TY - GEN

T1 - Directional Freezing of Cell-Seeded Electrospun Fiber Mats for Tissue Engineering Applications

AU - Mutsenko, Vitalii

AU - Chasnitsky, Michael

AU - Sirotinskaya, Vera

AU - Müller, Marc

AU - Glasmacher, Birgit

AU - Braslavsky, Ido

AU - Gryshkov, Oleksandr

N1 - Funding Information: Acknowledgements. This work was sponsored by Short-Term Research Grant from Minerva Foundation, the Israel Science Foundation (grant number 930/16) as well as Ways to Research II Program of the Leibniz University Hannover (60442522).

PY - 2020/11/30

Y1 - 2020/11/30

N2 - 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.

AB - 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.

KW - Cryopreservation

KW - Directional freezing

KW - Electrospun fiber mats

UR - http://www.scopus.com/inward/record.url?scp=85097615407&partnerID=8YFLogxK

U2 - 10.1007/978-3-030-64610-3_45

DO - 10.1007/978-3-030-64610-3_45

M3 - Conference contribution

AN - SCOPUS:85097615407

SN - 9783030646097

T3 - IFMBE Proceedings

SP - 391

EP - 398

BT - 8th European Medical and Biological Engineering Conference - Proceedings of the EMBEC 2020

A2 - Jarm, Tomaz

A2 - Cvetkoska, Aleksandra

A2 - Mahnič-Kalamiza, Samo

A2 - Miklavcic, Damijan

PB - Springer Science and Business Media Deutschland GmbH

CY - Cham

T2 - 8th European Medical and Biological Engineering Conference, EMBEC 2020

Y2 - 29 November 2020 through 3 December 2020

ER -