Using Beckman Coulter Instrumentation for Exosome Isolation and Characterization

Derived from the late endosome, exosomes are tiny microvesicles that are usually described as being less than 120nm in diameter. All types of cells release exosomes, which have been shown to play a major role in cancer metastasis.

While the biological function of exosomes is yet to be fully described, characterization and analysis of these small microvesicles is a quickly evolving research area. Lipids, proteins, and microRNA (miRNA) present in exosomes can regulate many genes of interest.

According to studies performed recently, exosomes can act as biomarkers for future diagnostic and clinical use in cancer and numerous other human diseases.

Exciting new study results have suggested the importance of exosomes in autoimmune syndromes, cardiovascular disease, neurodegenerative disorders, including Parkinson’s disease and Alzheimer’s disease, and infectious diseases such as diphtheria, tuberculosis and HIV.

In order to develop this field, a better and more efficient means of isolating and characterizing exosomes and other EVs is essential and experts have recently requested that standardized techniques are established.

EV isolation is a very laborious process, with rounds of differential centrifugation required, as well as a density gradient centrifugation step for acquiring highly purified vesicles. A standardized technique for genetic profiling of encapsulated miRNA is one of the downstream challenges involved.

This article demonstrates a workflow in which automated Biomek techniques were used for centrifugation layering and fractionation, total RNA extraction, amplification of cDNA, and clean-up for next generation sequencing (NGS). NGS results are given for both cancerous and benign cancerous colon cell lines.

Materials and methods

Preparing exosome-depleted media

Ultracentrifuged media is prepared by adding 500mL of standard HI-FBS equally to 6 Beckman Coulter Ultra-Clear 94mL centrifuge tubes with an adapter and then placing these tubes in a Beckman Coulter Type 45 Ti rotor, and spinning at 120,000 x g for 18 hours at 4˚C in a Beckman Coulter Optima XPN ultracentrifuge.

The supernatant in each tube was recovered and aliquoted to 50mL and kept in the -20˚C freezer for later use. 50mL of the centrifugally-depleted FBS was added to 450mL of RPMI 1640 and MEM media. The media was then supplemented with 100U/mL Penicillin-Streptavidin and 10mM HEPES.

Cell culturing

For cell culture, frozen stocks of CCD 841 CoN (colorectal carcinoma) and HCT 116 (normal colon) cell lines were thawed and suspended in the separate buffer types and introduced to six well culture plates. Cells were expanded as they reached confluency and added to T-175 flasks.

Both cell lines were trypsinized, resuspended in appropriate buffer, and centrifuged at 750xg for a period of 10min at a temperature of 20˚C in a Beckman Coulter Allegra X-15 R in an SX4750A rotor.

Cells were again resuspended in the appropriate buffer and 1mL was added to vials and mounted in the Vi-Cell for analysis. 1 mL of suspended cells was directly added to the Vi-Cell and measured for both viability and yield.

Exosome isolation by differential centrifugation

After cell pelleting, the cell culture media was subjected to filtration through a 0.45μm filter and then centrifuged at 2000xg for a period of 20min at a temperature of 4˚C. Using the Optima XPN ultracentrifuge fitted with an SW 32 Ti rotor, the supernatant was centrifuged at 10,000 xg for 30min to eliminate cell debris.

The supernatant was again recovered, filtered through a 0.22µm membrane, and spun at 100,000 xg for a period of 90min by means of an SW 41 Ti rotor in an Optima XPN ultracentrifuge.

This time, the supernatant was aspirated and the pellet recovered by resuspending in phosphate buffered saline. The resuspended sample was marked as ‘crude’ and remains stable at -20˚C temperature for a long period of time.

Density gradient centrifugation for additional refinement

Beckman Coulter’s Biomek 4000 Laboratory Automation Workstation was applied for additional sample purification. This offers a fast, reproducible, and consistent way for layering a centrifugation density gradient with the density and volumes depicted in Figure 2.

The crude exosome sample was layered on top of the gradient and centrifuged at 100,000 xg at a temperature of 4˚C temperature for 18h with the Optima XPN ultracentrifuge and an SW 41 Ti rotor.

Following the centrifugation step, the gradient was fractionated using the Biomek 4000 Laboratory Automation Workstation. Then, using liquid level tracking, 1mL fractions were acquired from the top, for a total of 13 fractions.

These fractions were then pelleted with a TLA 120.2 rotor in an Optima Max-XP bench-top centrifuge. The pellet obtained was resuspended again in phosphate buffered saline, examined for size, and depending on the predicted size and density of recovered exosomes, 7 to 9 fractions were combined, pelleted again with the TLA 120.2 rotor, and resuspended in a small amount of phosphate buffered saline.

Particle characterization

The Beckman Coulter DelsaMax was used to assess the size of the highly purified fractions. Hydrodynamic diameters Dh and translational diffusion coefficients D were established from an autocorrelation analysis of the scattered light at 514.5nm on a Beckman Coulter DelsaMax Pro.

Next, 20 DLS acquisitions of 5s each were ran without auto-attenuation with to and peak radius cut-offs between 0.5 to 150nm. From the DelsaMax software package, data was exported and placed into Origin and plotted.

RNA extraction

Using Qiagen’s miRNeasy kits, total RNA was extracted from the highly purified exosomes of both cell lines and both the crude and highly purified exosomes.

The concentration of the extracted RNA was examined using a Thermo Scientific NanoDrop 8000 and size was determined using an Agilent BioAnalyzer Pico Chip.

NGS library preparation, sequencing, and analysis

Quant-iT RiboGreen equipped with a SpectraMax i3 plate fluorometer was used to measure exosome RNA from each pair of fractions. Using the NEBNext Small RNA Library Preparation Kit for Illumina, automated on the Biomek 4000 automated liquid handler, 100ng of each exosome RNA samples were changed into Illumina compatible small RNA libraries.

SPRI-based size selection was used to perform size selection. After library construction, the libraries were assayed on a BioAnalyzer 2100 using a DNA High Sensitivity Chip.

An Illumina MiSeq with a 50 cycle single read sequencing run, was then used to sequence the libraries, after which, the data was analyzed with the Illumina BaseSpace Small RNA application.

Results and discussion

Exosomes are the tiniest subset of extracellular vesicles and are comprised of proteins, cell-specific receptors, tetraspanins, lipid rafts, and small RNA (Figure 1). They are involved in cell-to-cell communication and the message within their lipid-derived shell is essential to homeostasis.

Exosomes have recently been studied for their therapeutic properties, as a nano-delivery device to diseased cells. Although exosomes are derived from almost all cell types, bodily fluids and species, cell culture continues to be a widely used method for studying extracellular vesicles.

Figure 1. Schematic of an exosome budding from a cell and magnified to show major components. Image credt: Beckman Coulter

The Vi-Cell automated viability counter from Beckman Coulter was used to evaluate the viability and number of CCD 841 CoN and HCT 116 cells. The cells were highly viable at 98.4% and 97.3% for CCD 841 CoN and HCT 116 cell, respectively (Table 1) at a density of 0.82 x 108 and 1.52 x 108, respectively.

Table 1. Cell number and viability of two colon cell lines

Large aggregates, whole cells, soluble proteins and cell debris can be isolated from the target vesicles using a number of differential centrifugation steps and a density gradient. Figure 2 details these steps.

Figure 2. Typical centrifugation workflow and iodixanol gradient setup for stringent purification of exosomes from cell culture. Gradients were layered and fractionated using a Biomek 4000. Image credt: Beckman Coulter

Two paths are followed for the experimental technique: one where the isolation of exosomes is ended after the fourth centrifugal spin, and one that passes via the density gradient separation and two more pelleting steps.

For both the protocols, the starting material was equal, since initial cell input was aliquoted accordingly. In the density gradient workflow, the Biomek 4000 Workstation was used to layer the separation media as well as to fractionate the samples in order to decrease the variability between runs and reduce the hands-on time.

After the exosomes had been isolated, dynamic light scattering using Beckman Coulter’s DelsaMax Core was applied to size them. The highly purified exosomes, in all cases, were the appropriate size between 30 to 150nm, with some residual proteins and other particles of around 5nm. Figure 3 shows the data for HCT 116 crude exosomes.

Figure 3. Representative plot of DLS data acquired from purified exosomes

Then, the total RNA was extracted, measured, and sized. Many different sizes of RNA are present in exosomes, which is evident in the BioAnalyzer tracers (Figure 4). Image credt: Beckman Coulter

Figure 4. Electrophoretic BioAnalyzer trace of exosomal total RNA derived from CCD 841 CoN (top) and HCT 116 (bottom) cells isolated with (red) or without (blue) a density gradient. Image credt: Beckman Coulter

Broad peaks in the RNA were centered between 20 and 30 nucleotides, indicating a large population of miRNA. However, the RNA also had peaks indicating the presence of other RNA species such as ribosomal RNA, mRNA, and precursor RNA, as probed by the Pico RNA chip.

Surprisingly, the RNA acquired from the density gradient purified exosomes and the crude exosomes were a very similar size; however, the RNA concentration was considerably higher in that from the density gradient.

After isolating the RNA from the individual exosomal samples, Illumina-compatible Small RNA sequencing libraries were prepared using the NEBNext Small RNA Library Preparation Kit for Illumina on the Biomek 4000 Genomic Workstation.

Next, the Kapa Biosystems Illumina Library Quantification kit was used to measure the libraries. RNA from crude and density gradient preps from CCD and HCT cell lines were prepared into small RNA libraries for NGS and sequencing was performed on an Illumina MiSeq sequencer using a 50 cycle v2 sequencing kit.

After FASTQ generation and read trimming, sequencing analysis was easily carried out on BaseSpace using the Small RNA Application, which uses DESeq2 and miRDeep for differential expression and BowTie alignment to the hg19 human reference genome.

HCT 116 and CCD 841 CoN density gradient exosomal RNA produced 600,307 and 678,231 PassFilter read counts, respectively, while HCT 116 and CCD 841 CoN crude exosomal RNA produced 617,001 and 660,025 read counts, respectively.

The low variation between data sets and the large number of reads indicates that the isolated RNA is robust and that the yield is sufficiently high for sequencing.

There was considerable variation in RNA type between preparation techniques and cell lines (Figure 5). It is evident that, in the top plot, the total relative abundance of miRNA is lowest in the HCT 116 density gradient preparation technique, but that mature miRNA is the greatest in this prep.

Variation between preparation techniques was greatest in the HCT 116 cells. In the case of small RNA, there were significantly more exons in the density gradient prep; however, this translated to long, non-coding RNA, additional GtRNA, snRNA, piRNA, and snoRNA and precursor RNA for the crude preparation. In the case of the abundant RNA, human ribosomal RNA was the most prevalent in all the preparation techniques and cell lines.

Figure 5. Relative abundance chart of RNA type following FASTQ generation and read-trimming. Image credt: Beckman Coulter

Figure 6 shows the expression heat maps of the sequenced mature miRNA and precursor miRNA that were also plotted. The plot shows differential expression between preparation techniques within a cell line. Further analysis showed significant differential expression between normal and cancerous colon cell lines for many miRNA families (Figure 7).

Figure 6. Differential expression heat-map of preparation method within a single cell line. Both precursor miRNAs and mature miRNAs are plotted. Image credt: Beckman Coulter

Figure 7. Differential expression of miRNA read counts within a sequencing library between HCT116 and CCD 841 CoN cells. Image credt: Beckman Coulter

Fifteen gene families were found to be significantly differentially-expressed. mir-183, mir-182, and mir-1246 were all significantly up-regulated in the CCD 841 CoN colon cancer cell line. Actually, these three gene families have been found to be up-regulated in colon cancer, which is in agreement with our results.

Conclusion

Standardized, automated separation and characterization techniques are essential to the progression of this exciting, emerging field. The preferred choice for exosome isolation is density gradient ultracentrifugation, which generates highly purified sample preparations, but, reproducibility of the workflow is often lacking.

One of the many downstream assays for biomarker identification and exosome characterization is next generation small RNA sequencing. However, again, there is often significant variation in the protocols used. This article has described a solution using the various Beckman Coulter instruments to increase reproducibility, walk-away time, throughput, and accuracy of results (Figure 8).

Figure 8. Beckman Coulter’s standardized exosome workflow. Image credt: Beckman Coulter

The results show a workflow that is able to create high impact NGS data with just one MiSeq sequencing run, which can identify potential biomarkers and quantify differential expression against sample type.

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Acknowledgements

Produced from content authored by Chad Schwartz, Ph. D., Zach Smith, M.S. Beckman Coulter Life Sciences, Indianapolis, IN 46268

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