Investigation on compound leaching of DLP 3D printed materials to advance microfluidic cell culture Introduction Among other 3D printing methods DLP printing has gained significant interest for rapid prototyping of lab- and organ-on-a-chip devices, because of its fabrication speed and high resolution. Compared to conventional chip fabrication methods it streamlines the process from design to finished product eliminating labor-intensive steps such as photolithography, casting, and bonding (1). Despite its benefits, the resin materials used in DLP printing rely mainly on radical photo-initiator-based polymerization, which cannot achieve complete polymerization, particularly in bulk materials. Cytotoxic monomers, crosslinkers and photo initiators may remain in the material and leach out over time, negatively affecting the microphysiological systems (2). Therefore, we aim to develop a streamlined process to rapidly assess the suitability of resins for use in microfluidic cell culture. Materials and Methods We investigated the applicability of two ISO-certified biocompatible 3D printing resins (dental applications) and two non-ISO-certified resins (industrial applications). All resins were printed with an Asiga Max X43 UV (Asiga®) DLP printer with a modified build platform to achieve a transparent finish. The characterization process (Fig. 1) involved identifying the optimal post-processing condition through the evaluation of (a) the degree of curing using Raman spectroscopy on both the surface and inside the bulk material (wavelength = 488 nm) by comparing the peak ratio of acrylate monomer groups in the uncured material and -CH2 groups present in the cured material. Additionally, (b) the leaching of potentially cytotoxic components into cell culture medium was monitored over 7 days via HPLC. The second phase assessed material compatibility on a biological level by performing a 24-hour cell viability assay (PrestoBlue, live/dead staining) with primary human fibroblasts comparing the optimal condition to the uncured material as proof of concept. Finally, in long-term on-chip studies (Fig. 2 D) the relationship of cell stress (IL-6, caspase-3, p53) and cell adhesion (integrins, PXN, actin) to compound leaching should be investigated via RT-qPCR gene expression and HPLC. Results Raman spectroscopy studies on ISO-certifed material show that curing degrees of the material differ between the surfaces and the inner printed material (Fig. 2 C). The curing degree is regulated by treatments such as heat treatment and UV exposure where it was elucidated that curing degree decreases with the thickness of the printed material. Surprisingly, for non-ISO-certified material 1 the Raman Spectroscopy studies do not show differing curing degrees. This aligns with the PrestoBlue and live/dead data showing cell death on all conditions except for heat treatment in hot water (data not shown). HPLC studies on cell culture media in contact with 3D printed materials demonstrated the presence of leached residual printing compounds in conditions without post-processing (Fig. 2 A). The quantity also depends on the surface-volume ratio (for example print thickness: 1, 3, 5 mm). In 24 hour viability studies with all four materials survival rates of up to 80 % for the ISO-certified materials could be observed when post-processed by heat and UV treatment (Fig. 2 B) and survival rates between 50 - 60 % for the non-ISO-certified materials. Discussion Results show that polymerization efficiency is influenced by an interplay of material thickness, washing, UV and heat treatment steps which all affect the leaching of compounds. The ISO-certified materials in general perform better than non-ISO certified materials that require additional post-processing steps. The cell survival rates of the cell studies confirm the qualitative and quantitative observations from Raman spectroscopy and HPLC. Conclusions In conclusion, this approach highlights the necessity to evaluate compound leaching of DLP-printed materials prior to microfluidic cell culture as optimized post-processing steps are required to minimize adverse effects on MPS. Unlike previous studies, this approach emphasizes the long-term evaluation of compound leaching, coupled with molecular-biological analytics to assess material applicability and reusability, further enhancing sustainable rapid prototyping. References: (1) Fritschen, A. et al., 2022, 10.1039/D1BM01794B (2) Brooks, A. et al., 2024, 10.1002/app.56545