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Human induced Pluripotent Stem Cell (HiPSC) culture protocol for maintenance, passaging and cryostorage (Version 1.0) | ATLAS-D2K Center

PLEASE NOTE: ATLAS-D2K closed July 31, 2025 and this website is for reference purposes only.

Human induced Pluripotent Stem Cell (HiPSC) culture protocol for maintenance, passaging and cryostorage (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

The Genome Engineering and iPSC Center; Bendi Gong; Sanjay Jain

Keywords

[‘cell maintainence’, ‘human’, ‘induced pluripotent stem cells (iPSC)’]

Subjects

[‘Cell culture’, ‘Tissue culture’]

Release Date

2017-04-18

Abstract

Several methods have been adopted for maintenance and propagation of human induce pluripotent cells (hiPSCs). Each method has its merits and demerits. As participants of the Rebuilding a Kidney (RBK) consortium one of our goals is to characterize a subset of parent iPSC cell lines and derived reporter lines. Here we describe a protocol that we have implemented in propagating, maintaining, freezing and recovery of hiPSC cells that works consistently for a number of RBK lines. We have used this protocol for the RBK BJFF6, AN1.1, WTC11, WTC11-GCaMP6f and new ongoing collecting duct reporter lines. This protocol does not require feeder cells. The cells are grown on a Matrigel matrix in E8-flex media. The cells are initially recovered in media with ROCK inhibitor followed by growth in media alone that gives rise to healthy islands of iPSCs with sharp edges. Some useful tips are also included in the text.

Reagents

  • Essential 8TM Flex Medium (E8 Flex) (Cat. No. A2858501; Thermo/Gibco)
  • DMEM/F-12, without calcium and magnesium (Sigma)
  • PBS, without calcium and magnesium (Sigma)
  • Matrigel (Corning cat 354277)
  • L7™ hPSC Passaging Solution (Cat. No. FP-5013, Lonza)
  • Rock inhibitor (Y-276320; Tocris #1254)
  • KOSR (thermo fisher cat 10828-028)

Procedure

Preparation

Coat 6 well or desired plate with matrigel

  1. thaw 5 ml Matrigel on ice, aliquot enough 50-60ul to 15 ml tube, depend on the dilution factor for each batch of Matrigel
  2. Add 6 mL of cold DMEM/F-12 into the 15 mL tube.
  3. Mix well and transfer 1 ml to each well. Tip: Handle Matrigel on ice as it solidifies when warmed.
  4. Keep the flask at room temperature for at least 60 min to allow Matrigel to coat the surface.
  5. remove the coating solution and add 1 ml E8-Flex medium to each well. Tip. Thaw E8 Flex only at room temperature.
  6. L7™ hPSC Passaging Solution

Plating frozen cells on Matrigel coated plates

  1. Prepare 9ml of E8 Flex media with 10 µM rock inhibitor in a 15 ml conical tube. Tip: make sure E8 Flex is thawed at room temperature, do not use 37oC.
  2. Thaw HiPSC cells stored in liquid nitrogen in a 37oC water bath.
  3. Transfer to 9ml E8-Flex medium with 10uM rock inhibitor tube made in step 1.
  4. Collect the cells by centrifugation at 500g 5 min.
  5. Remove medium and resuspend the cells in 3 ml E8 Flex medium with 10uM rock inhibitor and evenly spread them on the coated Matrigel plates.
  6. Grow in a tissue culture incubator at 37oC, 5% CO2.
  7. Replace medium next day with E8 Flex medium without rock inhibitor.

After a few days without rock inhibitor you should see islands of iPSC with sharp edges

Passaging Pluripotent Stem Cells

Prepare

a) Coated Matrigel plates as above b) Warm E8 Flex medium at room temperature

  1. Remove and discard culture medium from the 6 well plate. Gently wash cells with 1X sterile PBS without Ca2+ and Mg2+.
  2. Remove and discard the washing solution.
  3. Add 1 ml of L7™ hPSC Passaging Solution.
  4. Leave the plates undisturbed for 5 minutes at room temperature.
  5. remove and discard the L7™ hPSC Passaging Solution.
  6. Add 3 ml E8 Flex hPSC medium, gentle detach the cell aggregates by pipet and sweeping, and seed directly onto a culture surface freshly coated with hPSC Matrix. Suggest split ratio of 1:6. Add up to 3ml E8-flex media to each well.
  7. Grow in a tissue culture incubator at 37oC, 5% CO2.
  8. Feed 24 h, can take a break of 2 days over the weekend.

Cyropreservation of iPSCs

  1. Follow the steps of cell harvest till cells suspended in E8 Flex medium
  2. Mix 1 ml E8-flex cell suspension with 1 ml of 80% KOSR 20% DMSO and Rock inhibitor (10uM)
  3. Aliquot1 ml in each cryo vial, freeze at -80oC overnight, then transfer to liquid nitrogen tank.

Consortium

(Re)Building a Kidney (RBK) Consortium