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Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology

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Abstract

The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett–Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and l-cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

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Abbreviations

BMP4:

Bone morphogenic protein 4

CDM:

Chemically defined medium

DoE:

Design of experiments

ESC:

Embryonic stem cells

hESC:

Human embryonic stem cells

LIF:

Leukaemia inhibitory factor

MEF:

Mouse embryonic fibroblast

mESC:

Mouse embryonic stem cells

MinRes IV:

Minimum run resolution IV

PTP:

Protein-tyrosine phosphatases

ROS:

Reactive oxygen species

SSC:

Sum of squares

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Acknowledgments

The work for this study was supported by the Federal Ministry of Education and Research (BMBF, FKZ 01GN0526, FKZ 01GN0529) and by the Federal Ministry of Economics and Technology (BMWi, FKZ KF2080802AJ9, FKZ KF2354401AJ9). The authors thank Annika Wulf-Goldenberg und Dr. Antje Siegert at the Max Delbrück Center (MDC), Berlin-Buch, Germany, for provision of mouse embryonic fibroblasts.

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Authors and Affiliations

  1. Division of Experimental Surgery, Biomedical Research Center, Berlin-Brandenburg Center for Regenerative Therapies, Charité Campus Virchow-Klinikum, Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany

    Fanny Knöspel, Marc Lübberstedt & Katrin Zeilinger

  2. Biochrom AG, Berlin, Germany

    Rudolf K. Schindler & Stephanie Petzolt

  3. Departments of Surgery and Bioengineering, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Jörg C. Gerlach

Authors
  1. Fanny Knöspel
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  2. Rudolf K. Schindler
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  3. Marc Lübberstedt
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  4. Stephanie Petzolt
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  5. Jörg C. Gerlach
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  6. Katrin Zeilinger
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Correspondence to Fanny Knöspel.

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Knöspel, F., Schindler, R.K., Lübberstedt, M. et al. Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology. Cytotechnology 62, 557–571 (2010). https://doi.org/10.1007/s10616-010-9307-8

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  • DOI: https://doi.org/10.1007/s10616-010-9307-8

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