(A-28) Mitigating reactive oxygen species production and increasing gel porosity improves cell viability and spreading in photocrosslinked gelatin-thiol hydrogels

Introduction:: Generating immunocompetent 3D culture models in microphysiological systems, particularly to incorporate lymphocytes in organized systems, will benefit tissue engineering efforts to model immune organs and/or integrate immunity into multiorgan-on-chip platforms. Photopatterning is a potentially powerful tool for generating immune-competent hydrogel cultures because it can recapitulate the spatial cell organization seen in vivo. Indeed, photocrosslinking of cell-laden gels is used both in photolithographic patterning methods as well as in many 3D bioprinters. However, free radicals produced during photocrosslinking are damaging to T cells, which are sensitive to reactive oxygen species (ROS). We have previously shown that T cells lose viability after several days in culture in photopatterned gels (Ortiz-Cárdenas et al, 2022), even in thiol-norbornene gels, which generate less ROS than methacrylate gels do. Furthermore, T cells and adherent cells require a porous environment for migration and spread, respectively, whereas common photocrosslinkable materials such as gelatin-thiol mixed with PEG-norbornene may generate dense, impermeable gels. Here, we identified strategies to reduce photopatterning-induced ROS production, increase porosity of photocrosslinked gels, and optimize multi-day viability of T cells and lymphatic fibroblasts in these gels.

Materials and Methods:: Jurkat T cells were purchased from ATCC, while human naïve CD4 T cells were isolated from platelet apheresis collars obtained from healthy donors (StemCell Technologies). Human lymphatic fibroblasts from lymph nodes were sourced from ScienCell. Based on the high efficiency and relatively low ROS production of thiol-norbornene photoclick chemistry compared to methacrylate chemistry, we selected thiol-modified gelatin (GelSH) as the polymer backbone and norbornene-functionalized polyethylene glycol (PEG-NB) as the linker for our photocrosslinked hydrogel system. Gel precursor was prepared from 5% GelSH, 10 mM PEG-NB and 1.7 mM LAP photoinitiator in 1x PBS. Cell-laden gel precursor was crosslinked by exposure to 385 nm light at an intensity of 25 mW/cm² for 10 seconds; these conditions were selected in preliminary experiments to minimize the dose of light during exposure. Gel morphology was visualized with scanning electron microscopy (SEM) at an accelerating voltage of 5 kV. Cellular ROS levels were measured with the DCFH-DA fluorogenic assay (Invitrogen) to identify the impact of photocrosslinking conditions (light and/or LAP) on cells. Cell viability was quantified using calcein AM/DAPI staining following exposure to light and/or LAP, and culture in photocrosslinked hydrogels.

Results, Conclusions, and Discussions:: First, we tested whether photoexposure induced ROS production in Jurkat cells using the DCFH-DA assay (2D wells). While cellular ROS levels did not increase in response to 385 nm light alone, ROS levels increased after 24-hour culture with LAP alone, and to a greater extent following exposure to light + LAP (Fig 1a). This trend was also observed for lymphatic fibroblasts and primary CD4 T cells (not shown). Next, we hypothesized that pre-treating cells with an antioxidant, ascorbic acid (AA), would mitigate light + LAP-induced ROS production, and tested a range of AA concentrations and treatment times. We found that AA was toxic at high doses, but with optimization of dose and treatment time, a non-toxic treatment condition was identified. Pre-treatment with 1.56 mM AA for 5 min mitigated light + LAP-induced ROS production in Jurkat cells (Fig 1b), lymphatic fibroblasts and CD4 T cells (not shown).

In addition to photocrosslinking-induced ROS production, we identified that GelSH gels were limited by their low porosity. Lymph node fibroblasts remained viable in the gel but were unable to spread (Fig 1c), whereas they spread in collagen/fibrinogen (Col/Fib) gels (Fig 1d). Similarly, the lymphocytes did not migrate in GelSH gels, but did so in Col/Fib (not shown). SEM images of GelSH gels (Fig 1e) revealed a densely packed morphology in sharp contrast to the more porous Col/Fib network (Fig 1f). Therefore, we doped GelSH precursor with varied collagen concentrations before crosslinking. Adding collagen to GelSH increased gel porosity (Fig 1g). Combining AA pretreatment with collagen doping of GelSH allowed lymphatic fibroblasts to spread (not shown) and rescued Jurkat cell viability over 3-day culture (Fig 1h).


AA pre-treatment protected cells from light + LAP-induced ROS and was non-toxic at optimized doses. Adding collagen increased GelSH gel porosity. Combining AA pretreatment with collagen doping preserved viability of Jurkat cells and enabled spreading of lymphatic fibroblasts in GelSH gels. Future work will explore long-term cell culture and test immune function in photocrosslinked gels. These findings will enable researchers utilizing photocrosslinking methods to develop immunocompetent 3D culture models that support viability and function of sensitive lymphoid cells.


Acknowledgements (Optional): : This work was supported with funding from the National Institute of Biomedical Imaging and Bioengineering (NIBIB) under award number U01EB029127 through the National Institutes of Health (NIH), with co-funding from the National Center for Advancing Translational Sciences (NCATS). Tochukwu Ozulumba was supported by a Research Award from the Global Infectious Diseases Institute at the University of Virginia.


References (Optional): : < 1. Ortiz-Cárdenas JE et al. Towards spatially-organized organs-on-chip: Photopatterning cell-laden thiol-ene and methacryloyl hydrogels in a microfluidic device. Organs-on-a-Chip. 2022 Dec 1;4:100018.