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Biomaterials

Biomaterials - Poster Session E

Poster B3 - Staining Techniques for Imaging and Viability Assessment of Cells within a 3D Bioprinted Hydrogel Scaffold

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

Introduction:  Bioprinting has recently emerged as a leading technology and research topic to help advance regenerative medicine. Hydrogels are commonly used materials for bioprinting and have applications in tissue engineering and wound healing. This study focuses on staining and imaging to assess the viability of living cells seeded within 3D printed hydrogel structures. To achieve this, we have developed a staining protocol to test a fluorescent cell stain at different print material concentrations and seeding densities within in a thin-walled cylindrical hydrogel print. This research not only helps advance the understanding of bioprinting but also provides insight into the survival of cells within these prints, which can be used for tissue engineering technologies in the future.

Materials and

Methods:  CellTracker Green CMFDA fluorescent probes are designed to pass through cell membranes and be retained in living cells through multiple generations. NIH3T3 fibroblasts were stained with CellTracker according to manufacturer's protocol by first diluting CMFDA with DMSO, then adding PBS to achieve a target concentration of 2.5 µM. Cells were incubated for 30 minutes then centrifuged to remove residual stain. Cells were resuspended in media to a measured concentration of 8e5 cells/ml then mixed with equal parts 4% sodium alginate, to make a 2% sodium alginate bioink with a cell concentration of 4e5 cells/ml, and then with distilled water to make a bioink with 1% sodium alginate and 2e5 cells/ml. A PRUSA i3 printer was used to print 3D cylinders into a 1% agar slurry with a 11mM calcium chloride crosslinker for shape retention. Prints were heft to set for 1.5 hours before being extracted. Printed structures were imaged under an Olympus IX81 Inverted Fluorescence Motorized Microscope with a blue filter at excitation wavelength of 492nm and emission wavelength of 517nm. Cylinder structures were imaged again after 24 hours incubation in media. To assess confluency, images were processed through a MATLAB code that uses an edge detection algorithm to find the perimeter of cells and label everything within the perimeter as a cell. Another MATLAB code was used to determine cell count within the image by looking for a local maxima in brightness to establish the center of individual cells.

Results, Conclusions, and Discussions: The results of the data collected thus far demonstrate the ability for NIH 3T3 fibroblasts to be stained using CellTracker Green CMFDA fluorescent probes within 3D printed cylindrical structures. With a concentration of 2.5 µM, cells were easily visible under fluorescence microscopy, as can be seen in the figures. Testing two concentrations of bioink allowed us to evaluate the effects of print quality on imaging and cell counting procedures. Images obtained under light microscopy reveal the presence of bubbles in both prints immediately after printing, which dissipated after 24hours more in the 1% bioink, as can be seen in Figure 2. Both the confluence and count algorithms identified cells, but there was no clear relationship between seeding densities and measured result.

The initial cell concentrations limited our ability to find significant relations between seeding densities and measured results. Cells were seeded at low densities, higher densities of 0.5e6 cells/ml, 1e6 cells/ml, and 2e6 cells/ml will be tested to evaluate viability and proliferation over time within the 3D printed structures and the effect of fluorescent probe concentration. Additional concentration levels of 5 µM and 10 µM will also be tested to analyze effects on visualization, retention of florescence over time, and potential toxicity affecting cell behavior. Further work will focus on using the 3D printed hydrogel scaffolding to determine their applications in tissue engineering.

Acknowledgements (Optional):