Cell Patterning Technology on Polymethyl Methacrylate through Controlled Physicochemical and Biochemical Functionalization

Abstract

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.

Keywords: cell patterning; cell-based microsystems; commercialization; microcontact printing; microfluidic device; polymethyl methacrylate.

Figure 1

Cartoon of cell patterning process on PMMA. (i) Generation of a hydrophilic zone on the PMMA surface through localized plasma treatment using a PDMS stencil. (ii) Microcontact printing of cell adhesion proteins. (iii) Pattern of a fibronectin dot array. (iv) Cell isles pattern.

Figure 2

Localized oxidation of PMMA: (A) Drawing of the process for localized oxidation of a single zone of the PMMA surface to generate a discrete hydrophilic zone, red spot. (B) Photographs for contact angle measurements of drops on PMMA surfaces untreated (left), after plasma treatment (center) and plasma treated with a PDMS layer on top during the plasma exposition (right) for contact angle measurements. (C) Plot of the normalized fluorescence intensity (left) and fluorescence images (right) of PMMA substrates with and without plasma treatment after micropatterning of BSA-TAMRA protein. Error bars mean ± SD (n = 3 samples).

Figure 3

Adhesion of HCT116 cells on PMMA-treated surface: (A) Brightfield microscopy images of HCT116 cells cultured before flow (left) and after flow (right). (B) Brightfield microscopy images of cells adhered to collagen (top row) and fibronectin (bottom row) after flow (left column) and 24 h afterwards (right column). (C) Plot of the percentage of cells adhered to collagen (Coll)- and fibronectin (Fn)-treated hydrophilic zones in the PMMA after rinsing. Error bars mean ± SD (n = 3 samples per experimental condition).

Figure 4

Patterning of cells on PMMA: (A) Brightfield images of PC3 patterns on 100 µm dots array of fibronectin with prior hydrophilic zone generation on cell cultures: polystyrene (PS) wells (top), glass cover (middle) and PMMA surface (bottom). (B) Brightfield images of the arrays of small cell colonies for hHF-MSCs, PC3 and HCT116 (top) and plot of the number of cells per spot (bottom). Error bars represent mean ± SD (n = 3 samples per experimental condition). (C) Brightfield microscopy images of a full PC3 array on 100 µm fibronectin dot patterns (left), pattern of PC3 on 100 µm dots, lollipop-like features and 50 µm dots (middle column, top to bottom) and fluorescence images of fluorescent BSA printed using the same features (right column).

Figure 5

Maintenance of cell patterns under flow conditions: (A) Schematic drawing of the syringe–device connection. (B) Photograph of the setup. (C) Schematic drawing of patterned cell maintenance inside the chip. (D) Brightfield microscopy images of PC3 cells on 50 µm patterns after patterning and after 20 h on flow (10 µL min⁻¹).

Figure 6

Single-cell patterning on PMMA: (A) Brightfield microscopy image of >200 single hHF-MSCs patterned in oscillation conditions. (B) Brightfield microscopy images of HeLa and hHF-MSCs patterns on 20 µm patterns after 30 min incubation.