A helping hand for morphologists

3D Scaffold for Cell Culturing – scanning electron microscopy

I have always quite enjoyed ‘doing’ tissue culture. Sitting at the laminar-flow cabinet, piles of culture dishes, changing media – passaging, what a fabulous word that is! There is something very pleasant about the manual, repetitive side of tissue culture that I have always found quite … well reassuring. The cultures are still alive; all is right with the world!

Now, as a morphologist, I did find that the view of a well or plate (as seen down a stereo light microscope) somewhat disappointing. However, the move to electron microscopy, especially transmission electron microacopy (TEM), brings its own set of culture-related problems. This is a necessary step though, of course, if one is interested in the ultrastructure of the in vitro cell and particularly if one is interested in cell-to-cell connections.

‘Macula and fascia adhaerens’, ‘desmosomes and hemidesmosomes’, ‘intercalated discs’, ‘tight junctions’ and ‘gap junction’s – the world of cellular junctions is a fascinating and important one with its own terminology. Understanding how these junctions function is a vital part of understanding how cells become tissues. And when cells are in culture they are an important measure of how well the culture is replicating the in vivo situation.

The problem as far as TEM is concerned is that cells in a tissue culture dish generally lead a pretty two-dimensional life; they are actually often (slightly) erroneously called monolayers. And if you want to embed these cells, for subsequent sectioning and electron microscopical investigation, you have two choices. You either attempt to section parallel to the surface of the culture in the hope of getting a decent filed of view or shrug and make do with the rather more limited information obtainable from a section perpendicular to the bottom of the dish. This is after you have gone through the tricky business of separating your cultures from the culture dish after the protracted process of resin embedding. And then you have to get handy with your hacksaw to find the part of the culture that interests you. And then mount that! Spotting cell junctions in sections isn’t easy!

One of the best ways of investigating cell junctions in TEM is to use freeze fracture replication (FFR). This involves freezing the sample (without fixation if possible) and then fracturing it (maybe with a knife!) before making a shadowed platinum/carbon replica of the surface. After digesting away the original biological material the ‘metal’ replica in the microscope can produce a fabulous detailed image of the junction. This is because the fracture plane passing through the frozen sample splits the lipid bilayer of cell and organelle membranes, leaving the membrane proteins as small isolated ‘intramembranous particles’; especially impressive when passing through a junction. But try to imagine doing this process with a cell culture! It is not easy, believe me. I have grown cells on cover slips and tried to fracture frozen cover slip sandwiches. I have slammed culture dishes against helium-cooled copper blocks and tried scraping them with knives. There must be easier ways.

And now we have 3D culturing. This, mainly, is tissue culture carried out on matrices that mimic the arrangement of the extracellular matrix allowing cells to grow into them. And this is exciting stuff!

Most 3D cell cultures seem to be much closer representations of the in vivo state than 2D cultures. 3D permits better assays and more physiologically relevant results. Matrices can be made of highly porous polystyrene scaffolds and these allow for the applications of similar techniques and assays currently in use with conventional 2D cultures. These matrices can also be resin embedded for TEM. Results seen very encouraging and it looks like cell-to-cell junctions are both easier to find and more common (another good indication of the 3D culture’s physiological ‘appropriateness’. Biomimetic scaffolds and coatings can also be obtained e.g. collagen and laminin and basement membrane extract. These are very physiologically pertinent materials and also particularly well suited for sectioning for electron microscopy. Again results look very encouraging and there is an additional bonus: as the matrices are in the order of 200 microns thick, it should be possible to carry out FFR painlessly and with rather spectacular results! This looks like the answer to a morphologist’s tissue culture dreams!

So if anybody is feeling flush: could I have a grant please!