TITLE:  iPSCs Maintenance and Banking
AUTHORS: Mahmoud ElAchwah, sol.diazdeleon, pangzh

[DESCRIPTION]
This protocols offers a thorough description of the maintenance and banking of induced pluripotent stem cells.

[STEPS]
SECTION: Diluting Matrigel
1. Thaw Matrigel stock 9-11 µg/µL stored at -80 °C on ice

SECTION: Diluting Matrigel
5. 
Dilute 400 µL Matrigel aliquot in 40 mL of MEM (Working Concentration 90-110 µg/mL)

SECTION: Diluting Matrigel
6. Mix well before use. Store at 4 °C protected from the light

SECTION: Matrigel Coating
7. Coat wells with 1 mL of Matrigel per well of 6 well plates or per 35 mm plate

SECTION: Matrigel Coating
8. Leave coated plates for  120 min to 240 min in the incubator at 37 °C

SECTION: Matrigel Coating
9. Store diluted matrigel at 4 °C

SECTION: Medium Preparation For Passaging
10. Prepare mTesr+ by adding 100 mL of mTesr+ 10x supplement into 400 mL of mTESR+ media and filter it. 

SECTION: Medium Preparation For Passaging
13. Take out 1 mL of acutase per well of 6 well plate or per 35mm plate in 15 mL conical tube to warm up at RT

SECTION: Medium Preparation For Passaging
14. Take out  3 mL of mTESR+ per well of 6 well plate or 35 mm plate (2 mL/well for plating and 1 mL/well for resuspension) to warm up at RT

SECTION: Washing iPSCs
15. Put iPSC plate out of the incubator at Room temperature
SECTION: Washing iPSCs
18. Aspirate Wash
SECTION: Detaching Adherent Cells
19. Add  1 mL of acutase per well of 6 well plate or per 35 mm plate
SECTION: Detaching Adherent Cells
20. Place plate in the incubator at 37 °C for 5 min 


SECTION: Centrifuging Cells
21. Get plate out of incubator and check that cells are detached, avoid pippeting too much.

Add 2 mL of MEM

SECTION: Centrifuging Cells
22. Transfer detached cells with MEM (3 mL total volume) into 15 mL conical tube

SECTION: Centrifuging Cells
23. Place conical tubes in centrifuge and spin at 1000 rpm for 5 min at Room temperature

SECTION: Preparing Seeding Medium
25. Add 1 µL of P per 1 mL of mTesr+ (1:1000 ratio)

SECTION: Preparing Seeding Medium
26. Mix mTESR+ with CETP
SECTION: Preparing Seeding Medium
28. Add 2 mL of mTesr+ containing CETP to each well

SECTION: Collecting Cells
29. Aspirate supernatant from 15 mL conical tube containing cells (should have pellet)

SECTION: Collecting Cells
31. Add 50 µL of cell solution per well in 6 well plate or per 35 mm plate (on top of the mTESR+ with CETP)

SECTION: Incubation
33. Ensure the presence of cells in each well by viewing the plate under the microscope (should see floating cells)
SECTION: Collecting Cells
32. Gently mix the plate to evenly spread the cells
SECTION: Incubation
34. Incubate plate in the incubator at 37 °C 5 min

SECTION: Washing iPSCs
17. Add 1 mL of MEM per well of 6 well plate or per 35 mm plate, rock the plate to wash
SECTION: Washing iPSCs
16. Aspirate old medium
SECTION: Changing iPSC medium
35. Aspirate old medium the next day to wash the CETP
SECTION: Changing iPSC medium
36. Add 2 mL of fresh mTESR+ to each well of the 6 well plate or per 35mm plate (no wash with MEM first day after splitting)

SECTION: Wash and iPSC Medium Change
37. Wash CETP from each well/plate with 1 mL of MEM 

SECTION: Wash and iPSC Medium Change
38. Aspirate wash
SECTION: Wash and iPSC Medium Change
39. Add 2 mL of fresh mTESR+ to each well of the 6 well plate or per 35 mm plate

No need to keep washing plates after the first wash, you can directly change medium

SECTION: iPSC Maintainance
40. Keep feeding cells everyday until they are ready for splitting again (cells are usually ready for another splitting in 2-3 days, when their confluency reach 70%)

Check cells under microscope to estimate confluency
SECTION: Diluting Matrigel
3. Store 400 µL Matrigel aliquots at -20 °C until use
SECTION: Diluting Matrigel
4. To prepare matrigel working solution: thaw 400 µL Matrigel aliquots on ice or at 4 °C

SECTION: Diluting Matrigel
2. Make 400 µL aliquots of Matrigel to avoid multiple freeze-thaw cycles using previously cooled tips and tubes
SECTION: Preparing Seeding Medium
27. Aspirate matrigel from coated plates
SECTION: Medium Preparation For Passaging
12. Take out 3 mL of MEM per well of 6 well plates or per 35 mm plate in a conical tube
(1 mL/well to wash old medium and 2 mL/well to wash out acutase) and leave at room temperature to warm up
SECTION: Preparing Seeding Medium
24. Add 1 µL of CET per 1 mL of mTESR+ (1:1000 ratio)
SECTION: Collecting Cells
30. Resuspend pellet with 1 mL of mTESR+ very gently to avoid dissociating iPSCs into single cells
SECTION: iPSC Freezing
41. For freezing purposes, follow the same protocol shown above for cell maintenance for the following sections: Washing iPSCs, Detaching Afdherent cells, centirfuging cells (steps 10-23)
SECTION: iPSC Freezing
42. Instead of collecting cells have freezing vials set and barcoded with iPSC line number, passage number, gene mutation and date
SECTION: iPSC Freezing
43. Resuspend pellet with 1 mL of mFreSR very gently
SECTION: iPSC Freezing
44. Add 500 µL of cell solution to freezing vials
SECTION: iPSC Banking
45. Store freezing vials at -80 °C in a cryogenic freezing container to prevent ice crystals from forming within the cells in the freezing vials
SECTION: iPSC Banking
46. After 24 hours, move freezing containers with vials from -80 °C to a nitrogen tank 
SECTION: Medium Preparation For Passaging
11. Add 1 mL of 50 Molarity (m) Primocin (final concentration 100ug/mL)