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Coculture assays allow the investigation of the role of endothelial cell and mural cell interactions in small vessel development and function. Different setups for coculture can be used to assay questions of interest. We include here methods for direct coculture, indirect coculture, and coculture in a three-dimensional extracellular matrix scaffold for studies of either a simple and direct association between the two cell types, the exchange of soluble molecules, or the interaction within a biomimetic tissue microenvironment.
Cocultures have been used successfully over the years to study the interactions between endothelial cells and cells collectively known as mural cells (smooth muscle cells and pericytes). Two-dimensional (2D) direct and indirect coculture methods in cell culture plates are useful to examine interactions that require direct contact (juxtacrine) or those that are made through diffusion of soluble factors (paracrine). Three-dimensional (3D) techniques involve the use of agarose, matrigel, or a fibrous decellularized extracellular matrix (ECM) scaffold to investigate interactions within a 3D-biomimetic environment. We have investigated through the use of cocultures the effect of heterotypic cell interactions on cell proliferation [1 – 3], migration [4, 5], apoptosis , activation of growth factors , cord formation [6, 7], and Notch signaling . Coculture set-ups have allowed us to examine signaling pathways that dictate the behavior of endothelial cells [2, 4 – 7] during vessel formation, and the maturation and stabilization of neovessels through communication with pericytes [1 – 3, 6, 8].
We describe here three different methods of coculture including direct coculture, indirect coculture using transwells, and coculture in a 3D matrix using an ECM scaffold . The methodologies include detailed culture conditions for primary human endothelial cells and an updated protocol for isolating and culturing bovine retinal pericytes [2, 10].
All materials should be sterilized by autoclaving, filtering, or ethylene oxide sterilization.
Work in a biosafety cabinet. Use a surgical mask to protect yourself from exposure to enzymes.
This work was supported by NIH grants R00EY021624 (JA) and R01EY005318 (PDA).
1Calf eyes yield a higher number of pericytes with a higher proliferative potential than cells isolated from adult eyes.
2Use glass flasks, not plastic, to make dilution.
3Bovine calf serum 12133C from Sigma induces increased proliferation of pericytes.
4Passage at 90 % confluence. Confluence at these passage ratios is achieved in 3–6 days.
5The dish prevents the eye from rolling and maintains sterility.
6Keep the spear moist with PBS to avoid the retina from sticking to the spear.
7The larger vessels in the retina, where mural cells are mainly smooth muscle cells, will be removed with this section. These vessels are visible with the naked eye. The remaining retina in the posterior segment will contain the pericyte-covered micro-vasculature (which is not visible with the naked eye).
8Be careful not to remove any underlying pigmented tissue (i.e., the tapetum lucidum) or the retinal pigment epithelium.
9If any loose pigmented tissue is observed, carefully remove it by aspiration (do with care as it can result in the aspiration of the entire retina).
10Wash medium can be aspirated (instead of poured), but once again should be done with care to avoid the aspiration of the retina.
11If “contaminants” are still visible after the EDTA treatment, keep the cells in BRP culture medium for a few more days and then repeat this step.
12The most common “contaminants” are endothelial cells and fibroblasts. If left unattended, endothelial cells can become the 70 % of the total cell population. Fibroblasts hardly reach populations higher than the 10 % of the total cell population.
13Arrest of cell growth is only necessary for direct culture, where endothelial cell growth is determined by final cell number including both endothelial cell and pericyte total count. This is not needed in coculture since cells are physically separated.
14Interactions between HRECs and BRPs are observed after 2–4 days.
15Image scaffold without sectioning as cells tend to detach from thinner sections during the washes of staining procedures.
16Imaging of scaffolds works very well with upright fluorescence microscopy. Note in Fig. 4 the out of focus (i.e. out of plane) location of several cells, due to the 3D character of this coculture method. vWF antibody (catalog number A0082) (Dako) at a 1:100–200 dilution clearly stains endothelial cells (Fig. 6b). We have not obtained good results working with lectin for staining of endothelial cells on SIS membranes. For pericytes, SMA antibody (catalog number C6198) (Sigma) works very well at a 1:400 dilution (Fig. 6b). Alternatively, live cells can be prelabeled before seeding on the chamber slide using a PKH26 cell membrane linker kit (catalog number PKH26GL) (Sigma) to label endothelial cells red and a PKH67 cell linker kit (catalog number PKH67GL) (Sigma) to label pericytes green. To label 1 × 10 6 live cells, detach cells from flask and wash twice with serum-free media. Add 0.5 μL of PKH dye to 50 μL of diluent-C and vortex. After second wash, resuspend cells in 50 μL diluent-C and mix with dye solution. Incubate for 3–5 min at room temperature. Add complete media (with serum) to quench reaction and wash two to three more times in complete media. Resuspend and culture as desired.