Rapid Collagen Assembly Enabled Direct Cell Printing of Functional Cardiac Tissues

Introduction: : Three-dimensional bioprinting enables precise spatial patterning of cells and biomaterials, offering transformative potential for patient-specific disease models and implantable tissues. Among natural bioinks, type I collagen is widely favored for its extracellular matrix (ECM) relevance, biocompatibility, and bioactivity. However, its low viscosity and slow gelation under physiological conditions hinder high-fidelity, cell-laden printing. Traditional methods rely on acidic pH, high collagen concentrations, or chemical crosslinking, which can compromise cell viability.
Macromolecular crowding (MMC) is a biocompatible strategy to accelerate polymer self-assembly by increasing molecular collisions and stabilizing intermediate structures. Our prior work demonstrated that MMC enables tunable 3D collagen architectures, including micro-bundles that support vasculature formation and molded strips for intestinal differentiation. However, these methods lack the geometric control and scalability required for complex tissue engineering.
To address these challenges, we propose TRACE (Tunable Rapid Assembly of Collagenous Elements) printing, a bioprinting method that combines MMC-induced rapid collagen self-assembly with embedded extrusion bioprinting. We hypothesize that TRACE enables immediate stabilization of low-viscosity, unmodified collagen at physiological pH without crosslinking and supports high-density, cell-laden constructs that retain complex geometry and bioactivity. As a functional demonstration, we applied TRACE to print ventricular cardiac tissues containing hiPSC-derived cardiomyocytes and fibroblasts. We further hypothesize that TRACE allows the formation of functional cardiac tissues exhibiting synchronized contractions, calcium propagation, fluid pumping, and drug responsiveness.
By addressing core limitations of collagen biofabrication, TRACE enables a scalable, biocompatible strategy for high-resolution, high-cell-density 3D bioprinting of functional human tissues, with broad applicability across diverse organ systems.

Materials and

Methods: : TRACE bioprinting integrates macromolecular crowder with an embedded support material into a shear-yielding granular support bath. The bath was prepared by mixing 0.7% agarose slurry with 200 mg/mL PEG 8000 using a dual-syringe connector. All components were sterilized via filtration or autoclaving and handled under biosafety conditions.
Bioinks were prepared using 2–3 mg/mL neutralized type I collagen (Corning), supplemented with 15% Geltrex for cardiac constructs. Cell-laden formulations included hiPSC-derived cardiomyocytes (4×10⁷ cells/mL) and human cardiac fibroblasts (4×10⁶ cells/mL). Acellular inks were supplemented with food dye or fluorescent beads for visualization. Rheological properties of both the bath and bioinks were assessed at 37 °C using cone-plate rheometer.
Bioprinting was performed using either CELLINK BioX6 or a Lulzbot syringe-based printer with customized temperature-controlled printheads (8 °C). Designs were created in SolidWorks and sliced via DNA Studio or Cura. Constructs were printed into TRACE baths using 25-gauge nozzles at speeds of 5–10 mm/s, stabilized for 3–5 minutes, then released and transferred into culture media.
Printed geometries were imaged and analyzed using ImageJ for dimensional fidelity. Cell viability was assessed via Live/Dead staining on Days 7 and 14. Functional assays for cardiac tissues were conducted between Days 7–77, including contractility video analysis, calcium imaging, and particle imaging velocimetry (PIV) at the ventricle outlet. Drug response was tested with isoproterenol and carbamylcholine. Tissue compaction and post-culture cell density were quantified. Immunofluorescence for α-actinin and N-cadherin was used to assess sarcomere organization and cell-cell junctions.

Results, Conclusions, and Discussions:: TRACE bioprinting enabled rapid gelation of low-concentration (2mg/mL) neutralized collagen, minimizing ink diffusion and preserving geometry. Compared to standard agarose granular baths, TRACE maintained structural fidelity while controls exhibited broadening and particle entrapment even after 30 minutes of gelation. When printed collagen was released after only 3 minutes post-printing, TRACE constructs retained their geometry, while controls collapsed due to incomplete gelation.
Immediate gelation allowed tunable resolution by adjusting flow rate and speed, achieving ~24 μm filaments in TRACE and ~7 μm in PEG-only baths. This enabled high-fidelity printing of complex millimeter-scale structures, including cubes, pyramids, a four-chambered heart, coronary tree, and stomach, all using 2 mg/mL neutralized collagen.
TRACE supported direct printing of cardiac constructs with hiPSC-derived cardiomyocytes and fibroblasts. Printed tissues compacted to physiological densities (~1.8×10⁸ cells/cm³), stabilized in shape within days, and formed aligned sarcomeres and N-cadherin-rich junctions. Unlike constructs printed in regular baths, TRACE tissues exhibited synchronized contractions, calcium transients, fluid pumping, and pharmacological responsiveness for over 11 weeks. PIV confirmed peak flow velocities of ~7.8 mm/s. Beyond cardiac tissues, TRACE enabled lumenized endothelial networks and hPSC-laden tissues retaining pluripotency, highlighting broader applicability. Together, these findings establish TRACE as a scalable, biocompatible platform for high-resolution, high-cell-density bioprinting of unmodified collagen.
Bioprinting presents unique challenges in balancing structural complexity with cellular viability. While recent advances have improved structural complexity and resolution, replicating functional human tissues remains limited. Collagen, the predominant ECM component, offers unmatched biological relevance but has long been hindered by poor printability under physiological conditions. TRACE addresses this by integrating MMC into a granular support bath to drive collagen self-assembly during embedded extrusion, eliminating the need for high viscosity, acidic pH, or chemical crosslinkers. Its simplicity, versatility, and compatibility with diverse cell types position TRACE as a robust strategy for building complex, functional human tissues.
As the field advances toward constructing cell-rich, functionally mature tissues, TRACE provides an accessible “print-and-function” workflow. Although perfusion and multi-material capabilities remain areas for future development, the collagen-rich, cell-laden printing strategy presented here is broadly applicable across organ systems and represents a significant advance toward clinically relevant tissue fabrication.

Acknowledgements and/or References (Optional):: X. Gong, Z. Wen, Z. Liang, H. Xiao, S. Lee, A. Rossello, Q. Xing, T. Wright, R. Y. Nguyen, A. Rossello, M. Mak, Instant Assembly of Collagen for Tissue Engineering and Bioprinting. Nature Materials, in press.