Poster Y9 - A Carbon Dioxide Laser Welding Approach to Manufacture Microplate Microfluidic Devices

Introduction: Rapid prototyping tools and techniques have the potential to speed the development of human-based cell culture devices that can replace animal models in drug and chemical testing. Computer numerical control (CNC) machining devices directly into ANSI/SLAS polystyrene (PS) microplates is one promising approach to creating throughput-compatible microfluidic devices that integrate directly into existing infrastructure and avoid incompatible materials [1].
Devices milled into the bottom of a well plate need to be sealed to create closed fluidics. Current bonding techniques use solvents such as acetonitrile to seal microfluidic devices with PS sheets [2]. However, this approach is prone to inconsistency due to issues like etching and leaking. To address this issue and maintain an easily translatable manufacturing process, our research focused on utilizing a relatively inexpensive and widely available 10.6µm, 60-watt carbon dioxide (CO2) laser-cutting machine to weld PS cell culture plates to clear PS sheets. By employing a systematic Design of Experiment (DoE) approach, we explored the optimal settings for laser power, speed, and density to achieve functional welds. We characterized these welds using a series of tests to quantify their integrity and understand the physical and chemical changes taking place. Using these methods, we have identified multiple parameter combinations that create reliable welds. Our initial work has demonstrated the broad utility of using a standard CO2 laser cutter to weld clear-clear PS for rapid prototyping of cell culture devices.

Materials and

Methods: Corning Falcon 96-well plates were CNC machined (Figure 1A) using a 1/32” diameter, 2-fluted square endmill. The laser welding setup included a CO2 laser head (VLS 6.60/75 Universal Laser Systems), a vacuum pump, and a manifold. The laser head operated at a focal length of 4.1 cm. The manifold was positioned on the laser bed with the vacuum hose running through the door, and the milled plate was placed on top of the manifold with the sheet tightly affixed using constant vacuum pressure (Figure 1B).
The laser settings (power, speed, and density) were varied based on the DoEs and the bond types were initially categorized through visualization (Figure 1C). Minitab Statistical Software facilitated the creation of the initial DoE using a Box-Behnken design methodology, covering the entire design space. A second DoE, also using Box-Behnken design, narrowed the design space further. Two additional experiments refined the working space, as represented in (Figure 1C).
The following tests were developed and conducted to characterize the weld's effectiveness.
• Peel Test – Performed a manual peel test to characterize the weld strength.
• Leak Test – Added water to the wells to detect any leakage after 48 hours.
• Vacuum Test – Applied vacuum to evaluate seal integrity at -585 mmHg pressure.
• Pressure Test – Pressurized the wells to 350 mmHg to determine bursting pressure.
• Seep Test – Assessed the weld integrity by holding under pressure at 150 mmHg and testing for leakage after one minute.

Results, Conclusions, and Discussions: The initial experimental design covered a broad parameter space ranging from no visual effects to overheating and burning the material. Based on the results of the hierarchical testing, the subsequent welding parameters were narrowed. Tests were conducted on the polystyrene sheet, and a pass-fail graph was generated after every test from the outcomes. Each test progressively narrowed down the settings. We identified a parameter space covering four of the 78 settings tested, which yielded positive results on every test. We conclude that CO2 laser welding can effectively create clear-clear PS-PS welds.
Welds were further characterized using Scanning Electron Microscopy (SEM) imaging. Mass spectroscopy was utilized to determine the residuals being formed to obtain a functional weld. To characterize the effects these residuals may have on cells, cell viability studies will be conducted. Further tests are going to be conducted on non-plasma treated plates and Cyclic Olefin Copolymer (COC) plates to determine if surface chemistry and olefin behavior alter the weld strength and cytotoxicity.
In conclusion, we have successfully harnessed CO2 laser welding technology to efficiently create human-based cell culture models with diverse applications and functionalities. The manufacturing of these devices is not equipment-heavy, making it a feasible option for traditional biological laboratories to adopt custom microfluidic device development into their research.

Acknowledgements (Optional): References
[1] Guckenberger DJ, de Groot TE, Wan AM, Beebe DJ, Young EW. Micromilling: a method for ultra-rapid prototyping of plastic microfluidic devices. Lab Chip. Jun 7 2015;15(11):2364-78. doi:10.1039/c5lc00234f
[2] Reynolds JI, Vitek RA, Geiger PG, Johnson BP. Engineering Epithelial–Mesenchymal Microtissues to Study Cell-Cell Interactions in Development. In: Dworkin S, ed. Craniofacial Development: Methods and Protocols. Springer US; 2022:201-213.

Acknowledgements
We thank Dr Jinxing Li and his lab members for allowing us to use the laser, The Center for Advanced Microscopy at Michigan State University for performing the SEM imaging, and Amy Albin of the Center for her assistance in interpreting the images.

Conflict of interest: BJ owns equity in Onexio Biosystems, LLC.