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Methods Mol Biol. 2017;1612:29-37. doi: 10.1007/978-1-4939-7021-6_3.

3D Cell Culture in Interpenetrating Networks of Alginate and rBM Matrix.

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Department of Mechanical Engineering, Stanford University, 452 Escondido Mall, Stanford, CA, 94305, USA.
Department of Mechanical Engineering, Stanford University, 452 Escondido Mall, Stanford, CA, 94305, USA.


Altered tissue mechanical properties have been implicated in many key physiological and pathological processes. Hydrogel-based materials systems, made with native extracellular matrix (ECM) proteins, nonnative biopolymers, or synthetic polymers are often used to study these processes in vitro in 3D cell culture experiments. However, each of these materials systems present major limitations when used in mechanobiological studies. While native ECM-based hydrogels may enable good recapitulation of physiological behavior, the mechanics of these hydrogels are often manipulated by increasing or decreasing the protein concentration. This manipulation changes cell adhesion ligand density, thereby altering cell signaling. Alternatively, synthetic polymer-based hydrogels and nonnative biopolymer-based hydrogels can be mechanically tuned and engineered to present cell adhesion peptide motifs, but still may not fully promote physiologically relevant behavior. Here, we combine the advantages of native ECM proteins and nonnative biopolymers in interpenetrating network (IPN) hydrogels consisting of rBM matrix, which contains ligands native to epithelial basement membrane, and alginate, an inert biopolymer derived from seaweed. The following protocol details the generation of IPNs for mechanical testing or for 3D cell culture. This biomaterial system offers the ability to tune the stiffness of the 3D microenvironment without altering cell adhesion ligand concentration or pore size.


3D culture; 3D materials systems; Alginate; Biomaterials; ECM stiffness; Hydrogels; Interpenetrating networks; Reconstituted basement membrane

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