Two leading international congresses in Iran in the era of COVID-19: 21st royan international twin congress, 4th international and 16th Iranian genetics congress.

In this report, we look at the challenges posed by the outbreak of COVID-19 and how the Executive Board of these two congresses succeeded in overcoming those challenges and holding two congresses. The approach for a large festival with different virtual setting components provided a suitable solution that led to exemplary achievements and created an appropriate model for future virtual or combined virtual and face-to-face events. These events proved that pandemic problems could not limit the organizers, pushing them to make better use of the facilities and turning this threat into an opportunity.

Micro-quantity straw as a carrier for cryopreservation of oligozoospermic semen samples: Effects of storage times and cryoprotectant.

Application of an appropriate freezing carrier is crucial for improving post-thaw recovery of oligozoospermic samples. In this study, our purpose is developing a user-friendly, easy handling and close micro-quantity (MQ) straw along with different freezing media, for cryopreservation of oligozoospermic samples. Twenty oligozoospermic semen samples were collected and mixed with glycerol egg yolk citrate (GEYC) or Spermfreeze® (SPF) medium. The mixture was loaded into MQ straws, sealed and stored in liquid nitrogen (LN) vapor. After freezing, the straws were transferred into cryotube and plunged into LN. Post-thawed sperm parameters including motion characteristics, viability, membrane and DNA integrity were evaluated one and three months after cryopreservation. The post-thawed sperm parameters were significantly reduced in GEYC and SPF medium compared to fresh samples. No statistically significant differences were seen in sperm characteristics between the two storage times (i.e. month 1 vs. month 3). Furthermore, GEYC medium yielded higher motility, viability and membrane integrity compared to SPF at both storage time-points. Sperm DNA integrity was also improved in GEYC group compared to SPF after 1 month of storage. The findings of our study showed that application of MQ straw along with GEYC, as the cryoprotectant, was beneficial for cryopreservation of low count semen samples.

Enhancing developmental rate and quality of mouse single blastomeres into blastocysts using a microplatform.

The present work reports the beneficial effects of using a microplatform on the development of mouse single blastomeres (SBs) to the blastocyst stage. Development of blastocysts from SBs separated from two- and four-cell stage embryos (two- and four-cell SBs) can provide a valuable supply both for couples who use fertility-assisted techniques and farm animals. As a step forward, we introduce three chips that provide the possibility of culturing SBs separately, in groups, and in the vicinity of the intact embryo (co-culture), while each well of the chips is assigned to an isolated SB. Two- and four-cell SBs co-cultured with intact embryos showed 97.1% and 76.6% developmental rates and up to 34.1% and 49.1% growth relative to the microdroplet method (control). We examined the quality of developed blastocysts by assessing the total cell number, the number of inner cell mass (ICM) according to the octamer-binding transcription factor 4 marker (OCT4), and trophectoderm (TE). Co-culture of SBs with an intact embryo in a chip with nanoscale culture medium volume also increased the cell population of the developed embryo. The ICM:TE ratio, which is the most important blastocyst quality parameter, also indicated that developed two-cell SBs have a higher degree of similarity to intact embryos despite fewer numbers of total cells.

In Vitro Development of Mouse Preantral Follicle with Using Amniotic Membrane Extract-Loaded Hydrogels.

An artificial ovary based on the alginate (ALG) hydrogel has been widely implemented to preserve prepubertal female fertility. However, this platform is not fully capable of successful an ovary microenvironment simulation for follicle development, holding great potential for its improvement. Therefore, this experimental study aimed to evaluate the effect of an amniotic membrane extract (AME) -loaded hydrogel on the mouse preantral follicles in vitro development. In order to have better follicle development, first, the impact of different concentrations of follicle-stimulating hormone (FSH) was evaluated on the mouse preantral follicles encapsulated in ALG. Later, the appropriate dose was adjusted for the follicles encapsulated in the ALG-AME hydrogel. Results demonstrated that 100 mIU/ml FSH showed a significant follicle survival rate compared with 10 mIU/ml FSH (P=0.005). According to MTT assay finding, the rate of weight loss, and rheology evaluations, ALG containing 1 mg/ml AME was identified as an optimal sample of follicle culture instead of other AME concentrations. Follicle diameter significantly increased in the ALG-AME 1 hydrogel compared with the ALG control group without AME (P=0.027). The storage modulus of ALG-AME 1 was 773 Pa and retained the follicle morphology for 13 days. No statistically substantial difference was seen in survival, antrum cavity formation, and competent oocyte in terms of the normal chromosomal arrangement and meiotic spindle rate in comparison with the control group. It can be concluded that ALG-AME 1 could not significantly impact the mouse preantral follicle.

Regulation of cell fate by cell imprinting approach in vitro.

Cell culture-based technologies are widely utilized in various domains such as drug evaluation, toxicity assessment, vaccine and biopharmaceutical development, reproductive technology, and regenerative medicine. It has been demonstrated that pre-adsorption of extracellular matrix (ECM) proteins including collagen, laminin and fibronectin provide more degrees of support for cell adhesion. The purpose of cell imprinting is to imitate the natural topography of cell membranes by gels or polymers to create a reliable environment for the regulation of cell function. The results of recent studies show that cell imprinting is a tool to guide the behavior of cultured cells by controlling their adhesive interactions with surfaces. Therefore, in this review we aim to compare different cell cultures with the imprinting method and discuss different cell imprinting applications in regenerative medicine, personalized medicine, disease modeling, and cell therapy.

Biological evaluation of polyvinyl alcohol hydrogel crosslinked by polyurethane chain for cartilage tissue engineering in rabbit model.

Polyvinyl alcohol (PVA) hydrogel chains were crosslinked by urethane pre-polymer (PPU) in order to fabricate a new substitute for cartilage lesions. The microscopy images showed that the cultured chondrocytes had spherical morphology on PVA-PPU sample after 4 weeks of isolation in vitro. The alcian blue and safranin O staining proved the presence of proteoglycan on the surface of PVA-PPU sample secreted by cultured chondrocytes. This was confirmed by the detection of sulfate ions in the wavelength dispersive X-ray (WDX) analysis. In addition, the expression of collagen type II and aggrecan were observed in chondrocytes cultured on PVA-PPU by RT-PCR. Moreover, the implantation of the PVA-PPU sample with autologous cultured chondrocytes revealed the formation of neocartilage tissue in a rabbit model during 12 weeks follow up. In conclusion, the results verified that isolated chondrocytes cultured on PVA-PPU retain their original phenotype and this composition can be considered as promising substrate for cartilage tissue engineering.

Engineered substrates incapable of induction of chondrogenic differentiation compared to the chondrocyte imprinted substrates.

It is well established that surface topography can affect cell functions. However, finding a reproducible and reliable method for regulating stem cell behavior is still under investigation. It has been shown that cell imprinted substrates contain micro- and nanoscale structures of the cell membrane that serve as hierarchical substrates, can successfully alter stem cell fate. This study investigated the effect of the overall cell shape by fabricating silicon wafers containing pit structure in the average size of spherical-like chondrocytes using photolithography technique. We also used chondrocyte cell line (C28/I2) with spindle-like shape to produce cell imprinted substrates. The effect of all substrates on the differentiation of adipose-derived mesenchymal stem cells (ADSCs) has been studied. The AFM and scanning electron microscopy images of the prepared substrates demonstrated that the desired shapes were successfully transferred to the substrates. Differentiation of ADSCs was investigated by immunostaining for mature chondrocyte marker, collagen II, and gene expression of collagen II, Sox9, and aggrecan markers. C28/I2 imprinted substrate could effectively enhanced chondrogenic differentiation compared to regular pit patterns on the wafer. It can be concluded that cell imprinted substrates can induce differentiation signals better than engineered lithographic substrates. The nanostructures on the cell-imprinted patterns play a crucial role in harnessing cell fate. Therefore, the patterns must include the nano-topographies to have reliable and reproducible engineered substrates.

Development of a novel cervix-inspired tortuous microfluidic system for efficient, high-quality sperm selection.

Microfluidic systems have been extensively studied in recent years as potential alternatives for problematic conventional methods of sperm selection. However, despite the widespread use of simple straight channels in these systems, the impact of channel geometry on selected sperm quality has not been thoroughly investigated. To explore this further, we designed and fabricated serpentine microchannels with different radii of curvature, inspired by the tortuous structure of the cervix. Our results showed that in the presence of gentle backflow, microfluidic channels with a 150 µm radius of curvature significantly enhanced the quality of selected sperms when compared to straight channels. Specifically, we observed significant improvements of 7% and 9% in total motility and progressive motility, respectively, as well as 13%, 18%, and 19% improvements in VCL, VAP, and VSL, respectively. Through careful observation of the process, we discovered a unique near-wall sperm migration pattern named boundary detachment-reattachment (BDR), that was observed exclusively in curved microchannels. This pattern, which is a direct consequence of the special serpentine geometry and sperm boundary-following characteristic, contributed to the superior selection performance when combined with a fluid backflow. After determining the best channel design, we fabricated a parallelized chip consisting of 85 microchannels capable of processing 0.5 ml of raw semen within 20 minutes. This chip outperformed conventional methods of swim-up and density gradient centrifugation (DGC) in terms of motility (9% and 25% improvements, respectively), reactive oxygen species (18% and 15% improvements, respectively), and DNA fragmentation index (14% improvement to DGC). Outstanding performance and advantages such as user-friendliness, rapid selection, and independence from centrifugation make our microfluidic system a prospective sperm selection tool in clinical applications.

Mouse ovarian follicle growth in an amniotic membrane-based hydrogel.

Three-dimensional cultures of follicles on ECM-based scaffolds can be an approach for women who become infertile after cancer treatments. Human amniotic membrane (HAM) is extensively employed in tissue engineering because of its unique properties. We cultured mouse pre-antral follicles in a hydrogel derived from decellularized amniotic membrane (DAM) combined with alginate (ALG) to improve ovarian follicle culture. HAM was decellularized. Quantitative (nuclear contents, collagen, glycosaminoglycan [GAG]) and qualitative (DAPI, H&E, Masson's trichrome, Alcian blue, scanning electron microscopy assessments were performed. Then, we created an amniotic membrane-based hydrogel (AMBH) and conducted AMBH characterization assays (rheology, MTS, degradation rate). Isolated mouse pre-antral follicles were cultured in 15 mg/mL AMBH (AMBH15), 30 mg/mL AMBH (AMBH30), or 45 mg/mL AMBH (AMBH45). ALG hydrogel was the control group. Follicular diameters, estradiol hormone secretion rate, follicular morphology, and the follicle antral and degeneration rate were examined. Quantitative and qualitative assays indicated successful decellularization. AMBH characterization assays showed that the ALG hydrogel had more appropriate gelation and slower degradation than AMBH. There was a statistically higher antral follicle formation rate in the AMBH45 group (p < .05) compared to the AMBH30 and AMBH15 groups and less (p < .05) degenerated follicles. There was no significant difference with the ALG group. Diameter and estradiol hormone secretion in the AMBH45 group were not significantly higher than the ALG group. Although decellularization was confirmed and the viscoelastic parameters of AMBH support follicle culture, there was no significant effect on ovarian follicle maturation compared to the ALG control group.

Correction: A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids.

Correction for 'A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids' by Tohid Rezaei Topraggaleh et al., Biomater. Sci., 2019, 7, 1422-1436, DOI: 10.1039/C8BM01001C.

Oxygen releasing materials: Towards addressing the hypoxia-related issues in tissue engineering.

Manufacturing macroscale cell-laden architectures is one of the biggest challenges faced nowadays in the domain of tissue engineering. Such living constructs, in fact, pose strict requirements for nutrients and oxygen supply that can hardly be addressed through simple diffusion in vitro or without a functional vasculature in vivo. In this context, in the last two decades, a substantial amount of work has been carried out to develop smart materials that could actively provide oxygen-release to contrast local hypoxia in large-size constructs. This review provides an overview of the currently available oxygen-releasing materials and their synthesis and mechanism of action, highlighting their capacities under in vitro tissue cultures and in vivo contexts. Additionally, we also showcase an emerging concept, herein termed as "living materials as releasing systems", which relies on the combination of biomaterials with photosynthetic microorganisms, namely algae, in an "unconventional" attempt to supply the damaged or re-growing tissue with the necessary supply of oxygen. We envision that future advances focusing on tissue microenvironment regulated oxygen-supplying materials would unlock an untapped potential for generating a repertoire of anatomic scale, living constructs with improved cell survival, guided differentiation, and tissue-specific biofunctionality.

An integrated microfluidic device for stem cell differentiation based on cell-imprinted substrate designed for cartilage regeneration in a rabbit model.

Separating cells from the body and cultivating them in vitro will alter the function of cells. Therefore, for optimal cell culture in the laboratory, conditions similar to those of their natural growth should be provided. In previous studies, it has been shown that the use of cellular shape at the culture surface can regulate cellular function. In this work, the efficiency of the imprinting method increased by using microfluidic chip design and fabrication. In this method, first, a cell-imprinted substrate of chondrocytes was made using a microfluidic chip. Afterwards, stem cells were cultured on a cell-imprinted substrate using a second microfluidic chip aligned with the substrate. Therefore, stem cells were precisely placed on the chondrocyte patterns on the substrate and their fibroblast-like morphology was changed to chondrocyte's spherical morphology after 14-days culture in the chip without using any chemical growth factor. After chondrogenic differentiation and in vitro assessments (real-time PCR and immunocytotoxicity), differentiated stem cells were transferred on a collagen-hyaluronic acid scaffold and transplanted in articular cartilage defect of the rabbit. After 6 months, the post-transplantation analysis showed that the articular cartilage defect had been successfully regenerated in differentiated stem cell groups in comparison with the controls. In conclusion, this study showed the potency of the imprinting method for inducing chondrogenicity in stem cells, which can be used in clinical trials due to the safety of the procedure.

Oxygen-rich Environment Ameliorates Cell Therapy Outcomes of Cardiac Progenitor Cells for Myocardial Infarction.

To some extent, cell therapy for myocardial infarction (MI) has supported the idea of cardiac repair; however, further optimizations are inevitable. Combined approaches that comprise suitable cell sources and supporting molecules considerably improved its effect. Here, we devised a strategy of simultaneous transplantation of human cardiac progenitor cells (CPCs) and an optimized oxygen generating microparticles (MPs) embedded in fibrin hydrogel, which was injected into a left anterior descending artery (LAD) ligating-based rat model of acute myocardial infarction (AMI). Functional parameters of the heart, particularly left ventricular systolic function, markedly improved and reached pre-AMI levels. This functional restoration was well correlated with substantially lower fibrotic tissue formation and greater vascular density in the infarct area. Our novel approach promoted CPCs retention and differentiation into cardiovascular lineages. We propose this novel co-transplantation strategy for more efficient cell therapy of AMI which may function by providing an oxygen-rich microenvironment, and thus regulate cell survival and differentiation.

Effects of Alginate Concentration and Ovarian Cells on In Vitro Development of Mouse Preantral Follicles: A Factorial Study.

BACKGROUND: In the present study, the effects of alginate (ALG) concentration and ovarian cells (OCs) on the development and function of follicles were simultaneously evaluated. MATERIALS AND METHODS: In the first step of this experimental study, preantral follicles were isolated from the ovaries of 2-week-old mice, encapsulated in the absence or presence of OCs in 0.5, 0.75 and 1% ALG hydrogels, and cultured for 14 days. The morphology, diameter, survival and antrum formation rates of the follicles and the maturation of the oocytes were evaluated during culture. In the second step, preantral follicles were cultured in the best chosen ALG concentration, in both the absence and presence of OCs. Following these steps, the amount of DNA fragmentation, the expression levels of connexin 37 and connexin 43 proteins, the secretion levels of estradiol, progesterone and androstenedione by the follicles and the quality of mature (MII) oocytes were assessed. RESULTS: Our data revealed that in the absence of OCs, follicles of 0.5% group showed a higher survival rate than the 0.75 and 1% groups (71.87 vs. 52.52 and 40%, respectively, P<0.05). Nonetheless, the antrum formation rate of the 1% group was higher and its oocyte degeneration rate was lower than that in the other groups. Furthermore, it was observed that co-culture of follicles with OCs relatively increased the follicle diameter, survival, antrum formation, and germinal vesicle (GV) to GV break down (GVBD)/MII transition rates. At last, the comparison of 0.5%-OCs and 0.5%+OCs groups indicated that the co-culture condition resulted in more progesterone production (1.8 ± 0.2 vs. 3.2 ± 0.4 ng/ml, respectively, P<0.05) and also decreased oocytes' cortical granule abnormalities (100 vs. 40% for 0.5%- OCs and 0.5%+OCs groups, respectively). CONCLUSION: The present study revealed that 0.5% ALG hydrogel is relatively suitable for preantral follicle culture, and in the presence of OCs, it mimics the natural ovarian condition better than the higher concentrations of ALG hydrogel.

Applicability of Hyaluronic Acid-Alginate Hydrogel and Ovarian Cells for In Vitro Development of Mouse Preantral Follicles.

OBJECTIVE: In the present study, the applicability of hyaluronic acid-alginate (HAA) hydrogel and ovarian cells (OCs) for the culture of mouse ovarian follicles were investigated and compared with those of alginate (ALG) and fibrin-alginate (FA) hydrogels. MATERIALS AND METHODS: In the first step of this experimental study, mechanically isolated preantral follicles from the ovaries of two-week-old mice were encapsulated in the absence or presence of OCs in ALG, HAA, and FA hydrogels and cultured for 14 days. The morphology, diameter, survival and antrum formation rates of the follicles and the maturation and quality of the oocytes were evaluated during culture. In the second step, preantral follicles were cultured similar to the first step, but for 13 days, and their gene expressions and hormonal secretion were assessed on the last day of culture. RESULTS: In the absence of OCs, higher numbers of ALG- and HAA-encapsulated follicles reached the antral stage compared to FA-encapsulated follicles (P<0.05). However, a higher percentage of HAA-developed oocytes resumed meiosis up to the germinal vesicle breakdown (GVBD)/metaphase II (MII) stages in comparison with ALG-developed oocytes (P<0.05). HAA-encapsulated follicles had significant overexpression of most of the growth and differentiation genes, and secreted higher levels of estradiol (E2) compared to ALG- and FA-encapsulated follicles (P<0.05). The co-culture condition increased the diameter of ALG-encapsulated follicles on day 13 of culture (P<0.05). It also increased the survival and maturation rates of ALG- and FA-encapsulated follicles, respectively (P<0.05). The co-culture condition improved cortical granule distribution in all groups, increased E2 and progesterone (P4) secretions in the ALG and FA groups, and androstenedione (A4) secretion in the FA group (P<0.05). CONCLUSION: The present study results show that HAA hydrogel is a promising hydrogel for follicle culture. OCs utilization could ameliorate the culture conditions regardless of the type of hydrogel.

Generation of Scalable Hepatic Micro-Tissues as a Platform for Toxicological Studies.

BACKGROUND: Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects of therapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to human hepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and the presence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this study was to generate the scalable and functional hepatic micro-tissues (HMTs). METHODS: The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cells and human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, using an air-driven droplet generator. RESULTS: The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoring glycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretion levels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involved in hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional (2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity to hepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450 enzymes confirmed that the HMTs can be used for in vitro drug screening. CONCLUSION: Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7, with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.

Tissue Engineering in Liver Regenerative Medicine: Insights into Novel Translational Technologies.

Organ and tissue shortage are known as a crucially important public health problem as unfortunately a small percentage of patients receive transplants. In the context of emerging regenerative medicine, researchers are trying to regenerate and replace different organs and tissues such as the liver, heart, skin, and kidney. Liver tissue engineering (TE) enables us to reproduce and restore liver functions, fully or partially, which could be used in the treatment of acute or chronic liver disorders and/or generate an appropriate functional organ which can be transplanted or employed as an extracorporeal device. In this regard, a variety of techniques (e.g., fabrication technologies, cell-based technologies, microfluidic systems and, extracorporeal liver devices) could be applied in tissue engineering in liver regenerative medicine. Common TE techniques are based on allocating stem cell-derived hepatocyte-like cells or primary hepatocytes within a three-dimensional structure which leads to the improvement of their survival rate and functional phenotype. Taken together, new findings indicated that developing liver tissue engineering-based techniques could pave the way for better treatment of liver-related disorders. Herein, we summarized novel technologies used in liver regenerative medicine and their future applications in clinical settings.

Vascular endothelial growth factor sustained delivery augmented cell therapy outcomes of cardiac progenitor cells for myocardial infarction.

Cell therapy has become a novel promising approach for improvement of cardiac functional capacity in the instances of ventricular remodeling and fibrosis caused by episodes of coronary artery occlusion and hypoxia. The challenge toward enhancing cell engraftment as well as formation of functional tissue, however, necessitated combinatorial approaches. Here, we complemented human embryonic stem cell-derived cardiac progenitor cell (hESC-CPC) therapy by heparin-conjugated, vascular endothelial growth factor (VEGF)-loaded fibrin hydrogel as VEGF delivery system. Transplantation of these cardiac committed cells along with sustained VEGF release could surpass the cardiac repair effects of each constituent alone in a rat model of acute myocardial infarction. The histological sections of rat hearts revealed improved vascularization as well as inclusion of hESC-CPC-derived cardiomyocytes, endothelial, and smooth muscle cells in host myocardium. Thus, co-transplantation of hESC-CPC and proangiogenic factor by a suitable delivery rate may resolve the shortcomings of conventional cell therapy.

Optogenetic Stimulation for Restoring Vision to Patients Suffering From Retinal Degenerative Diseases: Current Strategies and Future Directions.

Optogenetic strategies for vision restoration involve photosensitizing surviving retinal neurons following retinal degeneration, using emerging optogenetic techniques. This approach opens the door to a minimally-invasive retinal vision restoration approach. Moreover, light stimulation has the potential to offer better spatial and temporal resolution than conventional retinal electrical prosthetics. Although proof-of-concept studies in animal models have demonstrated the possibility of restoring vision using optogenetic techniques, and initial clinical trials are underway, there are still hurdles to pass before such an approach restores naturalistic vision in humans. One limitation is the development of light stimulation devices to activate optogenetic channels in the retina. Here we review recent progress in the design and implementation of optogenetic stimulation devices and outline the corresponding technological challenges. Finally, while most work to date has focused on providing therapy to patients suffering from retinitis pigmentosa, we provide additional insights into strategies for applying optogenetic vision restoration to patients suffering from age-related macular degeneration.

A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids.

Extracellular matrix-derived scaffolds provide an efficient platform for the generation of organ-like structures. Successful development of testicular organoids (TOs) with the capability of supporting complete spermatogenesis has not been reported yet. Here, we have developed an optimized method for the decellularization of ram testicular tissue fragments. Our findings showed that testicular fragments treated with a serial combination of Triton X-100 and SDS in PBS for 48 h resulted in the efficient removal of cellular materials and retention of the extracellular matrix (ECM) components. In order to fabricate testis-derived scaffolds (TDSs), the testicular ECM (T-ECM) was digested in acid/pepsin, followed by neutralization of pre-gel solution to form a hydrogel. Then, the hydrogels were freeze-dried and cross-linked using a chemical method. To reach the optimal concentration for the T-ECM in the fabrication of TDSs, the scaffold properties including porosity, pore size, swelling behavior, and degradation were evaluated. Our study suggested that 25 mg ml-1 of the T-ECM is the best concentration for the fabrication of macroporous TDSs for demonstrating lower pore size, homogeneously distributed pores, and a higher swelling ratio. Furthermore, inoculation of neonatal mouse testicular cells onto TDSs resulted in the generation of multicellular TOs in which the differentiation of spermatogonial cells into post-meiotic cells was confirmed. Hormonal analysis of TDSs revealed the functionality of TOs in the secretion of testosterone and inhibin B. The current study also demonstrated that macroporous TDSs could provide a novel platform for testicular tissue engineering and in vitro spermatogenesis.