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

Published on 1 July 2025

Application Note

Overview

ITS Animal-Free™ ITS Animal-Free (ITS AF), InVitria’s 100x cocktail of Insulin, Transferrin, and Selenium that is completely animal-origin-free, is essential for
culture of all mammalian cell types including iPSCs. ITS AF can be conveniently supplemented into custom media formulations to replace the core components of serum. This reduces variability, eliminates safety and regulatory hurdles, and allows users to focus on the finer points when optimizing custom media formulations. Here we show that InVitria ITS AF enhances iPSC proliferation and maintains pluripotency comparably to the leading competitors.

Key Findings

• Enables robust iPSC proliferation in serum-free systems
• Maintains ≥85% Oct4+/Sox2+/Nanog+ pluripotent population
• Performs equivalently to leading ITS products without optimization
• Animal-origin-free and blood-free, supporting GMP-ready workflows

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 – 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 Full Application Note