3D stem-like spheroids-on-a-chip for personalized combinatorial drug testing in oral cancer.

BACKGROUND: Functional drug testing (FDT) with patient-derived tumor cells in microfluidic devices is gaining popularity. However, the majority of previously reported microfluidic devices for FDT were limited by at least one of these factors: lengthy fabrication procedures, absence of tumor progenitor cells, lack of clinical correlation, and mono-drug therapy testing. Furthermore, personalized microfluidic models based on spheroids derived from oral cancer patients remain to be thoroughly validated. Overcoming the limitations, we develop 3D printed mold-based, dynamic, and personalized oral stem-like spheroids-on-a-chip, featuring unique serpentine loops and flat-bottom microwells arrangement. RESULTS: This unique arrangement enables the screening of seven combinations of three drugs on chemoresistive cancer stem-like cells. Oral cancer patients-derived stem-like spheroids (CD 44+) remains highly viable (> 90%) for 5 days. Treatment with a well-known oral cancer chemotherapy regimen (paclitaxel, 5 fluorouracil, and cisplatin) at clinically relevant dosages results in heterogeneous drug responses in spheroids. These spheroids are derived from three oral cancer patients, each diagnosed with either well-differentiated or moderately-differentiated squamous cell carcinoma. Oral spheroids exhibit dissimilar morphology, size, and oral tumor-relevant oxygen levels (< 5% O2). These features correlate with the drug responses and clinical diagnosis from each patient's histopathological report. CONCLUSIONS: Overall, we demonstrate the influence of tumor differentiation status on treatment responses, which has been rarely carried out in the previous reports. To the best of our knowledge, this is the first report demonstrating extensive work on development of microfluidic based oral cancer spheroid model for personalized combinatorial drug screening. Furthermore, the obtained clinical correlation of drug screening data represents a significant advancement over previously reported personalized spheroid-based microfluidic devices. Finally, the maintenance of patient-derived spheroids with high viability under oral cancer relevant oxygen levels of less than 5% O2 is a more realistic representation of solid tumor microenvironment in our developed device.

Facile Route for 3D Printing of Transparent PETg-Based Hybrid Biomicrofluidic Devices Promoting Cell Adhesion.

3D printing has emerged as a promising fabrication technique for microfluidic devices, overcoming some of the challenges associated with conventional soft lithography. Filament-based polymer extrusion (popularly known as fused deposition modeling (FDM)) is one of the most accessible 3D printing techniques available, offering a wide range of low-cost thermoplastic polymer materials for microfluidic device fabrication. However, low optical transparency is one of the significant limitations of extrusion-based microfluidic devices, rendering them unsuitable for cell culture-related biological applications. Moreover, previously reported extrusion-based devices were largely dependent on fluorescent dyes for cell imaging because of their poor transparency. First, we aim to improve the optical transparency of FDM-based microfluidic devices to enable bright-field microscopy of cells. This is achieved using (1) transparent polymer filament materials such as poly(ethylene terephthalate) glycol (PETg), (2) optimized 3D printing process parameters, and (3) a hybrid approach by integrating 3D printed microfluidic devices with cast poly(dimethylsiloxane) (PDMS) blocks. We begin by optimizing four essential 3D printing process parameters (layer height, printing speed, cooling fan speed, and extrusion flow), affecting the overall transparency of 3D printed devices. Optimized parameters produce exceptional optical transparency close to 80% in 3D printed PETg devices. Next, we demonstrate the potential of FDM-based 3D printing to fabricate transparent micromixing devices with complex planar and nonplanar channel networks. Most importantly, cells cultured on native 3D printed PETg surfaces show excellent cell attachment, spreading, and proliferation during 3 days of culture without extracellular matrix coating or surface treatment. Next, we introduce L929 cells inside hybrid PETg-PDMS biomicrofluidic devices as a proof of concept. We demonstrate that 3D printed hybrid biomicrofluidic devices promote cell adhesion, allow bright-field microscopy, and maintain high cell viability for 3 days. Finally, we demonstrate the applicability of the proposed fabrication approach for developing 3D printed microfluidic devices from other FDM-compatible transparent polymers such as polylactic acid (PLA) and poly(methyl methacrylate) (PMMA).