Preparation of alginate hydrogel microspheres with Microdroplet/Microsphere Generator

1.Reagents preparation:

(1) 2 M CaCl2 solution:
2.2197 g of CaCl2 (MCaCl2=110.984 g/mol) is dissolved into pure water. Fix the volume to 10 mL.
(2) 2% (wt%) sodium alginate solution:
0.2 g of sodium alginate powder and 9.8 g of pure water are mixed homogeneously and heated to 60℃ to be dissolved completely.
(3) 2 M NaOH solution:
1.6 g of NaOH (MNaOH= 39.997 g/moL) is dissolved into pure water. Fix the volume to 20 mL.
(4) 240 mM Ca-EDTA solution (pH=7.2):
1.2 mL of 2M CaCl2, 4.8 mL of 0.5 M EDTA and 5 mL of pure water are mixed homogeneously. Add 2M NaOH into the solution to adjust pH to 7.2, finally fix the volume to 10 mL.
[Note] The volume of NaOH added in the following table is for reference only. You should determine the volume of NaOH solution based on your own measurements.

(5) Preparation of the dispersed phase (1% sodium alginate, 120 mM Ca-EDTA):
A mixture of an equal volume of 240 mM Ca-EDTA solution and 2% sodium alginate solution is vortexed homogeneously to obtain the dispersed phase. Filter the solution with a 0.22 μm syringe filter.
(6) Preparation of 1% acetic acid solution in oil:
5 μL of glacial acetic acid and 495 μL of Droplet Generation Oil are mixed homogeneously with vortex to obtain a 1% acetic acid solution.

2.Preparation of microdroplets:

(1) Microfluidic devices set-up
For a more detailed installation and connection of Microdroplet/Microsphere Generator, refer to the User Guide V.1.0 Of Microdroplet/Microsphere Generator, section 2. Installation and connection of Microdroplet/Microsphere Generator. The connection is as follows (steps ③-⑦ are shown in the figure below):
① Connect the devices with PU tubing in the following order:
The air compressor — the air source processing device — the Microdroplet/Microsphere Generator
② Connect the device to the power supply and PC, respectively.
③ Connect A0 (the pressure channel 1 outlet) and A1 (the continuous phase reservoir), B0 (the pressure channel 2 outlet) and B1 (the dispersed phase reservoir) with PU tubing, respectively;
④ Connect A1 (the continuous phase reservoir) and A2 (the flow sensor channel 1), B1 (the dispersed phase reservoir) and B2 (the flow sensor channel 2) with PEEK tubing and 1/4-28 UNF, respectively;
⑤ Connect A2 (the flow sensor channel 1) and A3 (the continuous phase inlet of the PDMS chip), B2 (the flow sensor channel 2) and B3 (the dispersed phase inlet of the PDMS chip) with PEEK tubing and 1/4-28 UNF, respectively;
⑥ C is a combination of a PDMS standard chip and a chip holder, which is sealed by 6 silicone connector seals;
⑦ The PEEK tubing is inserted into the chip & chip holder outlet (D) for emulsion output.

(2) Installing FluidicLabSuite software and adding equipment:
Refer to the section 3.1 Installation of FluidicLabSuite Software in the User Guide V.1.0 Of Microdroplet/Microsphere Generator.
(3) Preparation of alginate microdroplets:
① 5 mL of Droplet Generation Oil and 1 mL aqueous solution into the corresponding reservoirs respectively:
Reservoir 1 (controlled by Pressure Channel 1): Droplet Generation Oil;
Reservoir 2 (controlled by Pressure Channel 2): the dispersed phase.
② For the use of the integrated camera and flow sensors, refer to the section 3.2 Equipment Addition of FluidicLabSuite Software in the User Guide V.1.0 Of Microdroplet/Microsphere Generator.
③ Turn on the air compressor and air source treatment device.
④ A centrifuge tube is placed at the emulsion outlet to collect the pre-waste liquid .
⑤ Set channel 1 pressure and channel 2 pressure by FluidicLabSuite control to exhaust the air in the PEEK tubing and the microchannels of chip;
Tips: Due to its high viscosity, we kindly suggest you set a higher pressure in the dispersed phases than in the continuous phase. For example,
Pressure Channel 1 (controlling the oil phase): 150 mbar;
Pressure Channel 2 (controlling the aqueous phase): 900 mbar.
⑥ After the PEEK tubing and the microchannels of chip are filled with liquid, switch the control mode from pressure control to flow rate control. The flow rates of channels 1 and 2 are set as 15 and 8 μL/min, respectively.
⑦ The smooth output of target flow rate can be quickly achieved through adjusting the feedback value.
⑧ After a few minutes, collect a drop of the emulsion onto a hydrophobic substrate base, and ensure the uniformity of droplet size with an optical microscope.
⑨ After that, collect the emulsion into a 1.5 mL centrifuge tube containing cross-linking reagent solution.
⑩ Collect the emulsion for 20 minutes, and 20 minutes of cross-linking time with gently vibration is allowed for cross-linking gelation.

3. Aftertreatments:

① Remove the oil phase (at the bottom of the tube) with a pipette.
② Add 2x volume of Drop-Surf Demulsifier to the microspheres. For every 100 μL microspheres, add 200 μL Drop-Surf Demulsifier.
③ Vortex the mixture for 20 seconds, then centrifuge it at 1000 rpm for 30 seconds. Remove the demulsifier at the bottom of the container.
④ Repeat steps ② and ③ 1~2 times, until all white microspheres on the top of the container change into transparent.
⑤ Add 3x volume of PBS buffer to the microspheres. For every 100 μL microspheres, add 300 μL PBS buffer.
⑥ Vortex the mixture for 20 seconds, then centrifuge 1000 rpm for 120 seconds. Remove PBS buffer at the upper of the container.
⑦ Repeat steps ⑤ and ⑥ 1~2 times.
⑧ Finally, the solidified alginate hydrogel microspheres are obtained and dispersed in PBS buffer.

4.Cleaning of Microdroplets/Microsphere of Generator:

The PEEK tubing, the flow sensors and the microchannels of the chip should be cleaned after the experiment. Otherwise, the reagents remaining in the flow channels could possibly damage the flow sensors and clog the microchannels of the chip. For details, please refer to the Instruction Card of Microdroplet/Microsphere Generator.

The alginate microdroplets have an average particle size of 111.04 μm, and have extremely high monodispersity (coefficient of variation: CV=2.01%). The picture and particle size distribution are shown as follow:

After cross-linking gelation, the average particle size of alginate hydrogel microspheres is 107.86 μm, and have extremely high monodispersity (CV=3.47%). The picture and particle size distribution are shown as follow:

Key points of the experiment

1.The cross-linking reagent solution (1% acetic acid in oil phase) is acidic (pH<5).
2.In this application note, the dispersed phase should not contain free Ca2+. For example, a medium containing free Ca2+ is not applicable for use in the dispersed phase.
3.The viscosity of the dispersed phase is higher than that of the continuous phase, thus, the pressure of the dispersed phase is higher than that of the continuous in the same flow rate. Due to its high viscosity, we kindly suggest you set a higher pressure in the dispersed phase than in the continuous phase.
4.Due to its low concentration, the final obtained microspheres may not be observed when dispersed in PBS buffer. You can add a small amount of free Ca2+ into the PBS buffer, or disperse the microspheres in water.

References

[1] Ching S. H., et al. Alginate gel particles–a review of production techniques and physical properties, Crit. Rev. Food Sci., 57, 1133-1152 (2015).

[2] Wang H , et al. The use of micro- and nanospheres as functional components for bone tissue regeneration , Tissue Eng. Part B Rev., 18, 24-39 (2012).

[3] Wan. J, Microfluidic-Based Synthesis of hydrogel particles for cell microencapsulation and cell-based drug delivery, Polymers, 4, 1084-1108 (2012).

[4] Rowley J.A., et al. Alginate hydrogels as synthetic extracellular matrix materials., Biomaterials, 20 , 45-53 (1999).

[5] Bhatia, S. N., et al. Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J. 13, 1883–1900 (1999).

[6] Zhang H., et al. Exploring microfluidic routes to microgels of biological polymers, Macromol. Rapid Comm.，28, 527-538 (2007).

[7] Utech S., et al. Microfluidic generation of monodisperse, structurally homogeneous alginate microgels for cell encapsulation and 3D cell culture，Adv. Heal. Mater., 4, 1628-1633 (2015).