Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology

In this protocol, we demonstrate an effective procedure for fabricating PLGA-based highly-open porous microspheres, which offer several advantages like convenience of packaging cells, improved cell retention, and minimal invasiveness. The resultant monodispersed PLGA-based highly-open porous microspheres possessed particle sizes of approximately 400 micrometers and open pores of approximately 50 micrometers with interconnecting windows for improved cell retention efficacy. These minimally invasive microcarriers may support a platform of an intricate 3D tumor model for drug screening and facile tools to construct cell laden micro tissues for tissue regeneration.

This approach is quite facile, as the microfluidic device can be assembled from polyvinyl chloride tubing, a glass capillary, and a needle. Demonstrating the procedure will be Sheng-Chang Luo, a doctoral student in the laboratory. Begin by constructing a co-flow microfluidic generator using a glass capillary, polyvinyl chloride tubes, and a 26 gauge dispensing needle.

Then connect the glass capillary to the end of the polyvinyl chloride tube, and pierce the connection between the two with a needle. For the droplet generation, place the other end of the polyvinyl chloride tube in the continuous phase, and cure with the ultraviolet glue to seal the gaps at the joint. Now take a 50 milliliter syringe to load the continuous phase, and a five milliliter syringe for emulsion formation.

Then set the flow rates of the continuous phase at two milliliters per minute, and the dispersion phases at 0.08 milliliters per minute. Place a 500 milliliter beaker in an ice bath, and fill it with a pre-cooled polyvinyl alcohol aqueous solution. To prepare the emulsion, immediately decant the aqueous gelatin solution into the DCM solution of PLGA, and emulsify using the ultrasonic device.

Then adjust the ultrasonic power to 400 watts, and the total processing time to 90 seconds, and constantly change the probe's position manually. Stabilize the resulted emulsion for syringe suction and droplet generation in approximately 20 minutes. After ultrasonic treatment, rapidly load the prepared emulsion in the five milliliter syringe in the microfluidic platform and introduce the unstable water oil emulsion into the microfluidic device as the discontinuous phase.

Simultaneously, use the aqueous solution of polyvinyl alcohol as the continuous phase. After injecting proper portions of the emulsion into the microfluidic device, collect the microsphere containing the emulsion at the bottom of the collecting beaker and place the sample at four degrees Celsius overnight for further stabilization. Normalize the stored microspheres to room temperature and eliminate the residual DCM by stirring with a glass rod for one hour at 60 RPM.

Then, carefully decant the collecting phase in a 500 milliliter beaker and wash off the residual polyvinyl alcohol from the surface of porous microspheres with deionized water thrice. Dissolve the gelatin wrapped in the PLGA backbone by placing the PLGA microspheres in the water bath at 37 degrees Celsius for 30 minutes. Then remove the residual gelatin by rinsing the resultant microspheres with the deionized water twice, and pre-cool for lyophilization at minus 80 degrees Celsius for 24 hours.

The PLGA-based highly-open porous microspheres displayed sizes of 350 to 500 micrometer with an arbitrary sized pore of 10 to 100 micrometers, and interconnected windows, which could facilitate high efficiency in oxygen metabolic waste transport. The bone marrow mesenchymal stem cells cultured within the PLGA-based highly-open porous microspheres adhered to the scaffolds in one day, representing extensive adhesion and migration capabilities of cells. The calcium-AM and propidium iodide staining showed that the cells are live and distributed uniformly.

It is essential to set the ultrasonic power and the probe's position. The reproducible production of PLGA-based highly-open porous micro spheres will pave the way to construct off the shelf graphs to induce regeneration or drug screening.