Non-Fouling Micro-Patterned Surface for the Study of Pore Contribution in Cell-Substrate Interactions

Optimization of the PLL-g-PEG coating on SiO2 substrate:

The first goal was developing an efficient method for PLL-g-PEG coating which provides a high density of PLL-g-PEG grafting on SiO2 substrate and effectively prevent cell adhesion. To achieve this goal, different concentrations of PLL-g-PEG in 10mM HEPES solution ranging from 0.1 to 0.9 mg/ml (0.1, 0.3, 0.5, 0.7, and 0.9 mg/ml) were placed dropwise on SiO2 substrate for different periods of time ranging from 30 to 115 minutes with increments of 15 minutes. The highest PLL-g-PEG grafting, which could efficiently prevent BSA adsorption on SiO₂ substrate, was achieved using 0.5mg/ml PLL-g-PEG solution for 75 minutes.

 

Development of non-fouling micro-pattern on SiO₂ substrate

Photolithography steps were followed to obtain a non-fouling micro-pattern on the SiO2 layer. this pattern was visualized using a coating of f-BSA above the patterned sample.

 

 

Cell adhesion and morphology on PLL-g-PEG coating

Preliminary experiment was required to verify that the developed coating method is effective, and PLL-g-PEG coating is efficiently non-fouling for cells. Cell culture on PLL-g-PEG coated substrate verified that this coating can effectively prevent cell adhesion.

 

Cell spreading and F-actin quantification

HUVECs were seeded on patterned substrates, and cells were stained using DAPI and Phalloidin after 24 h to visualize cell nuclei and stress fibers, respectively.

While mean cell area on SiO2 is around 1250 mm², this value has drastically decreased on patterned samples and this number goes to around 850 mm². This cell size reduction can originate from the frequent disruption on the SiO₂ substrate by PLL-g-PEG islands. However, it was shown that the same pattern of disruption on SiO₂ membranes by pores did not lead to cell spreading reduction.⁹ This contradictory behavior will be further discussed.

Mean fluorescent intensity of stained cytoskeleton in each cell was also measured using ImageJ software to quantify stress fiber production on the patterned and unpatterned substrates. It has been found that HUVECs can form less actin fibers on disrupted surface of patterned samples

Fibronectin fibrilogenesis

Fibronectin fibrilogenesis was also evaluated on the patterned and unpatterned samples. After cell culture for 24 h, cells were stained using 1:100 dilution of AlexaFluor 488 conjugated anti-fibronectin, Clone FN-3. The fibril length was found to be shorter on the PEG islands  compared to the control SiO2 due to their substrate disruption.¹⁰

 

Migration Assay

After 3 h of cell seeding on the samples, HUVECs were labeled using SiR-DNA which was uptaken by the cells within 1 h. Nuclei of labeled cells were tracked by obtaining images every 15 min for 24 h through SiR-DNA probe. As it has been shown in Figure 5, mean cell migration speed on the PLL-g-PEG-patterned are slightly more than migration speed on the unpatterned samples.

 

Comparison between PLL-g-PEG-patterned and porous SiO₂ substrates

These diverse behavior changes on the same pattern of disruption can stem from the fact that the surface disruption is not the only contributing factor in the mentioned cellular behavior on a porous membrane. In order to understand the mentioned observations, we should notice that a non-fouling patterned substrate can be considered as a disrupted surface, while a porous membrane is considered as a disrupted surface with pore edges.