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INTRODUCTION. Volumetric bioprinting (VBP) is a recently developed light-based biofabrication method enabling the rapid generation of complex 3D structures within seconds. Short printing times combined with freedom of design allow for the advancement of novel in vitro models and physiologically relevant constructs. However, a more in-depth understanding of the effects of light-based bioprinting techniques is needed. During printing, the bioresin volume is illuminated, triggering the production of free radicals needed for common photocrosslinking reactions (i.e. chain growth and thiol-ene). Albeit safe light doses have been identified, radicals include reactive oxygen species, which concentration needs to be kept below supra-physiological values in the proximity of cells to avoid DNA damage. Moreover, in light-based bioprinting, unreacted bioresin volumes (from the reaction vat) are typically discarded, resulting in the loss of valuable cells. Unraveling the impact of cell-light interactions in bioprinting is key for the design of clinically-viable constructs for biomedical applications. Herein, we investigated the cell response after VBP via single-cell transcriptomics and analyzed stress and health markers. Further, we evaluated the usability of recycled mesenchymal stromal cells (MSCs), retrieved from the excess bioresin in the printing vat.
METHODOLOGY. Viability, metabolic activity, and H2O2 production were assessed upon printing human bone marrow-derived MSCs with 10% wt GelMA and 0.1% wt lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Casted gels (crosslinked with 365nm or 405nm lamps), and enzyme-crosslinked, non-photoexposed gelatin were used as controls. Samples were printed with increasing light dosages (375-625 mJ/cm2). DNA damage was assessed through immunostaining of apoptosis and double-strand breaks markers (Caspase-3, gH2AX, and TP53BP1). To determine inter-cellular variability and screen in-depth for light-based cell effects, single-cell RNA analyses were performed with a library of ≥75000 target genes. Cells were sequenced without undergoing VBP, 1 hour post-printing, and after 7 days in culture post-printing. Finally, MSCs retrieved from the uncrosslinked GelMA were collected and re-plated after printing, to assess proliferation rate and differentiation ability (osteogenic, adipogenic, and chondrogenic lineages).
RESULTS. Viability was unchanged over seven days of culture for the printed structures, compared to the casted gels (>90%), and higher light dosages correlated with higher metabolic activities. The first assessment of H2O2 concentration showed no significant increase post-printing. No significant DNA-damage was measured with the different markers. Re-plated cells had increased metabolic activity post-printing. Initial results from the single-cell sequencing data have shown a mild activation of stress-related genes, mainly involved in the TNF-a and KRAS signaling pathways, 1 hour post-printing. Results from the sequencing of cells cultured 7 days post-printing will shed light on the effect of the maturation process on their transcriptional state, and on the time-dependent response in the transcriptome.
CONCLUSION. Volumetric bioprinting is a fast biofabrication method that allows for the generation of tissue constructs without hampering cell viability and functionality post-printing. High-throughput, single-cell transcriptomic assays have great potential to elucidate the safety and risks related to bioprinting technologies. These results give valuable insights on cell behavior post-printing, which are needed to develop the next generation of bioprinted in vitro models and patient-specific grafts.
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