Poster S16 - Assessment of CT26 Cancer Spheroids in a Novel 3D-Printed Microfluidic Platform

Introduction: In vitro testing platforms aim to bridge the divide between basic cell culture and in vivo conditions. Platforms capable of emulating in vivo conditions, however, are often expensive to produce in addition to being restrictive in their designs. Nonetheless, it is crucial to further develop these devices, as 2D culture and animal models often produce misleading results not representative of what in vivo human physiology. Animal models have also become less desirable due to ethical and financial reasons leading to the recent FDA Modernization Act 2.0, authorizing use of alternatives to animal studies in the advancement of new drug exploration and evaluation.
Here we describe a custom microfluidic platform capable of reproducing shear stress and flow conditions on 3D cellular constructs, to more faithfully mimic aspects of the tumor microenvironment. The microfluidic device is 3D-printed and ready for use within 90 minutes, and is scalable up to 90 channels within the space of a standard 96 well plate. This platform was used to image and assess the viability of cancer spheroids under flow and static conditions, both with and without cancer killing compounds such as Staurosporine dosed into the media.

Materials and

Methods: An Asiga Max X27 3D printer was used in conjunction with a photopolymerizable resin GR-1 purchased from Pro3dure Medical GmbH (Dortmund, Germany). A thin metal scraper and washer bottles with built in strainers were purchased alongside the Asiga 3D printer. A UV curing oven and Micro Meshes of 3600, 4000, and 6000 grit were used for post processing. Cole Parmer ID 0.02” tubing (95802-00), Tygon ID 0.02” tubing (06419-01), and Ismatec E Tygon tubing ID 0.13mm (96460-09) were all purchased along with 0.8mm metal connectors for system setup. A 12 channel IPC Ismatec Peristaltic Pump was used for all testing (ISM932).

CT26 colon cancer cells (CDL-2638), RPMI-1640 media, fetal bovine serum, and Trypsin EDTA solution 1X (30-2101) were purchased from ATCC. CellTracker Green CMFDA Dye (C7025), APC Annexin V (A35110), and UltraPure Agarose (16500500) were purchased from Invitrogen. PBS and DRAQ7 (D15106) were purchased through ThermoFisher Scientific. Staurosporine (S6942) was purchased from Sigma-Aldrich and was used to induce cell death within created spheroids. 3D Petri Dishes (24-96, 24-35) were purchased from Microtissues, Inc. A Zeiss LSM 780 confocal with a Zeiss EC Plan-Neofluor 10X and Argon 466nm Laser were used in conjunction with Zen 2.3 software for imaging.

Results, Conclusions, and Discussions: The 3D-printed platform was capable of spheroid capture and containment under flow conditions for up to a week. Furthermore, the 3D-printed platform was capable of producing sub-80 µm feature size, and only required 45 minutes on average to print, with minimal post-printing treatment required to ready them for cellular testing. Additional 3D-printed pieces such as tubing holders and custom conical tube caps helped improve the platform setup process.
Spheroids exposed to flow conditions showed higher delivery of DRAQ7 and Staurosporine into the spheroid compared with spheroids in static culture, resulting in higher levels of cell death compared to static controls. An IC50 curve was created to characterize the effect of Staurosporine on CT26 spheroids.

3D printing devices is more cost-effective and versatile when compared to traditional methods for microfluidic platform creation. Through the use of this novel microfluidic device and 3D cancer spheroids, this platform was capable of supporting perfusion of samples with compounds such as Staurosporine more effectively, when compared to traditional static culture.

The developed platform and associated protocols are foundational pieces towards devices capable of more personalized treatments and more efficient cancer therapeutic evaluation. The 3D printed device was capable of rapid cycle times compared to traditional methods, however, the overall setup time for experimentation was fairly long. Further design optimizations such as an increase in the size of the onboard bubble trap or integration of tubing connections could help improve both the workflow for setup and prospects for bubble avoidance.
Data collected from the flow vs static experimentation also displayed the complexity of balancing stain diffusion with interpreting cell death signaling. Specifically, active perfusion delivered DRAQ7 much more effectively and quickly than static diffusion. The result was high cell death signaling in the flow condition, which runs contrary to the expectation that flow extends overall cellular viability compared to static. Moving forward, use of cells which are capable of producing their own fluorescence would eliminate the need for most manual stains and concerns about equal stain diffusion.

Acknowledgements (Optional): I would like to thank my advisors as well as the Draper Scholar's Program.