Animals and Drugs
Male Naval Medical Research Institute [NMRI) mice were used in this examination. The animals were obtained from Animal Resources Department, University of Tabriz (Tabriz, Iran). The mice were kept in animal houses under standard conditions (at 22 ± 2 °C room temperature, 50–55% humidity, light / dark cycles: 12/12 hours per day). The chemicals that were used in this study were purchased from Sigma–Aldrich unless otherwise mentioned.
Testis dissociation and SSCs isolation
Ten male NMRI mice (6 days old; n=10) were euthanized with intraperitoneal injection 100 mg/Kg ketamine and 10 mg /Kg xylazine. Their testis was removed and placed into a petri dish containing culture medium and antibiotic. Testicular tissue cells were isolated with slight modifications according to the methods presented by . In summary, after washing the testicular tissue several times in PBS solution for cell isolation, the testicular capsule was removed, and then the testicular tissue was divided into approximately 1 mm³ using the mechanical procedure. Thereafter, the dissociated tissues were placed in a solution containing trypsin (0.5 mg / ml, Sigma-Aldrich, St Louis, MO, USA), collagenase I (0.5 mg / mL Sigma-Aldrich, St Louis, MO, USA), and hyaluronidase (0.5 mg / mL, Sigma-Aldrich, St Louis, MO, USA). Then, a cell was centrifuged (1500 rpm for 5 min) and washed in DMEM/F12 (Gibco, Grand Island, NY, USA) medium. Finally, the next digestion was accomplished for 15 min in a solution containing collagenase I (1mg/mL, Sigma-Aldrich, St Louis, MO, USA) DNase (0.5 mg/mL Sigma-Aldrich, St Louis, MO, USA,), and hyaluronidase (1.5 mg/mL, Sigma-Aldrich, St Louis, MO, USA) in the water bath at 37°C. The obtained cells were washed and prepared for culture after passing via a 70 µm cell strainer.
SSCs culture and proliferation
After enzymatic digestion, cell viability was evaluated with 0.4 % trypan blue solution in a 6-well plate with the culture medium DMEM/F12 containing 10% FBS (Gibco, Grand Island, NY, USA) with penicillin (100 U/mL) and streptomycin (100 µg/mL; Gibco, Grand Island, NY, USA). To isolate the SSCs which usually attach to the bottom of the culture dish later than the somatic cells, the supernatant was removed after 24 hours and centrifuged (1000 rpm for 5 minutes) to separate the non-attached cells. Then cells were cultured in DMEM/F12 medium containing 5 % Knockout serum replacement (KSR) (Gibco, Grand Island, NY, USA) GDNF (10ng/mL, Sigma Alderich St.Louis.MO, USA), LIF (103U/mL, Sigma Alderich St.Louis.MO, USA) EGF (20ng/mL, Sigma Alderich St.Louis.MO, USA) bFGF (10ng/mL, Sigma Alderich St.Louis.MO, USA) in gelatin (0.2 %; Sigma Alderich St.Louis.MO, USA) coated dishes, and then incubated at 35°C in a humidified atmosphere with 5 % CO2. The medium replaces every 48h. After two weeks since the initial culture, SSCs colonies were observed. After two weeks in culture, the Plzf protein was traced as a positive marker to confirm the identity of the colonies derived from the SSCs .
Immunocytochemistry for characterization of SSCs colonies
To characterize SSC colonies, Plzf expression was determined by immunocytochemistry (ICC). After fixation with paraformaldehyde (4%) permeability with 0.4 % Triton X100 (Sigma-Aldrich, St Louis, MO, USA) and blocking with 10% goat serum (Sigma-Aldrich, St Louis, MO, USA), cells were treated with rabbit polyclonal antibody anti-Plzf (sc28319;mouse monoclonal, 1:500, Santa Cruz, Houston, USA) and anti-GFR-α1 (pa519873;Rabbit polyclonal, 1:100, Thermo Fisher, Altrincham, UK) for 2 h at 37°C. After washing with PBS, a secondary antibody, Donkey Anti-Rabbit, labeled with fluorescent isothiocyanate (FITC) 1: 100 (Sigma-Aldrich, St Louis, MO, USA), was added for 3 h. Control cells were treated under similar conditions except for the removal of primary antibodies. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 1μg / mL; Sigma-Aldrich, St Louis, MO, USA) .
In this experimental study, testicular scaffolds were generated using the testes obtained from 6 mice models (6-8 weeks old). The tunica albuginea was removed from the testes of the mice using a 29-gage insulin syringe and washed with phosphate buffer saline [PBS, Sigma, USA) to remove the residual blood. The sections of 100 µm diameters of testis were immersed in % 0.5 (v/v) of sodium dodecyl sulfate (SDS, Sigma-Aldrich, St Louis, MO, USA) and % 0.5 (v/v) triton X-100 (TX-100; Sigma-Aldrich, St Louis, MO, USA) and then incubated for 18 h in water. Then, they were placed by PBS for 2 h to remove detergents. After washing, the decellularized tissue sections were sterilized in 70% ethanol for 1 hour and immersed in PBS for 2 hours. Before usage, the scaffolds were immersed in a culture medium for 24 hours .
Hematoxylin-eosin (H&E) staining was used to confirm the quality of DTM and evaluate cell migration to the scaffold after its recellularization. Each sample was fixed in formaldehyde [10%), dehydrated through increasing ethanol concentration, after clearing by xylen and embedding in paraffin, 5-μm thick sections were prepared with a microtome (Leica, Germany). Sections were deparaffinized, rehydrated, and stained via Hematoxylin & eosin (all from Merck Germany). Alcian blue staining (Merck, Germany) was conducted to assess the amount of glycosaminoglycan's (GAGs) based on previous described protocols . Masson's Trichrome staining was performed according to guidelines to determine the existence of collagen . Slides were observed under a light microscope (Labomed, USA), and images (nuclei are stained dark blue, cytoplasm are stained bright-red, as well as collagen is stained green or blue) were taken with a Ziess Camera.
DAPI staining for nuclei staining of decellularized tissue
Both confirmed the intact and decellularization of the testis by 4, 6-diamidino-2- phenylindol (DAPI) staining and DNA quantification. To assess the homogeneity of decellularization, the samples were collected from different sites of the scaffolds. In brief, DAPI solution (1μg/mL; Sigma-Aldrich, St Louis, MO, USA) was pipetted directly to each tissue section after fixing the tissues in formalin. They were kept in the dark room for 15 min. After washing with PBS, the samples were evaluated with an inverted fluorescence microscope.
DNA content in decellularized testicular tissue
For quantitative testing, the total amount of DNA from native tissue and decellularized testicular scaffold were extracted by a genomic DNA purification kit (Qiagen, UK). The purity and concentration of DNA were calculated by Nanodrop spectrophotometers (UV-visible; Thermo Nanodrop ND-1000) at 260 nm and agarose gel .
Synthesis of dual-crosslinked hydrogels: Natural ECM of decellularized testis and Hyaluronic acid hydrogel
For composite-cross-linked hydrogels, 10 mg of sodium hyaluronate powder was dispensed into a circular silicone rubber mold. The same volume of lyophilized testicular tissue extract was sandwiched between two rubber molds and tightly secured. Crosslinking solutions consisted of 0.2 M NaOH containing butanediol diglycidyl ether (BDDE). The concentration of cross linkers was measured in units of µL BDDE/g HA . This fluid was inserted into each rubber mold by a syringe equipped with a 25-gage needle. A second needle created an air escape, and caution was considered to stop bubble trapping. A volume of crosslinking fluid was applied to fill each mold. Molds were roughly 100 µL in volume and had an HA concentration of 10%. HA and crosslink solution were then incubated at 40ºC for 8 h. The cross-linked hydrogels were separated from the molds and relocated to a large amount of distilled deionized water or saline (0.9% NaCl) for swelling.
Cytocompatibility of scaffold by MTT assay
Following the scaffold was produced, its toxicity was tested to confirm that it did not contain any hazardous substances during the scaffold preparation and decellularization stages. After the culture of SSCs at a concentration of 5 ×104 in each well of 96-well plate, scaffold extract was added to cells in the culture medium, and after 24 h, 48 h, and 72 h since the inception of the culture, the cell was evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In brief, add 200 µl of MTT solution (0.5ng/ml) to each well and incubated for 4 h at 37 °C in a humidified atmosphere 5 % CO2 incubator, and after that, cells lysed and purple formazan crystal dissolved by adding 400 μL of DMSO (D8418, Sigma-Aldrich, St Louis, MO, USA) in each well for 30 min at room temperature. Finally, the purple formazan formed during the test step was measured at 540 nm with the microplate reader .
Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) evaluated the 3D structure of scaffold, quality, decellularization, and cell migration after recellularization. The tissues were fixed with 2.5% glutaraldehyde for 2 h, and then with 1% osmium tetroxide and dewatering by increasing the degrees of ethanol (30 to 100%). After dehydration, the tissues were placed on a grid, covered with a gold-palladium coating, and seen via SEM (FEI Quanta 200). The number and diameter of SSCs colonies in each field and pores size of the scaffold were analyzed using SEM .
Preparation of DTM and Reconstitution of testicular cells on it
Reconstituted testicular artificial tissue using a hyaluronic gel matrix 2% and 50 mg of Decellularized testes extract (1:1 v/v ratio) were placed in a 24-well culture dish in a thin layer form. Then, 1×105 cells were added to each well plate (Nunc, Roskilde, Denmark) to which collagen gel matrix and somatic cells adhere poorly. After gently agitating the gels for 1 minute, they were overlaid with DMEM/F12 medium supplemented with 5 % KSR, 5 % FBS. During culture, the period medium was replaced every 2 days. After two weeks of culture, the matrix gel was re-dissolved with 0.25% trypsin to release the embedded cells, and then cell viability and proliferation were determined after two weeks of initiation of culture .
The culture groups were classified into 2 categories include (1) 2D culture and (2) 3D culture. Each group were defined in a subgroup with different treatments include 1a (2D- D-Serine) or 2a (3D- D-Serine); D-Serine treatment, 1b [2D- Glutamic acid) or 2b (3D- Glutamic acid); Glutamic acid, 1c (2D- MK-801) or 2c; MK-801 (3D- MK-801). D-Serine and MK-801 were used 20 µM, and Glutamic acid was used 50 µM in a final concentration. SSCs cultured in each experimental group for 2 weeks for assessment of SSCs proliferation in different culture medium situations. Two normal groups included 2D and 3D without D-serine, and MK-801 were designed as control groups in the whole study phases.
Gene expression analysis
In the second weeks of proliferation culture, the total RNA of the cells cultured in 2D and 3D culture was extracted using guanidine / phenol solution (Qiazol-Qiagene USA). Then RNA was treated with DNase I (Fermentase, USA) to eliminate genomic contamination. The purity and concentration of RNA were determined using Nano Drop 2000 (Thermo scientific). For Complementary DNA (cDNA) synthesis, 1 µg total RNA was carried out by reverse transcription kit, using a Prime Script RT reagent kit (Hyper script RT-PCR- GeneAll) according to the manufacture instructions. PCR was carried out using mixed Master Mix and SYBR green in the stage one of thermal cycle (ABI stage one, USA). PCR program began with melting cycle of 15 min at 95°C. The stage was followed by 40 cycles: initial denaturation 30s at 95°C, annealing 30s at 60ºC, and extension 30s at 60ºC. The specific primers used to determine the expression levels of pre-meiotic genes including Plzf [Forward: 5' AGT GGG ATT GAT GAG GAG ATG G 3' and Reverse 5' AGT GGA GTG TAG GGA GAA GGA 3’) as SSCs specific markers and βactin (Forward 5' TCA GAG CAA GAG AGG CAT CC 3'and Reverse: 5' GGT CAT CTTCTC ACG GTT GG 3') as internal control gene. To investigate the presence of non-specific products and primer dimers, a melting curve analysis was performed. Furthermore, this process was replicated in triplicates for target and reference genes. All samples were normalized to the βactin as a reference gene using (∆∆CT) method .
Protein evaluation by Flow cytometry
The process of cell staining for flow cytometry was done based on pervious researchers by Kanatsu et al. . Furthermore, the cells fixed at 4% paraformaldehyde and permeabilized at 0.5% Triton X-100 (Invitrogen, UK) for 5 minutes before being blocked by a combination of 10% goat serum and Bovine serum albumin [BSA, 1 mg/ml). The incubation of cells was performed with the primary antibodies, including rabbit anti Plzf at 4°C overnight. Cells were incubated with a certain number of secondary antibodies (FITC, conjugated goat anti rabbit IgG) 60 min at 37° C in the dark. After washing with PBS, flow cytometric analysis was accomplished with a Becton Dickinson (BD) and was analyzed with Flowjo 7.6 software.
The data presented are expressed as mean ± standard error (mean ± SEM). Kolmogorov-Smirnov test was used to determine the normal distribution of data. An independent sample t-test was used for DNA content and MTT analysis. For multiple comparisons of data, One-way analysis of variance (ANOVA) followed by Tukey’s test used. Graph Pad Prism software version 8 (Graph Pad Software San Diego, CA) was used for data analysis. Data with less than < 0.05 were considered statically significant.