PA5 hOMCs growth and metabolic profile in cell cultures using Plastic and Plastic Plus microcarriers
A method previously developed by Santos et al 2011 was adopted to performed PA5 hOMCs microcarrier cell culture in spinner flasks. A total working volume of 80 mL in 100 mL Belco spinner flasks was used with 50% of media change every two days, for a total of 7 days. An initial seeding density of 6000 cells/cm2 was used, which is equivalent to 3.4x104 cells/mL. Figure 3 A shows that both Plastic and Plastic Plus microcarrier cultures experienced a decrease in cell concentration after day 1 from 3.4±0.03x104 cells/mL to 1.4±0.3x104cells/mL and 9.4±1.3x103 cells/mL, respectively. Cell number started increasing after day 3. On day 7, the concentration of cells in cultures employing Plastic microcarriers was 1.4±1.1x105 cells/mL and for Plastic Plus microcarriers was 4.9±1.8x104 cells/mL (statistically significant, p<0.05). Cell viability remained above 90% (Figure 3 A) throughout the culture period. Metabolic analysis of glucose, lactate and ammonia revealed similar concentration of these nutrient and metabolites for both conditions (Figure 3 B-D). On day 7, glucose concentration was of 10.5±1.7 mM and 11.5±1.6 mM, while lactate concentration was of 7.3±2.3 mM to 5.2±2.5 mM, for PA5 hOMCs grown on Plastic and Plastic Plus, respectively. The concentration of ammonia was of 0.7 mM for both conditions. Considering an exponential phase starting on day 3, specific consumption of glucose, specific production of lactate and ammonia, as well as yield of lactate per glucose were calculated. PA5 hOMCs grown of Plastic Plus led to a specific glucose consumption rate of 24.4±2.3 pmol.day-1cell-1, similar to the one measure for PA5 hOMCs grown on Plastic which was 20.4±4.7 pmol.day-1cell-1. Specific production rates for lactate and ammonia was 47.9±6.3 pmol.day-1cell-1 and 0.6±0.1 pmol.day-1cell-1, respectively for PA5 hOMCs grown on Plastic Plus. These results were on average 2 times higher than the specific production rates observed for PA5 hOMCs grown on Plastic microcarriers (24.6±7.2 pmol.day-1cell-1 and 0.3±0.05 pmol.day-1cell-1 for lactate and ammonia, respectively). The yield of lactate per glucose was 1.4±0.4 and 2.0±0.3 for PA5 hOMCs grown on Plastic and Plastic Plus microcarriers, respectively.