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ITS-Animal-Free™ Supplementation for iPSC Expansion and Pluripotency: A Platform Approach for Serum-Free Cell Expansion

Published on 26 June 2025

Application Note

Authors: Mark Stathos, PhD & Andrew Hamann, PhD
InVitria, Inc., USA

Overview

This application note evaluates the use of ITS Animal-Free™ (ITS AF)—a recombinant, animal-origin-free supplement containing insulin, transferrin, and selenium—for serum-free expansion of human induced pluripotent stem cells (iPSCs). The study demonstrates that ITS AF supports robust iPSC proliferation and maintains high levels of Oct4, Sox2, and Nanog expression, performing comparably to a leading competitor at all tested concentrations without requiring media optimization.

Key Findings

  • Enables robust iPSC proliferation in serum-free systems
  • Maintains ≥85% Oct4+/Sox2+/Nanog+ pluripotent population
  • Matches performance of competitor ITS across all doses
  • Pre-optimized, animal-origin-free formulation simplifies media development

Materials & Methods

Human iPSCs maintained in Essential 8™ media (ThermoFisher Scientific) were dissociated using Accutase® (ThermoFisher Scientific) and seeded into 96-well plates at 10,000 cells per well. Each well was coated with 0.5 µg/cm² N-terminal truncated Vitronectin™ (ThermoFisher Scientific) and supplemented with 10 µM Y-27632 ROCK inhibitor.

Cells were cultured in one of the following conditions:

  • E5 media (homemade Essential 8 without ITS)
  • E5 supplemented with 1–5x InVitria ITS AF™ or competitor ITS
  • Complete commercial Essential 8

Plates were incubated on an Incucyte SX5 system (Sartorius), and proliferation was monitored via confluence measurements taken every 2 hours. After 24 hours, the media was changed to remove ROCK inhibitor, and monitoring continued for an additional 24 hours.

At 48 hours, cells were harvested using Accutase® and stained for flow cytometry.

  • Viability was assessed using Zombie Aqua™ fixable dye (BioLegend).
  • Cells were fixed and permeabilized with the FOXP3 eBioscience™ buffer set (ThermoFisher Scientific).
  • Cells were stained with pluripotency markers:
    • Oct4 Alexa Fluor® 647
    • Sox2 Alexa Fluor 488
    • Nanog Alexa Fluor 594 (BioLegend antibody catalog)

Samples were analyzed using a CytoFLEX LX™ flow cytometer (Beckman Coulter).
Pluripotency was defined as the percentage of live cells triple-positive for Oct4, Sox2, and Nanog.

Featured Solution

 

ITS Animal-Free Blood-free cell culture media supplement, three bottles

ITS Animal-Free – Recombinant Insulin, Transferrin, and Selenium Supplement

ITS Animal-Free™ (ITS AF) is a chemically defined, animal-origin-free 100x liquid supplement designed to replace serum in cell culture. It supports robust proliferation and maintains pluripotency in iPSCs and other mammalian cell types. ITS AF eliminates the variability and regulatory concerns associated with serum-derived inputs, enabling more consistent, compliant, and scalable workflows in cell and gene therapy, regenerative medicine, and vaccine development.

Learn more about ITS AF

Download the Application Note

First page of application note showing ITS AF™ benefits for iPSC expansion and pluripotency with graphs comparing InVitria and competitor ITS performance
Explore the data: Download the full ITS AF™ Application Note to see how this animal-origin-free supplement supports iPSC expansion and pluripotency.

Footnotes

References

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