Metre-long cell-laden microfibres exhibit tissue morphologies and functions

Journal name:
Nature Materials
Year published:
Published online


Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core–shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.

At a glance


  1. Formation of a metre-long cell-laden microfibre.
    Figure 1: Formation of a metre-long cell-laden microfibre.

    a, A cell-containing ECM-protein/Ca-alginate core–shell hydrogel microfibre is continuously generated in a double-coaxial microfluidic device. The cells in the gelated ECM protein migrate and form a fibre-shaped cellular construct with cell-to-cell contacts. The Ca-alginate shell can be selectively removed with an enzymatic reaction. Various types of cell can be applied to the cell fibres. b, An image (top) and an explanatory illustration (bottom) of the double-coaxial laminar flow microfluidic device. The device was composed of pulled glass capillaries and connectors. A double-coaxial laminar flow consisting of a pre-gel solution of ECM proteins with cells (core), a pre-gel solution of Na-alginate (shell) and a CaCl2 solution (sheath) was generated in the device, and a cell-containing ECM-protein/Ca-alginate core–shell hydrogel microfibre was continuously formed. c, Sequential images of the behaviours of NIH/3T3 cells in the different types of ECM protein. NIH/3T3 cells in ACol and Fib formed cell fibres. In contrast, NIH/3T3 cells in PCol did not form cell fibres. d, An image of the NIH/3T3–ACol fibre at day 4. e, Viability of the cells in NIH/3T3 fibres. The results are shown as the mean±s.d. of three independent fibres. Scale bars, 1 mm (b); 100 μm (c); 500 μm (d).

  2. Intrinsic cellular morphologies and functions of cell fibres using primary cells.
    Figure 2: Intrinsic cellular morphologies and functions of cell fibres using primary cells.

    a,b, Image of a primary cardiomyocyte–Fib fibre (a) and its spontaneous contraction between A and A′ in a (b). c, A sliced CLSM image of a primary HUVEC–PCol fibre. The diameter of the tubular structure is 23±4.0 μm (mean±s.d., eight different points on three independent fibres). Actin, green; nuclei, blue. d, Reconstructed cross-sectional image along the line A–A′ in c. e, Fluorescence micrograph of a primary cortical cell–PCol fibre at day 35. Tuj1, green. f,g, Ca2+ imaging of the cortical cell fibre at day 14. The graph in g shows changes in the fluorescence intensity at 1–4 in f. ΔF/F0: relative concentration of Ca2+. Scale bars, 20 μm (a,c,d); 100 μm (e,f).

  3. Differentiation induction of primary NSC–PCol fibres.
    Figure 3: Differentiation induction of primary NSC–PCol fibres.

    a,b, RT–PCR analysis (a) and immunofluorescence staining (b) of NSC–PCol fibres before differentiation induction (day 0) and NSC–PCol fibres with differentiation induction (day 10 (+)) and without induction (day 10 (−), control) for 10 days. Tuj1, green; GFAP, magenta; nuclei, blue. c, Immunofluorescence staining of NSC–PCol fibres with differentiation induction for 77 days. The arrows indicate synaptic clusters (right). Tuj1, green; synapisin, magenta; nuclei, blue. Scale bars, 20 μm.

  4. Fibre-based assembly of higher-order 3D macroscopic cellular structures.
    Figure 4: Fibre-based assembly of higher-order 3D macroscopic cellular structures.

    a, Microfluidic handling of a cell fibre in culture medium. A cell fibre can be ejected, withdrawn, bridged and clamped using capillaries. b, Schematic of a microfluidic weaving machine working in culture medium. c,d, Fluorescence micrographs of centimetre-scale woven macroscopic tissue using three different cell fibres assembled by the weaving machine. e, Folded 3D macroscopic cellular structure made by post-processing the woven tissue. f, Schematic of the fabrication of a double helical tube structure using cell fibres. g, Image of the helical tube released from the glass rod. h,i, Micrographs of a co-cultured helical tube (NIH/3T3–ACol fibre and HepG2–PCol fibre). j, Comparison of albumin secretion from the co-cultured helical tube and the mono-cultured HepG2–PCol helical tube (control). The results are shown as the mean±s.d. of three independent tubes. *p<0.05, **p<0.005, unpaired Student’s t-test. All scale bars represent 1 mm except i, 100 μm.

  5. Transplantation of primary islet cell fibres into diabetic mice.
    Figure 5: Transplantation of primary islet cell fibres into diabetic mice.

    a, Optical image of a primary islet cell fibre with an alginate–agarose IPN hydrogel shell. bGlucose-induced insulin secretion of primary islet cell fibres. The results are shown as the mean±s.d. of four independent samples. c,d, Images of the implantation of a 20-cm-long primary islet cell fibre into the subrenal capsular space of a recipient mouse during (c) and after (d) the implantation. The fibre was precisely folded in the capsular space using a microcatheter. e, Changes in the blood glucose concentration of three mice receiving 20-cm-long primary islet cell fibres (solid lines) and three mice receiving dispersed islet cells (dashed lines). Each symbol indicates an individual recipient. Fifteen days after implantation, the implanted fibres were removed from the subrenal capsular space. Scale bars, 100 μm (a); 2 mm (c,d).


  1. Dzenis, Y. Spinning continuous fibers for nanotechnology. Science 304, 19171919 (2004).
  2. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297302 (2006).
  3. Mouritz, A. P., Bannister, M. K., Falzon, P. J. & Leong, K. H. Review of applications for advanced three-dimensional fibre textile composites. Compos. Part A 30, 14451461 (1999).
  4. Quinn, B. Textiles in architecture. Archit. Design 76, 2226 (2006).
  5. Vakoc, B. J. et al. Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging. Nature Med. 15, 12191223 (2009).
  6. Wedeen, V. J. et al. The geometric structure of the brain fiber pathways. Science 335, 16281634 (2012).
  7. Martini, F. H., Nath, J. L. & Bartholomew, E. F. Fundamentals of Anatomy & Physiology 9th edn (Pearson Education, 2012).
  8. Shin, S. et al. ‘On the fly’ continuous generation of alginate fibers using a microfluidic device. Langmuir 23, 91049108 (2007).
  9. Lee, K. H., Shin, S. J., Park, Y. & Lee, S. H. Synthesis of cell-laden alginate hollow fibers using microfluidic chip and microvascularized tissue-engineering applications. Small 5, 12641268 (2009).
  10. Sugiura, S. et al. Tubular gel fabrication and cell encapsulation in laminar flow stream formed by microfabricated nozzle array. Lab Chip 8, 12551257 (2008).
  11. Kang, E. et al. Digitally tunable physicochemical coding of material composition and topography in continuous microfibres. Nature Mater. 10, 877883 (2011).
  12. Yamada, M., Sugaya, S., Naganuma, Y. & Seki, M. Microfluidic synthesis of chemically and physically anisotropic hydrogel microfibers for guided cell growth and networking. Soft Matter 8, 31223130 (2012).
  13. Raof, N. A., Padgen, M. R., Gracias, A. R., Bergkvist, M. & Xie, Y. One-dimensional self-assembly of mouse embryonic stem cells using an array of hydrogel microstrands. Biomaterials 32, 44984505 (2011).
  14. Hu, M. et al. Hydrodynamic spinning of hydrogel fibers. Biomaterials 31, 863869 (2010).
  15. Zhang, S. et al. A self-assembly pathway to aligned monodomain gels. Nature Mater. 9, 594601 (2010).
  16. Kiriya, D. et al. Meter-long and robust supramolecular strands encapsulated in hydrogel jackets. Angew. Chem. Int. Ed. 51, 15531557 (2012).
  17. Zhang, Z. K., Li, G. Y. & Shi, B. Physicochemical properties of collagen, gelatin and collagen hydrolysate derived from bovine limed split wastes. J. Soc. Leath Tech. Ch. 90, 2328 (2006).
  18. Silver, F. H. & Trelstad, R. L. Type-I collagen in solution—structure and properties of fibril fragments. J. Biol. Chem. 255, 94279433 (1980).
  19. Endres, G. F. & Scheraga, H. A. Molecular weight of bovine fibrinogen by sedimentation equilibrium. Arch Biochem. Biophys. 144, 519528 (1971).
  20. Mckee, P. A., Mattock, P. & Hill, R. L. Subunit structure of human fibrinogen, soluble fibrin, and cross-linked insoluble fibrin. Proc. Natl Acad. Sci. USA 66, 738744 (1970).
  21. Li, R. H., Altreuter, D. H. & Gentile, F. T. Transport characterization of hydrogel matrices for cell encapsulation. Biotechnol. Bioeng. 50, 365373 (1996).
  22. Nur-E-Kamal, A., Ahmed, I., Kamal, J., Schindler, M. & Meiners, S. Three dimensional nanofibrillar surfaces induce activation of Rac. Biochem. Bioph. Res. Co. 331, 428434 (2005).
  23. Wang, H. B., Dembo, M. & Wang, Y. L. Substrate flexibility regulates growth and apoptosis of normal but not transformed cells. Am. J. Physiol.-Cell Ph. 279, C1345C1350 (2000).
  24. Nemir, S. & West, J. L. Synthetic materials in the study of cell response to substrate rigidity. Ann. Biomed. Eng. 38, 220 (2010).
  25. Discher, D. E., Janmey, P. & Wang, Y. L. Tissue cells feel and respond to the stiffness of their substrate. Science 310, 11391143 (2005).
  26. Murry, C. E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: Lessons from embryonic development. Cell 132, 661680 (2008).
  27. Shen, B. Q., Greenfield, P. F. & Reid, S. Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor. Cytotechnology 16, 5158 (1994).
  28. Orive, G. et al. Cell encapsulation: Promise and progress. Nature Med. 9, 104107 (2003).
  29. Lim, F. & Sun, A. M. Microencapsulated islets as bioartificial endocrine pancreas. Science 210, 908910 (1980).
  30. Chang, T. M. S. Therapeutic applications of polymeric artificial cells. Nature Rev. Drug. Discov. 4, 221235 (2005).
  31. Mironov, V. et al. Organ printing: Tissue spheroids as building blocks. Biomaterials 30, 21642174 (2009).
  32. McGuigan, A. P. & Sefton, M. V. Vascularized organoid engineered by modular assembly enables blood perfusion. Proc. Natl Acad. Sci. USA 103, 1146111466 (2006).
  33. Matsunaga, Y. T., Morimoto, Y. & Takeuchi, S. Molding cell beads for rapid construction of macroscopic 3D tissue architecture. Adv. Mater. 23, H90H94 (2011).
  34. Kojima, N., Takeuchi, S. & Sakai, Y. Establishment of self-organized system in rapidly formed multicellular heterospheroids. Biomaterials 32, 60596067 (2011).
  35. Du, Y., Lo, E., Ali, S. & Khademhosseini, A. Directed assembly of cell-laden microgels for fabrication of 3D tissue constructs. Proc. Natl Acad. Sci. USA 105, 95229527 (2008).

Download references

Author information


  1. Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan

    • Hiroaki Onoe,
    • Teru Okitsu,
    • Akane Itou,
    • Midori Kato-Negishi,
    • Riho Gojo,
    • Daisuke Kiriya,
    • Koji Sato,
    • Shigenori Miura,
    • Shintaroh Iwanaga,
    • Kaori Kuribayashi-Shigetomi,
    • Yukiko T. Matsunaga,
    • Yuto Shimoyama &
    • Shoji Takeuchi
  2. Takeuchi Biohybrid Innovation Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology (JST), Tokyo 153-8904, Japan

    • Hiroaki Onoe,
    • Teru Okitsu,
    • Akane Itou,
    • Midori Kato-Negishi,
    • Riho Gojo,
    • Daisuke Kiriya,
    • Koji Sato,
    • Shigenori Miura,
    • Shintaroh Iwanaga,
    • Kaori Kuribayashi-Shigetomi &
    • Shoji Takeuchi


H.O. and S.T. conceived the design of the study. H.O. fabricated cell fibres. T.O. and H.O. designed and conducted transplantation experiments of cell fibres to diabetic mice. A.I. contributed to the protein secretion analyses of cell fibres. M.K-N. and H.O. performed the Ca2+ imaging of the cortical cell fibre and the differentiation induction of the NSC fibre. R.G. and H.O. performed the knitting, reeling and weaving processes. H.O. and D.K. developed the double-coaxial microfluidic device and measured the mechanical strength of the cell fibres. K.S. performed the RT–PCR analysis and laser Raman scattering spectroscopy. S.M. examined the temporal changes of ECM proteins and the cell-to-cell contacts in the cell fibres. S.I. analysed Fourier-transform infrared spectra of the hydrogel materials. K.K-S. performed the folding of the cell fabric with H.O. Y.T.M. provided considerable advice on the initial direction of the research. Y.S. contributed to the development of the ECM-protein/Ca-alginate core–shell fibres. H.O., T.O. and S.T. wrote the paper. All authors discussed the results and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Information (4.03 MB)

    Supplementary Information


  1. Supplementary Information (1.42 MB)

    Supplementary Movie S1

  2. Supplementary Information (1.78 MB)

    Supplementary Movie S2

  3. Supplementary Information (2.05 MB)

    Supplementary Movie S3

  4. Supplementary Information (1.48 MB)

    Supplementary Movie S4

  5. Supplementary Information (1.21 MB)

    Supplementary Movie S5

  6. Supplementary Information (2.32 MB)

    Supplementary Movie S6

Additional data