TY - JOUR
T1 - Directional freezing for the cryopreservation of adherent mammalian cells on a substrate
AU - Bahari, Liat
AU - Bein, Amir
AU - Yashunsky, Victor
AU - Braslavsky, Ido
N1 - Funding Information:
This work was supported by a European Commission grants "FP7 Ideas" and "FP7 Nanosciences" and Yissumit grant from Yissum The Hebrew University technology transfer company. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2018 Bahari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2018/2/1
Y1 - 2018/2/1
N2 - 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 -80C 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 3.8C/min), followed by gradual cooling of the sample with decreasing rate of 0.5C/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.
AB - 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 -80C 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 3.8C/min), followed by gradual cooling of the sample with decreasing rate of 0.5C/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.
UR - http://www.scopus.com/inward/record.url?scp=85042191094&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0192265
DO - 10.1371/journal.pone.0192265
M3 - Article
C2 - 29447224
AN - SCOPUS:85042191094
SN - 1932-6203
VL - 13
JO - PLoS ONE
JF - PLoS ONE
IS - 2
M1 - e0192265
ER -