Zimmermann, Martina (2021)
The impact of S-sulfocysteine in cell culture media.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00014299
Dissertation, Erstveröffentlichung, Verlagsversion

Kurzbeschreibung (Abstract)

L-Cysteine is a critical amino acid required for the cultivation of mammalian cells such as Chinese hamster ovary cells (CHO). Due to low stability of cysteine and low solubility of the dimer cystine at neutral pH, S-sulfocysteine (SSC) was developed to substitute cysteine in highly concentrated feeds. The bioavailability of SSC, the positive impact but also the toxic response at high SSC concentrations was already described in previous studies. The underlying mechanism for cellular uptake and the root cause for the toxicity however remained unclear so far and were studied in the current work. Due to the structure similarity of SSC to cystine and glutamate, it was proposed that the cystine/glutamate antiporter (xc−) allow cellular uptake of SSC and was assessed via transporter inhibition using sulfasalazine and transporter overexpression using either sulforaphane or sulforaphane-N-acetylcysteine during fed-batch experiments. Following daily addition of 50 µM and 100 µM sulfasalazine, the extracellular SSC concentration was increased by 65% and 177% respectively, suggesting a reduced uptake due to xc− inhibition. In c ontrast, enhanced transporter activity through 15 µM sulforaphane and sulforaphane-Nacetylcysteine treatment, induced a 60% and 52% reduced extracellular SSC concentration, respectively. The inverse cellular response of CHO cells strongly suggests that SSC is transported via xc−. Additionally, elevated SSC uptake via sulforaphane treatment was linked to aggravated SSC toxicity indicating that the xc− antiporter activity impacts the toxic response by controlling the concentration of intracellular SSC. The second part of this study was aimed at assessing the impact of the feed formulation and cell line dependency regarding SSC toxicity via a combined approach of high throughput small-scale fed-batch experiments and multivariate data analysis (MVDA). A diverse SSC response was observed for eight different CHO cell lines and 79 different feed formulations indicating a clone dependent SSC response and a significant impact of the feed formulation on the toxicity. However, MVDA was not able to identify cell culture media components which impair the toxic response of SSC, suggesting that the feed formulation has an indirect impact on the SSC toxicity. The final section sought to clarify the root cause for toxicity after SSC application. For this purpose, SSC metabolization by CHO cells was investigated in vitro by spiking SSC to cell lysates. Critical intracellular metabolites were identified using LC-MS/MS, whereby significant responses to SSC treatment were e.g. glutathione depletion and the accumulation of S-sulfo-glutathione. These might support excessive persulfidation of cysteine-dependent key proteins through SSC metabolization, which was suggested to be the root cause of toxicity.

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