In vitro assays for investigating the immunomodulatory properties of human mesenchymal stromal cells

Abstract

Mesenchymal stromal cells (MSCs) are widely recognized for their immunomodulatory properties, which underpin their therapeutic potential in inflammatory and immune-mediated diseases. Although MSC therapies have consistently proven safe, clinical efficacy remains inconclusive, maybe due to incomplete understanding of MSC interactions with the immune environment. This review evaluates current trends in MSC immunomodulation research, based on 318 studies published since 2019 until medio 2024. The most frequently used assays included characterization, proliferation, and polarization, employing methods such as flow cytometry, enzyme-linked immunosorbent assays and colorimetric assays, and polymerase chain reaction. Many studies incorporated strategies for priming of MSCs or included immune cells, most commonly peripheral blood mononuclear cells, T cells, and macrophages. We identify key sources of variability and propose a minimum reporting checklist including MSC source, priming conditions, assay design, and immune cell characteristics. We further recommend implementation of multi-assay workflows combining phenotypic characterization with at least one functional assay. These measures may improve transparency, comparability across studies, and guide robust assay design.

1. Introduction

Mesenchymal stromal cells (MSCs) were previously coveted for their role in tissue homeostasis and maintenance of the stem cell niche, because of their ability to differentiate into various cell types. Furthermore, an important biological property was thought to be their ability to sense and home to damaged tissue in the local environment. However, in recent years, the focus has shifted, and now the primary role of MSCs is thought to be as orchestrators of the immune response [1, 2]. Inflammation is a complex state characterized by interplay between pro- and anti-inflammatory cytokines that regulate activity of leucocytes and surrounding tissue and plays a vital role in many acute and chronic progressive diseases.

Therefore, the ability to adequately regulate the immune response is crucial in disease development and progression, and as MSCs have both immunomodulatory and anti-inflammatory properties, there is increasing interest in exploring their therapeutic potential [3–10]. MSCs affect their environment through paracrine signalling and crosstalk with innate and adaptive immune cells, affecting numerous biological processes, e.g., enabling them to alter proliferation rate and polarization state. Thereby, they assist in maintaining homeostasis of the tissue environment [4, 11, 12]. Paracrine signalling is facilitated by the MSC secretome, which consists of soluble factors such as cytokines, chemokines, or growth factors as well as extracellular vesicles and apoptotic bodies [13, 14]. MSCs are highly dynamic and plastic cells that perform different functions depending on the environment to which they are exposed. They can trigger a response from the immune system in response to external stimuli, such as damage or infection, yet they can also respond to immune activation by becoming immunosuppressive, thereby preventing an excessive immune response [7, 11]. However, the exact mechanisms by which these functions are exerted remain unknown. Owing to their immunomodulatory properties, MSCs have been suggested as a novel therapy for numerous clinical indications [9, 11, 15–28]. Despite the large number of clinical trials in which MSC therapy has been proven safe, the results concerning its efficacy have been inconclusive, maybe due to the complexity of the interactions between the tissue environment of patients and MSCs, which are not yet fully understood [7, 17–19, 29]. Various assays have been used to explore different aspects of MSC characteristics, but currently, there is no consensus for the existing bioassays on either design nor endpoints and thereby variability is introduced. As such, the development of relevant assays following minimum reporting standards is crucial for advancing the understanding of MSCs in an immunological context. This review aims to evaluate current trends in types of assays used to assess the MSC immunomodulatory potential, identify key challenges, and serve as a guide for future research.