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Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues

Nature Protocols volume 4, pages 1522–1534 (2009)Cite this article

Abstract

This protocol describes a cell/hydrogel molding method for precise and reproducible biomimetic fabrication of three-dimensional (3D) muscle tissue architectures in vitro. Using a high aspect ratio soft lithography technique, we fabricate polydimethylsiloxane (PDMS) molds containing arrays of mesoscopic posts with defined size, elongation and spacing. On cell/hydrogel molding, these posts serve to enhance the diffusion of nutrients to cells by introducing elliptical pores in the cell-laden hydrogels and to guide local 3D cell alignment by governing the spatial pattern of mechanical tension. Instead of ultraviolet or chemical cross-linking, this method utilizes natural hydrogel polymerization and topographically constrained cell-mediated gel compaction to create the desired 3D tissue structures. We apply this method to fabricate several square centimeter large, few hundred micron-thick bioartificial muscle tissues composed of viable, dense, uniformly aligned and highly differentiated cardiac or skeletal muscle fibers. The protocol takes 4–5 d to fabricate PDMS molds followed by 2 weeks of cell culture.

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Figure 1: Fabrication of bioartificial muscle tissue constructs.
Figure 2: Fabrication of PDMS tissue molds.
Figure 3: Compaction of cell–hydrogel mixture with time in culture.
Figure 4: Control of tissue construct dimensions and structure.
Figure 5: Analysis of muscle cell alignment in tissue constructs.
Figure 6: Assessment of cell differentiation and distribution within the muscle tissue constructs.

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Acknowledgements

We thank Ava Krol and Lisa Satterwhite for their assistance with cell isolation. This study is supported by a national science scholarship from the Singapore Agency for Science, Technology and Research (A*STAR) to B.L.; an American Heart Association predoctoral fellowship (No. 0715178U) to N.B. (Nima Badie); and NIH grants HL080469 from the National Heart, Lung, and Blood Institute and AR055226 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases to N.B. (Nenad Bursac).

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Author notes

  1. Weining Bian and Brian Liau: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA

    Weining Bian, Brian Liau, Nima Badie & Nenad Bursac

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  1. Weining Bian
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Contributions

W.B. and B.L. contributed equally to this work. W.B., B.L. and N.B. (Nenad Bursac) jointly developed the protocol; B.L. optimized the fabrication of PDMS tissue molds; W.B. carried out the cell–hydrogel molding experiments and structural assessment of the resulting tissue constructs; N.B. (Nima Badie) developed the program for the analysis of cell alignment. All authors contributed the preparation of the paper.

Corresponding author

Correspondence to Nenad Bursac.

Supplementary information

Supplementary Video 1

Spontaneously twitching skeletal muscle tissue construct at culture day 7 (i.e., after 3 days of differentiation). (MOV 865 kb)

Supplementary Video 2

Spontaneously contracting cardiac muscle tissue construct at culture day 13. (MOV 312 kb)

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Bian, W., Liau, B., Badie, N. et al. Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues. Nat Protoc 4, 1522–1534 (2009). https://doi.org/10.1038/nprot.2009.155

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