Protocol | Published:

A modular approach to the design, fabrication, and characterization of muscle-powered biological machines

Nature Protocols volume 12, pages 519533 (2017) | Download Citation


Biological machines consisting of cells and biomaterials have the potential to dynamically sense, process, respond, and adapt to environmental signals in real time. As a first step toward the realization of such machines, which will require biological actuators that can generate force and perform mechanical work, we have developed a method of manufacturing modular skeletal muscle actuators that can generate up to 1.7 mN (3.2 kPa) of passive tension force and 300 μN (0.56 kPa) of active tension force in response to external stimulation. Such millimeter-scale biological actuators can be coupled to a wide variety of 3D-printed skeletons to power complex output behaviors such as controllable locomotion. This article provides a comprehensive protocol for forward engineering of biological actuators and 3D-printed skeletons for any design application. 3D printing of the injection molds and skeletons requires 3 h, seeding the muscle actuators takes 2 h, and differentiating the muscle takes 7 d.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    , & A review on stereolithography and its applications in biomedical engineering. Biomaterials 31, 6121–6130 (2010).

  2. 2.

    & Stereolithographic 3D bioprinting for biomedical applications. in Essentials of 3D Biofabrication and Translation (eds. Atala, A. & Yoo, J.J.) Ch. 6 (Academic Press) 89–121 (2015).

  3. 3.

    et al. High-resolution projection microstereolithography for patterning of neovasculature. Adv. Healthc. Mater. 5, 610–619 (2016).

  4. 4.

    A review of 3D printing in tissue engineering. Tissue Eng. Part B Rev. 22, 298–310 (2016).

  5. 5.

    , , & A review of rapid prototyping techniques for tissue engineering purposes. Ann. Med. 40, 268–280 (2008).

  6. 6.

    , , , & 3D biofabrication strategies for tissue engineering and regenerative medicine. Annu. Rev. Biomed. Eng. 16, 247–276 (2013).

  7. 7.

    & Creating living cellular machines. Ann. Biomed. Eng. 42, 445–459 (2014).

  8. 8.

    Biological soft robotics. Annu. Rev. Biomed. Eng. 17, 243–65 (2015).

  9. 9.

    , & Utilization and control of bioactuators across multiple length scales. Lab Chip 14, 653–670 (2014).

  10. 10.

    & Vertebrate Myogenesis 56, 191–213 (2015).

  11. 11.

    & Engineered skeletal muscle tissue for soft robotics: fabrication strategies, current applications, and future challenges. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 6, 178–195 (2014).

  12. 12.

    , , & Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues. Nat. Protoc. 4, 1522–1534 (2009).

  13. 13.

    et al. Three-dimensionally printed biological machines powered by skeletal muscle. Proc. Natl. Acad. Sci. USA 111, 10125–10130 (2014).

  14. 14.

    et al. Optogenetic skeletal muscle-powered adaptive biological machines. Proc. Natl. Acad. Sci. USA 113, 3497–3502 (2016).

  15. 15.

    et al. Muscular thin films for building actuators and powering devices. Science 317, 1366–70 (2007).

  16. 16.

    et al. A tissue-engineered jellyfish with biomimetic propulsion. Nat. Biotechnol. 30, 792–797 (2012).

  17. 17.

    et al. Multi-material bio-fabrication of hydrogel cantilevers and actuators with stereolithography. Lab Chip 12, 88–98 (2012).

  18. 18.

    et al. Development of miniaturized walking biological machines. Sci. Rep. 2, 857 (2012).

  19. 19.

    et al. Phototactic guidance of a tissue-engineered soft-robotic ray. Science 353, 158–162 (2016).

  20. 20.

    & Engineered skeletal muscle tissue networks with controllable architecture. Biomaterials 30, 1401–1412 (2009).

  21. 21.

    , , & The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle. Biomaterials 32, 3575–83 (2011).

  22. 22.

    et al. Formation and optogenetic control of engineered 3D skeletal muscle bioactuators. Lab Chip 12, 4976–85 (2012).

  23. 23.

    , & Use of flow, electrical, and mechanical stimulation to promote engineering of striated muscles. Ann. Biomed. Eng. 42, 1391–1405 (2014).

  24. 24.

    , , & Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines. Am. J. Physiol. Cell Physiol. 280, C288–C295 (2001).

  25. 25.

    & A swimming robot actuated by living muscle tissue. J. Neuroeng. Rehabil. 1, 6 (2004).

  26. 26.

    & Cell lines: valuable tools or useless artifacts. Spermatogenesis 2, 1–5 (2012).

  27. 27.

    , , , & Three-dimensional photopatterning of hydrogels using stereolithography for long-term cell encapsulation. Lab Chip 10, 2062–2070 (2010).

  28. 28.

    et al. Photopatterning of hydrogel scaffolds coupled to filter materials using stereolithography for perfused 3D culture of hepatocytes. Biotechnol. Bioeng. 112, 777–787 (2015).

  29. 29.

    et al. 3D printing enables separation of orthogonal functions within a hydrogel particle. Biomed. Microdevices 18, 49 (2016).

  30. 30.

    , & Vascularization is the key challenge in tissue engineering. Adv. Drug Deliv. Rev. 63, 300–311 (2011).

  31. 31.

    Light-emitting diodes (LEDs) in dermatology. Semin. Cutan. Med. Surg. 27, 227–238 (2008).

  32. 32.

    , & Application of high brightness LEDs in the human tissue and its therapeutic response. in Applied Biomedical Engineering (eds. Gargiulo, G.D. & McEwan, A.) Ch. 1 (InTech) 3–20 (2011).

  33. 33.

    et al. A novel bioreactor for stimulating skeletal muscle in vitro. Tissue Eng. Part C Methods 16, 711–718 (2010).

  34. 34.

    , , & Mechanical stimulation improves tissue-engineered human skeletal muscle. Am. J. Physiol. Cell Physiol. 283, C1557–C1565 (2002).

  35. 35.

    , & Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins: roles in skeletal muscle growth and differentiation. Gen. Comp. Endocrinol. 167, 344–351 (2010).

  36. 36.

    et al. Microfluidic platform for the formation of optically excitable, three-dimensional, compartmentalized motor units. Sci. Adv. 2, e1501429 (2015).

  37. 37.

    Manual Tracking. (2005).

Download references


We thank J. Sinn-Hanlon at the University of Illinois at Urbana-Champaign (UIUC) for image rendering of Figure 1a, and the Core Facilities at the Carl R. Woese Institute for Genomic Biology at the University of Illinois at Urbana-Champaign for assistance with sample preparation and imaging. We also thank V. Chan, P. Bajaj, S. Uzel, P. Sengupta, T. Saif, and R. Kamm for insightful discussions regarding this work. This work was funded by National Science Foundation (NSF) Science and Technology Center Emergent Behavior of Integrated Cellular Systems (EBICS) Grant CBET – 0939511. R.R. was funded by NSF Graduate Research Fellowship Grant DGE – 1144245. R.R. and C.C. were funded by an NSF Cellular and Molecular Mechanics and Bionanotechnology (CMMB) Integrative Graduate Education and Research Traineeship (IGERT) at UIUC (grant 0965918).

Author information


  1. Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Ritu Raman
  2. Micro and Nanotechnology Laboratory, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Ritu Raman
    • , Caroline Cvetkovic
    •  & Rashid Bashir
  3. Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Caroline Cvetkovic
    •  & Rashid Bashir
  4. Carle Illinois College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Rashid Bashir


  1. Search for Ritu Raman in:

  2. Search for Caroline Cvetkovic in:

  3. Search for Rashid Bashir in:


R.R. designed the modular muscle-ring protocol; R.R. performed and analyzed muscle seeding and functional assessment experiments; C.C. performed and analyzed muscle staining experiments and protein quantification experiments; R.R., C.C., and R.B. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Rashid Bashir.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Methods 1–3.

Zip files

  1. 1.

    Supplementary Data

    CAD files of muscle ring and muscle strip injection molds and symmetric and asymmetric one-ring, two-ring, and four-ring bio-bot skeletons; and FEA template file of one-ring bio-bot skeleton for computational modeling.


  1. 1.

    A modular approach to designing, fabricating, and controlling muscle-powered machines.

    Overview of major steps in protocol, including CAD design, 3D printing, muscle seeding, muscle differentiation, and bio-bot functional assessment.

  2. 2.

    Electrical stimulation of unconstrained muscle ring.

    Electrical stimulation (1 Hz) controls contraction in a muscle ring that is untethered to a bio-bot skeleton.

  3. 3.

    Directional locomotion in a bio-bot.

    Optical stimulation (4 Hz) drives directional locomotion of a one-leg asymmetric bio-bot in the direction of the longer pillar.

  4. 4.

    Rotational locomotion in a bio-bot.

    Optical stimulation (2 Hz) of one half of one muscle ring in a two-leg symmetric bio-bot drives rotational locomotion.

About this article

Publication history



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.