm proximal and 0.5‑µm distal resolution, 160 droplets per second, uniform cell densities up to 216 cells/mm², and >90 % viability after 14 days, demonstrating potential for regenerative‑medicine tissue constructs.

Abstract

The ability to bioengineer three-dimensional (3D) tissues is a potentially powerful approach to treat diverse diseases such as cancer, loss of tissue function, or organ failure. Traditional tissue engineering methods, however, face challenges in fabricating 3D tissue constructs that resemble the native tissue microvasculature and microarchitectures. We have developed a bioprinter that can be used to print 3D patches of smooth muscle cells (5 mm × 5 mm × 81 μm) encapsulated within collagen. Current inkjet printing systems suffer from loss of cell viability and clogging. To overcome these limitations, we developed a system that uses mechanical valves to print high viscosity hydrogel precursors containing cells. The bioprinting platform that we developed enables (i) printing of multilayered 3D cell-laden hydrogel structures (16.2 μm thick per layer) with controlled spatial resolution (proximal axis: 18.0 ± 7.0 μm and distal axis: 0.5 ± 4.9 μm), (ii) high-throughput droplet generation (1 s per layer, 160 droplets/s), (iii) cell seeding uniformity (26 ± 2 cells/mm2 at 1 million cells/mL, 122 ± 20 cells/mm2 at 5 million cells/mL, and 216 ± 38 cells/mm2 at 10 million cells/mL), and (iv) long-term viability in culture (>90%, 14 days). This platform to print 3D tissue constructs may be beneficial for regenerative medicine applications by enabling the fabrication of printed replacement tissues.