A Bioprinting Workflow for Live Cell Imaging and Mechanical Testing
Master thesis
Permanent lenke
https://hdl.handle.net/11250/3080906Utgivelsesdato
2023Metadata
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Sammendrag
Bioprinting, an emergent field bridging biology, chemistry and 3D printing technology offers to researchers innovative solutions to progress the study of tissue engineering, drug testing and cellular studies. This thesis presents a novel workflow for the live cell imaging and mechanical testing of 3D bioprinted constructs.
The workflow begins with cultivating PANC-1 cells in standard cell culture flasks or alternatively using 3D culture methods, such as hanging drops or spheroid culture plates. The cells are then encapsulated in one of two different hydrogel environments: Laminink 411, a product from CELLINK, composed of gelatin functionalised with methacrylate groups, alginate and several laminins; or a 3 mg/mL collagen solution made from TeloCol®-10. Laminink encapsulations were printed in the BIO X bioprinter whereas the collagen constructs were formed with the use of a mold. Laminink-PANC-1 hydrogels are solidified using ultra-violet (UV) and ionic crosslinking with CaCl2 before incubation, while collagen constructs are crosslinked by exposure to 37 ⁰C in an incubator. All hydrogel constructs were formed in Ibidi 35 mm imaging μ-dishes. Post-incubation, cell viability was assessed on a Leica SP8 microscope, using a dual fluorescence stain of calcein green and mitotracker red. Subsequent to imaging, mechanical testing was performed at earliest convenience. Hydrogels were taken to a Discovery HR 20 Hybrid Rheometer (DHR-2) where oscillation amplitude and frequency sweep measurements of the samples were taken.
An advantage of this workflow is that it offers a repeatable process to allow ongoing imaging and mechanical testing. Laminink hydrogels exhibited a resilient, non-degradable nature which allowed the imaging and mechanical workflow to be repeated after a second period of incubation. The results of the study revealed high cell viability for both Laminink and collagen hydrogels, as demonstrated by the confocal images, and the matrix stiffness of the constructs was determined through the Rheometry data obtained. In addition, utilization of the Raise3D Pro 2 and Formlabs 3+ 3D printers were essential to overcome technical challenges in the progress of this thesis. Specifically, they were used to design calibration plates for the BIO X, molds for collagen hydrogel crosslinking and producing static helix mixers to assist in the homogeneous encapsulation of cells within hydrogel networks.
In conclusion, the workflow presented demonstrates an advancement in the merging of advanced 3D bioprinting technology with biological and chemical explorations, broadening the positive future in biomedical research.