1. Introduction
Mesenchymal stem cells (MSCs) possess excellent therapeutic properties, including the ability to exert anti-inflammatory and immunosuppressive effects and to differentiate into tissue-specific cells.[1] In addition, MSCs are self-proliferating and can be prepared on a large scale after isolation from a variety of tissues.[2,3] Based on these superior features, MSCs have been used in the treatment of graft-versus-host disease,[4] autoimmune diseases,[5] and inflammatory diseases.[6] Although the therapeutic usefulness of MSC transplantation in patients with these diseases has been demonstrated in some cases, many studies have revealed that the survival rate and duration of MSCs after transplantation are extremely low and short, respectively.[7] Therefore, it is necessary to increase the survival of administered MSCs for more efficient MSC-based therapy.
Multicellular spheroids, which are three-dimensional cultured cell aggregates, are highly functionalized cells, because spheroid formation generally increases cellular function through extensive cell–cell interactions.[8] It has been reported that spheroid formation upregulates the expression of transporters in cancer cells.[9] Therefore, cancer cell spheroids have been used to screen for bioactive compounds, including anticancer agents. In addition, Bartosh et al. reported that multicellular spheroids derived from MSCs secrete large amounts of anti-inflammatory cytokines.[10] Additionally, our previous studies using poly(N-isopropylacrylamide)-coated polydimethylsiloxane-based microwell plates showed that spheroid formation improved the survival of mouse insulinoma NIT-1 cells after transplantation in mice,[11] enhanced albumin secretion from human hepatoblastoma HepG2 cells,[12] and forcibly polarized mouse macrophage-like J774.1 cells to M1-type macrophages with tumoricidal activity.[13] Furthermore, we demonstrated that compared to suspended mouse adenocarcinoma colon26 cells, multicellular spheroids of colon26 cells efficiently accumulated in the lung after intravenous injection in mice and established pulmonary tumor colonies.[14] Therefore, we hypothesized that spheroid formation could increase the survival of MSCs after intravenous injection and improve their therapeutic effects in lung diseases.
Here, we investigated the survival and function of MSC spheroids after intravenous injection in mice. First, the murine adipose-derived MSC line m17.ASC transfected with NanoLuc luciferase (NLuc) was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids. Thereafter, tissue distribution was evaluated by measuring NLuc activity after intravenous injection of m17.ASC spheroids or suspended m17.ASC cells. Finally, the anti-inflammatory effect of suspended m17.ASC cells or m17.ASC spheroids was evaluated in a lipopolysaccharide (LPS)-induced inflammatory mouse model.