(554b) Fabrication of PNIPAM Electrospun Nanofiber Substrates for Temperature-Mediated Cell Release

Thermoresponsive polymers (TRPs) are a relevant class of synthetic polymers for tissue engineering applications due to their ability to undergo a reversible phase transition at the lower critical solution temperature (LCST). One of the most well studied TRPs, poly(N-isopropyl acrylamide) (PNIPAM), has a biologically relevant LCST of 32°C, exhibiting a collapsed conformation above the LCST that facilitates protein adsorption and cell adhesion. Below the LCST, the hydrophobic-to-hydrophilic phase change mechanically lifts cultured cells and preserves critical cell-cell and cell-matrix interactions. PNIPAM chains have been successfully grafted to tissue culture plastic to enable the fabrication of cell sheets, biomaterial-free constructs for transplantation. One limitation of PNIPAM substrates, however, is the relatively long thermo-release of cells, which likely affects cell function and phenotype. A strategy to accelerate release kinetics is to increase the surface area to volume ratio of the substrate, thereby facilitating greater hydration of polymer chains with decreased temperature. Electrospinning is a relatively simple and inexpensive technique that enables the fabrication of polymer nanofibers with a high surface area to volume ratio and the potential for biofunctionalization or nanofiber alignment. Electrospun fibers also act as a biomimetic extracellular matrix, providing a three-dimensional architecture for cell adhesion and proliferation. Although promising for myriad of reasons, few studies have examined the capability of electrospinning PNIPAM substrates for cell culture applications.

The objective of this study was to optimize the electrospinning of PNIPAM to produce nanofiber scaffolds capable of mechanically releasing adherent cells below its LCST. A fractional factorial Design of Experiment was used to study the effect of concentration, collecting distance, flow rate, and voltage on average fiber diameter. Optimized parameters yielded uniform PNIPAM fibers between 400-600nm. Nonwoven electrospun mats of high molecular weight PNIPAM (300 kDa) were completely soluble in aqueous media, immediately dissolving upon introduction. To prevent dissolution, substrates were crosslinked. Briefly, the polymer solutions were prepared by dissolving a 10 w/v% solution of 100:15:0.3 PNIPAM:OpePOSS:EMI weight ratio in a 1:1 (vol/vol) solution of THF/DMF at room temperature. After electrospinning using the previously optimized parameters (22 gauge blunt needle tip (inner diameter 0.41 mm), collection distance of 13 cm, voltage of 15 kV, and flow rate of 0.1 mL/hr), samples were placed in a vacuum desiccator overnight, followed by an oven at 160°C for 4 hours to activate the crosslinking reaction. Crosslinked PNIPAM electrospun mats were then immersed in deionized water at 37°C to qualitatively asses their stability. Samples remained intact, with little change to fiber morphology, as verified by scanning electron microscopy. When the substrates were cooled to room temperature, the phase change was observed by a coloration change in the mat from opaque to transparent. Electrospun mats were shown to be quite robust, undergoing a thermoresponsive change each day for 7 days with no significant change to fiber morphology or diameter.

Cell studies were performed using L929 fibroblast cells cultured in low glucose DMEM with L-glutamine, supplemented with 10% FBS and 1% penicillin/streptomycin. Crosslinked electrospun scaffolds were prepared using a 1.0 cm diameter biopsy punch and fixed to the bottom of a 48-well plate using vacuum grease. Scaffolds were incubated in media overnight prior to cell seeding to allow for hydration of the PNIPAM matrix and protein adsorption. Cells were seeded at a density of 25,000 cells/cm² and allowed to attach for approximately 16 hours, at which point the media was exchanged with room temperature media to allow for the thermodynamic phase change of the PNIPAM. The supernatant was collected at various timepoints (10, 20, 30 minutes) and cells were counted using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS). At each timepoint, the cells remaining on the scaffold were also counted using the MTS assay. Results show that cells successfully thermo-released from electrospun mats, statistically comparable to the thermo-release of cells from the commercially available Thermo Scientific PNIPAM UpCell Surface. Cells did not release from a tissue culture plastic negative control.

Collectively, results show that PNIPAM can be successfully electrospun into ECM-mimicking substrates for cell culture applications. These crosslinked nanofiber mats are capable of supporting cell attachment, spreading, and release upon a temperature switch. Further optimization of the electrospinning process to allow for the collection of aligned, crosslinked PNIPAM substrates will allow for the fabrication of aligned cell sheets for applications such as vascular tissue engineering.