Fibrous Hydrogels for Cell Encapsulation: A Modular and Supramolecular Approach

Abstract

Artificial 3-dimensional (3D) cell culture systems, which mimic the extracellular matrix (ECM), hold great potential as models to study cellular processes under controlled conditions. The natural ECM is a 3D structure composed of a fibrous hydrogel that provides both mechanical and biochemical cues to instruct cell behavior. Here we present an ECM-mimicking genetically engineered protein-based hydrogel as a 3D cell culture system that combines several key features: (1) Mild and straightforward encapsulation meters (1) ease of ut I am not so sure.encapsulation of the cells, without the need of an external crosslinker. (2) Supramolecular assembly resulting in a fibrous architecture that recapitulates some of the unique mechanical characteristics of the ECM, i.e. strain-stiffening and self-healing behavior. (3) A modular approach allowing controlled incorporation of the biochemical cue density (integrin binding RGD domains). We tested the gels by encapsulating MG-63 osteoblastic cells and found that encapsulated cells not only respond to higher RGD density, but also to overall gel concentration. Cells in 1% and 2% (weight fraction) protein gels showed spreading and proliferation, provided a relative RGD density of at least 50%. In contrast, in 4% gels very little spreading and proliferation occurred, even for a relative RGD density of 100%. The independent control over both mechanical and biochemical cues obtained in this modular approach renders our hydrogels suitable to study cellular responses under highly defined conditions.

Fig 1. Tentative structure of a B^RGDC₂S^H₄₈C₂ fiber at neutral pH.

Drawing not to scale. B^RGD consists of 24 residues, and the entire protein sequence is 826 residues.

Fig 2. Partition coefficient of FITC-labelled dextran particles of different hydrodynamic radius in 0B gels of varying protein concentrations.

Fig 3. Rheological properties of hydrogels with different ratios of C₂S^H₄₈C₂ and B^RGDC₂S^H₄₈C₂, and different final protein concentrations.

(A) Time sweep for 100B material at 1%, 2% and 4% (w/v) of protein. (B) Final storage modulus dependency on protein concentration for gels with varying RGD domain density.

Fig 4. Strain sweep characteristics of C₂S^H₄₈C₂ and B^RGDC₂S^H₄₈C₂ gels.

(A) Storage modulus as a function of strain (γ) at various protein concentrations. (B) Maximal increase in storage modulus (strain stiffening) before gel rupture, in comparison with G’(γ = 0.1), for different concentrations of 100B material. (C) Strain at which gel ruptures (γ _max) as a function of protein concentration for 0B and 100B material.

Fig 5. Ability to recover after breakage of hydrogels with different ratios of C₂S^H₄₈C₂ and B^RGDC₂S^H₄₈C₂, and different final protein concentrations.

(A) Time sweep for 100B 2% material, with several breaking cycles: gel formation and recovery after strain-induced breakage repeated 5 times in ~5.5 h intervals. (B) Storage modulus after one breakage and subsequent recovery as a function of protein concentration for gels with varying RGD domain density. (C) Percentage of storage modulus recovered after one breakage as a function of protein concentration for gels with varying RGD domain density.

Fig 6. Cell metabolic activity.

Determined by the alamarBlue^® assay on day 1.

Fig 7. Morphology of fluorescently stained MG-63 cells encapsulated in silk-inspired protein scaffolds, with different total protein concentration and RGD domain density at day 3 and 7 of culturing.

Red: actin; blue: nuclei. Image size: 370 × 370 μm.

Fig 8. Quantitative determination of MG-63 cell number based on analysis of confocal images.

Significant differences between different days of cell culture for the same scaffold type are marked with horizontal lines; ^ significant difference relative to 50B 2% WP scaffold; * significant difference relative to 50B 1%; ** significant difference relative to 100B 4%; p < 0.05.

Fig 9. Determination of MG-63 cell distribution inside scaffolds, based on analysis of confocal pictures.

Significant differences between different days of cell culture for the same scaffold type are marked with horizontal lines; p < 0.05. Values below 1 indicate clustered distributions, equal to 1 –a random distribution, and above 1 –more ordered patterns.

Fig 10. Quantitative determination of MG-63 cell roundness, based on analysis of confocal images.

Significant differences between different days of cell culture for the same scaffold type are marked with horizontal lines; p < 0.05. The data analysis is focused on relative changes over time rather than on absolute values. Owing to a strong red background from the scaffold protein no data could be obtained for 50B 2% samples on day 1.