Three-Dimensional Cell Entrapment as a Function of the Weight Percent of Peptide-Amphiphile Hydrogels

Abstract

The design of scaffolds which mimic the stiffness, nanofiber structure, and biochemistry of the native extra-cellular matrix (ECM) has been a major objective for the tissue engineering field. Furthermore, mimicking the innate three dimensional (3D) environment of the ECM has been shown to significantly alter cellular response compared to traditional two dimensional (2D) culture. We report the development of a self-assembling, fibronectin-mimetic, peptide-amphiphile nanofiber scaffold for 3D cell culture. To form such a scaffold, 5 mol% of a bioactive PR_g fibronectin-mimetic peptide-amphiphile was mixed with 95 mol% of a diluent peptide-amphiphile (E2) whose purpose was to neutralize electrostatic interactions, increase the gelation kinetics and promote cell survival. Atomic force microscopy verified the fibrilar structure of the gels and the mechanical properties were characterized for various weight percent (wt%) formulations of the 5 mol% PR_g - 95 mol% E2 peptide-amphiphile mixture. The 0.5 wt% formulations had an elastic modulus of 429.0 ± 21.3 Pa while the 1.0 wt% peptide-amphiphile hydrogels had an elastic modulus of 808.6 ± 38.1 Pa. The presence of entrapped cells in the gels decreased the elastic modulus and the decrease was a function of the cell loading. While both formulations supported cell proliferation, the 0.5 wt% gels supported significantly greater NIH3T3/GFP fibroblast cell proliferation throughout the gels than the 1.0 wt% gels. However, compared to the 0.5 wt% formulations, the 1.0 wt% hydrogels promoted greater increase in mRNA expression and production of fibronectin and type IV collagen ECM proteins. This study suggests that this fibronectin-mimetic scaffold holds great promise in the advance of 3D culture applications and cell therapies.

Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium

“…There was no statistically significant difference between these two measurements, and therefore the entrapment of iPSCs in the Cell‐Mate3D gels had no effect on the mechanical properties of the gels. A decrease in the Young's modulus of a hydrogel after entrapment of cells has been previously demonstrated and was attributed to embedding cells that are softer than the gel and the disruption of interactions between the gel fibers [29]. Interestingly, Engler et al showed that mesenchymal stem cells differentiate to a neural lineage on a substrate stiffness of 0.1–1 kPa [26], and Leipzig and Shoichet showed that neural stem/progenitor cells will differentiate to neurons, astrocytes, and oligodendrocytes when cultured on substrates of stiffness between 0.6 and 7 kPa, whereas on stiffer substrates (≥10 kPa), they will differentiate to oligodendrocytes [30].…”

Biomaterials for 4D stem cell culture

“…PR_g peptide amphiphiles were diluted with another peptide, C 16 -GGGSSSESE (E 2 ), to screen charges and enable faster gelation. PR_g/E2 gels supported 3D culture and proliferation of fibroblasts (NIH3T3/GFP) over five days [53]. In seminal work, Stupp and coworkers designed another peptide amphiphile (PA) that contained a 16-carbon chain, 4 alanine residues, 3 glycine residues, and the sequence IKVAV, which is found in laminin and known to promote and direct neurite outgrowth.…”

A Silk Fibroin and Peptide Amphiphile‐Based Co‐Culture Model for Osteochondral Tissue Engineering

“…[30,31,45] Recently, peptide amphiphile hydrogels have been explored for this need due to their design flexibility, ease of synthesis, and similarity to natural ECM of cartilage. [33] PA hydrogels were successfully used in encapsulation processes for different cell types such as MC3T3-E1, [32,46] neural progenitor cells, [47] fibroblasts, [48] hBMSCs. [38] However, they have never been studied in detail for building cartilage layer of an osteochondral scaffold or an osteochondral system composed of multiple cell types in different layers.…”