Last month I wrote a post about exosome internalization by recipient cells. One of the topics I discussed was the lack of good quantitative data in the exosomal field, and what the current data tells us about the efficiency and capacity of exosome-mediate cell-to-cell communiation.
Today I came across an interesting paper in which the researchers try to get quantitative data of exosome secretion by the donor cells.
The paper, published in the journal “Small”, describes a unique method to collect exosomes from single cells. The idea here is to plate the cells through a mesh with small holes, so statistically only one cell enters and rests at the bottom (which they verify microscopically). They then remove the mesh and place on top of the cells a glass slide coated with antibodoes against known exosomal markers. The glass is placed 100micron above the cells. They then image the cells while simultaneously collect exosomes over time.
The exosomes bound to the glass are then labeled by quantum dots and the number of Qdots for each cell location is counted under the microscope. [The authors don’t mention if and how they verify that there is only one Qdot per exosomes. For clarity, lets assume also a 1:1 ratio].
They tested and show that the maximum capacity of of the exosome-Qdot complex in the 100x field of view of their microscope was ~1500 Qdots. To stay in range, they collected exosomes for 3 hrs, so that the highest measured level was ~500 exosomes. They measured the background signal (of empty places in the mesh) was about 3% of the signal, probably due to exosome diffusion side-ways.
And here comes the interesting part: they measured exosome secretion rate of three cell lines. Two lines (MCF7 & MDA-MB-231) showed exosomes secretion rate of ~60-65 exosomes per cell per hour. The third line (MCF10A) was more prolific, secreting ~170 exosomes per cell per hour.
In the previous post, I calculated that the researchers added ~100,000 exosomes per cell to the recipient cells. If the numbers from this paper here are correct, it is the equivalent to the number of exosomes secreted by 588 MCF10A cells for an hour.
To me, this says that those assays that use isolated concentrated exosomes with ratios of 1000s to 100s of thousands of exosomes/cell, are really way too high compared to what might actually happen.
Even if the paper here is under-estimating the number of exosomes/cell/hour – how much underestimation are we talking about? 2-fold? 10-fold? The assays described in the preious post used ~500-fold more exosomes that anticipated by the results here.
Of course, these are differnt cell lines (HEK293 were the cells used in the other paper), but how much differnc can there be? This needs to be tested.
A detailed protocol can be found at Bio-protocol, here.
Anyway, to me, this shows that the exosome field MUST obtain more quantitative data.
Chiu YJ, Cai W, Shih YV, Lian I, & Lo YH (2016). A Single-Cell Assay for Time Lapse Studies of Exosome Secretion and Cell Behaviors. Small (Weinheim an der Bergstrasse, Germany) PMID: 27254278