Research Article: Directional freezing for the cryopreservation of adherent mammalian cells on a substrate

Date Published: February 15, 2018

Publisher: Public Library of Science

Author(s): Liat Bahari, Amir Bein, Victor Yashunsky, Ido Braslavsky, Boris Rubinsky.


Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices.

Partial Text

Cell culture methods are routinely used in many fields and are indispensable for a variety of applications in basic research, clinical practice, medical diagnostics, and the pharmaceutical industry. Cell culturing is a labor-intensive and time-consuming process that involves multiple manipulations. Cryopreserving cells is an important part of the culturing process and is needed to preserve the original cellular characteristics during cell storage over long starches of time. For that, cryopreservation methods must provide significant survival rates and normal cell functionality after thawing for a wide range of cell types.

Cryopreservation methods can be classified as one of two main approaches: slow cooling or vitrification. In both methods, cells are surrounded by a freezing medium cooled below the glass transition temperature without inducing cell freezing. Vitrification, which employs flash cooling with a high concentration of CPAs, completely avoids the formation of ice in the sample. On the other hand, slow cooling uses much lower CPA concentrations permitting ice formation in the extracellular medium. These factors favor slow cooling during scaling up of cryopreservation methods applied to large tissues and organs [27]. In directional slow cooling, a sample is subjected to a temperature gradient in the direction of the sample movement which induces the controlled growth of ice crystals in the same direction, leading to the formation of uniformly distributed ice crystals throughout the sample [29, 34].

By combining directional freezing and gradual cooling at specific rates, we were able to present a proof-of-concept for successful cryopreservation of cells adherent to a substrate. Two types of computer controlled cooling stages, translational cryomicroscopic system and LN cooled cryostage, were developed for this goal. Different cooling regimes and freezing medium compositions were tested and optimized concluding in a new cryopreservation protocol presented herein. The protocol consists of the following key parameters: 10% DMSO, directional freezing with a translational velocity of 30 μm/sec (equivalent to cooling rate of 3.8°C/min), followed by gradual cooling at a rate of 1.2°C/min to -20°C and deeper gradual cooling at a rate of 0.5°C/min to -80°C. We found that the directional freezing phase, which facilitates formation of evenly distributed ice crystals in the extracellular medium should be carried out at faster rate than expected in cell suspension cryopreservation (1°C/min). In our interpretation this step reminiscent of the “ice seeding” practiced in embryo cryopreservation [36]. We suggest that the cooling rate in cell culture cryopreservation should be slowed down with the decrease of temperature, which is also in line with a protocol applied in embryo cryopreservation [36]. Our approach displays high reproducibility and high survival rates compared to non-directional slow freezing methods and other previously reported attempts of cryopreservation of cells in an adherent state [3, 12–18, 21]. Our method can be readily applied as a robust cryopreservation protocol for various in vitro applications. We believe that the demonstrated capacity will pave the way for cryopreservation of multicellular networks and complex tissues preserving contact between the cells and adhesion with the substrate allowing new cell banking opportunities and development of new off-the-shelf cell based assays.




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