Sep 19, 2020
Ultrafiltration and purification of conditioned media (Pall Jumbsosep and Izon qEV-10)
- 1Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health
- Translational Nanobiology Section
Protocol Citation: Joshua A Welsh, Julia Kepley, Bryce Killingsworth, Tim Traynor, Jennifer Jones 2020. Ultrafiltration and purification of conditioned media (Pall Jumbsosep and Izon qEV-10) . protocols.io https://dx.doi.org/10.17504/protocols.io.beqnjdve
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 07, 2020
Last Modified: September 23, 2020
Protocol Integer ID: 35310
Disclaimer
This protocol summarizes key steps for a specific type of method, which is one of a collection of methods and assays used for EV analysis in the NCI Translational Nanobiology Section at the time of submission of this protocol. Appropriate use of this protocol requires careful, cohesive integration with other methods for EV production, isolation, and characterization.
Abstract
Protocol for ultrafiltration of cell culture conditioned media using Pall Jumbosep products and purification of EVs from conditioned media concentrate using Izon qEV-10 size exclusions columns.
Guidelines
- All handling of conditioned media for isolation and purification of EVs should be done in sterile conditions
- Each additional 60 mL of conditioned media that is filtered through the Jumbsosep device insert increases the time required to concentrate subsequently added media to the device
Materials
MATERIALS
0.5M NaOH
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190136
Ethanol
For concentrating conditioned media:
- An unopened sterile 500 mL bottle of Gibco DPBS
- 70 mL of HPLC or other pure water
- 30 mL of Ethanol
- Pall Jumbosep filtrate receiver, sample reservoir, and 100k membrane insert
- 3 Eppendorf 5 mL low protein binding tubes
- Centrifuge inserts to hold Jumbosep devices
Table of available Pall Jumbsosep products (Jan 2020)
For concentrate purificiation with qEV column:
- Izon qEV-10 Column (70 nm pore size)
- New 500 mL of bottle Gibco sterile DPBS
- 14 x 5 mL LoBind Eppendorf tubes
- ~ 20 mL of filterd 20% ethanol
- ~ 5 mL of filtered 0.5 M NaOH
Preparing the Pall Jumbosep device
Preparing the Pall Jumbosep device
Make up 70% ethanol with HPLC or other pure water
Filter at least 20 mL of the 70% ethanol with a 0.02 µm syringe-driven filter, or other small pore size filter
Obtain a Jumbosep sample reservoir, a Jumbsosep filtrate receiver, and a Jumbosep insert
Snap the reservoir onto the receiver
Without contacting the inside of the reservoir or the insert, place the insert into the reservoir
Use the blunt end of an aspirating tip to firmly push the insert into place
Fill the reservoir with at least 10 mL of the prepared filtered 70% ethanol
Place the reservoir cap onto the reservoir
Swirl the device around to maximize ethanol contact and let sit for a 5 minutes
Spin the device (with an appropriate swinging bucket rotor and adaptor insert, along with another balancing device, as needed) at 2500 RCF for 5 minutes to concentrate ethanol down to the device's dead volume of 5 mL
To separate the assembled device, remove the reservoir cap, clasp hands behind the device, place both thumbs on the lip of the sample reservoir, and press upward and backward.
Use a tissue to blot any fluid which may be present on rim of reservoir and receiver
Flush the reservoir with ~10 mL of sterile PBS and aspirate all volume from the receiver
Assemble the device, add 60 mL of sterile PBS to the reservoir and spin at 3000 RCF for 5 minutes
Aspirate all volume from the receiver, and the device is ready to have conditioned media loaded onto it
Cell culture media clearing
Cell culture media clearing
Cell culture media is ready to be harvested from cells when they are 50% to 80% confluent, typically 48 hours after cells have been seeded into phenol-red-free, bovine EV-depleted complete media
Using a 50 mL serological pipet tip, transfer the complete volume of media in each flask of cells to a sterile 50 mL conical tube
Spin the tubes at 500 RCF for 10 minutes
Transfer the supernatants from the 50 mL conical tubes to a new set 50 mL conical tubes, leaving behind a small but visible pellet of cells and cell debris
Spin the tubes at 2500 RCF for 10 minutes
Transfer the supernatants from the 50 mL conical tubes (leaving behind 1 to 2 mL of fluid at the bottom of the tubes) to a sterile media storage bottle for temporary storage during the subsequent ultrafiltration process
Concentrating cleared conditioned media
Concentrating cleared conditioned media
Wipe down with 70% ethanol two 750 mL centrifuge bucket inserts and the insides of the 750 mL centrifuge buckets
Load conditioned media into the reservoir until the volume reaches the 60 mL mark
Spin the device at 3000 RCF for ~20 minutes checking to ensure only the ~5 mL dead volume of fluid remains in the sample reservoir after the spin
Disasemble the device, aspirate the filtrate, and reasemble the device before addition of conditioned media
Add conditioned media to the sample reservoir until it reaches the 60 mL mark
If you are trying to balance two devices but have less than 60 mL of media for each device, PBS can be added to one of the devices to ensure the volumes are equal
Spin at 3000 RCF for 30 minutes at room temperature or until only ~5 mL of fluid remains in the sample reservoir
Continue the process of adding additional cleared conditioned media until all media has been transferred to the sample reservoir, increasing the centrifuge time as necessary (typically 5 to 10 minutes more per additional spin)
If the last volume of media remaining to be filtered is not 60 mL, PBS can be added to the reservoir to fill it to the 60 mL marker
After the last spin with added cleared media, fill the sample reservoir with 60 mL of PBS, ensuring resuspension of any material adhered to the bottom of the reservoir and insert, and complete the final 3000 RCF spin at RT, so that there is a ~5 mL dead volume
Note
This is a wash step to remove any soluble protein <100 kDa that may be remaining in the 5 mL dead volume.
Being careful not to spill concentrate or create excessive bubbles, use a P1000 micropipette to thoroughly resuspend the concentrate which is sitting on top of the insert in the sample reservoir
Transfer the ~5 mL from the top of the sample reservoir to Eppendorf Low-protein binding 5.0 mL tubes, placing a maximum volume of 4 mL in each tube to prevent overflow
Label and store the conditioned media concentrate at 4 ˚C overnight, do not store for more than a short period of time without purifying the concentrate
Size exclusion chromatography of conditioned media concentrate
Size exclusion chromatography of conditioned media concentrate
Remove a new qEV-10 column from its 4˚C storage location at the start of work day to allow to warm to room temperature
Use a ring stand and metal clamp to position the column vertically
Rinse 5 mL of PBS from a new, unopened, room temperature bottle of PBS through the loading chamber into a waste container
Unscrew the top cap from the column
Fill the column with PBS until it fills the column
Press the loading chamber into the top of the column, ensuring a tight seal between the two units
Fill the loading chamber to the top with PBS
Unscrew the bottom cap from the column, PBS should flow through the column and into a waste container
Once around half the PBS volume in the loading chamber has flushed through the column, fill it back up to the top with PBS
While the column is being flushed, label 14, 5mL low protein-binding Eppendorf tubes with the a fraction number (1 through 14) and date. A marker can be used to highlight the 5.0 mL volume graduation on the tube
As soon as all of the loaded PBS has moved through the loading chamber, position the "Fraction 1" 5.0 mL tube under the colum. Gently vortex and invert the concentrated media and add it to the loading chamber 1 mL at a time, ensuring to pipette the media directly onto the reservoir filter and not the walls of the chamber. The time elapsed from the last drop of PBS eluting to loading the cell culture media concentrate onto the column should be as short as possible.
As soon as all of the loaded concentrated media has passed through the loading chamber, the loading reservoir can be filled to the top with PBS
Continue collecting fractions in 5.0 mL increments until all fractions have been collected, refilling the loading chamber to the top with PBS when the PBS level falls by about half
While PBS continues to flush through column, into a waste container, collected fraction tube lids can be closed and set aside
Once the entire volume of PBS in the loading chamber has run through, add 5 mL of filtered 0.5 M NaOH to the chamber and allow it to pass through the column. The NaOH can be added by pipetting along the walls of the reservoir to wash away any contaminants
Fill the buffer reservoir to the top with PBS and allow it all to pass through the column except for the last ~1 mL to prevent drying the column
Refill the buffer reservoir to the top with PBS
After the majority of the buffer has eluted use a pH test strip to ensure the eluting buffer is neutral
Fill the column up with 5 mL of filtered 20% EtOH and allow it to flush through column
Place the bottom cap back on the column
Pipette filtered 20% EtOH into the top of the column until it is filled to the top with EtOH.
Place the top cap back on the column
Allow the loading chamber to dry in the hood overnight
Return all column components to the box they came from and lable the box with date used, cell line used on, and volume of concentrated media loaded into the column.