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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster V8 - Optimizing Cellular Functionality in Hydrogels with Bioprinted Vascular Scaffold

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  3D bioprinting facilitates micrometer precise cell placement, enabling the engineering of functional tissues by strategically positioning cells within the tissue. The viability and functionality of embedded cellular constructs necessitate an optimal microenvironment within the gel matrix, crucial for their development and integration in a hydrogel matrix. This environment includes vasculature-like structures for effective transport of nutrients and signaling molecules. Using a commercial 3D printer, we fabricate these structures in hydrogel constructs with Pluronic F-127 as a sacrificial ink. Upon its removal, the formed channels can be filled with various components to closely mimic the function of natural vasculature.
We employ genetically modified HEK293T cells designed to express the fluorescent protein mScarlet-I in response to doxycycline. Mixed with GelMa, these cells are cast over the pre-formed channel structures. We investigate the proliferation and growth of these cells within the gel matrix and the induction by doxycycline as a proxy for nutrient uptake analysis.
To broaden our focus from analyzing nutrient uptake to observing more complex genetic circuits, we investigate the transfection of HEK293T cells in hydrogel. We use plasmid DNA encoding for the fluorescent protein mCherry and compare it to direct mRNA transfection for the same protein. Further, we explore transfection of pegRNA and mRNA for a Cas9 prime editor to enable targeted protein editing within the cells.
Our next step is to evaluate the impact of different vascular structures on these systems. We anticipate this will advance functional 3D-printed tissues, integrating cellular systems and offering new possibilities in tissue engineering.

Materials and

Methods:  Pluronic F-127 at a concentration of 40% (w/v) is used as a sacrificial ink to fabricate vasculature-like structures with a commercial 3D printer. GelMa (15%, w/v) with LAP (0.25%, w/v) as a photocrosslinker is cast on top and UV-crosslinked for structural integrity. Upon cooling to 4°C, the liquid-state Pluronic is removed, leaving channels that can be filled with the desired material. HEK293T cells, mixed with GelMa, are cast over the fabricated channels.
Doxycycline (500 ng/ml) is flushed through the channel, establishing an orthogonal gradient, which induces mScarlet-I expression in the modified cells. To visualize the diffusion fluoresceinis flushed through the constructed channels.
We utilize three different HEK293T cell lines: one genetically modified to express mScarlet-I upon doxycycline induction, another unmodified HEK293T cell line where a plasmid encoding for the fluorescent protein mCherry is transfected, and a third cell line modified to stably express a blue fluorescent protein that can be changed to green fluorescence upon prime editing. Cells were cultured in DMEM with 10% fbs.
Transfection of plasmid DNA was done with cation lipids and for mRNA with Lipofektamin 2000. The pegRNA and mRNA for the Cas9 prime editor were produced by in vitro transcription, capped with the Faustovirus Capping Enzyme and poly A tailed with PolyA polymerase.
Gelatin Methacryloyl (GelMA) was produced by dissolving Gelatin Type A in PBS, adding methacrylic anhydride (MAA) in a 1:1 ratio, and removing unreacted MAA through centrifugation. The pH was adjusted to 7.4 with NaHCO3, then dialyzed and lyophilized.

Results, Conclusions, and Discussions: Our study demonstrates that HEK293T cells are viable and proliferate within our custom-made GelMa matrix, forming noticeable cell clusters with a doubling time of approximately 2 days. Genetically modified cells cultivated in hydrogel respond to doxycycline induction more efficiently when applied through fabricated vascular channels, with higher fluorescent protein expression observed closer to these channels. Furthermore, we observed transfection efficiency differences between plasmid DNA encoding mCherry and the respective mRNA. In traditional adherent cultures, DNA transfection was more efficient, while in the GelMa matrix, mRNA transfection proved superior. This discrepancy likely results from reduced growth rate of cells in the hydrogel, making nuclear entry for DNA more challenging, whereas mRNA transfection bypasses this requirement. Also, we successfully transfected mRNA for a Cas9 prime editor into a modified HEK293T cell line. Future work involves applying this to our vascular structure systems, to show that more complex genetic networks could be engineered in hydrogel tissues.
Our study demonstrates the biocompatibility of the custom-made GelMa matrix, as evidenced by the ability of cells to proliferate, highlighting their capacity to thrive and create multicellular structures in this environment. We observed that mRNA transfection is more efficient in the GelMa matrix compared to plasmid DNA, likely due to slower cell division rates limiting nuclear access for DNA. This indicates that RNA-based transfection methods are better suited for applications involving cells in a 3D matrix. The necessity of advanced channel structures for effective nutrient and molecule transport is evident, as they are crucial for maintaining the viability and functionality of larger tissue constructs by ensuring uniform distribution of essential materials. Our findings suggest that combining mRNA transfection methods with adapted vascular structures can significantly enhance the development of fully functional 3D-printed tissues, supporting complex genetic circuit integration. We will optimize these methods and explore their long-term stability and functionality. Different channel geometries could be used to engineer more complex genetic networks that are dependent on different timescales.
To conclude, our work provides a foundational understanding of the interplay between transfection methods and structural design in 3D bioprinting, paving the way for advanced tissue engineering solutions.

Acknowledgements (Optional):  This work was funded by the Federal Ministry of Education and Research (BMBF) and the Free State of Bavaria under the Excellence Strategy of the Federal Government and the Länder through the ONE MUNICh Project Munich Multiscale Biofabrication.