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Efficient passage of human pluripotent stem cells on spider silk matrices under xeno-free conditions

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Abstract

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine and pharmaceutical development. Such applications require cell culture methods and reagents that are chemically defined, xeno-free, scalable, and low-cost. Herein, we describe non-mechanical passaging of hPSCs on spider silk films under chemically defined and xeno-free conditions. The cells were dissociated into single cells or small aggregates using Accutase or enzyme-free dissociation buffer and then passaged to spider silk films, where they expanded in monolayers until they covered the surface. Cells cultured over 10 passages on spider silk film remained karyotypically normal and pluripotent. In conclusion, a novel method for passaging dissociated hPSCs under conditions that are compatible with clinical applications is presented. The method is cost-efficient and may be useful for both research and clinical applications.

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Abbreviations

2D:

Two-dimensional

3D:

Three-dimensional

BAC:

Bacterial artificial chromosome

BMP4:

Bone morphogenetic protein 4

cTnT:

Cardiac troponin T

FGF2:

Basic fibroblast growth factor

FOXA2:

Forkhead box A2

hPSC:

Human pluripotent stem cell

hESC:

Human embryonic stem cell

hiPSC:

Human induced pluripotent stem cell

NKX2.5:

NK2 homeobox 5

OCT4:

Octamer-binding transcription factor 4

PAX6:

Paired box 6

SOX17:

SRY (sex determining region Y) Box 17

SSEA-1:

Stage-specific embryonic antigen 1

SSEA-4:

Stage-specific embryonic antigen 4

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Acknowledgments

The authors wish to acknowledge Spiber Technologies AB for providing matrices and for funding and Karolinska Institutets Research Funds for funding.

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Authors and Affiliations

  1. Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society (NVS), Center for Alzheimer Research, Karolinska Institutet, Huddinge, 14157, Stockholm, Sweden

    Siqin Wu, Jan Johansson & Anna Rising

  2. Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 7011, 75007, Uppsala, Sweden

    Jan Johansson & Anna Rising

  3. Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt 25, 10120, Tallinn, Estonia

    Jan Johansson

  4. Division of Obstetrics and Gynecology, Department of Clinical Sciences, Intervention and Technology, Karolinska Institutet and Karolinska University Hospital, Huddinge, 14186, Stockholm, Sweden

    Outi Hovatta

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  1. Siqin Wu
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  4. Anna Rising
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Corresponding author

Correspondence to Anna Rising.

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Conflict of interest

This study was funded by Spiber Technologies AB.

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Wu, S., Johansson, J., Hovatta, O. et al. Efficient passage of human pluripotent stem cells on spider silk matrices under xeno-free conditions. Cell. Mol. Life Sci. 73, 1479–1488 (2016). https://doi.org/10.1007/s00018-015-2053-5

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  • DOI: https://doi.org/10.1007/s00018-015-2053-5

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