Macromolecular crowding in animal component-free and/or xeno-free media for human mesenchymal stromal cell cultures: In vitro and preliminary in vivo studies

Mesenchymal stromal cells (MSCs) present great potential for therapeutic and reparative use in tissue engineering due to their self-renewal, multi-lineage differentiation and immunomodulatory properties. In the last twenty years, different strategies to expand the therapeutic potential of MSCs have been developed, however manufacturing and handling of these cells for their use as advanced therapy medicinal products remains insufficiently studied and available data are mainly related to non-industrial processes. Cell culture media containing undefined animal-derived components and prolonged in vitro culture periods in the absence of native extracellular matrix result in phenotypic drift of MSCs. Another limiting factor in the development of tissue engineered medicines is the prolonged in vitro expansion times required to reach the high cell numbers needed for therapeutic effects which is associated with loss of phenotype and immunomodulatory capability. Herein, we assessed whether animal component-free (ACF) or xeno-free (XF) media formulations maintain human bone marrow MSC (hBM-MSC) phenotypic characteristics more effectively than foetal bovine serum (FBS)-based media. In addition, we assessed whether tissue-specific extracellular matrix, induced via macromolecular crowding (MMC) during expansion and / or differentiation, can more tightly control hBMSC fate. Then, the effect of carrageenan in a xeno-free media on human umbilical cord derived MSCs (hUC-MSCs) was studied and in vitro and in vivo assessment of a non-animal sourced chitosan scaffold loaded with xeno-free umbilical cord mesenchymal stromal cells cultured under macromolecular crowding conditions was conducted. hBM-MSCs expanded in animal component-free media showed overall the highest phenotype maintenance, as judged by cluster of differentiation expression analysis. Contrary to FBS media, ACF and XF media increased cellularity over time in culture, as measured by total DNA concentration. While MMC with Ficoll™ increased collagen deposition of cells in FBS media, FBS media induced significantly lower collagen synthesis and/or deposition than the ACF and XF media. Cells expanded in FBS media showed higher adipogenic differentiation than ACF and XF media, which was augmented by MMC with Ficoll™ during expansion. Similarly, Ficoll™ crowding also increased chondrogenic differentiation. Of note, donor-to-donor variability was observed for collagen type I deposition and trilineage differentiation capacity of hBM-MSCs. Collectively, our data indicate that appropriate screening of donors, media and supplements, in this case MMC agent, should be conducted for the development of clinically relevant MSC medicines. Following appropriate in vitro experimentation, a splinted nude mouse wound healing model was used to assess wound closure and epidermal thickness of non-treated control, non-animal sourced chitosan scaffold, non-animal sourced chitosan scaffold loaded with xeno-free human umbilical cord mesenchymal stromal cells and non-animal sourced chitosan scaffold loaded with xeno-free human umbilical cord mesenchymal stromal cells cultured under macromolecular crowding conditions groups. Across all three donors, carrageenan supplementation significantly increased collagen deposition at day 5, day 8 and day 11 without affecting cell morphology, viability, DNA concentration and metabolic activity. Through freeze drying, a non-animal sourced chitosan sponge was developed with appropriate structural and mechanical properties for wound healing applications. In vitro biological analysis made apparent that neither the scaffold nor macromolecular crowding negatively impacted xeno-free human umbilical cord mesenchymal stromal cell metabolic activity and proliferation. In vivo biological analysis revealed no significant differences between the groups in wound closure and epidermal thickness, raising question about the suitability of the model. In any case, this work sets the foundations for the development of completely xeno-free tissue engineered medicines.

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University of Galway

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CC BY-NC-ND

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University of Galway Theses (PhD Theses)