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US-12624714-B2 - Three-dimensional shaped microparticles having protected regions for holding cells and uses thereof

US12624714B2US 12624714 B2US12624714 B2US 12624714B2US-12624714-B2

Abstract

A method of forming three-dimensional shaped microparticles in a microfluidic device includes flowing a mixture of a monomer and photoinitiator in a microfluidic channel having a plurality of pillars disposed therein to define a flow stream having a pre-defined shape and temporarily stopping the same. One or more portions of the flow stream are polymerized by passing polymerizing light through one or more masks and onto the flow stream, the polymerization process forming a plurality of three-dimensional shaped microparticles. The three-dimensional shape of the microparticle may be geometrically complex by using non-rectangular 2D orthogonal shapes for the flow and/or masked light source. The microparticles may include protected regions on which cells can be adhered to and protected from shear forces. The flow stream is restarted to flush out the newly formed microparticles and prepare the device for the next cycle of particle formation.

Inventors

  • Dino Di Carlo
  • Chueh-Yu Wu

Assignees

  • THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date
20260512
Application Date
20210607

Claims (15)

  1. 1 . A three-dimensional shaped microparticle having a longest dimension of less than about 200 μm and comprising a crosslinked hydrogel material, the three-dimensional shaped microparticle having a protected region formed on the three-dimensional shaped microparticle, wherein the protected region comprises a cellular binding moiety.
  2. 2 . The three-dimensional shaped microparticle of claim 1 , wherein the cellular binding moiety comprises one or more biotinylated regions.
  3. 3 . The three-dimensional shaped microparticle of claim 1 , wherein the three-dimensional shaped microparticle is asymmetrically shaped.
  4. 4 . The three-dimensional shaped microparticle of claim 1 , wherein the protected region comprises a notch.
  5. 5 . The three-dimensional shaped microparticle of claim 1 , wherein the protected region comprises a groove.
  6. 6 . The three-dimensional shaped microparticle of claim 1 , wherein the protected region comprises a valley.
  7. 7 . The three-dimensional shaped microparticle of claim 1 , wherein the protected region comprises a void or hole.
  8. 8 . The three-dimensional shaped microparticle of claim 1 , wherein the protected region comprises a flexible region.
  9. 9 . The three-dimensional shaped microparticle of claim 1 , further comprising one or more cells adhered to the three-dimensional shaped microparticle in the protected region.
  10. 10 . The three-dimensional shaped microparticle of claim 1 , further comprising a fluorescent species bound directly or indirectly to the three-dimensional shaped microparticle in the protected region.
  11. 11 . A method of using three-dimensional shaped microparticles comprising: providing a plurality of three-dimensional shaped microparticles having a longest dimension of less than about 200 μm and comprising a crosslinked polymer material, the three-dimensional shaped microparticles each having a protected region formed on the three-dimensional shaped microparticle; binding an adherent species to the three-dimensional shaped microparticles in the protected region including a fluorescent species bound directly or indirectly to the three-dimensional shaped microparticles; and subjecting the three-dimensional shaped microparticles to fluorescent imaging and/or detection.
  12. 12 . The method of claim 11 , wherein the adherent species comprises one or more cells.
  13. 13 . The method of claim 11 , wherein the adherent species comprises a marker or target.
  14. 14 . The method of claim 11 , further comprising imaging the three-dimensional shaped microparticles.
  15. 15 . The method of claim 11 , wherein subjecting the three-dimensional shaped microparticles to fluorescent imaging and/or detection comprises subjecting the three-dimensional shaped microparticles to flow cytometry.

Description

RELATED APPLICATION This Application is a continuation of U.S. application Ser. No. 15/763,765, now issued as U.S. Pat. No. 11,060,541, which is a U.S. National Stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2016/055011, filed Sep. 30, 2016, which claims priority to U.S. Provisional Patent Application No. 62/236,782 filed on Oct. 2, 2015, which is hereby incorporated by reference. Priority is claimed pursuant to 35 U.S.C. §§ 119, 371 and any other applicable statute. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Grant No. 1307550 from the National Science Foundation. The Government has certain rights in the invention. FIELD OF THE INVENTION The technical field generally relates to devices and methods that generate and utilize shaped three-dimensional (3D) microparticles with complex shapes, topologies, and material composition or properties. BACKGROUND Shaped microparticles with complex curvature, holes, and surface or composite heterogeneity enable unique applications in biomaterials, self-assembly, photonic crystals, and encoding. Microparticles with shape control and multi-functionality have been engineered to enable advanced technologies, including self-aligned flow cytometry and encoded-microparticle-based multiplexed detection. Drug delivery and cell uptake are known to be shape-dependent while assembly of tissue mimetic systems could benefit from 3D interlocking shapes and spatially engineered combinations of constituents. Moreover, for scaling up production of therapeutic compounds in cell bioreactors, particles shaped with pores or voids could enhance cell growth, achieving high surface area while protecting cells from high fluid shear stress. Additive layer-by-layer fabrication processes, such as 3D printing, have allowed for the manufacturing of complex three-dimensional and software-designed objects with holes or voids from a variety of materials, but mass-production of microscale objects or features are difficult to achieve. Optical lithography approaches that leverage microfluidic delivery of precursor photopolymer streams, such as stop-flow lithography (SFL), possess many of the advantages of 3D printing technologies, and have expanded fabrication to microscale objects (i.e. particles) with a relatively high manufacturing rate that is on an upward trajectory. In SFL, a flowing stream of oligomer is stopped prior to polymerizing an array of particles into it. See Dhananjay et al., Stop-flow lithography in a microfluidic device, Lab Chip, 7, 818-828 (2007). Although recent work has expanded optofluidic lithography approaches, initially limited to extrusions of 2D patterns, to a larger set of 3D shapes, the particle shapes that are achievable remains limited compared to 3D printers. Structure-induced flow deformation has been used to shape co-flows of UV-crosslinked polymer precursor streams and inert streams to create three-dimensional fibers and millimeter-scale particle shapes following flood or masked UV exposure subsequent to flow stoppage respectively. The flow deformation can be precisely predicted within a laminar flow regime, Reynolds number, Re<˜2000, where Re is the ratio of inertial to viscous effects in the channel. More recently, a process for the fabrication of shaped microfibers has been developed that combines software-enable inertial microfluidics and photopolymerization. See Nunes et al., Fabricating Shaped Microfibers with Inertial Microfluidics, Advanced Materials, Vol. 26, 3712-3717 (2014). This approach was also used to predict and design shaped fibers. When transitioning from fibers to shaped particles the velocity gradients within the channel require flow stoppage before polymerization, since the timescale for polymerization is also comparable or longer than the timescale of the flow, and the fluid will slip different amounts before solidifying, leading to elongated or distorted shapes. However, a significant challenge for inertial-microfluidics-based lithography is to shrink down the size of fabricated particles to the micrometer scale. Specifically, higher viscosity solutions of the polymer precursors that must be shaped at higher Reynolds number than standard stop-flow lithography lead to pressures that significantly flex the fluidic system leading to long capacitive times and the inability to quickly stop the shaped flow before it transits out of the channel. Prior attempts to address this problem while still achieving a high Reynolds number necessary to shape a flow and stop flow in a reasonable time was to enlarge the channel size. However, this led to particles that were fabricated at the millimeter scale and required a longer flow development and settling time due to longer timescales for viscous dissipation of fluid momentum. Methods are needed to achieve the production of shaped particles having a much smaller size. SUMMARY In one embodiment, a method of forming