Fabricating engineered tissues with spatially varied microenvironments via embedded 3D printing in a cell-dense suspension

Abstract The long-term goal of bioengineered tissues is to achieve precise cell type distribution, physiological cell density, perfusable vascular channels, and mature functionality. However, fabricating engineered tissue with the microenvironmental features of organs with physiological cell density remains a significant challenge in this field. To address this, several key obstacles must be overcome. First, vascularization is indispensable for engineered tissues; however, disturbances may occur when introducing vascular channels within pre-fabricated tissues. Second, maintaining fabrication precision becomes increasingly difficult during high-cell-density embedded printing. Third, the suspension bath used for embedded printing often fails to provide a suitable growth environment. Herein, we modified the rheological properties of the bioactive hydrogel by incorporating a thixotropic laponite nanoclay (LPN) and demonstrated that an optimized ratio of collagen methacrylate (ColMA) to LPN forms a self-healing suspension bath, which is enhanced by hydrogen bonding interactions and is capable of in situ crosslinking. This printing strategy was generalized as the embedded 3D printing in cell-dense suspension (EPICS). The self-healing properties of the EPICS remain unaffected even when encapsulating a near-physiological cell density of 10 ⁸ cells·mL ⁻¹ , and it provides precise control of the printing resolution from 1 mm to 100 µm. Compared with the model containing 10 ⁶ cells·mL ⁻¹ , the use of EPICS could create a robust hepatic model with mature liver markers and reduced apoptosis gene expression. Moreover, EPICS can efficiently fabricate spatially controlled perfusable channels, thereby mimicking the spatially varied microenvironments of hepatocellular carcinoma, highlighting its broad applications in therapeutics involving tissue and organ constructs.