Decellularization Methods Research Articles

Abstract 3831: Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms

Abstract Triple-negative breast cancer (TNBC) is an aggressive, heterogenous disease that lacks approved targeted therapeutics. Due to the complexity of tumor microenvironment (TME) interactions in TNBC, it is crucial to identify therapeutic targets that regulate the extracellular matrix (ECM). Traditional 2D TNBC models do not accurately mimic the cell-matrix interactions and structural matrix proteins that are key in maintaining the unique TME in individual patient tumors. Here, we introduce our decellularized TNBC patient-derived xenograft (PDX) models to address this gap in knowledge. Utilizing our decellularized models, we can investigate the unique ECM composition and cell-matrix interactions in PDX models from understudied patients with diverse clinical presentations. We can also use our decellularized tumor scaffolds as drug testing models. We utilize our tissue decellularization method on PDX tumor scaffolds derived from two of our established TNBC PDX models (TU-BcX-56S and TU-BcX-4QX). Decellularization of tumor scaffolds was confirmed by DNA quantification and histological analysis (H&E, Masson’s Trichrome, and Movat’s staining). MDA-MB-231 cells expressing GFP and luciferase were seeded onto scaffolds and treated with 100nM of Paclitaxel, a microtubule-stabilizing cytotoxic chemotherapeutic, or DMSO for 48 hours. Concordantly, cells in 2D culture were treated with 100nM of Paclitaxel or DMSO for 48 hours. Using the IVIS imaging system, we measured the bioluminescence of cells on our seeded scaffolds before treatment and at 24 and 48 hours after treatment. Cells in 2D culture showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Cells seeded on TU-BcX-56S scaffold also showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Interestingly, cells seeded on TU-BcX-4QX scaffold showed a decrease in bioluminescence in both the treatment and control groups at 24 and 48 hours, albeit there was no significant difference. Using qPCR, we investigated scaffold regulation of ECM gene composition in our treatment and control groups. These results demonstrate that the unique ECM composition and architecture of these scaffolds are important drivers of cell behavior. Here, we demonstrate that our decellularized PDX tumor scaffold models can be used as drug testing platforms. Furthermore, our models recapitulate key structural matrix proteins in the TNBC TME, can be used to investigate ECM onco-architecture and composition, and can be used in therapeutic discovery research. Citation Format: Maryl Wright, Khoa Nguyen, Elizabeth Martin, Melyssa Bratton, Bridgette Collins-Burow, Matthew Burow. Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3831.

In Vitro Tissue Reconstruction Using Decellularized Pericardium Cultured with Cells for Ligament Regeneration.

Recent applications of decellularized tissues have included the ectopic use of their sheets and powders for three-dimensional (3D) tissue reconstruction. Decellularized tissues are fabricated with the desired functions to employ them to a target tissue. The aim of this study was to develop a 3D reconstruction method using a recellularized pericardium to overcome the difficulties in cell infiltration into tight and dense tissues, such as ligament and tendon tissues. Decellularized pericardial tissues were prepared using the high hydrostatic pressurization (HHP) and surfactant methods. The pericardium consisted of bundles of aligned fibers. The bundles were slightly disordered in the surfactant decellularization method compared to the HHP decellularization method. The mechanical properties of the pericardium were maintained after the HHP and surfactant decellularizations. The HHP-decellularized pericardium was rolled up into a cylindrical formation. Its mechanical behavior was similar to that of a porcine anterior cruciate ligament in tensile testing. NIH3T3, C2C12, and mesenchymal stem cells were adhered with elongation and alignment on the HHP- and surfactant-decellularized pericardia, with dependences on the cell type and decellularization method. When the recellularized pericardium was rolled up into a cylinder formation and cultured by hanging circulation for 2 days, the cylinder formation and cellular elongation and alignment were maintained on the decellularized pericardium, resulting in a layer structure of cells in a cross-section. According to these results, the 3D-reconstructed decellularized pericardium with cells has the potential to be an attractive alternative to living tissues, such as ligament and tendon tissues.

The in vitro analysis of migration and polarity of blastema cells in the extracellular matrix derived from bovine mesenteric in the presence of fibronectin.

Cell migration is an essential process in embryonic development, wound healing, and pathological conditions. Our knowledge of cell migration is often based on the two dimentional evaluation of cell movement, which usually differs from what occurred in vivo. In this study, we investigated cellular migration from blastema tissue toward bovine decellularized mesentery tissue. In this regard, fibronectin (FN) was assessed to confirm cell migration. Therefore, we established a cell migration model using blastema cells migration toward the extracellular matrix derived from bovine mesenteric tissue. A physiochemical decellularization method was utilized based on freeze-thaw cycles and agitation in sodium dodecyl sulfate and Triton X-100 to remove cells from the extracellular matrix (ECM) of bovine mesenteric tissue. These types of matrices were assembled by the rings of blastema tissues originated from the of New Zealand rabbits pinna and cultured in a medium containing FN in different days in vitro, and then they are histologically evaluated, and the expression of the Tenascin C gene is analyzed. By means of tissue staining and after confirmation of the cell removal from mesenteric tissue, polarity, and migration of blastema cells was observed in the interaction site with this matrix. Also, the expression of the Tenascin C gene was assessed on days 15 and 21 following the cell culture process. The results showed that the three dimentional model of cellular migration of blastema cells along with the ECM could be a suitable model for investigating cell behaviors, such as polarity and cell migration in vitro.

Research Progress on the Immunogenicity and Regeneration of Acellular Adipose Matrix: A Mini Review.

Acellular adipose matrix (AAM) has received increasing attention for soft tissue reconstruction, due to its abundant source, high long-term retention rate and in vivo adipogenic induction ability. However, the current decellularization methods inevitably affect native extracellular matrix (ECM) properties, and the residual antigens can trigger adverse immune reactions after transplantation. The behavior of host inflammatory cells mainly decides the regeneration of AAM after transplantation. In this review, recent knowledge of inflammatory cells for acellular matrix regeneration will be discussed. These advancements will inform further development of AAM products with better properties.

Peripheral Nerve Decellularization for In Vitro Extracellular Matrix Hydrogel Use: A Comparative Study.

The rise of tissue-engineered biomaterials has introduced more clinically translatable models of disease, including three-dimensional (3D) decellularized extracellular matrix (dECM) hydrogels. Specifically, decellularized nerve hydrogels have been utilized to model peripheral nerve injuries and disorders in vitro; however, there lacks standardization in decellularization methods. Here, rat sciatic nerves of varying preparations were decellularized using previously established methods: sodium deoxycholate (SD)-based, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS)-based, and apoptosis-mediated. These nerves were characterized for cellular debris removal, ECM retention, and low cytotoxicity with cultured Schwann cells. The best preparations of each decellularization method were digested into dECM hydrogels, and rheological characterization, gelation kinetics, and confocal reflectance imaging of collagen fibril assembly were performed. It was determined that the SD-based method with nerve epineurial removal best maintained the overall ECM composition and mechanical properties of physiological peripheral nerves while efficiently stripping the scaffolds of tissue-specific cells and debris. This method was then utilized as a culture platform for quiescent Schwann cells and cancer-nerve crosstalk. Hydrogel-embedded Schwann cells were found to have high viability and act in a more physiologically relevant manner than those cultured in monolayers, and the hydrogel platform allowed for the activation of Schwann cells following treatment with cancer secreted factors. These findings establish a standard for peripheral nerve decellularization for usage as a dECM hydrogel testbed for in vitro peripheral nerve disease modeling and may facilitate the development of treatments for peripheral nerve disease and injury.

Assessing the biocompatibility of bovine tendon scaffold, a step forward in tendon tissue engineering.

Tendon is a collagen-enriched, tough, and intricately arranged connective tissue that connects muscle to the bone and transmits forces, resulting in joint movement. High mechanical demands can affect normal tissues and may lead to severe disorders, which usually require replacement of the damaged tendon. In recent decades, various decellularization methods have been studied for tissue engineering applications. One of the major challenges in tendon decellularization is preservation of the tendon extracellular matrix (ECM) architecture to maintain natural tissue characteristics. The aim of the present study was to create a decellularized bovine Achilles tendon scaffold to investigate its cytocompatibility with seeded hAd-MSCs (human adipose derived-mesenchymal stem cells) and blastema tissue in vitro. Here, we describe a reliable procedure to decellularize bovine Achilles tendon using a combination of physical and chemical treatments including repetitive freeze-thaw cycles and the ionic detergent SDS, respectively. The decellularization effectiveness and cytocompatibility of the tendon scaffolds were verified by histological studies and scanning electron microscopy for up to 30days after culture. Histological studies revealed hAd-MSC attachment and penetration into the scaffolds at 5, 10, 15 and 20days of culture. However, a decrease in cell number was observed on days 25 and 30 after culture in vitro. Moreover, migration of the blastema tissue cells into the scaffold were shown at 10 to 25days post culture, however, destruction of the scaffolds and reduction in cell number were observed on 30th day after culture. Our results suggest that this decellularization protocol is an effective and biocompatible procedure which supports the maintenance and growth of both hAd-MSCs and blastema cells, and thus might be promising for tendon tissue engineering.

Preliminary Study on the Antigen-Removal from Extracellular Matrix via Different Decellularization.

Due to the abundance of bioactive components, surficial decoration with cell-derived extracellular matrix (ECM) is a promising strategy to improve the biological functionality of the tissue engineering scaffolds. However, decellularization is necessary to remove antigenic components in the ECM that may trigger adverse immune response. Freeze-thaw (FT) cycles and treatment with Triton X-100/ammonium hydroxide (TN) are two commonly used decellularization methods for ECM, but their effects on both growth factor retention and antigen removal are still controversial. The objectives of this study are to compare the preservation of ECM texture and beneficial ingredients and the removal of cellular antigens by these two methods. First, the constructs combined bone marrow mesenchymal stem cell-derived ECM and poly(lactic-co-glycolic acid) (PLGA) membrane are prepared and decellularized using FT and TN treatments. Moreover, the effects of decellularization on the ultrastructure and the composition of ECM-decorated PLGA membrane are compared by scanning electron microscope observation and protein quantification. Furthermore, the ECM deposited on PLGA is stripped off and then implanted subcutaneously in rats, and the host macrophage and local lymphocyte responses were investigated. Finally, ECM-decorated porous PLGA scaffolds are implanted into rat calvarial defects, and the new bone formation is evaluated. Our results showed that both methods effectively removed DNA. TN treatment partially retained collagen, glycosaminoglycan, bone morphogenetic protein-2, and vascular endothelial growth factor, and better preserved structural integrity than FT treatment. ECM implants decellularized by both methods induced a mild host response after subcutaneous implantation. Although the total content of residual DNA in the two ECMs digested by the DNA enzyme seemed to be similar and very low, the interfaces between implanted materials and natural tissues in the TN group recruited lower numbers of CD68+ macrophages, CD68+CD86+ (M1) macrophages, and CD4+ T lymphocytes than that in FT group, implying that there exist other ECM antigens to influence immune response besides DNA. Furthermore, ECM-decorated scaffolds decellularized by TN treatment induced greater bone formation than that of bare scaffolds in vivo, demonstrating the effective retention of ECM bioactive components after decellularization. This study showed that TN treatment was a more effective and safer decellularization method than FT cycles. Impact statement Decellularization is a prerequisite for extracellular matrix (ECM) application, but there is still no standard for its selection. This study demonstrated that detergent treatment was more effective than freeze-thaw (FT) cycles in removing ECM antigens besides DNA, and the prepared ECM elicited a milder allogenic immune response, which ensured the safety of ECM. Moreover, detergent better preserved the ECM integrity than FT cycles, and effectively retained growth factors, and the decellularized ECM-decorated scaffolds significantly promoted bone repair, which ensured the effectiveness of ECM. This study provides the theoretical and experimental bases for the decellularization strategy of ECM-modified tissue engineering scaffolds.

Decellularization of human amniotic membrane using detergent-free methods: Possibilities in tissue engineering

The human amniotic membrane (HAM) is widely used as a natural scaffold in tissue engineering due to its excellent biological characteristics, including anti-microbial, anti-inflammatory, low immunogenicity, and pro-angiogenic properties. This study aimed to develop simple and cost-effective protocols for the decellularization of HAM (d-HAM) using detergent-free methods, i.e., mechanical force (brushing) and physical treatment (heating 45–55 °C). The effectiveness of the methods of interest was compared with a chemical-based approach (EDTA + NaOH + NH4Cl). The prepared d-HAMs were characterized using a series of physico-chemical, mechanical, and biological evaluations. The results from DAPI staining revealed that the chemical method could completely remove epithelial cells from HAM, while the two other approaches only reduced the number of epithelial cells. All three decellularization methods led to a sharp reduction (P < 0.001) in the DNA content of the tissue samples (< 50 ng/mg). Histological evaluations showed the preservation of the d-HAMs’ integrity along with the conservation of collagen and glycosaminoglycans (GAGs). Although the chemical method caused the lowest mechanical deterioration (3.55 MPa in ultimate tensile stress), the mechanical method preserved the highest hydroxyproline levels (3.13 mg/mL). On the other hand, the physical method (heating to 45 and 50 °C) encouraged cell proliferation more than the chemical and mechanical approaches. All of the samples proved to be suitable for cell attachment and could induce cell migration. In conclusion, the present study showed that the use of detergent-free protocols is applicable for the decellularization of HAM, and the obtained tissues may be considered as inexpensive dressings for numerous tissue engineering applications.

Kartogenin releasing decellularized umbilical cord Wharton's jelly scaffold promotes rotator cuff fibrocartilaginous interface regeneration

Fibrocartilaginous regeneration at the tendon-to-bone interface is a major challenge for rotator cuff tear repair. We fabricated a decellularized umbilical cord Wharton’s jelly (DUCWJ) scaffold by decellularization method and performed histological and proteomic analyses. We then synthesized a kartogenin-conjugated DUCWJ (DUCWJ-KGN) scaffold using an EDC/NHS catalyst. 1H NMR, Fluorescence staining, and FTIR confirmed that the carboxyl group of KGN was covalently coupled with the free amine groups in the Wharton’s jelly (WJ) matrix, and the in vitro release profiles of KGN from the DUCWJ-KGN scaffold showed sustained release for 6 weeks. Both the DUCWJ and DUCWJ-KGN scaffolds had excellent biocompatibility and biofunctionality, supporting cell attachment, migration, and proliferation, and DUCWJ-KGN scaffolds the KGN-loaded scaffold could induce higher chondrogenic markers expression. The DUCWJ-KGN scaffold could also promote fibrocartilaginous regeneration at the rotator cuff tendon-to-bone interface, which indicates that the DUCWJ-KGN scaffold is a promising enthesis to enhance fibrocartilaginous interface regeneration.

Decellularization in Tissue Engineering and Regenerative Medicine: Evaluation, Modification, and Application Methods.

Reproduction of different tissues using scaffolds and materials is a major element in regenerative medicine. The regeneration of whole organs with decellularized extracellular matrix (dECM) has remained a goal despite the use of these materials for different purposes. Recently, decellularization techniques have been widely used in producing scaffolds that are appropriate for regenerating damaged organs and may be able to overcome the shortage of donor organs. Decellularized ECM offers several advantages over synthetic compounds, including the preserved natural microenvironment features. Different decellularization methods have been developed, each of which is appropriate for removing cells from specific tissues under certain conditions. A variety of methods have been advanced for evaluating the decellularization process in terms of cell removal efficiency, tissue ultrastructure preservation, toxicity, biocompatibility, biodegradability, and mechanical resistance in order to enhance the efficacy of decellularization methods. Modification techniques improve the characteristics of decellularized scaffolds, making them available for the regeneration of damaged tissues. Moreover, modification of scaffolds makes them appropriate options for drug delivery, disease modeling, and improving stem cells growth and proliferation. However, considering different challenges in the way of decellularization methods and application of decellularized scaffolds, this field is constantly developing and progressively moving forward. This review has outlined recent decellularization and sterilization strategies, evaluation tests for efficient decellularization, materials processing, application, and challenges and future outlooks of decellularization in regenerative medicine and tissue engineering.

Interdisciplinary Methods for Zoonotic Tissue Acellularization for Natural Heart Valve Substitute of Biomimetic Materials.

The goal of this work was to create a bioactive tissue-based scaffold using multi-disciplinary engineering materials and tissue engineering techniques. Materials & methods: Physical techniques such as direct laser interference lithography and proton radiation were selected as alternative methods of enzymatic and chemical decellularization to remove cells from a tissue without degradation of the extracellular matrix nor its protein structure. This study was an attempt to prepare a functional scaffold for cell culture from tissue of animal origin using new physical methods that have not been considered before. The work was carried out under full control of the histological and molecular analysis. Results & conclusions: The most important finding was that the physical methods used to obtain the decellularized tissue scaffold differed in the efficiency of cell removal from the tissue in favour of the laser method. Both the laser method and the proton method exhibited a destructive effect on tissue structure and the genetic material in cell nuclei. This effect was visible on histology images as blurred areas within the cell nucleus. The finite element 3D simulation of decellularization process of the three-layer tissue of animal origin sample reflected well the mechanical response of tissue described by hyperelastic material models and provided results comparable to the experimental ones.

725 Case Series: New Porcine Placental ECM for Burn Injuries

IntroductionHuman amniotic membrane (HAM) has been used as a biologic dressing for burn wounds since 1955 but limited due to availability, size, and processing costs. In 2021 a new porcine placental product was FDA-approved overcoming challenges with human-sourced products. Our study is the first case series to report outcomes using porcine placental extracellular matrix (PPECM) in the use of adult burn patients.MethodsAdults with thermal burns resulting in partial-thickness burn wounds (PTBW) were consented and included in the study from 03/2021 to 09/2021. Patients with full-thickness injures, concomitant trauma, or adverse beliefs to porcine products were not included in the study. Serial still images and initial wound measurements were obtained intraoperatively and post-operatively. PPECM trial product processed with a proprietary decellularization method to produce single sheets up to 15x20cm was approved by the facility value assessment committee. Adverse events were defined a priori as infection, increased pain or itching relative to adjacent autografts, or failure to heal. Infection was defined as a PPECM treatment site requiring any change from standard of care or initiation of local or systemic antibiotics. Pain was assessed using a visual analogue scale. Itching was assessed at discharge and follow-up. Healing was assessed using the FDA guidance for wound closure with 2 consecutive visits 2 weeks apart demonstrating 100% epithelialization without drainage or dressing requirements.ResultsFour patients were treated during the study period with wounds involving the torso and major joints such as the hands/wrists and knees. None of the PPECM wounds demonstrated failure to heal or required revision excision, or autograft. None of the PPECM wounds had evidence of infection. PPECM wounds had decreased pain/itching relative to adjacent burn wounds which were treated with split-thickness autograft, autologous skin cell suspension, or allogeneic cultured skin substitute (VAS mean 1 vs 3.1). Healing was noted in all wounds at 1-week primary dressing removal with confirmation at 2-week interval follow-up.ConclusionsPPECM treatment of PTBW was not associated with adverse events and resulted in favorable outcomes clinically. The large size, ease of use, and lower costs relative to HAM is an intriguing alternative for PTBW. Comparative studies are needed in the field to determine best practices and overall value.

Novel Decellularization Method for Tissue Slices.

Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue’s vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices—with an emphasis on lung—while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies.

Determining the optimal pancreatic decellularization protocol, taking into account tissue morphological features

Introduction. Developing a tissue-engineered pancreatic construct (TEPC) involves a search for matrices/scaffolds capable of mimicking the structure and composition of the natural extracellular matrix (ECM), which is an important component of the tissue microenvironment. A cell-free, tissue-specific matrix obtained from pancreas decellularization seems to be the most suitable for creation of a TEPC. The choice of pancreatic tissue decellularization protocol should take into account the morphological characteristics of the original pancreas. Preservation of the architectonics and composition of the native tissue in the decellularized pancreas matrix (DPM), and the presence of native ECM components allow for creation of conditions for prolonged vital activity of functionally active islet (insulin-producing) cells when creating TEPC.Objective: to determine the optimal parameters for decellularization of deceased donor pancreas with fibrosis, lipomatosis, and without pronounced signs of fibrosis and lipomatosis.Materials and methods. We used the caudal part of the pancreas obtained after multiorgan procurement from deceased donors, which was unsuitable for transplantation. Tissue-specific matrix was obtained by a combination of physical and chemical methods of pancreatic decellularization. A freeze-thaw cycle protocol and two protocols using osmotic shock were used. Samples of initial pancreatic tissue and decellularized fragments were subjected to histological analysis.Results. It was shown that a physico-chemical method with freeze-thaw cycles is suitable for effective pancreatic decellularization in severe lipomatosis; a physico-chemical method using osmotic shock, but different protocol variants, is suitable for pancreas with diffuse fibrosis and for pancreas without pronounced signs of fibrosis and lipomatosis.Conclusion. For complete human pancreatic decellularization, the protocol should be correlated with histological features of the original tissue.

Introduction of an efficient method for placenta decellularization with high potential to preserve ultrastructure and support cell attachment.

The placenta, as a large discarded tissue and rich in extracellular matrix (ECM), is an excellent candidate for biological scaffolds in reconstructive medicine. Considering the importance of ECM structure in cell fate, the aim of this study was to achieve human placenta decellularization protocol that preserve the structure of scaffolds. Thus, human placenta was decellularized by four protocols and decellularization efficacy was compared by hematoxylin and eosin (H&E), 4',6-diamidino-2-phenylindole (DAPI) staining, and DNA measurement. Decellularized placenta structure preservation was assessed by Masson's trichrome staining, scanning electron microscopy (SEM), and immunofluorescence (IF) for collagen I, IV, and fibronectin. Finally, liquid displacement measured scaffolds' porosity. After culturing menstrual blood-derived stem cells (MenSCs) on placenta scaffolds, cell adhesion was investigated by SEM imaging, and cell viability and proliferation were assessed by MTT assay. According to H&E and DAPI staining, only protocols 1 and 3 could completely remove cells from the scaffolds. DNA measurements confirmed a significant reduction in the genetic material of decellularized scaffolds compared to native placenta. According to Masson's trichrome, IF, and SEM imaging, scaffold structure is better preserved in P3 than P1 protocol. Liquid displacement showed higher porosity of P3 scaffold than P1. SEM imaging confirmed cells adhesion to the decellularized placenta, and the attached cells showed good viability and maintained their proliferative capacity, indicating the suitability of the scaffolds for cell growth. Results introduced an optimized protocol for placenta decellularization that preserves the scaffold structure and supports cell adhesion and proliferation.

Decellularization Following Fixation of Explanted Aortic Valves as a Strategy for Preserving Native Mechanical Properties and Function.

Mechanical or biological aortic valves are incorporated in physical cardiac simulators for surgical training, educational purposes, and device testing. They suffer from limitations including either a lack of anatomical and biomechanical accuracy or a short lifespan, hence limiting the authentic hands-on learning experience. Medical schools utilize hearts from human cadavers for teaching and research, but these formaldehyde-fixed aortic valves contort and stiffen relative to native valves. Here, we compare a panel of different chemical treatment methods on explanted porcine aortic valves and evaluate the microscopic and macroscopic features of each treatment with a primary focus on mechanical function. A surfactant-based decellularization method after formaldehyde fixation is shown to have mechanical properties close to those of the native aortic valve. Valves treated in this method were integrated into a custom-built left heart cardiac simulator to test their hemodynamic performance. This decellularization, post-fixation technique produced aortic valves which have ultimate stress and elastic modulus in the range of the native leaflets. Decellularization of fixed valves reduced the valvular regurgitation by 60% compared to formaldehyde-fixed valves. This fixation method has implications for scenarios where the dynamic function of preserved valves is required, such as in surgical trainers or device test rigs.

Preparation and Properties of Decellularized Sheep Kidney Derived Matrix Scaffolds

Scaffolds from tissues or organs have nanoscale microstructures. Derived matrix scaffolds prepared by decellularized method can provide more cell attachment sites, which is conducive to cell adhesion, proliferation, differentiation and other physiological activities on scaffolds. In this study, the sheep kidney decellularized matrix scaffold was prepared by the method of decellularization. Due to the poor mechanical properties of the decellularized matrix, the cross linking method was adopted to enhance its mechanical properties. The decellularization efficiency of sheep renal matrix scaffolds was observed by scanning electron microscopy and histological staining, and the biocompatibility of the scaffolds was investigated by inoculating adipose derived stem cells. It was found that the scaffold had good decellularization effect and good pore structure.

Decellularization and Recellularization Methods for Avian Lungs: An Alternative Approach for Use in Pulmonary Therapeutics.

The shortage of compatible allogeneic organs and an increase in the number of patients requiring long-term lung assist devices while waiting for lung transplantation have motivated scientists to explore alternatives to bioengineer new lungs, including through decellularization and recellularization processes. A novel approach for bioengineering an extracorporeal membrane oxygenator is based on the parenchymal structure of avian lungs which utilizes a cross-current unidirectional flow of air and blood rather than bidirectional airflow, and thus eliminates dead-space ventilation. This provides more efficient gas exchange than mammalian lungs. The novel approach utilized is to decellularize avian lungs and then to recellularize with patient-derived human lung epithelial and vascular endothelial cells with the goal of creating a fully functional structure that can be used as a gas-exchange device. Here, we present avian lung decellularization and recellularization methods for chicken and emu lungs, in order to study both small- and large-scale avian lung models. For decellularization, a detergent-based protocol is utilized, and different techniques are used to validate the de- and recellularization of those lungs, including microscopy, mass spectrometry, and immunohistochemical analyses. For recellularization, techniques for seeding different human lung cell types into the decellularized scaffolds are presented.

Perfusion decellularization for vascularized composite allotransplantation.

Vascularized composite allotransplantation is becoming the emerging standard for reconstructive surgery treatment for patients with limb trauma and facial injuries involving soft tissue loss. Due to the complex immunogenicity of composite grafts, patients who undergo vascularized composite allotransplantation are reliant on lifelong immunosuppressive therapy. Decellularization of donor grafts to create an extracellular matrix bio-scaffold provides an immunomodulatory graft that preserves the structural and bioactive function of the extracellular matrix. Retention of extracellular matrix proteins, growth factors, and signaling cascades allow for cell adhesion, migration, proliferation, and tissue regeneration. Perfusion decellularization of detergents through the graft vasculature allows for increased regent access to all tissue layers, and removal of cellular debris through the venous system. Grafts can subsequently be repopulated with appropriate cells through the vasculature to facilitate tissue regeneration. The present work reviews methods of decellularization, process parameters, evaluation of adequate cellular and nuclear removal, successful applications of perfusion decellularization for use in vascularized composite allotransplantation, and current limitations.

A Fast and Efficient Decellularization Method for Tissue Slices.

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue's vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2-3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies. This protocol was validated in: Front Bioeng Biotechnol (2022), DOI: 10.3389/fbioe.2022.832178.