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TM Fig.5 OriCell Wistar Ratt ADSCs are d ifferentiated into chondrocy ytes and are sstained with Alcian A Blue. CRYOPR RESERVA ATION OF O CELL LS USIN NG OriCe ellTM CRYOPR RESERVA ATION MEDIA M OriCellTM NCR R Protein-F Free Cryoprreservation Medium (C Cat. No. NC CPF-10001) is a use freezing g medium. Its chemically-define ed and prottein-free prrotein-free,, ready-to-u fo ormulation has h been optimized to o stem cells s and prima ary cells, th hus greatly enhancing th he viability and integrity of these cells by prrotecting th hem from da amage durring the on ne-step free eze-thaw procedure. p Unlike other conventiional freezi ng media, which re equire a slow programmed freeze e, this prod duct allows the cells to o be directly y frozen at 80 0°C. Cryopreservation ge the cultu ure medium m with fresh h growth medium m 24 h hours before freezing N Note: Chang g. 1.. Collect ce ells that are e in the log garithmic growth phas se. Perform m a cell count to determin ne the viable cell dens ity. 2.. Centrifug ge the cells for 3-5 mi nutes at 25 50 x g and 20°C. Rem move and discard the supernattant using a pipette. 3.. Resuspen nd the cell pellet in th e OriCellTM NCR Protein-Free Cry yopreservation Medium m at a cell density d of 10 1 5-106 cellls/mL. 4.. Dispense e aliquots of the cell su uspension into cryogenic storage e vials that are properly labeled. 5.. Place the e vials direc ctly in a -80 0°C freezerr. After 24 hours, tran nsfer the fro ozen vials to liquid nitrogen fo or long-term m preservattion. IMPI0056A3 RA AWMD-01001 Page 12 of 1 4