decellularization

This study aimed to construct a decellularized mouse spleen scaffold and evaluate its cellular compatibility in vitro using murine bone marrow-derived mesenchymal stem cells (BM-MSCs).

A combination of physical, chemical, and enzymatic treatments was employed for mouse spleen decellularization. These included multiple freeze-thaw cycles, the ionic detergent sodium dodecyl sulfate (SDS), and enzymatic trypsin. Histological and scanning electron microscopy analyses were conducted up to 7 days post-culture to assess the impact of decellularization and cellular adaptation to the spleen scaffolds.

Histological studies revealed the attachment and penetration of BM-MSCs into the scaffolds on days 5-7 following cell seeding. Furthermore, cell migration into the scaffold was observed 5 days after the seeding process.

The decellularization approach utilized in this study proved to be effective and biocompatible, supporting the preservation and proliferation of BM-MSCs. These findings indicate its potential for spleen tissue engineering applications.

Liver transplantation is the gold standard treatment for end-stage liver failure, but the scarcity of organ donors is the main limiting factor for performing liver transplant surgery.

The objective was to evaluate hepatocytes’ phenotype and functionalities after co-culturing with endothelial (HUVEC) and stellate cells (LX2) in the decellularized liver.

The livers were decellularized with 1% sodium lauryl ester sulfate (SLES). Cell removal and preservation of extracellular matrix (ECM) ultrastructure were studied by staining, scanning electron, and Raman confocal microscopy. The cell viability was evaluated by MTT, and the functions of cells were assessed on a decellularized scaffold with/without co-culturing with HUVEC and LX2 cell lines. The results were then compared to cells with the same condition on collagen scaffolds.

The data confirmed that SLES prevented the destruction of the liver ECM ultrastructure along with nuclear material removal. Raman spectra confirmed DNA and cell debris removal. The decellularized liver was suitable for cell survival, but the proliferation rate was lower than those cultured in collagen. The tests showed that the function of individual cells on the decellularized scaffold was better than that in collagen scaffolds. Co-culturing with HUVEC and LX2 cell lines did not improve hepatocyte functions.

As a biocompatible scaffold, co-culturing hepatocytes with endothelial and stellate cells within the decellularized liver improved liver-specific functions.

Men’s infertility and lack of production of healthy and active sperm are concerns of recent years in mostcountries. Studies on the preparation of extracellular matrix (ECM) from decellularization of testis tissue and spermatogenesiscould provide proper results to solve some of the men’s infertility problems. This study aims to decellularize calftestis by different methods to reach a suitable scaffold and introduce it in spermatogenesis studies.

In this experimental study, calf testis were decellularized by a freeze-de freeze, 1% sodiumdeoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), 0.1% SDS-vacuum, 1% SDS, 1% SDS-vacuum, and Triton-X100 methods. The content of DNA, collagen, and glycosaminoglycan (GAG) was analyzed using the kit and stainingwith Hematoxylin-Eosin, Masson’s trichrome, Alcian blue, and Orcein methods. The morphology of the scaffolds wasanalyzed with a scanning electron microscope (SEM).

Methods of 1% SDS, 1% SDS-vacuum, and 1% SD completely removed the cells. The preservation of collagenand GAG was confirmed using the staining kit and methods. The use of a vacuum showed greater porosity inthe SEM images. Toxicity and hemolysis were not observed in the scaffolds.

Testis decellularization with 1% SDS and 1% SD, in addition to cell removal, could maintain the ECMstructure to a large extent without having cytotoxic and hemolysis effects.

The aim of the present study was to evaluate alterations in the vegf gene expression as an angiogenic factor in mouse embryo fibroblasts seeded on the decellularized liver fragments.

Liver tissue samples (n = 10) collected from adult male mice were randomly divided into decellularized and native control groups. Tissues were decellularized by treating with 1% Triton X-100 and 0.1% SDS for 24 hours and assessed by H&E staining and SEM. Then DNA content analysis and toxicity tests were performed. By centrifugation, DiI-labeled mouse embryo fibroblasts were seeded on each scaffold and cultured for one week. The recellularized scaffolds were studied by H&E staining, SEM, and LSCM. After RNA extraction and cDNA synthesis, the expression of the vegf gene in these samples was investigated using real-time RT-PCR.

Our observations showed that the decellularized tissues had morphology and porous structure similar to the control group, and their DNA content significantly reduced (p < 0.05) and reached to 4.12% of the control group. The MTT test indicated no significant cellular toxicity for the decellularized scaffolds. Light microscopy, SEM, and LSCM observations confirmed the attachment and penetration of embryonic fibroblast cells on the surface and into different depths of the scaffolds. There was no statistically significant difference in terms of vegf gene expression in the cultured cells in the presence and absence of a scaffold.

The reconstructed scaffold had no effect on vegf gene expression. Decellularized mouse liver tissue recellularized by embryonic fibroblasts could have an application in regenerative medicine.

Efficient production of functional and mature alveolar epithelial is a major challenge for developing any cellreplacement therapy for lung degenerative diseases. The extracellular matrix (ECM) pro-vides a dynamic environmentand mediates cellular responses during development and maintenance of tissue functions. The decellularized ECM(dECM) which retains its native-like structure and bio-chemical composition can provide the induction of embryonicstem cell (ESC) differentiation toward the tissue-specific lineages during in vitro culture. Therefore, the aim of this studywas to evaluate the effect of sheep lung dECM-derived scaffold on differentiation and further maturation of ESC-derivedlung progenitor cells.

This study was an experimental study. In the first step, a sheep lung was decellularizedto achieve dECM scaffolds and hydrogels. Afterwards, the obtained dECM scaffold was evaluated for collagen andglycosaminoglycan contents, DNA quantification, and its ultrastructure. Next, the three experimental groups: i. Sheeplung dECM-derived scaffold, ii. Sheep lung dECM-derived hydrogel, and iii. Fibronectin-coated plates were comparedin their abilities to induce further differentiation of human embryonic stem cells (hESCs)-derived definitive endoderm(DE) into lung progenitor cells. The comparison was evaluated by immuno-staining and real-time polymerase chainreaction (PCR) assessments.

We found that the dECM-derived scaffold preserved its composition and native porous structures whilelacking nuclei and intact cells. All experimental groups displayed lung progenitor cell differen-tiation as revealed by theRNA and protein expression of NKX2.1, P63 and CK5. DE cells differenti-ated on dECM-derived scaffold and dECMderivedhydrogel showed significant upregulation of SOX9 gene expression, a marker of the distal airway epithelium.DE cells differentiated on the dECM-derived scaffold compared to the two other groups, showed enhanced expressionof SFTPC (type 2 alveolar epithelial [AT2] cell marker), FOXJ1 (ciliated cell marker), and MUC5A (secretory cell marker)genes.

Overall, our results suggest that dECM-derived scaffold improves the differentiation of DE cells towardslung alveolar progenitor cells in comparison with dECM-derived hydrogel and fibronectin-coated plates.

Decellularized livers could provide an environment for liver-specific cell migration. Synthetic glucocorticoids induce liver development and hepatocyte differentiation as well as limit immune cell migration, which can both promote or inhibit fibrogenesis in the engineered liver. Although decellularized scaffolds provide a promising approach for liver regeneration, the effect of the implant on the donor's liver remained clear.

This study investigated the impact of the transplanted decellularized liver with/without prednisolone preloading on liver histopathology and functions.

Decellularized rat liver scaffolds were prepared by the perfusion method. After scaffolds characterization, they were grafted to partially hepatectomized rats in prednisolone-free and -loaded groups. After 2 and 4 weeks, the liver and grafts were removed and evaluated by Periodic acid Schiff staining and immunohistochemistry. Serological assessments were also performed on blood sera and compared with untreated control. The data were analyzed by ANOVA and LSD.

Both grafts were invaded by hepatocytes. No histopathological symptom was detected in the recipient's liver; however, oval cells were observed within the epithelium of the bile duct and in the surrounding connective tissue. No significant variation was observed in the levels of alkaline phosphatase(ALP), but the levels of albumin and total protein were significantly reduced in both groups that received the grafts after two weeks; however, after four weeks, total protein and albumin reached the average level.

decellularized liver transplantation with/without the drug is safe enough for the recipient liver to be considered a promising technique in regenerative medicine.

Organ transplantation is the last therapeutic choice for end-stage liver failure, which is limited by the lack of sufficient donors. Decellularized liver can be used as a suitable matrix for liver tissue engineering with clinical application potential. Optimizing the decellularization procedure would obtain a biological matrix with completely removed cellular components and preserved 3-dimensional structure. This study aimed to evaluate the decellularization efficacy through three anatomical routes.

In this experimental study, rat liver decellularization was performed through biliary duct (BD), portal vein (PV), and hepatic vein (HV); using chemical detergents and enzymes. The decellularization efficacy was evaluated by measurement of DNA content, extracellular matrix (ECM) total proteins, and glycosaminoglycans (GAGs). ECM preservation was examined by histological and immunohistochemical (IHC) staining and scanning electron microscopy (SEM). Scaffold biocompatibility was tested by the MTT assay for HepG2 and HUVEC cell lines.

Decellularization through HV and PV resulted in a transparent scaffold by complete cell removal, while the BD route produced an opaque scaffold with incomplete decellularization. H&E staining confirmed these results. Maximum DNA loss was obtained using 1% and 0.5% sodium dodecyl sulfate (SDS) in the PV and HV groups and the DNA content decreased faster in the HV group. At the final stages, the proteins excreted in the HV and PV groups were significantly less than the BD group. The GAGs level was diminished after decellularization, especially in the PV and HV groups. In the HV and PV groups the collagen amount was significantly more than the BD group. The IHC and SEM images showed that the ECM structure was preserved and cellular components were entirely removed. MTT assay showed the biocompatibility of the decellularized scaffold.

The results revealed that the HV is a more suitable route for liver decellularization than the PV and BD.

Decellularized greater omentum (GOM) is a good extracellular matrix (ECM) source for regenerative medicine applications. The aim of the current study was to compare the efficiency of three protocols for sheep GOM decellularization based on sufficient DNA depletion and ECM content retention for tissue engineering application. In this experimental study, in the first protocol, low concentrations of sodium dodecyl sulfate (SDS 1%), hexane, acetone, ethylenediaminetetraacetic acid (EDTA), and ethanol were used. In the second one, a high concentration of SDS (4%) and ethanol, and in the last one sodium lauryl ether sulfate (SLES 1%) were used to decellularize the GOM. To evaluate the quality of scaffold prepared with various protocols, histochemical staining, DNA, and glycosaminoglycan (GAGs) quantification, scanning electron microscopy (SEM), Raman confocal microscopy, Bradford assay, and ELISA were performed. A comparison of DNA content showed that SDS-based protocols omitted DNA more efficiently than the SLESbased protocol. Histochemical staining showed that all protocols preserved the neutral carbohydrates, collagen, and elastic fibers; however, the SLES-based protocol removed the lipid droplets better than the SDS-based protocols. Although SEM images showed that all protocols preserved the ECM architecture, Raman microscopy, GAGs quantification, total protein, and vascular endothelial growth factor (VEGF) assessments revealed that SDS 1% preserved ECM more efficiently than the others. The SDS 1% can be considered a superior protocol for decellularizing GOM in tissue engineering applications.

In testis, the extracellular matrix (ECM) in addition to the supportive role for cells in the seminiferous epithelium, is also essential for the accurate functioning of these cells. Thus, using a decellularized testicular ECM (DTECM), as a scaffold for three-dimensional (3D) culture of testicular cells can mimic native ECM for studying in vitro spermatogenesis.  The rat testis was decellularized via perfusion of 0.5% sodium dodecyl sulfate (SDS) for 48 hr, followed by 1% Triton X-100 for 6 hr, and then 1% DNase I for 1 hr. The efficiency of decellularization was evaluated by histology, immunohistochemistry (IHC), scanning electron microscopy (SEM), and MTT test. The prepared scaffolds were recellularized with testicular cells and cultured and assessed with hematoxylin-eosin (H&E) staining after two weeks.  Based on the H&E image, no trace of cell components could be observed in DTECM. IHC images demonstrated collagen types I and IV, laminin, and fibronectin were preserved. Masson’s trichrome and alcian blue staining revealed that collagen and glycosaminoglycans (GAGs) were retained, and the SEM image indicated that 3D testicular architecture remained after the decellularization process. Based on the results of the MTT test, DTECM was cytocompatible, and H&E images represented that DTECM supports testicular cell arrangements in seminiferous tubule-like structures (STLSs) and organoid-like structures (OLSs).   The results showed that the applied protocol successfully decellularized the testis tissue of the rat. Therefore, these scaffolds may provide an appropriate vehicle for in vitro reconstruction of the seminiferous tubule.

Since bone defects can result in different disabilities, many efforts have been made to bone tissue engineering. In this case, scaffolds play an important role as a key element of tissue engineering in providing three-dimensional structure for cell growth in vitro The aim of the present study was to provide the three-dimensional biological bioscaffold from the bovine femur dense bone and investigate the possibility of its potential for application in tissue engineering as biological 3D ECM bioscaffold via mesenchymal stem cells seeding and differentiation toward bone tissue. For the preparation of bioscaffolds, after cutting bovine femur bone into small pieces, demineralization and decellularization were done. Bioscaffolds biocompatibility was evaluated using an MTT assay. The morphological and cell adhesion characteristics of Bone marrow mesenchymal stem cells (BMSCs) on the bioscaffolds were evaluated using Scanning Electron Microscopy (SEM) technique. Finally, the cells were treated with an osteogenic differentiation medium and then evaluated for differentiation. Histological studies showed that the use of sodium dodecyl sulfate (2.5%) for 8 h eliminated the cells. Radiography and calcium oxalate test confirmed demineralization. MTT assay and SEM studies showed that the obtained bioscaffolds are biocompatible and could provide an optimum three-dimensional environment for cell adhesion and movement. Moreover, the Alizarin red staining showed a higher differentiation rate for BMSCs. In the present study, bone-derived 3D bioscaffold showed an important role in the growth and differentiation of BMSCs, due to the natural characteristics, cell adhesion properties, and potential to enhance differentiation toward bone tissue. It may have the potential for use as bioscaffold as supporting metrics for maintenance, growth in bone tissue engineering.

Biologic scaffolds composed of extracellular matrix (ECM) are frequently used for clinical purposes of tissue regeneration. Different methods have been developed for this purpose. All methods of decellularization including chemical and physical approaches leave some damage on the ECM; however, the effects of these methods are different which make some of these procedures more proper to maintain ECM structure than other methods. This review is aimed to introduce and compare new physical methods for the decellularization of different tissues and organs in tissue engineering. All recent reports and research that have used at least one physical method in the procedure of decellularization, were included and evaluated in this paper. The advantages and drawbacks of each method were examined and compared considering the effectiveness. This review tried to highlight the prospective potentials and benefits of applying physical methods for decellularization protocols in tissue engineering instead of the current chemical methods. These chemical methods are harsh in nature and were shown to be destructive and harmful to essential substances of ECM and scaffold structure. Therefore, using physical methods as a partial or even a whole protocol could save time, costs, and quality of the final acellular tissue in complicated decellularization procedures. Moreover, regarding the control factor that could be achieved easily with physical methods, optimization of different decellularization protocols would be quite satisfactory. Combined methods take advantage of both chemical and physical approaches.

Decellularization techniques have been widely used in tissue engineering recently. However, applying these methods which are based on removing cells and maintaining the extracellular matrix (ECM) encountered some difficulties for dense tissues such as articular cartilage. Together with chemical agents, using physical methods is suggested to help decellularization of tissues.

In this study, to improve decellularization of articular cartilage, the effects of direct and indirect ultrasonic waves as a physical method in addition to sodium dodecyl sulfate (SDS) as chemical agents with 0.1% and 1% (w/v) concentrations were examined. Decellularization process was evaluated by nucleus staining with hematoxylin and eosin (H and E) and by staining glycosaminoglycans (GAG) and collagen.

The H and E staining indicated that 1% (w/v) SDS in addition to ultrasonic bath for 5 h significantly decreased the cell nucleus residue to lacuna ratio by 66%. Scanning electron microscopy showed that using direct sonication caused formation of micropores on the surface of the sample which results in better penetration of decellularization material and better cell attachment after decellularization. Alcian Blue and Picrosirius Red staining represented GAG and collagen, respectively, which maintained in ECM structure after decellularization by ultrasonic bath and direct sonicator.

Ultrasonic bath can help better penetration of the decellularization material into the cartilage. This improves the speed of the decellularization process while it has no significant defect on the structure of the tissue.

Extracellular matrix (ECM) functions as a scaffold for tissue morphogenesis and regeneration, promotes the maintenance of differentiated tissue and plays an extremely indispensable role in cell proliferation and differentiation. As a corollary of this importance, studying the cell behaviors without considering the ECM is to some extent impossible. Deletion of cells from ECM obtains natural three dimensional matrices that ape the in vivo functions of ECM..
The current study aimed to verify whether acellular dermal matrix (ADM) is a suitable 3D-matrix for blastema tissue cells originated from pinna of rabbit and study different behaviors of these cells on ADM..
To prepare ADM, small pieces of skin were decellularized by repeated snap freeze-thaw and treatment with sodium dodecyl sulfate as a detergent. Decellularization was verified and proved by histological tests. Blastema rings were prepared by double punching the pinnas of male New Zealand White rabbits and cultured in vitro after assembling with ADM. Specimens were studied after 5, 10, 15, 20 and 25 days of culturing..
Migration of cells started on the fifth day. As a result of this migration, most cells were located on the surface of the scaffold and formed epiderm-like structure and some penetrated into the scaffold. By day 25, cells which had penetrated into the scaffold had various destinies such as becoming elongated spindle-shaped cells and creating blood vessel-like structures..
ADM can induce migration and probably differentiation of blastema tissue cells; therefore, it is a suitable 3D-model to study cell behaviors in vitro..