Type: Article
Testis-derived extracellular matrix scaffolds: Evaluation of fresh and frozen tissue decellularization for applications in male infertility therapy
Seyedeh Fezeh Hashemi Karouei 1, Tooba Mirzapour 1,*, Roghayeh Pourbagher 2, Mir Mohammad Reza Aghajani 3
1. Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran
2. Cellular and Molecular Cell Biology Research Center, Babol University of Medical Sciences, Babol, Iran.
3. Fatemeh Al-Zahra Specialized Infertility Treatment Center, Babol, Iran
Abstract: Differentiation of spermatogonial stem cells (SSCs) on decellularized biological scaffolds has emerged as a novel approach in tissue engineering and male infertility treatment. Testis-derived extracellular matrix (ECM) scaffolds provide a natural microenvironment that can support SSC culture and differentiation. In this study, we compared the efficiency of different detergents for the decellularization of fresh and frozen human testicular tissues. Human testicular tissue samples were divided into 2 groups: fresh and frozen at -80 °C. Cellular decellularization was performed using concentrations of 0.5% and 1% sodium dodecyl sulfate (SDS) and Triton x100 alone and in combination. The extent of cell removal and reduction in cell DNA content was examined using histological studies and one-way ANOVA statistical method. Frozen tissues exposed sequentially to 1% SDS for 24 h followed by 1% Triton X-100 for 24 h showed the highest efficiency in removing cellular components while maintaining ECM integrity, compared with fresh tissues. Moreover, frozen testicular matrices demonstrated greater resistance to detergent-induced damage than fresh matrices. Both detergent concentration and tissue condition (fresh vs. frozen) are critical determinants of scaffold quality in testicular decellularization. Optimizing these parameters may facilitate the development of natural testis-derived scaffolds for reproductive tissue engineering. In the future, seeding germ cells from patients with non-obstructive azoospermia onto these optimized scaffolds under differentiation-inducing conditions may provide a promising strategy for male infertility treatment.
Keywords: Decellularization, Sodium dodecyl sulfate, Triton x100, Male infertility, Extracellular matrix
Article Info.
Submitted: 29-9-2025; Revised: 02-10-2025; Accepted: 05-10-2025; Online:11-10-2025
Cite as:, T., Pourbagher, R., Aghajani, M.M.R. (2025) Testis-derived extracellular matrix scaffolds: Evaluation of fresh and frozen tissue decellularization for applications in male infertility therapy. Animal Reports 1(2): 118-136.
https://doi.org/10.64636/ar.30
Male infertility is a major reproductive health challenge worldwide, accounting for approximately 50% of all cases of infertility in couples (Bendayan et al.,2022; Abdelaal et al., 2021). In many of these cases, the quality or function of testicular tissue is impaired, and existing treatments do not provide an adequate response (Kaltsas et al., 2022). In recent years, tissue engineering has been considered as a novel approach to reconstruct and support testicular function (Socci et al., 2023; Omolaoye et al., 2022; Lin et al., 2021). One of the main approaches in tissue engineering is the use of decellularized bioscaffolds for the cultivation of stem cells isolated from dysfunctional testicular tissue (Garreta et al., 2017).
In the decellularization process, cells in the tissue are removed, while the extracellular matrix (ECM) components and 3D structures of the tissue are preserved as much as possible (Badylak et al., 2009). Due to their natural compounds and biological signals, these scaffolds provide a suitable environment for the growth and differentiation of stem cells (SSC) and can be effective in the treatment of male infertility (Hanai et al., 2020; Hsieh et al., 2021). De Kock et al., (2021) investigated the spermatogenic potential of human testicular cells using 3D bioprinting derived from a patient with non-obstructive azoospermia (NOA). The results showed an increase in germ cell markers as well as an increase in the expression of genes involved in spermatogonial stem cells in 3D printed tubules after 12 days of in vitro culture. In another study conducted by Kanbar et al., (2022) pig tissues and organs were cut to the appropriate thickness and exposed to carbon dioxide cell extraction technology. Decellularized ECM scaffolds obtained from decellularized tissues were freeze-dried and then cut into different sizes and thicknesses for use in laboratory methods and tissue engineering and placed in Petri dishes.
The findings demonstrated that integrating human mesenchymal stem cells with decellularized dermal matrix scaffolds can facilitate full restoration of skin architecture in a porcine wound model. However, the effectiveness and quality of these scaffolds are largely influenced by the specific methods employed during the decellularization process. (Kanbar et al., 2022). To date, various methods such as the use of physical, enzymatic, and chemical agents have been used to decellularize tissues. Stimulation and immersion are one of the applied techniques of the physical decellularization method. This method, in combination with the chemical method, helps the substance used for decellularization to better penetrate the tissue and reach the tissue cells. To achieve this, the target tissue or organ is placed in a chamber filled with a decellularizing solution. The duration of immersion, as well as the level of physical stimulation applied, are adjusted based on the tissue’s density and thickness (Rabbani et al., 2021; Belviso et al., 2022; Al-Hejailan et al., 2022).
Enzymatic techniques, such as the use of trypsin in combination with ethylenediaminetetra acetic acid, target cell adhesion to the ECM or deoxyribonuclease (DNAse) target the cell's nuclear material (Vockel et al., 2021; Gilpin et al., 2017; Narciso et al., 2022). The commonly used enzymes are nucleases and proteases that can degrade RNA and DNA by breaking nonspecific bonds within or at the ends of nucleic acids or denaturing proteins (Narciso et al., 2022). Chemical approaches utilize detergents such as SDS and sodium deoxycholate to disrupt and dissolve cellular membranes. Similarly, exposure to hypertonic or hypotonic solutions induces osmotic shock, leading to cell lysis. While osmotic shock effectively eliminates cells, it fails to remove the residual cellular debris within the matrix. This limitation must be taken into account when developing and optimizing decellularization protocols (Vockel et al., 2021).
According to previous studies, among the chemical agents, SDS and Triton X-100 have been the most widely used. The effectiveness of these detergents depends largely on their concentration and duration of use, with higher concentrations resulting in more complete cell removal but increasing the risk of matrix damage (Belviso et al., 2022; Al-Hejailan et al., 2022). Sodium dodecyl sulfate is the most widely applied detergent for cell lysis. It effectively disrupts both the plasma and nuclear membranes; however, it also carries the risk of protein denaturation and structural alteration of the extracellular matrix. Therefore, limiting the exposure time to SDS is essential in order to reduce potential protein damage and preserve matrix integrity. Despite these drawbacks, SDS remains highly efficient in eliminating nuclear and cytoplasmic residues (Rabbani et al., 2021). Triton X-100 is another detergent commonly utilized in cell lysis processes. It primarily disrupts lipid–lipid and lipid–protein associations while preserving protein–protein interactions. This property makes it effective for cell removal in various tissues; however, it is not suitable for tissues in which glycosaminoglycans represent a major structural component of the extracellular matrix (Rabbani et al., 2021; Belviso et al., 2022).
In addition to the type and concentration of detergents, the condition of the tissue sample (fresh or frozen tissue) can also affect the result of decellularization (De Kock et al., 2021; Kanbar et al., 2022). In recent years, more attention has been paid to decellularization of testicular tissue in studies related to in vitro differentiation of SSC cells (Narciso et al., 2022). In line with these studies, the use of SDS and Triton X-100 alone, as well as in combination with other enzymatic and non-enzymatic reagents, has led to the creation of several biocompatible scaffolds from testicular tissue (Topraggaleh et al., 2019). In their study, De Kock et al. (2021) reported that agitating testicular tissue fragments in 1% (w/v) SDS for 24 hours achieved superior decellularization and DNA removal while maintaining key extracellular matrix proteins, compared to treatment with 1% (v/v) Triton X-100. In a separate investigation, Topraggaleh et al., (2019) successfully generated decellularized scaffolds from ram testicular tissue. These scaffolds, characterized by their large pores, were subsequently seeded with neonatal mouse testicular cells. Although the resulting organoids did not replicate the native testicular architecture, they were capable of producing hormones and generating post-meiotic cells (Topraggaleh et al., 2019). In another study, Akbarzadeh et al., (2019) first introduced the most efficient detergent for decellularizing testicular tissue, then determined the optimal time required for the decellularization process with this combination. In this way, an efficient method was presented for producing a 3D bioscaffold from testicular tissue that retained the characteristics of the natural organ and could be used in tissue engineering studies.
This study highlights key innovations in tissue engineering. By optimizing the decellularization process, it becomes possible to decellularize a wider range of human- and animal-derived tissues, reducing the need for strict donor-patient matching in graft transplantation. Additionally, advances in recellularization are essential to ensure uniform distribution of appropriate cell types, alongside adequate delivery of nutrients and oxygen to support cell survival. The primary objective of this study was to examine and compare the effects of varying detergent concentrations on the efficiency of testicular tissue decellularization. Secondly, the freshness and freezing of the tissue were also affecting the quality of decellularization of the resulting bioscaffolds. The production of decellularized testicular matrices can be used in the future to culture testicular cells from individuals with non-obstructive azoospermia and to relieve cell arrest and resume spermatogenesis in vitro and can be considered as a suitable therapeutic strategy for the treatment of male infertility in the future.
All human studies and protocols of this study were conducted after approval and receipt of the ethics code by the Ethics Committee of the University of Guilan with the ethics ID (IR.GUILAN.REC.1403.32). After receiving consent from the patients and approval from the Pathology Department of Shahid Beheshti Hospital, Babol City, complete testicular tissue samples obtained from orchidectomy were received from 4 patients with testicular cancer. Then, observing hygienic conditions, they were transferred into a container containing sterile culture medium and within 30 minutes, along with dry ice, were transferred to the cell culture laboratory of the Fatemeh Al-Zahra Infertility Center in Babol city for research work.
The testicular tissue was placed in a sterile plate under the class II laminar hood, and then the tunica albuginea layer was completely separated from the testicular tissue using forceps and scissors. The tissue was cut into 1 cm pieces and transferred to a 15 ml Falcon tube. Then 5 cc of PBS buffer was added and centrifuged for 5 minutes at 2000 rpm to remove blood and unwanted particles. Then, the supernatant was discarded, and the testicular tissue pieces were transferred to 1.5 ml microtubes using a sampler. The samples were divided into three parts. The first part included fresh testicular tissue, which was exposed to different concentrations of detergents according to different groupings to decellularize them. The second part was placed as a frozen sample at -80 °C for 24 hours before decellularization. Then, the cell removal process was performed on them according to different groupings. One section was also considered as a control group. This group was not exposed to any detergent for cell removal. Sodium didecyl sulfate (SDS) with concentrations of 1% and 0.5% along with Triton X-100 with concentrations of 1% and 0.5% was used for cell removal and increased the removal rate of testicular tissue DNA. To perform this process, the microtube containing testicular tissue samples in each group was placed on a mechanical shaker at a speed of 70 rpm at room temperature after adding the appropriate detergent to reduce cellular components and DNA content with better quality.
Each of the testicular tissues of the first and second sections was divided into 6 groups. These 6 groups were as follows:
Group 1: Cells were exposed to 1% SDS at room temperature on a mechanical shaker for 24 hours.
Group 2: Cells were exposed to 1% Triton for 24 hours at room temperature on a mechanical shaker.
Group 3: Cells were exposed to 0.5% SDS for 24 hours at room temperature on a mechanical shaker.
Group 4: Cells were exposed to 0.5% Triton for 24 hours at room temperature on a mechanical shaker.
Group 5: Cells were first exposed to 1% SDS for 24 hours and then to 1% Triton for another 24 hours on a mechanical shaker at room temperature.
Group 6: Cells were first exposed to 0.5% SDS for 24 hours and then to 0.5% Triton for another 24 hours on a mechanical shaker at room temperature.
For the second part, which included frozen testicular tissue samples, the samples were first removed from the freezer and placed at room temperature. Then, the 6 above groupings were also performed to decellularize the tissues of this part. All analyses were repeated three times. In each group, after the samples were exposed to detergent, in order to completely remove detergent and cell particles, the samples were washed with PBS.
In order to analyze the DNA content in fresh and frozen decellularized samples, as well as control samples, the tissues were cut into pieces with an average weight of 20 mg. For quantitative assessment of DNA content, the samples were first lyophilized. DNA was then extracted from 10 mg of dry tissue using a DNA extraction kit (Qiagen FPKT023.0050) following the manufacturer’s protocol. In this way, in brief, 20 mg of tissue was selected and cut into small pieces with a scalpel blade and added to a container containing 200 μl of PreLysis solution, 20 μl of proteinase K, and 5 μl of ribonuclease A. Then, the pieces were vortexed vigorously for 10 seconds at 55°C to complete digestion. Finally, 200 μl of Wash Solution I was added and the samples were centrifuged at 10,000 rpm for 1 minute. Subsequently, 500 μl of Wash Solution II was added, followed by centrifugation at 10,000 rpm for 2 minutes. After removing the filter section of the collection tube, 50 μl of Elution Buffer was applied directly to the center of the membrane. It was kept at room temperature for 2-3 minutes, and then centrifuged at 10,000 RPM for 1 minute. Finally, the collection tube was discarded and the collection was transferred to a new 1.5 ml microtube. At this stage, the concentration of DNA content in tissues was determined using a UV-Vis NanoDrop spectrophotometer (C, Thermo Scientific, Venlo 2000) and the results were expressed as ng/mg of dry weight (Topraggaleh et al., 2019).
At the end of this section, based on the results of DNA content analysis, the group with the lowest DNA content was selected.
To evaluate the quality of decellularized ECM, four aspects of ECM were measured: cell removal, removal of genetic material, preservation of protein content (type 4 collagen) and preservation of mechanical properties (glycosaminoglycan).
Control scaffolds tissue and decellularized samples were fixed in 10% formalin for 24 hours at room temperature. Following fixation, the specimens were rinsed with PBS and dehydrated through a graded series of alcohols, with each concentration applied for 20 minutes. Then, they were embedded in paraffin molds and serial sections with a thickness of 5 μm were prepared from them. The deparaffinized samples were exposed to descending alcohol grades (dehydration) and xylol. They were then stained with hematoxylin and eosin (H&E) to perform normal histological analysis and quantify cell nuclear debris, and to compare cell decellularization in treated and control tissues. Hematoxylin stains the cell nuclei in a bluish-purple color, and eosin stains the extracellular matrix and cytoplasm in pink.
DAPI staining (Sigma-Aldrich) was employed to evaluate the effectiveness of the decellularization process, verify cell removal, and identify any remaining cellular material. Samples were taken from various regions of the scaffolds to assess decellularization. DAPI directly binds to DNA, enabling visualization of cell nuclei.
The selected decellularized scaffolds and control tissue were fixed in 10% formalin solution at room temperature for 24 hours. After fixation, the samples were washed in PBS and dehydrated by placing in ascending alcohol grades (herbal alcohol for 20 minutes) and embedded in paraffin molds and 5 μm thick sections were obtained from them. The samples were deparaffinized and dehydrated again and for histological analysis and quantitative determination of DNA content, hematoxylin/eosin staining was performed on the samples. To investigate the presence of ECM proteins, including collagen IV in the scaffold, immunofluorescence method was applied on prepared paraffin slides. First, the tissue sections were transferred to an oven at 60 °C to remove paraffin. Then the slides were exposed to xylol and cleared. They were dehydrated and washed with descending alcohol grades. After this step, the slides were placed in 10 mM citrate solution with pH=6 for 20 minutes to remove excess antigens. Then, the samples were placed in Triton X-100 for 40 minutes to increase the permeability of the cells to the antibodies. After this step, primary antibody such as anti-collagen type 4 antibody COL4A1 (5E10): sc-517572) (with appropriate dilution was added to the scaffold and native tissue as a control sample and were left in this state for one day. After washing with PBS, the secondary antibody Goat Anti-Mouse IgG(H+L)(CY3 conjugated) E-AB-1011,) was added to the scaffold and native tissue and after 2 hours of incubation, the samples were washed with PBS. Finally, DAPI staining was performed to create contrast and the slides were examined using a fluorescent microscope (BM-600 LED EPI FLURESCENT/Germany-AXIOM) (Khazaei et al., 2024).
The Alcian Blue Staining Kit (ab150662) was utilized to verify the presence of glycosaminoglycans in both the decellularized scaffolds and the control tissues. For the staining procedure, 5 μm-thick paraffin sections were first heated at 60°C for 30 minutes. The sections were then dehydrated using xylene followed by graded alcohols (100%, 95%, 70%) and rinsed with distilled water. Subsequently, the sections were treated with 0.5% periodic acid for 10 minutes, washed again with distilled water, and finally incubated in 1% Alcian Blue solution at pH 2.5 for 30 minutes. Then, they were washed with running water and a final wash was performed with distilled water to remove excess dye. In this staining, due to the removal of cells and the low number of nuclei in the scaffolds of different groups, the complementary dye (Nuclear Fast Red) was not used. The steps of dehydrating and mounting were similar to the previous staining. All slides were analyzed with a light microscope for qualitative examination, and the intensity of staining and distribution of the dye in different parts of the scaffold were evaluated (Khazaei et al., 2024).
The PAS staining technique is used to stain tissues that contain relatively high amounts of polysaccharides such as glycogen and mucous substances (glycoproteins, glycolipids, and mucins). In this study, for PAS staining, 5 μm-thick paraffin sections were initially incubated in an oven at 60°C for 30 minutes. The sections were then dehydrated using xylene and graded alcohol (100%, 95%, 70%) and rinsed with distilled water. Subsequently, the sections were treated with 0.5% periodic acid solution for 10 minutes at room temperature, followed by another rinse with distilled water. The 0.5% periodic acid solution was prepared by dissolving 0.5 g of periodic acid powder in 100 mL of distilled water. Then, the samples were placed in Schiff Reagent solution for 15 minutes and then washed well with distilled water until the pink-violet color was determined. After that, the samples were placed in a 0.3% sodium borate solution (0.3 g in 100 cc of water) for 15 seconds and then rinsed four times with distilled water. After staining with hematoxylin and washing with water, the paraffin-embedded samples were dehydrated. Then, they were cleared with xylene and, after mounting the slides with Entalan glue, they were examined with a light microscope (Nag et al., 2023).
Since these fragments are used for the culture of testicular cells from infertile individuals, the toxicity of residual decellularizing detergents on cells (in the selected group) was investigated by MTT assay. For this experiment, selected decellularized testicular scaffolds and fresh testicular tissue (as a control) were thoroughly washed with sterile PBS and then incubated for 24 hours at 37°C in a culture medium composed of DMEM High Glucose supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 20 ng/mL GDNF, and 10 ng/mL bFGF. The scaffolds and control tissues were cut into approximately 10 mm pieces using sterile scissors and placed into the wells of a 96-well plate. Subsequently, 1.5 × 105 mouse fibroblasts were seeded into each well on the testicular scaffolds. Following the protocol, 500 μL of culture medium containing 0.5 mg/mL 3-[4,5-dimethyl(thiazol-2-yl)-3,5-diphenyl]tetrazolium bromide (MTT) was added to each well and incubated for 3 hours at 37°C to allow for the formation of formazan crystals. After removing the culture medium, the formazan crystals were dissolved in 150 μL of dimethyl sulfoxide (DMSO, Sigma, USA). The optical density (OD) of the supernatant was then measured using a microplate reader (SYNERGY HTX BioTek) at 570 nm.
All experiments were performed with 3 replications. Data obtained from treatment and replication groups were evaluated using SPSS software and one-way ANOVA test and followed by Tukey post-hoc tests. The significance level was 0.05 and the confidence level was 95%.
In this study, the effect of cell-degradation in different groups was measured using quantitative and qualitative assays. To this end, DNA quantification and hematoxylin/eosin staining were conducted to perform histological analysis of decellularized testicular fragments (DTF). In order to determine the minimum DNA content, fresh and frozen decellularized tissues were separately compared with the control sample (intact tissue). In the first step, an independent sample t-test was used to directly compare the two fresh and frozen decellularized groups. The results showed that the mean DNA content in the fresh group was 1.3086 and in the frozen group it was 0.5896. The t-test showed a significant difference (p < 0.001) between the two groups. Therefore, it can be concluded that the DNA content in the frozen group is significantly lower than that in the fresh group. This finding suggests that the freezing process leads to a decrease in the DNA content in decellularized samples. To obtain an overall estimate of the difference between the groups and to obtain the best subgroup of the group Freeze, One-Way ANOVA test was used. Post-hoc Tukey HSD tests indicated that in the frozen testicular tissue section, the lowest DNA content was observed in subgroup 5 (SDS/Triton1%) (Fig. 1). There was a significant difference between this group and the control group (p < 0.05) (Fig. 1). After that, groups 2 and 3 had the lowest DNA content (p < 0.05). The highest DNA content was also observed in group 2.
Fig. 1. Exposure of frozen testicular tissue samples to Triton X100 and SDS detergents at 1% and 0.5%, alone or in combination. The results of statistical analysis showed that the lowest amount of DNA was observed in the group exposed to sodium dodecyl sulfate/Triton 100X (1%) (p < 0.05).
The results of ANOVA in the decellularization section of fresh testicular tissue showed that the lowest amount of DNA was observed in group 5. There was also a significant difference in this group than the control group in terms of DNA content. After that, the lowest amount of DNA was observed in group 2 and then group 1 (p < 0.05). In this section, the highest amount of DNA content was in group 6.The DNA content of all treatment groups were less than 50 ng/mg of dry weight.
Fig. 2. Exposure of fresh testicular tissue samples to Triton X100 and SDS detergents at 1% and 0.5%, alone or in combination. The results of statistical analysis showed that the lowest amount of DNA was observed in the group exposed to sodium dodecyl sulfate/Triton 100X (1%). (p < 0.05)
Based on the results, group 5 of the frozen section was selected as the selected group with the lowest DNA content. Since in addition to reducing DNA content, the preservation of the matrix structure is also considered as an indicator for producing a successful scaffold, in this section, histological studies conducted to investigate the amount of collagen, matrix glycosaminoglycans, and basement membrane glycogen are discussed.
Fig. 3. Dapi staining of seminiferous tubules in human testis tissue a) Before decellularization b) After decellularization
Histological studies of hematoxylin and eosin-stained slides showed that among the scaffolds decellularized with Triton X-100 and SDS detergents at concentrations of 1% and 0.5% in frozen and fresh conditions, scaffolds in group 5 from the frozen section had the lowest number of nuclei compared to other samples. On the other hand, matrix properties were largely preserved in this group.
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Fig 4. Histopathological evaluation of testes by H&E staining: (a) testicular tissues in the control group b) decellularized testicular tissues in group scaffold 5. In this group the number of nuclei has been decreased rather than control groups (Scale Bar:50 µm.
Examination of histological slides in different groups and counting stained nuclei in different fields of view and in different replicates of each group showed that in group 5 the number of intact cells or nuclei decreased, and fewer blue dots were observed in the slides of this group (Fig. 4).
In this study, immunohistochemical staining was compared against collagen type 4 in decellularized and control scaffolds. The results showed that in selected group 5, the distribution of collagen type 4 was more uniform and dense. This condition provides a better environment for cell interaction with the matrix. On the other hand, in this group, the fibrous network of the extracellular matrix was preserved more continuously, which can provide better support for cells during the re-culture. These results are probably due to the combined use of SDS and Triton, which helped to effectively remove cells and preserve the fibrous structures of the matrix. Considering the importance of collagen type 4 in cell adhesion and proliferation, group 5 scaffolds seem to be a more suitable option for re-culture of cells due to their structural integrity and better preservation of the matrix (Fig. 5).
Fig. 5. Immunohistochemical staining results of the preservation of collagen type IV in a) Control group, b) S5 group. Immunohistochemical staining results showed that in the S5 group, the distribution of type 4 collagen was condensed and continuous, probably due to the effects of SDS on the extracellular matrix and sufficient compensation by Triton X-100 in a short time. Sale Bar in control group =10µm. in other groups= 50 µm.
The results showed that in the scaffold 5 group, the effect of detergents on the removal of glycosaminoglycans around the seminiferous tubules was less and a unified tissue was observed around the tubules, which somewhat made it closer to the control tissue (Fig. 6).
Fig. 6. The preservation of glycosaminoglycan in decellularized scaffolds was evaluated using immunohistochemistry with an HRP-conjugated antibody and DAB staining. a) In control group; b) In Group scaffold 5. Group scaffold 5 exhibited a more uniform and dense glycosaminoglycan distribution, forming a continuous fibrous network, indicative of superior ECM preservation. Sale Bar in control and treatment groups = 50µm.
The basis of the PAS reaction is the oxidation of carbohydrates in biological samples in the presence of a specific Schiff solution, which results in the identification of carbohydrates and carbohydrate-rich compounds of the basement membrane. According to the results, in decellularized tissue, detergents such as SDS and Triton X100, with the concentrations and timing used in this study, removed cells from the tissue but preserved the glycogen structure, tissue clicocalyx, and ultimately the basement membrane (Fig. 7).
Fig. 7. The preservation of glycogen polysaccharide and glycocalyx in decellularized scaffolds was evaluated using Periodic Acid Schiff staining (PAS). a) In control group; b) In Group scaffold 5. Group scaffold 5 exhibited a more uniform and dense glycogen distribution, indicative of ECM preservation. Sale Bar in control and treatment groups= 50µm.
Based on the results, the scaffold group 5 from the frozen section was considered as selected group in this study. To assess the impact of the detergents employed during the decellularization process on cell viability, an MTT assay was conducted. In this experiment, fibroblast cells were cultured in proximity to decellularized testicular tissue fragments from group 5, which had undergone freezing and thawing, and the results were compared with those of the control group. The results showed that the viability of cells in the vicinity of the decellularized scaffold (88% viability) was not significantly different from the control group (P>0.05) and was close to the viability in the control group (92% viability). This finding indicates that the residues of detergents used in the decellularization process did not have a significant toxic effect on fibroblast cells.
Given the complexity of the spermatogenesis process and the high sensitivity of germ cells to the culture medium, recreating the natural testicular environment has been proposed as one of the effective strategies for the successful differentiation of these cells. In this regard, the use of biological scaffolds based on extracellular matrix (ECM) can preserve the structural, biochemical, and mechanical properties of the original tissue and provide a similar in vivo environment for cellular differentiation. The present study was conducted with the aim of designing a three-dimensional biological model of testicular tissue using decellularized scaffolds (Al-Hejailan et al., 2022).
In this study, different protocols for decellularization of mouse testicular tissue were compared. The results showed that the combined use of SDS and Triton X-100 at a concentration of 1% has the best efficiency in terms of effective removal of cellular DNA, preservation of ECM components including type IV collagen, fibronectin, glycosaminoglycans, and basement membrane glycogen, and preservation of the three-dimensional architecture. These results are in complete agreement with the findings of Topraggaleh et al., who showed that the stepwise use of ionic and nonionic detergents improves decellularization and preserves the ECM structure in testicular tissue (Topraggaleh et al., 2019).
Comparison of our protocol with other studies, such as Liang et al., (2022) Al-Hejailan et al., (2022), showed that the success of the decellularization process depends on the choice of detergents and timing. For example, the use of ammonia in some studies () resulted in severe ECM destruction, whereas our method, by avoiding strong chemicals and using gentle mechanical agitation, achieved a balance between cell removal and preservation of the fibrous structure (Narciso et al., 2022). In the present study, decellularization of fresh and frozen testicular tissue was performed using different concentrations (1% and 0.5%) of detergents. The results showed that in the presence of 1% SDS and Triton X-100, the decellularization process in frozen tissue occurred more effectively than in fresh tissue. This could be due to structural changes in the tissue during the freezing and thawing process, which probably increased the permeability of detergents and facilitated cell removal.
Previous studies have employed varying concentrations of SDS and Triton X-100 for tissue decellularization. De Kock et al. (2021) reported that agitating testicular tissue fragments in 1% (w/v) SDS for 24 hours resulted in more effective decellularization and DNA removal, while better preserving key extracellular matrix proteins, compared to treatment with 1% (v/v) Triton X-100. Nag et al. (2023) successfully decellularized mouse kidneys to produce a gelatinous extracellular matrix, which was subsequently used to generate human pluripotent stem cell-derived renal organoids. Their approach involved detergent-mediated decellularization using a combination of 0.5% (w/v) SDS and 1% (v/v) Triton X-100, along with mechanical agitation for a duration of 60 hours. Rabbani et al. (2020) concluded that chemical decellularization methods are inherently harsh and can damage the extracellular matrix and compromise scaffold integrity. Consequently, incorporating physical methods, either partially or as part of a combined protocol, may help preserve tissue quality while reducing time and costs in complex decellularization procedures.
Yoshimasa et al. (2023) applied a stepwise decellularization protocol, immersing tissue in PBS containing 0.01% SDS for 6 hours at 4°C, followed by PBS with 0.1% SDS for 6 hours at 4°C, and finally re-immersing in PBS with 1% SDS for 6 hours at room temperature. They concluded that decellularized endometrial scaffolds promote endometrial stromal regeneration and are effective in repairing extensively damaged uterine endometrium by preventing adhesions.
Therefore, there are different methods for decellularizing different tissues. In each decellularization method, one or more decellularizing agents may be applied to the target tissue, which affect specific ECM components differently. To assess the quality of the resulting scaffold, it is necessary to analyze the effects of the decellularizing agent on most of the ECM components. While analyses often focus solely on interstitial ECM components, such as type I collagen and elastin, evaluating the basement membrane is crucial, particularly in tissue engineering and cell culture contexts, since it significantly influences cell adhesion and differentiation. Therefore, in the present study, the effect of SDS and Triton X100 at different concentrations on the nature of collagen, fibronectin, and basement membrane polysaccharides was investigated.
Decellularization protocols have many limitations. Most of them require long periods of decellularization, ranging from 6 to 7 hours to several days. In addition, many existing methods do not preserve some ECM components. A significant reduction in elastin, collagen, glycosaminoglycans, laminins, and proteoglycans has been observed during the ECM decellularization process. On the other hand, most of the previous methods are not suitable for decellularization of an entire organ or thick sections of an organ. (Al-Hejailan et al., 2022, Gilpin & Yang 2017, Narciso et al., 2022).
Thus, identifying optimal decellularization methods is essential to produce an effective extracellular matrix using minimal chemical concentrations and within a short duration. Such methods preserve critical structural, biochemical, and biomechanical properties, maintain the matrix’s intrinsic function, minimize host immune responses, and generate spaces that facilitate host cell infiltration and proliferation (Kanbar et al., 2022). In this study, the thickness of the tissue to be decellularized was minimized and the effective time for decellularization was reduced to 24 to 48 hours. In agreement with our study, studies such as Akbarzadeh et al., (2019) and Al-Hejailan et al. (2022) have also highlighted the importance of choosing the type and duration of detergent treatment in maintaining the ECM structure and reducing damage caused by decellularization agents.
One of the key achievements of this study was the reduction of the residual DNA content in all scaffolds studied, which reached values below 50 ng/mg, as confirmed by DNA assay kits. In group 5 decellularization (frozen tissue), the value was 0.82 ± 0.1 and in group 5 decellularization (fresh tissue), the value was 7.26 ± 1.50 ng/mg dry weight of scaffold. This result indicated the high efficiency of our two-step protocol. Comparison of the present study with the method of Baret et al. (2012), who achieved DNA content equivalent to 60 to 70 ng/mg, demonstrates the superiority of our method, which may be related to the precise sequence of detergents (SDS, Triton X-100, and the short two-step time used. Topraggaleh et al. (2019) achieved a value of 80 ng/mg with a simpler combination of SDS and Triton X-, which showed the relative inefficiency of their method in decellularizing dense tissues such as the testis. They supplemented their work by using the same 1% SDS in distilled water and were able to achieve better decellularization.
The results of the present study on DNA reduction are consistent with the results of Skolasinski et al., (2017) who showed that DNA content above 50 ng/mg can induce an inflammatory response in engineered tissues. In individuals with non-obstructive azoospermia, cell division is arrested at one stage for various reasons. Various methods have been developed to treat this type of male infertility, but many of these methods are in the animal testing or early clinical trials stages (Zhang et al., 2022; Goericke-Pesch et al.,2022; Cerván-Martín et al., 2020). The isolation of cells from testicular tissue from these individuals and then the culture in vitro on a decellularized testicular matrix (DTM) (as a three-dimensional environment to break cell arrest) in the presence of appropriate culture media can lead to the resumption of spermatogenesis in vitro in these individuals and may be considered as a suitable therapeutic strategy for the treatment of infertility in the future. In the present study, decellularization of testicular tissue was performed in both fresh and frozen samples. The results showed that the frozen tissue had better conditions for decellularization.
In agreement with our study, Gharenaz et al. (2020) showed that storage of decellularized scaffolds at low temperatures, especially using the slow freezing method, preserves the effectiveness of scaffold performance better than storage at 4 °C. On the other hand, Khazaei et al. (2024) showed that the use of freeze/thaw cycles with detergents such as 1% SDS or SDC resulted in effective cell removal while preserving the ECM matrix. Also, Afshari et al. (2025) succeeded in effective cell removal and preserving the ECM structure by performing three freeze–thaw cycles on rat testicular tissue and using detergents such as Triton X-100 and SLES. In another study, Gharenaz. et al. (2020) also found that the slow freezing method has long-term applications for the preservation of mouse testicular scaffolds.
Overall, although some concerns have been raised about the effect of the freeze-thaw process on the fine structure of the tissue, the available evidence suggests that decellularization of frozen testicular tissue preserves the quality of the scaffold, increases the efficiency of the process, and preserves the biological properties of the matrix. Based on evidence, freezing before decellularization (freeze–thaw or freeze–de–freeze) increases the permeability of the cell membrane to decellularizing agents. On the other hand, the use of frozen tissues allows for standardization and reduction of biological variations, because the researcher can freeze the samples immediately after harvest and store them under the same conditions to perform decellularization at the appropriate time.
Although decellularized testicular scaffolds provide a promising and biocompatible 3D environment to support the proliferation and differentiation of spermatogonial stem cells, several key challenges remain to extend this technology to clinical applications in men with azoospermia. The need to standardize decellularization protocols, quantify and evaluate residual ECM components, preserve or load biological growth factors, restore mechanical properties, and develop appropriate strategies for coculture of germ cells with supporting cells (Sertoli/Leydig) are some of the issues that need to be addressed. Looking forward, the integration of improved decellularization strategies with human induced pluripotent stem cell technology holds significant promise. Such progress could expand the availability of functional tissues and organs for three-dimensional culture, ultimately advancing the potential for successful clinical transplantation. However, current approaches remain limited, particularly for larger tissues and organs, emphasizing the need for more effective techniques. Systematic addressing of these challenges could significantly increase the chances of achieving functional sperm production in vitro and applications for reproductive rehabilitation (Goericke-Pesch et al., 2022; Rosa et al., 2025 (.
In this study, we optimized a decellularization protocol for human testicular tissue. One-way ANOVA analysis of DTFs showed that freezing testicular tissue at -80 °C for 24 hours and then placing it in a serial combination of Triton X-100 and SDS in PBS for 48 hours was the most efficient method to remove cellular material and preserve tissue architecture. In the absence of effective therapies for non-obstructive azoospermia, decellularized testicular matrix (DTM) allows researchers to investigate the specific composition of the testicular extracellular matrix (ECM) and its role in spermatogenesis. Furthermore, this type of matrix provides a natural structural framework that supports the in vitro reorganization of isolated testicular cells. This line of research represents a critical step toward preserving male fertility and holds potential as a therapeutic approach for non-obstructive azoospermia.
Acknowledgments
The authors would like to thank the staff of the Developmental Biology Laboratory at Babol University of Medical Sciences for their valuable assistance with the cell culture procedures.
Authors contribution
The first draft of the manuscript was written by Seyedeh fezeh Hashemi karouei. Study conception and design, material preparation and data collection were performed by Tooba Mirzapour. Mir Mohammad Reza Aghajani collected the data. Data analysis, review and editing were performed by Roghayeh Pourbagher. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
This research did not receive financial support from the Special Scientific Research Fund.
Ethical approval
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Guilan, Iran with the ethics ID (IR.GUILAN.REC.1403.32).
Informed consent
Informed consent has been obtained from all individual participants involved in the study and approved by Pathology Department of Shahid Beheshti Hospital, Babol City, Iran.
Conflict of Interest
The authors declare that there are no conflicts of interest regarding this study.
Data availability
Data can be provided by corresponding author upon reasonable request.
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