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Chronic respiratory diseases often necessitate lung transplantation due to irreversible damage. Organ engineering offers hope through stem cell-based organ generation. However, the crucial sterilization step in scaffold preparation poses challenges. This study conducted a systematic review of studies that analysed the extracellular matrix (ECM) conditions of decellularised lungs subjected to different sterilisation processes. A search was performed for articles published in the PubMed, Web of Sciences, Scopus, and SciELO databases according to the PRISMA guidelines. Overall, five articles that presented positive results regarding the effectiveness of the sterilisation process were selected, some of which identified functional damage in the ECM. Was possible concluded that regardless of the type of agent used, physical or chemical, all of them demonstrated that sterilisation somehow harms the ECM. An ideal protocol has not been found to be fully effective in the sterilisation of pulmonary scaffolds for use in tissue and/or organ engineering.

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The recellularization of tissues after decellularization is a relatively new technology in the field of tissue engineering (TE). Decellularization involves removing cells from a tissue or organ, leaving only the extracellular matrix (ECM). This can then be recellularized with new cells to create functional tissues or organs. The first significant mention of recellularization in decellularized tissues can be traced to research conducted in the early 2000s. One of the landmark studies in this field was published in 2008 by Ott, where researchers demonstrated the recellularization of a decellularized rat heart with cardiac cells, resulting in a functional organ capable of contraction. Since then, other important studies have been published. These studies paved the way for the widespread application of recellularization in TE, demonstrating the potential of decellularized ECM to serve as a scaffold for regenerating functional tissues. Thus, although the concept of recellularization was initially explored in previous decades, these studies from the 2000s marked a major turning point in the development and practical application of the technology for the recellularization of decellularized tissues. The article reviews the historical advances and limitations in organ recellularization in TE over the last two decades.

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Liver transplantation represents the only definitive treatment for patients with end-stage liver disease. However, the shortage of liver donors provokes a dramatic gap between available grafts and patients on the waiting list. Whole liver bioengineering, an emerging field of tissue engineering, holds great potential to overcome this gap. This approach involves two main steps; the first is liver decellularization and the second is recellularization. Liver decellularization aims to remove cellular and nuclear materials from the organ, leaving behind extracellular matrices containing different structural proteins and growth factors while retaining both the vascular and biliary networks. Recellularization involves repopulating the decellularized liver with appropriate cells, theoretically from the recipient patient, to reconstruct the parenchyma, vascular tree, and biliary network. The aim of this review is to identify the major advances in decellularization and recellularization strategies and investigate obstacles for the clinical application of bioengineered liver, including immunogenicity of the designed liver extracellular matrices, the need for standardization of scaffold fabrication techniques, selection of suitable cell sources for parenchymal repopulation, vascular, and biliary tree reconstruction. In vivo transplantation models are also summarized for evaluating the functionality of bioengineered livers. Finally, the regulatory measures and future directions for confirming the safety and efficacy of bioengineered liver are also discussed. Addressing these challenges in whole liver bioengineering may offer new solutions to meet the demand for liver transplantation and improve patient outcomes.

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BACKGROUND: Bioscaffolds and cells are two main components in the regeneration of damaged tissues via cell therapy. Umbilical cord stem cells are among the most well-known cell types for this purpose. The main objective of the present study was to evaluate the effect of the pretreatment of the foreskin acellular matrix (FAM) by monophosphoryl lipid A (MPLA) and Lactobacillus casei supernatant (LCS) on the attraction of human umbilical cord mesenchymal stem cells (hucMSC). METHODS AND RESULTS: The expression of certain cell migration genes was studied using qRT-PCR. In addition to cell migration, transdifferentiation of these cells to the epidermal-like cells was evaluated via immunohistochemistry (IHC) and immunocytochemistry (ICC) of cytokeratin 19 (CK19). The hucMSC showed more tissue tropism in the presence of MPLA and LCS pretreated FAM compared to the untreated control group. We confirmed this result by scanning electron microscopy (SEM) analysis, glycosaminoglycan (GAG), collagen, and DNA content. Furthermore, IHC and ICC data demonstrated that both treatments increase the protein expression level of CK19. CONCLUSION: Pretreatment of acellular bioscaffolds by MPLA or LCS can increase the migration rate of cells and also transdifferentiation of hucMSC to epidermal-like cells without growth factors. This strategy suggests a new approach in regenerative medicine.

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Background: Osteochondral regeneration has long been recognized as a complex and challenging project in the field of tissue engineering. In particular, reconstructing the osteochondral interface is crucial for determining the effectiveness of the repair. Although several artificial layered or gradient scaffolds have been developed recently to simulate the natural interface, the functions of this unique structure have still not been fully replicated. In this paper, we utilized laser micro-patterning technology (LMPT) to modify the natural osteochondral "plugs" for use as grafts and aimed to directly apply the functional interface unit to repair osteochondral defects in a goat model. Methods: For in vitro evaluations, the optimal combination of LMPT parameters was confirmed through mechanical testing, finite element analysis, and comparing decellularization efficiency. The structural and biological properties of the laser micro-patterned osteochondral implants (LMP-OI) were verified by measuring the permeability of the interface and assessing the recellularization processes. In the goat model for osteochondral regeneration, a conical frustum-shaped defect was specifically created in the weight-bearing area of femoral condyles using a customized trephine with a variable diameter. This unreported defect shape enabled the implant to properly self-fix as expected. Results: The micro-patterning with the suitable pore density and morphology increased the permeability of the LMP-OIs, accelerated decellularization, maintained mechanical stability, and provided two relative independent microenvironments for subsequent recellularization. The LMP-OIs with goat's autologous bone marrow stromal cells in the cartilage layer have securely integrated into the osteochondral defects. At 6 and 12 months after implantation, both imaging and histological assessments showed a significant improvement in the healing of the cartilage and subchondral bone. Conclusion: With the natural interface unit and zonal recellularization, the LMP-OI is an ideal scaffold to repair osteochondral defects especially in large animals. The translational potential of this article: These findings suggest that such a modified xenogeneic osteochondral implant could potentially be explored in clinical translation for treatment of osteochondral injuries. Furthermore, trimming a conical frustum shape to the defect region, especially for large-sized defects, may be an effective way to achieve self-fixing for the implant.

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BACKGROUND: Type 2 diabetes mellitus (T2DM) is increasing at an alarming rate, and only 50% of patients with T2DM achieve or maintain adequate glycemic control with pharmacological therapies. Metabolic surgery demonstrated superior efficacy compared to medical therapy but is unfeasible for most patients with T2DM. Duodenal mucosal resurfacing (DMR) by hydrothermal mucosal ablation, recellularization via electroporation therapy (ReCET), and photodynamic therapy are novel endoscopic procedures that use thermal, electrical, and photochemical energy, respectively, to ablate and reset dysfunctional duodenal mucosa. We assessed the data on the effects of these techniques on glycemic control and nonalcoholic fatty liver disease (NAFLD). METHODS: We systematically searched independently and in duplicate English and non-English language publications through January 31st, 2024. Outcomes assessed were an improvement in different metabolic health parameters and the safety of duodenal mucosal ablation (DMA) procedures. Outcomes were presented descriptively. FINDINGS: We selected 12 reports reporting results from 3 randomized and 6 uncontrolled trials (seven evaluating DMR, two evaluating ReCET, all with a low risk of bias) for a total of 317 patients enrolled. DMA reduced HbA1c, fasting plasma glucose, and liver fat. When combined with newer antidiabetic drugs, it allowed insulin discontinuation in up to 86% patients. No major safety signal emerged. CONCLUSIONS: All DMA techniques improve glucose homeostasis; DMR and ReCET appear to be safe in patients with T2DM. If confirmed by future randomized trials and by trials with histological endpoints in NAFLD, then DMA appears to be a promising alternative or complement option to medications for T2DM and NAFLD treatment. FUNDING: This study received no funding.

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Tissue engineering heart valves (TEHVs) are expected to address the limitations of mechanical and bioprosthetic valves used in clinical practice. Decellularized heart valve (DHV) is an important scaffold of TEHVs due to its natural three-dimensional structure and bioactive extracellular matrix, but its mechanical properties and hemocompatibility are impaired. In this study, DHV is cross-linked with three different molecular weights of oxidized hyaluronic acid (OHA) by a Schiff base reaction and presented enhanced stability and hemocompatibility, which could be mediated by the molecular weight of OHA. Notably, DHV cross-linked with middle- and high-molecular-weight OHA could drive the macrophage polarization toward the M2 phenotype in vitro. Moreover, DHV cross-linked with middle-molecular-weight OHA scaffolds are further modified with RGD-PHSRN peptide (RPF-OHA/DHV) to block the residual aldehyde groups of the unreacted OHA. The results show that RPF-OHA/DHV not only exhibits anti-calcification properties, but also facilitates endothelial cell adhesion and proliferation in vitro. Furthermore, RPF-OHA/DHV shows excellent performance under an in vivo hemodynamic environment with favorable recellularization and immune regulation without calcification. The optimistic results demonstrate that OHA with different molecular weights has different cross-linking effects on DHV and that RPF-OHA/DHV scaffold with enhanced immune regulation, anti-calcification, and recellularization properties for clinical transformation.

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Reconstructive techniques to repair severe tissue defects include the use of autologous fasciocutaneous flaps, which may be limited due to donor site availability or lead to complications such as donor site morbidity. A number of synthetic or natural dermal substitutes are in use clinically, but none have the architectural complexity needed to reconstruct deep tissue defects. The perfusion decellularization of fasciocutaneous flaps is an emerging technique that yields a scaffold with the necessary composition and vascular microarchitecture and serves as an alternative to autologous flaps. In this study, we show the perfusion decellularization of porcine fasciocutaneous flaps using sodium dodecyl sulfate (SDS) at three different concentrations, and identify that 0.2% SDS results in a decellularized flap that is efficiently cleared of its cellular material at 86%, has maintained its collagen and glycosaminoglycan content, and preserved its microvasculature architecture. We further demonstrate that the decellularized graft has the porous structure and growth factors that would facilitate repopulation with cells. Finally, we show the biocompatibility of the decellularized flap using human dermal fibroblasts, with cells migrating as deep as 150 µm into the tissue over a 7-day culture period. Overall, our results demonstrate the promise of decellularized porcine flaps as an interesting alternative for reconstructing complex soft tissue defects, circumventing the limitations of autologous skin flaps.

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Duodenal mucosa ablation (DMA) is a novel approach to treat diabetes, consisting of endoscopic ablation of dysfunctional diabetic duodenal mucosa, which, following the healing response, is replaced by normally functioning mucosa. Two techniques, duodenal mucosal resurfacing (DMR) and recellularization via electroporation therapy (ReCET), recently showed promise in type 2 diabetes mellitus (T2DM) patients.

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Muscle tissue engineering is a promising therapeutic strategy for volumetric muscle loss (VML). Among them, decellularized extracellular matrix (dECM) biological scaffolds have shown certain effects in restoring muscle function. However, researchers have inconsistent or even contradictory results on whether dECM biological scaffolds can efficiently regenerate muscle fibers and restore muscle function. This suggests that therapeutic strategies based on dECM biological scaffolds need to be further optimized and developed. In this study, we used a recellularization method of perfusing adipose-derived stem cells (ASCs) and L6 into adipose dECM (adECM) through vascular pedicles. On one hand, this strategy ensures sufficient quantity and uniform distribution of seeded cells inside scaffold. On the other hand, auxiliary L6 cells addresses the issue of low myogenic differentiation efficiency of ASCs. Subsequently, the treatment of VML animal experiments showed that the combined recellularization strategy can improve muscle regeneration and angiogenesis than the single ASCs recellularization strategy, and the TA of former had greater muscle contraction strength. Further single-nucleus RNA sequencing (snRNA-seq) analysis found that L6 cells induced ASCs transform into a new subpopulation of cells highly expressing Mki67, CD34 and CDK1 genes, which had stronger ability of oriented myogenic differentiation. This study demonstrates that co-seeding ASCs and L6 cells through vascular pedicles is a promising recellularization strategy for adECM biological scaffolds, and the engineered muscle tissue constructed based on this has significant therapeutic effects on VML. Overall, this study provides a new paradigm for optimizing and developing dECM-based therapeutic strategies.

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The present study assessed the supportive roles of the decellularized human ovarian tissue in homing of mouse fetal ovarian cells into the scaffold as well as the formation of the follicular-like structure. The human ovarian cortical tissues were decellularized by three freeze-thaw cycles and then, treated with Triton X-100 for 15 h and 0.5% sodium dodecyl sulfate for 72 h. After isolation and preparation of mouse fetal ovarian cells (19 dpc) they were seeded into the decellularized scaffolds and cultured for 7 days, then using a light microscope, laser confocal scanning microscope, and scanning electron microscope these scaffolds were studied. Analysis of gene expression related to oocyte and follicular cells such as Ddx4, Nobox, Gdf9, and Connexin37 was assessed by real-time RT-PCR and the DDX4 and GDF9 proteins were detected by immunohistochemistry. The result showed that the human ovarian tissue was decellularized properly and the tissue elements and integrity were well preserved. After 7 days of in vitro culture, the fetal ovarian cells attached and penetrated into different sites and depths of the scaffold. The formed organoid within the scaffold showed large round, small polyhedral, and elongated spindle cells similar to the follicle structure. The molecular analysis and immunohistochemistry were confirmed an increase in the expression of genes and proteins related to oocyte and follicular cells in these reconstructed structures. In conclusion, the recellularization of human ovarian scaffolds by mouse fetal ovarian cells could support the follicular-like structure formation and it provides an in vitro model for follicle reconstitution and offers an alternative approach for clinical usage.

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Pancreatic bioengineering is a potential therapeutic alternative for type 1 diabetes (T1D) in which the pancreas is decellularized, generating an acellular extracellular matrix (ECM) scaffold, which may be reconstituted by recellularization with several cell types to generate a bioartificial pancreas. No consensus for an ideal pancreatic decellularization protocol exists. Therefore, we aimed to determine the best-suited detergent by comparing sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), and Triton X-100 at different concentrations. Murine (n=12) and human pancreatic tissue from adult brain-dead donors (n=06) was harvested in accordance with Institutional Ethical Committee of the University of São Paulo Medical School (CEP-FMUSP) and decellularized under different detergent conditions. DNA content, histological analysis, and transmission and scanning electron microscopy were assessed. The most adequate condition for pancreatic decellularization was found to be 4% SDC, displaying: a) effective cell removal; b) maintenance of extracellular matrix architecture; c) proteoglycans, glycosaminoglycans (GAGs), and collagen fibers preservation. This protocol was extrapolated and successfully applied to human pancreas decellularization. The acellular ECM scaffold generated was recelullarized using human pancreatic islets primary clusters. 3D clusters were generated using 0.5×104 cells and then placed on top of acellular pancreatic slices (25 and 50 μm thickness). These clusters tended to connect to the acellular matrix, with visible cells located in the periphery of the clusters interacting with the ECM network of the bioscaffold slices and continued to produce insulin. This study provided evidence on how to improve and accelerate the pancreas decellularization process, while maintaining its architecture and extracellular structure, aiming at pancreatic bioengineering.

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Objectives: Tissue engineering approaches via repopulation of acellular biological grafts provide an exciting opportunity to generate lung grafts for transplantation. Alveolar type 2 (AT2) cells are a promising cell source for re-epithelialization. There are however inherent limitations with respect to their survival and growth, thus impeding their usability for tissue engineering applications. This study investigates the use of mesenchymal stromal cells to support primary AT2 cells for recellularization of mouse lung scaffolds. Methods: AT2 cells and bone marrow-derived mesenchymal cells (BMC) were co-delivered to decellularized mouse lung scaffolds. Recellularized lungs were evaluated for cell surface coverage, viability, and differentiation at 1 and 4 days after cell seeding. Recellularization was evaluated via histological analysis and immunofluorescence. Results: Simultaneous delivery of AT2 and BMC into acellular lung scaffolds resulted in enhanced cell surface coverage and reduced AT2 cell apoptosis in the recellularized scaffolds at Day 1 but not Day 4. AT2 cell number decreased after 4 days in both of AT2 only and codelivery groups suggesting limited expansion potential in the scaffold. After retention in the scaffold, AT2 cells differentiated into Aqp5-expressing cells. Conclusions: Our results indicate that BMC support AT2 cell survival during the initial attachment and engraftment phase of recellularization. While our findings suggest only a short-term beneficial effect of BMC, our study demonstrates that AT2 cells can be delivered and retained in acellular lung scaffolds; thus with preconditioning and supporting cells, may be used for re-epithelialization. Selection and characterization of appropriate cell sources for use in recellularization, will be critical for ultimate clinical application.

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A significant hurdle for kidney tissue engineering is reproducing the complex three-dimensional structure of the kidney. In our study, a stepwise approach of generating a reproducible Xeno kidney scaffold from a goat kidney is described, which can be implanted and recellularized by host cells. We have proposed a combination of sodium dodecyl sulfate and Triton-X-100-based protocol to generate a reproducible Xeno kidney scaffold, which was then analyzed by histology, DNA quantification, SEM, and renal angiography. Further, a small portion from the cortico-medullar region of the acellular scaffold was implanted in the rat's kidney subcapsular pocket for a period of 1 month, to check the recruitment of host cells into the scaffold. Post implantation, the extracellular matrix of the scaffold was well preserved and it did not induce any damage or inflammation in the native kidney. Implantation of the Xeno scaffold resulted in apparent early vascularization which helped in the recruitment of the host cells, which was characterized by histology, immunohistochemistry, and scanning electron microscopy. Implanted Xeno scaffold showed AQP-1, Nephrin, α-SMA, and VEGF expression in proximal tubules and renal glomerulus. Importantly, Ki-67 and WTAP-expressing cells were also observed near proximal tubules suggesting a high level of proliferation in the scaffold. Thus, showing the potential of Xeno kidney development that can be recellularized by the host cell to engineer into a functional kidney.

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Allogeneic transplant organs are potentially highly immunogenic. The endothelial cells (ECs) located within the vascular system serve as the primary interface between the recipient's immune system and the donor organ, playing a key role in the alloimmune response. In this study, we investigated the potential use of recipient-derived ECs in a vein recellularization model. In this study, human iliac veins underwent complete decellularization using a Triton X-100 protocol. We demonstrated the feasibility of re-endothelializing acellular blood vessels using either human umbilical cord vein endothelial cell or human venous-derived ECs, with this re- endothelialization being sustainable for up to 28 days in vitro. The re-endothelialized veins exhibited the restoration of vascular barrier function, along with the restoration of innate immunoregulatory capabilities, evident through the facilitation of monocytic cell transmigration and their polarization toward a macrophage phenotype following transendothelial extravasation. Finally, we explored whether recellularization with EC of a different donor could prevent antibody-mediated rejection. We demonstrated that in chimeric vessels, allogeneic EC became a target of the humoral anti-donor response after activation of the classical immune complement pathway whereas autologous EC were spared, emphasizing their potential utility before transplantation. In conclusion, our study demonstrates that replacement of EC in transplants could reduce the immunological challenges associated with allogeneic grafts.

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Introduction: The decellularization and recellularization is a promising approach for tissue engineering and regenerative medicine. However, the decellularization process depletes important components like glycosaminoglycans (GAGs), affecting cell attachment and causing immunogenicity. Studies have explored various surface modification strategies to enhance recellularization. Methods: To optimize the decellularization method, we employed whole kidney perfusion and slice kidney immersion/agitation techniques. The decellularized extracellular matrix (dECM) was then analyzed using hematoxylin and eosin (H&E) staining, scanning electron microscope (SEM), and DNA quantification. To enhance cell proliferation efficiency, albumin coating and rotating culture were applied. Also, we evaluated in vitro blood clot formation on the albumin-coated dECM by immersing it in blood. Results: After decellularization, the unique structures of the kidney were preserved whether cellular components were removed. Subsequently, we utilized albumin coating and rotating culture for recellularization, and observed that albumin-coated dECM not only promoted high cell proliferation rates but also prevented blood clot formation. Conclusion: The albumin-coated dECM promoted cell proliferation and reduced blood clot formation in vitro. Also, dynamic culture condition using rotating culture allowed for improved cellular penetration into the dECM, leading to a conductive environment for enhanced tissue infiltration. This new approach suggests that the combined utilization of albumin coating and rotating culture conditions can improve the efficiency of recellularization.

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Biomaterial templates play a critical role in establishing and bioinstructing three-dimensional cellular growth, proliferation and spatial morphogenetic processes that culminate in the development of physiologically relevant in vitro liver models. Various natural and synthetic polymeric biomaterials are currently available to construct biomimetic cell culture environments to investigate hepatic cell-matrix interactions, drug response assessment, toxicity, and disease mechanisms. One specific class of natural biomaterials consists of the decellularized liver extracellular matrix (dECM) derived from xenogeneic or allogeneic sources, which is rich in bioconstituents essential for the ultrastructural stability, function, repair, and regeneration of tissues/organs. Considering the significance of the key design blueprints of organ-specific acellular substrates for physiologically active graft reconstruction, herein we showcased the latest updates in the field of liver decellularization-recellularization technologies. Overall, this review highlights the potential of acellular matrix as a promising biomaterial in light of recent advances in the preparation of liver-specific whole organ scaffolds. The review concludes with a discussion of the challenges and future prospects of liver-specific decellularized materials in the direction of translational research.

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Acute Liver Failure (ALF) is a life-threatening illness characterized by the rapid onset of abnormal liver biochemistries, coagulopathy, and the development of hepatic encephalopathy. Extracorporeal bioengineered liver (BEL) grafts could offer a bridge therapy to transplant or recovery. The present study describes the manufacture of clinical scale BELs created from decellularized porcine-derived liver extracellular matrix seeded entirely with human cells: human umbilical vein endothelial cells (HUVECs) and primary human liver cells (PHLCs). Decellularized scaffolds seeded entirely with human cells were shown to adhere to stringent sterility and safety guidelines and demonstrated increased functionality when compared to grafts seeded with primary porcine liver cells (PPLCs). BELs with PHLCs were able to clear more ammonia than PPLCs and demonstrated lower perfusion pressures during patency testing. Additionally, to determine the full therapeutic potential of BELs seeded with PHLCs, longer culture periods were assessed to address the logistical constraints associated with manufacturing and transporting a product to a patient. The fully humanized BELs were able to retain their function after cold storage simulating a product transport period. Therefore, this study demonstrates the manufacture of bioengineered liver grafts and their potential in the clinical setting as a treatment for ALF.

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Corneal opacity is a leading cause of vision impairment and suffering worldwide. Transplantation can effectively restore vision and reduce chronic discomfort. However, there is a considerable shortage of viable corneal graft tissues. Tissue engineering may address this issue by advancing xeno-keratoplasty as a viable alternative to conventional keratoplasty. In particular, livestock decellularization strategies offer the potential to generate bioartificial ocular prosthetics in sufficient supply to match existing and projected needs. To this end, we have examined the best practices and characterizations that have supported the current state-of-the-art driving preclinical and clinical applications. Identifying the challenges that delimit activities to supplement the donor corneal pool derived from acellular scaffolds allowed us to hypothesize a model for keratoprosthesis applications derived from livestock combining 3D printing and decellularization.

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Introduction: Two-dimensional cell cultures have contributed substantially to lung cancer research, but 3D cultures are gaining attention as a new, more efficient, and effective research model. A model reproducing the 3D characteristics and tumor microenvironment of the lungs in vivo, including the co-existence of healthy alveolar cells with lung cancer cells, is ideal. Here, we describe the creation of a successful ex vivo lung cancer model based on bioengineered lungs formed by decellularization and recellularization. Methods: Human cancer cells were directly implanted into a bioengineered rat lung, which was created with a decellularized rat lung scaffold reseeded with epithelial cells, endothelial cells and adipose-derived stem cells. Four human lung cancer cell lines (A549, PC-9, H1299, and PC-6) were applied to demonstrate forming cancer nodules on recellularized lungs and histopathological assessment were made among these models. MUC-1 expression analysis, RNA-seq analysis and drug response test were performed to demonstrate the superiority of this cancer model. Results: The morphology and MUC-1 expression of the model were like those of lung cancer in vivo. RNA sequencing revealed an elevated expression of genes related to epithelial-mesenchymal transition, hypoxia, and TNF-α signaling via NF-κB; but suppression of cell cycle-related genes including E2F. Drug response assays showed that gefitinib suppressed PC-9 cell proliferation equally well in the 3D lung cancer model as in 2D culture dishes, albeit over a smaller volume of cells, suggesting that fluctuations in gefitinib resistance genes such as JUN may affect drug sensitivity. Conclusions: A novel ex vivo lung cancer model was closely reproduced the 3D structure and microenvironment of the actual lungs, highlighting its possible use as a platform for lung cancer research and pathophysiological studies.