(F1215) HIGH-DENSITY EXPANSION AND METABOLIC CHARACTERIZATION OF HUMAN ADIPOSE AND WHARTON'S JELLY MESENCHYMAL STEM CELLS IN SCALABLE MICROCARRIER SUSPENSION CULTURE

Abstract: Mesenchymal stem cells (MSCs) are widely used in regenerative therapies for various conditions but face challenges in large-scale production due to the limitations of conventional plate culture methods. This study proposes a scalable, lab-scale microcarrier culture system to expand MSCs efficiently using commercial media supplemented with fetal bovine serum (FBS) or human platelet lysate (hPL) as a xenofree alternative. The metabolism of cultured cells was assessed by monitoring glucose, pyruvate, lactate, ammonia, and amino acid consumption/production.

Human Wharton’s Jelly MSCs (WJ-MSCs) and adipose-derived stem cells (ASCs) were successfully expanded in an agitated suspension culture using Cytodex beads. In FBS- or hPL-supplemented cultures, up to 200 million cells were obtained from ASCs in a single flask, representing a 28-fold increase in cell number. For WJ-MSCs, a significantly lower yield was observed with hPL (60 million cells), compared to FBS (over 200 million cells), likely due to the formation of spheroid-like structures instead of attachment to microcarriers. Despite this, culturing WJ-MSCs in xenofree suspension cultures was still possible.

Metabolic analysis revealed high specific consumption/production rates (qₛ) for glucose, pyruvate, lactate, glutamine, glutamate, and ammonia. Essential amino acids were consumed, except for lysine, which showed negative qₛ values during the lag phase. No metabolites were depleted during cultivation, indicating efficient nutrient utilization.

This culture system is scalable, producing up to 250 million cells in a single flask, equivalent to 40 adherent T175 flasks. It provides a cost-effective, contamination-resistant solution for MSC expansion. Further optimization of microcarrier adhesion, particularly for WJ-MSCs, and refined feeding strategies based on metabolic profiling will improve culture performance and reduce media usage. Additionally, the cells retained their pluripotency, as evidenced by phenotypic markers and differentiation potential, confirming the effectiveness of this method for MSC expansion and potential therapeutic applications.

Funding Source: Supported by ANID-FONDECYT #11230208, ANID-Basal funds for CeBiB #AFB240001 and #FB0001, and the ANID-National Master’s Grant #22240802

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