Mar 19, 2026
Human Induced Pluripotent Stem Cell (hiPSC) Culture and Passaging Protocol
- Elena Coccia1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Human Induced Pluripotent Stem Cell (hiPSC) Culture and Passaging Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly523pvx9/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: February 12, 2026
Last Modified: March 19, 2026
Protocol Integer ID: 243123
Keywords: hiPSC, cell culture, passaging, Geltrex, StemFlex medium, human induced pluripotent stem cell, induced pluripotent stem cell, pluripotent stem cell, stemflex medium, using geltrex, hipsc, cell differentiation, minimizing cell differentiation
Abstract
This protocol is designed to maintain and passage human induced pluripotent stem cells (hiPSCs) using Geltrex-coated plates and StemFlex medium, ensuring optimal growth conditions and minimizing cell differentiation.
Materials
Geltrex (Coating solution for cell culture)
StemFlex Medium (Gibco, #A3349401)
Accutase (STEMCELL Technologies, #07920)
ROCK Inhibitor Y-27632 (Tocris, #1254)
Reagents:
Cell Dissociation Accutase - Enzyme for dissociating adherent cells
Inhibitor Y-27632 - ROCK inhibitor to enhance cell survival
Troubleshooting
Problem
Low cell viability after thawing
Solution
Ensure proper thawing technique and use of ROCK inhibitor.
Problem
Cells not adhering to plates
Solution
Check Geltrex coating and ensure proper incubation time.
Safety warnings
Handle all reagents and materials in a biosafety cabinet. Wear appropriate personal protective equipment (gloves, lab coat, goggles). Dispose of all biological waste according to institutional guidelines.
Cell Culture Preparation
Prepare Geltrex-coated plates and StemFlex medium for hiPSC culture.
Geltrex Coating
1. Dilute Geltrex in cold DMEM/F12 to a final concentration of 1:100. 2. Add 3 mL of diluted Geltrex to each 100 mm plate. 3. Incubate at 37 °C for 1 hour to allow coating.
StemFlex Medium Preparation
1. Thaw StemFlex medium at room temperature. 2. Supplement with 10 µM Y-27632.
Cell Passaging
Passage hiPSCs when they reach 70-80% confluency.
Cell Dissociation
1. Aspirate the old medium from the plates. 2. Rinse cells with 3 mL of PBS without Ca2+ and Mg2+. 3. Add 3mL of Accutase to each plate. 4. Incubate at RT for 3 minutes until cells are dissociated. 5. Gently pipette to ensure single-cell suspension. 5. Pipette the cells into 10ml of StemFlex + Y-27632.
Cell Replating
1. Centrifuge the cell suspension at 300 g for 5 minutes. 2. Discard the supernatant and resuspend the cell pellet in 1 mL of StemFlex medium containing 10 µM Y-27632. 3. Transfer the cell suspension to new Geltrex-coated plates.