01834nas a2200193   4500000000100000000000100001008004100002260001500043100002200058700001900080700002400099700001700123700002200140700001900162245008800181856007000269520128700339022001401626       2025                            d  c2025-10-081 aAlexandre Martins1 aSylvie Klieber1 aCharlotte Le Graët1 aEric Leclerc1 aCécile Legallais1 aRachid Jellali00aIn situ formation and culture of cell spheroids in a low-binding 3D-printed biochip  uhttps://pubs.rsc.org/en/content/articlelanding/2025/lc/d5lc00503e3 aOrgan-on-a-chip and microfluidic systems offer new ways to overcome limitations from traditional in vitro models in preclinical studies. However, the lack of standardization and important non-specific binding of tested drugs to devices commonly made of polydimethylsiloxane (PDMS) still slow down their full integration into industrial research pipelines. The goal of this study is to develop a standardized 3D-printed biochip with low-binding properties using perfluoropolyether (PFPE), allowing long-time dynamic cultures of in situ formed cellular spheroids. We first documented the non-specific binding of molecules relevant for pharmaceutical companies and mechanical and surface properties of PFPE as compared with PDMS. A new microstructured biochip was then designed and 3D-printed in PFPE to offer a 400 μL chamber containing 384 microwells. The 3D-printing fabrication protocol has been detailed considering key parameters such as UV exposure time or postcuring. Finally, 384 HepG2/C3a spheroids were formed per chip under dynamic conditions and maintained for 11 days. The high viability, functionality and polarization of the spheroids cultured in these printed PFPE biochips showed the relevance of this new microphysiological system as an alternative to PDMS devices.  a1473-0189