The present study proposed to compare the impact of agitation mode (static, orbital, and mechanical) on the culture of mesenchymal stem cells extracted from the Wharton's jelly of umbilical cords (WJ‐MSC), in a clinical grade culture medium, using human platelet lysate and different xeno‐free microcarriers. Attachment, expansion, and detachment performances were characterized by a new dedicated tool of microscopic image posttreatment, allowing an in situ cell counting without detachment step. Results showed that performances in static mode were not necessarily representative of those obtained in dynamic mode. Moreover, impacts on nutrient consumptions and metabolite productions were identified, such as a higher glutamine consumption when Cytodex‐1 microcarriers were used. The detachment strategy used was relatively efficient for Star‐Plus, Plastic‐Plus, and Hillex II, but not sufficient for Cytodex‐1. Despite Cytodex‐1 presented promising attachment and expansion performances, Star‐Plus and Plastic‐Plus showed a better compromise, respectively, for the orbital and the mechanical agitation modes.

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