Organ Decellularization Research Articles

Decellularization and Their Significance for Tissue Regeneration in the Era of 3D Bioprinting.

Three-dimensional bioprinting is an emerging technology that has high potential application in tissue engineering and regenerative medicine. Increasing advancement and improvement in the decellularization process have led to an increase in the demand for using a decellularized extracellular matrix (dECM) to fabricate tissue engineered products. Decellularization is the process of retaining the extracellular matrix (ECM) while the cellular components are completely removed to harvest the ECM for the regeneration of various tissues and across different sources. Post decellularization of tissues and organs, they act as natural biomaterials to provide the biochemical and structural support to establish cell communication. Selection of an effective method for decellularization is crucial, and various factors like tissue density, geometric organization, and ECM composition affect the regenerative potential which has an impact on the end product. The dECM is a versatile material which is added as an important ingredient to formulate the bioink component for constructing tissue and organs for various significant studies. Bioink consisting of dECM from various sources is used to generate tissue-specific bioink that is unique and to mimic different biometric microenvironments. At present, there are many different techniques applied for decellularization, and the process is not standardized and regulated due to broad application. This review aims to provide an overview of different decellularization procedures, and we also emphasize the different dECM-derived bioinks present in the current global market and the major clinical outcomes. We have also highlighted an overview of benefits and limitations of different decellularization methods and various characteristic validations of decellularization and dECM-derived bioinks.

Macroscopic and microscopic features of pancreatic scaffold generated by SDS-based decellularization using multiple needle injections

Background: Pancreatic tissue engineering requires a scaffold in addition to cells and signaling. An adequate scaffold no longer contains cells but retains its extracellular matrix. Biological scaffolds from organ or tissue decellularization are widely used because they can retain the extracellular matrix essential for cell proliferation and differentiation. Pancreatic scaffolds were developed for pancreatic tissue engineering through various decellularization techniques. Available decellularization techniques have advantages and disadvantages, and the development of new techniques is necessary. This study aims to characterize pancreatic scaffold generated by SDS-based decellularization using multiple needle injections. Method: The rat pancreas was isolated and injected with multiple SDS with graded concentration (0,1-1%) using a 1 cc syringe. Scaffold characterization was performed using hematoxylin-eosin (H&E) staining. Results: The results obtained were the loss of cells in the pancreatic scaffold with intact extracellular matrix. Macroscopic appearance after decellularization showed a translucent, white, and flabby pancreas. Microscopic image after decellularization showed pancreatic tissue with cell loss but visible extracellular matrix. Conclusion: This study can produce an SDS-based decellularized pancreatic scaffold using multiple needle injections with adequate macroscopic and microscopic characteristics.

Nerve regeneration using decellularized tissues: challenges and opportunities.

In tissue engineering, the decellularization of organs and tissues as a biological scaffold plays a critical role in the repair of neurodegenerative diseases. Various protocols for cell removal can distinguish the effects of treatment ability, tissue structure, and extracellular matrix (ECM) ability. Despite considerable progress in nerve regeneration and functional recovery, the slow regeneration and recovery potential of the central nervous system (CNS) remains a challenge. The success of neural tissue engineering is primarily influenced by composition, microstructure, and mechanical properties. The primary objective of restorative techniques is to guide existing axons properly toward the distal end of the damaged nerve and the target organs. However, due to the limitations of nerve autografts, researchers are seeking alternative methods with high therapeutic efficiency and without the limitations of autograft transplantation. Decellularization scaffolds, due to their lack of immunogenicity and the preservation of essential factors in the ECM and high angiogenic ability, provide a suitable three-dimensional (3D) substrate for the adhesion and growth of axons being repaired toward the target organs. This study focuses on mentioning the types of scaffolds used in nerve regeneration, and the methods of tissue decellularization, and specifically explores the use of decellularized nerve tissues (DNT) for nerve transplantation.

Developing Bovine Brain-Derived Extracellular Matrix Hydrogels: a Screen of Decellularization Methods for Their Impact on Biochemical and Mechanical Properties.

Tissue models that recapitulate the key biochemical and physical aspects of the brain have been highly pursued in neural tissue engineering. Decellularization of native organs offers the advantage of preserving the composition of native extracellular matrix (ECM). Brain ECM has distinct features which play a major role in neural cell behavior. Cell instructive ligands and mechanical properties take part in the regulation of cellular processes in homeostasis and diseases. One of the main challenges in decellularization is maintaining mechanical integrity in reconstituted hydrogels and achieving physiologically relevant stiffness. The effect of the decellularization process on different mechanical aspects, particularly the viscoelasticity of brain-derived hydrogels, has not been addressed. In this study, we developed bovine brain-derived hydrogels for the first time. We pursued seven protocols for decellularization and screened their effect on biochemical content, hydrogel formation, and mechanical characteristics. We show that bovine brain offers an easily accessible alternative for in vitro brain tissue modeling. Our data demonstrate that the choice of decellularization method strongly alters gelation as well as the stiffness and viscoelasticity of the resulting hydrogels. Lastly, we investigated the cytocompatibility of brain ECM hydrogels and the effect of modulated mechanical properties on the growth and morphological features of neuroblastoma cells.

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

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

Capturing effects of blood flow on the transplanted decellularized nephron with intravital microscopy

Organ decellularization creates cell-free, collagen-based extracellular matrices that can be used as scaffolds for tissue engineering applications. This technique has recently gained much attention, yet adequate scaffold repopulation and implantation remain a challenge. Specifically, there still needs to be a greater understanding of scaffold responses post-transplantation and ways we can improve scaffold durability to withstand the in vivo environment. Recent studies have outlined vascular events that limit organ decellularization/recellularization scaffold viability for long-term transplantation. However, these insights have relied on in vitro/in vivo approaches that need enhanced spatial and temporal resolutions to investigate such issues at the microvascular level. This study uses intravital microscopy to gain instant feedback on their structure, function, and deformation dynamics. Thus, the objective of this study was to capture the effects of in vivo blood flow on the decellularized glomerulus, peritubular capillaries, and tubules after autologous and allogeneic orthotopic transplantation into rats. Large molecular weight dextran molecules labeled the vasculature. They revealed substantial degrees of translocation from glomerular and peritubular capillary tracks to the decellularized tubular epithelium and lumen as early as 12 h after transplantation, providing real-time evidence of the increases in microvascular permeability. Macromolecular extravasation persisted for a week, during which the decellularized microarchitecture was significantly and comparably compromised and thrombosed in both autologous and allogeneic approaches. These results indicate that in vivo multiphoton microscopy is a powerful approach for studying scaffold viability and identifying ways to promote scaffold longevity and vasculogenesis in bioartificial organs.

Effect of different decellularization protocols on reendothelialization with human cells for a perfused renal bioscaffold of the rat

BackgroundScaffolds for tissue engineering can be received by whole organ decellularization while maintaining the site-specific extracellular matrix and the vascular tree. One among other decellularization techniques is the perfusion-based method using specific agents e.g. SDS for the elimination of cellular components. While SDS can disrupt the composition of the extracellular matrix and impair the adherence and growth of site-specific cells there are indications that xenogeneic cell types may benefit from protein denaturation by using higher detergent concentrations. The aim of this work is to investigate the effect of two different SDS-concentrations (i.e. 0.66% and 3%) on the ability of human endothelial cells to adhere and proliferate in an acellular rat kidney scaffold.Material and methodsAcellular rat kidney scaffold was obtained by perfusion-based decellularization through the renal artery using a standardized protocol including SDS at concentrations of 0.66% or 3%. Subsequently cell seeding was performed with human immortalized endothelial cells EA.hy 926 via the renal artery. Recellularized kidneys were harvested after five days of pressure-controlled dynamic culture followed sectioning, histochemical and immunohistochemical staining as well as semiquantitative analysis.ResultsEfficacy of decellularization was verified by absence of cellular components as well as preservation of ultrastructure and adhesive proteins of the extracellular matrix. In semiquantitative analysis of recellularization, cell count after five days of dynamic culture more than doubled when using the gentle decellularization protocol with a concentration of SDS at 0.66% compared to 3%. Detectable cells maintained their endothelial phenotype and presented proliferative behavior while only a negligible fraction underwent apoptosis.ConclusionRecellularization of acellular kidney scaffold with endothelial cells EA.hy 926 seeded through the renal artery benefits from gentle decellularization procedure. Because of that, decellularization with a SDS concentration at 0.66% should be preferred in further studies and coculture experiments.

An Impact of Hypoxia and Macromolecular Crowders on Extracellular Matrix Deposition by Human Endometrial Mesenchymal Stromal Cells

The last decades are characterized by intensive development of extracellular matrix (ECM) biology. ECM binds cells in an integral tissue and controls the cell functions – from proliferation and differentiation to migration and apoptosis. Bioactive properties of ECM provide the wide perspectives of using in bioengineering and regenerative medicine. In this context, the ECM production by decellularization of organs, tissues or cell cultures is a key technology. To date, a problem of a rapid and large-scale production of bioactive ECM by cultured cells remains very relevant. Optimization of the ECM deposition conditions by human endometrial mesenchymal stromal cells (MESCs) had not been studied yet. Here, we investigated an impact of macromolecular compounds (crowders) – ficoll and PEG on efficiency of crucial ECM proteins deposition depending on both concentration and molecular weight of crowders under normoxia and hypoxia. According to immunofluorescence analysis, among all studied crowders, ficoll 400 had a potent effect on the production of ECM core proteins – fibronectin, type IV collagen and, in a lower rate, type III collagen. The MESCs incubation under hypoxia promoted the formation of a properly organized ECM structure as well as increase in efficiency of ECM protein deposition. Of note, in these conditions ficoll 400 accelerated the ECM production only in а low serum medium. Together, combination of ficoll 400, low serum medium and hypoxia provides the optimal conditions for ECM synthesis. The present work demonstrates for the first time the phenomenon of macromolecular crowding in the context of improving the conditions for deposition and organization of ECM by MESCs.

Angiogenesis and Re-endothelialization in decellularized scaffolds: Recent advances and current challenges in tissue engineering.

Decellularization of tissues and organs has recently become a promising approach in tissue engineering and regenerative medicine to circumvent the challenges of organ donation and complications of transplantations. However, one main obstacle to reaching this goal is acellular vasculature angiogenesis and endothelialization. Achieving an intact and functional vascular structure as a vital pathway for supplying oxygen and nutrients remains the decisive challenge in the decellularization/re-endothelialization procedure. In order to better understand and overcome this issue, complete and appropriate knowledge of endothelialization and its determining variables is required. Decellularization methods and their effectiveness, biological and mechanical characteristics of acellular scaffolds, artificial and biological bioreactors, and their possible applications, extracellular matrix surface modification, and different types of utilized cells are factors affecting endothelialization consequences. This review focuses on the characteristics of endothelialization and how to optimize them, as well as discussing recent developments in the process of re-endothelialization.

Easy-to-Assembly System for Decellularization and Recellularization of Liver Grafts in a Bioreactor.

Decellularization of organs creates an acellular scaffold, ideal for being repopulated by cells. In this work, a low-cost perfusion system was created to be used in the process of liver decellularization and as a bioreactor after recellularization. It consists of a glass chamber to house the organ coupled to a peristaltic pump to promote liquid flow through the organ vascular tree. The rats' liver decellularization was made with a solution of sodium dodecyl sulfate. The recellularization was made with 108 mesenchymal stromal/stem cells and cultivated for seven days. The decellularized matrices showed an absence of DNA while preserving the collagen and glycosaminoglycans quantities, confirming the efficiency of the process. The functional analyses showed a rise in lactate dehydrogenase levels occurring in the first days of the cultivation, suggesting that there is cell death in this period, which stabilized on the seventh day. Histological analysis showed conservation of the collagen web and some groups of cells next to the vessels. It was possible to establish a system for decellularization and a bioreactor to use for the recellularization method. It is easy to assemble, can be ready to use in little time and be easily sterilized.

Tissue engineering of decellularized pancreas scaffolds for regenerative medicine in diabetes.

Diabetes mellitus is a global disease requiring long-term treatment and monitoring. At present, pancreas or islet transplantation is the only reliable treatment for achieving stable euglycemia in Type I diabetes patients. However, the shortage of viable pancreata for transplantation limits the use of this therapy for the majority of patients. Organ decellularization and recellularization is emerging as a promising solution to overcome the shortage of viable organs for transplantation by providing a potential alternative source of donor organs. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have been performed, and show promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we provide an overview of the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with an outlook on the remaining work that needs to be done in order to realize the goal of using this technology to create bioengineered pancreata for transplantation in diabetes patients. STATEMENT OF SIGNIFICANCE: Pancreas or islet transplantation is a means of providing insulin-independence in diabetes patients. However, due to the shortage of viable pancreata, whole-organ decellularization and recellularization is emerging as a promising solution to overcome organ shortage for transplantation. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have shown promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we highlight the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with future work that needs to be done in order to realize clinical translation of bioengineered pancreata for transplantation in diabetes patients.

Advances in Recellularization of Decellularized Liver Grafts with Different Liver (Stem) Cells: Towards Clinical Applications.

Liver transplantation is currently the only curative therapy for patients with acute or chronic liver failure. However, a dramatic gap between the number of available liver grafts and the number of patients on the transplantation waiting list emphasizes the need for valid liver substitutes. Whole-organ engineering is an emerging field of tissue engineering and regenerative medicine. It aims to generate transplantable and functional organs to support patients on transplantation waiting lists until a graft becomes available. It comprises two base technologies developed in the last decade; (1) organ decellularization to generate a three-dimensional (3D) extracellular matrix scaffold of an organ, and (2) scaffold recellularization to repopulate both the parenchymal and vascular compartments of a decellularized organ. In this review article, recent advancements in both technologies, in relation to liver whole-organ engineering, are presented. We address the potential sources of hepatocytes and non-parenchymal liver cells for repopulation studies, and the role of stem-cell-derived liver progeny is discussed. In addition, different cell seeding strategies, possible graft modifications, and methods used to evaluate the functionality of recellularized liver grafts are outlined. Based on the knowledge gathered from recent transplantation studies, future directions are summarized.

A Systematic Review of Tissue Engineering of a Total Artificial Heart: Focus on Perfusion Decellularization

Introduction: A leading cause of death worldwide is heart disease, and in the United States alone, 6.2 million individuals live with heart failure. The recent ability to recreate the vascular network of cardiac Extracellular Matrix (ECM) suggests the feasibility of engineering whole heart constructs. Tissue engineering method such as perfusion decellularization allows for effective removal of nearly all cellular composition of a heart while maintaining the native mechanical integrity of the scaffold. This scaffold can be reseeded with stem cells in the hopes of generating a functional heart. The goal of this systematic review was to assess the various perfusion decellularization methods and its effects upon the biologic scaffold. Methodology: A comprehensive systematic literature review search was carried out through Pubmed, MEDLINE and Google Scholar using the search terms “whole heart decellularization”, “whole organ decellularization” and “perfusion decellularization”. The inclusion criteria for this research were as follows: scholarly or peer-reviewed studies, published within the last 20 years in the English language, and primary studies in which whole cardiac organ decellularization was performed. Chosen articles were those examining the various decellularization methods and its effects upon the biologic scaffold. Excluded were studies regarding heart valve decellularization, tissue decellularization, cardiac patch, tissue recellularization, articles in foreign languages, articles dated prior to 2001, literature reviews, systematic reviews, and duplicate articles. Results: Chemical agents such as acid and bases cause hydrolytic degradation of biomolecules which could reduce ECM strength and eliminate growth factors from the matrix. Compared to other detergents such as Triton X-100, sodium dodecyl sulfate yields a more complete removal of nuclear material. A combination of these various approaches has shown an increased efficacy of the decellularization process. The decellularization of a large solid organ such as the heart requires several sophisticated steps. Perfusion decellularization is achieved via anterograde or retrograde perfusion of the intrinsic vascular network of the heart by utilizing decellularizing agents (i.e., chemicals and/or enzymes). Hodgson et al., 2017 demonstrates a combined strategy in which the decellularization of porcine hearts is accomplished in 24 hours and results in 98% DNA removal with only 6 hours of detergent exposure. Conclusion: There have been significant advances to address the heart disease epidemic. A promising approach is perfusion decellularization which allows for complete DNA content removal while maintaining the ECM scaffold integrity. This scaffold is then re-cellularized with stem cells in the hopes of creating an artificial heart. There are several challenges that need to be surpassed before bio-artificial hearts can be used to replace in vivo function. Future research is directed towards optimizing types of cells and cell sources used to repopulate decellularized hearts, seeding strategies and bioreactor systems to provide in vitro conditions required for organ maturation.

Characterization of a decellularized rat larynx: comparison between microscopy techniques

BackgroundNo effective method has yet been developed to efficiently reconstruct the larynx and restore its function. Decellularization has recently been tested for this purpose with very promising results. The goal of decellularization is to remove cells leaving an intact scaffold made of an extracellular matrix (ECM). Although the use of hematoxylin/eosin and Masson trichrome stains is widely accepted to highlight tissue structure, the methods based on evaluation of collagen and elastin are considered highly variable. The aim of this study was to develop a whole organ decellularization protocol and compare the qualitative and quantitative efficiency of some microscopy techniques for collagen and elastin detection in paraffin-embedded tissues. MethodsH&E, Masson Trichrome and DAPI staining as well as DNA quantification were used to evaluate decellularization efficiency. Van Gieson stain, Picrosirius Red stain (PRS) and multiphoton laser scanning microscopy (MPM) were carried out for collagen detection and quantitative assessment. Polarized PRS was used to investigate collagen network, and Weigert stain and MPM were used to detect and estimate elastin content. ResultsThe decellularization process removed the cellular components without affecting glycosaminoglycan, collagen and elastin content. Concerning collagen quantification, Van Gieson stain underestimated collagen content, while PRS, apparently less fading, did not reach reliable results when used as quantitative method. MPM effectively quantified collagen content. Collagen fibers were visualized much better under polarized light microscopy, allowing to underline that decellularization process affects the homogeneity of 3D collagen network. Concerning elastin detection, Weigert stain and MPM produced overlapping results. ConclusionsAn efficient protocol to decellularize the whole larynx was developed, allowing the removal of cells without affecting ECM integrity. The results supported the use of non-polarized PRS to highlight collagen, even the thin fibers, second harmonic generation for major fibrillar collagens and polarized PRS for 3D collagen network. Concerning elastin, Weigert stain and MPM showed similar results, thus the use of MPM, rather than that of the Weigert stain, may be suitable to avoid the additional time and costs of a histological staining.

Scaffolds Based on Silk Fibroin with Decellularized Rat Liver Microparticles: Investigation of the Structure, Biological Properties and Regenerative Potential for Skin Wound Healing.

The development of advanced biomaterials and constructs for accelerated recovery of damaged tissues is a key direction in regenerative medicine. Biocompatible scaffolds based on natural biopolymers are widely used for these tasks. Organ decellularization enables obtaining a cell-free extracellular matrix (ECM) with preserved composition and biological activity. The objectives of the present work were combining these two approaches for the development of a composite scaffold based on silk fibroin and ECM microparticles and assessing its structure, biological properties, and regenerative potential. ECM microparticles were obtained by grinding the decellularized matrix of Wistar rat liver in liquid nitrogen. Scaffolds in the form of films were prepared by the casting method. The sinuous and rough topography of the scaffold surface was assessed by the scanning probe nanotomography (SPNT) technique. The inclusion of ECM microparticles in the composition did not affect the elasticity and tensile strength of the scaffolds. The obtained scaffold was non-toxic to cells, maintained high levels of adhesion and proliferation of mouse 3T3 fibroblast and Hep-G2 cells, and showed high regenerative potential, which was studied in the experimental model of full-thickness rat skin wound healing. The wound healing was accelerated by 1.74 times in comparison with the control.

P-466 Decellularization of whole organ human cervix: Physio and biochemical aspects of decellularized extracellular matrix

Abstract Study question Can the whole organ-cervix be decellularized while maintaining its fiber ultrastructure and extracellular matrix (ECM) functionality? Summary answer Cervix after decellularization could maintain the physical properties, ECM was found to be intact and functional. What is known already ECM-rich bioscaffolds are obtained from the process that utilizes the decellularization of tissues and organs. Cervical bioengineering is needed for clinical cervicovaginal reconstruction and tissue constructs can help women with cervical abnormalities. Study design, size, duration Human cervices were obtained from hysterectomized samples from 3 women undergoing surgery for complete hysterectomy during January 2021 to December 2021 Participants/materials, setting, methods Human cervices were obtained from hysterectomized samples from 3 women undergoing surgery and were subjected to whole organ decellularization. Various physical, chemical methods were employed to remove cellular remnants for a period of 10 days via perfusion of ionic and detergent solutions. Validation of complete decellularization was done via hematoxylin and eosin (H&E) staining, DNA quantification. ECM functionality was observed. Main results and the role of chance Well-organized ECM was obtained. Effective complete decellularization was dictated and verified by cervical tissue density, histopathological studies, DNA quantification and biochemical tests. H&E staining reflected the absence of any nuclear material in the endocervical and ectocervical tissues. Tissue microarchitecture and descending uterine artery were intact and could be helpful in the nutrient and gas exchange. DNA was found to be around 70 ng/mg tissue weight. Cell-free tissue-derived scaffolds were obtained along with a combination of growth factors and proteins. Limitations, reasons for caution Acellular cervical scaffold as a whole organ with vasculature intact for site-specific transplantation is a bigger question and still unaddressed. Wider implications of the findings Our study creates numerous prospects for in-vitro and in-vivo whole cervix tissue engineering. Site-specific tissue holds a promising avenue for the reconstruction of cervical abnormalities. Trial registration number Not applicable

Decellularization Detergents As Methodological Variables in Mass Spectrometry of Stromal Matrices.

Collagens, elastin, fibrillin, decorin, and laminin are key constituents of the extracellular matrix and basement membrane of mammalian organs. Thus, changes in their quantities may influence the mechanochemical regulation of resident cells. Since maintenance of a native stromal composition is a requirement for three-dimensional (3D) matrix-based recellularization techniques in tissue engineering, we studied the influence of the decellularization detergents on these proteins in porcine kidney, liver, pancreas, and skin. Using a quick thawing/quick microwave-assisted decellularization protocol and two different detergents, sodium dodecyl sulfate (SDS) vs Triton X-100 (TX100), at identical concentration, variations in matrix conservation of stromal proteins were detected by liquid chromatography-mass spectrometry coupled to light and scanning electron microscopies, in dependence on each detergent. In all organs tested except pancreas, collagens were retained to a statistically significant level using the TX100-based protocol. In contrast fibrillin, elastin (except in kidney), and decorin (only in liver) were better preserved with the SDS-dependent protocol. Irrespective of the detergent used, laminin always remained at an irrelevant level. Our results prompt attention to the type of detergent in organ decellularization, suggesting that its choice may influence morphoregulatory inputs peculiar to the type of 3D bioartificial mammalian organ to be reconstructed. Impact statement Simple change of the protocol's main detergent leads to a very substantial difference in the panel of the stromal proteins detected by qualitative and semiquantitative mass spectrometry in acellular porcine matrices. This remarkable methodological variable promises to yield proteomic reference panels in a number of different species-specific acellular matrices allowing for selective retainment of peculiar mechanochemical inputs, to differently address the development of the seeded cells in relation to the type of organ to be bioartificially reconstructed.

A new strategy for the decellularization of whole organs by hydrostatic pressure.

Tissue engineering has been able to develop novel decellularization-recellularization techniques, which facilitates the research for the generation of functional organs. This is based in the initial obtention of the organ's extracellular matrix (ECM). Therefore, any improvement in the decellularization process would have a positive impact in the results of the recellularization process. Nevertheless, commonly the methods and equipment employed for this process are expensive and thus limit the access of this technique to various research groups globally. To develop a decellularization technique with the exclusive use of hydrostatic pressure of detergent solutions, to have an easily accessible and low-cost technique that meets the basic requirements of acellularity and functionality of the ECM. This experimental study was performed in 10 male Wistar rats, obtaining the liver to carry out serial washes, with 1%, 2%, and 3% Triton X-100 solutions and 0.1% SDS. The washes were performed by using a gravity perfusion system (GPS), which assured us a continuous hydrostatic pressure of 7.5 mmHg. The obtained ECM was processed using stains and immunostaining to determine the residual cell content and preservation of its components. The staining showed a removal of cellular and nuclear components of approximately 97% of the acellular ECM, with an adequate three-dimensional pattern of collagen and proteoglycans. Furthermore, the acellular ECM allowed the viability of a primary hepatocyte culture. The use of the GPS decellularization technique allowed us to obtain an acellular and functional ECM, drastically reducing experimentation costs.

Improving hemocompatibility of decellularized liver scaffold using Custodiol solution

Organ decellularization is one of the most promising approaches of tissue engineering to overcome the shortage of organs available for transplantation. However, there are key hurdles that still hinder its clinical application, and the lack of hemocompatibility of decellularized materials is a central one. In this work, we demonstrate that Custodiol (HTK solution), a common solution used in organ transplantation, increased the hemocompatibility of acellular scaffolds obtained from rat livers. We showed that Custodiol inhibited ex vivo, in vitro, and in vivo blood coagulation to such extent that allowed successful transplantation of whole-liver scaffolds into recipient animals. Scaffolds previously perfused with Custodiol showed no signs of platelet aggregation and maintained in vitro and in vivo cellular compatibility. Proteomic analysis revealed that proteins related to platelet aggregation were reduced in Custodiol samples while control samples were enriched with thrombogenicity-related proteins. We also identified distinct components that could potentially be involved with this anti-thrombogenic effect and thus require further investigation. Therefore, Custodiol perfusion emerge as a promising strategy to reduce the thrombogenicity of decellularized biomaterials and could benefit several applications of whole-organ tissue engineering.

Optimization of Complete Rat Heart Decellularization Using Artificial Neural Networks.

Whole organ decellularization techniques have facilitated the fabrication of extracellular matrices (ECMs) for engineering new organs. Unfortunately, there is no objective gold standard evaluation of the scaffold without applying a destructive method such as histological analysis or DNA removal quantification of the dry tissue. Our proposal is a software application using deep convolutional neural networks (DCNN) to distinguish between different stages of decellularization, determining the exact moment of completion. Hearts from male Sprague Dawley rats (n = 10) were decellularized using 1% sodium dodecyl sulfate (SDS) in a modified Langendorff device in the presence of an alternating rectangular electric field. Spectrophotometric measurements of deoxyribonucleic acid (DNA) and total proteins concentration from the decellularization solution were taken every 30 min. A monitoring system supervised the sessions, collecting a large number of photos saved in corresponding folders. This system aimed to prove a strong correlation between the data gathered by spectrophotometry and the state of the heart that could be visualized with an OpenCV-based spectrometer. A decellularization completion metric was built using a DCNN based classifier model trained using an image set comprising thousands of photos. Optimizing the decellularization process using a machine learning approach launches exponential progress in tissue bioengineering research.