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Bacteria were grown in Rhodococcus surfactant (RS) medium supplemented with 3%% (v/v) n-hexadecane at 160 rpm, 28 °C for 48 h (Ivshina et al. 2013).
After 1 hour, the resulting 3D bioconstructs were incubated in standard conditions of cultivation in MesenPRO RS Medium.
Therefore, cells plated in T75 culture flasks (Nunc, Thermo Scientific, Waltham, MA, USA) were incubated in MesenPRO RS Medium, at 37°C in a humidified atmosphere of 5% CO2 and 95% air, with growth media changed every 48 h.
hADSCs were isolated as previously described [ 22] and seeded on top of G-A and G-A-PAA biomatrices at an initial density of 6 × 10 cells/cm after propagation in MesenPRO RS Medium.
The stromal cells were recovered from cryofreeze in MesenPro RS Medium (Invitrogen 12746-012) supplemented with 200 mM l-glutamine (Invitrogen 25030-081) and 1% penicillin-streptomycin solution (Invitrogen 15140-122).
Cells were isolated from human lipoaspirate tissue, then they were expanded for one passage in MesenPRO RS Medium (Invitrogen, Life Technologies, Foster City, CA, USA), a low (2%) serum concentration medium that reduces ASCs doubling times and finally they were cryopreserved from primary cultures.
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It is generally accepted that medium Paracos-R (medium viscosity cement, Smith & Nephew, Memphis, USA) has more efficient elution characteristics than other cement types [7, 8].
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Deflagellated cells were diluted 10-fold into R medium at 22°.
A 5-mL aliquot was taken and diluted to 50 mL in R medium as "pretreatment" sample.
For live cell imaging, cells were incubated with Hoechst 33342 for 30 min at 37°C and then washed with no Phenol Red medium.
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