TITLE: iNDI Maintenance protocol of iPSCs Version 1 
AUTHORS: michael.ward  , Erika  Lara Flores, Mark  Cookson

[DESCRIPTION]
IPSC maintenance protocol 
Matrigel procedure for coating plates
Vitronectin procedure for coating plates
Thawing iPSC
Splitting and Passaging iPSC
Freezing iPSC

[STEPS]
SECTION: Matrigel Coating
1. Aliquot concentrated Matrigel:
Gradually thaw a 5ml bottle of Matrigel on ice in a Styrofoam container
Pre-chill labeled Eppendorf tubes by placing in a cool rack on ice. 
Before pipetting concentrated Matrigel into pre-chilled tubes, chill a 1 ml pipet tip by pipetting ice-cold KnockOut™ DMEM/F-12 up and down several times, then immediately use the tip to aliquot Matrigel.
Prepare aliquots of 500 µL of concentrated Matrigel and freeze down at-80 °C  

 


SECTION: Vitronectin Coating
3. Aliquot concentrated Vitronectin 0.5 mg/ ml0.5 mg/mL 
Thaw a vial of Vitronectin on ice in a Styrofoam container.
Prepare aliquots of 60 µL of concentrated Vitronectin and freeze down at -80 °C 


SECTION: Vitronectin Coating
4. Coating plates with Vitronectin solution 5 µg/mL 
Reconstitute a 60 µL aliquot of Vitronectin into a 15 ml conical tube containing 6 mL of PBS, gently resuspend by pipetting up and down, do not vortex.
Add half of the normal culture volume of Vitronectin solution to the culture surface (i.e. 1 ml per well of a 6-well plate).
Gently rock plate to spread the Vitronectin solution evenly across the plate.
Place in 37 °C incubator for     before use it.
Aspirate Vitronectin and add culture medium (E8).






SECTION: Thawing iPSC
5. Remove iPSC stock cryovial from liquid nitrogen and thaw in 37 °C bead bath. Thaw quickly by gently swirling until a small piece of frozen material remains. Spray the vial with 70% ethanol before transferring to a biological safety cabinet.
 



SECTION: Thawing iPSC
6. Gently add the thawed cell suspension dropwise to a conical tube containing 10 mL culture medium or PBS, rinse cryovial with 1ml of medium and add the rinse to the tube, gently mix cells by swirling.


SECTION: Thawing iPSC
7. Centrifuge tube 5 min at 200 - 300 x g at Room temperature 


SECTION: Thawing iPSC
8. Aspirate the supernatant and gently resuspend cells in culture medium (E8) supplemented with 50 nanomolar (nM) Chroman I and transfer to Matrigel or Vitronectin-coated plates.

 


SECTION: Thawing iPSC
9. Gently rock plate to evenly distribute cells.

SECTION: Thawing iPSC
10. Return plate to 37 °C incubator.


SECTION: Thawing iPSC
11. Next day replace the media with fresh E8 medium (2 mL /well of a 6-well plate).


SECTION: Thawing iPSC
12. When the well is 80% confluent pass to expand stock.

SECTION: Splitting/Passaging iPSC
13. EDTA
SECTION: Splitting/Passaging iPSC
13.1. Aspirate culture medium and wash with PBS 1X.

SECTION: Splitting/Passaging iPSC
13.2. Aspirate PBS and add half of culture volume (1ml/well of a 6-well plate) of EDTA 0.5 millimolar (mM) in PBS.

 


SECTION: Splitting/Passaging iPSC
13.3. Incubate for 3 min at 37 °C or 8 min at Room temperature 

 


SECTION: Splitting/Passaging iPSC
13.4. Aspirate EDTA solution, the cells colonies should remain attached so be careful not to disturb them.


SECTION: Splitting/Passaging iPSC
13.5. Add 1 mL of culture medium supplemented with 50 nanomolar (nM) Chroman I to cells to dissociate by pipetting two or three times.


SECTION: Splitting/Passaging iPSC
13.6. Typically splitting ratios for 6 well plates are between 1:6 and 1:12, so, add the desire volume of culture medium to the cells and discard any excess of cells or re-plate into a new Matrigel or Vitronectin-coated well.


SECTION: Splitting/Passaging iPSC
14. Accutase

SECTION: Splitting/Passaging iPSC
14.1. Aspirate culture medium and wash with PBS 1X.

SECTION: Splitting/Passaging iPSC
14.2. Aspirate PBS and add half of culture volume of Accutase. 
SECTION: Splitting/Passaging iPSC
14.3. Transfer to 37 °C incubator for 8 min 

 

SECTION: Splitting/Passaging iPSC
14.4. Meanwhile aspirate Matrigel/Vitronectin from plates and add culture medium E8 supplemented with 50 nanomolar (nM) Chroman I. 

SECTION: Splitting/Passaging iPSC
14.5. When Incubation is ready, tilt the plate and pipet the accutase solution two to three times up and down the culture surface to break the colonies.

SECTION: Splitting/Passaging iPSC
14.6. Quench the Accutase adding half of the culture volume of PBS.


SECTION: Splitting/Passaging iPSC
14.7. Transfer to a new conical tube and rinse with more PBS the culture surface, combine with the cell solution in the tube.


SECTION: Splitting/Passaging iPSC
14.8. Centrifuge 5 min at 200 - 300 x g at Room temperature 


SECTION: Splitting/Passaging iPSC
14.9. Aspirate supernatant.


 



SECTION: Splitting/Passaging iPSC
14.10. Resuspend the cell pellet in culture medium E8 supplemented with 50 nanomolar (nM) Chroman I.



SECTION: Splitting/Passaging iPSC
14.11. Count cells and plate cells the desire amount into a Matrigel-coated plates.


SECTION: Splitting/Passaging iPSC
14.12. Gently rock plate to evenly distribute cells.
SECTION: Splitting/Passaging iPSC
14.13. Return plate to 37 °C incubator.


SECTION: Splitting/Passaging iPSC
14.14. The next day replace the medium with fresh E8 (2ml/well of a 6-well plate)

SECTION: Splitting/Passaging iPSC
14.15. When the well is 80% confluent pass to achieve an assay or to expand stock.


 



SECTION: Freezing iPSC
15. Prepare freezing medium as combining Knockout™ Serum Replacement with 10% DMSO.
SECTION: Freezing iPSC
16. Leftovers of EDTA or Accutase dissociation procedure could be cryopreserved by centrifuging as in step 14.8 and resuspending the cell pellet with freezing medium.


SECTION: Freezing iPSC
17. When freezing cells from a well/plate, prepare the cells as for EDTA split.



SECTION: Freezing iPSC
18. Aspirate EDTA and gently dissociate cells with freezing medium.


SECTION: Freezing iPSC
19. Transfer 1 mL of cell suspension to a 1 mL cryovial and freeze in a CoolCell freezing container at -80 °C 
  

SECTION: Freezing iPSC
20. Next day transfer the cryovials to liquid nitrogen for long term storage.
SECTION: Matrigel Coating
2. Coating plates with Matrigel solution:
Reconstitute a 500 µL aliquot of Matrigel in 50 mL of cold KnockOut™ DMEM/F-12, pipet  500 µL cold media into the aliquot of Matrigel tube and pipet up and down several times, then transfer what has thawed to the tube containing  50 mL of cold KnockOut™ DMEM/F-12, repeat until the frozen aliquot of Matrigel has been completely transferred to the 50 ml of cold media. Mix inverting several times. 

 
Add half of the normal culture volume of Matrigel solution to the culture surface (i.e., 1 ml per well of a 6-well plate).
Gently rock plate to spread the Matrigel solution evenly across the plate.
Place in 37 °C incubator for 30 min to 60 min before use it.

 
Aspirate Matrigel and add culture medium (E8).

There is another option for coating plates besides Matrigel. Vitronectin is a recombinant human protein that provides a defined surface for feeder-free culture of iPSC. When used with E8 medium, vitronectin has been proven to maintain pluripotency and normal growth characteristics in multiple iPSC lines.