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Browsing by Subject "jäännöskosteus"

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  • Nevala, Laura (2010)
    The objective of the research was to study the effects of different polymers, sugars, and cell handlings on the viabilities of ARPE-19 and ARPE-19-SEAP-2-neo cells after freeze-drying. Also the residual moisture content of the freeze-dried samples and the amount of intracellular sugar after incubation in trehalose medium were measured. The mixtures used in the study contained different polymers (e.g. polyvinylpyrrolidone, alginate, polyethylene glycol) and sugars (sucrose, trehalose and mannitol). Some cells were incubated or heated in trehalose medium before freeze-drying in order to increase the amount of intracellular sugar. The aim of the heating was also to increase the heat shock protein formation in the cells. The samples were mainly freeze-dried in 24 well plates. The same freeze-drying parameters were used in all freeze-drying runs. The freeze-drying cycle lasted 38.5 hours (freezing 2.5 h, primary drying 32 h, and secondary drying 4 h). In the freezing stage the samples were froze to -40 °C and the freezing rate was 1 °C/minute. In the primary drying stage the shelf temperature was mainly -35 °C and the pressure was 150 mTorr. The viabilities of the samples after freeze-drying were determined by measuring the fluorescence after 3 and 6 hours from addition of the Alamar Blue indicator dye. The residual moisture contents were measured by thermogravimeter. According to the results, mixture containing glycerol (9%) and PEG 10 000 (18%) was the best lyoprotectant in the study (70% viability after 3 hours). However, the viability decreased significantly (24% viability) in the measurement after 6 hours. Similar viability decrease was observed among all lyoprotectant mixtures used in the study. Extracellular sugars rarely had positive effect on the viability results. The 12 days incubation in 150 mM trehalose medium before freeze-drying affected positively to the post freeze-drying viability. Shorter incubation time or heating did not induce the same effect. Intracellular sugar measurements revealed, that the amount of intracellular trehalose was multiple after 12 days of incubation in 150 mM trehalose medium compared to the cells that were not incubated. The residual moisture contents of the samples varied between 0-7%. The residual moisture content of the sample containing glycerol 9% and PEG 10 000 18% was 1,5%.