Dose-response immunomodulatory effects of Mesenchymal stem cell-derived culture-conditioned media in acute Graft-versus-Host Disease

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Mohini Mendiratta, Meenakshi Mendiratta, Sujata Mohanty, Sandeep Rai, Ritu Gupta, Vatsla Dadhwal, Sameer Bakhshi, Deepam Pushpam, Mukul Aggarwal, Aditya Kumar Gupta, Ranjit Kumar Sahoo

Published: 01 Dec 2025, Last Modified: 10 Nov 2025Regenerative TherapyEveryoneRevisionsCC BY-SA 4.0
Abstract: BackgroundMesenchymal stem cell-based therapy faces challenges that have driven interest in MSCs-derived culture-conditioned media (CCM) as a cell-free alternative. Our study aims to optimize the dose and collection timing of CCM to enhance its therapeutic efficacy in aGVHD, while also standardizing co-culture conditions for CD3+ T-cell interaction with CCM.Material and methodsHuman MSCs were isolated from BM and WJ and subsequently preconditioned under hypoxic conditions (1 % O2) for 24 h in a tri-gas incubator. Culture-conditioned media (CCM) were collected from both naive (MSCs) and hypoxia-preconditioned MSCs (MSCsHYP) at 24, 48, and 72 h and filtered using a 0.2 μm membrane filter. CD3+ T-cell were isolated from PBMNCs derived from aGVHD patients. These T-cell were co-cultured at varying densities (2∗106, 5∗106, and 10∗106 cells/ml) with different concentrations of CCM (25 %, 50 %, and 100 %), and cell proliferation was assessed using the MTS assay. Furthermore, CD3+ T-cell proliferation and activation status were evaluated in a 2D co-culture model of CD3+ T-cell and CCM using flow cytometry.ResultsOur findings revealed that CCM collected at 48 h, at a 50 % concentration, exerted the most pronounced inhibitory effect on CD3+ T-cell proliferation, particularly at a density of 5∗106 cells/ml, irrespective of the MSCs source. Hypoxia preconditioning significantly enhanced the immunomodulatory effects, with WJ-MSCsHYP-CCM demonstrating superior efficacy in suppressing T-cell proliferation, increasing the CD4+/CD8+ T-cell ratio, and reducing CD4+ T-cell activation compared to BM-MSCsHYP-CCM.ConclusionThese results emphasize the critical role of optimizing CCM collection timing and concentration to maximize therapeutic potential. Our study paves the way for the development of standardized, scalable, and effective cell-free therapies for aGVHD.