This video will show you how to adapt PSCs
adapted to feeder-based or feeder-free media to Essential 8 Medium and Vitronectin. Essential 8™ Medium is a fully defined,
feeder-free medium formulated for the growth and expansion of human pluripotent stem cells.
It was originally developed as E8 Medium in the laboratory of James Thomson, and validated
by Cellular Dynamics International. Unlike other feeder-free media such as mTeSR and
StemPro hESC SFM, Essential 8™ Medium does not require the presence of BSA (bovine serum
albumin) that contributes to lot-to-lot variability. In addition, other serum-free media consist
of more than 20 components, adding complexity, time, and cost, while Essential 8™ Medium
is comprised of only eight components. PSCs grown using mTeSR™ Medium on Matrigel™-coated
culture vessels can be thawed directly into Essential 8™ Medium on vitronectin-coated
culture vessels. PSCs maintained in KnockOut™ Serum Replacement
on feeders prior to freezing should be thawed into KSR Medium on MEF feeder layer. Allow
the cells to recover, and then passage into Essential 8™ Medium on vitronectin-coated
culture vessels, using EDTA as the passaging reagent. When thawing directly into Essential 8, place
10 mL of Essential 8™ Medium in a 50-mL tube and warm to room temperature. Next, equilibrate an appropriate quantity
of vitronectin-coated 6-well plates to room temperature. Remove the vial of PSCs from liquid nitrogen
storage using metal forceps. Transfer it on dry ice to the cell culture hood. Immerse the vial in a 37°C water bath without
submerging the cap. Swirl the vial gently. When only an ice crystal remains, remove the
vial from the water bath. Spray the outside of the vial with 70% ethanol
and place it in hood. Pipet cells gently into a sterile 50-mL conical
tube using a 5-mL sterile pipette. Slowly add 10 mL of Essential 8™ Medium
drop-wise to cells in the 50-mL conical tube. While adding the medium, gently move the tube
back and forth to mix the PSCs. This reduces osmotic shock to the cells. Rinse the vial with 1 mL of Essential 8™
Medium and add to the 50-mL tube with cells. Slowly transfer the contents to a 15mL tube Centrifuge the cells at 200 g for 5 minutes. Aspirate and discard the supernatant. Resuspend the cell pellet in 2 mL of Essential
8™ Medium by gently pipetting the cells up and down in the tube a few times. Aspirate the vitronectin from the pre-warmed
vitronectin-coated plates. Slowly add the PSC suspension into the vitronectin-coated
plate, plating one vial of thawed cells per well of the 6-well plate. Move the dish in several quick figure-eight
motions to disperse cells across the surface of the wells. Place the dish gently into the 37°C, 5% CO2
incubator and incubate the cells overnight. The next day, replace the spent medium with
fresh complete Essential 8™ Medium. Replace the medium daily thereafter until
the cells are approximately 85% confluent.

Leave a Reply

Your email address will not be published. Required fields are marked *