TITLE: Culture of established induced pluripotent stem cell lines
AUTHORS: Cellular Generation and Phenotyping

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the method for thawing, passaging and cryopreserving established feeder-free induced pluripotent stem cell lines. In this culture method, cells are kept as aggregates throughout the process. Cells are cultured on vitronectin matrix (full length xeno-free or truncated protein) in E8 or TeSR-E8 media. Cells can be cultured with or without Penicillin-Streptomycin in the media. Cell lines cultured using these protocols are suitable to use for downstream applications including differentiation and CRISPR screens. </div></div>

[STEPS]
?. [iPSC Thawing]
PreparationCoat a 6 well plate with vitronectin and incubate according to the manufacturer's instructions. Prepare complete E8 or TeSR-E8 media according to the manufacturer's instructions.
?. [iPSC Thawing]
Prepare thawing media by supplementing culture media with rock inhibitor (Y-27632) to a final concentration of 10μM.
?. [iPSC Thawing]
Add 8ml of thawing media per cryovial being thawed into a sterile 15ml falcon tube(s).
?. [iPSC Thawing]
Partially thaw the frozen cryovial(s) of iPS cells in a   water bath until there is a small ice crystal remaining.
37 °C
?. [iPSC Thawing]
Add 1ml of thawing media dropwise to each cryovial. Collect the full volume of media and cell suspension and add to the Falcon tube prepared in step 3. iPSCs should be kept as small clumps as much as possible to increase survival efficiency; minimize the amount of pipetting when thawing cells to reduce the number of single cells in the cell suspension.
?. [iPSC Thawing]
Centrifuge at 120 rcf for 3 min at room temperature.
Centrifuge: 120 34, 3 min
?. [iPSC Thawing]
Aspirate the vitronectin from the labware surface and replace with 1ml of thawing media per well. Do not allow surfaces to dry out.
?. [iPSC Thawing]
Aspirate the supernatant and gently re-suspend each cell pellet in 1ml of thawing media. Plate the cell suspension into the 1ml of thawing media in the well(s).
?. [iPSC Thawing]
Agitate the plate gently within a tissue culture incubator set at  , 5% CO2 to ensure even distribution of cells across the well.
37 °C
?. [iPSC Thawing]
The cells will take 2-24 hours to attach to the surface. Make sure the plate is not disturbed during this time. 24 hours after plating, do a full media change to remove the rock inhibitor.
?. [iPSC Passaging]
PreparationCoat a 6 well plate(s) with vitronectin and incubate according to the manufacturer's instructions.
?. [iPSC Thawing]
Media change the cells every 24 hours with culture media until they reach 70-80% confluency; at this point they need to be passaged.
?. [iPSC Passaging]
Prepare fresh 0.5mM EDTA by diluting UltraPure 0.5M EDTA, pH 8.0 with DPBS(-/-). Store at room temperature and use on day of preparation only.
?. [iPSC Passaging]
Aspirate the spent medium from cells and wash with 2ml of DPBS(-/-) per well.
?. [iPSC Passaging]
Aspirate DPBS(-/-) and add 1-2ml of 0.5mM EDTA per well. Rock the dishes to cover the surface of the cells and incubate for 4 min at room temperature.
?. [iPSC Passaging]
Observe the colonies under a microscope until colonies display shiny ‘halos’ around the edges and holes appear throughout the colonies.
?. [iPSC Passaging]
Aspirate the EDTA solution and add 2ml of culture medium per well.
?. [iPSC Passaging]
Collect and dispense medium across the labware surface up to 3 times to detach cells. If colonies are still attached, repeat wash with same volume of fresh media. Avoid creating bubbles. Collect the cells in a falcon tube.
?. [iPSC Passaging]
Aspirate victronectin from new labware surfaces and replace with 1ml of pre-warmed culture media. Do not allow surfaces to dry out.
?. [iPSC Passaging]
Add required amount of culture medium to cell suspension depending on desired split ratio. Aim for a split ratio where the cells are passaged approximately every 4-5 days. The split ratio may have to be adjusted depending on cell quality.
?. [iPSC Passaging]
Plate 1ml of cell suspension into each new well.
?. [iPSC Passaging]
Agitate the plate gently within a tissue culture incubator set at  , 5% CO2 to ensure even distribution of cells across the well.
37 °C
?. [iPSC Cryopreservation]
When colonies are compact and roughly 70% confluent they are ready to be frozen. One confluent well of a 6 well plate should be frozen down into 5 cryovials.
?. [iPSC Passaging]
The cells will take 2-24 hours to attach to the surface. Media change the cells every 24 hours. Culture the cells until they reach 70-80% confluency; at this point they can passaged again for further expansion.
?. [iPSC Cryopreservation]
Make fresh freezing media using 10% DMSO in KSR (for example 1mL DMSO in 9mL KSR) and store at 4°C until required.
?. [iPSC Cryopreservation]
Aspirate the spent medium from the labware and wash with 2ml of DPBS(-/-) per well.
?. [iPSC Cryopreservation]
Aspirate the DPBS(-/-) and add 2ml of 0.5mM EDTA per well.
?. [iPSC Cryopreservation]
Rock the dishes to cover the surface of the cells and incubate for 4-8 min at room temperature. Observe the colonies under a microscope until colonies display shiny ‘halos’ around the edges and holes appear throughout the colonies.
?. [iPSC Cryopreservation]
Aspirate the EDTA solution and add 2ml of culture medium per well for cell collection.
?. [iPSC Cryopreservation]
Remove the cells by gently washing the labware surface with the media up to 3 times. If >50% of colonies are still attached, repeat wash with same volume of fresh media. Avoid creating bubbles. Collect the cells into an appropriate Falcon tube.
?. [iPSC Cryopreservation]
Centrifuge the Falcon tube at 120rcf for 1 min.
Centrifuge: 120 34, 1 min
?. [iPSC Cryopreservation]
Aspirate the supernatant and re-suspend pellet in enough of the freezing media (prepared in step 3) to give a total volume of 1mL per cryovial.
?. [iPSC Cryopreservation]
Dispense 1mL of cell suspension into each cryovial. Place the vials in a CoolCell or similar freezing container and store at  -80°C. Note: Once in freezing media, cells should be transferred to the -80°C freezer as quickly as possible to prevent cell death.
?. [iPSC Cryopreservation]
After 24-48 hours at -80°C, transfer vials from the CoolCell into Liquid Nitrogen storage. The vials can be stored in liquid nitrogen indefinitely.
?. [iPSC Cryopreservation]
Prepare fresh 0.5mM EDTA by diluting UltraPure 0.5M EDTA, pH 8.0 with DPBS(-/-). Store at room temperature and use on day of preparation only.