Spermatogenesis, the process of sperm production, relies heavily on the intricate interplay between Sertoli and Leydig cells within the seminiferous tubules of the testis. Sertoli cells, acting as nurse cells, form the blood-testis barrier and secrete essential factors for germ cell development. Leydig cells, located in the interstitial tissue, are the primary source of testosterone, another key element in spermatogenesis. The bidirectional communication between these two cell types is crucial for maintaining testicular function and successful spermatogenesis. Dysfunction in this crosstalk can lead to various male reproductive disorders, including cryptorchidism, hypospadias, and hypogonadism. Existing 2D and even some 3D in vitro models fail to adequately capture the complexities of this interaction and the multicellular nature of testicular tissue, particularly when using human cells. This study aimed to overcome these limitations by developing a sophisticated 3D human testis-on-a-chip model that accurately reflects the reciprocal interactions between Sertoli and Leydig cells, as well as other important testicular cell types. The model was designed to improve upon previous attempts that largely used animal cells or failed to fully replicate the functional interplay between key cell types within the seminiferous tubules.

Numerous studies have attempted to create in vitro models of the testis to study spermatogenesis and toxicology. However, most previous studies have focused on animal models, and those employing human cells often fall short of fully capturing the complex interplay between Sertoli and Leydig cells and other testicular components. Traditional 2D cell culture systems lack the three-dimensional structure and cell-cell interactions found in vivo. While some 3D models have been developed, many rely on animal cells or fail to adequately represent the dynamic reciprocal communication between Sertoli and Leydig cells, ultimately limiting their predictive capabilities for human reproductive health and toxicology. The need for a human-cell-based model that accurately simulates the complex testicular microenvironment and the reciprocal endocrine crosstalk between Sertoli and Leydig cells is apparent from the limitations of current approaches. This study aims to address this gap by establishing a robust and physiologically relevant in vitro model.

The researchers developed a 3D testis-on-a-chip platform using a polydimethylsiloxane (PDMS) microfluidic device fabricated from a 3D-printed mold. The chip was designed with distinct chambers for Sertoli cells and Leydig cells, interconnected by channels lined with vascular endothelial cells to facilitate the exchange of hormones and other signaling molecules. An additional chamber was included to incorporate macrophages, representing the immune cell component of the testicular microenvironment. Human Sertoli and Leydig cells, isolated from a patient undergoing orchiectomy, were embedded within a natural polymer mixture of collagen and hyaluronic acid, further strengthened with fibrinogen and thrombin to enhance mechanical stability and biocompatibility. The cells were carefully characterized to confirm their identity and functionality before being incorporated into the chip. The mechanical properties of the resulting 3D tissue construct were extensively analyzed using techniques such as scanning electron microscopy (SEM), compressive stress testing, and rheological analysis. The long-term viability and metabolic activity of the embedded cells were assessed using live/dead assays and CCK-8 assays, respectively. Immunofluorescence staining was performed to verify the maintenance of cell-specific characteristics and expression of key biomarkers. ELISAs were used to measure the secretion of ABP (androgen-binding protein) from Sertoli cells and testosterone from Leydig cells. The responsiveness of the cells to hormonal stimulation (testosterone and LH, respectively) was evaluated by assessing cell viability and metabolic activity, as well as the expression of relevant markers. To identify biomarkers of male reproductive toxicity, RNA sequencing was performed on Sertoli and Leydig cells exposed to dioxin (TCDD), a known toxicant. SERPINB2 was identified as a promising biomarker. This was verified using real-time PCR and western blotting. SERPINB2 knockdown experiments were conducted to determine the role of SERPINB2 in mediating toxic responses, including apoptosis, growth, and migration. Finally, a fluorescent reporter system was developed by conjugating fluorescent proteins (mCherry or GFP) to the SERPINB2 promoter. This reporter system was integrated into the testis-on-a-chip to provide an intuitive and quantitative measure of male reproductive toxicity in response to various substances.

The researchers successfully created a 3D human testis-on-a-chip that accurately mimicked the complex multicellular environment and reciprocal endocrine crosstalk within the seminiferous tubules. The 3D tissue architecture exhibited suitable mechanical properties for cell survival and functionality. Various embedded cells (Sertoli cells, Leydig cells, vascular endothelial cells, and macrophages) maintained viability, metabolic activity, and expression of cell-specific markers for extended periods. The chip accurately replicated key physiological functions, including the secretion of ABP and testosterone in response to hormonal stimulation. RNA sequencing revealed SERPINB2 as a reliable biomarker for predicting male reproductive toxicity, confirmed by real-time PCR, western blotting, and SERPINB2 knockdown experiments. The integrated fluorescent reporter system demonstrated the ability to quantitatively assess toxicity by measuring SERPINB2-mediated fluorescence intensity in response to various toxicants. Importantly, the system demonstrated that SERPINB2-mediated fluorescence intensity increased in both the Sertoli and Leydig cells in response to a broad range of tested toxicants, suggesting its utility as a universal biomarker.

This study presents a significant advancement in in vitro models of the testis, addressing the limitations of existing 2D and 3D systems. The human testis-on-a-chip successfully replicates the complex cellular interactions and hormonal dynamics crucial for spermatogenesis and provides a highly sensitive and reproducible platform for assessing male reproductive toxicity. The identification of SERPINB2 as a universal biomarker and the development of a quantitative fluorescent reporter system represent major improvements in toxicity screening methodologies. This model significantly reduces the reliance on animal models and has the potential to revolutionize drug discovery and toxicology research related to male reproductive health.

The development of a 3D human testis-on-a-chip incorporating a SERPINB2-based fluorescent reporter system represents a significant leap forward in male reproductive toxicology and research. The model accurately simulates the complex interactions between key cell types and provides a highly sensitive method for evaluating the toxicity of materials. Future research could explore the use of this platform to study the effects of various environmental toxins, hormonal imbalances, and genetic mutations on testicular function and spermatogenesis. Expansion of the model to include other testicular cell types and further refinement of the reporter system could also enhance its predictive capabilities.

While this study provides a robust and advanced testis-on-a-chip model, some limitations should be acknowledged. The study used cells from a single donor, limiting the generalizability of the findings. Further studies with cells from multiple donors are needed to validate the model's consistency and reproducibility. The current model focuses on a specific set of toxicants. A more comprehensive analysis with a wider range of chemicals would strengthen the utility of the SERPINB2 biomarker and the reporter system. Long-term studies beyond 28 days are needed to fully understand the model's long-term stability and maintenance of cell function.