Exosome Capture Update: A Tale of Two Pumps

The week before last, I finally switched back to my exosome experiments with some hope of repeating my January successes with my new clampable format and two pump system. After failing for several hours (and breaking 7 membranes), I was finally able to get the system to work and get a sample for SEM.

(A quick side note on my failures that may prove to be important. I think that there may be a lifetime to using the PDMS chunk multiple times. I had used the same piece for at least 10 experiments and on this experiment, it did not seem to want to work, so I completely started fresh and it finally worked. There was a sealing issue that I was having and a new PDMS piece seemed to solve the problem. This may be important when considering reusing some components of the devices, so it’s something to think about if you want to start working with them.)

Now back to the good stuff. I was able to pass my plasma sample (from the frozen stock that I have) over the membrane and successfully fix and extract it. I then put it through a critical point drying procedure, as I thought that this may help improve stability. It turns out that something in this process did, and for the first time ever, I have a fully intact membrane to look at. (This sample is still sitting in a box on my bench, fully intact, so I could theoretically do further imaging.) However, as it was also the first time that I had fixed anything on the surface with these experiments, the results were quite interesting.

As we can see in Figure 1, there are what appear to be exosomes on the surface of the membrane. I did some measurements of the particles (the white stuff, which means it’s organic) and they were of the appropriate size range that we would expect and have seen. One thing to note is that the pores are approximately 50 nm, which is 20 nm larger than is given for the size.

Figure 1: NPN membrane with exosomes on the surface. Note how the pore size measurements are 50 nm.

I’m not entirely sure as to why the pore diameters are larger than spec, but I do know that previous chips had the appropriate diameter pores, so there must be another explanation for this phenomenon. The particles appear to have a morphology similar to exosomes, but they are not lodged in the pores as we have seen previously. This may be an artifact of the two pump system and indeed some exosomes may well be pulled through the pores, but for a sample size of n = 1, it is hard to say for certain why they are not in the pores.

One other very interesting feature of this sample was the presence of large, string like structures all over the surface. In fact, it was very difficult to find a place that did not have these strings covering it. As you can see in Figure 2, they appear almost like strands of pearl necklaces, with “beads” along their length. What these are is a mystery, but it is possible that they could be neutrophil endothelial traps that were present in the plasma sample or cell free DNA that is wrapped around histones. Since I’ve never had a fully intact membrane or fixed the sample before this result is very intriguing and will definitely require more exploration.

Figure 2: Possible NET or histone bound cfDNA from a frozen plasma sample.

This sample provided very interesting results and they all are very important to investigate further. Indeed, there are many components in these plasma samples that may be of interest and perhaps we just haven’t seen them before because I have not been capable of rescuing a membrane intact. However, given this success I believe that we are going to start seeing a lot more interesting things on the surface that we are capturing, especially with the two pump setup.

I am currently working on another sample where I am trying to confirm the identity of these particles and prove that they are indeed exosomes. I performed immunogold labeling of one of my samples and on Wednesday I’ll be attempting a FIB lift-out of part of the membrane for TEM and confirmation of the presence of the immunogold label. If this is successful, then we will have proven that we can indeed capture exosomes, so fingers crossed! I’ve included a couple more images of these results, but for now stay tuned for a TEM update!