Instant assembly of unmodified collagen for multiscale functional tissue engineering

Introduction: : Engineering functional cellular tissues holds significant potential in regenerative medicine. Collagen type I, the primary scaffolding material in biological tissues, is widely used in tissue engineering due to its excellent biocompatibility and bioactivity. However, its unmodified form suffers from inherently slow gelation kinetics—typically requiring 30–40 minutes under physiological conditions—limiting its use in precise or rapid tissue construction, particularly during cell-laden fabrication processes like bioprinting. This slow transition from liquid to solid restricts the ability to create well-defined architectures and compromises cellular retention and organization during scaffold formation. Overcoming this limitation without chemical modification—which often diminishes bioactivity—remains a longstanding challenge. Here, we present a novel strategy, Tunable Rapid Assembly of Collagenous Elements (TRACE), which uses macromolecular crowding (MMC) via bioinert polyethylene glycol (PEG) to drive instant collagen assembly. TRACE enables near-instant gelation of unmodified collagen under cell-compatible conditions. This approach retains collagen’s natural functionality while unlocking new capabilities for multiscale biofabrication, organoid engineering, and 3D bioprinting.

Materials and

Methods: : Neutralized, cell-compatible collagen type I solution (2 mg/mL) was delivered in a physiological buffer containing a PEG-based macromolecular crowding agent. Gelation speed was characterized via turbidity and rheological measurements. Using MMC-induced rapid gelation, collagen structures were fabricated at multiple length scales: (1) Microscale fibers were created by pipetting collagen into PEG-containing buffers. This simple yet effective strategy was used to fabricate scaffolds for microvascular networks. (2) Using TRACE, macroscopic structures—including high–aspect ratio, strip-like constructs—were rapidly fabricated with human induced pluripotent stem cells (iPSCs), which were then differentiated into intestinal lineages in situ. (3) A novel TRACE support bath was developed for embedded bioprinting of biofunctional engineered tissues using cell-laden, unmodified collagen bioinks. Cell viability and organization were assessed via immunofluorescence imaging. For TRACE-printed cardiac chambers, cardiac function was evaluated using video microscopy to quantify rhythmic contraction and fluid displacement. All experiments were replicated across multiple batches of collagen, including both commercially available and decellularized sources.

Results, Conclusions, and Discussions:: MMC triggered the polymerization of collagen solutions within seconds when delivered into a PEG solution. This principle was used to design a versatile, multiscale method for collagen-based biofabrication. At the microscale, hierarchically organized, cell-sized microbundles formed immediately upon pipetting. These bundles supported robust endothelial cell connectivity, viability, and vascular network formation with long-term angiogenic potential. At larger scales, the rapid collagen gelation prevented cell sedimentation and enabled the formation of iPSC-laden or intestinal stem cell-laden organoids with uniform cell density, which could be differentiated or matured into structured mini-intestinal tissues, forming well-organized epithelial layers and exhibiting regional differentiation markers consistent with functional gut development. Furthermore, TRACE enabled millimeter- to centimeter-scale printing of complex, high-integrity, and functional cellular tissues using cell-compatible collagen bioinks. Bioprinted cardiac mini-chambers remained viable for 77 days with preserved tissue-level geometry and cellular architecture, demonstrating spontaneous beating and the ability to displace fluid—a hallmark of functional myocardium. In conclusion, TRACE provides a robust, scalable, and chemically inert platform for collagen biofabrication across multiple length scales. It eliminates longstanding bottlenecks in collagen-based cellular tissue engineering by enabling rapid, native-preserving assembly. Importantly, the method is compatible with living cells, fabrication techniques, and collagen sources, supporting its broad utility. This approach opens new directions for constructing physiologically relevant tissues for developmental biology, disease modeling, and regenerative therapy.

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.