Method Article

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

DOI:

10.3791/69473

⸱

November 14th, 2025

 ,  ,  ,  ,  ,  ,  , 
1State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, 2School of Power and Mechanical Engineering, The Institute of Technological Science, Wuhan University, 3Department of Oral Implantology, School and Hospital of Stomatology, Wuhan University

In This Article

Summary

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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

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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

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Cells have the ability to sense and respond to mechanical stimuli within their external microenvironment1,2,3, 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 tumors5,6,7. A comprehensive investigation of these mechanical factors is essential for understanding the regulatory mechanisms governing cell behavior. These mechanisms are pivotal in processes such as tissue development, disease progression, and the process of regeneration and repair8,9.

To simulate this complex mechanical microenvironment in vitro, hydrogels or elastomers are widely used to create substrates with varying stiffness by adjusting the degree of cross-linking or component ratio3,10,11,12,13. However, these methods often result in discrete stiffness changes that fail to reproduce the continuous, microscale stiffness gradients that accurately mimic physiological conditions13,14,15,16,17. Recent studies have explored techniques such as optical cross-linking (e.g., using a photomask to regulate UV exposure)18, microfluidic multi-channel mixing19,20, and diffusion-driven means21 to construct stiffness gradients. Unfortunately, these approaches can be technically complex and introduce additional variables, such as mesh size22, swelling23,24, and topography25. Introduction of disturbance factors complicates the interpretation of cellular responses to stiffness. A bilayer PDMS system, utilizing micropillar arrays or checkerboard patterns as a support structure, has been adopted to create cell culture substrates with stiffness gradients26,27. These approaches rely on discrete underlying supports beneath a chemically homogeneous membrane to achieve isotropic stiffness modulation, providing valuable platforms for investigating cell adhesion and spreading. However, the native cellular microenvironment in vivo is often highly anisotropic in its mechanical properties28,29. This directional mechanical signaling is postulated to profoundly influence fundamental biological events. Therefore, current biofabrication strategies are limited in their ability to replicate the subtle cell-stiffness interactions observed in vivo, which depend on accurately mimicking microscale stiffness gradients and spatial heterogeneity within cell-loaded substrates.

Previous research has demonstrated that cells can perceive the effective stiffness of rigid objects even when they are not in direct cellular contact30. Based on this study, we introduce a novel bilayer polydimethylsiloxane (PDMS) cell culture substrate that generates controlled stiffness gradients at spatial scales ranging from 5 to 50 µm, aligning with the size range relevant to subcellular and single-cell mechanosensing. The substrate consists of two distinct layers: a rigid PDMS underlayer featuring tunable geometric microstructures and a uniform, flat soft PDMS overlayer that serves as the cell-contacting surface. This design 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 modulation.

In contrast to traditional hydrogel-based substrates, our approach enables single-variable mechanical studies without interference from swelling, mesh size, or surface topology. The substrate is straightforward to fabricate using standard soft lithography and is highly reproducible31. It is also compatible with high-resolution microscopy, which makes it particularly well-suited for live-time imaging and quantitative studies of cellular responses to microscale stiffness cues. This manuscript presents the complete fabrication protocol, details the parameter control strategy, and demonstrates the substrate's utility with representative cell culture experiments. Ultimately, this approach provides a robust and accessible in vitro tool for exploring cell responses to a microscale stiffness gradient.

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Protocol

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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 coating. Apply 3-5 mL of photoresist to the wafer center.
  3. To obtain a feature height of 20 µm, apply the following spin protocol: spin for 10 s at 7.1 × g, followed by spin at 410 × g for 30 s. To obtain a feature height of 30 µm, apply the following spin protocol: spin for 10 s at 7.1 × g, followed by spin at 170 × g for 30 s. Use a scalpel carefully to remove any edge bead that may form during the spin coating.
  4. Soft bake in the following manner: set the hotplate to 95 °C, allow the wafer to sit on hot plate for 13 min. Remove the wafer from the hotplate and allow it to cool to room temperature naturally, avoiding a sudden temperature drop.
  5. Align the photomask on the photoresist-coated wafer and expose to UV light with a total dose of 120 mJ/cm2.
  6. Postexposure bake in the following manner: using two hotplates (65 °C and 95 °C), allow the photoresist-coated wafer to sit on the 65 °C hotplate for 1 min before transferring it to the 95 °C hotplate and leaving it there for 5 min. Move the wafer onto a stack of microfiber tissues and allow it to cool down to room temperature.
  7. Develop the wafer by transferring it to a Petri dish filled with PGMEA, and submerge the entire wafer in the developer. Accelerate the development by manual agitation.
    NOTE: PGMEA is flammable and irritant to the eyes and respiratory tract. Dispose of it as "flammable organic waste" in dedicated containers.
  8. After development, take out the wafer from the developer and wash it in isopropanol. If a milky white flow forms in the isopropanol close to the sample surface (the development is not complete), immerse the sample back in the developer again to complete the development process. Wash the developed sample in isopropanol until no milky white flow forms close to the sample surface. Once fully developed, hard bake the photoresist by placing the wafer on a 150 °C hotplate for 15 min.
  9. Fluorinate the wafer for the following soft lithography processes: expose the wafer to oxygen plasma under vacuum for 30 s (100 W) to activate the surface, and then place it in a vacuum desiccator. Drop 30 µL of trichloro(1H,1H,2H,2H-tridecafluoro-n-octyl) silane onto a glass slide placed inside the vacuum desiccator. Evacuate the desiccator for 2 min and maintain the vacuum condition for 30 min. Bake the wafer at 90 °C for 1 h to complete the silanization process.
    NOTE: Strictly control the ambient relative humidity below 60% throughout the process. Excessive moisture can induce quick hydrolysis of the silane, which generates white non-volatile polysiloxane precipitates. The precipitates will impair the uniformity of silane vapor distribution, resulting in a failed silanization. Carry out the treatment in a hood as trichloro(1H,1H,2H,2H-tridecafluoro-n-octyl) silane reacts violently with water, releasing toxic HCl fumes. Neutralize with dilute alkali before disposal as fluorinated/chlorinated organic waste. The silanization process achieves a 90% success rate.

2. Fabrication

NOTE: The soft lithography process used to fabricate the bilayer polydimethylsiloxane (PDMS) substrate can be divided into two distinct steps: 1) The preparation of both the structured underlayer and the top layer, 2) the bonding of the two PDMS layers. The overall manufacturing yield is over 80%.

  1. PDMS preparation
    1. For the structured underlayer, prepare the PDMS precursor by mixing the prepolymer and curing agent in a ratio of 10:1 by weight, followed by degassing under vacuum. Pour 1.5 mL of the PDMS precursor (10:1) onto the SU8 master mold, covering the entire surface, followed by degassing under vacuum and resting for 30 min. Carefully move it to an oven and cure at 90 °C for 1 h. Demold the structured PDMS underlayer from the master mold.
    2. For the top layer, prepare the PDMS precursor by mixing the prepolymer and curing agent in a ratio of 30:1 by weight and degassing under vacuum. Pour the PDMS precursor on a fluorinated silicon wafer. To obtain a thickness of 9 µm, apply the following spin coating protocol: spin at 7.1 × g for 10 s, followed by a spin at 2,300 × g for 60 s.
    3. After spin coating, visually inspect the PDMS film under ambient light to check for uniformity -- specifically examining for any visible defects such as streaks, bubbles, or uneven edge buildup. If the film appears uniformly transparent with consistent reflectivity across the entire wafer surface, proceed to the next step; if not, repeat the spin coating process with a new wafer. Place the coated wafer on a level surface to ensure a homogeneous PDMS film thickness. Carefully move it to an oven and cure at 90 °C for 3 h.
      ​NOTE: Films with a thickness of 9.11 ± 0.34 µm were obtained.
  2. Bonding
    1. Place the structured PDMS underlayer (10:1) with patterned side facing down onto the PDMS-top-layer film, ensuring good contact between the two layers without bubbles at the interface.
    2. Move the assembly into the oven and cure at 90 °C for 3 h.
    3. Allow the assembly to cool down to room temperature on a stack of microfiber tissues. Detach the final double-layer structure from the wafer, and store in a Petri dish for further use.
      NOTE: If bubbles form between the top layer and the underlayer during peeling, it typically indicates incomplete adhesion between the two layers. Ensure thorough degassing of the PDMS precursor before spin-coating, and verify that the film surface is free of dust or contaminants prior to the application of the PDMS underlayer for full contact. Ensure the silicon wafer is level during spin-coating, assembly, and curing. Film tearing may occur due to the uneven silanization of the wafer or non-uniform PDMS thickness. To solve the problem, strictly control the environmental humidity during silanization under 60% RH. The silanization process achieves a 90% success rate.

3. Characterization

  1. Scanning electron microscopy (SEM)
    1. Cut the sample with a sharp blade to obtain clean edges.
    2. Mount the samples on 90° and 0° SEM stages using conductive carbon tape to enable cross-sectional and surface characterization, respectively.
    3. Sputter coat the samples with gold for 120 s to enhance SEM imaging quality and collect SEM images of the samples.
  2. Atomic Force Microscopy (AFM)
    NOTE: Select ScanAsyst-Air probe for quantitative nanomechanical mapping of modulus.
    1. Position the AFM cantilever tip at the center of the view field. Position the laser spot onto the very end of the cantilever by aligning the optical lever system.
    2. Prior to PeakForce Quantitative Nanomechanical Mapping (PeakForce QNM) measurements, calibrate the AFM cantilever to determine the following essential parameters: Cantilever Spring Constant: 0.40135 N/m; Deflection Sensitivity: 28.993 nm/V (determined by performing a force curve on a rigid reference sample); Tip Radius: ~21 nm (calibrated using the relative method against a reference sample with known modulus).
    3. Within the Scan panel of the Scan Parameters List, configure the following initial scan settings: Set the Scan Size to 70 µm for samples featuring periodic stripes with 50 µm spacing. Set the Scan Size to 50 µm for samples featuring periodic stripes with 20 µm spacing. Set Samples/Line and Lines to 256 (resulting in a 256 x 256 pixel image).
    4. Open Engage Settings in the Microscope and set the Instrument's default Peak Force Engage Setpoint in Engage Parameters.
    5. Click the Engage icon on the Workflow Toolbar and continue to monitor the real-time signals during the automated approach.
    6. Ensure ScanAsyst Auto Control is set to Individual mode. Adjust the PeakForce Setpoint to an appropriate value to achieve the desired Deformation range: for the top layer, PeakForce Setpoint is 0.020 nN; for the structured underlayer, PeakForce Setpoint is 5 nN.
    7. Initiate the scan by clicking the top to bottom or bottom to top buttons.
    8. Force Curve Monitoring and Correction: If force curves appear distorted or synchronization issues are observed (e.g., irregular shapes, inconsistent baselines), right-click within the Force Monitor window and select Auto Config to automatically correct the force curve alignment and sensitivity.
    9. Within the modulus calculation channel settings, select DMT model to fit the retraction portion of the force curve for the calculation of the elastic modulus.
    10. Click the capture button to acquire and save the high-resolution topography and nanomechanical property map.
    11. Perform post-acquisition analysis using the AFM image processing and analysis software. Open the saved AFM data files and extract modulus values by selecting regions across the sample surface; calculate average modulus, standard deviation, and distribution profiles for both the top layer and underlayer using the software's built-in statistical analysis tools. Export processed modulus maps and quantitative data.
    12. Number of AFM scans per sample: Perform three independent full-area scans for each sample. Ensure each scan covers a region matching the sample's structural feature size, 70 µm × 70 µm for samples with 50 µm-spaced periodic stripes, and 50 µm × 50 µm for those with 20 µm-spaced stripes. For modulus measurements of the underlying structure (10:1 PDMS) and the top layer film (30:1 PDMS), implement the following sampling strategy to ensure data reliability.
      1. Top layer film (30:1 PDMS): measure the modulus 2x independently with each measurement consisting of two non-overlapping 5 µm × 5 µm scans.
      2. Underlying structure (10:1 PDMS): Given its two distinct periodic stripe configurations (20 µm and 50 µm spacing), measure the modulus 2x independently for each configuration.

4. In vitro cell culture experiment

NOTE: PDMS is a hydrophobic material that needs to be coated to promote cell adhesion before cell culture, and the appropriate coating method should be selected according to the cell type. Ethical approval must be obtained if primary cells are used for the cellular experiment.

  1. Poly-D-lysine (PDL) coating
    1. Sterilize the substrate by alcohol immersion for 10 min; then wash for 3 x 5 min with PBS.
    2. Dissolve PDL (molecular weight 150,000 to 300,000 Da) using PBS at a concentration of 0.1 mg/mL. For the 24-well plate size substrate, add 1 mL of PDL working solution and place it in a 37 °C incubator for 4 h to fully coat the substrate surface.
    3. After the coating is completed, remove the PDL working solution, and remove any residual PDL by washing 3x with PBS. Use the substrate for subsequent cell experiments. If the coating is not used immediately, dry it and store at 4 °C.
      NOTE: A successful coating presented a homogeneous, featureless surface under phase-contrast microscopy, with no visible crystalline precipitates or drying artifacts. Substrates with any signs of crystallization or non-uniform coating were discarded.
  2. Isolation and culture of primary mouse bone marrow stromal cells (mBMSCs)
    1. Isolate primary mouse bone marrow stromal cells (mBMSCs) from the tibia and femur of mice.
    2. Culture the cells in a growth medium consisting of α-MEM supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic, and 1% sodium pyruvate.
    3. Seed cells at passage 3 onto the substrates obtained in step 4.1.3 at a density of 5,000 cells/cm2 to minimize potential confounding effects from cell-cell contact.
  3. Immunofluorescence staining and imaging
    1. Following 16 h of culture (37 °C, 5% CO2), fix the cells with 4% paraformaldehyde for 10 min at room temperature and wash 3x with PBS. Then, permeabilize the cells with 0.1% Triton X-100 for 15 min at room temperature and wash 3x with PBS.
    2. For cytoskeletal visualization, stain the F-actin using phalloidin (1:500 dilution) for 20 min at room temperature, and wash 3x with ample PBS to remove any residual dye.
    3. Mount the substrate with simultaneous staining of cell nuclei. Apply a drop of glycerin with DAPI to cover the cells. Thereafter, carefully place a coverslip on top of the substrate and seal the margins using nail polish. Avoid bubbles as much as possible.
    4. Acquire fluorescence images capturing cellular morphology and nuclear positioning using an inverted fluorescence microscope.

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Results

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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...

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Discussion

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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...

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Disclosures

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The authors declare that they have no competing financial interests.

Acknowledgements

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This study was financially supported by the Fundamental Research Funds for the Central Universities of China (No. 2042024YXB018 to W. Ji).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
 4% paraformaldehydeServicebio TechnologyG1101
AcetoneShanghai Aladdin10000418
Anti-AntiGibco15240-062Antibiotic-Antimycotic
Antifade Mounting Medium with DAPIBeyotime BiotechnologyP0131glycerin with DAPI 
Cell Culture PlateNEST70200124 wells
DesiccatorSichuan Shubo(Group) Co.,Ltd913354-30SNInner diameter=180mm
Fetal Bovine SerumHyCloneSV30208.02
Inverted fluorescence microscopeOlympusDP74
IsopropanolShanghai Aladdin80109218GC≥99.7%
Mask alignerSichuan Nanguang Vacuum Technology Co.,LtdH94-37
MEM Alpha modificationHyCloneSH30265.01
Nanoscope AnalysisBrukerVersion 3.0AFM image processing and analysis software.
Opera Phenix Plus High Content Imaging SystemPerkin Elmer2400L23248 Equipped with 10x air objective
PGMEASuzhou NanoMicro Technology Co.,Ltd171011-1Developer Negative Photoresist; SPEC: UL; Percent:min99.7%
PhalloidinUelandy YP0052L594-Phalloidin
Phosphate Buffered Saline solutionHyCloneSH30256.01
Plasma CleanerPlasma technology (Germany)Flecto 10 
Poly-D-lysineBeyotime BiotechnologyST508CAS Number 27964-99-4
Scanning Probe MicroscopeBrukerDimension IconUses Nanoscope control software, including PeakForce Quantitative Nanomechanical Mapping (PF-QNM)and Point-and-Shoot Ramping experimental workspaces.
Silicon Tip on Nitride LeverBrukerSCANASYST-AIRProbe features a single, V-shaped, Al reflex coated cantilever; k= 0.4 N/m (nominal), f0 = 70 kHz.
Silicon wafersShenzhen Rigorous Technology Co.,Ltd-50.8±0.3mm diameter, (100)oriented, 525±25 μm thickness
Sodium PyruvateGibco11360-070
Spin coaterInstitute of Microelectronics of the Chinese Academy of SciencesKW-4A
SU-8 3025Suzhou NanoMicro Technology Co.,Ltd-Negative Photoresist; 72.3% Solids; Viscosity 4400; Density 1.143g/ml
SYLGARD 184 Silicone Elastomer KitThe Dow Chemical Company01673921
Trichloro(1H,1H,2H,2H-tridecafluoro-n-octyl)silaneShanghai Aladdin78560-45-9
Triton X-100BioFroxx1139ML1000.1% Triton X-100 for permeabilization
Trypsin-EDTAGibco25200-0720.25% Trypsin-EDTA (1x)
Vacuum OvenMemmert (Germany) VO 200 

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Stiffness GradientsMicrofabrication SubstratesBone Marrow Stromal CellsCell MigrationPDMS SubstrateRidge Groove MicrostructuresAtomic Force MicroscopyScanning Electron MicroscopyCell MechanotransductionElastic Moduli

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