1x10^7 Human embryonic kidney cell spheroids were grown for 7 days in suspension over agarose prepared with FBS supplemented DMEM containing 50mM HEPES, pH 7.2, and 1xGlutamax-1 (Invitrogen). Following growth, media was removed and the spheroids were stored on the surface of the agarose in sealed flasks for 2,4, and 6 week intervals at room temperature in the dark. The samples were compared against unstored controls of both spheroids and monolayer (at 30-40% confluence)samples. All samples were conducted in triplicate. For each storage timepoint, a set of unrehydrated samples were analyzed, and an additional set of rehydrated samples were analyzed after allowing for growth in a treated tissue culture flask, to assess how well the samples returned to a normal, pre-storage state. Rehydration was conducted with DMEM (Invitrogen) containing 20%FBS and antibiotics (100U penicillin, 100ug/mL streptomycin, and 0.25ug Fungizone), with HEPES and Glutamax as stated previously.
