Isolation of functional human Leydig cells: A differential-adhesion approach with multi-modal phenotypic and steroidogenic validation
https://doi.org/10.15605/jafes.041.S1
- Author:
Yee Jia Heng
1
;
Pooi Pooi Leong
1
;
Omar Ahmed Fahmy Ahmed
2
;
Waye Hann Kang
3
Author Information
1. Department of Preclinical Sciences, M. Kandiah Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman
2. Department of Urology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia
3. Department of Medicine, M. Kandiah Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman
- Publication Type:Journal Article
- MeSH:
Leydig Cells
- From:
Journal of the ASEAN Federation of Endocrine Societies
2026;41(S1):3-
- CountryPhilippines
- Language:English
-
Abstract:
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.
- Full text:2026072808320939281OP_A003.pdf
