Self‐Orienting Hydrogel Micro‐Buckets as Novel Cell Carriers

Abstract Hydrogel microparticles are important in materials engineering, but their applications remain limited owing to the difficulties associated with their manipulation. Herein, we report the self‐orientation of crescent‐shaped hydrogel microparticles and elucidate its mechanism. Additionally, the microparticles were used, for the first time, as micro‐buckets to carry living cells. In aqueous solution, the microparticles spontaneously rotated to a preferred orientation with the cavity facing up. We developed a geometric model that explains the self‐orienting behavior of crescent‐shaped particles by minimizing the potential energy of this specific morphology. Finally, we selectively modified the particles’ cavities with RGD peptide and exploited their preferred orientation to load them with living cells. Cells could adhere, proliferate, and be transported and released in vitro. These micro‐buckets hold a great potential for applications in smart materials, cell therapy, and biological engineering.


Characterization
Microfluidic experiments were performed on Axio Observer A1 inverted microscope (Zeiss, ×10 air objective) with a Zyla 5.5 sCMOS camera (Andor) at 50 fps. Size distributions, selforientation and cell loading were obtained by confocal laser scanning microscopy (CLSM, Zeiss LSM 710, ×10 and ×20 air objectives and a ×40 oil immersion objective). Mercury-arc light source (HXP 120 V,120 W) with a band pass filter 300-400 nm (peak intensity at 365 nm) was used to supply UV light. Particles morphology was checked by JEOL 6010 Scanning Electron Microscope (SEM) after freeze-drying in liquid nitrogen. Cell transport, proliferation, release and viability were obtained by Andor Inverted Microscope (Zeiss, ×20 air objective).

Fabrication of microfluidic device
The device was fabricated by PDMS (Dow Corning, Sylgard 184 elastomer kit) using soft lithography. The non-planar chip was bonded by two pieces of PDMS after oxygen plasma treatment. The channel height is about 300 μm, width is about 500 μm. The width of nozzles is about 40 μm. The device was connected to individual syringe pumps (Harvard Apparatus, 11PicoPlus) via tube (PEEK® 0.5/1.6 mm inner/outer diameter).

Formation of the crescent-shaped hydrogel microparticles
All solutions were prepared using demineralized water. Dextran (28.6% W/W) and RGD peptide (4 mg/mL) were dissolved in HEPES buffer (50 mM, pH=7) solution. PEGDA (28.6% W/W) and LAP (10 mg/mL) were dissolved in demineralized water. Surfactant (span 80, 3% w/w) was dissolved in hexadecane. These three phases were independently injected into the microfluidic device by syringe pumps. As shown in Figure S1a and S1b, Dextran and polyethylene glycol diacrylate (PEGDA) with photo initiator were used as inner phase and middle phase, respectively. Co-flows of dextran and PEGDA break up into highly monodisperse phase separated ATPS droplets in hexadecane flow. The volumetric flow rate of dextran phase was 0.03~0.1 μL/min and that of PEGDA phase was 0.1~0.12 μL/min. The volumetric flow rate of hexadecane was 12~15 μL/min.
Fluorescein methacrylate was added into PEGDA phase, and the fluorescence images ( Figure S1c and S1d) indicate that PEGDA forms the outside (the crescent part), and dextran forms the inside of the particles (the spherical part). The hydrogel particles were cross-linked by on chip UV irradiation and collected into vials. Firstly, hexadecane in the vial was removed by pipetting. Secondly, the particles were washed with THF (3 times) and water (5 times) to remove surfactant and dextran. Lastly, the hydrogel particles were washed with ethanol (3 times). Then the hydrogel particles were prepared for the bio experiment after

Measuring of ds and dl
From the bright-field microscopy image of the crescent-shaped hydrogel microparticle, the rotation of particle causes an ellipse (white dash, Figure S2). Since, the short diameter (ds, red line) and the long diameter (dl, white line) of the ellipse can be measured by the software.

Modelling
We established a geometrical model (as shown in Figure S3a) to calculate the center of mass and potential energy of the crescent-shaped hydrogel microparticle at different rotation angle.
We assume that the crescent-shaped hydrogel microparticles are formed by two spheres with two centers (2 and 3) and radiuses (R and r). A two-dimensional coordinate system was established based on the side view of the particle. The X axis is the tangent line of the big sphere of the particle, while the Y axis is along the connection between the centers of the two spheres. Therefore, the bottom point of the particle is placed at the origin of the coordinate system. Meanwhile, the rotation angle of particle is θ. From the top view, the rotation angle can be calculated as cos(θ)=ds/dl (short diameter divided by long diameter). The specific coordinates of Y0-Y6 in the side view are given in Figure S3b. Volume of the crescent-shaped hydrogel microparticle (Equation 1): The mass center of the particle (G, θ=0º) can be estimated by the integration along the Y axis: Height of mass center (Equation 2): Figure S4. The height of mass center changes for various cavity sized particles at different rotation angle.
When the particle rotates an angle of θ around the Y2 point, the center of mass G will raise and the corresponding potential energy will also increase as: Potential energy change after rotation θ (Equation 3): The density of PEGDA hydrogel is measured around 1.225 g/mL by Archimedes' immersion method.
7 Figure S5. Potential energy change of particle (Fd: Fp=2: 3) with different (a) radius (R) and (b) density.   Figure S8, the RGD peptide in PEGDA phase was much more than that in dextran phase and the partition coefficient of RGD peptide in ATPS (Kp) was calculated about 2.4. It indicates that the RGD peptide prefers PEGDA phase than dextran phase. Therefore, during the cross-linking reaction, RGD peptide can diffuse from dextran phase to PEGDA phase and be immobilized in the interior of the PEGDA hydrogel particles through the thiol-ene click reaction. Figure S8. 1 H NMR of RGD peptide in ATPS of PEGDA phase (a) and dextran phase (b).
Cell staining. NIH/3T3 Cells were colored by CellTracker TM red CMTPX (Molecular Probes, C34552). Cells were detached from the T-flasks by 2 mL 0.25% Trypsin/2.21 mM EDTA and placed into 8 mL growth medium, containing 5 mL DMEM supplemented with 10% (v/v) NCS and 0.5% (v/v) Pen-Strep. 10 µL CellTracker™ Red CMTPX DMSO solution was also added into the growth medium, and cells are incubated at 37°C in 5%/95% CO2/Air atmosphere. After 45 minutes, growth medium was removed by centrifugation.
Cell Culture. Cells were cultured in 25 cm 2 tissue culture flasks (T-flasks) and immersed in 5 mL growth medium. The crescent-shaped hydrogel microparticles were sterilized with 70% ethanol and washed with DPBS (3 times) and growth medium (3 times), respectively. Then particles with growth medium were added into sterile 8-well cell culture plate. Cells were trypsinized, stained and centrifuged to remove the growth medium. Cells were resuspended in new growth medium (around 5*10 4 cells/mL) and added in the 8-well cell culture plate containing a layer of crescent-shaped hydrogel particles (as shown in Figure S9). The 8-well cell culture plate was placed in incubator.    14 Figure S14. Cell proliferation and release in the hydrogel microparticle. Scale bar 50 μm.
Cell proliferation was observed with time increase. After 3 days, cells migrated from cavity to outside. After 7 days, a few cells show up on the glass slide of culture plate, most cells still stayed in the hydrogel microparticle. After 13 days, cells were completely released from the microparticles by gentle agitation of the particle-cell suspensions for around 20 to 30 min.
After cell release, the crescent hydrogel microparticle reoriented to the cavity facing up. Figure S15. Cell viability of the cells after being released from the gel buckets. The cells were cultured from 1 day to 4 days after release, the cell viability was around 93%. Calcein AM (green) and propidium iodide (PI, red) were used to check cell live and death.