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Maria Luz Alonso-Alonso, M T Garcia-Gutierrez, Jose-Carlos Pastor, Ivan Fernandez-Bueno, Girish K Srivastava; Biotech approach for enhanced neuroprotection based on adipose-MSCs and drugs properties. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1122.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal Pigment Epithelial (RPE) layer damage studies are crucial for retinal degeneration diseases. Stem cells therapy is a promising option for neuroprotection but in some cases, it needs enhanced neuroprotection without stem cell injection. Our approach attempts to achieve this goal using the adipose Mesenchymal Stem cells (adipose-MSCs) and drugs properties. For achieving this goal, our present study was to optimize culture time to obtain effective condition medium (CM) and further to look its best storage temperature.
Adipose-MSCs and ARPE19 were cultured under standard culture condition (5% CO2, 37°C) in completed DMEM low glucose with glutamine and completed DMEM/F12 respectively. Further they were synchronized by 24 h of serum starvation. Adipose-MSCs based CMs were collected at 24, 48 and 72 h, and one part was stored at 4°C for 5-3 days. Other part was stored for a month at 37°C, 4°C and -80°C. 96 well plated synchronized ARPE19 cells were incubated in standard culture medium (control 1), fibroblast CM (FCM; control 2) or 50% CMs (experiment samples). A 50% dilution of CMs was necessary to compare CM collected at different culture time, and hence with different concentrations of nutrients, waste products and factors secreted by the cells. At 1, 3 and 6 days, Alamar Blue® assay (proliferation) and at 7 days, Viability/cytotoxicity Assay Kit for Animal Live & Dead Cells® was performed and data was statistically analyzed. The results provided an effective CM, which was further studied for determining storage temperature following same protocols.
The results showed ARPE19 cells proliferated higher in 24h CM than control 1. ARPE19 proliferated higher in FCM than experiment samples. However, there was not statistical significance. It could be due to 50% dilution of CM. ARPE19 proliferation was significant lower (P<0.001) in 24h CM stored at 37°C than 4 and -80°C.
Highest proliferation of ARPE19 was in 24h CM. This CM maintained its effects after 1 month storage at -80°C and 4°C. Further study and analysis with drugs properties will support to develop a CM that could show enhanced neuroprotection for retinal degeneration diseases treatment.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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