Abstract
Three-dimensional (3D) cell culture systems mimic the structural complexity of the tissue microenvironment and are gaining increasing importance as they resemble the extracellular matrix (ECM)–cell and cell–cell physical interactions occurring in vivo. Several scaffold-based culture systems have been already proposed as valuable tools for large-scale production of spheroids, but they often suffer of poor reproducibility or high costs of production. In this work, we present a reliable 3D culture system based on collagen I-blended agarose hydrogels and show how the variation in the agarose percentage affects the physical and mechanical properties of the resulting hydrogel. The influence of the different physical and mechanical properties of the blended hydrogels on the growth, size, morphology, and cell motility of the spheroids obtained by culturing three different breast cancer cell lines (MCF-7, MDA-MB-361, and MDA-MB-231) was also evaluated. As proof of concept, the cisplatin penetration and its cytotoxic effect on the tumor spheroids as function of the hydrogel stiffness were also investigated. Noteworthily, the possibility to recover the spheroids from the hydrogels for further processing and other biological studies has been considered. This feature, in addition to the ease of preparation, the lack of cross-linking chemistry and the high reproducibility, makes this hydrogel a reliable biomimetic matrix for the growth of 3D cell structures.
Highlights
Three-dimensional (3D) cell culture models, such as multicellular spheroids and organoids, have been demonstrated to mimic several biological processes in vitro much better than monolayer cell cultures [1,2]
We developed collagen hydrogels blended with variable amounts of agarose and we investigated the possibility to grow within them tumor spheroids of three different breast cancer cell lines, i.e., the luminal estrogen receptor positive cells, MCF-7 and MDA-MB-361, and the triple negative model, MDA-MB-231
Three agarose–collagen (A-C) hydrogels with different weight ratios were tested in this study
Summary
Three-dimensional (3D) cell culture models, such as multicellular spheroids and organoids, have been demonstrated to mimic several biological processes in vitro much better than monolayer cell cultures [1,2]. There are essentially two methods to produce uniform-size multicellular spheroids: scaffold free, in which cells are prevented from adhering to the substrate but not to each other, forming spheroids, and scaffold- or matrix-based, in which cells are embedded into a three-dimensional biomaterial, such as a hydrogel [3] The latter has the advantage of providing a physical structure comparable, in terms of stiffness and viscoelasticity, to the in vivo extracellular matrix (ECM), being able to reproduce cell–cell interactions, and cell–matrix interplays directly related to phenomena associated with cell growth (cell fate), such as cytoskeletal organization, gene expression, nutrients diffusion, pH, and hypoxia [4]. The most common are MatrigelTM, derived from Engelbreth-HolmSwarm mouse tumor sarcoma, and other basement membrane-rich matrices These hydrogels contain matrix membrane proteins, hormones, and soluble growth factors whose composition may vary among different batches. Hydrogels with these features are made of natural, synthetic, or hybrid materials, such as alginate, agarose, collagen, hyaluronic acid, and polyethylene glycol [1,9]
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