Mixed lymphocyte reaction assay
Mixed Lymphocyte Reaction (MLR) assays are traditionally used to investigate if an external agent stimulates or inhibits T-cell proliferation. To perform a MLR assay, responder T-cells from one individual are co-incubated with effector T-cells from another individual (heterologous T-cells). In case of a MHC-mismatch between the individuals, the responder T-cells will recognize the MHC molecules of the effector T-cells as “foreign” and will start to proliferate. The effector T-cells can also be stimulated by other mitogens such as the plant mitogen concanavalin A (ConA) (Dwyer and Johnson, 1981).
When MSCs are added to these stimulated T-cells, it has been noted that they supress the proliferation of the T-cells (Di Nicola et al., 2002; Bartholomew et al., 2002; Le Blanc et al., 2003; Tse et al., 2003). This was one of the first indications that MSCs display an immunomodulatory effect (da Silva Meirelles et al., 2009; Spees et al., 2016). This immunomodulatory capacity has also been investigated for equine MSCs. Carrade et al., (2012) found that equine MSCs derived from several tissue sources possess similar immunomodulatory profiles as human and rodent MSCs. Additionally, Colbath et al. (2017) reported that allogeneic bone marrow derived equine MSCs have a similar immunomodulatory potency as autologous MSCs.
Therefore, we have developed a MLR assay to investigate the immunomodulatory properties of our product portfolio. For this MLR assay we mark the responder T-cells with a fluorescent dye which lights up green when it is exposed to a specific light frequency. These responder T-cells are then stimulated with a plant mitogen (ConA) to induce proliferation. When the T-cells start to divide the dye is distributed over their daughter cells, so the dye is serially diluting with every cell division. Therefore, the amount of proliferation of T-cells can be measured by looking at the decrease of colour. Thus, if we want to investigate the immunomodulatory properties of a product, the MSCs of interest are added to the stimulated responder T-cells and co-incubated for several days. Appropriate positive and negative controls are included to see if the test is performed successfully. At the end of the incubation period, the amount of T-cell proliferation is measured using flow cytometry, enabling us to see whether or not the product has supressed the T-cell proliferation.
We have also developed a modified MLR assay, so we cannot only investigate the immunomodulatory capacities of our products, but also their immunogenicity. In this modification the effector T-cells, or in our case the plant mitogen ConA, is replaced by the MSCs as external stimulator for the responder T-cells. This type of modification has been applied frequently in the past to assess immunogenicity of stem cells (Pigott et al., 2013; Paterson et al., 2014; Schnabel et al., 2014). If the MSCs are recognized as “foreign” by the responder T-cells, they will start to proliferate. Therefore, this assay enables us to investigate whether or not our products induce a cellular immune response and thus elucidates their safety profile.
We use explant cultures to investigate the mode of action and the functionality of our MSC based products. In explant cultures, small pieces of tissue of organ are harvested and cultured in its entirety (Bonnomet et al, 2012). Thus, small samples of for example cartilage or tendon tissue are collected and cultured in vitro. For our purposes, lesions are created in the cartilage or tendon tissue, so these mimic an in vivo trauma (e.g. cartilage defect or tendon tear). Then, a MSC based product under investigation meant for the treatment of a joint disease or tendon problems is added to the cartilage or tendon explant cultures respectively. The MSCs and explants are co-incubated for a fixed period of time, so the cells have time to interact with the tissue. At the end of the incubation period the tissue explants are collected and by means of different histological and immunohistological stains the function of the MSCs can be assessed (e.g. have the cells attached? Where are they located in the tissue? Do they produce certain components essential for the extracellular matrix repair? Etc.). Additionally, the fluid in which the explants were cultured together with the stem cells can be collected and analysed using ELISA‘s. This latter method allows us to assess if the stem cells produces certain pro-or anti-inflammatory cytokines or growth factors, which can explain how they aid in the tissue regeneration in patients.
By using explant culture in the past, we have demonstrated that chondrogenic induction of MSCs significantly facilitates homogenous adhesion of the stem cells to cartilage and enhances lesion penetration by the cells (Spaas et al., 2015).
Cell differentiation assays
MSCs are characterised by their ability for self-renewal and their capacity to differentiate into several cell types of the mesodermal lineage (Fülber et al., 2016). Therefore, the differentiation potential of our cells is assessed by using trilineage cell differentiation assays already at an early stage of our manufacturing process to ensure a high and uniform quality of our products. During this trilineage differentiation assay, native MSCs are differentiated towards chondrocytes, osteocytes and adipocytes using culture media supplemented with specific growth factors, which differ depending on the wanted cell type. Successful differentiation of the MSCs is confirmed using histological stains and evaluation of the cell morphology. Non-differentiated MSCs are used as a control reference. For example, when the MSCs are differentiated towards adipocytes they acquire a more round shape and produce little fat droplets which can be stained using Oil Red O staining. Non-differentiated MSCs will retain their spindle shape and stain negative on Oil Red O (Spaas et al., 2013). Therefore, by using this trilineage differentiation assay we can evaluate and confirm the functional capacity of our MSCs.
MSCs can be characterised immunophenotypically by the presence or absence of certain cell surface protein markers (De Schauwer et al., 2012, Spaas et al., 2013). Thus, an assessment of the expression levels of these cell surface markers can be used to investigate if a cell type of interest are actually MSCs or not. Such an assessment is crucial to ensure the quality of a MSC based product, since too many impurities (e.g. other cell types, cellular debris) in a cell based product could affect its efficacy and safety. Indeed, MSCs are reported to be immunoprivileged, partially due to their low levels of rejection proteins, but other cell types possess high levels of these proteins (Paterson et al., 2014; Daar, 1984; Murphy et al., 2012). Therefore, it is important to screen for the presence of these cell types with high levels, since they can cause inflammatory reactions in a lesser or greater extent. For these reasons, our products are evaluated using flow cytometry assays. The MSC product of interest is mixed with group of antibodies labelled with fluorescent proteins and targeted against a defined set of cell surface markers typically present or absent on MSCs (e.g. rejection proteins). If the product contains impurities the expression levels of these markers will be out of the specified range. This test therefore enables us to provide qualitative, impurity free products.
The exact mode of action by which MSCs exert their function is still unclear (Spees et al., 2016). However, paracrine signalling, cell-cell contact, events of cell fusion or the secretion of extracellular vesicle have been proposed to be responsible for the beneficial effect seen after MSC administration (da Silva Meirelles et al., 2009; Spees et al., 2016). These mechanisms would be the reason that MSCs display angiogenic, anti-inflammatory and immunomodulatory actions under certain circumstances and that they can stimulate local cell survival and proliferation (da Silva Meirelles et al., 2009; Spees et al., 2016). Since there is no consensus on which parameter has to be investigated to assess the potency of a MSC product, we have developed custom assays. Depending on the MSC product and the expected mode of action, different evaluation techniques are chosen. One of the potency assay techniques includes a polymerase chain reaction (PCR) assay, by which the upregulation of mRNA of a cartilage specific protein is monitored. The upregulation of this mRNA has been linked to a more pronounced clinical improvement (Broeckx et al., 2014) and an increased adherence capacity of the cells to cartilage, which allows the MSCs to remain local and exert their function (Spaas et al., 2015).