078 Improving standarised freezing protocols

Abstract

In applications like cell and tissue banking, the method of freezing is used for long term storage of biological specimens at low temperatures. Lowering the temperatures reduces the metabolism of cells and therefore can prohibit their quality loss over time. During the freezing process different kinds of factors have been identified as cell damaging mechanisms [1]. However, a correct setting of process parameters including the cooling rate, warming rate, nucleation temperature, (Tn) and type and concentration of cryoprotective agents can reduce cellular damage and result in high quality output after freezing. For comparison of the qualities of different biological specimens, freezing protocols and viability analyses need to be standardised. Most of the earlier studies did not account for the control of all process parameters, especially the nucleation temperature [1]. Therefore, the aim of this study was to control all possible process parameters during freezing of human endothelial cells using different experimental methods. A standardised freezing protocol was developed which is based on our earlier experimental results [2]. The protocol includes a cooling rate of 5 K/min, heating rate of 100 K/min, 5% (v/v) dimethyl sulfoxide and a nucleation temperature of −8 °C. The first method used was a biological freezer (CM2000, Carburos Metalicos) where 1 mL cell suspensions were frozen in cryovials. For the control of the nucleation temperature a new setup was developed which is based on nucleation via Peltier elements [3]. A second method used a cryomicroscopic system including a freezing chamber (FDCS, Linkam) and a digital camera (Retiga, QImaging). Therefore, another new setup was constructed to control Tn. In this setup nucleation is induced by a liquid nitrogen cooled copper rod which is positioned towards the specimen by a step motor system. Both constructions allowed the control of the nucleation temperature and contribute to a further standardisation of freezing protocols. After freezing of the endothelial cells, the quality of the protocol was determined using staining methods based on trypan blue, calcein AM and ethidium homodimer. As a general quality test the absolute viability was determined which is defined as the ratio of viable cell number after freezing to the total cell number before the freezing protocol. Using this method, the viability of endothelial cells after freezing was 90%. To achieve a standardisation of freezing protocols we propose the necessity to control all process parameters including the nucleation temperature and the use of the absolute viability as a general method for quality comparison of different cellular specimens after freezing. [1] P. Mazur, S.P. Leibo, E.H.Y. Chu: A two-factor hypothesis of freezing injury, Experimental Cell Research 71 (1972) 2, 345–35 [2] I. Bernemann, N. Hofmann, R. Spindler, A. Szentivanyi, B. Glasmacher: Strategy to improve cryopreservation protocols, CryoLetters 29 (2008) 1, 73–88 [3] R. Spindler, B. Rosenhahn, N. Hofmann, B. Glasmacher: Video analysis of osmotic cell response during cryopreservation, Cryobiology 64 (2012) 3, 250–260 Source of funding: This work is supported by funding from the Deutsche Forschungsgemeinschaft for the Cluster of Excellence REBIRTH (EXC 62/1). Conflict of interest: None declared. glasmacher@imp.uni-hannover.de