Stability of Minimum Essential Medium functionality despite L-glutamine decomposition

Abstract

L-Glutamine (L-Gln) instability in liquid media is a well-known fact. Also, negative effect of ammonia, one of the L-Gln degradation products, on viability of many cell cultures and on replication of different viruses has been described. However, negative effects of ammonia have been reported in doses excessively exceeding those that could be generated in regularly used liquid culture media due to spontaneous L-Gln breakdown (below 2 mM). Traditional virus vaccine production processes have been established and registered involving L-Gln containing media use. Eventual culture media replacement in the regular production process belongs to the major regulative changes that require substantial financial expenses. The aim of this study was to evaluate the effect of storage of Minimum Essential Media with Hanks salts on their relevant biological functions during virus vaccine production process in relation to L-Gln decrease. Our results show a cell type dependent effect of spontaneous L-Gln degradation during medium storage. They also suggest that for cell cultures used in measles, mumps, and rubella virus production the media retain their functionality in respect to cell viability or virus growth over a certain time window despite L-Gln degradation.

Keywords: Apoptosis; Cell viability; L-Glutamine; Measles; Minimum Essential Medium (MEM); Mumps; Necrosis; Rubella.

Fig. 1

Experimental procedure for investigation of MEM-H(0) stability. Influence of medium age on replication of mumps (A), measles (B) and rubella (C) viruses was investigated in the virus harvests prepared by the described protocol. Influence of medium age on the quality of cell cultures was investigated by the analysis of cells from the cultures that were subjected to the same procedure, only without infection (mock-infected); these were named harvest equivalents

Fig. 2

Typical chromatograms of MEM-H(N)-FBS (A), and MEM-H(0) (B-2) that were used for l-Gln quantification. Each amino acid peak was identified by spiking the medium with the pure respective amino acid. B-1 represents chromatogram of calibration solution consisting of l-Gln (Gln in the chromatogram) and internal standard d-Glu (Glu in the chromatogram) necessary for calculation of response factor (F)

Fig. 3

l-Gln concentration decrease in three batches of MEM-H(N)-FBS during the storage of 1 year at 2–8 °C. Each point represents individual ln (c(l-Gln_t)/c(l-Gln₀)) value. The l-Gln decomposition followed first order kinetics, whose rate constant was calculated from the slope of the line describing relationship between ln (c(l-Gln_t)/c(l-Gln₀)) values and storage period in days. Unopened, originally filled and stored samples are denoted with empty circles, while samples reopened several times before analysis are denoted with grey circles

Fig. 4

Representative examples of cell growth promotion results. A The growth of Vero cells was unaffected by the 9-month long storage of MEM-H(N)-FBS. B The growth of WI-38 cells was influenced by the 3-month long storage of MEM-H(N)-FBS. Dashed lines denote the log phase of the cell growth curves

Fig. 5

l-Gln concentration decrease in five individual samples of the MEM-H(0) incubated at 37 °C. Each point represents individual ln (c(l-Gln_t)/c(l-Gln₀)) value. The l-Gln decomposition followed first order kinetics, whose rate constant was calculated from the slope of the line describing relationship between ln (c(l-Gln_t)/c(l-Gln₀)) values and incubation period in days

Fig. 6

The influence of MEM-H(0) age on the ability of cell cultures to produce viruses evaluated by comparison of mumps virus titers (harvested from CEF) as well as measles and rubella virus titers harvested from MRC-5 maintained either in fresh (empty columns) or 9-month old (stored) (dashed columns) medium. Mean ± standard deviation from analyzed duplicates is given

Fig. 7

The influence of MEM-H(0) age on the measles virus stability. Virus harvests prepared according to the scheme in Fig. 1 either in fresh or in 9-month old (stored) medium, as indicated on x-axis, were diluted prior titration 1:2 (higher pair of columns) or 1:300 (lower pair of columns) either in fresh (empty columns) or in 9-month old medium (dashed columns). Mean ± standard deviation from analyzed duplicates is given

Fig. 8

The influence of the MEM-H(0) age on the viability of CEF. Cell cultures were incubated in either fresh (empty columns) or 9-month old (stored) (dashed columns) medium according to the mumps virus harvesting regime and analysed by MTT assay (A), and by flow cytometric determination of necrotic (B) and apoptotic (C) cells percentage. Standard deviations are shown (n = 4 in A; n = 3 in B and C), asterisk indicates the statistically significant differences between the two media (t statistic: p < 0.05)

Fig. 9

The influence of the MEM-H(0) age on the viability of MRC-5 cells incubated according to the measles virus harvesting regime. Cell cultures were incubated in either fresh (empty columns) or 9-month old (stored) (dashed columns) medium and analysed by MTT assay (A), and by flow cytometric determination of necrotic (B) and apoptotic (C) cells percentage. Standard deviations are shown (n = 4 in A; n = 3 in B and C), asterisk indicates the statistically significant differences between the two media (t statistic: p < 0.05)

Fig. 10

The influence of the MEM-H(0) age on the viability of MRC-5 cells incubated according to the rubella virus harvesting regime. Cell cultures were incubated in either fresh (empty columns) or 9-month old (stored) (dashed columns) medium and analysed by MTT assay (A), and by flow cytometric determination of necrotic (B) and apoptotic (C) cells percentage. Standard deviations are shown (n = 4 in A; n = 3 in B and C), asterisk indicates the statistically significant differences between the two media (t statistic: p < 0.05)