I think that diffusion of small molecules through the 0.4 µm pores in both directions should be possible. We use membranes with 3 µm and 8 µm pore size for transmigration assays with monocytes, endothelal cells (both in the upper chamber) and MCP-1 as chemoatractant in the lower chamber. Even when the endothelial cell layer is 100% confluent and has a cobblestoned apperarance the MCP-1 in the lower chamber induces transmigration. That means that MCP-1 reaches the monocytes in the upper champber by passing the pore membrane an the tight endothelial cell layer.

Hi,

it depends on the conditions of your monolayer. E.g. no diffusion of medium occurs in a confluent monolayer of epithelial cells sealed by tight junctions. Thus if your endothelial cells achieve sealing at confluence molecules in the lower compartment will contact only the basal side of endothelial cells

In addition, the cross talk between cells in the upper and lower compartment will be also influenced by the culture conditions. I grow epithelial cells in the upper compartment in air/liquid interface conditions. Obviously under these conditions there is no diffusion between the upper and lower compartment

Nicholas,

I am not sure I fully understand your question. I try anyway.

If you grow one cell type in the insert, and the other cell type (submerged) in the lower compartment of the well, you will only address interactions that involve soluble/excreted factors. You will miss the cell-cell interaction aspects.

I know of test formats involving endothelial cells, where these endothelial cells are grown on the down-site of the insert. They attach sufficiently strong to allow for mechanistic studies and assay development for e.g. toxicity testing. The second cell type (in the example lung alveolar cells) in your case muscle cells are grown in the insert. This allows proper cell-cell contact provide that you use inserts with a larger poor size (e.g. 0.4 um). This poor size allows for cell migration but will not result in cell leakage provide that the endothelial cell barrier is closed.

Whether the endothelial cells in this setup are able to interact with the apical side of the cells in the insert I do not know, but the question is whether this is relevant for real life. I do know that the endothelial cells are required to get a proper alveolar barrier. This seems to indicate that both cell types interact/communicate sufficiently to establish an in vivo relevant alveolar-endothelial barrier. Furthermore, some cytokines can be detected aboce and/or underneath this barrier.

My experience comes from the test developed by Maria Iris Hermanns at Mainz University. I am sure there are more of these tests around.

Alternatively one could consider to grow both cell types inside the insert, but this I would consider as physiologically irrelvant for the cell types you intend to work with.

I think that diffusion of small molecules through the 0.4 µm pores in both directions should be possible. We use membranes with 3 µm and 8 µm pore size for transmigration assays with monocytes, endothelal cells (both in the upper chamber) and MCP-1 as chemoatractant in the lower chamber. Even when the endothelial cell layer is 100% confluent and has a cobblestoned apperarance the MCP-1 in the lower chamber induces transmigration. That means that MCP-1 reaches the monocytes in the upper champber by passing the pore membrane an the tight endothelial cell layer.

Andreas,

good to know. Thank you for this comment.

The monolayer of endothelial cells cultured on inserts is permeable for large size molecules, e.g. fluorescently labelled dextran. Pore size 0.4µm is large enough not only for the transport of the molecules, but even for viruses (e.g. 0.1µm filters are used to eliminate KSHV virions in culture).

I think a real co-culture of endothelial cells and muscle myotubes is difficult because of different medium requirements. Endothelial cells are very sensitive in this regard.

I want to thank all of you for your input, this has been helpful. I initially considered growing both cell types together, but as has been mentioned, this is not only physiologically irrelevant, but also probably impossible due to the different medium requirements. After some consideration, I think that if I grow the ECs in the lower compartment, my system will work. I won't have to be concerned with what passes through the tight junctions and what doesn't, and once myoblasts fuse, they aren't really a monolayer anymore.

Hi

Doń.t Forget that endothelial cells are polarized when they are confluent. So,it´s mean that in your configuration ,sécrétion of your muscle myotubules Will reach the luminal side of the mono layer instead of the abluminal and that is not very physiologicm

Nicholas, keep us informed.

I disagree Romeo. If we were talking about smooth muscle cells I would agree, however, I am using skeletal muscle myotubes. In vivo, these would be spatially distant from endothelial cells, and any crosstalk would occur through the bloodstream. This is my assumption, at least. At any rate, I am more interested in the effect of factors originating from the endothelial cells affecting the myotubes, rather than vice versa.

I totally agree with your remark and I was telling the same in my last comment.

But In your configuration ,the problem will be the same. If your EC are in the bottom of the well,if the Secretion is polarized,all your secreted molecules could be trapped in the basal matrix and then never reach your myotubules.

You are also be aware about 2 others problems

1 - only free concentration of a factor can act on a receptor,so try to use essential medium without serum to avoid plasma proteins binding

2- to be sure that the interactions are specific and not due to a modification of the medium(metabolisation,acidification etc) by the culture of two cell types, you need a control with an other cell types (SMC or fibroblasts instead of EC

Thank you Romeo, the addition of a control is a good idea, and we do typically use a reduced serum medium for experiments, although not serum-free. Regarding the configuration, I think physiologically, anything secreted toward the basal side of the ECs would never reach a skeletal muscle cell, so I am only interested in factors coming from the luminal side, that might be expected to be released into the blood.

Be aware of increased apoptosis rates of endothelial cells if you reduce the serum content in the medium over longer time points.