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Biomanufacturing

Biomanufacturing - Poster Session A

Poster B3 - Batch Manufacturing of Polyelectrolyte Microcapsules Through Use of Electrospray Technologies.

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Constructing a tissue from smaller constituent or modular units can facilitate the assembly of geometrically complex structures that better model native structures. Such module-based tissue elements with embedded vascular channels may serve as superior, physiologically-relevant model tissues. Polyelectrolyte microcapsules have been demonstrated to be a viable tool in developing cell-laden tissue modules which can be appropriately handled and introduced into more complex biomanufacturing modalities. However their application in the generation of high cell density tissues requires their diameters to be uniformly generated at a scale of 300 𝜇m or smaller to prevent viability loss from diffusion limitations.
Here, we expand on modifications to a previously reported method for developing glycosaminoglycan (GAG)-stabilized chitosan membranes using an electrospray (ES) apparatus. Using an improved system design, we have been able to better study the effects of key production parameters on the batch production of cell-laden capsules. These parameters include voltage, working distance between electrodes, and the viscosity and extrusion rate of the GAG-phase.

Materials and

Methods:  The ES-apparatus consists of: a high voltage (HV) power supply, a magnetic stirrer, and a syringe pump for precise flow of the GAG solution to a stainless-steel needle. The needle is connected to the negative electrode of the HV system. A 3D printed support scaffold system was used to secure the needle through which a GAG solution is extruded over the center of 50 mL beaker containing stirred chitosan (0.6 wt% in 1% acetic acid). Droplets generated at the needle tip are collected in the stirred chitosan and ionic complexation between the oppositely charged polymers forms a polyelectrolyte-complex membrane at the liquid-liquid interface. Biomaterials and/or cells dispersed in the GAG phase become entrapped in the resulting complex membrane. A thin metal ring was placed around the beaker and served as the positive electrode of the system. The working distance, defined as the vertical distance between this ring electrode and the needle tip can be adjusted using the needle support scaffold.
Preliminary studies were carried out using a GAG solution containing chondroitin sulfate (4 wt%, 30-40 kDA, CS) and carboxymethyl cellulose (1.5 wt%, CMC). This solution was electrosprayed using a range of voltages (8-14 kV), extrusion rates (4-10 mL/hr), and working distances (12-20 mm). A 24G needle was used for all experiments. Capsule membranes were imaged, and their diameters measured using ToupView software, and the resulting data analyzed using SigmaPlot.

Results, Conclusions, and Discussions: Work with previous iterations of the ES system suggested that once an appropriate working distance could be identified, the electrospraying of smaller diameter capsules could be achieved using a combination of higher voltages and lower extrusion rates. A working distance of 15 mm was identified as the largest distance between electrodes that would still allow the GAG solution to be atomized and deposited in the stirred chitosan as spherical droplets. When working with the CMC/CS GAG formulation, repeated experiments showed that increasing the voltage difference resulted in smaller capsule diameters, with the trend being observed across three distinct extrusion rates. The majority of these parameter combinations resulted in a range of capsule diameters with bimodal, non-normal distributions around a mean value. Decreasing extrusion rate while keeping voltage constant also showed a decrease in capsule diameter.
The improvements to the apparatus allowed for the generation of capsules using a GAG solution of hyaluronic acid (1 wt%, HA) with chondroitin sulfate (2 wt%, CS), which likewise showed similar trends in capsule diameter as extrusion rate was decreased from 10 to 4mL/hr at 14 kV. Across all tested conditions, capsules were mostly uniform in circularity. The system has therefore been shown to be robust in the ability to tailor key parameters to repeatedly generate hollow capsules, though ongoing work still needs to be done to bring the diameter down below the desired 300 microns.
Higher extrusion speeds are preferred for the encapsulation of cells to prevent loss in viability due to restrictively long manufacturing time. The need to identify the corresponding voltages, working distances and needle gauges for extrusion speeds at or above 10 mL/hr is an ongoing point of study. Studies to encapsulate rat bone marrow mesenchymal stem cells (bmMSCs) and evaluate their short and long time viability and differentiation inside capsules are ongoing.
The cell-laden capsules produced via ES can therefore serve as effective modular units in the development of tissue constructs for physiological modeling using lab-on-a-chip and bioreactor approaches for organs of interest like brain, liver, and kidney.

Acknowledgements (Optional):  We appreciate the support of the Michigan Space Grant Consortium Graduate Fellowship program (Grant Number 80NSSC20M0124) for this support in funding the proof of concept work.