nCS1 – Extracellular vesicles Application Note
Microﬂuidic Resistive Pulse Sensing (MRPS) validated as a rapid and practical method for evaluating EV enrichment techniquesIntroduction
For extracellular vesicles (EVs) to deliver on their promise as commercially valuable therapeutics requires improved techniques for their isolation and enrichment. However, the development of these techniques has been hindered by a lack of practical technologies for accurate EV quantiﬁcation.
In this study, Microﬂuidic Resistive Pulse Sensing (MRPS) is validated as a rapid, practical tool for characterizing the size exclusion chromatography (SEC) method of EV puriﬁcation.The MRPS technique
Microﬂuidic Resistive Pulse Sensing (MRPS) has recently emerged as a powerful new technique for measuring the size and concentration of extracellular vesicles. MRPS uses electrical sensing to count and size particles directly and one-by-one, without the use of any optics or mathematical conversion from diﬀusion behaviour to size. As a result, MRPS is independent of the material properties of the particles, and measures samples accurately no matter their polydispersity.
These fundamental strengths of the MRPS technology are particularly important for measurements of extracellular vesicles, since EV samples have low index of refraction contrast with their surrounding medium, and as biological particles are inherently polydisperse. A direct comparison of Spectradyne’s nCS1 to Nanoparticle Tracking Analysis (NTA) is shown in Figure 1 below, and shows the misleading results that can be obtained when using optical methods to quantify EVs.
Figure 1. Size and concentration of urinary vesicles measured by Spectradyne’s nCS1, tunnelling electron microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). The size distribution as measured by Spectradyne’s nCS1 agrees exactly with the gold standard, TEM, showing an approximate power-law distribution of size vs. concentration that is commonly found in EV samples. The signiﬁcant loss of sensitivity of optical methods to small particles is highlighted in the NTA data, which shows a false peak at 130 nm and a discrepancy in concentration of 5 orders of magnitude at 50 nm diameter.Practical EV Quantification
Spectradyne’s nCS1 delivers signiﬁcant practical beneﬁts that make it ideally suited for routine and quantitative EV analysis:
- Only 3 microliters sample required
- Results in minutes
- Pre-calibrated, single-use cartridges
- No cleaning required between runs
- No user-adjustable parameters
For this study in particular, the small sample volume required for analysis was a critical enabler of the measurements, since only a few tens of microliters of each sample was produced for a given set of process conditions.Methods
DMSC25 mesenchymal stem/stromal cells were cultured to 70% conﬂuence in growth media. Cells were then cultured for 2 days in chemically deﬁned, vesicle-free medium. Conditioned medium (50 ml) was then concentrated by sequential ultracentrifugation and resuspended in SEC buﬀer and applied to a GE NAP-5 column for further puriﬁcation. Fractions were collected and total EV concentration measured using MRPS on the size range of 70 nm – 400 nm. UV absorption, an orthogonal technique to MRPS, was used to quantify the total protein in each fraction. Results of each of the techniques were compared.Results
As expected, MRPS measurements showed a clear peak in total particle concentration in column fractions 3-5, in which EVs are known to elute. Importantly however, particle size distributions obtained by MRPS showed that each eluted fraction contained a broad range of particle sizes spanning the full measured range of 70 nm – 400 nm, and that elution in diﬀerent fractions did not signiﬁcantly aﬀect the size distribution proﬁles.
Figure 2. Concentration vs size distributions for each eluted fraction measured with the nCS1. In each fraction, a broad distribution of particle sizes is observed whose proﬁle is independent of elution time.
Signiﬁcant diﬀerences were observed between the two techniques for measurements of the non-EV fractions: a peak in total protein was detected in fractions 7 and 8, while no corresponding peak in particle concentration was observed, suggesting the protein in these fractions was not bound in the form of solid particles (Figure 3).
Figure 3. Measurements of nanoparticle concentration correlate strongly with total protein in fractions 1-6, in which exosomes are expected to elute. In fraction 7, nCS1 measurements indicate that protein eluting from the column is not associated with particles in this size range, but may instead be free in solution.Conculsion
MRPS was validated as a practical tool for characterising EV puriﬁcation methods. In addition to demonstrating good agreement with orthogonal techniques, MRPS provided important insight about the limitations of SEC as a size-separation technique—broad particle size distributions were observed in each fraction. MRPS is therefore an important tool for accurate characterisation of EV puriﬁcation methods as they are being developed.