EXPECT: A thermosensitive embedded bioprinting platform for guided spatial cell organization

Complex tissue engineering requires precise spatial cell organization, but static or isotropic hydrogels hinder long-term pattern maintenance due to random cell migration. We developed EXtrusion Patterned Embedded ConstruCT (EXPECT), a thermosensitive hydrogel embedding medium for 3D bioprinting, integrating Carbopol® 940 and gelatin for rheological properties and print fidelity, with poly (N-isopropylacrylamide)-graft-chondroitin sulfate (pNIPAAm-CS) for biocompatibility and temperature-responsive behavior (∼32 ​°C lower critical solution temperature ( LCST )). Rheological and small-angle X-ray scattering (SAXS) analyses confirmed EXPECT's self-healing printability and reversible LCST-driven transitions from hydrophobic (above ∼32 ​°C) to hydrophilic (below ∼32 ​°C) states. Temperature actuation (15 ​min at 25 ​°C every ∼5 days, otherwise 37 ​°C) in 10 ​mm toroid channels embedded within EXPECT guided cellular organization of cells seeded in these channels. In chondrogenic medium, actuated single mesenchymal stromal cells (MSCs) showed ∼50 ​% narrower patterns by day 7, sustained to day 36 ( p ​< ​0.001 vs. static, which widened to 137 ​± ​20 ​%). Actuated MSC spheroids elongated, forming bipedal shapes and fusing into extended patterns (length 480 ​± ​158 ​μm, p ​< ​0.0001) over 36 days. In 14-day human umbilical vein endothelial cells (HUVEC)-MSC co-cultures (10:1), actuation reduced pattern width by 27.5 ​% ( p ​= ​0.0236), promoted early protrusions, and decreased cell circularity (vs. 2 ​% increase in static, p ​= ​0.0173), indicating enhanced elongation and potential vascularization. EXPECT's dynamic, actuation-mediated control of anisotropic cell organization overcomes limitations of static hydrogels, offering significant potential for engineering complex, organized tissues in regenerative medicine. • Periodic temperature shifts reduce random migration and guide cell organization. • EXPECT maintains MSC viability and differentiation potential over a 60-day period. • EXPECT sustains MSC alignment and condensation for up to 36 days in 3D culture. • MSC spheroids elongate and fuse into columnar structures within EXPECT. • EXPECT supports early vascular stabilization in HUVEC-MSC co-cultures.