Directional Freezing of Cell-Seeded Electrospun Fiber Mats for Tissue Engineering Applications

As novel tissue engineered constructs (TECs) are developed, current tissue banking practices need better control over ice formation and growth to prevent cryodamage to cells and a scaffold. Directional solidification demonstrates benefits in adhered cells and native tissues cryopreservation through controlled heat transfer. Therefore, this study aims to investigate the feasibility of using this technique for cryopreservation of cell-seeded electrospun fiber mats as model TECs. Fiber mats were produced using blend electrospinning of polycaprolactone (PCL, 200 mg/ml) and poly-L-lactic acid (PLA, 100 mg/ml) dissolved in 2,2,2-Trifluoroethanol. The fiber size and morphology was characterized using scanning electron microscopy. Specific heat measurements were conducted using differential scanning calorimetry. The square-shaped fiber mats were seeded under static conditions with HeLa cells and cultivated for 24 h. Samples were directionally frozen in a sandwich format either in 10% DMSO or culture medium with the sample movement at 30 μm/s through the predetermined temperature gradients along a 2.6 mm slit. After directional solidification, samples were gradually frozen at 1 K/min down to −80 ℃. Crystal shape was visualized using cryomicroscopic system. Before freezing and 24 h after thawing, cell viability was assessed using live-dead assay. Within randomly orientated PCL-PLA fibers, HeLa cells exhibited typical shape and attachment with higher than 90% viability prior to freezing. While up to 80% of HeLa cells were alive on fiber mats after freezing using DMSO with or without directional solidification step. The demonstrated controlled freezing may assist optimizing the freezing of more sensitive cells. The results suggest that directional freezing becomes a viable option for cryopreservation in tissue engineering applications.