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

Background: Colorectal cancer (CRC) is the second most common cancer in Malaysia. Awareness of risk factors, symptoms and warning signs of CRC will help in early detection. This paper presents the level of CRC awareness among the urban population in Malaysia. Method: A cross-sectional study was conducted from November 2015 till December 2016 at three government clinics in the Klang Valley. The validated Bowel Cancer Awareness Measure questionnaire in both English and Malay was used. The mean knowledge scores for the warning signs and risk factors of CRC in different socio-demographic groups were compared using ANOVA in SPSS version 23. Statistical significance was set at p<0.05 and a 95% confidence level. Results: Of the 426 respondents, 29.1% were unable to recall the warning signs and symptoms of CRC. Average recall was less than two warning signs and symptoms (mean 1.62, SD 1.33). The mean total knowledge score for CRC was 9.91 (SD 4.78), with a mean knowledge scores for warning signs and risk factors at 5.27 (SD 2.74) and 4.64 (SD 2.78), respectively. Respondents with a higher level of education were found to have higher level of knowledge regarding the warning signs of CRC. There was a significant positive association between knowledge score for warning signs and level of confidence in detecting warning signs. Regarding the total knowledge score for CRC, 3.3% of respondents scored zero. For warning signs and risk factors, 8.2% and 8.5% of respondents had zero knowledge scores, respectively. Conclusions: Generally, awareness of CRC is poor among the urban population of Klang Valley. Greater education and more confidence in detecting warning signs are significantly associated with better knowledge of warning signs. CRC awareness programs should be increased to improve awareness.