Abstract
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.
Highlights
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
Increment of the velocity increased the branching of the ice crystals and decreased their width, in line with previous reports describing the directional solidification process [24, 31]
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
Summary
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. Cryopreservation methods must provide significant survival rates and normal cell functionality after thawing for a wide range of cell types. Cells are most commonly cryopreserved while dispersed in specialized freezing solutions. Preservation protocols involve detaching adherent cells from a substrate using a proteolytic enzyme (e.g., trypsin) and adding ‘cryoprotective agents’ (CPAs). This step is followed by a slow freezing protocol (1 ̊C/min) and storage at -80 ̊C or -196 ̊C.
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