IP Library › Granted Patent US 12,496,767
Granted Patent B2
US 12,496,767 · App. 18/010,993 · Granted Dec 16, 2025

Digital assembly of spherical hydrogel voxels to form 3D lattice structures

Inventors: Liheng Cai (Charlottesville, VA); Jinchang Zhu (Charlottesville, VA)
Assignee: University of Virginia
B29C64/106B29C64/209B29C64/227B29C64/393B29C64/40B81C1/00095B29L2031/756B33Y10/00B33Y30/00B33Y50/02B33Y70/00B81C2203/03
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Quick Facts
Patent No.
US 12,496,767
App. No.
18/010,993
Granted
Dec 16, 2025
Kind
B2
Abstract

Systems and methods for 3D bioprinting of hydrogel voxels enable microfluidics-assisted digital assembly of spherical particles (DASP). The systems include a 3D motion system, a microfluidic printhead coupled to the 3D motion system, an extrusion device fluidly coupled to the microfluidic printhead, and a sacrificial support matrix. The sacrificial support matrix is designed to support the hydrogel voxels during printing and cross-link the hydrogel voxels. The system includes bio-inks comprising hydrogel compositions having independently controllable viscoelasticity and mesh size. The bio-inks are extruded by the extrusion device and microfluidic printhead to produce the hydrogel voxels. Exploiting the microfluidic printhead enables printing individual spherical hydrogel voxels with diameters from 150 micrometers (μm) to 1200 μm. Positioning and interconnection of the hydrogel voxels can be precisely controlled. The systems and methods produce free-standing 3D structures and can be used for producing functional tissue mimics.

Claims (31)

1 . A method of assembling hydrogel voxels to form a structure, the method comprising:

depositing a plurality of hydrogel voxels within a sacrificial support matrix, wherein the hydrogel voxels are spherical; and

cross-linking the plurality of hydrogel voxels to form the structure to comprise a one dimensional line, a two dimensional array, or a free-standing three dimensional lattice; wherein each of the spherical hydrogel voxels in the structure are interconnected and are distinguishable from each other when viewed through optical microscopy;

wherein the sacrificial support matrix is self-healing.

2 . The method of claim 1 , wherein the structure is a free-standing three dimensional lattice.

3 . The method of claim 1 , wherein depositing the plurality of hydrogel voxels comprises:

positioning a microfluidic printhead within the sacrificial support matrix; and

mechanically extruding a hydrogel composition through the microfluidic printhead.

4 . The method of claim 3 , comprising mechanically extruding the hydrogel composition at an injection speed of from 40 nanoliters per second to 680 nanoliters per second for each of the plurality of hydrogel voxels.

5 . The method of claim 3 , wherein depositing the plurality of hydrogel voxels comprises:

positioning the microfluidic printhead at a first position in the sacrificial support matrix;

extruding a first volume of the hydrogel composition at the first position;

moving the microfluidic printhead past a second position in the sacrificial support matrix by a distance of at least 3.5 mm;

moving the microfluidic printhead back to the second position; and

extruding a second volume of the hydrogel composition at the second position.

6 . The method of claim 5 , wherein the sacrificial support matrix is aqueous.

7 . The method of claim 3 , further comprising adjusting a movement speed of the microfluidic printhead, a distance between adjoining hydrogel voxels of the interconnected voxels, a concentration of calcium ions in the sacrificial support matrix, or combinations of these to modify the spacing, cross-linking, or both of the plurality of hydrogel voxels.

8 . The method of claim 1 , comprising depositing the plurality of hydrogel voxels at a center-to-center distance between the hydrogel voxels of from 0.8 to 1.7 times the average diameter of the plurality of hydrogel voxels.

9 . The method of claim 1 , wherein cross-linking the plurality of hydrogel voxels comprises contacting the plurality of hydrogel voxels with calcium ions pre-dissolved in the sacrificial support matrix.

10 . The method of claim 9 , further comprising, after contacting the plurality of hydrogel voxels with the calcium ions pre-dissolved in the sacrificial support matrix, washing the plurality of hydrogel voxels with a calcium solution, wherein the washing further cross-links the plurality of hydrogel voxels and dissociates the sacrificial support matrix to leave the free-standing structure.

11 . The method of claim 1 , wherein after depositing the plurality of hydrogel voxels within the sacrificial support matrix, the plurality of hydrogel voxels swell and have an average diameter of from 300 to 1200 micrometers.

12 . The method of claim 1 , wherein each of the hydrogel voxels comprises a hydrogel composition comprising an aqueous solution of a hydrogel and at least one cell.

13 . The method of claim 1 , wherein a storage modulus G′ and a loss modulus G″ of the sacrificial support matrix are not reduced for a period of 200 seconds after applying an instant shear strain of 1000% for 1 second to the sacrificial support matrix.

14 . The method of claim 1 , wherein the sacrificial support matrix comprises fragmented gelatin microparticles and calcium ions pre-dissolved in the fragmented gelatin microparticles.

15 . A method of assembling hydrogel voxels to form a structure, the method comprising:

depositing a plurality of hydrogel voxels within a sacrificial support matrix, wherein the hydrogel voxels are spherical and the plurality of hydrogel voxels are deposited at a center-to-center distance between the hydrogel voxels of from 0.8 to 1.7 times the average diameter of the plurality of hydrogel voxels; and

cross-linking the plurality of hydrogel voxels to form the structure to comprise a one dimensional line, a two dimensional array, or a free-standing three dimensional lattice; wherein each of the spherical hydrogel voxels in the structure are interconnected and are distinguishable from each other when viewed through optical microscopy.

16 . The method of claim 15 , wherein:

the sacrificial support matrix is self-healing; and

a storage modulus G′ and a loss modulus G″ of the sacrificial support matrix are not reduced for a period 200 seconds after applying an instant shear strain of 1000% for 1 second to the sacrificial support matrix.

17 . The method of claim 15 , wherein the sacrificial support matrix is aqueous.

Assignments (2)
CONFIRMATORY LICENSE Recorded Feb 12, 2025
From: UNIVERSITY OF VIRGINIA
To: NATIONAL SCIENCE FOUNDATION
Reel/Frame 070189/0057 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 22, 2023
From: CAI, LIHENG; ZHU, JINCHANG
To: UNIVERSITY OF VIRGINIA
Reel/Frame 064664/0833 →
Continuity (2)
Provisional Application 63040881 · Jun 18, 2020
Related Publication 20230271377A1 · Aug 31, 2023
References Cited (68)
US 9168328B2 · Sun · 2015 [cited by examiner]
US 10150258B2 · Feinberg · 2018 [cited by examiner]
US 11192292B2 · Fernandez-Nieves · 2021 [cited by examiner]
US 11931969B2 · Hudson · 2024 [cited by examiner]
US 20170199507A1 · Murphy et al. · 2017 [cited by applicant]
US 20170307598A1 · Skardal · 2017 [cited by examiner]
US 20170361534A1 · Fernandez-Nieves et al. · 2017 [cited by applicant]
US 20190275746A1 · Huang et al. · 2019 [cited by applicant]
US 20190307923A1 · Gatenholm et al. · 2019 [cited by applicant]
WO 2019199971A1 · 2019 [cited by applicant]
WO 2021003270A1 · 2021 [cited by applicant]
S. V. Murphy et al.; “3D bioprinting of tissues and organs”; Nature Biotechnology, vol. 32, No. 8; Aug. 2014; pp. 773-785. [cited by applicant]
J. P. Vacanti et al.; Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation; The Lancet, Molecular medicine, Jul. 1999, 354; pp. 32-34. [cited by applicant]
Ali Khademhosseini et al.; “Microscale Technologies for Tissue Engineering” Advances in Tissue Engineering; 2008, 103, pp. 349-369. [cited by applicant]
R. L. Truby et al.; “Printing soft matter in three dimensions”; Nature; Dec. 15, 2016; vol. 540, pp. 371-378. [cited by applicant]
I. T. Ozbolat et al.; “Current advances and future perspectives in extrusion-based bioprinting”; Biomaterials, 2016, 76, 321-343. [cited by applicant]
T. Bhattacharjee et al.; “Writing in the granular gel medium”; Science Advances, Sep. 25, 2015, pp. 1-6. [cited by applicant]
Thomas J. Hinton et al.; “Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels”; Science Advances, Oct. 23, 2015; pp. 1-10. [cited by applicant]
A. Lee et al.; “3D bioprinting of collagen to rebuild components of the human heart”; Science, Aug. 2, 2019; 365, pp. 482-487. [cited by applicant]
Yu Shrike Zhang et al.; “Advances in engineering hydrogels”; Science, 356, 500 May 5, 201. [cited by applicant]
Mark W. Tibbitt, et al.; “Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture”; Biotechnology and Bioengineering; vol. 103, No. 4, Jul. 1, 2009, pp. 655-633. [cited by applicant]
Jason A. Burdick et al.; “Hyaluronic Acid Hydrogels for Biomedical Applications”; Adv. Mater., 2011, Mar. 25; 2023, (12). [cited by applicant]
Jeanie L. Drury et al.; “Hydrogels for tissue engineering: scaffold design variables and applications”; Biomaterials, 2003, 24, 4337-4351. [cited by applicant]
Kuen Yong Lee et al.; “Hydrogels for Tissue Engineering”; Chemical Reviews; vol. 101, No. 7, Jul. 2001; 1869-1879. [cited by applicant]
David B. Kolesky et al.; “3d Bioprinting of Vascularized, Heterogeneous Cell-Laden Tissue Constructs”; Advanced Materials; 2014, 26, 3124-3130. [cited by applicant]
Mark A. Skylar-Scott et al.; “Voxelated soft matter via multimaterial multinozzle 3D printing”; Nature, vol. 575; Nov. 14, 2019; 330-335. [cited by applicant]
Florence G. Downs et al.; “Multi-responsive hydrogel structures from patterned droplet networks”; Nature Chemistry, vol. 12, Apr. 2020; pp. 363-371. [cited by applicant]
Joseph T. Muth et al.; “Embedded 3D Printing of Strain Sensors within Highly Stretchable Elastomers”; Advanced Materials, 2014, 26, 6307-6312. [cited by applicant]
Stefanie Utech, et. al.; “Microfluidic generation of monodisperse, structurally homogeneous alginate microgels for cell encapsulation and 3D cell culture”; , Adv. Healthc. Mater., Aug. 5, 2015, 4, (11) 1628-1633. [cited by applicant]
Abigail K. Grosskopf et al.; “Viscoplastic Matrix Materials for Embedded 3D Printing”; ACS Applied Materials & Interfaces; 2018, 10, 23353-23361. [cited by applicant]
Christopher B. Highley et al.; Direct 3D Printing of Shear-Thinning Hydrogels into Self-Healing Hydrogels; Advanced Materials, 2015, 27, 5075-5079. [cited by applicant]
Manuel Schaffner et al.; 3D printing of bacterial into functional complex materials; Science Advances, 2017, 3, Dec. 1, 2017; pp. 1-9. [cited by applicant]
Pragya Mittal et al.; “Codon usage influences fitness through RNA toxicity”; Proc. Natl. Acad. Sci. U. S. A., Aug. 21, 2018, vol. 115, No. 34, pp. 8639-8644. [cited by applicant]
Takanori Takebe et al.; “Organoids by design”; Science, 364, Jun. 7, 2019, pp. 956-959. [cited by applicant]
Zev J. Gartner et al.; “Programmed assembly of 3-dimensional microtissues with defined cellular connectivity”; Proc. Natl. Acad. Sci. U. S. A., Mar. 24, 2009, vol. 106, No. 12., pp. 4606-4610. [cited by applicant]
M. M. Stanton et al.; “Bioprinting of 3D hydrogels”; Lab Chip, Royal Society of Chemistry; 2015, 15, pp. 3111-3115. [cited by applicant]
Shia-Yen Teh et al.; “Droplet microfluidics”; Lab Chip, The Royal Society of Chemistry; 2008, 8, 198-220. [cited by applicant]
Mira T. Guo, et al.; “Droplet microfluidics for high-throughput biological assays”; Lab Chip, The Royal Society of Chemistry, 2012, 12, pp. 2146-2155. [cited by applicant]
Wen Li et al.‘Microfludic fabrication of microparticles for biomedical applications’ Chem. Soc. Rev., Royal Society of Chemistry, 2018, 47, pp. 5646-5683. [cited by applicant]
Hyun-Wook Kang et al.; “A 3D bioprinting system to produce human-scale tissue constructs with structural integrity” I Nature Biotechnology, vol. 34, No. 3, Mar. 2016, pp. 312-319. [cited by applicant]
Samuel Clark Ligon et al.; “Polymers for 3D Printing and Customized Additive Manufacturing”; Chemical Reviews, 117, 10212-10290 (2017). [cited by applicant]
Shifeng Nian et al.; Three-Dimensional Printable, Extremely Soft, Stretchable, and Reversible Elastomers from Molecular Architecture-Directed Assembly. Chemical Materials 2021, 33, pp. 2436-2445. [cited by applicant]
Ferry P.W. Melchels et al.; “A review on stereolithography and its applications in biomedical engineering”; Biomaterials 31, 6121-6130 (2010). [cited by applicant]
Brian Derby; “Inkjet Printing of Functional and Structural Materials: Fluid Property Requirements, Feature Stability, and Resolution”; Annu Rev. Mater. Res. 40, 395-414 (2010). [cited by applicant]
Mark A. Skylar-Scott et al.; “Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels”; Science Advances, 5 Sep. 6, 2019 pp. 1-13. [cited by applicant]
Q. D. Nguyen et al.; Measuring the Flow Properties of Yield Stress Fluids. Annu. Rev. Fluid Mech. 24, 47-88 (1992). [cited by applicant]
Arif Z. Nelson et al.; “Embedded droplet printing in yield-stress fluids”;Proc. Natl. Acad. Sci. vol. 117, No. 11; Mar. 17, 2020, pp. 5671-5679. [cited by applicant]
Andrew C. Daly, et al.; “3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels”; Nature Communications, 12, 1-13 (2021). [cited by applicant]
Jianyu Li et al.; “Designing hydrogels for controlled drug delivery”; . Nat. Rev. Mater. 1, Dec. 1-18, 2016. [cited by applicant]
Jos Malda et al.; “25th Anniversary Article: Engineering Hydrogels for Biofabrication”; Advanced Materials 25, 5011-5028 (2013). [cited by applicant]
Naomi Paxton et al.; “Proposal to assess printability of bioinks for extrusion-based bioprinting and evaluation of rheological properties governing bioprintability”; Biofabrication. 9 (2017). [cited by applicant]
Johnson H.Y. Chung et al.; “Bio-ink properties and printability for extrusion printing living cells”; Biomaterials Science, 1, 763-773 (2013); The Royal Society of Chemistry. [cited by applicant]
Jordan S. Miller, et al.; “Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues”; Nature Materials, vol. 11, Sep. 2012, 768-774. [cited by applicant]
David B. Kolesky et al.; “Three-dimensional bioprinting of thick vascularized tissues”; Proc. Natl. Acad. Sci. U. S. A. Volume 113, No. 12, 3179-3184, Mar. 22, 2016. [cited by applicant]
Bagrat Grigoryan et al.; “Multivascular networks and functional intravascular topologies within biocompatible hydrogels”; Science. 364, 458-464, May 3, 2019; pp. 1-7. [cited by applicant]
Franklin Lim et al.; Microencapsulated Islets as Bioartificial Endocrine Pancreas. Science, vol. 210, No. 21, pp. 908-910, Nov. 1980. [cited by applicant]
Ronit Satchi-Fainaro et al.; “Polymer Therapeutics for Cancer: Current Status and Future Challenges”; Advances in Polymer Science (2006), vol. 193, pp. 1-65. [cited by applicant]
Kuen Yong Lee et al.; “Alginate: Properties and biomedical applications”; Prog. Polym. Sci. 2012, Jan. 37, 106-126. [cited by applicant]
He Wen et al.; Characterization of Human Sclera Barrier Properties for Transscleral Delivery of Bevacizumab and Ranibizumab. J. Pharm. Sci. Mar. 2013; 102 (3), 892-903. [cited by applicant]
Gabriel Villar et al.; “A Tissue-Like Printed Material”; Science, vol. 340, pp. 48-Apr. 5, 53, 2013. [cited by applicant]
Gabriel Villar et al.; “Formation of droplet networks that function in aqueous environments”. Nat. Nanotechnol. 6, 803-808 (2011). [cited by applicant]
Lingzhi Cai et al.; “An unbounded approach to microfluidics using the Rayleigh-Plateau instability of viscous threads directly drawn in a bath”; . Proc. Natl. Acad. Sci. U. S. A. Nov. 12, 2019, vol. 116, No. 46, pp. 229… [cited by applicant]
Hing Jii Mea et al.; “On-demand modulation of 3D-printed elastomers using programmable droplet inclusions”; Proc. Natl. Acad. Sci. U. S. A. 117, No. 26, pp. 14790-14797, Jun. 30, 2020. [cited by applicant]
Bugra Ayan et al.; “Aspiration-assisted bioprinting for precise positioning of biologics”; Science Advances, Mar. 6, 2020, 6, 1-17. [cited by applicant]
Xuewen Du et al.; “Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials”; Chemical Reviews, 115, 13165-13307 (2015). [cited by applicant]
Eric A. Appel et al.; “Supramolecular polymeric hydrogels”; Chem. Soc. Rev. 41, 6195-6214 (2012); The Royal Society of Chemistry. [cited by applicant]
Michael Rubinstein et al.; “Polymer Physics”; Oxford University Press, Oxford, UK, 2003. [cited by applicant]
International Search Report and Written Opinion issued Nov. 5, 2021 in related International Application No. PCT/US2021/037811 filed Jun. 17, 2021. [cited by applicant]