<p><strong>Contamination challenge:</strong> Helped us address a major issue with fungal contamination in our CF cell cultures, which were unstable and prone to infection.</p>
<p><strong>Contamination challenge:</strong> Helped us address a major issue with fungal contamination in our CF cell cultures, which were unstable and prone to infection.</p>
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<p><strong>Introduction through iGEM Hamburg:</strong> Collaborated with Prof. Ignatova, a leading expert in CF research, to deepen our understanding of the disease.</p>
<p><strong>Introduction through iGEM Hamburg:</strong> Collaborated with Prof. Ignatova, a leading expert in CF research, to deepen our understanding of the disease.</p>
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<p><strong>Support from Dr. Oliver Dräger:</strong> Hands-on support from Dr. Dräger helped us perform patch-clamp experiments using HEK cells provided by Uni Leuven.</p>
<p><strong>Support from Dr. Oliver Dräger:</strong> Hands-on support from Dr. Dräger helped us perform patch-clamp experiments using HEK cells provided by Uni Leuven.</p>
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<p><strong>Consultation because of test system:</strong> HEK cells with corresponding CFTR mutation derived from Leuven were successfully cultured in our lab for testing purposes.</p>
<p><strong>Consultation because of test system:</strong> HEK cells with corresponding CFTR mutation derived from Leuven were successfully cultured in our lab for testing purposes.</p>
<p><strong>Chitosan properties:</strong> Provided valuable insights into chitosan, a cationic polymer with strong potential to stabilize RNA in lipid nanoparticle (LNP) formulations due to its robust protection against RNases and heat stability.</p>
<p><strong>Chitosan properties:</strong> Provided valuable insights into chitosan, a cationic polymer with strong potential to stabilize RNA in lipid nanoparticle (LNP) formulations due to its robust protection against RNases and heat stability.</p>
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<p>24 h, 48 h and 72 h post-transfection, we observed in the conditions with Lipofectamine alone, or combined with DNA or RNA, no fluorescence, indicating unsuccessful transfection. Similarly, no fluorescence was seen in cells treated with LNPs alone or in combination with DNA or RNA. When LNPs were combined with Minicircle DNA, clear fluorescence was observed, indicating successful transfection and expression of our eYFP reporter under this condition (figure X). However, a strong background fluorescence from the OptiMEM medium was observed, complicating the analysis.</p>
<p>24 h, 48 h and 72 h post-transfection, we observed in the conditions with Lipofectamine alone, or combined with DNA or RNA, no fluorescence, indicating unsuccessful transfection. Similarly, no fluorescence was seen in cells treated with LNPs alone or in combination with DNA or RNA. When LNPs were combined with Minicircle DNA, clear fluorescence was observed, indicating successful transfection and expression of our eYFP reporter under this condition (figure X). However, a strong background fluorescence from the OptiMEM medium was observed, complicating the analysis.</p>
<p>Overall, among all the tested conditions, the LNP formulation with Minicircle DNA was the only combination that resulted in noticeable fluorescence, suggesting it to be the most effective transfection method for HEK293 cells in this experiment.</p>
<p>Overall, among all the tested conditions, the LNP formulation with Minicircle DNA was the only combination that resulted in noticeable fluorescence, suggesting it to be the most effective transfection method for HEK293 cells in this experiment.</p>
Figure X Fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 72 h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B.
Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification after 72 h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. For Lipofectamine (Lipo) + Minicircle DNA (Mini DNA) only the fluorescence image is shown.