Date of Award
Master of Science
In 2009 and 2011, it was shown that micropores in the cell membrane that form during inkjet cell printing are a potential vector for gene transfection1,2. These papers showed that porcine aortic endothelial cells and Chinese hamster ovary cells were coprinted with green fluorescent protein-coding (GFP) plasmids onto collagen or gelatin "biopaper" substrates. Printed cells showed a viability of ~90% with transfection efficiencies ranging from 2-30%1,2. Using inkjet-mediated gene transfection (IMGT) to insert a plasmid coding for the Yamanaka stem cell induction factors, it may be possible to offset common drawbacks of the induction process, such as low efficiency, throughput, and teratoma formation through transgene reactivation.
Based on initial concerns that substrates may interfere with IMGT due to potential absorption or destruction of printed DNA, a protocol was developed for printing cells without using any substrate such as collagen gels or gelatin coating. Human foreskin fibroblasts (HFF), STO fibroblasts, and Btc-6 Beta cells were printed into varying amounts of culture medium, to absorb shock, in a 48-well plate, and observed for 7 days after printing. After a recuperation period, which ranged from 12 to 24 hours, printed cells, even those that were printed into no culture medium at all, began growing normally, however, printed Beta cells grew slower than manually seeded controls, not reaching confluency in the wells for up to 10 days post-print.
HFFs and Beta cells were coprinted with a GFP plasmid to test IMGT on these cells. Of the two, only the Beta cells showed any fluorescence post-print, with transfection efficiencies up to 13.2%. Beta cells were then coprinted with, T7-VEE-OKS-iG a plasmid coding for Oct4, Sox2, Klf4, and Glis-1, which are common factors used to induce pluripotency in somatic cells3. Bioink containing cells and plasmid DNA in PBS was printed into wells containing growth medium. At 14 days post-transfection, liposome-transfected controls and printed cells displayed morphological changes and resistance to selection antibiotics, however they did not form typical IPS cell colonies, which may indicate that beta cells are not receptive to nonviral transfection methods for induction.
These results demonstrate that while HFFs, STO fibroblasts and BTC-6 Beta cells can remain viable post-print without a biopaper substrate, and that IMGT can be used to transfect Beta cells with a GFP-coding plasmid. IMGT is not currently suitable for transfecting a single plasmid containing four transcription factors for induction into beta cells. This is likely due to the large size of the plasmid (16.8 kb), compared to the size of the generated micropores. It may be possible, however, to generate IPS cell using IMGT as smaller vectors become available. IMGT should not be ruled out, due to the fact that different cell types may display varying levels of membrane disruption, thus potentially allowing for larger plasmids to be transfected.
Received from ProQuest
Gilbert Paquian Jr.
Paquian Jr., Gilbert, "Stem Cell Induction via Inkjet-Mediated Gene Transfection" (2016). Open Access Theses & Dissertations. 717.