Generation of Polyacrylamide and Silicone Extracellular Matrix Substrates with Defined Stiffness for Cell Biology Applications

Aisha Almsoud; Upama Nyaupane; Kashish Jain; Ghmkin Hassan; Johnathan N. Lakins; Valerie M. Weaver; FuiBoon Kai

doi:10.3791/70626

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Method Article

Generation of Polyacrylamide and Silicone Extracellular Matrix Substrates with Defined Stiffness for Cell Biology Applications

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DOI:

10.3791/70626

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June 26th, 2026

Aisha Almsoud¹^,² , Upama Nyaupane¹^,² , Kashish Jain³ , Ghmkin Hassan³ , Johnathan N. Lakins³ , Valerie M. Weaver⁴^,⁵^,⁶ , FuiBoon Kai¹^,²

¹Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, ²Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, ³Department of Surgery, University of California, San Francisco, ⁴Center for Bioengineering and Tissue Regeneration, University of California, San Francisco, ⁵UCSF Helen Diller Comprehensive Cancer, University of California, San Francisco, ⁶Department of Bioengineering and Therapeutic Sciences, Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California, San Francisco

Summary

Although extracellular matrix (ECM) stiffness is a key regulator of cellular behavior, the underlying sensing and transduction mechanisms remain unclear. To address this, we provide protocols for creating polyacrylamide- and silicone-based substrates with tunable stiffness to advance our understanding of cell-ECM crosstalk.

Abstract

The extracellular matrix (ECM) is a critical regulator of cell behavior, with ECM stiffness serving as a key mechanical cue that governs cellular signaling, morphology, and fate. However, experimental investigation of stiffness-dependent cellular responses is often limited by the availability of reproducible, cost-effective, and accessible culture platforms with precisely tunable mechanical properties. This protocol describes polyacrylamide (PA)- and silicone-based methods for fabricating ECM substrates with tunable stiffness. The procedures outline substrate preparation, surface functionalization, and ECM protein conjugation to ensure consistent cell adhesion across a physiologically relevant stiffness range. PA-based substrates are compatible with downstream biochemical assays, including protein and RNA extraction, as well as high-resolution fluorescence imaging. Silicone-based substrates are optimized for total internal reflection fluorescence (TIRF) microscopy, enabling visualization of cell-ECM interactions at the basal membrane. In addition, soft substrates can be adapted to support spheroid cultures positioned within a consistent imaging plane, facilitating consistent image acquisition. These methods provide robust, cost-effective, and reproducible platforms for systematically probing how ECM stiffness regulates cellular processes, advancing mechanistic insight into cell-ECM crosstalk in both physiological and disease contexts.

Introduction

The extracellular matrix (ECM) is a fundamental structural and biochemical scaffold that defines the mechanical properties of tissues and supports their biological functions. For instance, elastic tissues such as the lungs and arteries are enriched in collagen- and elastin-based ECM that provides the mechanical resilience required for repeated stretch-recoil cycles during respiration. In contrast, the central nervous system contains glycosaminoglycan-rich ECM that creates a highly compliant environment optimized for neuronal differentiation and signaling¹. Across diverse tissues within the body, tissue stiffness spans several orders of magnitud....