ORIGINAL RESEARCH article
Front. Bioeng. Biotechnol.
Sec. Cell and Gene Therapy
Volume 13 - 2025 | doi: 10.3389/fbioe.2025.1632810
This article is part of the Research TopicAdvances in Stem Cell Engineering: Paving the Way for Regenerative MedicineView all 5 articles
Alternating 2D and 3D Culture Reduces Cell Size and Extends the Lifespan of Placenta-Derived Mesenchymal Stem Cells
Provisionally accepted
- 1The Pennsylvania State University (PSU), University Park, United States
- 2University of California Davis, Davis, United States
- 3Cleveland Clinic, Cleveland, United States
- 4Mayo Clinic Arizona, Scottsdale, United States
- 5University of New Haven, West Haven, United States
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Mesenchymal stem cells (MSCs) hold great promise for treating a variety of human diseases; however, their clinical translation is hindered by challenges in large-scale expansion while preserving therapeutic potency and maintaining small cell size.Conventional 2D Culture on rigid substrates induces MSC senescence and enlargement, compromising their function and biodistribution. Here, we present an alternating 2D/3D culture strategy that combines adherent monolayer expansion with transient spheroid formation to mitigate these limitations. Using placenta-derived MSCs, we demonstrate that spheroid culture significantly reduces cell size and enhances immunomodulatory function. Optimal spheroid conditions were identified, and extracellular matrix supplementation and chemically defined media further improved cell viability. The alternating 2D/3D protocol slowed MSC enlargement and senescence over multiple passages while preserving anti-inflammatory activity. To address scalability, we developed RGD-functionalized alginate hydrogel tubes (AlgTubes) that support dynamic transitions between adherent and spheroid states, enabling the implementation of the alternating culture strategy in a continuous and scalable format. This work demonstrates a promising strategy for MSC manufacturing.
Keywords: Mesenchymal Stem Cells, alternating 2D/3D culture, Spheroid culture, Cell Size, senescence
Received: 21 May 2025; Accepted: 07 Aug 2025.
Copyright: © 2025 Pan, Han, Yang, Wu, Wang, Xie, Zhu, Wang and Lei. 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:
Shue Wang, University of New Haven, West Haven, United States
Yuguo Lei, The Pennsylvania State University (PSU), University Park, United States
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