IZON qEV10 Columns Isolate Extracellular Vesicles User Manual

IZON qEV10 Columns Isolate Extracellular Vesicles

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Izon Science Ltd
PO Box 9292
Addington
Christchurch 8024

New Zealand
Telephone: +64 3 357 4270
Email: [email protected]
Website: www.izon.com

DEFINITIONS AND WRITING CONVENTIONS

Make sure to follow the precautionary statements presented in this guide. Safety and other special notices will appear in boxes and include the symbols detailed in Table 1.

Table 1: Safety and Hazard Symbols

| This symbol indicates general advice on how to improve procedures or recommends measures to take in specific situations.
---|---
****| This symbol indicates where special care should be taken.

Table 2: Terminology Used in this Manual

Buffer volume
(Previously void volume)| The volume of liquid that corresponds to the volume before the Purified Collection Volume (PCV); this volume is collected automatically into the central well. The buffer volume is different for each column type.
Chromatography| A method used primarily for separation of the components of a sample. The components are distributed between two phases; one is stationary while the other one is mobile. The stationary phase is either a solid, a solid-supported liquid, or a gel. The stationary phase may be packed in a column, spread as a layer or distributed as a film. The mobile phase may be gaseous or liquid.
Column volume| The combined volume of packed material and buffer volume (can be referred to as the bed volume).
Degassing| Degassing involves subjecting a solution to vacuum to “boil” off excess dissolved gas e.g. applying a vacuum to a flask.
Flow rate| The volumetric flow in mL/min of the carrier liquid.
Purified Collection Volume (PCV)| The volume immediately succeeding the buffer volume, containing particles of interest purified from the loaded sample. The PCV is different for each column type.

SAFETY AND HAZARDS

Refer to the Safety Data Sheet for the classification and labelling of hazards and associated hazard and precautionary statements. The The Safety Data Sheet for qEV Isolation columns is located at www.izon. com/resources

Hazards

qEV SMART columns are a laboratory product. However, if biohazardous samples are present, adhere to current Good Laboratory Practices (cGLP) and comply with any local guidelines specific to your laboratory and location.

Disposal of Biohazardous Material
The qEV SMART column contains < 0.1% sodium azide, which is potentially fatal if swallowed or in contact with skin. Please review
the following guidelines and precautions prior to each use of the qEV SMART column:

Prevention:

1. Do not get into eyes, on skin, or on clothing.
2. Wash skin thoroughly after handling.
3. Do not eat, drink, or smoke when using this product.
4. Avoid release of product into the environment.
5. Wear protective gloves and clothing; follow general laboratory precautions.

Response

1. IF SWALLOWED: immediately call a POISON CONTROL CENTRE/Doctor.
2. IF ON SKIN: Gently wash with plenty of soap and water and immediately call a POISON CONTROL CENTRE/Doctor.
3. Remove immediately any contaminated clothing and wash before reuse.
4. Collect any spillage and dispose of appropriately.

For more information, see the MSDS Documentation for Izon qEV SMART columns: www.izon.com/resources

Sodium azide can be fatal if swallowed or in contact with skin. It can cause damage to neurological organs through prolonged or repeated exposure. It is very toxic to aquatic life with long-lasting effects.

Be sure to adhere to the following guidelines and comply with any local guidelines specific to your laboratory and location regarding use and disposal.

General Precautions:

• Always wear laboratory gloves, coats, and safety glasses with side shields or goggles.
• Keep your hands away from your mouth, nose, and eyes.
• Completely protect any cut or abrasion before working with potentially infectious or hazardous material.
• Wash your hands thoroughly with soap and water after working with any potentially infectious or hazardous material before leaving the laboratory.
• Remove watches and jewelry before working at the bench.
• The use of contact lenses is not recommended due to complications that may arise during emergency eye-wash procedures.
• Before leaving the laboratory, remove protective clothing.
• Do not use a gloved hand to write, answer the telephone, turn on a light switch, or physically engage with people without gloves.
• Change gloves frequently.
• Remove gloves immediately when they are visibly contaminated.
• Do not expose materials that cannot be properly decontaminated to potentially infectious or hazardous material.
• Upon completion of the tasks involving potentially infectious or hazardous materials, decontaminate the work area with an appropriate disinfectant or cleaning solution (1:10 dilution of household bleach is recommended).

Dispose of the following potentially contaminated materials in accordance with laboratory local, regional, and national regulations:

• Biological samples
• Reagents
• Used reaction vessels or other consumables that may be contaminated.

Storage

Rapid changes in temperature should be avoided, as this can introduce bubbles into the gel bed.
Unused qEV columns can be stored at room temperature. Used qEV columns can be stored at room temperature providing they have been cleaned according to the instructions in this document and stored in 20% ethanol or 0.05% w/v sodium azide. If the appropriate solutions are not available for storage at room temperature, then columns can be stored at +4 to +8 °C after use.

Disposal

Waste buffer should be disposed of in a safe manner. Sodium azide accumulation over time in copper pipes can result in an explosion.

INTRODUCTION TO SIZE EXCLUSION CHROMATOGRAPHY

Overview

qEV Size Exclusion Chromatography (SEC) columns separate particles based on their size as they pass through a column packed with a porous, polysaccharide resin. As the sample passes through the column under gravity, smaller particles enter the resin pores on their way down and their exit from the column is delayed (Figure 1C). As the sample exits the column, sequential volumes are collected. Particles will be distributed across the volumes based on their size, with the largest particles exiting the column first and the smallest particles exiting the column last.

The packed column is equilibrated with a buffer, which fills the column. The total column volume is occupied by both the solid resin (stationary phase) and the liquid buffer (the mobile phase). As the particles do not bind to the resin, the buffer composition will not significantly affect the resolution (the degree of separation between peaks).

Figure 1: Process of SEC (A) Schematic picture of a resin bead with an electron microscopic enlargement. (B) Schematic drawing of sample molecules diffusing into the pores of the resin beads. (C) Graphical description of separation: (i) sample is applied to the column; (ii) the smallest particles (yellow) are more delayed than the largest particles (red); (iii) the largest particles are eluted first from the column. Band broadening causes significant dilution of the particle zones during chromatography. (D) Schematic chromatogram. From: GE Healthcare and Biosciences. (n.d.). Size Exclusion Chromatography Principles and Methods [Brochure]. Uppsala, Sweden. Accessed June 2019.

Intended Use

Izon qEV columns isolate extracellular vesicles from biological samples. qEV10 columns are equipped with RFID chips for use with the Izon Automatic Fraction Collector (AFC). These chips will not impact manual use. The column is intended to be used in research laboratories by professional personnel for research use only. The qEV column is not intended for diagnostic purposes and should not be used to make treatment decisions.
qEV columns are designed to isolate and purify vesicles from most biological samples, including:

• Serum
• Plasma
• Saliva
• Urine
• Cerebrospinal Fluid (CSF)
• Cell culture media

NOTE: most ‘raw’ samples cannot be directly run on qEV columns and analysed with TRPS without some preparation such as centrifugation and concentration steps. Contact the Izon Support Centre for recommendations and protocols.

Comparison of qEV/35nm and qEV/70nm series

All qEV columns are available in one of two isolation ranges, the
qEV/35nm series and the qEV/70nm series. The qEV/35nm series of columns generally perform better when the target particle to be isolated is less than 110 nm in diameter, while the qEV/70nm series of columns generally perform better when the target particle to be isolated is greater than 110 nm in diameter (see Table 3). For optimal recovery of particles between 35 and 350 nm a qEV/35nm series column is recommended. For optimal recovery of particles between 70 and 1000 nm a qEV/70nm series column is recommended.

Table 3: Specifications of qEV/35nm and qEV/70nm Series

| qEV/35nm SERIES| qEV/70nm SERIES
---|---|---
Target Particle Size (nm)| 35 nm to 350 nm| 70 nm to 1000 nm
Optimum Recovery Range (nm)| Particles < 110 nm| Particles > 110 nm

qEV10 Specifications

Table 4: qEV10 Specifications

• Loading higher sample volumes results in a lower level of purity in the later vesicle volumes, greater overlap between protein and EV elution peaks, and a higher protein peak within the PCV. Loading lower sample volumes results in a higher dilution factor of the sample. The optimal recommended sample volume for purity on the qEV10
  is 10 mL.

• Values for human plasma samples only.

qEV10 Performance Characteristics

As shown in Figure 3 and 4 below, particles less than 70 nm typically elute later than the PCV on the qEV/70nm, whereas particles larger than 35 nm are captured in this zone on the qEV/35nm column. A higher recovery in the PCV of particles larger than 70 nm occurs on the qEV/70nm series columns compared with the qEV/35nm series (Fig. 4). Proteins typically elute slightly earlier on the qEV/35nm series. Higher protein levels in the PCV are mainly due to an increase in EV bound proteins.

Different samples may give slightly different elution profiles and purity, hence an initial measurement of EV concentration and protein contaminants in collected fractions is recommended.

Figure 3: Comparison of plasma protein elution and recovery levels of 69 nm liposomes between a qEV10/35nm and a qEV10/70nm.

Figure 4: Comparison of plasma protein elution and recovery levels of 200 nm particles between a qEV10/35nm and a qEV10/70nm.

qEV10 EV Elution Profile

The elution of vesicles typically peaks at 10 mL ± 5 mL after the buffer volume, for a 10 mL sample volume and collecting 5 mL fractions. Figure 5 shows the elution of vesicles when 10 mL of plasma sample is loaded onto a qEV10/35nm column.

The majority of EVs typically elute in 20 mL after the buffer volume. If higher purity is desired, collect only the first 15 mL. The user therefore chooses between maximizing recovery by collecting a bigger volume or maximizing purity by collecting a lesser volume.

The elution of plasma protein is slower, eluting predominantly from 25 – 70 mL after the buffer volume. Any vesicles recovered beyond 20 mL contain higher protein contamination and may be less suitable for downstream analysis because of their lower purity.

Indicative protein elution profiles can be obtained by monitoring the absorbance at a wavelength of 280 nm. An accurate measurement of the level of protein can be obtained using a colorimetric protein assay.

Figure 5: Typical elution profile for a qEV10/35nm column with 10 mL of plasma loaded; proteins elute later than extracellular vesicles (EVs) and similarly sized particles >60 nm. The vesicle concentration was measured using TRPS and relative protein levels by absorbance at 280 nm.

qEV10 Sample Input Volume Effects and

Recovery Rates
Effect of Sample Input Volume on EV Elution Profile
Loading higher sample volumes results in a lower level of purity in the later vesicle volumes, greater overlap between protein and EV elution peaks, and a higher protein peak within the PCV. Figure 6 shows the effect of loading 5 mL and 10 mL of plasma. Note the delay in elution of EVs in the 10 mL sample.

The optimal recommended sample volume for purity on the qEV10 is 10 mL, which consistently results in vesicles eluting in the 20 mL PCV. Loss of vesicles occurs with sample volumes over 10 mL as the vesicle elution is broadened. EVs collected outside of the 20 mL PCV are not recommended for downstream analysis where high purity is required.

Figure 6: The effect of loading larger volumes is shown in the extracellular vesicles (EVs) and similarly sized particles >60 nm profiles from loading 5 mL and 10 mL of plasma.

Effect of Resin Type on Protein Elution Profile
The PCV on a qEV10 contains very little protein, with protein levels increasing in later volumes, as shown in Figure 7. Higher protein levels for the qEV10/35nm are due in part to higher recovery of smaller EVs in the PCV (refer to Fig. 2).

Figure 7: Percentages of protein cf. starting quantity of protein from loading 10 mL of plasma onto a qEV10. Pink bars are the PCV. qEV10/70nm bars are dark pink and dark blue. qEV10/35nm bars are light pink and light blue.

MANUAL OPERATING INSTRUCTIONS

The following section provides instructions for the manual use of qEV columns. For use of qEV columns with the Automatic Fraction Collector (AFC) instrument, please see the full AFC User Manual at: support.izon.com

Operational Recommendations

The following recommendations are provided to ensure optimal performance of the qEV column:

• Centrifuge samples prior to loading onto the column. To avoid clogging of column frits, it is recommended to filter or centrifuge the biological sample to remove large particulate matter.
  • Centrifuge samples at 1,500 x g for 10 minutes to remove any cells and large particles.
  • Gently move the supernatant to a new tube and centrifuge again at 10,000 x g for 10 min.
  • For macrovesicle isolation, use lower g-forces for the second  centrifugation step.
• Samples can be concentrated before application to the column or after isolation if needed. It is possible to concentrate samples both before and/or after use of the qEV column, however Izon offers multiple column sizes to reduce the need for pre-analytical sample concentration. If concentration protocols are needed, please consider the following recommendations:
  • Concentration of some sample types may result in the formation of precipitates and protein aggregates, especially for urine samples. Concentrated samples should be centrifuged at 10,000 x g for 10 minutes prior to loading onto a qEV column.
  • Izon recommends using Merck Millipore concentration devices (Amicon® Ultra Centrifugal filters; C7715). Use according to manufacturer’s recommendations.
  • Concentration of samples after purification with qEV may result in the loss of some EVs on the membrane.
• Single-use columns are advisable where the vesicles will be analyzed for nucleic acids. Use of a single-use column, such as the Izon qEV single, will reduce the possibility of cross-contamination.
• Ensure that the sample buffer has been prepared appropriately. To maintain the functionality of EVs, the flushing buffer should be of the same temperature as the sample buffer. SEC can also be used to exchange the buffer of a sample.
  • Sample buffer temperature should be within the operational temperature of 18-24 ˚C (65-75 ˚F).
  • Sample buffers should be degassed and room temperature to avoid air bubbles forming in the gel bed. Rapid changes in temperature, for example removing packed columns from a cold room and applying buffer at room temperature, can introduce air bubbles in the packed bed, resulting in poorer separation.
  • Use a buffer with an ionic strength of 0.15 M or greater to avoid any unwanted ionic interactions between the solute molecule and the matrix.
  • Only use freshly filtered (0.22 μm) buffer to avoid introducing particulate contamination.
  • qEV columns come equilibrated in filtered PBS containing <0.1% w/v sodium azide.

Column Setup and Equilibration

1. Equilibrate the column and the sample buffer to be within the operational temperature range of 18-24 ˚C.
   Do not remove the column caps until the column has reached operational temperature.
   Sample buffers should be degassed and room temperature to avoid air bubbles forming in the gel bed.

2. Attach the column in an upright position to a stand ready for use.

3. Rinse the reservoir with buffer.

4. Before connecting the reservoir to the column, add 5 mL of buffer to the reservoir and wait for the frit to wet and buffer to start running through.

   • If frit is slow to wet, apply some pressure to the reservoir top with the palm of your hand to aid the flow.

5. Allow buffer to run until it stops at the frit.

6. Remove the column connector cap, top up the connector with buffer, and firmly attach (a good seal is critical) the loading reservoir to the connector being careful to avoid trapping air bubbles in the connector.

7. Add buffer to the reservoir.

8. Remove the bottom cap and allow the buffer to start running through the column.

9. If you are using PBS as your buffer then your column is ready to use. If your downstream application is affected by 0.05% sodium azide, flush the column with at least two column volumes of PBS. If an elution buffer other than PBS is to be used, equilibrate the column with at least three column volumes of the new buffer.

Only use freshly filtered (0.22 µm) buffer to avoid introducing particulate contamination

Sample Loading

1. To avoid clogging of column frits, it is recommended to filter or centrifuge the biological sample to remove large particulate matter. See Section 4.1: Operational Recommendations for more information.

2. Continue to allow buffer to run through the column. The column will stop flowing when all of the buffer has entered the loading frit.

3. Load the prepared centrifuged sample volume onto the loading frit.
   Avoid stopping the column flow during the run for long periods of time to ensure accurate EV separation.

4. Immediately start collecting the buffer volume (this includes the volume displaced by loading the sample).

5. Allow the sample to run into the column. The column will stop flowing when all of the sample has entered the loading frit.

6. Top up the column with buffer and continue to collect the buffer volume.

7. Once the buffer volume is collected, continue to collect the Purified Collection Volume (PCV).
   To collect accurate volumes, only load the required volume to the top of the column, wait for the volume to run through until the flow stops and repeat.

Column Flush and Storage

1. After the desired fractions have been collected, the column should be cleaned and sanitized to remove residual proteins. Rinse the column with 140 mL of buffer directly after finishing fraction collection, then wash the column with 5 mL of 0.5 M NaOH, then flush with 140 mL of buffer to return the column pH to normal before loading another sample.
   Simply flushing with a large volume of buffer after fraction collection is not sufficient to clean the column completely and there may be some carry-over from previous samples.

2. If storing the column for future use, it should be stored in a bacteriostatic agent such as PBS containing 0.05% w/v sodium azide, or 20% ethanol. Columns stored in 20% ethanol should be flushed with two column volumes of DI water after cleaning, then flushed with two column volumes of 20% ethanol for storage. Columns stored in buffer should be flushed with two column volumes of buffer.
   Avoid adding 20% ethanol to buffer inside the column as this can precipitate salt inside the gel bed and damage the column.

3. Columns can be stored at room temperature after use, providing they have been cleaned according to the instructions above. If the appropriate solutions are not available then columns can be stored at +4 to +8 ˚C after use.

Restoring Column Flow After a Blockage due to Airlock in the Junction

1. Place the bottom cap on the column.
2. Remove the loading reservoir.
3. Unscrew the column cap and add buffer to the top frit until the buffer is level with the top edge of the column.
4. Screw the column cap back on forcing buffer up through the connector junction.
5. Add 2 mL of buffer to the loading reservoir and allow buffer to run through until it stops at the frit.
6. Carefully attach the loading reservoir to the connector being careful to avoid trapping any air bubbles in the connector.
7. Add more buffer to the loading reservoir before removing the bottom cap.
8. The column should begin to flow again.

RESOURCES

Protocols for EV Isolation from Common Sources

See Izon Support Centre support.izon.com for application notes and typical protocols for common EV samples. If you are unsure of what to do to prepare your sample, please contact [email protected] for assistance.

EV Analysis Using TRPS

Izon recommends TRPS analysis for determination of particle size, concentration, and zeta potential. The Izon TRPS Reagent Kit includes coating solutions for pre-coating the pore, minimizing non-specific binding and provides for stable and accurate sizing and concentration analysis.
For TRPS analysis of the EVs, Izon recommends an initial dilution of
1/5 or 1/10 in electrolyte. Optimize the dilution to achieve a rate at the highest operating pressure of approximately 200 to 1600 particles per minute to avoid pore blockage.
See Izon Support Centre support.izon.com for more information on the analysis of EVs with TRPS.

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