Monday, June 29, 2009

Chocolate cells

After finding the 1696 article on medicinal uses of chocolate I was chatting with Kira in the coffee room at work. We got to a point where we decided that may be we should try feeding chocolate to adipocytes (fat cells), though now I can't remember what all the connections were. Naomi is an expert on adipocytes and kindly said that we were welcome to use some of these cells that she would extract from stored tissue. The aim would be simple, we'd incubate the cells in the presence of some chocolate and hopefully they would expand in size as they took in the fat from the chocolate. A nice circular idea whereby the cells, which had been discarded to remove 'fat' would get fed fat. Kira was also interested in embellishing them with gold or other precious objects. After some research I reckoned that the easiest way was to add so cationic gold (positively charged) that would adhere to the outside of the cells through an electrostatic interaction as the cells are slightly negatively charged. Rachel generously said we could visualise them on the scanning electron microscope (SEM) too, which meant we'd be able to see the gold and the structure of the cells.

So first off Kira and I spent the afternoon trying to get chocolate into solution. I had hoped that just warming it to 37°C would be OK but this had a tendency to sit as gloop at the bottom of the tube. After a lot of fiddling we decided upon grating some high cocoa solids content chocolate (85% Green & Blacks in these experiments but later Kira sourced some French 100% from Cortes Ingles in Valencia) and melting and mixing it into standard serum supplemented media.

Adipocytes in culture

Then Naomi kindly supplied us with some cells, but the transport between labs caused massive infection and they all died, so we decided to go to them and spent a couple of days over at Aston. We helped to process the tissue and extract the individual cells before adding the chocolate solution. We gave them an overnight incubation to see if they would take up any fat. The next morning the chocolate stimulated cells certainly looked different from the non-chocolate stimulated ones, but it was more that they had a fine coating of particulates rather than having swelled in size.

To add the gold we had to tether the cells. They were floating atop the liquid in which they were growing so using poly-D-lysine coated coverslips (making use of electrostatic interactions again) we captured the adipocytes. Once isolated we coated with the gold solution, taking only 10min, before fixing in preparation for the SEM. Further dehydration was required, as the SEM works under partial or near vacuum, before we could 'see' the fat cells. Rachel and I had a quick look-see under the low vacuum instrument before using the higher powered one over in the microscopy suite with Naomi. And ta-dah!!! We found lots of things, mostly bits of chocolate particulate but also a fat cells that had adhered and then subsequently lost most of its cell contents. It looked like the nucleus had been ripped from the cells, looking like a fried egg missing its yolk. Using back scatter electrons we could also see the gold - looking like constellations sprinkled across the membrane!

Whole fat cell missing its insides

So what does all this mean I hear you cry? Well, according to Naomi, its the first time anyone's tried to visualize adipocytes under SEM and hopefully we can use further experiments to characterise these cells. So from a scientific point of view this 'Friday afternoon experiment' has been incredibly useful. On other fronts, this has been totally mind blowing - I've never used such high powered microscopes before and seeing a cell in such high magnifications is really quite something, and add in the gold stars/sparkles and I'm very excited! So the cells didn't do what we expected, but then as Prof Einstein said 'If we knew what we were doing it wouldn't be research' and as such trying something different has brought out something new and exciting, not to say broadened all out horizons.

Magnification of the mambrane using back scatter electrons to see the gold labelling, like little stars

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