Introduction: Leydig cells (LCs) are the principal source of testosterone in males, underpinning male reproductive health and androgendependent physiology. Precise isolation of viable human LCs is essential for mechanistic steroidogenesis research and cell-based therapeutic development. Existing protocols rely on density-gradient centrifugation, which is technically demanding and often compromises yield and viability. Here, we describe a simplified density-gradient-free approach using differential adhesion to enrich functional human LCs from testicular tissue. Methodology: Human testicular fragments (~5 mm³) were minced and enzymatically digested with collagenase IV (2 mg/mL) at 37°C for 20 min under gentle agitation (100 RPM). The suspension was filtered (45 µm) and plated onto poly-L-lysine-coated T25 flasks. After 24 hours, non-adherent cells were removed by PBS washing. Viability exceeded 90% by trypan blue exclusion. Cells were maintained in DMEM/F12 with 10% FBS, 1% Antibiotic-Antimycotic, and 10 ng/mL luteinizing hormone (LH) to preserve the mature LC phenotype. Characterization employed immunofluorescence and flow cytometry using antibodies against SF-1, StAR, LHCGR, PDGFRA, and TEM-1. Testosterone secretion was quantified by ELISA under basal conditions and LH-stimulated conditions (10 ng/mL). Results: The protocol yielded ~1.25 × 10⁶ LCs per gram of tissue with >80% purity. Adherent HLCs displayed dense cytoplasmic lipid granules and intercellular networks consistent with active steroidogenesis. Flow cytometry confirmed 84.7% StAR⁺ cells, indicative of a robust steroidogenic population. A distinct progenitor subpopulation (PDGFRα⁺/TEM-1⁺) comprising ~28% of primary cultures, suggests retention of regenerative capacity. Basal testosterone secretion averaged 8.88 ng/mL per 24 hours, confirming preserved functional activity post-isolation. Conclusion This differential-adhesion protocol efficiently isolates functional human LCs without density-gradient media. Multimodal validation integrating immunofluorescence, flow cytometry, and ELISA confirms both phenotypic identity and steroidogenic competence, providing a reproducible and accessible platform for LC research and translational applications in male hypogonadism and androgen replacement.
Leydig Cells

Objective: To explore the efficacy of earthworm's coelomic fluid against gentamicin-induced hepatic and renal toxicity in rats. Methods: The animals were divided randomly into three groups (n = 6 per group): control, gentamicin, and Allolobophora caliginosa coelomic fluid-treated groups. Toxicity was established after injection of gentamicin daily for 8 days at a dose of 100 mg/kg. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total proteins, albumin, creatinine, urea, uric acid, malondialdehyde, glutathione, catalase and histopathology of tissues were investigated in the study. Results: Allolobophora caliginosa coelomic fluid significantly decreased urea, creatinine, uric acid, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and malondialdehyde levels while significantly increasing levels of total proteins, albumin, glutathione and catalase. The histopathological investigation showed partial restoration of renal and hepatic architecture. Conclusions: This study shows the potency of Allolobophora caliginosa coelomic fluid in improving the biochemical and histopathological changes induced by gentamicin in the liver and kidney of the rats.