Abstract
Mechanical stimulation by the extracellular matrix (ECM) controls physiological and pathological cellular responses, such as stem cell differentiation, organogenesis, and tumor progression. Polyacrylamide (PA) gels have been widely used to study cell-ECM mechanical interactions. Typically, sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-SANPAH) is used as a protein crosslinker in these gels. However, its low solubility, unstable binding with proteins, and high cost are barriers to its application. The objective of this study was to improve and simplify the preparation of PA gels using an economical crosslinker, N-hydroxysuccinimide-acrylamide (NHS-AA) ester, to enable increased stability in protein coating. By exposing excess NHS to the gel surface, we found an optimal ratio of NHS-AA ester:AA to obtain NHS-AA ester-containing PA gels with a uniform ECM protein coating and stiffness similar to that of sulfo-SANPAH-containing PA gels. The biological behavior of MCF7 and MCF10A cells were similar on NHS-AA ester and sulfo-SANPAH gels. Acini formation in Matrigel overlay culture were also consistent on NHS-AA ester and sulfo-SANPAH gels. This novel PA gel preparation method using NHS-AA ester can effectively replace the sulfo-SANPAH method and will be immensely useful in the evaluation of cell-ECM mechanical interactions.
Highlights
Mechanical stimulation by the extracellular matrix (ECM) controls physiological and pathological cellular responses
Excess surface crosslinker permits the saturated binding of a constant amount of ECM protein, and the amount of protein coating the surface is proportional to the amount of applied ECM protein (Beer et al, 2015)
We have developed a new preparation method for PA gels using NHS-AA ester
Summary
Mechanical stimulation by the extracellular matrix (ECM) controls physiological and pathological cellular responses. Various cell culture materials are used to investigate the effects of ECM stiffness on cellular responses, including biomaterials such as collagen (Parenteau-Bareil et al, 2010), Matrigel (Kleinman et al, 1986), polysaccharide (Baldwin and Kiick, 2010), polyacrylamide (PA) (Kandow et al, 2007), polyethylene glycol (PEG) (Zhu, 2010), and self-assembling peptides (Koutsopoulos, 2016). PA gels are most widely used to study cellECM mechanical interactions, because of their convenient usage, biocompatibility, and reproducibility of stiffness (Kandow et al, 2007; Tilghman et al, 2010; Dupont et al, 2011; Wen et al, 2014; Tsou et al, 2016; Domura et al, 2017; Martín et al, 2017).
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