TITLE: CELL STORAGE-02-Freezing and Thawing Protocol for Suspension Cell Lines
AUTHORS: Marco Cosentino, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Mariagiulia Albizzati, Marco Ferrari, Franca Marino, Alessandra Luini, Alessandra Luini

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">In our institute, cell lines are stored after freezing procedure that provides for a constant temperature lowering by 1 degree/minute. This procedure is useful to minimizes ice crystals formation during freezing process, this allow to reduce the cells damage and, in turn increasing cells viability after thawing. After storage at -80°C or in liquid nitrogen (in relation to subsequent applications or the expected freezing time) cells are thawed rapidly in a water bath at 37°C and the cryopreserving-medium is immediately remove by gentle centrifugation in order to reduce as soon as possible, the DMSO toxic effect.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Currently, in our institute, the freezing/thawing procedure is validated for the following cell lines:</div></div><br/><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">PC-12</span><span>, Rat adrenal phaeochromocytoma, small irregularly shaped cells morphology, suspension growth mode.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>After freezing/thawing procedure, cells quality must be assessed by cell morphology evaluation (using Optical Microscopy technique) and time required to reach confluence in the T25 cm2 culture flask, which must not exceed the times indicated in the</span><span style = "font-weight:bold;"> Table 1</span><span>. </span></li><li style = "counter-reset:ol0;list-style-type:disc;">After quality test cells must be resuspended according to specific protocols (for details see Table 2).</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span>   </span></li><li style = "counter-reset:ol0;list-style-type:disc;"><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">List of published work by our institute that using this procedure</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Cosentino M, Marino F, Rasini E, Legnaro M, Bombelli R, Luini A, Pacchetti B. </span><span style = "font-weight:bold;">Improved solubility and increased biological activity of NeoSol</span><span style = "font-weight:bold;vertical-align:super;">™</span><span style = "font-weight:bold;">RCL40, a novel Red Clover Isoflavone Aglycones extract preparation.</span><span> Biomed Pharmacother. 2019 Mar;111:91-98. doi: 10.1016/j.biopha.2018.12.065. Epub 2018 Dec 19. PMID: 30579257.</span></li></ul></div><br/><div class = "text-block"><span>After freezing/thawing procedure, cells quality must be assessed by cell morphology evaluation (using Optical Microscopy technique) and time required to reach confluence in the T25 cm2 culture flask, which must not exceed the times indicated in the</span><span style = "font-weight:bold;"> Table 1</span><span>. </span></div><div class = "text-block">After quality test cells must be resuspended according to specific protocols (for details see Table 2).</div><br/><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/d3gfbevpp.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/d3gjbevpp.jpg" /></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">List of published work by our institute that using this procedure</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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[STEPS]
?. [CELL FREEZING PROCEDURE FOR SUSPENSION CELLS (PC-12)]
At confluence remove cell suspension from the T25 cm2 culture flask and recover in 15 mL conical tube.
?. [CELL FREEZING PROCEDURE FOR SUSPENSION CELLS (PC-12)]
Centrifuge for  at .
Centrifuge: 200 34
0 Room temperature
?. [CELL FREEZING PROCEDURE FOR SUSPENSION CELLS (PC-12)]
Remove supernatants and resuspend the cell pellet in  of cryopreserving medium (90% FBS + 10% DMSO).
1 mL
?. [CELL FREEZING PROCEDURE FOR SUSPENSION CELLS (PC-12)]
Immediately aliquot of cell suspension into a cryogenic vial.
1 mL
?. [CELL FREEZING PROCEDURE FOR SUSPENSION CELLS (PC-12)]
Place the cryogenic vial(s) in cotton wool, and then place it in a polystyrene box.
?. [CELL FREEZING PROCEDURE FOR SUSPENSION CELLS (PC-12)]
Put the polystyrene box in -80°C freezer.
?. [CELL FREEZING PROCEDURE FOR SUSPENSION CELLS (PC-12)]
After a minimum of transfer the cryogenic vials into a box for storage in -80°C freezer.
?. [CELL THAWING PROCEDURE]
Remove cryogenic vial from -80°C and keep it at  in water bath, this procedure should take approximately 1-2 minutes or until ampoule is slightly thawed.
37 °C
?. [CELL THAWING PROCEDURE]
Resuspend the cells into appropriate cell culture medium (Table 2) and start quality control procedure (Table 1).
?. [CELL THAWING PROCEDURE]
Clean the cryogenic vial outside with 70% EtOH, open the cryogenic vial and addof prewarmed complete medium for specific cells lines (Table 2).
1 mL
?. [CELL THAWING PROCEDURE]
Transfer the cell suspension into a 15 mL conical tube containing of prewarmed complete medium for specific cells lines.
10 mL
?. [CELL THAWING PROCEDURE]
Centrifuge for at .
Centrifuge: 200 34
37 Room temperature