TITLE: Ultrafiltration and purification of conditioned media (Pall Jumbsosep and Izon qEV-10)  
AUTHORS: Joshua Welsh, Julia Kepley, Bryce Killingsworth, Tim Traynor, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Preparing the Pall Jumbosep device]
Make up 70% ethanol with HPLC or other pure water
?. [Preparing the Pall Jumbosep device]
Filter at least 20 mL of the 70% ethanol with a 0.02 µm syringe-driven filter, or other small pore size filter
?. [Preparing the Pall Jumbosep device]
Obtain a Jumbosep sample reservoir, a Jumbsosep filtrate receiver, and a Jumbosep insert
?. [Preparing the Pall Jumbosep device]
Snap the reservoir onto the receiver
?. [Preparing the Pall Jumbosep device]
Without contacting the inside of the reservoir or the insert, place the insert into the reservoir
?. [Preparing the Pall Jumbosep device]
Use the blunt end of an aspirating tip to firmly push the insert into place
?. [Preparing the Pall Jumbosep device]
Fill the reservoir with at least 10 mL of the prepared filtered 70% ethanol
?. [Preparing the Pall Jumbosep device]
Swirl the device around to maximize ethanol contact and let sit for a 5 minutes
?. [Preparing the Pall Jumbosep device]
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
?. [Preparing the Pall Jumbosep device]
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.
?. [Preparing the Pall Jumbosep device]
Flush the reservoir with ~10 mL of sterile PBS and aspirate all volume from the receiver
?. [Preparing the Pall Jumbosep device]
Assemble the device, add 60 mL of sterile PBS to the reservoir and spin at 3000 RCF for 5 minutes
?. [Preparing the Pall Jumbosep device]
Aspirate all volume from the receiver, and the device is ready to have conditioned media loaded onto it
?. [Preparing the Pall Jumbosep device]
Place the reservoir cap onto the reservoir
?. [Preparing the Pall Jumbosep device]
Use a tissue to blot any fluid which may be present on rim of reservoir and receiver
?. [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
?. [Cell culture media clearing]
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
?. [Cell culture media clearing]
Spin the tubes at 500 RCF for 10 minutes
?. [Cell culture media clearing]
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
?. [Cell culture media clearing]
Spin the tubes at 2500 RCF for 10 minutes
?. [Cell culture media clearing]
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]
Wipe down with 70% ethanol two 750 mL centrifuge bucket inserts and the insides of the 750 mL centrifuge buckets
?. [Concentrating cleared conditioned media]
Load conditioned media into the reservoir until the volume reaches the 60 mL mark
?. [Concentrating cleared conditioned media]
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
?. [Concentrating cleared conditioned media]
Disasemble the device, aspirate the filtrate, and reasemble the device before addition of conditioned media
?. [Concentrating cleared conditioned media]
Add  conditioned media to the sample reservoir until it reaches the 60 mL mark
?. [Concentrating cleared conditioned media]
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
?. [Concentrating cleared conditioned media]
Spin at 3000 RCF for 30 minutes at room temperature or until only ~5 mL of fluid remains in the sample reservoir
?. [Concentrating cleared conditioned media]
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)
?. [Concentrating cleared conditioned media]
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
?. [Concentrating cleared conditioned media]
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
This is a wash step to remove any soluble protein
?. [Concentrating cleared conditioned media]
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
?. [Concentrating cleared conditioned media]
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
?. [Concentrating cleared conditioned media]
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 ]
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
?. [Size exclusion chromatography of conditioned media concentrate ]
Use a ring stand and metal clamp to position the column vertically
?. [Size exclusion chromatography of conditioned media concentrate ]
Rinse 5 mL of PBS from a new, unopened, room temperature bottle of PBS through the loading chamber into a waste container
?. [Size exclusion chromatography of conditioned media concentrate ]
Unscrew the top cap from the column
?. [Size exclusion chromatography of conditioned media concentrate ]
Fill the column with PBS until it fills the column
?. [Size exclusion chromatography of conditioned media concentrate ]
Press the loading chamber into the top of the column, ensuring a tight seal between the two units
?. [Size exclusion chromatography of conditioned media concentrate ]
Fill the loading chamber to the top with PBS
?. [Size exclusion chromatography of conditioned media concentrate ]
Unscrew the bottom cap from the column, PBS should  flow through the column and into a waste container
?. [Size exclusion chromatography of conditioned media concentrate ]
Once around half the PBS volume in the loading chamber has flushed through the column, fill it back up to the top with PBS
?. [Size exclusion chromatography of conditioned media concentrate ]
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
?. [Size exclusion chromatography of conditioned media concentrate ]
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.
?. [Size exclusion chromatography of conditioned media concentrate ]
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
?. [Size exclusion chromatography of conditioned media concentrate ]
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
?. [Size exclusion chromatography of conditioned media concentrate ]
While PBS continues to flush through column, into a waste container, collected fraction tube lids can be closed and set aside
?. [Size exclusion chromatography of conditioned media concentrate ]
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
?. [Size exclusion chromatography of conditioned media concentrate ]
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
?. [Size exclusion chromatography of conditioned media concentrate ]
Refill the buffer reservoir to the top with PBS
?. [Size exclusion chromatography of conditioned media concentrate ]
After the majority of the buffer has eluted use a pH test strip to ensure the eluting buffer is neutral
?. [Size exclusion chromatography of conditioned media concentrate ]
Fill the column up with 5 mL of filtered 20% EtOH and allow it to flush through column
?. [Size exclusion chromatography of conditioned media concentrate ]
Place the bottom cap back on the column
?. [Size exclusion chromatography of conditioned media concentrate ]
Pipette filtered 20% EtOH into the top of the column until it is filled to the top with EtOH.
?. [Size exclusion chromatography of conditioned media concentrate ]
Place the top cap back on the column
?. [Size exclusion chromatography of conditioned media concentrate ]
Allow the loading chamber to dry in the hood overnight
?. [Size exclusion chromatography of conditioned media concentrate ]
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.