A simple method to determine evaporation and compensate for liquid losses in small-scale cell culture systems

Abstract

Objectives: Establish a method to indirectly measure evaporation in microwell-based cell culture systems and show that the proposed method allows compensating for liquid losses in fed-batch processes.

Results: A correlation between evaporation and the concentration of Na⁺ was found (R² = 0.95) when using the 24-well-based miniature bioreactor system (micro-Matrix) for a batch culture with GS-CHO. Based on these results, a method was developed to counteract evaporation with periodic water additions based on measurements of the Na⁺ concentration. Implementation of this method resulted in a reduction of the relative liquid loss after 15 days of a fed-batch cultivation from 36.7 ± 6.7% without volume corrections to 6.9 ± 6.5% with volume corrections.

Conclusion: A procedure was established to indirectly measure evaporation through a correlation with the level of Na⁺ ions in solution and deriving a simple formula to account for liquid losses.

Keywords: Evaporation; Fed batch; GS-CHO cells; Miniature shaken bioreactor; Reproducibility.

Fig. 1

Growth profile (a), viability (b), titre (c), and liquid loss through evaporation (d) of GS-CHO cells grown as batch culture in the micro-Matrix at a shaking speed of 220 rpm, a working volume of 3.5 mL, and active control of temperature (37 °C), DO (30%), and pH (7.2). Error bars represent one standard deviation about the mean (n = 3)

Fig. 2

Concentration of the electrolytes Na⁺ (a), K⁺ (b), and Ca²⁺ (c) depending on the relative evaporation (filled square), respective linear correlations (line) during a batch culture with GS-CHO cells grown in the micro-Matrix. The shaking speed was set to 220 rpm at a working volume of 3.5 mL. The DO was controlled at 30% and the pH at 7.2. Na⁺: y = 0.56·x − 60.4, R² = 0.95; K⁺: y = 6.94·x − 55.6, R² = 0.92; Ca²⁺: 42.9·x + 2.1, R² = 0.45. Error bars represent one standard deviation about the mean (n = 3)

Fig. 3

Growth profile (a, d), viability (b, e), and titre (c, f) of GS-CHO cells grown as fed-batch culture in the micro-Matrix at a shaking speed of 280 rpm, a working volume of 3 mL, and active control of temperature (37 °C), DO (30%), and pH (7.2). In run 1 (a–c) no compensation for evaporation was performed, whereas run 2 (d–f) was subjected to this procedure five times. Arrows indicate the time points of dH₂O additions to counteract evaporation based on the sodium level measured

Fig. 4

Endpoint values of the relative liquid loss (a) and osmolality (b) after 15 days of two fed-batch cultivations with GS-CHO cells grown in the micro-Matrix. In run 1 no compensation for evaporation was performed, whereas run 2 was subjected to this procedure five times. The shaking speed for both runs was set to 280 rpm, the working volume was 3 mL, temperature was controlled at 37 °C, DO at 30%, and pH at 7.2