CRYOPRESERVATION OF CELL-SEEDED ELECTROSPUN MATERIALS: TOWARDS BIOBANKING OF TISSUE-ENGINEERED CONSTRUCTS

Cryopreservation of tissue-engineered constructs (TECs) is very important to provide such ready-to-use products for regenerative medicine and clinical application upon demand. Although cryopreservation of isolated cells seems to be well established, there are still a number of challenges associated with the cryopreservation of native and artificial tissues due to adherent cell state, limited heat and mass transfer as well as inadequate cryopreservation protocols. Here, we aim at developing an approach for efficient cryopreservation of electrospun TECs based on multipotent stromal cells (MSCs). Blend electrospun fibre mats (fibre diameter 0.8±0.2 µm, thickness 100±10 µm) were produced from polycaprolactone and polylactic acid (PCL-PLA, ratio 100:50) using electrospinning. The fibre mats (diameter 16 mm) were UV sterilised and seeded with MSCs (5×104 cells/cm2). The cells were cultivated on fibre mats for 7 days under static conditions and then frozen using 1 K/min cooling rate in a controlled rate freezer with different formulations of cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO) and its combination with sucrose (with and without pre-culture with sucrose for 24 h). The viability of cells growing on fibre mats was monitored for 2 weeks after seeding and 24 h after thawing. The results indicate that PCL-PLA fibre mats are biocompatible with MSCs (viability higher than 82%). Pre-culture with sucrose before freezing as well as its addition to DMSO-containing freezing medium significantly improved cell viability after thawing. Moreover, duration of equilibration of cell-seeded fibre mats with the CPAs before freezing affected cell viability post-thaw. We showed that it is feasible to effectively cryopreserve electrospun TECs using controlled technological steps. This work could serve as a solid background for further development of efficient cryopreservation methods for biobanking of electrospun constructs for vascular or corneal tissue engineering.