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Proactively revolutionize the in vitro testicular toxicity testing

High quality canine and rat Sertoli Cells available for researcher

Sertoli cells play critical roles in regulating spermatogenesis and testis development by providing structural and nutritional support. This study aimed to develop a standard protocol for canine Sertoli cell isolation and culture; and characterize its biological features, functionality, and application of compound toxicity testing. Canine testicles were received from the neuter clinic, and three-step of enzymatic digestion was applied to isolate Sertoli cells. We characterized the growth and purity of Sertoli cells with the expression of SOX9, GATA4, and Clusterin. In addition, we selected cadmium as a model toxicant to evaluate the toxic responses in the newly established Sertoli cells using High-content Analysis (HCA). With our optimized protocol, the purity of isolated Sertoli cells was above 95%, as determined with Sertoli cell-specific protein markers of SOX9 and GATA4. More importantly, primary Sertoli cell populations could be expanded rapidly in vitro, passaged (up to seven), and cryopreserved. The HCA-based assay revealed that cadmium at 1 μM induced both disruptions of cytoskeletal and DNA damage responses. Furthermore, we established an HCA assay with the newly isolated and optimized culture of canine Sertoli cells to evaluate the epigenetic markers of histone modification. We found cadmium-induced differential changes in histone modifications H3Me3K9, H3Me3K36, H4Me3K20, and H4acK5. In summary, we have established the standardized protocol to produce canine Sertoli cells with Sertoli cell-specific phenotype. The isolation and expansion of large quantities of canine Sertoli cells will provide broad applications in studying male infertility, reproductive toxicology, testicular cancer, and cell therapy.

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Mar 2023

High-content Image based Analysis of Endocrine Disruptors

We published a munuscript titled "High-content analysis of testicular toxicity of BPA and its selected analogs in mouse spermatogonial, Sertoli cells, and Leydig cells revealed BPAF induced unique multinucleation phenotype associated with the increased DNA synthesis". The purpose of this study is to examine the selective toxicity of BPA, BPS), BPAF, and TBBPA on these testicular cells, including Sertoli, Leydig and spermatogonial cells. We developed a high-content image-based single-cell analysis and measured a broad spectrum of adverse endpoints related to the development of reproductive toxicology, including cell number, nuclear morphology, DNA synthesis, cell cycle progression, early DNA damage response, cytoskeleton structure, DNA methylation status, and autophagy. We introduced an HCA index and spectrum to reveal multiple HCA parameters and observed distinct toxicity profiling of BPA and its analogs among three testicular types. The HCA spectrum shows the dynamic, chemical-specific, dose-dependent changes of each HCA parameter. Each chemical displayed a unique dose-dependent profile within each type of cell. All three types of cells showed the highest response to BPAF at 10 μM across all endpoints measured. BPAF targeted spermatogonial cell more significantly at 5 μM. BPS more likely targeted Sertoli cell and Leydig cell and less at spermatogonia cells. TBBPA targeted spermatogonia, Sertoli cells, and less at Leydig cells. BPA is mainly targeted at Sertoli cells, followed by Ledig cells, and less at spermatogonial cells. Most importantly, we observed that BPAF induced a dose-dependent increase in spermatogonia cells, not in Sertoli and Leydig cells. In summary, our current HCA assays revealed the cell-type-specific toxicities of BPA and its analogs in different testicular cells. Multinucleation induced by BPAF, along with increased DNA damage and synthesis at low doses, could possibly have a profound long-term effect on reproductive systems.

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Jan 2023