REVIEW article
Front. Toxicol.
Sec. In Vitro Toxicology
Volume 7 - 2025 | doi: 10.3389/ftox.2025.1667573
This article is part of the Research TopicAdvancing In Vitro Cell Culture Practices: Achieving Truly Animal-Free Experiments and Scientifically Reliable and Reproducible MethodsView all 11 articles
Advancements In Vitro Culture of Human Pluripotent Stem Cells: Progress, Challenges, And Future Directions: Comprehensive Review
Provisionally accepted
Luis Villa-Diaz1,2,3*
Niraj Chaudhary2- 1Oakland University, Rochester, United States
- 2Deparment of Biological Sciences, Oakland University, Rochester, United States
- 3Department of Bioengineering, Oakland University, Rochester, United States
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The advancement of human pluripotent stem cell (hPSC) culture systems has revolutionized the landscape of preclinical drug discovery and toxicological evaluation. Progressing innovations from feeder-dependent and xenogeneic matrices to chemically defined, xeno-free, and fully synthetic platforms have addressed long-standing challenges in reproducibility, safety, and clinical translation. Developments in recombinant extracellular matrix proteins, synthetic peptide substrates, and polymer-based coatings have enabled the generation of Good Manufacturing Practice (GMP)-compliant, scalable hPSC cultures while minimizing biological variability and immunogenic risks. Integration of automation, artificial intelligence (AI), and three-dimensional (3D) bioprocessing technologies aims at further enhancement of standardization, quality control, and throughput. In the context of pharmaceutical research, hPSC-derived cellular models now underpin high-throughput drug screening and mechanistic toxicological assays, offering superior human relevance compared to traditional animal models. Despite these advances, barriers such as cellular immaturity, inter-batch variability, and limited regulatory acceptance persist, underscoring the need for further protocol standardization and technological refinement. This review provides a comprehensive analysis of current animal-free hPSC culture platforms, critically examines their strengths and limitations, and discusses future directions for advancing their application in drug discovery and predictive toxicology. The ongoing evolution of hPSC technologies promises to accelerate the development of safer, more effective therapeutic agents and to reshape the future of human disease modeling and pharmacological research.
Keywords: Human pluripotent stem cells, Feeder-free culture, xeno-free substrates, Drug Discovery, Toxicology
Received: 16 Jul 2025; Accepted: 20 Oct 2025.
Copyright: © 2025 Villa-Diaz and Chaudhary. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Luis Villa-Diaz, luisvilladiaz@oakland.edu
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.