CELL STORAGE-01-Freezing and Thawing Protocol for Adherent Cell Lines v1

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. Currently, in our institute, the freezing/thawing procedure is validated for the following cell lines: MCF7, Human Caucasian breast adenocarcinoma, epithelial-like morphology, adherent growth mode. HUVEC, Human Umbilical Vein Endothelial Cells, endothelial morphology, adherent growth mode. A549, Human Caucasian lung carcinoma, epithelial-like morphology, adherent growth mode. Synovial fibroblasts, Human synoviocytes, fibroblast-like cells morphology, adherent growth mode. 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 Table 1. After quality test cells must be resuspended according to specific protocols (for details see Table 2). List of published work by our institute that using this procedure Coelho M, Imperatori A, Chiaravalli AM, Franzi F, Castiglioni M, Rasini E, Luini A, Legnaro M, Marino F, Ribeiro L, Cosentino M. Beta1- and Beta2-Adrenoceptors Expression Patterns in Human Non-small Cell Lung Cancer: Relationship with Cancer Histology. J Neuroimmune Pharmacol. 2019 Dec;14(4):697-708. doi: 10.1007/s11481-019-09879-6. Epub 2019 Oct 16. PMID: 31620969. Marino F, Schembri L, Rasini E, Pinoli M, Scanzano A, Luini A, Congiu T, Cosentino M. Characterization of human leukocyte-HUVEC adhesion: Effect of cell preparation methods. J Immunol Methods. 2017 Apr;443:55-63. doi: 10.1016/j.jim.2017.01.013. Epub 2017 Feb 3. PMID: 28167274. Spagnuolo P, Rasini E, Luini A, Legnaro M, Luzzani M, Casareto E, Carreri M, Paracchini S, Marino F, Cosentino M. Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: an in vitro study in MCF-7 cells. Fitoterapia. 2014 Apr;94:62-9. doi: 10.1016/j.fitote.2014.01.027. Epub 2014 Feb 5. PMID: 24508860. Capellino S, Cosentino M, Luini A, Bombelli R, Lowin T, Cutolo M, Marino F, Straub RH. Increased expression of dopamine receptors in synovial fibroblasts from patients with rheumatoid arthritis: inhibitory effects of dopamine on interleukin-8 and interleukin-6. Arthritis Rheumatol. 2014 Oct;66(10):2685-93. doi: 10.1002/art.38746. PMID: 24965369. 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. 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.Pharmacol Res. 2007 Aug;56(2):140-7. doi: 10.1016/j.phrs.2007.05.001. Epub 2007 May 22. PMID: 17572100.