Speaker
Description
Introduction
Bioprinting offers the opportunity to reproduce tissue structures in-vitro to support the reconstruction of functional tissue constructs. Among the different bioprinting techniques, two photon polymerization (2PP) allows the generations of the smallest features up to the submicron range. However, the low volume throughput is the main drawback to obtaining constructs of a relevant size.[1] Moreover, the materials for cell encapsulation using 2PP can be printed in low concentrations, with consequently low viscosities that over long printing times can cause cell sedimentation.[2] When scaling up, ensuring adequate cell spreading within the structure is pivotal to allow high cell viability and functionality.[3] In this work, these challenges of applying 2PP to bioprinting with cells are addressed, using fibroblasts and photorosslinkable gelatin as reference substrates.
Methods
Gelatin was functionalized with norbornene (NB) moieties according to Van Hoorick et al. [4] and used with a thiolated PEG as a crosslinker. A gellifier was added to enable printing in a solid state, after measuring its sol/gel transition temperature via rheological measurements. The polymer blend solution was mixed with L929 fibroblasts to a final concentration of 2x106cells/mL, and the bioink was printed using an UpNano NanoOne printer.
The processability window of the ink was identified by printing a reference “multigrid” structure and varying the main process parameters in a full factorial DoE, followed by the estimation of shape fidelity and swelling via image analysis on confocal images after 1 day of incubation at 37 °C in PBS. To estimate the printing time, a full factorial DoE was prepared by printing cubes of different sizes at varying printing parameters.
Within this window, the effect of different lattice geometries and pore size on cell viability was tested using Live/Dead staining with fluorescence imaging 1 day post-printing. Cytoskeleton remodelling in larger structures (side 500 µm) with different porosities was assessed at day 7 via confocal imaging after actin-F/DAPI staining.
Results and discussion
The addition of the gellifier avoided cell sedimentation of the cells in bulk polymer, which enables the production of constructs with higher thickness (500 µm) with a homogeneous cell distribution of the cells along the z-axis.
The processability window of the material at varying process parameters was identified in a wider printing domain than previously reported, and a simple model for the printing time was used to exclude the printing parameters corresponding to a volume throughput higher than 1 mm3/hr. This reduced printing time while maintaining resolution.
Finally, a wider region of the processability window was identified as compatible with the bioprinting process. Moreover, the introduction of micro (pore size<cell size) and macro (pore size>cell diameter) porosity showed to improve both cell viability, cytoskeleton remodelling, and cell spreading, compared to bulk, non-porous structures obtained with the same printing parameters.
These strategies are promising for the scale-up and production of relevant tissue structures with viable cells present throughout the hydrogel.
References
[1] doi: 10.1016/j.tibtech.2022.10.009.
[2] doi: 10.1007/s42242-022-00183-6.
[3] doi: 10.1002/advs.202306470.
[4] doi: 10.1002/marc.201800181.
Acknowledgements
This work was funded by UKRI EPSRC programme grant EP/W017032/1
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