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


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.

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Figure 1: Bio-bot design process overview.
Figure 2: Cellular orientation and morphology in muscle rings versus strips.
Figure 3: Muscle differentiation protocol.
Figure 4: Calculation and finite element analysis (FEA) verification of passive and active tension force production.
Figure 5: Immunohistochemistry of engineered muscle tissues.
Figure 6: Muscle-ring force production.


  1. Melchels, F.P.W., Feijen, J. & Grijpma, D.W. A review on stereolithography and its applications in biomedical engineering. Biomaterials 31, 6121–6130 (2010).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  5. Peltola, S.M., Grijpma, D.W., Melchels, F.P.W. & Kellomaki, M. A review of rapid prototyping techniques for tissue engineering purposes. Ann. Med. 40, 268–280 (2008).

    CAS  Article  Google Scholar 

  6. Bajaj, P., Schweller, R.M., Khademhosseini, A., West, J.L. & Bashir, R. 3D biofabrication strategies for tissue engineering and regenerative medicine. Annu. Rev. Biomed. Eng. 16, 247–276 (2013).

    Article  Google Scholar 

  7. Kamm, R.D. & Bashir, R. Creating living cellular machines. Ann. Biomed. Eng. 42, 445–459 (2014).

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  9. Chan, V., Asada, H.H. & Bashir, R. Utilization and control of bioactuators across multiple length scales. Lab Chip 14, 653–670 (2014).

    CAS  Article  Google Scholar 

  10. Sambasivan, R. & Tajbakhsh, S. Vertebrate Myogenesis 56, 191–213 (2015).

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

    CAS  Article  Google Scholar 

  12. Bian, W., Liau, B., Badie, N. & Bursac, N. Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues. Nat. Protoc. 4, 1522–1534 (2009).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  20. Bian, W. & Bursac, N. Engineered skeletal muscle tissue networks with controllable architecture. Biomaterials 30, 1401–1412 (2009).

    CAS  Article  Google Scholar 

  21. Hinds, S., Bian, W., Dennis, R.G. & Bursac, N. The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle. Biomaterials 32, 3575–83 (2011).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  23. Rangarajan, S., Madden, L. & Bursac, N. Use of flow, electrical, and mechanical stimulation to promote engineering of striated muscles. Ann. Biomed. Eng. 42, 1391–1405 (2014).

    Article  Google Scholar 

  24. Dennis, R.G., Kosnik, P.E., Gilbert, M.E. & Faulkner, J.A. Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines. Am. J. Physiol. Cell Physiol. 280, C288–C295 (2001).

    CAS  Article  Google Scholar 

  25. Herr, H. & Dennis, R.G. A swimming robot actuated by living muscle tissue. J. Neuroeng. Rehabil. 1, 6 (2004).

    Article  Google Scholar 

  26. Kaur, G. & Dufour, J.M. Cell lines: valuable tools or useless artifacts. Spermatogenesis 2, 1–5 (2012).

    Article  Google Scholar 

  27. Chan, V., Zorlutuna, P., Jeong, J.H., Kong, H. & Bashir, R. Three-dimensional photopatterning of hydrogels using stereolithography for long-term cell encapsulation. Lab Chip 10, 2062–2070 (2010).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  30. Novosel, E.C., Kleinhans, C. & Kluger, P.J. Vascularization is the key challenge in tissue engineering. Adv. Drug Deliv. Rev. 63, 300–311 (2011).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  32. Moreira, M.C., Prado, R. & Campos, A. 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. Donnelly, K. et al. A novel bioreactor for stimulating skeletal muscle in vitro. Tissue Eng. Part C Methods 16, 711–718 (2010).

    CAS  Article  Google Scholar 

  34. Powell, C.A., Smiley, B.L., Mills, J. & Vandenburgh, H.H. Mechanical stimulation improves tissue-engineered human skeletal muscle. Am. J. Physiol. Cell Physiol. 283, C1557–C1565 (2002).

    CAS  Article  Google Scholar 

  35. Duan, C., Ren, H. & Gao, S. 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).

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  37. Cordeli, F. Manual Tracking. (2005).

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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

Authors and Affiliations



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.

Corresponding author

Correspondence to Rashid Bashir.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cellular alignment and circularity analysis

Starting with a fluorescent image of cellular nuclei, convert the image to binary. To calculate circularity, apply a threshold to the binary image and use the Analyze Particles feature in ImageJ to bin and plot the circularity (1 = circular; 0 = linear) for each data set. To compute alignment, perform FFT analysis on similarly-oriented images and plot the results (radial sums) as a function of degrees. (See Supplementary Methods for more information.) Circularity plots represent all data points from Figure 2c (n=2312 total nuclei for muscle rings; n=2702 for muscle strips). Data from normal distributions represent mean values ± standard deviations; * = p < 0.05. FFT Alignment plots represent individual curves for muscle ring and strip samples; averaged data are shown in bold black lines on each plot (and plotted together for comparison in Figure 2b).

Supplementary Figure 2 External stimulation of muscle rings

(A) Stimulation setup for optical pulse stimulation of bio-bots. (B) Representative optical pulse train signal. (C) Stimulation setup for electrical pulse stimulation of bio-bots. (D) Representative electrical biphasic pulse signal.

Supplementary Figure 3 Muscle ring exercise training regimen

Protocol for stimulating bio-bots using a static mechanical stimulus (imposed by tethering the bio-bot to an underlying glass coverslip) starting Day 1, immediately after ring transfer, and a dynamic optical stimulus (imposed by the apparatus shown in Supplementary Figure 1 starting Day 4, after transferring the bio-bots to differentiation medium.

Supplementary Figure 4 Modulus as a Function of Energy Dose

Plot of Young’s Modulus for PEGDA 700 g mol-1 as a function of the UV energy dose imposed by the laser of the SLA during fabrication.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Methods 1–3. (PDF 879 kb)

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. (ZIP 673 kb)

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. (MP4 28823 kb)

Electrical stimulation of unconstrained muscle ring.

Electrical stimulation (1 Hz) controls contraction in a muscle ring that is untethered to a bio-bot skeleton. (MP4 2968 kb)

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. (MOV 20777 kb)

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. (MOV 29692 kb)

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Raman, R., Cvetkovic, C. & Bashir, R. A modular approach to the design, fabrication, and characterization of muscle-powered biological machines. Nat Protoc 12, 519–533 (2017).

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