Determining the shelf life of culture media: an analysis of its impact on cell culture effectiveness

Introduction: Medium 199, developed by J.F. Morgan in 1950, was one of the first synthetic media for in vitro culture of mammalian cells, and it may present variations in its composition depending on the cell line.Additionally, commercial media are typically prepared and supplemented in-house with Fetal Bovine Serum, Antibiotics, and L-glutamine.After medium production, it is crucial to evaluate the shelf life of this solution to ensure quality, reduce costs, and optimize storage.Objectives: Thus, the objective of this study was to evaluate the viability/cell count and the population doubling level (PDL) of VERO cells maintained in culture, with batches of supplemented Medium 199 used up to approximately 40 days (Supplemented Medium, single batch, up to 40 days -MS40d) versus the control medium (Supplemented Medium, up to 15 days -MS15d).Methodology: For this, 2 control bottles (175cm, MS15d) and 8 test bottles (MS40d), both with Vero cells were used.Quantitative data were subjected to the Shapiro-Wilk test for normality; parametric (F-Test, T-Test, Bartlett, and ANOV A) and non-parametric (Levene, Wilcoxon, Kruskal-Wallis, and Dunn) tests to assess equality between groups, adopting a significance level of 5%.Results: No statistical differences were found in the number of VERO cells present in the two groups evaluated (MS15d versus MS40d) (Bartlett, p = 0.988 and ANOV A, p = 0.998).Although the PDL presented a non-normal distribution (Shapiro-Wilk, p = 0.0016), there were no statistically significant differences between the bottles according to: Levene, p-value = 0.87 and Kruskal-Wallis: p = 0.959 or in the distribution of data in the tested groups (Wilcoxon, p = 0.304).Furthermore, there was no change in cell viability between the tested groups (MS15d versus MS40d). Conclusion:No statistically significant differences were observed in cell count, PDL, or cell viability, indicating that MS40d (up to 40 days post-supplementation) preserves the essential quantitative and qualitative characteristics of Vero cell culture.These findings support the extended use of supplemented Medium 199, optimizing resource utilization without compromising cell integrity.