Rapid Prototyping of Thermoplastic Microfluidic 3D Cell Culture Devices by Creating Regional Hydrophilicity Discrepancy

Abstract Microfluidic 3D cell culture devices that enable the recapitulation of key aspects of organ structures and functions in vivo represent a promising preclinical platform to improve translational success during drug discovery. Essential to these engineered devices is the spatial patterning of cells from different tissue types within a confined microenvironment. Traditional fabrication strategies lack the scalability, cost‐effectiveness, and rapid prototyping capabilities required for industrial applications, especially for processes involving thermoplastic materials. Here, an approach to pattern fluid guides inside microchannels is introduced by establishing differential hydrophilicity using pressure‐sensitive adhesives as masks and a subsequent selective coating with a biocompatible polymer. Optimal coating conditions are identified using polyvinylpyrrolidone, which resulted in rapid and consistent hydrogel flow in both the open‐chip prototype and the fully bonded device containing additional features for medium perfusion. The suitability of the device for dynamic 3D cell culture is tested by growing human hepatocytes in the device under controlled fluid flow for a 14‐day period. Additionally, the study demonstrated the potential of using the device for pharmaceutical high‐throughput screening applications, such as predicting drug‐induced liver injury. The approach offers a facile strategy of rapid prototyping thermoplastic microfluidic organ chips with varying geometries, microstructures, and substrate materials.

The tubing connectors were adhered to the inlets and outlets between gel loading and gelation steps.After gelation the resulting chips are ready for perfusion experiments by flowing culture medium solutions across medium lane using a peristaltic pump.Scale bar: 2 mm.

Figure S2 .
Figure S2.Effect of different coating configurations on hydrogel flow behavior.A)Photographs shows flow test results (before gelation) using chips (channel height: 120 µm) with different coating configurations using (i-iii).~1.5 μL 0.35% rat collagen I solution; and (iv-v).~1.5 μL 0.4% bovine collagen I solution, as hydrogel solution.Scale bar: 1 mm.B) Summary of hydrogel flow test using chips with different coating configurations.

Figure S3 .
Figure S3.Effect of gamma irradiation on hydrogel flow behavior.A) Photographs showing results after loading 0.4% bovine collagen I solution into chips after sterilization by gamma irradiation at 25 kilogray (kGy).Numbers in the images are loading volumes.Scale bar: 2 mm.B) Photographs showing flowing and gelation results after loading ~1.5 μL 0.4% bovine collagen I solution into chips after sterilization by gamma irradiation at 25 kGy (channel height: 120 μm).

Figure S4 .
Figure S4.Effect of substrate materials and channel heights on hydrogel flow behavior.Photographs showing flowing and gelation results after loading 0.4% bovine collagen I solution into non-sterilized chips (channel height: 120 μm) with different types of plastic substrates.A) The top substrate was either PMMA or PC slide with ECM lanes coated by 1% PEG, the bottom substrate was pristine TC-PS slide without surface modifications.B) The top substrate was TC-PS slide with ECM lanes coated by 1% PEG, the bottom substrate was glass slide with ECM lanes coated by 1% PEG.C) The top substrate was TC-PS slide with ECM lanes coated by 1% PEG, the bottom substrate was TC-PS slide with ECM lanes coated by 1% PEG (channel height: 240 μm).Scale bar: 2 mm.

Figure S5 .
Figure S5.Effect of UV exposure on gel flow behavior and sterilization efficacy.A) Photographs showing the results of hydrogel flow tests where the ECM channel of the top substrate and the entire bottom substrate were coated by PVP.Both substrates were sterilized by UV at 254 nm with different energy levels.In all cases, the hydrogel solution only filled the ECM channel without significant overflow into the medium channel.Scale bars: 2 mm (top) and 250 μm (bottom).B) Summary on the effect of UV sterilization at different energy levels on hydrogel

Figure S6 .
Figure S6.Gel Stability under perfusion.Optical microscope images showing gel and cell morphology under continuous flow of culture medium at 100 μl/hour.The chip was not sterilized, and the experiment was conducted on a microscope stage.The images were taken at different time points during flow test.Scale bar: 250 μm.

Figure S7 .
Figure S7.Effect of different channel geometries on the occurrence of channel blockage.Bar plot comparing the percentage of chips with debris between chip 1.1 and chip 2.1.

Figure S8 .Figure S9 .
Figure S8.Comparing albumin production and troglitazone toxicity in 2D and 3D plate controls.A) Normalized albumin secretion levels in the culture medium at indicated days of culture in HepG2 2D and 3D plate controls.B) Albumin levels were measured from the HepG2 2D culture medium at 72 hours post-treatment at 7× Cmax and 20× Cmax concentration.Data represents mean ± SEM.N=3, one-way ANOVA with Tukey's post-hoc multiple comparisons correction, ****, p<0.0001.

Figure S10 .
Figure S10.Morphological analysis of CellPainting images.Bar graph showing feature importance ranking color-coded by either intensity-based features or geometry features.Note that the top-ranking features are intensity features.