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

Introduction: Multitude studies have shown perception is an integral factor associated with smoking, However, no such tool was available in Malay language. In this study, we established a Bahasa Malaysia version of PTSQ (BM-PTSQ) and tested the validity and reliability among secondary school adolescents. Methods: The English version of PTSQ originally consists of 12 items. It was translated into Bahasa Malaysia and back-translated again into English to check for consistency. After face validity (face-to-face query) was determined among 20 secondary school adolescents, only 10 items were included in the survey. Construct validity was established from 407 school adolescents through random selection in the same locality. More than 60% of the respondents were female, a majority (67.3%) were schooling in rural areas. Then, the reliability of the questionnaire was determined with Cronbach's alpha. Results: EFA has grouped PTSQ into two components, they are associated with either knowledge or attitude towards smoking. The variance and Cronbach's alpha for the first and second component were 38.24% and 0.861 (7 items) and 21.62% and 0.661 (3 items) respectively. Conclusion The PTSQ showed good validity and reliability for measurement of perception in smoking among school adolescents in Malaysia, thus this is a viable measurement tool. More importantly this study shows an urgent need to improve the smoking education among adolescents in Malaysia.