Microfabrication of Substrates with Microscale Stiffness Gradients to Guide Bone Marrow Stromal Cell Migration

Summary

This protocol details the fabrication of a bilayer polydimethylsiloxane (PDMS) substrate via soft lithography. The method utilizes geometrically modulated microstructures in a rigid underlayer combined with a soft flat top layer to generate microscale effective stiffness gradients, enabling cellular mechanobiology studies without topographic interference.

Abstract

Understanding how cells respond to mechanical signals is crucial for elucidating the mechanisms involved in tissue development and disease progression. However, existing in vitro cell culture substrates often fail to replicate the physiological stiffness gradients at the cellular scale while also eliminating confounding topographical cues. In this study, we present a decoupled stiffness model utilizing a bilayer polydimethylsiloxane (PDMS) substrate. This substrate consists of a soft, flat top film suspended over a rigid underlayer featuring ridge-and-groove microstructures. The top layer effectively transmits stiffness variations in the underlying structure while maintaining uniform topography and chemistry at the cell contact surface. Stiffness modulation is achieved by varying the width of the alternating ridges and grooves, which are spaced equally. Scanning electron microscopy confirmed the flat surface morphology and consistent contact topography. Atomic force microscopy demonstrated that stiffness variations were dependent on the microstructure: for 20 μm patterns, the elastic moduli were approximately 950 kPa for ridges and 850 kPa for grooves; for 50 μm patterns, these values were around 1070 kPa and 950 kPa, respectively. Mouse primary bone marrow cells adhered well and spread on the substrate, showing a preference for nuclear localization toward the stiffer ridge regions in the 50 µm pattern (61.49%, p < 0.05), thereby confirming effective cellular perception of the mechanical gradient. In summary, this protocol offers a reproducible method to construct a cell culture substrate with microscale stiffness gradients, minimizing the chemical or topography interference, enabling investigations into cell mechanotransduction.

Introduction

Cells have the ability to sense and respond to mechanical stimuli within their external microenvironment^1,2,3, which are crucial for regulating various cellular processes, including adhesion, migration, proliferation, and differentiation⁴. In vivo, the mechanical properties of the tissue microenvironment exhibit significant spatial heterogeneity, with variations in matrix stiffness observed both in physiological contexts - such as at the junctions of bone and muscle - and in pathological conditions, such as within tumors⁵

Protocol

Primary mouse bone marrow stromal cells (mBMSCs) were isolated from the tibia and femur of mice (No. WP20230204, approved by the Ethics Committee of Wuhan University Center for Animal Experimentation).

1. Master mold preparation

NOTE: Our protocols are specific to epoxy-based negative photoresist used during this research.

1. Place two clean silicon wafers (50.8 ± 0.2 mm, <100> oriented, 525 ± 25 µm) on the hotplate for a few minutes to dehydrate.
2. Take one silicon wafer from the hotplate and cool it down to room temperature before proceeding with the spin....

Results

Photolithography was used to fabricate reusable silicon master molds featuring parallel ridge-and-groove microstructures. Two designs were created, featuring groove width (ridge width is identical to groove width) of 20 and 50 μm, respectively. Each patterned area measured 7 mm × 7 mm. The precision and reusability of the silicon molds significantly simplified the process of repeated substrate fabrication. The master mold was then used to replicate the microstructures in PDMS. A hard PDMS formulation was molded against t.......

Discussion

This study presents a novel approach to engineer a bilayer PDMS-based substrate designed to introduce localized stiffness variations while maintaining a topographically uniform featureless surface. This approach allows for mechanical modulation solely through variations in substructure geometry, while maintaining consistent surface chemistry and topography. Consequently, it effectively decouples the stiffness signal from other physical and chemical cues, offering a more biologically relevant system for stiffness modulati.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% paraformaldehyde	Servicebio Technology	G1101
Acetone	Shanghai Aladdin	10000418
Anti-Anti	Gibco	15240-062	Antibiotic-Antimycotic
Antifade Mounting Medium with DAPI	Beyotime Biotechnology	P0131	glycerin with DAPI
Cell Culture Plate	NEST	702001	24 wells
Desiccator	Sichuan Shubo(Group) Co.,Ltd	913354-30SN	Inner diameter=180mm
Fetal Bovine Serum	HyClone	SV30208.02
Inverted fluorescence microscope	Olympus	DP74
Isopropanol	Shanghai Aladdin	80109218	GC≥99.7%
Mask aligner	Sichuan Nanguang Vacuum Technology Co.,Ltd	H94-37
MEM Alpha modification	HyClone	SH30265.01
Nanoscope Analysis	Bruker	Version 3.0	AFM image processing and analysis software.
Opera Phenix Plus High Content Imaging System	Perkin Elmer	2400L23248	Equipped with 10x air objective
PGMEA	Suzhou NanoMicro Technology Co.,Ltd	171011-1	Developer Negative Photoresist; SPEC: UL; Percent:min99.7%
Phalloidin	Uelandy	YP0052L	594-Phalloidin
Phosphate Buffered Saline solution	HyClone	SH30256.01
Plasma Cleaner	Plasma technology (Germany)	Flecto 10
Poly-D-lysine	Beyotime Biotechnology	ST508	CAS Number 27964-99-4
Scanning Probe Microscope	Bruker	Dimension Icon	Uses Nanoscope control software, including PeakForce Quantitative Nanomechanical Mapping (PF-QNM)and Point-and-Shoot Ramping experimental workspaces.
Silicon Tip on Nitride Lever	Bruker	SCANASYST-AIR	Probe features a single, V-shaped, Al reflex coated cantilever; k= 0.4 N/m (nominal), f0 = 70 kHz.
Silicon wafers	Shenzhen Rigorous Technology Co.,Ltd	-	50.8±0.3mm diameter, (100)oriented, 525±25 μm thickness
Sodium Pyruvate	Gibco	11360-070
Spin coater	Institute of Microelectronics of the Chinese Academy of Sciences	KW-4A
SU-8 3025	Suzhou NanoMicro Technology Co.,Ltd	-	Negative Photoresist; 72.3% Solids; Viscosity 4400; Density 1.143g/ml
SYLGARD 184 Silicone Elastomer Kit	The Dow Chemical Company	01673921
Trichloro(1H,1H,2H,2H-tridecafluoro-n-octyl)silane	Shanghai Aladdin	78560-45-9
Triton X-100	BioFroxx	1139ML100	0.1% Triton X-100 for permeabilization
Trypsin-EDTA	Gibco	25200-072	0.25% Trypsin-EDTA (1x)
Vacuum Oven	Memmert (Germany)	VO 200