27. Advances towards standardised freezing protocols

Abstract

In the field of cryopreservation cells are stored at cryogenic temperatures in order to maintain their quality after thawing. Lowering the temperature reduces the metabolism of cells and therefore can prevent their loss of quality over time. During the freezing process different parameters have been identified as cell damaging mechanisms [1] . Thus, a correct setting of process parameters including the cooling and warming rate, nucleation temperature ( T n ), sample geometry, as well as 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 (ECs) and mesenchymal stem cells (MSCs) of the marmoset monkey 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) DMSO and a nucleation temperature of −8 °C. The first method used was a μ -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 commercially available cryomicroscopic setup to visualise the cells in suspension which involves a LINHAM cryostage FDCS 196. and a digital camera (Retiga, QImaging). The temperature of the samples was controlled from 40 to −196 °C. The manual cell seeding process required the removal of the lid of the cryostage when a cooled copper rod touches the supercooled sample. To overcome this limitation, we developed a new seeding device for cryomicroscopic investigations of aqueous samples. This device applies a copper rod which is cooled via liquid nitrogen flushed heat exchanger. A USB camera is used to identify the position of the copper rod tip during an initial local calibration procedure which is electronically stored. Thus, the system can automatically calculate the distance to the sample. Nucleation is induced by a liquid nitrogen cooled copper rod which is positioned towards the specimen by a step motor system. The device allows for the control of the nucleation temperature at cooling rates up to 10 K/min with an accuracy of ±0.2 K. Both construction designs allow the control of the nucleation temperature and contribute to a further standardisation of freezing protocols. After cell freezing, 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 freezing. Using this method, the viability of ECs after freezing was 90%. After thawing, MSCs were tested for proliferation and differentiation into the osteogenic and adipogenic lineage. To achieve a standardisation of freezing protocols we propose 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 freeze/thawing.