TITLE: Culturing iPSC cells in Essential 8 medium
AUTHORS: rachel.bates, Matthew Gegg

[DESCRIPTION]
Culturing of iPSC lines in essential 8 medium, thawing and passaging techniques. 

[STEPS]
SECTION: Preparing plates and medium for cells
1. Essential 8 media (Thermo, Cat # A1517001). This consists of 500 mL  of basal medium and 10 mL  of supplement. Media with supplement has a shelf life at 4 °C  for 2 weeks. Unless culturing large numbers of cells it is best to make up 100 mL  aliquots (or whatever volume you think you will require over two weeks).

SECTION: Preparing plates and medium for cells
2. Defrost 10 mL of supplement in fridge overnight. Aliquot in to 2 mL aliquots and store at -20 °C. Never defrost supplement in water bath.

SECTION: Preparing plates and medium for cells
3. Mix 2 2 mL ml supplement with 98 mL l E8 media. 

SECTION: Preparing plates and medium for cells
4. Warm up complete media at room temperature , not a water bath. 
SECTION: Preparing plates and medium for cells
5. Stock vitronectin is 500 g/ml. Require 5 g/ml to coat plates therefore dilute stock 1/100 in PBS.  Vitronectin (Thermo, Cat# A14700)

SECTION: Preparing plates and medium for cells
6. Need 1 mL  vitronectin solution/6 well (scale up or down as required for different sized dishes: need ~ 5 g/cm2).

SECTION: Preparing plates and medium for cells
7. Incubate at room temperature for 1 hour. The plates can then be used immediately or wrapped up in cling film and stored in fridge for one week. Do not let dry them out. Plates need to be warmed up to room temperature before cells are seeded in them. Aspirate away vitronectin solution just prior to seeding cells. No need for wells to be washed with PBS.

SECTION: Thawing iPSC cells
8. Rapidly defrost vial from liquid nitrogen store in 37 °C water bath. Add 4 mL  complete E8 media dropwise to defrosted cells in 30 ml white-capped tube and centrifuge at 200 x g for 5 minutes. 

SECTION: Thawing iPSC cells
9. Aspirate media and resuspend very gently in 1 mL  of complete E8 media. Do not triturate more than twice. Transfer to vitronectin coated well and top up with 1 mL complete E8 media. Swirl in figure of eight and check under microscope that you can see clumps of cells.

SECTION: Thawing iPSC cells
10. If cell pellet quite large it is best to seed in to two or three 6 wells.
SECTION: Thawing iPSC cells
11. Incubate in incubator at 37 °C, 5% CO2. Change media next day. Should see small colonies of cells and very few single cells.
SECTION: Thawing iPSC cells
12. Change media daily (2 ml/6 well) until 80-90% confluent.
SECTION: Passaging iPSC
13. Cells should grow in colonies and with very few differentiated cells. When 80-90% confluent it is time to passage.
SECTION: Passaging iPSC
14. Wash cells in 6 well with 2 mL  room temperature PBS. Aspirate.

SECTION: Passaging iPSC
15. Add 1 ml of 0.5 mM EDTA in PBS (room temperature) per 6 well (stock EDTA is 500 mM so dilute 1/1000 in PBS; store at room temperature). Incubate at room temperature for 5 minutes.
SECTION: Passaging iPSC
16. Follow progress of cells under microscope. Colonies should become detached from any differentiating cells at the periphery of the colonies but still remain attached to the plate (if holes begin to appear in the middle of colonies remove EDTA immediately, even if before 5 minutes has elapsed).
SECTION: Passaging iPSC
17. Carefully aspirate EDTA solution and add 1 ml of complete E8 media. Gently ‘blast’ off colonies with 1 ml pipette. Two or three triturations should be sufficient as you do not want to break up the colonies too much. Remove media containing colonies from well and add to 7 ml of complete E8 media and gently mix with pipette. Plate two ml of cells/vitronectin coated 6 well. This is a 1:4 passage. Do not dilute iPSC more than this.
SECTION: Passaging iPSC
18. Only passage two or three wells at a time as detached colonies can quickly re-adhere to plates.
SECTION: Passaging iPSC
19. Change media daily until ready for passage again.