Abstract #5024: Detection of hypoxia response of engineered 9L glioma cells that were encapsulated in nanoporous microcontainers fabricated with electron-beam lithography.
Authors: Joonbum Kwon, Yang Su, Li Liu, Ralph Mason, Krutarth Trivedi, Wenchuang Hu, Jeong-Bong Lee, Barjor Gimi
Journal: Cancer Research (2009)

We fabricated nanoporous, cell-encapsulating microcontainers for potential use in cell based therapy using a novel approach involving electron beam (E-beam) lithography. The nanoporous surface can immunoprotect the encapsulated cells by preventing the entry of large molecules of the immune system. The passage of small molecules is permitted through these nanopores, thereby providing nutrition to the encapsulated cells and bi-directional ion transport, as well as allowing the egress of biotherapeutic molecules secreted by the encapsulated cells. Such biotherapeutic molecules may include enzymes for enzyme/prodrug therapy, and anti-tumor toxins. The microcontainer has: 1) a hollowed cubic base capable of encapsulating cells and comprising a female structure, and 2) a nanoporous lid with a male structure that seals the device after it is filled with its cellular payload. The base was formed with the SU-8 polymer whereupon first, the walls of the base were patterned using photolithography and then a female structure was formed atop the walls to accommodate a corresponding male structure formed in the lid. The lid was patterned with SU-8 islands and a male structure to seal the device. To fabricate the lid, we first deposited a Cr adhesion layer. Next, we formed 350 nm thin SU-8 2000.5 islands. Then, PMMA A2 photoresist was spun on the 350 nm SU-8 membrane, patterned and baked. A nanopore array was formed on PMMA using E-beam lithography. 200 A Al was deposited on the patterned PMMA as a protection layer. Next, the 350 nm SU-8 membrane was etched by O2 plasma using reactive ion etching. The Al protection layer and the PMMA layer were then removed. Finally, 100 \#956;m thick SU-8 2075 was spun and patterned along with 35 \#956;m SU-8 2025 to form the male structure. We transfected 9L rat glioma cells to express the bioluminescent protein \#8216;luciferin\#8217; under the HRE promoter. To ascertain their oxygenation condition post encapsulation, we encapsulated these 9L-3HRE-luc cells in 1) the aforementioned microcontainers with the nanoporous lid, 2) microcontainers with a non-porous lid. Unencapsulated cells were used as controls. Bioluminescence imaging (BLI) was performed 48 h post encapsulation. BLI revealed no signal from the unencapsulated controls. Cells from microcontainers with nonporous lids yielded significantly more BLI signal than microcontainers with nanoporous lids at 48 h, suggesting that the nanopores facilitated the oxygenation of encapsulated cells. However, the existence of some basal BLI signal from nanoporous microcontainers indicates that our nanopore array should be expanded so as to fully oxygenate the encapsulated cells. Our microcontainers support the oxygenation of encapsulated cells and can be used for allo- and xeno-grafts in cell based therapy. We acknowledge NIH-R01-EB007456. Address correspondence to BG at barjorg@yahoo.com  Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5024.