TITLE: CELL STORAGE-01-Freezing and Thawing Protocol for Adherent 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">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><br/><div class = "text-block">Currently, in our institute, the freezing/thawing procedure is validated for the following cell lines:</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;">MCF7</span><span>, Human Caucasian breast adenocarcinoma, epithelial-like morphology, adherent growth mode.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">HUVEC</span><span>, Human Umbilical Vein Endothelial Cells, endothelial morphology, adherent growth mode.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">A549</span><span>, Human Caucasian lung carcinoma, epithelial-like morphology, adherent growth mode.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Synovial fibroblasts</span><span>, Human synoviocytes, fibroblast-like cells morphology, adherent 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 cm</span><span style = "vertical-align:super;">2</span><span> 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;"><span>After quality test cells must be resuspended according to specific protocols (for details see </span><span style = "font-weight:bold;">Table 2</span><span>).</span></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 style = ":;"> </span></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 style = ":;"> </span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"></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>Coelho M, Imperatori A, Chiaravalli AM, Franzi F, Castiglioni M, Rasini E, Luini A, Legnaro M, Marino F, Ribeiro L, Cosentino M. </span><span style = "font-weight:bold;">Beta1- and Beta2-Adrenoceptors Expression Patterns in Human Non-small Cell Lung Cancer: Relationship with Cancer Histology.</span><span> J Neuroimmune Pharmacol. 2019 Dec;14(4):697-708. doi: 10.1007/s11481-019-09879-6. Epub 2019 Oct 16. PMID: 31620969.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Marino F, Schembri L, Rasini E, Pinoli M, Scanzano A, Luini A, Congiu T, Cosentino M. </span><span style = "font-weight:bold;">Characterization of human leukocyte-HUVEC adhesion: Effect of cell preparation methods</span><span>. J Immunol Methods. 2017 Apr;443:55-63. doi: 10.1016/j.jim.2017.01.013. Epub 2017 Feb 3. PMID: 28167274.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Spagnuolo P, Rasini E, Luini A, Legnaro M, Luzzani M, Casareto E, Carreri M, Paracchini S, Marino F, Cosentino M. </span><span style = "font-weight:bold;">Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: an in vitro study in MCF-7 cells. </span><span>Fitoterapia. 2014 Apr;94:62-9. doi: 10.1016/j.fitote.2014.01.027. Epub 2014 Feb 5. PMID: 24508860.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Capellino S, Cosentino M, Luini A, Bombelli R, Lowin T, Cutolo M, Marino F, Straub RH. </span><span style = "font-weight:bold;">Increased expression of dopamine receptors in synovial fibroblasts from patients with rheumatoid arthritis: inhibitory effects of dopamine on interleukin-8 and interleukin-6.</span><span> Arthritis Rheumatol. 2014 Oct;66(10):2685-93. doi: 10.1002/art.38746. PMID: 24965369.</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, Ferrari M, Rasini E, Bombelli R, Luini A, Legnaro M, Delle Canne MG, Luzzani M, Crema F, Paracchini S, Lecchini S. </span><span style = "font-weight:bold;">Estrogenic activity of 7-hydroxymatairesinol potassium acetate (HMR/lignan) from Norway spruce (Picea abies) knots and of its active metabolite enterolactone in MCF-7 cells.</span><span>Pharmacol Res. 2007 Aug;56(2):140-7. doi: 10.1016/j.phrs.2007.05.001. Epub 2007 May 22. PMID: 17572100.</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 cm</span><span style = "vertical-align:super;">2</span><span> 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"><span>After quality test cells must be resuspended according to specific protocols (for details see </span><span style = "font-weight:bold;">Table 2</span><span>).</span></div><br/><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/d3gpbevpp.jpg" /></div><br/><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/d3gtbevpp.jpg" /></div><br/><br/><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:justify"></div><style>
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							</style></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Coelho M, Imperatori A, Chiaravalli AM, Franzi F, Castiglioni M, Rasini E, Luini A, Legnaro M, Marino F, Ribeiro L, Cosentino M. </span><span style = "font-weight:bold;">Beta1- and Beta2-Adrenoceptors Expression Patterns in Human Non-small Cell Lung Cancer: Relationship with Cancer Histology.</span><span> J Neuroimmune Pharmacol. 2019 Dec;14(4):697-708. doi: 10.1007/s11481-019-09879-6. Epub 2019 Oct 16. PMID: 31620969.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Marino F, Schembri L, Rasini E, Pinoli M, Scanzano A, Luini A, Congiu T, Cosentino M. </span><span style = "font-weight:bold;">Characterization of human leukocyte-HUVEC adhesion: Effect of cell preparation methods</span><span>. J Immunol Methods. 2017 Apr;443:55-63. doi: 10.1016/j.jim.2017.01.013. Epub 2017 Feb 3. PMID: 28167274.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Spagnuolo P, Rasini E, Luini A, Legnaro M, Luzzani M, Casareto E, Carreri M, Paracchini S, Marino F, Cosentino M. </span><span style = "font-weight:bold;">Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: an in vitro study in MCF-7 cells. </span><span>Fitoterapia. 2014 Apr;94:62-9. doi: 10.1016/j.fitote.2014.01.027. Epub 2014 Feb 5. PMID: 24508860.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Capellino S, Cosentino M, Luini A, Bombelli R, Lowin T, Cutolo M, Marino F, Straub RH. </span><span style = "font-weight:bold;">Increased expression of dopamine receptors in synovial fibroblasts from patients with rheumatoid arthritis: inhibitory effects of dopamine on interleukin-8 and interleukin-6.</span><span> Arthritis Rheumatol. 2014 Oct;66(10):2685-93. doi: 10.1002/art.38746. PMID: 24965369.</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, Ferrari M, Rasini E, Bombelli R, Luini A, Legnaro M, Delle Canne MG, Luzzani M, Crema F, Paracchini S, Lecchini S. </span><span style = "font-weight:bold;">Estrogenic activity of 7-hydroxymatairesinol potassium acetate (HMR/lignan) from Norway spruce (Picea abies) knots and of its active metabolite enterolactone in MCF-7 cells.</span><span>Pharmacol Res. 2007 Aug;56(2):140-7. doi: 10.1016/j.phrs.2007.05.001. Epub 2007 May 22. PMID: 17572100.</span></li></ul></div><br/><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Marino F, Schembri L, Rasini E, Pinoli M, Scanzano A, Luini A, Congiu T, Cosentino M. </span><span style = "font-weight:bold;">Characterization of human leukocyte-HUVEC adhesion: Effect of cell preparation methods</span><span>. J Immunol Methods. 2017 Apr;443:55-63. doi: 10.1016/j.jim.2017.01.013. Epub 2017 Feb 3. PMID: 28167274.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Spagnuolo P, Rasini E, Luini A, Legnaro M, Luzzani M, Casareto E, Carreri M, Paracchini S, Marino F, Cosentino M. </span><span style = "font-weight:bold;">Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: an in vitro study in MCF-7 cells. </span><span>Fitoterapia. 2014 Apr;94:62-9. doi: 10.1016/j.fitote.2014.01.027. Epub 2014 Feb 5. PMID: 24508860.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Capellino S, Cosentino M, Luini A, Bombelli R, Lowin T, Cutolo M, Marino F, Straub RH. </span><span style = "font-weight:bold;">Increased expression of dopamine receptors in synovial fibroblasts from patients with rheumatoid arthritis: inhibitory effects of dopamine on interleukin-8 and interleukin-6.</span><span> Arthritis Rheumatol. 2014 Oct;66(10):2685-93. doi: 10.1002/art.38746. PMID: 24965369.</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, Ferrari M, Rasini E, Bombelli R, Luini A, Legnaro M, Delle Canne MG, Luzzani M, Crema F, Paracchini S, Lecchini S. </span><span style = "font-weight:bold;">Estrogenic activity of 7-hydroxymatairesinol potassium acetate (HMR/lignan) from Norway spruce (Picea abies) knots and of its active metabolite enterolactone in MCF-7 cells.</span><span>Pharmacol Res. 2007 Aug;56(2):140-7. doi: 10.1016/j.phrs.2007.05.001. Epub 2007 May 22. PMID: 17572100.</span></li></ul></div><br/><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Spagnuolo P, Rasini E, Luini A, Legnaro M, Luzzani M, Casareto E, Carreri M, Paracchini S, Marino F, Cosentino M. </span><span style = "font-weight:bold;">Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: an in vitro study in MCF-7 cells. </span><span>Fitoterapia. 2014 Apr;94:62-9. doi: 10.1016/j.fitote.2014.01.027. Epub 2014 Feb 5. PMID: 24508860.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Capellino S, Cosentino M, Luini A, Bombelli R, Lowin T, Cutolo M, Marino F, Straub RH. </span><span style = "font-weight:bold;">Increased expression of dopamine receptors in synovial fibroblasts from patients with rheumatoid arthritis: inhibitory effects of dopamine on interleukin-8 and interleukin-6.</span><span> Arthritis Rheumatol. 2014 Oct;66(10):2685-93. doi: 10.1002/art.38746. PMID: 24965369.</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, Ferrari M, Rasini E, Bombelli R, Luini A, Legnaro M, Delle Canne MG, Luzzani M, Crema F, Paracchini S, Lecchini S. </span><span style = "font-weight:bold;">Estrogenic activity of 7-hydroxymatairesinol potassium acetate (HMR/lignan) from Norway spruce (Picea abies) knots and of its active metabolite enterolactone in MCF-7 cells.</span><span>Pharmacol Res. 2007 Aug;56(2):140-7. doi: 10.1016/j.phrs.2007.05.001. Epub 2007 May 22. PMID: 17572100.</span></li></ul></div><br/><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Capellino S, Cosentino M, Luini A, Bombelli R, Lowin T, Cutolo M, Marino F, Straub RH. </span><span style = "font-weight:bold;">Increased expression of dopamine receptors in synovial fibroblasts from patients with rheumatoid arthritis: inhibitory effects of dopamine on interleukin-8 and interleukin-6.</span><span> Arthritis Rheumatol. 2014 Oct;66(10):2685-93. doi: 10.1002/art.38746. PMID: 24965369.</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, Ferrari M, Rasini E, Bombelli R, Luini A, Legnaro M, Delle Canne MG, Luzzani M, Crema F, Paracchini S, Lecchini S. </span><span style = "font-weight:bold;">Estrogenic activity of 7-hydroxymatairesinol potassium acetate (HMR/lignan) from Norway spruce (Picea abies) knots and of its active metabolite enterolactone in MCF-7 cells.</span><span>Pharmacol Res. 2007 Aug;56(2):140-7. doi: 10.1016/j.phrs.2007.05.001. Epub 2007 May 22. PMID: 17572100.</span></li></ul></div><br/><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><br/></div>

[STEPS]
?. [CELL FREEZING PROCEDURE FOR ADHERENT CELLS (MCF7, HUVEC, A549, SYNOVIAL FIBROBLAST)]
Add of complete medium to inactivate trypsin, recover the cell suspension  in 15 mL conical tube and centrifuge for at .
4 mL
Centrifuge: 200 34
0 Room temperature
?. [CELL FREEZING PROCEDURE FOR ADHERENT CELLS (MCF7, HUVEC, A549, SYNOVIAL FIBROBLAST)]
At confluence remove culture medium from the flasks, wash twice the cell monolayer with  of sterile PBS.
5 mL
?. [CELL FREEZING PROCEDURE FOR ADHERENT CELLS (MCF7, HUVEC, A549, SYNOVIAL FIBROBLAST)]
Add  (for T25 cm2 culture flasks) of trypsin/EDTA for 5-10 minutes, observe the cells under a microscope to make sure that cells are detached.
1 mL
?. [CELL FREEZING PROCEDURE FOR ADHERENT CELLS (MCF7, HUVEC, A549, SYNOVIAL FIBROBLAST)]
Remove supernatants and resuspend the cell pellet in  of cryopreserving medium (90% FBS + 10% DMSO).
1 mL
?. [CELL FREEZING PROCEDURE FOR ADHERENT CELLS (MCF7, HUVEC, A549, SYNOVIAL FIBROBLAST)]
Immediately aliquot of cell suspension into a cryogenic vial.
1 mL
?. [CELL FREEZING PROCEDURE FOR ADHERENT CELLS (MCF7, HUVEC, A549, SYNOVIAL FIBROBLAST)]
Place the cryogenic vial(s) in cotton wool, and then place it in a polystyrene box.
?. [CELL FREEZING PROCEDURE FOR ADHERENT CELLS (MCF7, HUVEC, A549, SYNOVIAL FIBROBLAST)]
Put the polystyrene box in -80°C freezer.
?. 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]
Clean the cryogenic vial outside with 70% EtOH, open the cryogenic vial and add  of 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  atat
Centrifuge: 200 34
0 Room temperature
?. [CELL THAWING PROCEDURE]
Resuspended the cells into appropriate cell culture medium (Table 2) and start quality control procedure (Table 1).