Fabricating Tunable Polyacrylamide Hydrogels To Study Matrix Stiffness Effects On Stem Leydig Cells

Summary

This protocol describes the fabrication of polyacrylamide hydrogels with tunable stiffness to study the effects of matrix mechanics on stem Leydig cell behavior in a controlled two-dimensional culture system.

Abstract

Extracellular matrix (ECM) stiffness critically regulates stem cell behavior. Previously, we demonstrated that pathological increases in matrix stiffness during aging disrupt stem Leydig cell (SLC) homeostasis, leading to a decline in testosterone. Building on this discovery, we here present a detailed protocol—originally developed in our laboratory—for fabricating polyacrylamide (PA) hydrogels with tunable stiffness to model the testicular microenvironment in vitro. This method enables reproducible casting of gels across a stiffness range of 1–100 kPa, covering physiological to pathological conditions. Key steps include precise mixing of acrylamide/bis-acrylamide, gel swelling equilibration, surface activation with Sulfo-SANPAH, and collagen coating to support SLC adhesion and culture. We provide optimized formulations for target stiffnesses and troubleshooting guidance for common issues such as incomplete polymerization and poor cell attachment. This system allows systematic investigation of how substrate stiffness modulates SLC proliferation, differentiation, and steroidogenic function under defined 2D conditions. Beyond reproductive biology, it also serves as a valuable platform for mechanobiological studies in other cell types and for screening therapeutics targeting stiffness-related dysfunction.

Introduction

The mechanical properties of the extracellular matrix (ECM) play a pivotal role in regulating stem cell fate, including proliferation, differentiation, and functional maintenance^1,2,3,4. In the testis, age-related ECM stiffening has been implicated in the decline of testosterone production. However, the underlying mechanobiological mechanisms remain poorly understood, partly due to a lack of standardized in vitro models that faithfully recapitulate the physiological stiffness range of the testicular niche. The physiological....

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat‑sen University (Approval No. SYSU-IACUC-2026-B1278) and were performed in accordance with the institutional guidelines.

1. Isolation and culture of primary mouse SLCs

1. Dissect testes from C57BL/6 mice and carefully remove the tunica albuginea.
2. Mince the testes into small pieces.
3. Dissociate interstitial cells from seminiferous tubules by incubating in 1 mg/mL collagenase type IV in DMEM/F12 at 37 ºC for 15 min.
4. Add DMEM/F12 containing 10% fetal bovine serum to stop collagenase ....

Results

Successful execution of this protocol yields polyacrylamide hydrogels with defined stiffness (ranging from 1 to 100 kPa) that remain firmly attached to the culture plate and support robust SLC adhesion and spreading. Figure 1 provides a schematic overview of the entire workflow, from gel casting to cell seeding, enabling users to visualize the key stages of the protocol.

Mechanical validation of polyacrylamide hydrogels (target stiffness: 1, 5, 15, 30, 50, and 100.......

Discussion

The mechanical properties of the extracellular matrix play a fundamental role in regulating stem cell behavior, yet the tools to systematically study these effects in reproductive cells remain limited. Our previous work demonstrated that pathological matrix stiffening during aging disrupts stem Leydig cell (SLC) homeostasis through the Piezo1/ROS/Gli1 axis, leading to testosterone decline¹⁰. Building on this discovery, we developed this polyacrylamide hydrogel protocol to provide a standardized, r.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	Sigma-Aldrich	Cat#A6283	Diluent for rat tail collagen (0.02 M)
Acryiamide	Sangon Biotech	Cat#A100341-0500	Monomer for polyacrylamide gel preparation
Ammonium persulfate (APS)	Sangon Biotech	Cat#A100486-0100	Initiator for gel polymerization
B27 supplement	Invitrogen	Cat#A1486701	Defined supplement enriched for antioxidants and hormones; supports neuronal and stem cell survival (2% v/v)
Basic fibroblast growth factor (bFGF)	Invitrogen	Cat#13256029	Mitogenic growth factor; promotes SLC proliferation and stemness (20 ng/mL)
Bis-acrylamide	Shanghai yuanye Bio-Technology	Cat#S14002	Crosslinker for polyacrylamide gels
Chicken embryo extract	US Biologicals	Cat#C3999	Rich source of growth factors and nutrients; supports SLC growth and viability (5% v/v)
Collagen, Type I, from rat tail	YEASEN	Cat#40125ES50	Coats the Sulfo-SANPAH-activated polyacrylamide hydrogel surface to promote SLC adhesion and culture.
Collagenase Type IV	Gibco	Cat#17104-019	1 mg/mL in DMEM/F12 for testicular tissue digestion
CYP11A1 antibody	GeneTex	Cat#GTX56293	For western blotting
Dexamethasone	Sigma-Aldrich	Cat#D1756	Glucocorticoid receptor agonist; supports SLC proliferation and stemness maintenance (1 nM)
DMEM/F12 medium	Gibco	Cat#11320033	Basal medium for digestion, washing, and SLC culture
Epidermal growth factor (EGF)	PeproTech	Cat#AF-100-15	Stimulates SLC proliferation and maintains undifferentiated state (20 ng/mL)
Fetal Bovine Serum (FBS)	VISTECH	Cat#SE100-011	Used at 10% to stop collagenase activity
Fiji	N/A	https://imagej.net/Fiji	Image analysis software
GADPH antibody	Proteintech	Cat#60004-1-Ig	For western blotting
Gel digestion enzymes	Accurate Biotechnology	Cat#GXDLFA	Enzyme mixture for testicular tissue digestion and SLC isolation
HEPES	Cytiva	Cat#SH30237.01	Buffer to maintain pH 7.0–7.4
HSD3β antibody	Santa Cruz	Cat#sc-515120	For western blotting
Influx Cell Sorter	BD	https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/BD_Influx_tech_specs.pdf
Insulin-Transferrin-Sodium Selenite (ITS)	Sigma-Aldrich	Cat#11074547001	Promotes cell survival, glucose uptake, and antioxidant defense (5 μg/L)
KnockOut serum replacement (KSR)	Gibco	Cat#10828-028	Serum substitute for SLC culture medium
LIF (Leukemia Inhibitory Factor)	Millipore	Cat#LIF1010	Cytokine that maintains stem cell pluripotency and self-renewal (1 ng/mL)
Mice testicles	This paper	N/A	Isolated from C57BL/6 mice
Mouse Testosterone ELISA Kit	Fine Biotech	Cat#40203ES80	For quantification of testosterone in culture supernatants
Mouse: C57BL/6	Shenzhen TopBiotech	N/A	Mouse strain used for testis collection
Mouse: Testicular Stem Leydig Cells (SLCs)	This paper	N/A	Primary cells isolated from mouse testes
N2 supplement	Invitrogen	Cat#17502001	Defined serum-free supplement supporting neural and stem cell cultures (1% v/v)
Non-essential amino acids	HyClone	Cat#SH30050.03	Provides nitrogen sources for protein synthesis and cell metabolism (1% v/v)
Oncostatin M (OSM)	PeproTech	Cat#300-10T	Cytokine involved in SLC differentiation regulation and Leydig cell maturation (20 ng/mL)
Phosphate-Buffered Saline (PBS)	Gibco	Cat#10010023	Washing and resuspension buffer
Platelet-derived growth factor (PDGF)	PeproTech	Cat#100-14B	Supports cell proliferation, migration, and survival (20 ng/mL)
Prism 9.0	GraphPad	https://www.graphpad.com/	Software for statistical analysis and graphing
StAR antibody	Proteintech	Cat#67130-1-Ig	For western blotting
Sulfo-SANPAH	MACKLIN	Cat#102568-43-4	Heterobifunctional crosslinker for gel surface activation
TEMED	Phygene	Cat#PH0341	Catalyst for polymerization
β-Mercaptoethanol	Invitrogen	Cat#21985023	Reduces oxidative stress and supports cell growth in culture (0.1 mM)