Glucose and lactate were determined via YSI 2300 STAT Plus Glucose & Lactate Analyzer (YSI Life Science). Materials and methods == CHO cells producing an antibody were cultured in chemically defined serum-free medium TC-BN.CHO (Teutocell AG) with addition of 6 mM glutamine. Three cultivations (37C, pH 7.1, 40% DO, 120 rpm) were performed in 2l-bioreactor systems with supplementation of 10 mg/l insulin or 0.1 NRA-0160 mg/l LongR3. The third culture was untreated and served as reference. Samples were taken every 24 h. Viable cell density and cell viability were measured using Cedex (Roche). Glucose and lactate were determined via YSI 2300 STAT Plus Glucose & Lactate Analyzer (YSI Life Science). Quantitation of antibody production was determined using POROSA columns (Invitrogen). N-Glycan abundance was analyzed by HPAEC-PAD method [2]. For RNA samples ‘Total RNA NucleoSpin Kit’ (Macherey-Nagel) was used. Quality and quantity of RNA were determined using NRA-0160 Nano Drop 1000 (Peqlab) and Bioanalyzer (Agilent). An in-house developed customized cDNA microarray with 41,304 probes was applied for transcriptome analysis. RNA was labeled using Agilent LIQUA Kit, one-color. Processing of microarray data was performed in ArrayLims and EMMA2 [3]. Raw data were standardized using Feature Extractor (Agilent) and LOWESS normalization. == Results == Cultivation data illustrated that maximal cell density was higher in cultivations with insulin and LongR3compared to that without growth factor. Additionally, glucose consumption and lactate production was slightly higher in cultivations with these supplements but time point of glutamine depletion Mouse monoclonal to MTHFR was similar in all reactors after similar cultivation time (Figure1A). Furthermore, product quantity and product quality was not influenced by growth factor addition. The most abundant glycoforms after 7 days of cultivation were G0F with about 50% and G1F with about 40% in all cultivation set-ups (Table1). == Figure 1. == (A)Time chart of viable cell density (VCD), cell viability (CV) and extracellular metabolites [glucose (Glc), lactate (Lac), glutamine (Gln)].(B)Number of significantly up- and down-regulated genes on day 5 in selected pathways (compared to day 3). == Table 1. == N-Glycan abundance [%] after 7 days of cultivation. For transcriptome analysis samples on day 5 were compared with those on day 3. Therefore, the following settings were used in statistical tests: a two-sample t-test with a p-value 0.01, signal intensity 6 (for A1 or A2) and intensity ratio 0.6 or -0.6 (for M1 or M2). Transcriptome data showed that LongR3supplementation resulted in the highest transcription change (1259 up- and 1689 down-regulated). Insulin supplementation resulted in second highest transcriptomic change (1026 up- and NRA-0160 1404 down-regulated) and reference cultivation led to lowest changes (344 up- and 301down-regulated). Supplemented cultures showed a higher transcription change in the selected pathways, like pentose phosphate pathway, TCA and glycolysis, than the reference culture, too. In LongR3containing cultures even more genes from these pathways were higher changed (Figure1B). == Conclusions == Data on cell growth and productivity as well as omics results were brought together to achieve a deeper insight into cellular processes and their influence on productivity and product quality. Cultivation data showed faster growth, glucose consumption and lactate formation for cultivations with insulin and LongR3compared to reference culture. However, antibody titer and glycan profiles were almost similar in all cultures. This indicates that supplementation with insulin or LongR3does not have an enhancing effect on product quality and quantity in antibody production with our CHO-K1 cells. Additionally, transcriptome data showed that growth factor supplementation resulted in a higher transcription change than in reference cultivation. Thus, for more understanding of the influence of insulin or LongR3supplementation on cultured CHO cells, further analysis of pathway regulation with full details is required. == Acknowledgements == The project NRA-0160 is co-funded by the European Union (European Regional Development Fund – Investing in your future) and the German federal state North Rhine-Westphalia (NRW). == References ==.
