Optiprep virus purification protocol

Note that the protocol below, which uses the broad spectrum nuclease Benzonase, is most appropriate if overall yield of recombinant capsids is the primary goal. If the primary goal is harvest of only the infectious pseudovirions, it may be preferable to use the Revised Production protocol. Collect producer cells by trypsinization about 48 hours after transfection Note 5. If there are floating cells in the culture, collect them by centrifugation.

Resuspend the cells in 10ml of DMEM and transfer to a conical tube with collected floaters, if applicable. Rinse the flask with another few ml of fresh DMEM Expect about 25 million cells from a confluent 75 cm 2 flask. Spin down the cells and discard the supernatant.

Partially resuspend the cell pellet in residual fluid by gently agitating the tube. Using a 2 ml pipet, transfer the suspension into a siliconized see Note 6 1. Rinse the original tube with 0. Repeat the DPBS rinse, if necessary. Spin down the cells in the siliconized tube and discard supernatant. This step of the protocol, in which cells are suspended in PBS and lysed with detergent, is surprisingly important.

If lysis is performed at too low a density, proteins in the lysate aggregate non-specifically. This results in entrapment of virions in large aggregates that can be lost during the initial low-speed clarification. The aggregation also seems to interfere with pseudovirus infectivity presumably due to sequestration of the pseudovirions inside large particles.

The large aggregates also drag free DNA and free reporter proteins e. Surprisingly, the line between an appropriately high-density lysate and an inappropriately low-density lysate is very sharp. It appears that cells that have replicated the transfected plasmids to very high copy number swell up like beach balls. Because of this problem, cell counts are less reliable than simply performing a gross estimate the volume of the cell pellet.

To estimate the volume of the cell pellet, load 1 ml of DPBS or other fluid into a 1ml pipet i. Dispense the liquid into a dummy tube until the volume in the dummy tube matches the height of cell pellet.

It may be necessary to shake the bubble out of the bottom of the dummy tube. Record the volume that has been dispensed out of the 1ml pipet. This represents the volume of the cell pellet. Add 1. For polyomaviruses, add 1.

Suspend the cells by briefly vortexing the tube. Add 0. See Improved Maturation protocol for details. If making dye-conjugated capsids, do NOT add ammonium sulfate, since it will quench the amine-reactive group on the fluorochrome. It may be helpful to mix the tube by inversion once or twice during the course of the incubation. Since it is possible for contaminating microbes to grow during the maturation period, it is essential to use aseptic techniques while preparing the cell lysate.

If the cell suspension is dense enough, there should be very little clear fluid above the settled cell suspension the next morning. A lot of clear fluid visible above the cell suspension may be a sign that the cell density was too low and nonspecific protein aggregation may have occurred. Although freezing the matured lysate does not result in significant loss of infectious titer, freeze-thawed lysates sometimes show greater amounts of contaminating cellular proteins after Optiprep purification.

In this section, PsV is purified by ultracentrifugation through an Optiprep step gradient. The section also outlines methods for biochemical analysis of the purified PsV. Use 50 ml conical centrifuge tubes to allow easier syringe draws see below. Pour Optiprep gradients in thin wall polyallomer 5 ml tubes e. If necessary, pour a balance gradient.

The gradients can be allowed to diffuse at room temperature for 1 to 4 hours. Gently layer clarified cell lysate Subheading 3. Spin for 3. Other types of rotors can be used successfully, for example SW The L1 band may be faintly visible as a light gray layer a little over a third of the way up the gradient. Collect gradient fractions by puncturing the bottom of the tube slightly off center with a syringe needle.

If a 25 gauge syringe needle is used, perform a simple in-and-out puncture without any rocking or rotating of the needle. A gauge syringe creates a very small hole with frustratingly slow flow rates. It is therefore necessary to gently rotate the gauge syringe after puncturing. Drip fractions into siliconized microcentrifuge tubes. The simplest method for screening fractions is to look for the presence of encapsidated DNA in core fractions of the gradient.

Optional: supplement the Picogreen working mixture with 0. Mix the Picogreen with the fraction samples either by trituration or by patting and swirling the plate. There should be a small histogram of DNA signal in the core fractions of the gradient, with a larger ramp of DNA signal unencapsidated DNA toward the top of the gradient.

The total transducing units generated using second and third generation packaging systems. It is commonplace to concentrate lentivirus by ultracentrifugation to obtain sufficient viral titres to transduce cells at a high MOI and remove contaminating impurities for sensitive in vivo applications. We found that concentrating lentivirus resulted in a fold reduction in virus yield, suggesting that significant amounts of virus are lost or lose infectivity during ultracentrifugation Figure 3 A.

This suggests that concentration removes impurities in the virus supernatant, which can enhance the transduction efficiency. Virus titre measured before and after concentration by ultracentrifugation. The level of transduction using un-concentrated and concentrated lentivirus supernatant as measured by flow cytometry. Virus titre measured following centrifugation of viral supernatants at decreasing centrifugal speeds.

Assessment by flow cytometry of transduction by lentivirus following concentration with decreasing centrifugal speeds. Virus titre measured following concentration of lentivirus using ultracentrifugation, Optiprep and Acrodisc methods. The level of transduction of each of the concentrated lentivirus stocks as measured by flow cytometry.

Concentrating lentivirus with reduced centrifuge speeds has previously been shown to increase the titre of lentivirus [ 22 ]; however whether this equates to increased transduction efficiency in primary T cells has never been demonstrated. Having determined that concentrating virus at a lower speed results in improved lentivirus titres and transduction efficiency, we next determined whether we could improve lentivirus infectivity further by density gradient concentration Optiprep or ultrafiltration Mustang Q Acrodisc.

These results suggest that centrifuging lentivirus at lower speeds negates the use of more costly and time-consuming purification techniques and is sufficient to remove impurities that may otherwise reduce transduction efficiency. This suggests that the strength of TCR signalling may be important for increased transduction efficiency. Therefore, we began to modify a standard protocol used to transduce primary T cells [ 16 ] see material and methods for protocol.

Our next step in optimisation was to determine whether the use of HS increased transduction efficiency. When human serum was used as a growth supplement, a significant increase in transduction was observed when compared to FCS cultured cells Figure 4 B , a modification that also led to increased activation marker expression data not shown. One explanation for increased transduction efficiency could be that more cells are cycling, making them more amenable to transduction.

Virus was generated using the standard production and concentration protocol. C and D. High transduction of lentivirus in primary human T cells. Left panel shows representative histogram from one donor of NGFR expression following transduction with lentivirus. Right panel shows the percentage NGFR expression from three lentivirus preparations in nine independent experiments.

Transduction efficiency was determined 24 hrs post-transduction using flow cytometry to determine the percentage of NGFR expression. Lentiviral transduction of exogenous genes into primary cells is technically challenging, requiring the preparation of high titre viral stocks and optimal culture conditions.

The generation of lentivirus requires the transfection of a stable producer cell line, such as T cells. We have shown that transfection of T cells with a second-generation lentivirus system produces virus with sufficiently high titre to transduce primary T cells, compared to the third-generation system.

While this approach increased viral titre, the viral supernatants did not generate sufficiently high transduction efficiency. To increase the virus transduction efficiency we sought to concentrate the virus supernatant, thereby allowing for the use of a high MOI whilst at the same time reducing contaminating material in the supernatant.

We determined that it was possible to increase virus transduction efficiency by concentration using ultracentrifugation. Transduction efficacy could be improved further by reducing the centrifugal speed from the standard protocol 90, g to 20, g. In addition to virus generation we also showed that transduction efficiency can be improved by modifications to cell culture conditions, specifically we showed that the strength of TCR stimulation and the time of transduction following stimulation affects transduction efficiency.

Interestingly, we also showed that lentivirus can transduce unstimulated T cells to similar efficiency to stimulated primary T cells; however the expression of the transgene is low. Overall, we demonstrate an easy and robust protocol to generate and transduce lentivirus at high efficiency in primary T cells.

This robust method is simple to achieve and does not require additional expensive laboratory equipment. APC and AK performed the experiments and drafted the manuscript.

APC performed the primary T cell transductions and analysed the data. AK generated lentivirus. BG participated in the design of the study and helped draft the manuscript. FMB conceived of the study, and participated in its design.

All authors apart from FMB read and approved the manuscript. Virus recovery was measured before and after centifugation of lentivirus at decreasing centrifugal speeds. Virus recovery was then expressed as a percent of the total TU of starting unconcentrated lentivirus. Figure S2. Virus recovery is reduced following purification using Optiprep and Acrodisc.

Virus recovery was measured before and after ultracentrifugation, Optiprep and Acrodic methods. We thank Prof. Trono for the pMD2. G and psPAX vectors. This work was supported by the Kennedy Trust for Rheumatology Research.

National Center for Biotechnology Information , U. BMC Biotechnol. Published online Nov Author information Article notes Copyright and License information Disclaimer. Corresponding author. Particle yield can be dramatically low in pressure-driven concentrating devices compared to centrifuge concentrating devices Fig. Non-specific absorption to both cellulose and biomax membranes of the Stirred Cell causes this reduction.

This is supported with washing the membranes with ethanol or sodium hydroxide. When the membrane was fully restored with sodium hydroxide, particle yield would remain low; however, an ethanol wash resulted in maximized particle yield Fig. This is most likely due to the large surface area This loss of particles does not occur with Centricons due to the lower surface area of the membrane 19 cm 2 , and a reverse centrifugation step allowing particles to be spun off the membrane.

Given this, when working with volumes of 50— mL of conditioned media, a centrifuge-based concentrator is the most appropriate device. Some applications may require the use of larger volumes of CCM. We found that pressure-driven concentrating is more appropriate with volumes in excess of mL due to the higher flow rate, and that exosome loss is only observed with the first 50— mL of CCM. Previous studies on comparing exosome isolation techniques have been carried out 6 , 13 , 20 , but there is limited information regarding recent precipitation-based isolation techniques.

Density gradient purification is a technique repeatedly shown to provide the highest degree of purity 6 , 12 , Interestingly, separation of particles with a density gradient revealed a large abundance of particles in lower density fractions, particularly fractions 3—5 Fig. This heterogeneity has been described by others 21 , showing the presence of particles with different structural and biochemical features due to different mechanisms of biogenesis.

For the determination of an isolation method that delivers the purest exosome isolation, the combination of both particle and protein concentration analysis is required as either alone is insufficient to determine the overall performance of an isolation technique. The importance of using both particle and protein concentration is indicated as precipitation protocols produced the highest yield of particles, yet had the lowest ratio of particles to protein, potentially due to co-isolation of contaminants Fig.

It is a possibility that high particle low protein measurements could be attained if exosomes were damaged during the isolation protocol and lost some protein cargo. A key message from these data is that protein concentration is not a good measure of exosome yield, and though common organelle markers such as Calnexin are used to assess purity of exosomes they are insufficient to indicate that isolation techniques are devoid of contaminates.

The utility of all exosome isolation methods are dependent on their performance when applied to human clinical samples, particularly when the focus of exosomes as novel biomarkers is being considered.

Modern precipitation protocols have been purported as alternative methods to ultracentrifugation because they require very little starting sample from human biofluids combined with high-throughput options. Isolating exosomes from plasma is further complicated due to viscosity and density issues 5 , 22 , thereby limiting the purity obtained from ultracentrifugation protocols. Recently, a single-step SEC isolation of exosomes from human plasma has been described Using a similar approach, we find similar results in the efficiency of qEV SEC columns to separate exosome vesicles from contaminating plasma proteins.

SEC, however, does not concentrate samples and therefore requires a second step. Instead of pelleting exosomes with an ultracentrifugation step, we used protein concentrating devices to rapidly concentrate exosomal fractions. This provides an efficient means of isolating and concentrating exosomes from human plasma, while avoiding ultracentrifugation Fig.

SEC purification using qEV columns significantly outperformed both precipitation protocols when the particle protein ratio was considered Fig. This is also supported by analysis of Flotillin Flotillin-1 could not be detected in both precipitation protocols but was present in the SEC isolation samples. Furthermore, the purity of exosome isolations is important and can be classified through a number of methods, including the presence of contaminating extracellular proteins Though no contaminating extracellular protein is shown in the in vitro precipitation isolations, Fig.

This furthers the assumption that precipitation protocols are prone to heavy contamination with plasma proteins and are therefore limited in their utility for proteomic analysis of exosomes from human plasma. Isolation techniques that have been well characterized are crucial for the analysis of exosomes as biomarkers.

In conclusion, we have investigated the influence of repeated ultracentrifugation of CCM on the integrity of exosomes and found that it is detrimental to achieve the highest recovery of particles. Modern ultrafiltration devices provide a more rapid and overall higher yield of exosomes when compared to ultracentrifugation. The steps of differential centrifugation also have an impact on the input before density gradient purification 19 , 25 , a problem that may bias subsequent analysis that ultrafiltration avoids.

Therefore, adoption of concentrating protocols will provide improved analysis of exosomes. We show that ultrafiltration coupled with SEC is a method that provides particle purity comparable to density gradient purification and is applicable to isolating a high yield of exosomes from CCM and human plasma in an efficient time frame.

These data should inform the community in developing optimal techniques for exosome extraction and research. The authors thank all Tumour Microenvironment Laboratory members for critical advice and proof-reading of the manuscript.

We thank Sarah Ellis for her assistance with the electron microscopy. The authors declare no conflict of interest. National Center for Biotechnology Information , U. Journal List J Extracell Vesicles v. J Extracell Vesicles. Published online Jul Richard J. Wong , 1 Adrian P. Christina S. Adrian P. Author information Article notes Copyright and License information Disclaimer. Lobb et al. This article has been cited by other articles in PMC.

Associated Data Supplementary Materials Optimized exosome isolation protocol for cell culture supernatant and human plasma. Optimized exosome isolation protocol for cell culture supernatant and human plasma. Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. Keywords: exosomes, cancer, plasma, cell culture, isolation, purification. Size exclusion purification Five-hundred microlitres of clarified CCM, or 1 mL of processed plasma centrifuged at 1, g and 10, g for 10 and 20 minutes, respectively was overlaid on qEV size exclusion columns Izon followed by elution with PBS.

Electron microscopy Exosomes were visualized using transmission electron microscopy TEM according to Thery et al. Western blot analysis Western blots were performed as previously described 8 , 9. Statistical methods Statistical analyses were performed using Student's t-test and one-way analysis of variance. Open in a separate window. Centrifugal concentration provides optimal particle yield from CCM Currently, the main protein concentrating devices available are either pressure-driven Stirred Cell or centrifugation-based Centricon.

Ultrafiltration is a faster alternative to ultracentrifugation To investigate the exact differences between ultracentrifugation and ultrafiltration on exosome yield and quality, we used a combination of particle analysis and protein assessment of positive markers.

Repeated ultracentrifugation reduces particle yield and recovery Next, we examined if repeated ultracentrifugation rounds would reduce the quality and recovery of exosomes purified with a density gradient. The choice of isolation method impacts on particle concentration and protein yield Exosomes were prepared from concentrated CCM of SK-MES-1 cells using 4 different isolation techniques.

Discussion EVs, including exosomes, are present in human biofluids such as plasma 1 , 14 , Supplementary Material Optimized exosome isolation protocol for cell culture supernatant and human plasma: Click here for additional data file. Optimized exosome isolation protocol for cell culture supernatant and human plasma: Click here for additional data file. Acknowledgements The authors thank all Tumour Microenvironment Laboratory members for critical advice and proof-reading of the manuscript.

Conflict of interest and funding The authors declare no conflict of interest. References 1. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta. Cancer becomes wasteful: emerging roles of exosomes dagger in cell-fate determination.