IMPACT: This project uses rapid prototyping, 3D printing, and cell seeding to solve the most common post-operative complications that arise from the current methods of urethral elongation during phalloplasty. OBJECTIVES/GOALS: Post-op fistula and stricture formation occur in up to 50% of phalloplasty patients. These complications arise from the mismatch between skin and uroepithelium, or from scarring secondary to ischemia. Here we describe the fabrication of a novel vascularized urothelial flap for phalloplasty that contains discrete urothelial and vascular channels. METHODS/STUDY POPULATION: A custom designed 3D negative mold, with a urethral channel and a vascular inlet and outlet channel was prototyped in Adobe Fusion 360 and printed on a Prusa i3 MK3S printer in PLA. A 2mm diameter pluronic sacrificial macrofiber was used to connect the channels to form a vascular loop, and 1% type-I collagen was extruded over the mold. After solidifying, the scaffold was demolded and seeded with grade I urothelial carcinoma (SW780 cells, at 5-10 x 106 cells/mL) in the urethral channel, and adenovirus-infected E4 endothelial cells (at 3x106 cells/mL) in the

OBJECTIVES/GOALS: To determine if decellularized costal cartilage (DCC), which could theoretically be obtained “off the shelf,” would provide similar results to autologous cartilage grafts previously studied in this lab, thereby widening the application of our novel nipple engineering approach to all patients undergoing nipple reconstruction. METHODS/STUDY POPULATION: PLA scaffolds (diameter: 1.0 cm, height: 1.0 cm) were printed using a PRUSA 3D printer and sterilized. Lamb costal cartilage was minced (1 mm3) or zested (<0.2 mm3) and then decellularized. The quality of decellularization was assessed using DNA quantification and histological analysis. DCC was then packed into PLA scaffolds and implanted subcutaneously into immunocompetent Sprague Dawley rats using a CV flap technique. The constructs were explanted and evaluated up to 6 months after implantation. RESULTS/ANTICIPATED RESULTS: All nipple reconstructions showed well-preserved diameter and projection due to persistence of the external scaffolds at 1, 3, and 6 months. Mass and volume of engineered tissue was well-preserved over all timepoints. Compared to implantation values, engineered zested nipples demonstrated a 12% mass increase and a 22% volume increase at 6 months. Minced nipples illustrated a similar mass and volume gain with a 21% increase in mass and a 13% increase in volume at 6 months secondary to infiltration of fibrovascular tissue and growth through scaffold wall pores, respectively. Histologic analysis demonstrated a mild inflammatory infiltrate 1 month after implantation which was replaced by fibrovascular tissue by 3 months that remained stable through 6 months. The processed DCC structure remained unchanged over time. DISCUSSION/SIGNIFICANCE: Using acellular ovine xenograft within bioabsorbable scaffolds, we have engineered neonipples that maintain their volume for at least 6 months. DCC architecture is well-preserved with minimal evidence of immune-mediated degradation. By using DCC, this novel approach to nipple engineering may be applied to any patient requiring reconstruction.

Purpose: While tissue engineering offers the promise of revolutionary innovation, scalable three-dimensional tissue culture is limited by the diffusion of nutrients and oxygen making media perfusion obligatory. Unfortunately, the cost of bioreactors for large construct tissue culture can be prohibitive, with a typical perfusion chamber costing several thousand dollars, and even small petri-dish-sized devices costing hundreds of dollars each. We have developed a low-cost perfusion setup that seals collagen-based perfusable cellular constructs within a sterile PDMS well between coverslips, allowing for repeated live-imaging of perfused 3D engineered tissues. Herein we describe fabrication of this novel system and validate its utility. Methods: Molds and frames were designed on 3D-modeling software (Fusion 360) and printed on a Prusa i3 MK3S 3D printer in poly(lactic acid) (PLA). Molds were filled with poly(dimethyl siloxane) (PDMS), which was cured to form chambers, bubble traps, mason jar lid chambers, and media reservoir lid adapters. In total, the tissue culture chamber device, mason jar lid inset, media reservoir lid, and bubble trap require 4, 1, 2, and 4 unique printed components, respectively. Results: Each perfusion chamber can be assembled for under 8 USD per device and reused repeatedly. The current model has a tissue chamber custom-built with 18x10x4 (LxWxH)mm 3 dimensions, but this chamber can be readily customized to experiment-specific dimensions. These devices allow cellular hydrogel constructs to be maintained in a sterile environment after assembly, perfused at varying rates to expose cells to different levels of shear stress, and the cells can be intermittently imaged with light, fluorescent or confocal microscopy - an unparalleled benefit for monitoring of experiments and collection of timepoint imaging data. The perfusion circuit consists of autoclavable glass and PDMS components, including a bubble trap, a crucial component of the circuit for preventing air bubbles that can damage cells and block microchannels, and a lid adapter, which allows 50 mL conical tubes to serve as self-oxygenating media reservoirs. Media changes can be performed via peristaltic pump perfusion or with syringe-based cell culture techniques for static culture. Constructs have been perfused within standard incubators for up to 14 days demonstrating normal cell viability without contamination or evidence of infection, with longer perfusion culture intervals (>1 month) currently being tested. Conclusion: The increasing accessibility of 3D-modeling and 3D-printing has enabled rapid prototyping of devices to address the problems that we face as surgeon-scientists. We have developed a low-cost tissue-engineering perfusion circuit that facilitates 3D-tissue-culture while allowing for repeated live-imaging as the cultured tissue develops. The instructions for our setup can be utilized to replicate o...