(B-69) Functional Fusion of Colloidosome-based Synthetic Cells into Microfluidic Devices

Introduction:: T cell-based immunotherapy is a promising approach for cancer treatment. Previously, T cells have been isolated from cancer patients, engineered and cultured ex vivo, and injected back into patients so that these proliferated T cells can identify cancer cells and eliminate them. However, ex vivo expansion of therapeutic T cells causes them to overproliferate, resulting in poor quality T cells with exhausted phenotypes. Therefore, we aim to implement a synthetic antigen-presenting cell technology, integrated into a microfluidic device that achieves physiological expansion of therapeutic-competent T cells. We chose to produce silica colloidosomes, which are self-assembled and cell-shaped colloidal particles that form a microcapsule, as artificial cell types. We hypothesized that their silica-based membrane will allow for siliane-mediated stable integration into polydimethylsiloxane (PDMS) microfluidic devices. This approach would enable an environment where T cells can proliferate in a controlled manner, as the surface of colloidosomes can be functionalized with specific molecules to ensure that the antigen is presented to the T cells in a regulated manner. Additionally, the use of microfluidic devices can allow us to mimic the mechanical and microanatomical properties of lymph nodes at different stages of inflammation and by this achieve a more physiological T cell expansion of potentially less exhausted cells.

Materials and Methods:: We created a mold for the microfluidic devices using the computer-aided design software, OpenSCAD. We applied 3D printed acrylnitril-butadien-styrol (ABS) copolymer negative molds for 3D microfluidic devices and lithography-based 3D resin printing for 2D microfluidic devices.
PDMS was mixed with a curing agent, poured onto the molds, and cured to create microfluidic devices. Next, a silane coating was introduced into the device channels for functionalization, and then was washed with water.
Colloidosomes were produced from water in oil pickering emulsions in a dodecane dispersant. After emulsification, colloidosomes were cross-linked by tetramethyl orthosilicate (TMOS) and transferred into an aqueous phase by sequential ethanol washes. We first optimized colloidosome assembly to achieve high-densities of spherical colloidosomes with reduced background contaminants. Colloidosomes were characterized by light microscopy to analyze the efficiency of colloidosome production, in comparison to colloidosome or silica nanoparticle aggregation. Scanning electron microscopy was used to assess colloidosome surface properties and formation rate. The TMOS-based cross-linking was also used for stable incorporation of the colloidosomes into the silane-functionalized microfluidic devices. Incorporation of the colloidosomes and formation of colloidosome-meshes within the microfluidic channels was assessed by light and electron microscopy. Two groups of colloidosome-integrated microfluidic devices were analyzed using electron microscopy: device channels with silane coating, and devices without silane coating. Within those two groups, we analyzed devices that were washed with ethanol and PBS after colloidosome integration, and devices that were left unwashed.

Results, Conclusions, and Discussions:: Through light microscopy analysis, we confirmed that the amount of colloidosomes that we produced were optimized in comparison to levels of background contaminants, such as nanoparticle aggregates. Additionally, light microscopy analysis confirmed the spherical, cell-shaped morphology of the colloidosomes. After integrating the colloidosomes into microfluidic devices, electron microscopy images confirmed that colloidosomes adhered to the channel walls when the walls were functionalized with silane coating. In the absence of silane coating, colloidosomes did not adhere to the channel walls. These results support our hypothesis that silica-based colloidosomes can be immobilized into microfluidic devices through silane-mediated adhesion. Additionally, when analyzing electron microscope images of colloidosomes integrated into silane-coated microfluidic devices, we saw that devices that had been treated with ethanol and PBS washes had more prominent, large colloidosomes than devices without the washing steps. Ultimately, these results indicate that silane chemistry mediates colloidosome adhesion to PDMS microfluidic device channels. By successfully developing a method for functional fusion of colloidosome-based synthetic cells within microfluidic devices, we have taken an important step towards achieving physiological expansion of therapeutic-competent T cells for cancer treatment. As next steps, methods for tuning the colloidosome mesh-size will need to be developed. Further, we will implement an n-hydroxysuccinimide-ester-based functionalization of the colloidosomes to immobilize T cell antigens and co-stimulatory elements on the colloidosomes. These devices will then be applied for incubation of primary human CD8+ T cells to assess their ex vivo expansion.

Acknowledgements (Optional): : Thank you to the Cooperative International Science and Engineering Internships program at the University of California, Santa Barbara for giving me opportunity to conduct research at the Leibniz Institute for New Materials in Saarbrücken, Germany. I would also like to thank Dr. Oskar Staufer (Leibniz Institute for New Materials) for helping me navigate this research project, and understand the scientific principles behind techniques such as microfluidic device production, scanning electron microscopy, and colloidosome assembly.

References (Optional): : 1. University of California, Santa Barbara.
2. INM - Leibniz Institute for New Materials.