Media matters: Impact of expansion media on the phenotype of mesenchymal stromal cells
Fitzgerald, Joan
Fitzgerald, Joan
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Publication Date
2023-03-02
Type
Thesis
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Abstract
Despite a long history of investigation and sustained efforts in clinical testing with large numbers of clinical trials being initiated, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration (FDA). There are a multitude of barriers impeding the clinical progression of MSC therapies, to the forefront of these is the lack of standardised and consistent manufacturing protocols as well as the lack of biologically meaningful cell characterisation tools and standard release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, amongst other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action. This work analysed the influence of five widely used culture media formulations on the isolation efficiency, growth parameters, trilineage differentiation potential and immunomodulatory potential of human bone marrow derived MSCs (BM-MSCs) and revealed significant differences in these parameters in response to the composition of the culture medium. However, analysis of surface immunophenotype showed that despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs; CD73, CD90 and CD105, a clear demonstration of the futility of the current standard. Further characterisation of the surface immunophenotype using a high-content screening panel of 243 antibodies revealed that the culture medium also influenced the surface proteome, with one third of markers assessed exhibiting variable expression profiles in response to culture medium and donor. Calculation of correlation coefficients between the aforementioned functional in vitro data and this phenotypic analysis identified a number of putative BM-MSC surface markers related to biological function for further investigation. Principal component analysis confirmed that BM-MSCs isolated and expanded in a proprietary serum-free medium previously developed in this laboratory, Purstem 2 (PS2), displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate (PL) and fetal bovine serum (FBS)-containing media. Given the safety and reproducibility issues surrounding the use of undefined media supplements such as PL and FBS, there is a need to move towards serum-free and chemically-defined culture media. PS2 medium supports the isolation and expansion of BM-MSCs from whole bone marrow but does not support isolation from the mononuclear cell (MNC) fraction of marrow only or of adipose derived stromal cells (ASCs). To address these shortcomings and develop a new iteration of the PS2 medium, growth factor and extracellular matrix molecule receptors expressed by BM-MSCs were identified and from these, candidate growth factors platelet derived growth factor (PDGF)-AA, PDGF-BB and epidermal growth factor (EGF) and an attachment factor fibronectin were selected for analysis. The three growth factors were added alone and in combinations to the existing PS2 and tested for their ability to isolate ASCs from the stromal vascular fraction (SVF) of adipose tissue with fibronectin coating of the tissue culture plates. Isolation and propagation of ASCs was successful in all media formulations containing PDGF-BB. Assessment of cell morphology, growth kinetics, trilineage differentiation propensity, immunomodulatory potential and surface immunophenotype demonstrated equivalent or greater performance in the serum/xeno-free media compared to 5% PL controls. However, these media did not support BM-MSC isolation from the MNC fraction of bone marrow only, indicating distinct biological differences between MSCs derived from different tissues. This highlights a deficit in our understanding of the biology of MSCs derived from different tissue sources and the need for bespoke culture media.
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NUI Galway