Exosome isolation a microfluidic road-map

Lab on a Chip

FRONTIER

Cite this:DOI:10.1039/c5lc00240k

Received 28th February 2015,Accepted 28th April 2015DOI:10.1039/c5lc00240k https://www.wendangku.net/doc/7f4966524.html,/loc

Exosome isolation:a microfluidic road-map ?

A.Liga,a A.D.

B.Vliegenthart,b W.Oosthuyzen,b J.W.Dear b and M.Kersaudy-Kerhoas *a

Exosomes,first isolated 30years ago,are nanoscale vesicles shed by most types of cells.The nucleic acid rich content of these nanoparticles,floating in virtually all bodily fluids,has great potential for non-invasive molecular diagnostics and may represent a novel therapeutic delivery system.However,current isolation techniques such as ultracentrifugation are not convenient and do not result in high purity isolation.This represents an interesting challenge for microfluidic technologies,from a cost-effective perspective as well as for enhanced purity capabilities,and point-of-care acquisition and diagnosis.In this frontier review,we

present the current challenges,comment the first microfluidic advances in this new field and propose a roadmap for future developments.This review enables biologists and clinicians familiar with exosome enrichment to assess the performance of novel microfluidic devices and,equally,enables microfluidic engineers to educate themselves about this new class of promising biomarker-rich particles and the chal-lenges arising from their clinical use.

1Introduction

Extracellular vesicles (EVs)are membrane-bound structures that are released by most,if not all,cell types.Due to their “cargo ”of protein,RNA and DNA from their cell of origin,EVs offer a minimally-invasive ‘window ’into the intra-cellular world.1,2In the literature,differing nomenclature and classifi-cation of EVs has been used based on size,density,morphol-ogy,lipid composition,protein composition,and subcellular origin.3Exosomes are distinguished from other EV subtypes by their unique intra-cellular biogenesis,in contrast with other EVs,such as microparticles,that are shed directly from the cell membrane.3Exosomes are reported to measure 20–100nm and appear cup-shaped when visualized by transmis-sion electron microscopy.Exosomes are first formed as intra-luminal vesicles by budding into early endosomes and multi-vesicular bodies (MVBs).Subsequently,the MVBs either fuse with lysosomes or with the plasma membrane and the endo-somes are released into the extra-cellular milieu to become exosomes.4Due to this specific formation process,exosomes contain a cargo that,to some extent,mirrors the cell of origin.

The observation that the proteome and transcriptome of exosomes changes according to the cell of origin 5,6and the

ability to access exosomes from virtually any biological fluid (including saliva,lymph,urine,milk,blood,synovial and amniotic fluids 7)forms the rationale for the use of exosomes as non-invasive biomarkers for disease and toxicity.8–10

Exosomes can also behave as inter-cellular signals by transferring functional mRNA and microRNA between cells in vitro .2,11The importance of exosomal signalling in vivo remains to be determined.This signalling capacity may enable exosomes to be used as a non-immunogenic RNA delivery system that can cross natural divisions such as the blood –brain barrier.12,13

2.Conventional exosome capturing

Current methods for exosome ‘capturing ’concentrate exo-somes,but do not isolate them.Contamination of exosome preparations with non-exosomal proteins and other EV sub-types can lead to spurious biomarker discovery studies and incorrect conclusions about exosome biological activity.14The isolation of a pure population of exosomes would facili-tate studies in exosome biology,in particular regarding their physiological functions and their roles in various pathologies.While next-generation deep sequencing (NGS)provides fast profiling of miRNA or other biomarkers in biological fluids,including from exosomes,liquid biopsy sample prep-aration is sometimes crucial to reliably differentiate between diseased and healthy patients.15,16In this case,the isolation of exosomes can improve the sensitivity of biomarker ampli-fication and reduce the number of false-negative results.16The most common method used for concentrating exosomes

a

Heriot-Watt University,Institute of Biological Chemistry,Biophysics and

Bioengineering,Edinburgh,United Kingdom.E-mail:m.kersaudy-kerhoas@https://www.wendangku.net/doc/7f4966524.html, b

University of Edinburgh/British Heart Foundation Centre for Cardiovascular Science,Pharmacology,Toxicology and Therapeutics,Queen's Medical Research Institute,Edinburgh,United Kingdom

?Electronic supplementary information (ESI)available.See DOI:10.1039/c5lc00240k

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is ultracentrifugation,a method that includes differential centrifugation steps reaching speeds of up to 200000×g .However,this procedure is time consuming (4–5hours),17requires specialist laboratory equipment that is not available in routine hospital laboratories,and is inefficient with regard to exosome yield (5–25%recovery).18Variations on this method,such as adding in a sucrose gradient centrifu-gation step,lead to higher purity of extracted exosomes.However,the more complicated the sample processing the longer the time from sample to exosome preparation.The capital cost of an ultracentrifuge is typically around $50–100k and the running cost is around $3k per year.Addition-ally it requires training and a dedicated team.Exosomes can be isolated in as little as two hours but isolation time ranges from 2to 10hours depending on the biofluid and protocol.It may be difficult for standard hospital laboratories and resource-poor countries to acquire and maintain such equip-ment,thus denying exosome-based diagnostics to a substan-tial part of the population.A promising method for EV selective concentration relies on antibody-coated magnetic beads.This technique results in higher recovery and purity –at least two-fold more exosome markers and proteins mea-sured in the exosome concentrate compared to ultracentrifu-gation.19The use of magnetic beads also facilitates the use of standard analysis methods,such as flow cytometry,immuno-blot and electron microscopy on exosome-bead complexes.20However,it is important to highlight that the magnetic beads only attach to exosomes that contain the targeted protein that might not be present in all the exo-somes in the sample resulting in a biased analysis.Recently,commercial precipitation kits like ExoQuick ?and Total Exosome Isolation ?precipitation solution have become available.The advantages of these kits are that they are easy to use with only 1or 2steps and do not require any expen-sive equipment or advanced technical know-how.The disad-vantage is that they commonly require a lengthy overnight incubation step and their mode-of-action has not been disclosed.The purity of the acquired exosomes has been reported to be inferior compared to OptiPrep density gradi-ent centrifugation,as demonstrated by lower enrichment of exosomal marker proteins and more contaminating proteins such as extra-cellular argonaute 2complexes.21Chemicals originating from the precipitation kit may contaminate the prep and these commonly undisclosed chemicals may affect the apparent biological activity of the exosomes.Micro-filtration techniques and other size exclusion methods can suffer from clogging and vesicle trapping issues and often entail shear stress-induced damages.This paper focuses on the potential of microfluidic devices to concentrate,and even isolate,exosomes,describing the most recent techno-logical developments and highlighting pro and cons of each of these methods that promise to deliver rapid and easy iso-lation.In this review we have chosen to focus on the diag-nostics rather therapeutic use of exosomes,as microfluidic solutions will be most beneficial,or at least of immediate benefit,in the diagnostic area.

3.Microfluidic technologies for exosome isolation

The advent of microfluidic technologies has allowed the development of new exosome manipulation techniques which,though still at an early stage of development (most of these were developed between 2012and 2015),are proving themselves extremely convenient in terms of reagent vol-umes,product purity and isolation time (cf.ESI ?Table for detailed comparison of techniques).The techniques devel-oped so far for microfluidic-based exosomal purification can be classified in three categories including (i)trapping exo-somes with an immune-affinity approach,(ii)sieving (e.g.nanoporous membranes)or (iii)trapping exosomes on porous structures (e.g.nanowire-on-micropillars).Sieving approach differs from the others by the possibility to start from whole blood without previous treatment.However,all methods require further off-chip additional steps for sample preparation,such as plasma extraction,or reagent mixing.223.1Immunological separation

Immuno-chip.Chen et al.demonstrated the first immune affinity approach for the capture of exosomes within a micro-fluidic device.23The separation principle relies on receptors on the exosome outer surface which enable specific collection according to their origin and function,and allow their isola-tion from other shed membrane particles and lipid struc-tures.The device features a planar structure with herring-bone engravings to enhance mixing (cf.Fig.1a).Collected exosomes are characterised in situ ,after several washing steps,or lysed for DNA extraction.23Chen et al.demonstrated a faster method (~1h),with respect to the traditional tech-nologies,that used smaller amounts of reagents (100–400μl).Total RNA amount,extracted from exosomes captured on-chip using CD63antibodies (common exosomal marker)from 400μL serum samples,was around 30ng for non-small cell lung cancer patients.Further analyses demonstrated the quality and quantity of on-chip exosomal RNA was sufficient for the evaluation of tumor-derived RNA.

Immuno-chips and on-chip fluorescent characterisation.Kanwar et al.22used the same principle,modifying the prior design so as to carry out “on chip ”exosome quantification by a fluorescent assay method on a standard read-out plate reader.The device,called Exochip,features several circular wells connected by narrow channels,in order to enhance mixing (Fig.1b –d).Additionally,the extended retention time leads to a stronger interaction between the exosomes and the functionalised surface.Besides being specially adapted for further analysis,the device can also be easily scaled up,sim-ply adding more rows of wells in the same chip.The protein yield of the ExoChip exosomes was 15–18μg of total protein and 10–15ng of total nucleic acid from 400μL serum sam-ples.A higher level of fluorescence on the chip was measured in exosomes obtained from patients with pancreatic cancer compared to healthy volunteers.This was in line with the

Lab on a Chip

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relatively higher protein expression of CD63and Rab5mea-sured in the exosomes from the cancer patients (western blot).A panel of miRNAs measured in the extracted exosomes was also able to distinguish between cancer patients and healthy volunteers.He et al.24developed a cascading chip where exosome isolation and enrichment,chemical lysis,pro-tein immuno-precipitation and sandwich immunoassay assisted by chemi-fluorescence detection are integrated in a microfluidic circuit.The on-chip isolation protocol takes less than 1.5hours and only requires 30μl of human plasma premixed with antibody-labelled magnetic beads.The analy-sis of type 1insulin growth factor receptor (IGF-1R)concen-tration in on-chip exosomes,though 100times lower than that recovered from ultracentrifugation,showed a clear overexpression of the marker in 5non-small-cell lung cancer patients compared to 6healthy volunteers confirming the diagnostic potential of this approach.As mentioned in section 2,one of the main limitations of immunological anal-ysis is its focus on only one particular surface protein at a time.

Immuno-chips and sensing.Several groups are proposing to use immunological techniques in combination with differ-ent techniques,allowing for better separation,or detection,or both.In this regard,Im et al.25devised an on-chip nano

plasmonic exosome sensor (nPLEX),in which surface plasmon resonance (SPR,a technique based on the oscilla-tions of electrons at an interface stimulated by incident light)is used through nanohole arrays patterned on a metal film.Each array is functionalised with different affinity ligands specific for a particular surface protein,which overcomes the issue highlighted in the previous section.In the first chip generation each of 12microfluidic channels were coupled with 3arrays (12×3),giving the possibility to monitor and display bonds with 36different proteins.A second generation of chips is being developed,implementing 33×33arrays thus allowing increased data collection from 1089sites.Thanks to the high sensitivity of the plasmonic sensors,it is possible to display in real time the intensity of exosome-site bonds,and “map ”the activity of each array,thus leading to a quantitative analysis of exosome content.Exosomes were present in large quantities (>109exosomes per mL)in ascitic fluid from 20ovarian cancer patients and 10non-cancer patients.Therefore the nPLEX device could function with samples simply collected through a 0.2μm membrane filter.Receiver operator characteristic curve analysis of exosomes using paired protein levels per exosome of epithelial cell adhesion markers (EpCAM)and CD24resulted in a promis-ing area under the curve of 0.97.This device performs well with exosome-rich ascites samples,but due to its sensitivity and high-throughput has potential as a point-of-care diagnos-tic tool on more conventional liquid biopsies such as blood,if combined with an integrated sample preparation.Further-more,its use could be broadened to diagnose a range of ill-nesses or even to monitor treatment response.Another exam-ple of enhanced on-chip immunological separation followed by detection was proposed by Vaydianathan et al.with an original approach named “nanoshearing ”aimed at reducing non-specific fouling as well as providing inline detection.26The use of this electrodynamic anti-fouling technique led to a 3-fold enrichment in detection sensitivity relatively to a nor-mal hydrodynamic flow.

Immuno-binding and inertial solution exchange.Finally,Dudani et al.,27used an off-chip immunological approach to bind exosomes to 20μm polystyrene beads followed by on-chip inertial focusing to enrich the bead-exosome complex from the original sample into a buffer.The device was tested with blood samples (after RBC lysis)spiked with centrifuged melanoma and breast cancer culture supernatant.This mixture was incubated for 4h with polystyrene beads functionalised with exosome-specific antibodies and then processed through the chip.There,inertial lift forces push the beads toward the centre of a channel with multiple out-lets,transferring them into a wash buffer and allowing their selective separation.A single centrifugation step removed the larger cell debris and the exosomes can then be eluted from the beads and characterized.The device features a relatively high throughput (70μl min ?1,five-fold higher than most other microfluidic techniques)and demonstrated the enrich-ment of exosomes but still needs a thorough molecular char-acterisation of the retrieved material.The 4h needed

for

Fig.1(a)The first chip realised for microfluidic exosome isolation featured a 19mm ×20μm ×4.5cm chamber with herringbone groves 10μm deep to enhance contact between microparticles and chip surface.The device is pictured here is filled with supernatant fluid and attached to a syringe pump.Reproduced from ref.23with the permission of the Royal Society of Chemistry.(b)A modified version of the same scheme:single channel Exochip,the device has the same dimensions of a standard glass-side (W =0.75mm,L =73mm,P =9mm,H =100μm).Up to 12channels could fit within the dimensions of a microtiter-plate.(c)Experimental setup using a 3channels Exochip (d)Exochip working scheme:exosomes counting is made possible through a simple 3steps procedure involving:(i)blood serum infusion within the chip (pre-coated with exosome-specific capture antibodies,anti-CD63),(ii)staining with specifically functionalised (Di-O)fluorescent dye,(iii)on chip analysis through conventional counting methods.Reproduced from ref.22with the permission of the Royal Society of Chemistry.

Lab on a Chip Frontier

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bead incubation makes it unsuitable for acute care applications.

In conclusion,it is important to highlight that immuno-logical methodologies are the only ones,so far developed,which can be directed towards the isolation of a pure exo-some population.Other methods relying on physical proper-ties (size,density,surface charge)lead to higher percentages of contaminants (similar micro-particles with different origin,proteins).

3.2Sieving

Davies et al.28developed a different approach to exosome col-lection,by sieving EVs directly from whole blood through a membrane and driving filtration either by pressure or electro-phoresis (cf.Fig.2).The pressure-driven solution leads to a shorter separation time while the electric field produces a higher purity of the extracted vesicles,proteins being less affected by electric fields than phospholipidic vesicles,due to their lower negative charge.29In the authors'opinion,the non-selectivity of this device with respect to vesicle type can be seen as an advantage compared to the over-specific collec-tion offered by immune-affinity related methods,which can lead to biased data analysis.A significant drawback is the low exosome recovery (unspecified in the text but shown to be next to 2%in a graph)while the device seems to perform well regarding separation time.The device reached a satura-tion point after 3–4μL of filtrate was extracted when pressure driven filtration was used.However this volume was enough to assess the exosomal cargo by western blot and RT-PCR.When electro-driven filtration was used,the average yield was 79ng RNA per 100μg of protein from a 100μL sample,com-pared to 187ng of RNA per 100μg of proteins from a 5mL sample obtained by centrifugation.3.3.Trapping on porous structures

Wang et al.7devised a third approach consisting of trapping EVs through a porous microstructure.They designed a

ciliated micropillar structure forming a microporous silicon nano-wire which was able to selectively trap particles in the range of 40–100nm (Fig.3).According to Wang et al.this method allows the selective collection of intact phospholipidic “exosome-like ”vesicles.Though the trapping step is relatively fast (~10min),to proceed with imaging and characterisation,it is necessary to dissolve the silicon nano-wire in PBS buffer overnight,thus making the recovery proce-dure time consuming.This device was tested with a solution containing 83nm and 120nm lipid vesicles and 500nm nanoparticles.The highest retention (60%)was obtained for 83nm vesicles when up to 30μL of sample was injected (based on fluorescent intensity measured with a plate reader,starting concentration undisclosed),followed by 120nm vesi-cles (45%),while the retention of 500nm bead was only 10%.The retention rate of 83and 120nm lipid vesicles decreased when more sample volume was injected,probably due to a saturation effect.The device was not validated with clinical samples,and no analysis of cargo protein or RNA was performed.

4.Road map for microfluidic exosomal isolation

The increased interest in the role of exosomes in biomarker discovery and diagnostics has highlighted the pressing need for new technologies to isolate exosomes in biofluids rapidly and accurately with minimal sample preparation.Here we discuss 5areas of consideration for future

developments.

Fig.2Schematics for two sieving modes for exosome filtration.The membrane is realised by in situ photopatterning of a porous polymer monolith.Sieving can be performed by (a)applying pressure on an on-chip dead end filter or (b)by applying an electric field to drive the exosomes through the porous membrane in a hybrid solution of cross flow and electrophoresis sorting.Vesicles damage through the device was shown to be undetectable by analysing haemolysis and DNA was not contaminated by intracellular proteins.Adapted from ref.28with the permission of the Royal Society of

Chemistry.

Fig.3Ciliated structures for exosome isolation and capture by Wang et al.The hierarchical geometry of the device does not allow cells larger than 1μm to access the wired area,due to the distance between the pillars (b)being too narrow (900nm).Smaller cell debris can enter the micropillar area but are excluded by the ciliated nano structure (a),which forms pores with diameters ranging between 30and 200nm,in order to selectively trap exosomes and small EVs.Smaller proteins and molecules are free to pass through the device without being captured.Reproduced from ref.7with the permission of the Royal Society of Chemistry.

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4.1.From research to clinical setting:increased speed and lower cost

Ultracentrifugation is currently used in the research setting,but not yet regularly used in the clinical setting.If an exosome-based diagnostic was to be used in real-life clinical medicine,one could envisage that in a non-urgent situation,such as the diagnosis of cancer,the use of a centralised labo-ratory with an ultracentrifugation unit could be feasible,pro-viding the extraction is reproducible.However if standardised performance is demonstrated using microfluidic chips,it would open the door for faster,reliable,cost-effective exo-some-based diagnostics for emergencies in acute care set-tings (e.g.,myocardial infections and drug toxicity)as well as non-emergency illnesses (cancer diagnostics).Furthermore,this could allow a more accurate exosomes analysis,since studies have indicated that there is a significant loss in exo-somes number from biofluids such as urine,even when frozen at ?80°C 30and within 2h.31If exosomes are to be used for acute care,the device would ideally allow the biolo-gist or clinician to rapidly and confidently isolate exosomes from biofluids within a 30minute time frame after sample collection.

4.2.Future directions,technological considerations and potential challenges for the development of microfluidic technologies

All prototypes presented in this review have used a single isolation strategy and none are free from issues.Future microfluidic devices might therefore benefit from hybrid approaches using two or more isolation techniques,for example,a first separation based on size could eliminate the largest blood cells from a blood samples,followed by an immunological separation to access a specific exosome pop-ulation.The challenge,as with all microfluidic development,is to provide a solution to a real clinical question.Two routes are likely to emerge:the first will explore the exo-some biology in the research area,and will require refined isolation.For this route,the advantage of microfluidics will be to provide cheaper alternative to ultracentrifugation with low capital investment.The second route will focus on the development of tools to facilitate liquid biopsy preparation for next generation sequencing.The challenge will lie in the rapid extraction from complex matrices.To fit in a clinical work flow the devices will need to be more robust and cost-effective as their current counterparts.The challenges in the development will lie in (i)reducing the cost of the devices,a consideration which will need to be taken into account at the design stage,and which might involve alleviating the need for structures below 80μm,at which point photoli-thography might be replaceable by rapid-prototyping and high volume manufacturing processes.(ii)Secondly,achieving the right throughput will be crucial and might necessitate stacking several microfluidic units on the same device.

4.3.Achieving specificity and reproducibility

As discussed above,in some cases,devices will need to reli-ably provide an exosome preparation specific to an organ of origin.If a device has on-chip technologies such as protein specific markers for sorting exosomes from specific organs it allows the clinician access to a relevant exosome population specifically to his site of interest.Current methods of isola-tion are not delivering on reproducibility of exosome recov-ery,arguably due to variations in sample handling.Ideally,microfluidic chips should be tested by different laboratories to assess reproducibility and consistency and demonstrate standardised performance.

4.5.Characterisation and biological validation

At present,the methods of quantifying exact or ideal exo-somal yield are still very semi-quantitative.There is an unmet need for quantitative and qualitative assessment of exosomes and the definition of clear biological controls.32We have demonstrated that nanoparticle tracking analysis is a promis-ing method for quantifying exosomes in biofluids,such as urine 31and it would therefore be interesting to compare the yield of current and future devices using this method.How-ever,there is no “one fits all ”solution for the characterisa-tion of exosomes.The characterisation required will depend on the clinical application.Spiking-in labelled exosomes offers a reliable method to determine the yield,either via fluorescence measuring or RT-PCR.Furthermore,it is recommended to assess not only the presence of the selected markers (yield)but also the absence of contaminants (specificity).32

5.Conclusion

Through highly precise fluid control and high surface to vol-ume ratios,microfluidics has the power to differentiate,cap-ture,enrich and isolate particles of very similar sizes and shapes.Despite being in its infancy,several microfluidic developments have demonstrated effective exosome separa-tion,leading to robust clinical diagnostics.However none of the first prototypes are free from issues,in particular they often require prior sample preparation based on macro-scale procedures.Among the aforementioned methods,immuno-logical separation leads to high specificity,and can be performed in a fairly short time in most cases (~1.5h).Trap-ping exosomes in porous structure,such as ciliated micro-pil-lars,seemingly leads to high purity exosomes recovery,but the recovery procedure is currently time consuming (over 1day).Sieving is a particularly interesting method as it can be performed directly on whole blood,without any treatment before the separation,but it is not exempt from drawbacks,such as low recovery and the possibility of damage to vesicles due to shear stress.Some isolation techniques demonstrated for microvesicles (typically of diameter above 100nm)might also be suitable for exosome recovery.33,34For example,Rho et al.have demonstrated a rapid and sensitive microvesicle

Lab on a Chip Frontier

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detection in packed red blood cell units using filter-assisted filtration,magnetic labelling and target-specific detection via miniaturised nuclear magnetic resonance system.33Detection and characterisation are an essential part of the capture-to-diagnosis path,and some microfluidic researchers have focused all,or part,of their efforts towards the development of detection techniques which could be the subject of another focused review.25,35–37Some of these techniques pro-pose the direct (preparation free)detection of exosomal RNA from raw cell medium,which could be potentially applicable to bodily fluids.37The applications of microfluidic technology to bodily fluids for exosome isolation have not been described extensively with few devices using clinical samples,and microfluidic engineers should seek to establish strong collaboration with biologists and clinicians to demonstrate the full potential of the technology.While current ultracentri-fugation techniques require significant capital and mainte-nance costs,microfluidic-based exosome isolation techniques are promising and cost-effective,and could be deployed in a large number of clinics and hospitals,as well as in resource-scarce settings.

Acknowledgements

AL is funded by a James Watt Scholarship.JWD acknowledges the support of NHS Research Scotland via NHS Lothian.MKK acknowledges the Royal Academy of Engineering for fellow-ship funding.The authors would like to thank the anony-mous reviewers for valuable insights that enhanced the origi-nal manuscript.

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