In the realm of cell-based assays and tissue engineering, the development of biomimetic polymers has revolutionized the field. However, one challenge that researchers face is the need for effective three-dimensional scaffold constructs. To address this, the crosslinking of electrospun nanofibrous biopolymer constructs, particularly gelatin, plays a pivotal role in achieving enhanced stability and applicability. In this blog article, we discuss a novel and versatile approach for fabricating and crosslinking electrospun gelatin constructs with tunable porosity and high aspect ratio nanofibers originally presented by our collaborators, Ghassemi and Slaughter (Biopolymers, 2018). The goal of this study was to overcome solubility and storage stability issues while promoting cell viability in cell-based assays.

Fabrication and Characterization of Gelatin Nanofibrous Constructs
We produced electrospun materials using solutions of gelatin/genipin or pure gelatin nanofibrous constructs with well-defined, smooth surfaces. The diameter analysis reveals that the gelatin/genipin constructs have nanofibers with an average diameter of 448 ± 364 nm, while the pure gelatin constructs possess nanofibers with an average diameter of 257 ± 57 nm. Since the original publication, we have been able to scale production onto full roll-to-roll commercial electrospinning systems.

Crosslinking Approaches for Enhanced Stability
Uncrosslinked electrospun gelatin nanofibrous constructs possess desirable morphological features, but they suffer from poor storage stability and aqueous solubility. In our pursuit of achieving insoluble gelatin nanofibrous constructs suitable for cell-based assays, we explored three crosslinking approaches: dehydrothermal, genipin-EDC/Sulfo-NHS, and EDC/Sulfo-NHS. Through mechanical characterization, we observed that the gelatin nanofibrous constructs crosslinked using EDC/Sulfo-NHS exhibit improved strength, stiffness, and fiber morphology retention. Moreover, these constructs exhibit no dissolution in aqueous solutions and showcase excellent storage stability even at temperatures ranging from 22°C to -80°C.

Biocompatibility and Cell Viability
To evaluate the biocompatibility of the as-crosslinked gelatin nanofibrous construct, we assessed the viability and proliferation of PC12 cells. The results demonstrated a highly biocompatible scaffold, with 90% cell viability and promoted cell proliferation. This highlights the potential of the crosslinked gelatin nanofibrous construct as an effective substrate for cell-based assays and tissue engineering applications.

The fabrication and crosslinking of electrospun gelatin nanofibrous constructs provide an enticing solution for enhancing scaffold stability and promoting cell viability in the fields of tissue engineering and cell-based assays. The approach presented offers versatility in creating constructs with tunable porosity, high aspect ratio nanofibers, and improved mechanical properties. By overcoming the challenges of solubility and storage stability, these crosslinked gelatin constructs pave the way for more reliable and efficient applications in regenerative medicine and biomaterial research.