Cryogenic 3D printing of ice structures with embedded cells using 70 pL droplets

Three-dimensional (3D) cryogenic bioprinting has emerged as a promising technique for fabricating cell-laden structures by freezing bioinks during printing. This method enables precise spatial arrangement of cells while preventing cell degradation during the structural fabrication. However, conventional cryogenic bioprinting often requires cryoprotective agents (CPAs), which may induce cytotoxicity. To address this issue, CPA-free cryogenic 3D printing was examined and evaluated for constructing cell-embedded ice structures. This approach employs inkjet printing to eject ultra-small 70 pL droplets onto a liquid nitrogen-cooled substrate, achieving ultrafast freezing that suppresses ice crystal formation. The effects of droplet ejection frequency on ice structure formation and cell viability were systematically investigated. Ice pillars were printed at frequencies of 5, 20, 50, 100, and 200 Hz, and their morphologies were analyzed. At 5 Hz, droplets remained distinctly separated, forming a well-defined layered structure, whereas at 20 Hz, they partially merged while maintaining a high aspect ratio. In contrast, at 50 Hz and above, incomplete freezing between droplets resulted in lower aspect ratios and structural instability. Cell viability after freezing and thawing showed no clear difference between 5 Hz and 20 Hz frequency ejections, but was lower than that achieved with conventional monolayer freezing. This decrease in viability is considered to result from a combination of factors, including insufficient cooling in the upper regions of the ice pillars, slower warming during thawing, and localized recrystallization. Additionally, overhanging ice structures were successfully fabricated by adjusting droplet spacing, demonstrating the feasibility of support-free complex ice structure printing. These findings highlight the potential of cryogenic 3D printing for producing intricate ice-based architectures. Further optimization of cooling conditions and droplet control is essential to improve the survival rate of embedded cells and enhance the applicability of cryogenic 3D bioprinting in biofabrication and tissue engineering.