Jul 07, 2023

Public workspaceRegular maintenance of human pluripotent stem cells

DOI
dx.doi.org/10.17504/protocols.io.bp2l69obdlqe/v1
  • Narayana Yadavalli1,2,3,4,5,
  • Shawn M. Ferguson1,2,3,4,6,5
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
  • 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Berrak Ugur
Open access
DOI: dx.doi.org/10.17504/protocols.io.bp2l69obdlqe/v1
Protocol Citation: Narayana Yadavalli, Shawn M. Ferguson 2023. Regular maintenance of human pluripotent stem cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69obdlqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 25, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82448
Keywords: hiPSCs, E8 media, Matrigel, EDTA, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol describes the regular maintenance and passaging human iPSCs.
Attachments
mqjubj3up.docx
mqjubj3up.docx
16KB
Materials
Reagents required

  1. E8 Media (Gibco)
  2. 0.5M EDTA (Gibco), working solution 0.5mM EDTA prepared in sterile PBS solution
  3. Y-27632 Rock inhibitor (Tocris Bioscience)
  4. Matrigel (corning)
Day 1
Day 1
Pre coat a 6 well dish with Matrigel matrix for Duration24:00:00 or 1 hour.

1d
Thaw the frozen iPSCs by placing the vial in Temperature37 °C water bath for Duration00:02:00 .

2m
After thawing, spray the tube with 70% ethanol and place in biosafety cabinet.
Aspirate the cells into 15 ml falcon tube and add Amount4 mL cold E8 media containing rock inhibitor.

Pipetting
Spin down the tube at Centrifigation0.3 rcf, 00:03:00 .

3m
Centrifigation
Aspirate the supernatant and resuspend in fresh E8 media containing rock inhibitor.
Remove Matrigel matrix and dispense appropriate number of cells onto Matrigel coated dishes.
Day 2
Day 2
Remove the E8 media containing rock inhibitor and feed cells with E8 media without rock inhibitor.
Feed every day with fresh media till plate gets 60-70% confluency.
Cells were passaged every 5 days with E8.


iPSC passaging with 0.5 mM EDTA solution: Day 1
iPSC passaging with 0.5 mM EDTA solution: Day 1
Pre coat a 6 well dish with Matrigel matrix for Duration24:00:00 or 1 hour.

1d
Remove the culture media.
Rince 1X with sterile PBS.
Wash
Add Amount1 mL of EDTA solution.

Pipetting
Place in the incubator for 5-7 minutes.
Incubation
Bring the plate into biosafety cabinet and add Amount2 mL E8 media containing rock inhibitor.

Pipetting
Dispense all the cell suspension into 15 ml falcon tube.
Spin down the tube at Centrifigation0.3 rcf, 00:03:00 .

3m
Centrifigation
Aspirate the supernatant and resuspend in fresh E8 media containing rock inhibitor.
Remove Matrigel matrix and dispense appropriate number of cells onto Matrigel coated dishes.
iPSC passaging with 0.5 mM EDTA solution: Day 2
iPSC passaging with 0.5 mM EDTA solution: Day 2
Remove the E8 media containing rock inhibitor and feed cells with E8 media without rock inhibitor.
Cells were passaged every 5 days with E8.