Protocol for synthesis and mechanical characterization of polyacrylamide hydrogels of varying stiffness for cell culture applications
7 affiliations
- 1Molecular Imaging and Photonics Division, Chemistry Department, Faculty of Sciences, KU Leuven, Celestijnenlaan 200F 3001 Heverlee, Belgium
- 2Molecular Materials group, Institute for Molecules and Materials, Faculty of Science, Radboud University, Heyendaalseweg 135, Nijmegen AJ 6525, the Netherlands
- 3Molecular Materials group, Institute for Molecules and Materials, Faculty of Science, Radboud University, Heyendaalseweg 135, Nijmegen AJ 6525, the Netherlands; Institute for Chemical Immunology, Nijmegen AJ 6525, the Netherlands
- 4Chronic Disease Programme, UFIEC, Instituto de Salud Carlos III, Madrid 28220, Spain
- 5Chronic Disease Programme, UFIEC, Instituto de Salud Carlos III, Madrid 28220, Spain; CIBER frailty and healthy aging (CIBERFES) 28029 Madrid, Spain
- 6Molecular Materials group, Institute for Molecules and Materials, Faculty of Science, Radboud University, Heyendaalseweg 135, Nijmegen AJ 6525, the Netherlands. Electronic address: [email protected]
- 7Molecular Imaging and Photonics Division, Chemistry Department, Faculty of Sciences, KU Leuven, Celestijnenlaan 200F 3001 Heverlee, Belgium. Electronic address: [email protected]
Abstract
Summary The impact of mechanical cues on cell behavior is increasingly being recognized, rendering hydrogel platforms that mimic the extracellular matrix indispensable in in vitro cell biology research. Here, we present a step-by-step protocol for synthesis and rheological characterization of polyacrylamide (PAAm) hydrogels with varying stiffnesses, produced as large circular unattached gels customizable in shape and size. We outline methods for their use in cell culture and downstream applications involving secretome or cell analysis, and protein visualization by fluorescence microscopy. This protocol is based on the recent work of Shi & Janmey who describe a novel and straightforward method for the production of large PAAm hydrogels for bulk cell culture and mechanobiology studies. 1 Their procedure results in one large gel that is not attached to a supporting surface and therefore can be transferred and/or stamped to generate PAAm gels of custom shapes and sizes. The aim of this step-by-step procedure is therefore not to improve the reported protocol, but to create a clearly outlined and repeatable protocol that enables a smooth implementation in any lab for a diverse audience. In addition, our protocol describes besides harvesting of cells also the collection of secretome for downstream biochemical analyses, as well as immunofluorescence labeling using antibodies that can readily be multiplexed for optimization of labeling conditions. Graphical abstract
Related
- Self-Assembling Peptide Hydrogels for Mucosal Drug Delivery: Synthesis, Triggering Strategies, and Therapeutic Applications
- Development and Characterization of Carrageenan-Based Hydrogels Enriched with Calendula officinalis Extract for Topical Skin Applications
- Effect of a Fiber Raw Material Structure on the Properties of Cellulose/Polyacrylamide Double-Network Antifreezing Conductive Hydrogels Prepared in an AlCl₃/ZnCl₂ Aqueous System.
- Double-network silica–polyacrylamide composite aerogels with enhanced mechanical integrity and multifunctional adsorption performance
- Supported gold nanoparticles – synthesis, characterization and applications in the multistep synthesis of valuable chemicals
BibTeX
@article{cresens2026protocol,
title = {{Protocol for synthesis and mechanical characterization of polyacrylamide hydrogels of varying stiffness for cell culture applications}},
author = {Charlotte Cresens and Samet Aytekin and Behrad Shaghaghi and Lotte Gerrits and Ana Montero‐Calle and Rodrigo Barderas and Paul H. J. Kouwer and Susana Rocha},
journal = {STAR Protocols},
year = {2026},
month = jun,
doi = {10.1016/j.xpro.2026.104606},
url = {https://doi.org/10.1016/j.xpro.2026.104606},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Protocol for synthesis and mechanical characterization of polyacrylamide hydrogels of varying stiffness for cell culture applications
AU - Charlotte Cresens
AU - Samet Aytekin
AU - Behrad Shaghaghi
AU - Lotte Gerrits
AU - Ana Montero‐Calle
AU - Rodrigo Barderas
AU - Paul H. J. Kouwer
AU - Susana Rocha
PY - 2026
DA - 2026/06/01
JO - STAR Protocols
DO - 10.1016/j.xpro.2026.104606
UR - https://doi.org/10.1016/j.xpro.2026.104606
AB - Summary The impact of mechanical cues on cell behavior is increasingly being recognized, rendering hydrogel platforms that mimic the extracellular matrix indispensable in in vitro cell biology research. Here, we present a step-by-step protocol for synthesis and rheological characterization of polyacrylamide (PAAm) hydrogels with varying stiffnesses, produced as large circular unattached gels customizable in shape and size. We outline methods for their use in cell culture and downstream applications involving secretome or cell analysis, and protein visualization by fluorescence microscopy. This protocol is based on the recent work of Shi & Janmey who describe a novel and straightforward method for the production of large PAAm hydrogels for bulk cell culture and mechanobiology studies. 1 Their procedure results in one large gel that is not attached to a supporting surface and therefore can be transferred and/or stamped to generate PAAm gels of custom shapes and sizes. The aim of this step-by-step procedure is therefore not to improve the reported protocol, but to create a clearly outlined and repeatable protocol that enables a smooth implementation in any lab for a diverse audience. In addition, our protocol describes besides harvesting of cells also the collection of secretome for downstream biochemical analyses, as well as immunofluorescence labeling using antibodies that can readily be multiplexed for optimization of labeling conditions. Graphical abstract
SN - 2666-1667
ER -