Quantitative activation suppression assay to evaluate human bone marrow–derived mesenchymal stromal cell potency

Abstract

Background With the increasing use of cell therapies involving immune modulatory cells, there is a need for a simple standardized method to evaluate and compare the suppressive potency of different cell products. We used the Karpas 299 (K299) cell line as the reference suppressor cell to develop a standardized suppression assay to quantitate immune-modulatory capacity of bone marrow derived mesenchymal stromal cells (BM-MSC). Methods Healthy donor CD4 T cells were co-cultured with the K299 cell line or with third party BM-MSC. After stimulating with anti-CD3/CD28 beads, CD154 activation and proliferation of CD4 T cells were measured to calculate suppression. Results The K299 cell line reproducibly suppressed both the activation and proliferation of healthy donor CD4 T cells in a dose-dependent manner. A rapid (16h) assay based on activation-suppression was selected for development. In replicate testing, there was an inherent variability of suppression of 11% coefficient of variation (CV) between different responder T cells. Suppression by BM-MSC on different responders correlated with suppression by K299. We therefore used the K299 suppression as the reference to define suppression potency of BM-MSC in K299 Suppression Units (KSU). We found that inter-donor variability, passage number, method of manufacture, and exposure of BM-MSC to steroids or interferon gamma all affected BM-MSC potency of suppression. Conclusion This method provides a platform for standardizing suppressor function to facilitate comparison between laboratories and for use as a cell product release assay.

Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity

“…Additionally, we used this assay to demonstrate consistent enhanced immunosuppression for MSC lines that were stimulated with IFN-γ, as has been reported previously (8,13,14). Using high-content imaging of MSCs following IFN-γ stimulation, morphological features were identified that significantly correlated with immunosuppressive capacity and predicted the immunosuppressive capacity of other MSC and non-MSC lines, as well as the effects of IFN-γ pretreatment on immunosuppressive capacity.…”

“…S2). These results suggest that the presence of a "reference" immunosuppressive cell line in each experiment would be beneficial to make direct comparisons of MSC lines tested in different experiments (8). A similar strategy could also facilitate comparing experimental results from different laboratories by using results obtained for shared "cell reference materials" to normalize results between laboratories (5, 30).…”

“…Another significant challenge facing development of MSC-based therapies is the lack of well-defined, robust assays for assessing their immunosuppressive function. Current assays for assessing MSC immunosuppressive capacity exist; however, they often rely on quantification of only a few markers in a single culture condition (8,9,20). Based on these limitations, we developed a robust, quantitative method of assessing the immunosuppressive capacity of human bone marrow-derived MSCs and further demonstrated that morphological features of IFN-γ-stimulated MSCs can be used to predict their overall immunosuppressive capacity.…”

“…A major challenge in the development of consistently effective MSC-based immunosuppressive therapies is that MSC lines derived from different donors and manufacturing processes (i.e., cell expansion) can possess markedly dissimilar immunosuppressive function (3,5,6). Although methods exist to assess MSC immunosuppression in vitro, they are often based on only a few measured outcomes, assay culture conditions, and donor MSC samples (5,(7)(8)(9). To improve upon these methods, we developed an experimental and analytical approach to quantify MSC-mediated immune suppression using principal-component analysis (PCA) to integrate multiple measurements of T-cell activation assessed at a range of MSC densities.…”

Potency Analysis of Mesenchymal Stromal Cells Using a Combinatorial Assay Matrix Approach

“…Several studies address the potential utility of a cellular universal ruler for MSC potency assays (Bloom et al, 2015; Deans, 2015; Salem et al, 2015; Tanavde et al, 2015; Viswanathan et al, 2014). However, MSCs’ mechanism of action in vivo may well involve the effect of a plurality of effector pathways with synergistic and overlapping functionalities whose integrative effects are not completely understood when examined for use in distinct clinical uses.…”