Breast Tumor Cell Survival and Morphology in a Brain-like Extracellular Matrix Depends on Matrix Composition and Mechanical Properties.

Triple-negative breast cancer (TNBC) is the most invasive type of breast cancer with high risk of brain metastasis. To better understand interactions between breast tumors with the brain extracellular matrix (ECM), a 3D cell culture model is implemented using a thiolated hyaluronic acid (HA-SH) based hydrogel. The latter is used as HA represents a major component of brain ECM. Melt-electrowritten (MEW) scaffolds of box- and triangular-shaped polycaprolactone (PCL) micro-fibers for hydrogel reinforcement are utilized. Two different molecular weight HA-SH materials (230 and 420 kDa) are used with elastic moduli of 148 ± 34 Pa (soft) and 1274 ± 440 Pa (stiff). Both hydrogels demonstrate similar porosities. The different molecular weight of HA-SH, however, significantly changes mechanical properties, e.g., stiffness, nonlinearity, and hysteresis. The breast tumor cell line MDA-MB-231 forms mainly multicellular aggregates in both HA-SH hydrogels but sustains high viability (75%). Supplementation of HA-SH hydrogels with ECM components does not affect gene expression but improves cell viability and impacts cellular distribution and morphology. The presence of other brain cell types further support numerous cell-cell interactions with tumor cells. In summary, the present 3D cell culture model represents a novel tool establishing a disease cell culture model in a systematic way.

Rheological and Biological Impact of Printable PCL-Fibers as Reinforcing Fillers in Cell-Laden Spider-Silk Bio-Inks.

The development of bio-inks capable of being 3D-printed into cell-containing bio-fabricates with sufficient shape fidelity is highly demanding. Structural integrity and favorable mechanical properties can be achieved by applying high polymer concentrations in hydrogels. Unfortunately, this often comes at the expense of cell performance since cells may become entrapped in the dense matrix. This drawback can be addressed by incorporating fibers as reinforcing fillers that strengthen the overall bio-ink structure and provide a second hierarchical micro-structure to which cells can adhere and align, resulting in enhanced cell activity. In this work, the potential impact of collagen-coated short polycaprolactone-fibers on cells after being printed in a hydrogel is systematically studied. The matrix is composed of eADF4(C16), a recombinant spider silk protein that is cytocompatible but non-adhesive for cells. Consequently, the impact of fibers could be exclusively examined, excluding secondary effects induced by the matrix. Applying this model system, a significant impact of such fillers on rheology and cell behavior is observed. Strikingly, it could be shown that fibers reduce cell viability upon printing but subsequently promote cell performance in the printed construct, emphasizing the need to distinguish between in-print and post-print impact of fillers in bio-inks.

Primary Glial Cell and Glioblastoma Morphology in Cocultures Depends on Scaffold Design and Hydrogel Composition.

3D cell cultures better replicate the in vivo environment compared to 2D models. Glioblastoma multiforme, a malignant brain tumor, highly profits from its cellular environment. Here, the U87 glioblastoma cell line in the presence/absence of primary astrocytes is studied. Thiolated hyaluronic acid (HA-SH) hydrogel reinforced with microfiber scaffolds is compared to Matrigel. Hyaluronic acid is a major extracellular matrix (ECM) component in the brain. Poly(ɛ-caprolactone) (PCL) scaffolds are written by meltelectrowriting in a box and triangular shaped design with pore sizes of 200 µm. Scaffolds are composed of 10-layers of PCL microfibers. It is found that scaffold design has an impact on cellular morphology in the absence of hydrogel. Moreover, the used hydrogels have profound influences on cellular morphology resulting in spheroid formation in HA-SH for both the tumor-derived cell line and astrocytes, while cell viability is high. Although cocultures of U87 and astrocytes exhibit cell-cell interactions, polynucleated spheroid formation is still present for U87 cells in HA-SH. Locally restricted ECM production or inability to secrete ECM proteins may underlie the observed cell morphologies. Thus, the 3D reinforced PCL-HA-SH composite with glioma-like cells and astrocytes constitutes a reproducible system to further investigate the impact of hydrogel modifications on cellular behavior and development.