Fig 1.
Overview of a 6-well plate and the proposed solutions.
Left: Non-adherent PEG coated surface in the central region of the well, where flow is disturbed. This solution allows to selectively grow cells in the periphery where flow is physiological (laminar pulsatile). After cell culture, a more homogeneous sample can be harvested for various biochemical assays (Western blot, RT-PCR, FACs, -omics) or stained using our second solution, shown on the right. Right: Multi-staining platform insert, which can be applied after sample fixation. Five individual pockets, one in the center and four in the periphery, allow up to 16 parallel stainings, considering four stainings per pocket and a comparison between a peripheral pocket and the one in the center.
Fig 2.
Step-by-step description of the method to block cell adhesion in the center of the well using a PEG-hydrogel.
(1) Placing a drop of TMSPMA at the center of the well. (2) Covering the droplet with a glass coverslip for incubation. (3,4) Incubation with PEG Mw3400 and Irgacure 2959 using a fresh coverslip and curing through UV exposure. (5,6) Coating of the well with a 0.2% gelatin solution and washing. (7) Sterilization with UV light. (8) Cell culturing on an orbital shaker.
Fig 3.
PEG-gel patch preventing cell adhesion in the center of the well.
SMC-EC co-culture in a 6-well plate with a patch of PEG-gel in the center. a) The top half of the well is shown as a schematic, while the bottom half shows ECs stained with VE-cadherin immunostaining. Widefield fluorescent images were stitched into a large mosaic. Scale bar 5 mm. b) Zoomed-in images of the white square showing SMCs stained with cell tracker (left) and ECs with VE-cadherin immunostaining (right). Scale bar 500 μm.
Fig 4.
Assembly of a multi-staining platform insert.
a) Overview schematics depicting the different pieces of the staining platform and assembly order. b) Assembly steps: (1) The pressing piece with the gasket is inserted into the well and placed directly on top of the fixed cells; (2) the locking frame is placed on top, and the centering lugs ensure proper alignment. The four locking pieces were previously inserted into the locking frame with the sharp edge facing upwards and the spring plungers inserted into the holes of the locking frame; (3) using circlip pliers, the retaining ring is compressed to fit into the groove of the locking frame, pressing the locking pieces out towards the wall of the well; (4) the spring plungers are tightened to apply vertical force; (5) the individual compartments are filled with staining reagents. c) Photographs of the different pieces and materials used for the assembly of a multi-staining platform insert. d) Photograph of the aluminum mounting frame, containing a glass-bottom 6-well plate with an inserted multi-staining platform.
Fig 5.
VE-cadherin and DAPI stainings performed with the multi-staining platform.
A confluent EC layer was exposed to shear on an orbital shaker with 2 mL fluid and 135 rpm rotation speed, for three days. Images were acquired using a widefield fluorescent light microscope. VE-cadherin immunostaining (green) shows the plasma membrane of HUVECs. DAPI staining (blue) shows cell nuclei. a) and b) show the border of one pocket and silicone grease that is used for sealing. c) Shows an image of cells in the central compartment, which are randomly aligned because they have been exposed to disturbed flow. d) Shows an image of cells in the bottom peripheral compartment, which are aligned along the flow direction.
Table 1.
Advantages and disadvantages of orbital shakers and how we addressed some of the associated challenges.