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In recent years, innovative cell-based biosensing systems have been developed, showing impact in healthcare and life science research. Now, there is a need to design mass-production processes to enable their commercialization and reach society. However, current protocols for their fabrication employ materials that are not optimal for industrial production, and their preparation requires several chemical coating steps, resulting in cumbersome protocols. We have developed a simplified two-step method for generating controlled cell patterns on PMMA, a durable and transparent material frequently employed in the mass manufacturing of microfluidic devices. It involves air plasma and microcontact printing. This approach allows the formation of well-defined cell arrays on PMMA without the need for blocking agents to define the patterns. Patterns of various adherent cell types in dozens of individual cell cultures, allowing the regulation of cell–material and cell–cell interactions, were developed. These cell patterns were integrated into a microfluidic device, and their viability for more than 20 h under controlled flow conditions was demonstrated. This work demonstrated the potential to adapt polymeric cytophobic materials to simple fabrication protocols of cell-based microsystems, leveraging the possibilities for commercialization.