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Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product–free environment

Nature Protocols volume 7, pages 1366–1381 (2012)Cite this article

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Abstract

The protocols described here are comprehensive instructions for deriving human embryonic stem (hES) cell lines in xeno-free conditions from cryopreserved embryos. Details are included for propagation, cryopreservation and characterization. Initial derivation is on feeder cells and is followed by adaptation to a feeder-free environment; competent technicians can perform these simplified methods easily. From derivation to cryopreservation of fully characterized initial stocks takes 3–4 months. These protocols served as the basis for standard operating procedures (SOPs), with both operational and technical components, that we set to meet good manufacturing practice (GMP) and UK regulatory body requirements for derivation of clinical-grade cells. As such, these SOPs are currently used in our current GMP compliant facility to derive hES cell lines ab initio, in an animal product–free environment; these lines are suitable for research and potentially for clinical use in cell therapy. So far, we have derived eight clinical-grade lines, which will be freely available to the scientific community after submission/accession to the UK Stem Cell Bank.

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Figure 1: Establishment of ICM outgrowth and hES cell line.
Figure 2
Figure 3
Figure 4: Growth of hES cells under xeno-free feeder-free conditions.
Figure 5: Characterization of an hES cell line.

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Acknowledgements

This work was supported by the Medical Research Council, UK (grants G0801061 and G0701172). We thank our collaborators at the Cytogenetics Department and the Clinical Transplantation Laboratory, Guy's Hospital, for genetic analyses and HLA typing; A. Huhn and P. Sharpe at the Dental Institute and C. Hobbs, Histology Manager at the Wolfson Centre for Age-Related Diseases, School of Biomedical Sciences, King's College London, for teratoma-related work; and J.-R. Fantino from Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, Marseille, for editing movies. We also want to thank Y. Khalaf, director of the Assisted Conception Unit of Guy's and St. Thomas' National Health Service (NHS) Foundation Trust, and his staff for supporting the research program. We are especially indebted to the patients who donated embryos.

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Authors and Affiliations

  1. Division of Women's Health, Embryonic Stem Cell Laboratories, Guy's Assisted Conception Unit, King's College School of Medicine, London, UK

    Emma Stephenson, Laureen Jacquet, Cristian Miere, Victoria Wood, Neli Kadeva, Stefano Codognotto, Peter Braude & Dusko Ilic

  2. Assisted Conception Unit, Guy's and St. Thomas' Hospital Trust, London, UK

    Glenda Cornwell, Yaser Dajani & Peter Braude

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  1. Emma Stephenson
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Contributions

E.S., P.B. and D.I. designed the protocol, carried out the work, analyzed the results and prepared the manuscript. L.J. and C.M. designed the protocol, carried out the work and analyzed the results. V.W., N.K. and S.C. carried out the work. G.C. consented the patients. Y.D. recorded an ICM isolation movie.

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Correspondence to Dusko Ilic.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Time-lapse movie showing development of human 2PN embryo into fully expanded blastocyst. (MOV 7985 kb)

Supplementary Video 2

Micromanipulator set-up. (MOV 8376 kb)

Supplementary Video 3

Zona Pellucida (ZP) drilling with a stream of acid Tyrode's solution. ICM, inner cell mass. Asterisk (*), thinning ZP and forming a hole through which ICM can be aspirated. (MOV 4178 kb)

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Stephenson, E., Jacquet, L., Miere, C. et al. Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product–free environment. Nat Protoc 7, 1366–1381 (2012). https://doi.org/10.1038/nprot.2012.080

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