Bioinspired Microfabrication

Developments in tissue engineering strategies for cell-based microgel fabrication.

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May 05, 2017
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The development of well-designed biological substitutes in human healthcare, with the overarching goal to adequately replace the loss/functional failure of an organ or tissue is known as regenerative medicine. Bioinspired reconstruction of natural tissue/organ structures that usually form during gradual human development, is an obvious interdisciplinary challenge. Tissue engineering, broadly combines the principles of life sciences, synthetic chemistry, materials science, physics, medicine and engineering, to develop materials and create functional substitutes, for tissue or organ replacement and therapeutic drug delivery. Based on the desired outcome/expected biological behaviour, varieties of tissue engineering approaches exist (Figure 1).

In a predominant approach to create organs for implantation, a scaffold or structural framework resembling native biology was formed to which biological cells adhered; this was achieved via cell-seeding de-cellularized tissues to construct lung1, liver2 and heart3,4 organs. Recent advances in tissue engineering include; scaffold-less organ bioprinting based on principles of self-assembly inspired by early human development5, three-dimensional printing strategies (acellular/cell-based bioprinting)6, recently developed bio-integrated robots in-part based on 3D printing7, self-assembly of micron-scale materials/molecules to create macrostructures8,9, and hydrogel-based10 or non-hydrogel-based11 therapeutic initiatives (Figure 1).

Figure 1: Ongoing strategies in tissue engineering for regenerative medicine with a few notable interconnections (green arrows), the focus in context is on hydrogel/microgels (blue) that contribute to organ, tissue and drug development.

Current strategies for tissue and organ development can either mimic native biology (reverse-engineering)2,3 or instill capacity for non-natural functional behaviours (forward-engineering)7. As a whole, cross-disciplinary approaches commonly contribute to synthetically recapitulate native architecture or fabricate biocompatible carriers for drug delivery. This post is a brief update on engineering strategies for hydrogel microfabrication; a biomaterial with a dual role in both organ re-construction and therapeutics development, in regenerative medicine.

The self-assembly of smaller molecules to build a three dimensional construct is “bottom-up” tissue engineering, while scaffold/mold-based cell-seeding is a “top-down” approach8,12. A classic example of bottom-up engineering is when self-assembling molecules form nanoscale structures, to then form macroscale gels8 (Figure 2A). On the same note, amphiphilic molecules have self-assembled to form micelles, vesicles and hydrogels while encapsulating pharmaceuticals in the form of small molecules, peptides and DNA for controlled delivery, with a variety of currently existing commercial applications9 (Figure 2B).

Bottom up tissue engineering and amphiphilic molecules illustrated

Figure 2: A) Illustrated ‘bottom-up’ tissue engineering via self-assembly of molecules from nanoscale to macroscale, B) (i) common therapeutics ranging from nanoscale to microscale and (ii) self-assembled structures for their therapeutic delivery (blue – hydrophilic, orange – hydrophobic). Images modified from reference8,9.

However, such delivery systems to accommodate cells, protein, antibody therapeutics and small interfering RNA (siRNA) for pharmaceutical applications, are still under development13. In tissue engineering research therefore, a variety of microfabrication approaches based on microfluidics, micromolding and electrospinning techniques specifically exist for hydrogel-based cell encapsulation (table 1)13.

Table 1: A few, ongoing methods under optimization to microfabricate cell containing hydrogels13.


Applications with examples


Soft lithography (replica molding/self-assembly)

Microtransfer molding: encapsulating fibroblasts in a liquid prepolymer hydrogel


Photolithography (hydrogel precursor cross-linked with light induced radicals)

Free radical polymerization of polymers and biomaterials (can be made cell-compatible for cell-based hydrogels)


Microfluidics (well controlled spherical gel particles for cell encapsulation )

Cell encapsulation with excellent fidelity; enable experiments that require tightly controlled cellular microenvironments in the lab.


Electrospraying and Spinning

Coupling bio-electrospraying with droplet based microfluidics for controlled live-cell encapsulation.



Cell-hydrogel based tissue spheroid dispensation using robotic bioprinters, for in-lab self-assembly and 3 dimensional organ construction.


In general, cell-embedded hydrogels for biological applications should meet a few key requirements, which include9,13:

1)Noncytotoxic-biocompatibility and potential biodegradability, 2) high loading capacity for in vivo therapeutic applications and 3) hydrophilicity (water-loving nature).

During hydrogel based cell encapsulation, the type of cell encapsulated is also important. Primary cardiomyocytes are more sensitive to their environments based on temperature, shear stress and pH than fibroblasts, making them less compatible with microfluidics methods compared to soft and photolithography based encapsulation13.

Structurally, hydrogels are three dimensional hydrophilic, polymeric networks, synthetically cross-linked by the exposure of polymer precursors to chemical stimuli (enzymes, functional groups) or physical stimuli (ionic interactions, temperature changes) to form a material that swells on exposure to water13. As an example of physical-stimuli based crosslinking, biocompatible polymer alginate hydrogels (derived from brown seaweed) utilize divalent Ca2+ cations13. A very recent article further elaborates on advanced engineering strategies for hydrogel fabrication19. The biomaterial can form macroscopic networks or be confined to smaller dimensions to form microgels via microfabrication.

In principle, during cell-based hydrogel microfabrication, researchers 1) optimize existing technologies (instead of introducing entirely new methods), 2) account for the sensitivity of living cells and 3) ensure all components are compatible for seamless integration18.

Existing techniques for cell encapsulation within microscale hydrogels generally yield high polymer-to-cell ratios13 while sometimes failing to capture any cells at all, resulting in empty microgel capsules. A recently developed method for hydrogel based cell-encapsulation at the microscale, achieves single cell-encapsulation with over 90% efficiency10. Briefly, a pure population of cell-embedded microgels in biocompatible alginate polymer is produced by starting with cell exposure to a suspension of calcium carbonate nanoparticles that mediate cross-linking, followed by removal of excess nanoparticles from cells prior to their combination with the soluble alginate polymer within a microfluidic device, to generate efficient single-cell encapsulated microgels (highlighted in red, Figure 3).

Deterministic encapsulation of single cells in thin tunable microgels

Figure 3: a) Cells exposed to CaCo3 nanoparticles -> excess particles washed out -> alginate polymer combined in a microfluidic device -> generate single cell-encapsulated microgels (red box, complete encapsulation), b) an enlarged view of the water-in-oil emulsion process within microfluidic device, c) confocal image of a fluorescently labelled encapsulated single-cell (green-alginate, red-actin cytoskeleton, blue-nucleus). Images from reference10,13,14

Notably, the new method does not construct a novel prototype for the expected outcome, instead optimizes engineering principles for a practically efficient approach than before, albeit based on pre-existing methods. The unprecedented step here was to remove excess nanoparticles on cells prior to polymer interaction, to ensure complete cell encapsulation. The process is also extremely fast, enabling encapsulation of "one thousand cells per second" inside the device. These cells were viable in culture for up to 3 days and able to retain structural integrity in circulation within a pre-clinical animal model10. The decreased material-to-cell ratio enhanced the ability of encapsulated cells to respond to external stimuli, with potential implications for controlled cellular function of microgel-based single-cell injections in physiological environments.

The team at Wyss Institute where these microgel-based cells were developed and experimentally validated, assume the method may improve cell-based therapies for precision tissue engineering, assist niche modelling to match physiological conditions and enable ‘micro-tissue’ formation by cell by cell construction, giving control over the composition of engineered tissues. Ongoing developments in tissue engineering strategies via combined methods as briefly detailed, may evolve overall to effectively recapitulate native biology and move closer towards clinically enabled precision tissue engineering.

Poster Image: Three-dimensional image of single-cell encapsulated in alginate microgel from Making Every Cell Matter, Wyss Institute at Harvard University.


  1. Ott, H. C. et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med 16, 927-933, (2010).
  2. Uygun, B. E. et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med 16, 814-820, (2010)
  3. Guyette, J. P. et al. Bioengineering Human Myocardium on Native Extracellular Matrix: Novelty and Significance. Circulation Research 118, 56-72, (2016)
  4. Ott, H. C. et al. Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Nat Med 14, 213-221, (2008)
  5. Karoly, J. et al. Tissue engineering by self-assembly and bio-printing of living cells. Biofabrication 2, 022001 (2010).
  6. Sears, N. A., Seshadri, D. R., Dhavalikar, P. S. & Cosgriff-Hernandez, E. A Review of Three-Dimensional Printing in Tissue Engineering. Tissue Eng Part B Rev 22, 298-310 (2016).
  7. Raman, R., Cvetkovic, C. & Bashir, R. A modular approach to the design, fabrication, and characterization of muscle-powered biological machines. Nat. Protocols 12, 519-533, (2017).
  8. Elbert, D. L. Bottom-up tissue engineering. Current opinion in biotechnology 22, 674-680, (2011).
  9. Branco, M. C. & Schneider, J. P. Self-assembling materials for therapeutic delivery. Acta biomaterialia 5, 817-831, (2009).
  10. Mao, A. S. et al. Deterministic encapsulation of single cells in thin tunable microgels for niche modelling and therapeutic delivery. Nat Mater 16, 236-243, (2017).
  11. Mahoney, M. J. & Saltzman, W. M. Transplantation of brain cells assembled around a programmable synthetic microenvironment. Nature biotechnology 19, 934-939, (2001).
  12. Catros, S. et al. Layer-by-layer tissue microfabrication supports cell proliferation in vitro and in vivo. Tissue Eng Part C Methods 18, 62-70, (2012).
  13. Selimović, Š., Oh, J., Bae, H., Dokmeci, M. & Khademhosseini, A. Microscale Strategies for Generating Cell-Encapsulating Hydrogels. Polymers 4, 1554 (2012).
  14. Khademhosseini, A. et al. Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment. J Biomed Mater Res A 79, 522-532, (2006).
  15. Fisher, J. P., Dean, D., Engel, P. S. & Mikos, A. G. Photoinitiated polymerization of biomaterials. Ann Rev Mater Res 31, 171-181, (2001).
  16. Atencia, J. & Beebe, D. J. Controlled microfluidic interfaces. Nature 437, 648-655, (2005).
  17. Hong, J., deMello, A. J. & Jayasinghe, S. N. Bio-electrospraying and droplet-based microfluidics: control of cell numbers within living residues. Biomed Mater 5, (2010).
  18. Mironov, V., Kasyanov, V. & Markwald, R. R. Organ printing: from bioprinter to organ biofabrication line. Current Opinion in Biotechnology 22, 667-673, (2011).
  19. Zhang, Y. S. & Khademhosseini, A. Advances in engineering hydrogels. Science 356, (2017).

Go to the profile of Thamarasee Jeewandara

Thamarasee Jeewandara

Research Associate , CUNY/Einstein Coll. Med.

Bioengineering, biochemistry and molecular biology

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