Aqueous Processed Biopolymer Interfaces for Single-Cell Microarrays

Abstract

Single-cell microarrays are emerging tools to unravel intrinsic diversity within complex cell populations, opening up new approaches for the in-depth understanding of highly relevant diseases. However, most of the current methods for their fabrication are based on cumbersome patterning approaches, employing organic solvents and/or expensive materials. Here, we demonstrate an unprecedented green-chemistry strategy to produce single-cell capture biochips onto glass surfaces by all-aqueous inkjet printing. At first, a chitosan film is easily inkjet printed and immobilized onto hydroxyl-rich glass surfaces by electrostatic immobilization. In turn, poly(ethylene glycol) diglycidyl ether is grafted on the chitosan film to expose reactive epoxy groups and induce antifouling properties. Subsequently, microscale collagen spots are printed onto the above surface to define the attachment area for single adherent human cancer cells harvesting with high yield. The reported inkjet printing approach enables one to modulate the collagen area available for cell attachment in order to control the number of captured cells per spot, from single-cells up to double- and multiple-cell arrays. Proof-of-principle of the approach includes pharmacological treatment of single-cells by the model drug doxorubicin. The herein presented strategy for single-cell array fabrication can constitute a first step toward an innovative and environmentally friendly generation of aqueous-based inkjet-printed cellular devices.

Keywords: biointerface; biopolymer; inkjet printing; microarray; single-cell.

Figure 1

Schematic illustration of single-cell array fabrication and application: (a) layer-by-layer printing steps from chitosan coating deposition on glass to EPEG grafting and collagen microarrays patterning and (b) single human cancer cells capture on the printed biointerface, followed by cell treatment with doxorubicin and analysis by confocal microscopy at the single-cell level.

Figure 2

Stroboscopic image of 10 pL (nominal volume) chitosan ink droplet pinching-off at the nozzle at 40 V and room temperature. The drop elongates as a function of time while modifying the drop front (red circles). The arrow in the last step indicates the drop tail (about 800 μm length).

Figure 3

XPS analysis of C 1s peaks for chitosan film before (a) and after (b) EPEG grafting. Tapping mode AFM images of 5 μm × 5 μm area of chitosan film before (c) and after (d) EPEG grafting.

Figure 4

Printing condition for collagen ink ejection: (a) single short-pulse waveform optimized for aqueous inkjet printing at the femtoliter-scale with a pulse length of 1.3 μs and (b) stroboscopic images of collagen ink droplets ejected by a nozzle of a diameter of 10.5 μm at 30 V jetting voltage, recorded from 6 to 61 μs.

Figure 5

Effect of the t_D on the dimension of sessile droplets of 0.08% w/v collagen ink: (a) representative sessile droplet size for each t_D, ejected at 30 V (red) and 40 V (black). The error bars represent the experimental error associated with the diameter measurement via optical microscopy; (b) white-field images of droplets microarrays printed at 40 V and (c) at 30 V. The graph in panel a also shows printing instability regions (double arrows), where breakup phenomena induce droplet fragmentation and satellites formation, as shown in the corresponding pictures of wet droplets arrays (satellites are indicated by arrows in panels b and c). Scale bars 100 μm.

Figure 6

H1975 cell array on collagen patterns of different spot sizes. The collagen arrays in panel a were printed by a 1 pL ejecting-cartridge at t_D values of 0.6, 5.0, and 10.0 μs (left to right), while the arrays in panel b were printed by a 10 pL ejecting-cartridge at t_D values of 10.0 and 23 μs (left to right). The number of cells per spot clearly increases with the spot diameter, starting from a nonspreading condition (22 μm) to obtain cell consortia patterns (50–67 μm). Scale bars 50 μm. The single-cell yield (panel c) was calculated on 8 × 8 spots collagen microarrays (spot diameter 32 μm). The mean percentages were calculated using three replicate samples. Error bars indicate the standard deviation. The biological evolution up to 48 h after the adhesion, distinguished in single-cells, double-cells, and empty spots, counted at 1 h (blue), 24 h (black), and 48 h (red) after the adhesion.

Figure 7

Representative fluorescence images of Dox (red) intracellular localization after 1 h in H1975 cells overlaid on the transmission channel (gray) to show complete cell morphology: (a–d) single-cell arrays and (e,f) conventional cell culture. Framed cells in panels c and e are shown in panels b,d, and f, respectively. Scale bars 20 μm in panels a–d, 50 μm in panels e and f.