"Glia-like" cells of fibroblast morphology are present in cultures from injured human brain tissue.

OBJECTIVE: Astrocytes undergo morphological and molecular changes in response to numerous pathological conditions. BACKROUND: Increased expression of glial fibrillary acidic protein (GFAP) has been reported as a characteristic feature of reactive astrocytes. However, GFAP-positive cells occur rarely in adult human brain cultures. These cultures are mostly composed of flat GFAP-negative "glia-like" cells, which remain poorly characterized in relation to reactive astrogliosis. METHODS: We examined the cultures from macroscopically injured and normal brain tissue from patients with brain trauma, gliomas, or brain metastases. Immunofluorescence and immunohistochemical methods were used for reactive astrocytes detection. RESULTS: The intensity of GFAP-positive staining was higher in reactive astrocytes in the brain tissue surrounding gliomas or metastases and lower in brain tissue damaged by traumatic injury. We did not observe any correlation between GFAP-positive reactive astrocytes in cultures and brain tissue. However, we found rapidly proliferating spindle-shaped cells in cultures prepared from injured brain tissue. CONCLUSION: Present data demonstrate the unexplained phenomenon of disparate cell morphologies in cultures when prepared either from macroscopically normal or injured human brain tissue. While normal cultures are mainly comprised of flat cells, the cultures from severely damaged brain tissue may be entirely composed of spindle-shaped cells usually classified as fibroblasts. We suggest that this spindle-shaped cellular morphology is not specific for fibroblasts, but it rather can be interpreted as the most favorable shape for rapid cell proliferation under culture conditions. After brain trauma, unknown processes may be triggered, such as induced cell proliferation which can be revealed under culture condition. Accordingly, we conclude that spindle-shaped cells are activated precursors of glial cells (Fig. 3, Ref. 15).

Anencephaly in Slovakia and Czech Republic: embryogenesis, risk factors, epidemiology and preventative approaches.

Anencephaly, a fatal anomaly of the central nervous system, belongs to the group of defects of the neural tube (NTDs). It is considered the most common congenital NTD, characterized by concurrent absence of a significant portion of the brain and cranial vault. This deformity occurs between days 23 and 26 after fertilization due to improper closure of the neural tube at its cranial end. Many genetic, epigenetic, and non-genetic factors (nutritional, environmental and geographical factors, parental socioeconomic status) contribute to the etiology of this disease. Despite significant advances in treatment and preventive measures, NTDs continue to pose a significant health and financial burden on patients and society as a whole. This study aimed to examine the incidence of anencephaly in Slovakia compared to the Czech Republic between 2012 and 2020. The authors seek to elucidate the reasons behind the higher incidence of this disease in Slovakia as compared to the Czech Republic, explore the male predominance of anencephaly in Slovakia, and investigate whether the prevention standards used in Slovakia differ from those employed in other countries (Tab. 1, Fig. 2, Ref. 129). Keywords: neural tube defects, anencephaly, risk factors, folic acid, food fortification.

The importance of Merkel cells in the development of human fingerprints.

Human Merkel cells (MCs) were first described by Friedrich S. Merkel in 1875 and named "Tastzellen" (touch cells). Merkel cells are mainly located in the basal layer of the epidermis and are concentrated in touch-sensitive areas. Their density varies among different anatomical sites. Increased concentration was observed in the palms of hands with a predominance in the finger pads and also in the soles and toes. They can be classified according to the function as mechanoreceptive, endocrine, and chemo-sensitive cells. In the development of primary ridges which establish the future fingerprint patterns is assumed that Merkel cells have a significant importance in this process. At about the 7th week EGA, they first time appear in the volar skin and start to occupy the place of future primary ridges at 10 weeks EGA. It will be interesting to study their presence or absence in individuals suffering with abnormal dermatoglyphics and also to study whether the skin diseases associated with altered dermatoglyphics display some deviation regarding the distribution and density of MCs in primary ridges (Fig. 2, Ref. 40). Text in PDF www.elis.sk Keywords: Merkel cells, development, primary ridges, fingerprints, CK-20.

Remote conditioning as protective strategy influencing ubiquitin-mediated stress response in the rabbit spinal cord after ischemia/reperfusion.

The aim of presented study was to investigate the model of non-invasive method of remote conditioning induced by compression of left forelimb with a tourniquet in three cycles of 2 min of ischemia each followed by 2 min of reperfusion and its influence on the rabbit spinal cord ischemia/reperfusion injury via ubiquitin-mediated stress response. Ubiquitin immunoreaction in spinal cord motor neurons as well as detection of neuronal survival in ventral horns of spinal cord were evaluated. Significantly increased (p < 0.001) number of ubiquitin positive neurons was registered in all remote conditioned groups versus both spinal cord ischemia (SC-ischemia) groups. Our results indicate that remote conditioning significantly attenuated degeneration of motor neurons in all conditioned groups versus SC-ischemia groups in each time point. According to our results, we concluded that the remote conditioning induced by transient limb ischemia is relevant stimulus that provides potent neuroprotection in a model of spinal cord ischemia/ reperfusion injury.

Fabrication and In Vitro Characterization of Novel Hydroxyapatite Scaffolds 3D Printed Using Polyvinyl Alcohol as a Thermoplastic Binder.

This paper presents a proof-of-concept study on the biocolonization of 3D-printed hydroxyapatite scaffolds with mesenchymal stem cells (MSCs). Three-dimensional (3D) printed biomimetic bone structure made of calcium deficient hydroxyapatite (CDHA) intended as a future bone graft was made from newly developed composite material for FDM printing. The biopolymer polyvinyl alcohol serves in this material as a thermoplastic binder for 3D molding of the printed object with a passive function and is completely removed during sintering. The study presents the material, the process of fused deposition modeling (FDM) of CDHA scaffolds, and its post-processing at three temperatures (1200, 1300, and 1400 °C), as well it evaluates the cytotoxicity and biocompatibility of scaffolds with MTT and LDH release assays after 14 days. The study also includes a morphological evaluation of cellular colonization with scanning electron microscopy (SEM) in two different filament orientations (rectilinear and gyroid). The results of the MTT assay showed that the tested material was not toxic, and cells were preserved in both orientations, with most cells present on the material fired at 1300 °C. Results of the LDH release assay showed a slight increase in LDH leakage from all samples. Visual evaluation of SEM confirmed the ideal post-processing temperature of the 3D-printed FDM framework for samples fired at 1300 °C and 1400 °C, with a porosity of 0.3 mm between filaments. In conclusion, the presented fabrication and colonization of CDHA scaffolds have great potential to be used in the tissue engineering of bones.

Blocking effect of ferritin on the ryanodine receptor-isoform 2.

Iron, an essential element for most living organism, participates in a wide variety of physiological processes. Disturbance in iron homeostasis has been associated with numerous pathologies, particularly in the heart and brain, which are the most susceptible organs. Under iron-overload conditions, the generation of reactive oxygen species leads to impairment in Ca2+ signaling, fundamentally implicated in cardiac and neuronal physiology. Since iron excess is accompanied by increased expression of iron-storage protein, ferritin, we examined whether ferritin has an effect on the ryanodine receptor - isoform 2 (RYR2), which is one of the major components of Ca2+ signaling. Using the method of planar lipid membranes, we show that ferritin induced an abrupt, permanent blockage of the RYR2 channel. The ferritin effect was strongly voltage dependent and competitively antagonized by cytosolic TEA+, an impermeant RYR2 blocker. Our results collectively indicate that monomeric ferritin highly likely blocks the RYR2 channel by a direct electrostatic interaction within the wider region of the channel permeation pathway.

Stem Cells and Their Derivatives-Implications for Alveolar Bone Regeneration: A Comprehensive Review.

Oral and craniofacial bone defects caused by congenital disease or trauma are widespread. In the case of severe alveolar bone defect, autologous bone grafting has been considered a "gold standard"; however, the procedure has several disadvantages, including limited supply, resorption, donor site morbidity, deformity, infection, and bone graft rejection. In the last few decades, bone tissue engineering combined with stem cell-based therapy may represent a possible alternative to current bone augmentation techniques. The number of studies investigating different cell-based bone tissue engineering methods to reconstruct alveolar bone damage is rapidly rising. As an interdisciplinary field, bone tissue engineering combines the use of osteogenic cells (stem cells/progenitor cells), bioactive molecules, and biocompatible scaffolds, whereas stem cells play a pivotal role. Therefore, our work highlights the osteogenic potential of various dental tissue-derived stem cells and induced pluripotent stem cells (iPSCs), the progress in differentiation techniques of iPSCs into osteoprogenitor cells, and the efforts that have been made to fabricate the most suitable and biocompatible scaffold material with osteoinductive properties for successful bone graft generation. Moreover, we discuss the application of stem cell-derived exosomes as a compelling new form of "stem-cell free" therapy.

Recent Overview of the Use of iPSCs Huntington's Disease Modeling and Therapy.

Huntington's disease (HD) is an inherited, autosomal dominant, degenerative disease characterized by involuntary movements, cognitive decline, and behavioral impairment ending in death. HD is caused by an expansion in the number of CAG repeats in the huntingtin gene on chromosome 4. To date, no effective therapy for preventing the onset or progression of the disease has been found, and many symptoms do not respond to pharmacologic treatment. However, recent results of pre-clinical trials suggest a beneficial effect of stem-cell-based therapy. Induced pluripotent stem cells (iPSCs) represent an unlimited cell source and are the most suitable among the various types of autologous stem cells due to their patient specificity and ability to differentiate into a variety of cell types both in vitro and in vivo. Furthermore, the cultivation of iPSC-derived neural cells offers the possibility of studying the etiopathology of neurodegenerative diseases, such as HD. Moreover, differentiated neural cells can organize into three-dimensional (3D) organoids, mimicking the complex architecture of the brain. In this article, we present a comprehensive review of recent HD models, the methods for differentiating HD-iPSCs into the desired neural cell types, and the progress in gene editing techniques leading toward stem-cell-based therapy.

Recent Progress in the Regeneration of Spinal Cord Injuries by Induced Pluripotent Stem Cells.

Regeneration of injuries occurring in the central nervous system, particularly spinal cord injuries (SCIs), is extremely difficult. The complex pathological events following a SCI often restrict regeneration of nervous tissue at the injury site and frequently lead to irreversible loss of motor and sensory function. Neural stem/progenitor cells (NSCs/NPCs) possess neuroregenerative and neuroprotective features, and transplantation of such cells into the site of damaged tissue is a promising stem cell-based therapy for SCI. However, NSC/NPCs have mostly been induced from embryonic stem cells or fetal tissue, leading to ethical concerns. The pioneering work of Yamanaka and colleagues gave rise to the technology to induce pluripotent stem cells (iPSCs) from somatic cells, overcoming these ethical issues. The advent of iPSCs technology has meant significant progress in the therapy of neurodegenerative disease and nerve tissue damage. A number of published studies have described the successful differentiation of NSCs/NPCs from iPSCs and their subsequent engraftment into SCI animal models, followed by functional recovery of injury. The aim of this present review is to summarize various iPSC- NPCs differentiation methods, SCI modelling, and the current status of possible iPSC- NPCs- based therapy of SCI.

Recently Discovered Interstitial Cell Population of Telocytes: Distinguishing Facts from Fiction Regarding Their Role in the Pathogenesis of Diverse Diseases Called "Telocytopathies".

In recent years, the interstitial cells telocytes, formerly known as interstitial Cajal-like cells, have been described in almost all organs of the human body. Although telocytes were previously thought to be localized predominantly in the organs of the digestive system, as of 2018 they have also been described in the lymphoid tissue, skin, respiratory system, urinary system, meninges and the organs of the male and female genital tracts. Since the time of eminent German pathologist Rudolf Virchow, we have known that many pathological processes originate directly from cellular changes. Even though telocytes are not widely accepted by all scientists as an individual and morphologically and functionally distinct cell population, several articles regarding telocytes have already been published in such prestigious journals as Nature and Annals of the New York Academy of Sciences. The telocyte diversity extends beyond their morphology and functions, as they have a potential role in the etiopathogenesis of different diseases. The most commonly described telocyte-associated diseases (which may be best termed "telocytopathies" in the future) are summarized in this critical review. It is difficult to imagine that a single cell population could be involved in the pathogenesis of such a wide spectrum of pathological conditions as extragastrointestinal stromal tumors ("telocytomas"), liver fibrosis, preeclampsia during pregnancy, tubal infertility, heart failure and psoriasis. In any case, future functional studies of telocytes in vivo will help to understand the mechanism by which telocytes contribute to tissue homeostasis in health and disease.

Delayed post mastectomy breast reconstructions with allogeneic acellular dermal matrix prepared by a new decellularizationmethod.

Acellular dermal matrix (ADM) is a tissue graft of allogeneic origin from post-mortem tissue donors prepared by an innovative decellularization process. The newly developed non-toxic and low cost decellularization process of cadaver origin dermis included ADM in breast reconstruction procedures proved to help coverage of the lower-pole of breast expanders or implants. As the results have shown, it did help to eliminate autologous dermis donor site morbidity along with shortening the operation time by avoiding elevation of additional muscle or fascia during the operation. Main aims of this article include histology evaluation of allogeneic acellular dermal matrix prepared by a new decellularization method and presentation of clinical results of its use. A total of 22 patients underwent 26 ADM based breast reconstructions. The mean patient's follow up was 12.6 months. Average total size of ADM used for one breast was 273 cm2. Post-operative complications occurred in 3 patients including one expander infection, one expander extrusion and one expander pocket disfiguration. Microscopic analysis of tissue samples has confirmed incorporation of the acellular dermal matrices into the surrounding connective tissue without any noticeable immune reaction. In a majority of the ADM samples we found pseudocapsullar formation on implant side of samples without acute or chronic inflammatory cells. The use of ADM prepared by new preparation method in expansive post mastectomy breast reconstruction was associated by a relatively low complication rate resulting in good outcomes.

Induction of pluripotency in long-term cryopreserved human neonatal fibroblasts in feeder-free condition.

A novel approach for stem cell generation is the attempt to induce conversion of the adult somatic cells into pluripotent stem cells so called induced pluripotent stem cells (iPSCs) by introducing specific transcription factors. iPSCs have two essential cell characteristics, they are pluripotent and posses long term cell-renewal capacity. Additionally, iPSCs can be derived from patient-specific somatic cells, thus bypassing ethical and immunological issues. The aim of our study was to reprogram long-term cryopreserved human neonatal fibroblasts by new method using lipid nano-particle technology (Lipofectamine 3000 reagent transfection system) in combination with Epi 5 reprogramming vectors. Obtained iPSCs were characterized by several sophisticated methods of molecular biology and microscopy. Distinct colonies of iPSCs started to appear by day 20 after reprogramming. The presence of iPSCs colonies was proved by alkaline phosphatase (AP) live staining. After manual picking the colonies and their subsequent passaging, they did not lose ability to form embryoid bodies, they were positive for AP, Tra-1-60, and SSEA-5. Moreover, obtained iPSCs expressed pluripotency markers Oct4, Sox2 and Nanog, and the expression levels of chondrogenic, osteogenic and adipogenic markers were significantly higher in comparison to control (p < 0.05). In summary, we have demonstrated that long-term cryopreserved human neonatal fibroblasts can be reprogrammed into iPSCs and after further analysis concerns on their biological safety they may be used as patient-specific cells in regenerative medicine.

Toxicity testing and drug screening using iPSC-derived hepatocytes, cardiomyocytes, and neural cells.

Unexpected toxicity in areas such as cardiotoxicity, hepatotoxicity, and neurotoxicity is a serious complication of clinical therapy and one of the key causes for failure of promising drug candidates in development. Animal studies have been widely used for toxicology research to provide preclinical security evaluation of various therapeutic agents under development. Species differences in drug penetration of the blood-brain barrier, drug metabolism, and related toxicity contribute to failure of drug trials from animal models to human. The existing system for drug discovery has relied on immortalized cell lines, animal models of human disease, and clinical trials in humans. Moreover, drug candidates that are passed as being safe in the preclinical stage often show toxic effects during the clinical stage. Only around 16% drugs are approved for human use. Research on induced pluripotent stem cells (iPSCs) promises to enhance drug discovery and development by providing simple, reproducible, and economically effective tools for drug toxicity screening under development and, on the other hand, for studying the disease mechanism and pathways. In this review, we provide an overview of basic information about iPSCs, and discuss efforts aimed at the use of iPSC-derived hepatocytes, cardiomyocytes, and neural cells in drug discovery and toxicity testing.

The functional morphology and role of cardiac telocytes in myocardium regeneration.

Key morphological discoveries in recent years have included the discovery of new cell populations inside the heart called cardiac telocytes. These newly described cells of the connective tissue have extremely long cytoplasmic processes through which they form functionally connected three-dimensional networks that connect cells of the immune system, nerve fibers, cardiac stem cells, and cardiac muscle cells. Based on their functions, telocytes are also referred to as "connecting cells" or "nurse cells" for cardiac progenitor stem cells. In this critical review, we provide a summary of the latest research on cardiac telocytes localized in all layers of the heart - from the historical background of their discovery, through ultrastructural, immunohistochemical, and functional characterizations, to the application of this knowledge to the fields of cardiology, stem cell research, and regenerative medicine.

Functional histology and possible clinical significance of recently discovered telocytes inside the female reproductive system.

PURPOSE: Key discoveries of recent years comprise detection of telocytes. These cells of connective tissue have extremely long cytoplasmic processes through which they form functionally connected spatial networks. Through their processes they mutually contact and functionally connect also cells of the immune system, nerve fibres and smooth muscle cells. They are located in various parts of the female reproductive system where they can perform specifically significant functions, which are summarized in our literature review. METHODS: Literature regarding "telocytes" and "interstitial Cajal-like cells" was reviewed using scientific databases PubMed/Medline, SCOPUS, and Web of Knowledge. RESULTS: Among other things telocytes regulate peristaltic muscle movements in the uterine tubes. Their decreased activity, e.g., in inflammatory diseases or endometriosis, causes disorders of a transport function through the uterine tubes resulting in infertility or tubal pregnancy. In the uterine myometrium they are, first, responsible for regulation of muscle contraction (in expelling menstrual blood or in childbirth) and, second, they participate also in immune surveillance during embryo implantation. They likely control also uterine involution post partum. Their function in the vagina has not been elucidated yet, but probably they participate in production of slow contraction waves during sexual intercourse. In the mammary gland their function may be to regulate cellular proliferation and apoptosis, thus they may play a role also in the development and growth of tumours. In the placenta, they may monitor and regulate blood flow through chorionic villi and they participate in aetiopathogenesis of preeclampsia. CONCLUSION: However, all above-mentioned functions of telocytes are purely hypothetic and have been published only recently. Therefore, only further research will demonstrate whether this recently discovered cell population will really play a key role in all processes mentioned, or whether it is just an effort of scientists to clarify unknown cause of some diseases in gynaecology and obstetrics. Our literature review is completed by our own original photomicrographs documenting telocytes in various organs of the female reproductive system.

The significance of electron microscopic examination of gingiva in cases of Hunter syndrome and hereditary gingival fibromatosis.

INTRODUCTION: Electron microscopy has been for decades a basic morphological method still used in diagnostic protocols of some pathological conditions affecting the ultrastructure of cells and extracellular matrix. The aim of this study was an ultrastructural description of gingiva of patients with Hunter syndrome and hereditary gingival fibromatosis. PATIENTS AND METHODS: Gingival biopsies were obtained during surgical periodontal treatment from a 9-year-old boy with Hunter disease (with enzyme replacement therapy with recombinant human idursulphase) and a 15-year-old girl with hereditary gingival fibromatosis. Gingival samples obtained from the upper anterior region were processed and examined with transmission electron microscope. RESULTS: In the case of Hunter syndrome due to the genetic lack of one lysosomal enzyme, an intercellular accumulation of glycosaminoglycans occurs. Within the gingiva of a patient with Hunter syndrome we observed membrane-bound storage vesicles in the cytoplasm of fibroblasts, endothelial cells of capillaries, surface epithelial cells, mast cells, and macrophages. Despite a long-term enzyme replacement therapy which improves clinical manifestations of Hunter syndrome, on the cellular level we still found marked accumulations of glycosaminoglycans in the cytoplasm of different cells as well as in the extracellular matrix. Hereditary gingival fibromatosis is a benign, slowly progressive and non-inflammatory gingival enlargement with a predominance of randomly oriented collagen fibrils in the gingival lamina propria. Some of these fibrils exhibited loops. Another unusual ultrastructural finding is the presence of empty perinuclear space in the cytoplasm of epithelial cells. The origin and significance of these non-membrane bound spaces are unknown. CONCLUSION: In both genetically determined diseases, the electron microscopic examination may be useful, and physicians get relevant information about the progress of illness.

Recent advances in iPSC technologies involving cardiovascular and neurodegenerative disease modeling.

Cardiovascular and neurodegenerative diseases are the most common health threats in developed countries. Limited cell derivation and cell number in cardiac tissue makes it difficult to study the cardiovascular disease using the existing cardiac cell model. Regarding the neurodegenerative disorders, the most potential sources of cell therapeutics such as fetal-derived primary neurons and human embryonic stem cells (ESCs) are associated with ethical or technical limitations. The successful derivation of human-induced pluripotent stem cells (iPSCs) by de-differentiation of somatic cells offers significant potential to overcome hurdles in the field of the replacement therapy. Human iPSCs are functionally similar to human embryonic stem cells, and can be derived autologously without the ethical challenges associated with human ESCs. The iPSCs can, in turn, be differentiated into all cell types including neurons, cardiac cells, blood and liver cells, etc. Recently, target tissues derived from human iPSCs such as cardiomyocytes (CMs) or neurons have been used for new disease modeling and regenerative medicine therapies. Diseases models could be advantageous in the development of personalized medicine of various pathological conditions. This paper reviews efforts aimed at both the practical development of iPSCs, differentiation to neural/cardiac lineages, and the further use of these iPSCs-derived cells for disease modeling, as well as drug toxicity testing.

Induced pluripotent stem cells and their implication for regenerative medicine.

In 2006 Yamanaka's group showed that stem cells with properties similar to embryonic stem cells could be generated from mouse fibroblasts by introducing four genes. These cells were termed induced pluripotent stem cells (iPSCs). Because iPSCs avoid many of ethical concerns associated with the use of embryonic material, they have great potential in cell-based regenerative medicine. They are suitable also for other various purposes, including disease modelling, personalized cell therapy, drug or toxicity screening and basic research. Moreover, in the future, there might become possible to generate organs for human transplantation. Despite these progresses, several studies have raised the concern for genetic and epigenetic abnormalities of iPSCs that could contribute to immunogenicity of some cells differentiated from iPSCs. Recent methodological improvements are increasing the ease and efficacy of reprogramming, and reducing the genomic modification. However, to minimize or eliminate genetic alternations in the derived iPSC line creation, factor-free human iPSCs are necessary. In this review we discuss recent possibilities of using iPSCs for clinical applications and new advances in field of their reprogramming methods. The main goal of present article was to review the current knowledge about iPSCs and to discuss their potential for regenerative medicine.

The effect of salivary gland extract of Lucilia sericata maggots on human dermal fibroblast proliferation within collagen/hyaluronan membrane in vitro: transmission electron microscopy study.

BACKGROUND: Lucilia sericata maggots are applied to chronic wounds to aid healing when conventional treatments have failed. After their application into a necrotic wound, they potentially influence wound healing with a combination of specific proteinases that are involved in the remodeling of extracellular matrix. These proteases cause changes in fibroblast adhesion and spread upon extracellular matrix protein surfaces, affecting integrity of the protein surfaces-especially fibronectin-while maintaining cell viability. OBJECTIVE: This study focused on in vitro monitoring of the effect of homogenate substances prepared from maggot salivary gland of L sericata on the ultrastructure of human neonatal fibroblasts. METHODS: Collagen/hyaluronan membrane was used as the synthetic substitute of extracellular matrix. The cultured human neonatal fibroblasts B-HNF-1 were seeded on the surface of the collagen/hyaluronan membrane and cultured with maggot salivary gland extract (SGE) at a concentration of 2.4 glands/1 mL. RESULTS: The authors observed increased cell metabolism and protein production (euchromatic nucleus, voluminous nuclear membrane, large reticular nuclei, distended and filled cisterns of rough endoplasmic reticulum, Golgi apparatus with saccules, and vesicles packed with fine fibrillar material) after incubating the cells in culture medium with SGE. CONCLUSION: The authors believe that increased cell metabolism and protein production corresponded with formation of microfibrillar net used for migration of fibroblasts in culture, but mainly for proper production of extracellular matrix. The authors suggest that their results may help explain the effect of SGE on wound healing and support implementation of maggot therapy into human medicine.

Decellularization of the human urethra for tissue engineering applications.

Recently, several scaffolds have been introduced for urethral tissue engineering. However, acellular human urethral scaffold harvested from deceased donors may provide significant advantages compared to synthetic, composite, or other biological scaffolds. This study aims to develop the protocol for decellularization of the human urethra that preserves substantial extracellular matrix (ECM) components, which are essential for subsequent recellularization mimicking the natural environment of the native ECM. A total of 12 human urethras were harvested from deceased donors. An equal part of every harvested urethra was used as a control sample for analyses. The protocol design was based on the enzyme-detergent-enzyme method. Trypsin and Triton X-100 were used to remove cells, followed by DNase treatment to remove DNA residues. Subsequently, the specimens were continually rinsed in deionized water for seven days. The efficiency of decellularization was determined by histochemistry, immunohistochemical staining, scanning electron microscopy (SEM), and DNA quantification. Histological analysis confirmed cell removal and preservation of urethral structure after decellularization. The preservation of collagen IV and fibronectin was confirmed by histologic examination and immunohistochemical staining. SEM confirmed the maintenance of the ultrastructural architecture of ECM and fibers. DNA content in decellularized urethra was significantly lower compared to the native sample (P < 0.001), and so the criteria for decellularized tissue were met. Cytotoxicity analysis data showed that the matrix-conditioned medium did not contain soluble toxins and had no significant inhibitory effect on cell proliferation, providing evidence that the decellularized samples are not toxic. This study demonstrates the feasibility of the enzyme-detergent-enzyme-based decellularization protocol for removing cellular components and maintaining urethral ECM and its ultrastructure. Moreover, obtained results provide solid ground for recellularization and urethral tissue engineering, which will follow.