Beyond Passage Numbers: How Culture Conditions and Population-Doubling Metrics Reporting Shape the Quality of
Carolina Quintero-Gil1, Wendy V Jaraba-Álvarez2, Catalina Machuca-Acevedo1
1BioXtech, Medellín 050021, Colombia.
Abstract:
Mesenchymal stromal cells (MSCs) are central to regenerative medicine and advanced therapies. However, the absence of consensus on reporting kinetic parameters, such as population doubling level (PDL), population doubling time (PDT), and the reliance on passage number alone obscures biological age and manufacturing history, and limits correlation of potency with expansion dynamics. Here, we clarify the distinctions among passages, PDL, PDT, and replication rate; we synthesize evidence that identical passage numbers can conceal multifold differences in cumulative doublings, with downstream effects on transcriptomic stability, and immunomodulatory performance. We further highlight culture determinants, oxygen tension, seeding density, media formulation, surface/bioreactor systems, and early niche mimetic stimuli, that shape proliferative kinetics and cellular aging trajectories in WJ-MSCs. Critically, we propose extracellular vesicles (EVs) as sensitive functional readouts of bioprocess stress and expansion history: EV quantity can increase while functional bioactivity declines, and EV miRNA cargo captures cell state programs not evident from minimal identity markers. To address these gaps, we recommend a reporting framework that incorporates: (1) culture conditions, (2) passage number and PDL at harvest, and (3) functional consequences of expansion. Adopting kinetic metrics beyond passage number will harmonize data capture and enable pooled analyses, accelerating clinical translation while safeguarding patient outcomes.
Insights
Standardizing mesenchymal stromal cell (MSC) expansion metrics like population doubling level (PDL) and time (PDT) is crucial. This ensures consistent cell manufacturing, potency, and accelerates advanced therapy translation for better patient outcomes.
Area of Science:
- Cell Biology
- Regenerative Medicine
- Biotechnology
Background:
- Mesenchymal stromal cells (MSCs) are vital for regenerative medicine.
- Current reliance on passage number for MSCs obscures biological age and manufacturing history.
- This lack of standardized kinetic parameters limits potency correlation and data comparability.
Purpose of the Study:
- To clarify distinctions between passage number, population doubling level (PDL), and population doubling time (PDT).
- To highlight culture determinants influencing MSC proliferative kinetics and aging.
- To propose extracellular vesicles (EVs) as functional readouts of bioprocess stress and expansion history.
Main Methods:
- Comparative analysis of passage number versus cumulative doublings.
- Investigation of culture determinants (oxygen, seeding density, media, systems) on WJ-MSCs.
- Characterization of extracellular vesicles (EVs) quantity, bioactivity, and miRNA cargo.
Main Results:
- Identical passage numbers can mask significant differences in cumulative doublings, impacting transcriptomic stability and immunomodulatory function.
- Culture conditions profoundly shape MSC proliferation and aging.
- EV quantity may increase while bioactivity declines; EV miRNA cargo reflects cell state.
Conclusions:
- Standardized reporting of MSC kinetic parameters (PDL, PDT) beyond passage number is essential.
- A comprehensive reporting framework including culture conditions, passage number, PDL, and functional outcomes is recommended.
- Adopting kinetic metrics will harmonize data, accelerate clinical translation, and improve patient safety.
