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Attachment of stem cells to scaffold particles for intra-cerebral transplantation

Nature Protocols volume 4, pages 1440–1453 (2009)Cite this article

Abstract

Cell-replacement therapy and tissue regeneration using stem cells are of great interest to recover histological damage caused by neuro-degenerative disease or traumatic insults to the brain. To date, the main intra-cerebral delivery for these cells has been as a suspension in media through a thin needle. However, this does not provide cells with a support system that would allow tissue regeneration. Scaffold particles are needed to provide structural support to cells to form de novo tissue. In this 16-d protocol, we describe the generation and functionalization of poly (D,L-lactic-co-glycolic) acid (PLGA) particles to enhance cell attachment, the attachment procedure to avoid clumping and aggregation of cells and particles, and their preparation for intra-cerebral injection through a thin needle. Although the stem cell–scaffold transplantation is more complicated and labor-intensive than cell suspensions, it affords de novo tissue generation inside the brain and hence provides a significant step forward in traumatic brain repair.

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Figure 1: Time line for procedures.
Figure 2: Fabrication of microparticles.
Figure 3: Confirmation of fibronectin coating using immunolabelling and confocal microscopy.
Figure 4: Effects of motion and particle engineering on cell attachment.
Figure 5: Comparison of the spreading and attachment characteristics of cells.
Figure 6: Defining stereotactic coordinates using magnetic resonance (MR) images.
Figure 7: De novo tissue generation assessed by immunohistochemistry.

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Acknowledgements

This work was supported by a BBSRC project grant (BB/D014808/1), NIBIB Quantum Grant (1 P20 EB007076-01), EU FP VII (201842-ENCITE) and the generous support by the Charles Wolfson Charitable Trust Foundation. We would like to thank Dr. Natalia Gorenkova for assisting with the transplantations and Dr. Mieke Heyde for generating early versions of the PLGA particles. M.M. is the recipient of a RCUK fellowship.

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Authors and Affiliations

  1. Department of Neuroscience, Institute of Psychiatry, Kings College London, London, UK

    Ellen Bible, Jack Price & Michel Modo

  2. Division of Advanced Drug Delivery, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, UK

    David Y S Chau & Kevin M Shakesheff

  3. Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, Nottingham, UK

    Morgan R Alexander

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  1. Ellen Bible
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  5. Kevin M Shakesheff
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Contributions

E.B.: design of experiments, cell culturing, transplantation, MRI, histology, drafting of manuscript, final approval of manuscript; D.Y.S.C.: design of experiments, microparticle generation and characterization, drafting of manuscript, final approval of manuscript; M.R.A.: plasma polymerization, final approval of manuscript; J.P.: design of experiments, funding, final approval of manuscript; K.M.S.: design of experiments, funding, final approval of manuscript; M.M.: design of experiments, funding, drafting of manuscript, final approval of manuscript.

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Correspondence to Michel Modo.

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Competing interests

The authors declare competing financial interests. Kevin Shakesheff is CSO of Regentec. Jack Price is a consultant to ReNeuron Ltd.

Supplementary information

Supplementary Fig. 1

In-house developed plasma polymerization reactor. (A) Photograph of plasma polymerization reactor including key valves and control units. The plasma reactor consists of T-shaped borosilicate chamber, enclosed within stainless steel endplates and sealed with Viton O-rings. (B) Photograph of plasma polymerization reactor in action coating a sample. The pink/purple discharge is characteristic of the plasma formed from allylamine. (PDF 494 kb)

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Bible, E., Chau, D., Alexander, M. et al. Attachment of stem cells to scaffold particles for intra-cerebral transplantation. Nat Protoc 4, 1440–1453 (2009). https://doi.org/10.1038/nprot.2009.156

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