Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for sheer stress application using the orbital shaker model. A common way to study effects of flow on endothelium is to culture endothelial cells in multi-well culture plates on an orbital shaker. The orbital shaker induces a wave that rotates around the well.

Cells at the center of the well experience the kind of flows that are thought to lead to atherosclerosis. Cells closer to the edge experience flow that is thought to be protective. Properties of the cells in each region are assumed to depend on the sheer stresses that they experience.

However, there is a potential floor in this logic. Endothelial cells release mediators in a flow-dependent fashion. These mediators will reach a uniform concentration in the swirling medium and will therefore affect cells in regions other than the one where they were released.

That may corrupt or hide true effects of shear on cell behavior. The effect is not limited to the swirling well system. It may occur even in simple models, like the parallel plate flow chamber.

It can be avoided by growing cells in only one part of the system. Here, we describe methods for permitting cell adhesion only in the center or only at the edge of the swirling well. Fabrication of stainless steel module.

Fabricate the stainless steel module from a grade 316 stainless steel according to an engineering drawing provided. 3D printing on PDMs. Prepare a 3D CAD model of PDMs mold using SOLIDWORKS according to the engineering drawing provided.

Export the CAD model to an STL file and import the STL file into Cura 2.6.2. Slice the model into layers with a print speed of 50 millimeters per second and infill density of 60%Export the file as a GCode. Uploaded it to an Ultimaker 3D printer for printing.

Use polylactic acid as the printing material. Casting of PDMS ring. Mix the PDMS base and curing agent well with the ratio of 90.9%base and 9.1%curing agent.

Pour the well-mixed solution into the 3D-printed mold. Remove bubbles in a vacuum degassing chamber. Cure it for one hour in an 80-degrees furnace.

Allow the PDMS ring to cool to room temperature. Then remove the cured PDMS ring carefully from the mold. Preparation of 1%Pluronic F-127.

Weigh out five grams of Pluronic F-127. Pour it into a glass bottle. Then add a hundred mil of Milli-Q water into the glass bottle.

This gives a 5%Pluronic F-127 solution. Ensure that all Pluronic F-127 powder is submerged in the water. Close the cap and autoclave it using a liquid sterilization cycle program.

Let the solution cool to room temperature before use. Add 10 mil of 5%Pluronic F-127 solution to 40 mil of autoclaved Milli-Q water to make 1%Pluronic F-127 solution. Perform the dilution in a biosafety cabinet hood.

Store both 1%and 5%Pluronic F-127 at room temperature. Coating of 6-well plate. Autoclave stainless steel module, PDMS ring, and tweezers before use.

Perform all subsequent procedures in a BSC hood and observe aseptic techniques to ensure sterility. Place the PDMS ring in a 6-well using tweezers. Use the external rim of the PDMS ring to align the PDMS ring concentrically within the well.

Note, only non tissue culture-treated well plates should be used. Place the stainless steel module on top of the PDMS ring using tweezers. Insert the tips of the internal retaining ring pliers into the grip holds of the retaining ring.

Squeeze the holder to reduce the diameter of the retaining ring. Put it into the 6-well, press it firmly on the stainless steel module, and release the pliers to secure the PDMS ring in the well. Add one mil of five microgram per microliter fibronectin into the center or the edge of the well, depending on the region of interest, through the opening of PDMS ring and stainless steel module.

Swirl the plate to ensure the fibronectin solution covers all the region of interest. Incubate for 30 minutes at 37 degrees in a humidified incubator under 95%air and 5%CO2. Remove the fibronectin solution from the well.

And now wash twice with PBS. Once done, completely remove the PBS from the well. Remove the retaining ring, stainless steel module, and PDMS ring from the well.

Add 1.5 mil of 1%Pluronic F-127 into the well and incubate for one hour at room temperature to passivate the uncoated surface. Remove the Pluronic F-127 solution from the well and wash three times with PBS. Once washed, use the coated well immediately or store at four degrees for up to two weeks with the layer of PBS in the coated well.

Seeding of endothelial cells. Count cells using a hemocytometer and seed 180, 000 cells into a coated 6-well plate in 1.5 mil of pre-warm cell culture medium. Shake the well plate laterally to ensure that the cells distribute evenly in the well.

Leave it in a 37-degrees humidified incubator under 95%air and 5%CO2 overnight. Shear stress application using an orbital shaker. Cells should reach confluence after three days of growth.

Replace the medium with 1.9 mil of pre-warn cell culture medium to achieve a height of two millimeters. Place a well plate on the platform of an orbital shaker in a humidified incubator and swirl it at 150 RPM for three days. Optional, after two days of shear, cytokines can be added to the cell culture medium to investigate the interplay between cytokines and sheer stress.

After treatment, shear the cells for another day. In this study, TNF-alpha was used to activate the cells. Perform analysis after three days of shear stress application.

Microscope images show that Pluronic F-127 prevented HUVEC adhesion to the region without fibronectin coating. No HUVECs were attached to the part of the well surface that had not been pretreated with fibronectin prior to passivation of Pluronic F-127 after 24 hours, in figure A, and 72 hours, in figure C, of growth. Without Pluronic F-127 passivation, HUVECs were attached to the surface without fibronectin, 24 hours after seeding, in figure B, and are proliferated further by 72 hours, in figure D.The scale bar is equal to 500 micrometers.

Nuclear Red stain shows the morphology of sheared HUVECs, A, in the center, and B, at the edge of a full well. Scale bar is equal to a hundred micrometers. A and B also show cell outlines delineated by immunostaining of ZO-1.

Note the alignment and elongation of cells at the edge but not at the center. It shows no significant difference in nuclear shape index, indicating roundness, between HUVECs grown in full well and segmented wells were seen for untreated or TNF-alpha-treated HUVECs. Cells were more elongated near the edge of the well.

A tendency for a greater elongation in TNF-alpha-treated HUVECs was not consistently significant across locations. No significant difference was observed between full and segmented wells in density number of A, untreated, and B, TNF-alpha-treated HUVECs at different radial locations. In both cases, there were more cells per unit area at the edge than at the center of the well.

In conclusion, the method we have outlined here allows you to grow cells in just one region of the swirling well or indeed, of any plastic-based cell culture system. That means cells in one part of the well experiencing one type of flow are not influenced by mediators released from cells in another region experiencing different flows. Not only that, we can look at cells grown in one region with or without cells being grown in other regions.

That allows the effects of such soluble mediators to be demonstrated. Testing the effects of conditioned medium on naive cells permits the same thing. By analyzing condition medium, the mediators can be identified.