TITLE: WTc11 iPSC Culture and Maintenance
AUTHORS: Connor Ludwig, Ruilin Tian, Martin Kampmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains general culture and maintenance of the WTc11 iPSC cell line. </div></div>

[STEPS]
?. [Prepare complete StemFlex Medium]
Thaw the frozen StemFlex Supplement 10X at  for ~ or overnight at  to .
0 Room temperature
2 °C
8 °C
IMPORTANT! Do not thaw the frozen supplement at 37°C.
?. [Prepare complete StemFlex Medium]
Mix the thawed supplement by gently inverting 3−5 times.
?. [Prepare complete StemFlex Medium]
Aseptically transfer 50 mL of StemFlex Supplement 10X to the bottle of StemFlexTM Basal Medium (450 mL fill).
?. [Prepare complete StemFlex Medium]
Gently invert the bottle several times to obtain 500 mL of homogenous complete medium.
Following reconstitution, complete StemFlexTM Medium can be stored at 2°C to 8°C for up to 2 weeks or  aliquoted and stored at   to   for up to 6 months. Alternatively, usage size aliquots of the supplement can be made and frozen at   to   for up to 6 months. Avoid multiple freeze-thaw cycles.
?. [Feeding WTc11 iPSC ]
Feed the PSCs the day after seeding followed by every-other-day thereafter.
If the cells are to be left without feeding for two days (for example, over a weekend), then double the feed volume (i.e., 4 mL added per well of 6-well plate).
?. [Passaging WTc11 iPSC]
Coat desired wells/plates with diluted Matrigel and incubate at  for - using the following table for volumes to add per well:  ABCDEFG1Per:96-well24-well12-well6-well10-cm dish15-cm dish2Volume to add:40 μL200 μL0.5 mL1 mL5 mL10 mL
37 °C
ABCDEFG1Per:96-well24-well12-well6-well10-cm dish15-cm dish2Volume to add:40 μL200 μL0.5 mL1 mL5 mL10 mL
Matrigel may be re-used during this time only to coat additional plates. Original plates shouldhave PBS or media to prevent the matrix from drying out. Matrigel coated plates must be used within 14 days of coating.
?. [Passaging WTc11 iPSC]
iPSCs should be split when cells are ~ 80% confluent.
?. [Passaging WTc11 iPSC]
Tilt cell-containing plate towards you and aspirate existing media.
?. [Passaging WTc11 iPSC]
Wash wells once with ample PBS (about 2x amount of media).
?. [Passaging WTc11 iPSC]
Add accutase to well(s) using the following table for volumes per well and incubate at  for ; add another - if cells have not mostly lifted/dissociated.   ABCDEFG1Per:96-well24-well12-well6-well10-cm dish15-cm dish2Volume to add:20 μL100 μL250 μL0.5 mL2 mL4 mL
37 °C
ABCDEFG1Per:96-well24-well12-well6-well10-cm dish15-cm dish2Volume to add:20 μL100 μL250 μL0.5 mL2 mL4 mL
?. [Passaging WTc11 iPSC]
Add ample PBS to accutase-containing well(s) to dilute accutase using the following table for volumes per well:   ABCDEFG1Per:96-well24-well12-well6-well10-cm dish15-cm dish2Volume to add:200 μL1 mL2.5 mL5 mL10 mL20 mL
ABCDEFG1Per:96-well24-well12-well6-well10-cm dish15-cm dish2Volume to add:200 μL1 mL2.5 mL5 mL10 mL20 mL
?. [Passaging WTc11 iPSC]
Pipette up and down gently to mechanically release remaining cells, collect, and add to appropriately-sized conical tubes.
?. [Passaging WTc11 iPSC]
Spin cells at  for  at .
Centrifuge: 200 34
37 Room temperature
?. [Passaging WTc11 iPSC]
Carefully aspirate supernatant from pelleted conicals.
?. [Passaging WTc11 iPSC]
Add appropriate volume of StemFlex + Rock inhibitor at  to conicals according to pellet size for counting.
Rock inhibitor should only be used when cells are individualized or in small colonies (typically the first two days after passaging); the presence of Ri at higher densities results in cell stress/death, and in general, Rock inhibitor greatly reduces proliferation.
For first time use of Rock inhibitor, it is suggested to aliquot at 10mM [1000x], diluting in DPBS, and use on cells at concentration  .
?. [Passaging WTc11 iPSC]
Triturate to resuspend cells in StemFlex + Rock inhibitor and remove 10μL and add this volume to a 1.5mL Eppendorf tube.
Be careful to minimize contact of pipette with the side of the conical wall.
?. [Passaging WTc11 iPSC]
Count cells and calculate desired number of cells to seed, and dilute this volume with additional StemFlex + Rock inhibitor to plate using the following table for volume to add per well:  ABCDEFG1Per:96-well24-well12-well6-well10-cm dish15-cm dish2Volume to add:50-100 μL250-500 μL0.75-1 mL1.5-2 mL8-12 mL15-25 mL
ABCDEFG1Per:96-well24-well12-well6-well10-cm dish15-cm dish2Volume to add:50-100 μL250-500 μL0.75-1 mL1.5-2 mL8-12 mL15-25 mL
For general passaging where exact cell number seeded is not important, adding resuspended cells at 1:100, 1:50, and 1:20 the final well volume typically provides near-confluency in 7 days, 5 days, and 3 days, respectively.
?. [Passaging WTc11 iPSC]
iPSCs can be frozen in StemFlex + 10% DMSO.
?. [Passaging WTc11 iPSC]
Thaw Matrigel on ice and dilute in pre-chilled Knockout DMEM for a final volume of .