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The impact of S-sulfocysteine in cell culture media

Zimmermann, Martina (2021):
The impact of S-sulfocysteine in cell culture media. (Publisher's Version)
Darmstadt, Technische Universität,
DOI: 10.26083/tuprints-00014299,
[Ph.D. Thesis]

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.

Item Type: Ph.D. Thesis
Erschienen: 2021
Creators: Zimmermann, Martina
Status: Publisher's Version
Title: The impact of S-sulfocysteine in cell culture media
Language: English
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.

Place of Publication: Darmstadt
Collation: v, 147 Seiten
Divisions: 07 Department of Chemistry
07 Department of Chemistry > Fachgebiet Biochemie
Date Deposited: 08 Jun 2021 08:56
DOI: 10.26083/tuprints-00014299
Official URL: https://tuprints.ulb.tu-darmstadt.de/14299
URN: urn:nbn:de:tuda-tuprints-142992
Referees: Kolmar, Prof. Dr. Harald ; von Hagen, Prof. Dr. Jörg
Refereed / Verteidigung / mdl. Prüfung: 2 November 2020
Alternative Abstract:
Alternative abstract Language

Die Aminosäure L-Cystein wird zur Kultivierung von Säugetierzellen wie chinesische Hamster ovarial (CHO) Zellen benötigt. Aufgrund der geringen Stabilität von Cystein und der geringen Löslichkeit von Cystin bei neutralem pH, wurde S-Sulfocystein (SSC) für den Einsatz in hochkonzentrierten Feeds entwickelt. Die Bioverfügbarkeit und der positive Einfluss von SSC wurde bereits mehrfach publiziert, wobei ebenfalls eine Toxizität bei hohen SSC Konzentrationen beobachtet wurde. Sowohl die zelluläre Aufnahme von SSC und die Ursache der Toxizität sind bisher ungeklärt und wurden in dieser Arbeit untersucht. Aufgrund struktureller Ähnlichkeit von SSC zu Cystin und Glutamat wurde vermutet, dass der Cystin/Glutamate-Antiporter (xc−) die SSC Aufnahme ermöglicht. Dies wurde einerseits durch die Hemmung des Transporters mittels Sulfasalazin und andererseits über Transporter Überexpression mittels Sulforaphan oder Sulforaphan-N-acetylcystein untersucht. Durch die tägliche Zugabe von 50 µM und 100 µM Sulfasalazin während Fed-Batch Experimenten wurde die extrazelluläre SSC-Konzentration um 65% bzw. 177% erhöht, was auf eine verringerte Aufnahme aufgrund der xc− Hemmung hinweist. Im Gegensatz dazu wurde eine erhöhte Transporteraktivität durch die Zugabe von 15 µM Sulforaphan und Sulforaphan-N-acetylcystein detektiert, indem die extrazelluläre SSC Konzentration um 60% bzw. 52% verringert wurde. Diese entgegengesetzte Wirkung stützt die Hypothese, dass SSC mittels xc− transportiert wird. Des weiteren wurde die Toxizität von SSC durch die Sulforaphan-bedingt erhöhte SSC Aufnahme verstärkt. Dies weist darauf hin, dass insbesondere die SSC Konzentration aber auch die xc− Antiporteraktivität die toxische Reaktion beeinflussen. Der zweite Teil dieser Studie zielte darauf ab, Auswirkungen der Feed-Formulierung auf die SSC bedingte Toxizität und die Abhängigkeit verschiedener Zelllinien mittels Hochdurchsatz Fed-Batch Experimenten und multivariater Datenanalyse (MVDA) zu untersuchen. Die unterschiedliche Auswirkung von acht CHO-Zelllinien und 79 verschiedenen Feed-Formulierungen deutet sowohl auf eine klonabhängige SSC Toxizität als auch einen signifikanten Einfluss der Feed-Formulierung hin. Die Identifizierung von Medienkomponenten, welche die SSC Toxizität fördern war nicht möglich, so dass die Feed-Formulierung nur einen indirekten Einfluss auf die SSC Toxizität haben mag. Der letzte Abschnitt diente dazu die Metabolisierung von SSC durch CHO-Zellen zu untersuchen, indem Zelllysate mit SSC versetzt wurden. Die mittels LC-MS/MS identifizierten Metabolite wurden genutzt um neue Hypotheses zu formulieren. Die Erschöpfung von Glutathion und Akkumulation von S-sulfo-Glutathion wurden als mögliche Schlüsselmetabolite identifiziert und könnten eine übermäßige Persulfidierung von Cysteinabhängigen Schlüsselproteinen durch die Metabolisierung von SSC fördern.

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