2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

Abstract

Tissue Engineering Part AAhead of Print AbstractsFree Access2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023Published Online:10 Apr 2023https://doi.org/10.1089/ten.tea.2023.29041.abstractsAboutSectionsPDF/EPUB Permissions & CitationsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Session Number: 3 Emerging Technology Highlights ‐ Melt Electrowriting and Extracellular VesiclesWednesday, April 12, 2023, 10:00 AM ‐ 11:30 AM1 ‐ Organized Melt‐electrowritten Tubular Scaffolds To Build Vascularised Kidney Proximal TubulesA. van Genderen1, M. de Ruijter2, E. Al‐Jehani1, V. Kersten1, M. G. Valverde1, J. Malda2, T. Vermonden1, R. Masereeuw1, J. Jansen3, M. Castilho4, S. Mihăilă1;1Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, NETHERLANDS, 2Department of Orthopaedics, Utrecht Medical Center Utrecht, Utrecht University, Utrecht, NETHERLANDS, 3Institute of Experimental Medicine and Systems Biology, RWTH Aachen University, Aachen, GERMANY, 4Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, NETHERLANDS.*Purpose/Objectives: Kidney tubular engineering requires tubular structures of small sizes (≤ 1 mm) to mimic curvature of native kidney proximal tubule, which is key for cell functionality and maturation. We created highly porous rhombus tubules to engineer both a vascular and an epithelial tubular scaffold for the formation of a vascularized proximal tubule.*Methodology: A custom‐built MEW device was used to fabricate small polycaprolactone tubular scaffolds (inner ∅ = 1 mm) with defined rhombus microarchitectures (winding angles of 30°, 50° and 70°). Human conditionally immortalized proximal tubule epithelial cells (ciPTEC) and glomerular endothelial cells (ciGEnC) were seeded alone or in co‐culture, and evaluated for monolayer formation and tightness, cell directionality, polarization, and extracellular matrix (ECM) deposition.*Results: Engineered tubular scaffolds with variable rhombus microarchitectures were successfully manufactured by fine controlling key instrument parameters. Both cell lines formed tight, homogenous monolayers within the tubular scaffolds. CiPTEC and ciGEnC formed a healthy ECM with low collagen I and high collagen IV content. The 30° winding angles caused preferential cell alignment along the scaffold fiber direction for ciPTEC and ciGEnC. By decreasing the pore size of the tubular scaffolds, ciGEnC could form a tight monolayer. Finally, a co‐culture model was successfully established.*Conclusion/Significance: MEW tubes with highly controlled fibrous microarchitectures advance both ciPTEC and ciGEnC cell organization and ECM deposition. Our results show the first prototype of a vascularized scaffold, where the basement membrane deposited by ciPTEC form a support for ciGEnC to adhere and grow on.Session Number: 4 Micro/nano Biofabrication of Organ‐on‐chips, Organoids and Disease ModelsWednesday, April 12, 2023, 10:00 AM ‐ 11:30 AM2 ‐ 3d Bioprinted Liver‐on‐a‐chip For Drug Cytotoxicity ScreeningJ. Huh, H. Kang, J. S. Copus, C. E. Bishop, S. Soker, S. Murphy, T. D. Shupe, J. J. Yoo, A. Atala, S. Lee;Wake Forest Institute for Regenerative Medicine, Winston Salem, NC.*Purpose/Objectives: Liver‐on‐a‐chip (LOC) is a 3D in vitro hepatic microphysiological system aiming to recreate the conditions of hepatic cells under the dynamic physicochemical environment. Conventional LOC fabrication uses an assembly of multiple chip parts and requires many manual processing steps, resulting in limitations in reproducibility from sample to sample. This study aimed to automate the fabrication process of LOC for high‐throughput drug cytotoxicity screening.*Methodology: We utilized a 3D integrated tissue and organ printing (ITOP) system to allow the one‐step fabrication of LOC by co‐printing of human liver carcinoma cell (HepG2) organoids and a polymeric chip with inner fluidic channels. After LOC was moved to the chip platform inside the incubator, it was dynamically cultured using a peristaltic pump. Its cell viability and metabolic activities after drug administration were further assessed.*Results: The liver organoids in the printed chip showed high cell viability (>70%), an increase in organoid size, consistent ATP production, and an increase in albumin production for up to 14 days. The effect of acetaminophen (APAP), which is a nonsteroidal anti‐inflammatory drug, was examined in LOC. Compared to the non‐treated group, the APAP‐treated LOC group showed a significant loss in cell viability (< 40%), lower ATP production, and smaller organoid size at 7 days in culture.*Conclusion/Significance: This demonstrated the potential of 3D bioprinted LOC to serve as a high throughput in vitro liver model for the accurate study of in vivo biological processes, such as monitoring the tissue response to administered drugs.3 ‐ Use Of Vascularized Heart‐on‐a‐chip Platform To Identify Extracellular Vesicles From HUVEC As An Anti‐inflammatory Treatment For SARS‐COV‐2 Acute MyocarditisR. Lu, N. Rafatian, M. Radisic;University of Toronto, TORONTO, ON, CANADA.*Purpose/Objectives: Myocarditis is characterized by inflammation of the heart, which results in poor heart function. Interest in viral‐associated myocarditis peaked during the COVID‐19 pandemic, yet reliable biomarkers for the early detection and treatment of myocarditis are an unmet clinical need. This motivates the need to develop an advanced model that enables the synergistic integration of endothelial cells, immune cells, and functional cardiac tissues to deepen the understanding of SARS‐CoV‐2‐induced acute myocarditis in vitro settings.*Methodology: To mimic SARS‐CoV‐2 infection under more physiologically relevant conditions, we repurposed a pre‐established vascularized heart‐on‐a‐chip system to capture the complex cascade of viral infection and subsequent myocardial inflammation in the presence of circulating immune cells.*Results: We demonstrated that PBMCs and CD3+ T‐lymphocytes infiltrated cardiac tissue from the vascular compartment. Local infiltration of immune cells stimulates the release of proinflammatory cytokines, which leads to electromechanical dysfunction of cardiac tissue. Myocardial inflammation also caused the release of ccf‐mtDNA in both an in vitro cardiac tissue model and in COVID‐19‐positive patients, demonstrating that fragmentation of myocardial mitochondria is an important pathophysiological mechanism leading to myocarditis. Using a myocarditis‐on‐a‐chip model, we demonstrated the cardioprotective effects of HUVEC‐EVs in alleviating SARS‐CoV‐2‐induced myocardial contractile deficits and abnormal intracellular calcium handling. Our results demonstrated that HUVEC‐EVs suppressed inflammatory responses by suppressing NF‐kB and IFN activation, which in turn mitigated mitochondrial stress by inhibiting the NLRP3 inflammasome.*Conclusion/Significance: Collectively, our study suggests that miRNAs may serve as promising immunomodulatory agents and that the transfer of anti‐inflammatory miRNAs helps maintain mitochondrial energetics.4 ‐ Investigating Glioblastoma Tumor Cell Invasion And Treatment Using A Blood Brain Barrier On A ChipT. DePalma1,2, A. Skardal1,2;1Biomedical Engineering, The Ohio State University, Columbus, OH, 2The Ohio State University and Arthur G. James Comprehensive Cancer Center, Columbus, OH.*Purpose/Objectives: The blood brain barrier (BBB) plays a critical role in the progression and treatment of glioblastoma (GBM). Current preclinical models of GBM are limited because they fail to accurately replicate the function of the BBB and the extreme heterogeneity of the GBM tumor. Here, we use an in vitro model of the human BBB to study how different GBM cell populations behave and impact BBB function with the goal of informing the development of novel therapeutic and drug delivery mechanisms.*Methodology: Microfluidic devices are cast using PDMS. Hydrogels composed of hyaluronic acid and gelatin are added to the device with human astrocytes. Brain pericytes and brain endothelial cells are seeded in a molded lumen within the hydrogel. Fluid flow is initiated using a rocker plate. GBM cell conditioned media and/or tumor cells are added to the device after the BBB has formed. Several distinct populations of tumor cells and glioma stem cells (GSC) are used.*Results: Our results indicate that GBM tumor cells with higher levels of drug resistance tend to induce more severe changes in BBB function. These cells also induce high levels of astrocyte reactivity and the release of inflammatory factors which could be a driver of this BBB dysfunction. We are currently evaluating the efficacy and transport of various treatments.*Conclusion/Significance: These experiments highlight the importance of studying GBM tumor heterogeneity when designing therapies. Future studies using patient derived cells in this platform could pave the way for the development of personalized treatments for GBM.5 ‐ RCAN1‐4 Suppresses Metastatic Invasion And Tumor Cell Proliferation In A 3D Thyroid Metastasis‐on‐a‐Chip ModelK. Nairon1, N. Rajan2, M. Ringel2, A. Skardal1;1Biomedical Engineering, The Ohio State University, Columbus, OH, 2The Ohio State University, Columbus, OH.*Purpose/Objectives: Regulator of calcineurin 1, isoform 4 (RCAN1‐4) has been shown suppress metastasis in thyroid cancer. RCAN1‐4 knockdown increases proliferation in vitro and metastatic burden in vivo, evidence that the gene regulates later steps in the metastatic cascade and potentially impacts metastatic dormancy. To study RCAN1‐4 in thyroid‐to‐lung metastasis, a metastasis‐on‐a‐chip (MOC) model was developed using control and RCAN1‐4 knockdown clones of thyroid cancer cell lines hTh74 and FTC236.*Methodology: Fluorescently tagged cancer cells were circulated through microfluidic channels, running parallel to lung hydrogels and allowing tumor cell‐lung tissue interactions. Imaging and quantification measured tumor cell invasion distance and number for control and knockdown clones of each cell line. Proteomics of hTh74 and FTC236 lines identified changes to invasion and dormancy‐linked pathways.*Results: RCAN1‐4 knockdown increased invasion distance into lung hydrogels, as well as number of invaded cells. Differing endogenous RCAN1‐4 expression between the cell lines was reflected in a greater difference between knockdown and control hTh74 invasion as compared to FTC236, aligning with in vivo observations. RCAN1‐4 knockdown did not change the time between tumor cell introduction to the system and attachment to lung sites, implying that RCAN1‐4's metastasis suppressing functions may be more related to motility and proliferation than extravasation.*Conclusion/Significance: Here, we demonstrated alignment of metastasis‐on‐a‐chip results with in vivo observations while exploring more in‐depth analyses of RCAN1‐4's metastasis‐suppressing roles. This model aims to overcome difficulties in monitoring distinct metastatic steps and kinetics in animal models, as well as provide a fully human alternative for improved translation.6 ‐ 3d Perfusable Vascularized Model To Study Dengue Virus Ns1 Induced Endothelial Permeability And DysfunctionM. Rajput, A. Medina, S. Costmire, M. Song, M. Ferrer, E. Lee;NCATS/NIH, Rockville, MD.*Purpose/Objectives: Dengue virus which infects approximately 400 million people worldwide each year can lead to fatal vascular damage, from vascular leakage, leads to hypovolemic shock and excessive immune cell activation. The dengue virus non‐structural protein 1 (NS1) is secreted and circulated in blood stream during infection and linked to disease severity, however, there is no current 3D model which fully recapitulates pathogenesis to understand pathophysiology. In this study, we aimed to model dengue induced pathogenesis using hydrogel based vascularized model to develop potential therapeutics.*Methodology: Using 64‐chip Mimetas Organograft plate platform we developed a reproducible, Perfusable vascular on chip model by combining three different cell types: human primary endothelial cells, lung fibroblast, and pericytes in fibrinogen hydrogel. Then, tested recombinant NS1 protein's dose dependent and time dependent effect on 3D vascularized model and documented vascular damage by different assay read outs.*Results: Our findings reveal that different concentration of rNS1 in a time dependent manner alters the permeability of vascular network and leads to differential disruption of endothelial glycocalyx layer, resulting in vascular leak, determined by perfusing 70kDa dextran dye. The vascular damage was further validated by the change in morphometric parameters: decrease in vessel density, branching index, vessel length and vessel intensity and expression of endothelial glycocalyx components and vascular related genes in comparison to control.*Conclusion/Significance: The results suggested the potentiality of secreted viral protein to modulate the endothelial barrier in a tissue ‐specific manner and can provide the potential targets for antiviral therapies and vaccine development.Session Number: 6 Complex Organ Decellularization: How Close are We to Translation? Wednesday, April 12, 2023, 2:15 PM ‐ 3:45 PM8 ‐ Decellularized Human Pancreatic Extracellular Matrix‐based Physiomimetic Microenvironment For Human Islet CultureA. ASTHANA, D. CHAIMOV, R. Tamburrini, L. N. Byers, G. Orlando;WFIRM, Winston Salem, NC.*Purpose/Objectives: Combining the biophysical and mechanical properties of inert encapsulation materials like alginate with the biochemical niche provided by the extracellular matrix (ECM), could provide a physiomimetic pancreatic microenvironment for maintaining long‐term islet viability and function in culture.*Methodology: We have developed a proprietary protocol for the production of human pancreatic decellularized extracellular matrix (dECM). Our method retains the matrisome, while clearing cellular proteins. In order to test the effect of the resulting dECM on the viability and function of pancreatic cells, primary human islets were embedded in dECM‐alginate capsules and cultured for 58 days. Glucose‐Stimulated Insulin Secretion (GSIS), morphology and live/dead imaging were performed on days 5, 9, 19, 33, 44 and 58 of culture.*Results: It was found that incorporating dECM (0.1 mg/ml) within alginate microcapsules provides biochemical cues essential for maintaining long‐term islet morphology, viability and function. Results indicated a significant increase in Glucose Stimulation Index (GSI) and total secreted insulin in islets encapsulated in dECM‐alginate capsules, compared to control groups ‐ free islets and islets encapsulated in dECM‐free alginate, starting on day 33 of culture. Moreover, islets in dECM‐alginate capsules maintained GSI levels similar to that observed in free islets at the first time point. The dECM was also tested in vitro with respect to nanosafety and itwas found to exhibit excellent hemocompatibility, immunocompatibility and cytocompatibility.*Conclusion/Significance: Our human pancreatic dECM is biocompatible and its addition within alginate capsules preserves the ability of human islets to produce insulin in a glucose‐responsive manner over long‐term culture.9 ‐ Decellularization Of Heterogeneous Tissue: Mechanical And Structural Characterization Of Decellularized Trachea ComponentsA. M. Greaney1, A. B. Ramachandra1, Y. Yuan2, A. Korneva1, J. D. Humphrey1, L. E. Niklason3;1Department of Biomedical Engineering, Yale University, New Haven, CT, 2Department of Pulmonary, Critical Care & Sleep Medicine, Yale School of Medicine, New Haven, CT, 3Department of Anesthesiology, Yale School of Medicine, New Haven, CT.*Purpose/Objectives: The repair of long‐segment, circumferential airway damage has proven a formidable challenge to clinicians and engineers for over 100 years. Decellularized cadaveric tracheas have been applied clinically as replacement scaffolds in a small number of compassionate use cases. However, decellularized tracheas tend to collapse in vivo, causing significant complications. We set out to characterize the biaxial (axial and circumferential) mechanical properties of native and decellularized rat tracheas and how deviations from native mechanics relate to changes in extracellular matrix (ECM) composition and structure.*Methodology: A custom biaxial mechanical testing system was used to perform axially isometric distension tests and isobaric extension tests on tracheas across a range of physiologic applied forces, to capture material behaviors and properties of each component of the heterogeneous trachea (cartilage, trachealis muscle, connective tissue). Native tissues were compared to tracheas decellularized by two different protocols, including one used clinically. Western blot and histology were performed to measure changes in ECM protein between conditions.*Results: Decellularization reduced collagen‐II, collagen‐III, laminin, and GAGs in the tissue, and disrupted the structure of large collagen fibers and longitudinal elastin. These changes had complex effects on the mechanics of the cartilage, trachealis muscle, and connective tissue, but overall represented significant deviations from native.*Conclusion/Significance: We present a robust method for the biaxial mechanical evaluation of engineered tracheas, which better captures tissue properties under physiologic conditions. Our findings provide powerful lessons on the complex effects of decellularization on tissues with heterogeneous ECM structure.10 ‐ Bioengineered Liver Crosslinked With Nanographene Oxide Enables Efficient Liver Regeneration Via MMP Suppression And ImmunomodulationD. Kim1, J. Ryu1,2, K. Kang1;1Adult Stem Cell Research Center and Research Institute for Veterinary Science, Seoul National University, Seoul, KOREA, REPUBLIC OF, 2Institute of Bio & Nano Convergence, Biogo Co. LTD, Seoul, KOREA, REPUBLIC OF.*Purpose/Objectives: As an alternative to living donor organs for liver transplantation, bioengineered livers have been constructed with decellularized extracellular matrix (dECM) scaffolds retaining a biomimetic 3D microenvironment. However, liver dECM is vulnerable to mechanical deformation and enzymatic destruction after transplantation, thereby detrimentally affecting graft survival. In this study, a new strategy to modify the biophysical properties of dECM by using nano‐graphene oxide (NGO) is proposed to fabricate transplantable bioengineered livers.*Methodology: The mouse bioengineered livers (MBLs) were reconstructed with NGO‐crosslinked dECM scaffolds (NGO‐MBLs) closely resembled normal liver structures, including parenchyma and endothelium. To validate in vivo regenerative capacity, the MBLs were transplanted into the liver failure mouse models: acute model induced by partial hepatectomy and chronic model induced by thioacetamide administration.*Results: NGO crosslinking not only reinforced the mechanical properties of the scaffolds, but also directly inhibited the catalytic activities of matrix metalloproteinases (MMPs). In both models, transplanted NGO‐MBLs exhibited dominant accumulation of M2c‐like macrophages expressing TIMP‐1, which helped the implants to remain relatively intact. These constructive phenotypes with type 2 immune responses mediated by the NGOs contributed to the retention of more functional cells in the NGO‐MBLs and resulted in a high regenerative capacity.*Conclusion/Significance: In conclusion, we successfully established a highly durable and biocompatible MBLs with superior regenerative potential through NGO crosslinking of dECM scaffolds. Thus, this study lays the cornerstone for creating clinically applicable human‐sized bioengineered livers in the future.11 ‐ Decellularized Composite Allografts Of Human Digits ‐ A Step Towards Non‐immunogenic Composite Allograft TransplantationM. E. McCarthy1,2,3, I. Filz von Reiterdank1,2,4, L. Charles5,6,7, K. Uygun1,2, A. G. Lellouch5,6, C. L. Cetrulo, Jr.5,6,2, B. E. Uygun1,2;1Department of Surgery, Center for Engineering in Medicine and Surgery, Boston, MA, 2Shriners Hospitals for Children in Boston, Boston, MA, 3Department of General Surgery, Lahey Hospital and Medical Center, Burlington, MA, 4Division of Surgical Specialties, Department of Plastic Surgery and Hand Surgery, University Medical Center Utrecht, Utrecht, NETHERLANDS, 5Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Boston, MA, 6Center for Transplantation Sciences, Vascularized Composite Allotransplantation Laboratory, Boston, MA, 7Department of Plastic Surgery, European George Pompidou Hospital, Paris, FRANCE.*Purpose/Objectives: Vascularized composite allograft (VCA) transplantation has been achieved with the hand, face, abdominal wall, and penis. However, the need for life‐long immunosuppressive therapy significantly contributes to recipient morbidity and mortality. Decellularization and recellularization technology is one method to circumvent the recipient's immune response. Decellularization protocols remove cellular material from the grafts, leaving only extracellular matrix (ECM). Decellularized composite allografts‐ which we have termed DCAs‐ can then be recellularized with the recipient's cells, resulting in non‐immunogenic biologic grafts.*Methodology: In this study, we have decellularized whole human digits, one thumb and one‐second digit, which had been in cold storage for four months. In brief, digits were thawed and continuously perfused with detergents for nine days. Perfusion pressure was monitored, and flow rates adjusted to maintain pressures of 50‐60mmHg.*Results: After nine days, there was a visible decrease in digit opaqueness. X‐ray of the digits with contrast agent showed an intact vascular network. DNA quantification of the digits showed significantly lower DNA content in DCA subcutaneous tissue (58.65 ± 20.72 vs 32.01 ± 14.13 ng/mg wet weight, p < 0.05) and bone (33.70 ± 2.55 vs 10.04 ± 4.07 ng/mg wet weight, p < 0.001). Notably, tissue is accepted as decellularized with DNA content of less than 50ng/mg.*Conclusion/Significance: Our results indicate that decellularization of whole digits from long‐term freezer storage is feasible and represents an essential step in developing non‐immunogenic VCA transplantation. Future work will focus on histologic and microscopic analysis of decellularized VCAs, comparison to native tissue, and recellularization with primary human cells.12 ‐ Alpha Gal Sugar Epitope Can'T Be Hidden Or Removed: Stays ImmunogenicK. V. KURAVI1, L. T. Sorrells1, F. Rahaman1, S. Choudhary2, S. Commins2, J. R. Bianchi1;1Revivicor Inc. (Subsidiary of United Therapeutics), Blacksburg, VA, 2Div. of Allergy, Immunology, and Rheumatology, University of North Carolina, Chapel Hill, NC.*Purpose/Objectives: The epitope, galactose‐α1,3‐galactose (α‐gal) is ubiquitously present on cells and tissues of most mammals. All immunocompetent humans generate anti‐α‐gal IgG/ IgM antibodies against α‐gal sugar epitope. Allergists have recently identified an additional population with high titers of anti‐ α ‐gal IgE (alpha‐gal syndrome (AGS)) due to a tick bite. Subsequent exposure to α‐gal containing food or medical products causes adverse events like anaphylactic shock, shortness of breath, etc. Bovine and porcine derived biomedical products are widely used as cardiovascular bio prosthesis, wound dressings, hemostatic agents, bone grafts, and medications.*Methodology: (1) to detect presence and /or quantify α‐gal epitopes in bovine or porcine derived medical products including demineralized bone material, cardiovascular bioprosthesis and therapeutic drugs, (2) investigate immunogenicity of these products via IgE reactivity from AGS patient serum Alpha gal epitope detection/quantitation in porcine or bovine derived medical products was determined via an indirect α‐gal ELISA. AGS patient serum IgE binding was determined by IHC and western blot. Porcine α‐gal knockout (devoid of α‐gal) derived native tissue or products served as negative controls for all assays.*Results: ELISA and IHC data indicates presence of α‐gal in all bovine/porcine derived products irrespective of their source, preparation, or form. The relative amount of α‐gal present in these products exceeded 10,000,000,000 epitopes per mg.*Conclusion/Significance: AGS patient sera anti‐gal IgE showed reactivity to source tissue material of these products. Porcine α‐gal knockout products or tissue material could be less immunogenic and serve as an alternative to standard pig derived products.13 ‐ Decellularization Of The Human Urethra For Tissue Engineering ApplicationsM. Kuniakova1,2, S. Ziaran1,3, Z. Varchulova Novakova1, M. Klein1,4, K. Bevizova5, I. Varga1,4, L. Danisovic1,2;1National Institute of Rheumatic Diseases, Piestany, SLOVAKIA, 2Institute of Medical Biology, Genetics and Clinical Genetics, Comenius University, Faculty of Medicine, Bratislava, SLOVAKIA, 3Department of Urology, Comenius University, Faculty of Medicine, Bratislava, SLOVAKIA, 4Institute of Histology and Embryology, Comenius University, Faculty of Medicine, Bratislava, SLOVAKIA, 5Institute of Anatomy, Comenius University, Faculty of Medicine, Bratislava, SLOVAKIA.*Purpose/Objectives: Recently, various scaffolds have been introduced for urethral tissue engineering. However, acellular urethral scaffold harvested from deceased donors may provide significant advantages in comparison to synthetic, composite, or other biological scaffolds. This study aims to develop the protocol for decellularization of the human urethra that preserves substantial extracellular matrix (ECM) components.*Methodology: Fifteen human urethras were harvested from deceased donors. Equal part of every sample was used as a control sample for analyses. The protocol design was based on the enzyme‐detergent‐enzyme method. Solution of trypsin (0.25%) and Triton X‐100 (1%) was used to remove cells, followed by DNAse treatment to remove DNA residues. Subsequently, the specimens were continually rinsed in deionized water for 7 days. The efficiency of decellularization was determined by histochemistry, immunohistochemical staining, scanning electron microscopy (SEM), and DNA quantification.*Results: Histological analysis confirmed cell removal and preservation of urethral structure after decellularization. The preservation of collagen IV and fibronectin was confirmed by histologic examination and immunohistochemical staining. SEM confirmed the maintenance of ultrastructural architecture of ECM. DNA content in decellularized urethra was significantly lower compared to the native sample. Cytotoxicity analysis data showed that the matrix‐conditioned medium did not contain soluble toxins and do not have any significant inhibitory effect on cell proliferation, providing evidence that the decellularized samples are not toxic.*Conclusion/Significance: This study demonstrates the feasibility of the enzyme‐detergent‐enzyme‐based decellularization protocol for removing cells and maintaining urethral ECM and its ultrastructure. Moreover, obtained results provide solid ground for recellularization and urethral tissue engineering.14 ‐ Decellularization: Leveraging A Tissue Engineering Technology For Food ProductionJ. Jones;Engineering, Boston College, Chestnut Hill, MA.*Purpose/Objectives: Edible plant leaves are particularly useful as scaffolds for the development of cultured meat. Recently, we demonstrated that decellularized spinach leaves can serve as scaffolds to grow and differentiate cells for cultured meat products. Decellularization of leaf scaffolds provides an effective substrate for cell adhesion while maintaining the plant's structural composition, preserving its vascular network for perfusion. However, conventional decellularization methods use solutions that are not safe for use in food.*Methodology: This study modified the protocols to incorporate detergents that are regulated by the FDA for use in food and eliminated the use of organic solvents for cuticle removal.*Results: The average DNA content for both groups was similar, at 1.3 ± 0.07 ng/mg and 1.3 ± 0.05 ng/mg, respectively. The importance of cuticle removal was tested by removing hexanes from the protocol. Groups that included the cuticle removal step exhibited an average DNA reduction of approximately 91.7%, while groups that omitted the step exhibited an average reduction of approximately 90.3% (p = ns), suggesting that the omission did not impede decellularization. Primary bovine satellite cells were then cultured on the surface of spinach leaves decellularized using the regulated protocol. After a 7‐day incubation period, the cells grown on the surface of the regulated scaffolds had an average viability of 97.4%.*Conclusion/Significance: The results suggest that the regulated protocol is effective for cultured meat production and adheres to food safety guidelines. This method is a safer option for producing lab‐grown meat and alternative protein products.15 ‐ The Decellularized Human Dermal Matrix Is A Promising Biomaterial For The Construction Of Stand‐alone Biological Scaffolds For Cardiac RegenerationF. Di Meglio1, A. Sacco1, V. Romano1, I. Belviso1, G. Ricci2, A. Catizone3, D. Nurzynska4, F. Schonauer1, C. Amarelli5, O. Casciello1, C. Castaldo1;1Public Health, University of Naples “Federico II”, Naples, ITALY, 2Experimental Medicine, Università della Campania Luigi Vanvitelli, Naples, ITALY, 3Anatomical Histological Medical Legal Sciences and Locomotor Apparatus, Sapienza University of Rome, Rome, ITALY, 4Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Naples “Federico II”, Baronissi, ITALY, 5Cardiovascular Surgery and Transplants, Monaldi Hospital, Naples, ITALY.*Purpose/Objectives: Tissue engineering (TE) aims at restoring tissue