## 1 Introduction  
In vitro cell culture systems have become indispensable tools in life science research. Historically, two-dimensional (2D) culturing on plastic plates has been favored due to its simplicity and cost-effectiveness. However, no substrate in living organisms closely resembles the rigidity of plastic. Furthermore, cells naturally function or behave in a three-dimensional (3D) environment, influenced by cell-cell contacts and interactions with the extracellular matrix (ECM). Thus, throughout the history of cell culture experiments, there has been a significant demand for developing 3D cell culture techniques that more accurately mimic in vivo cell behavior. A prominent example of this is the spheroid culture method [1]. The culturing of mouse pluripotent cells in suspension, leading to the formation of embryyoid bodies (EB), can be considered the genesis of cell spheroid culture [2]. Notably, the 3D EB culture method through spheroid formation maintained the tri-lineage differentiation potential of pluripotent cells, facilitating embryonic development mimicry in vitro, which was challenging in 2D cultures [3; 4; 5]. Building on this success, spheroid cultures have been adapted for various somatic cells, leading to advancements in cellular biology and tissue engineering applications. Mesenchymal stem cells (MSCs), identified from bone marrow, are renowned for their self-renewal and multi-lineage differentiation properties [6; 7]. Anyway, recent studies demonstrated that MSCs exhibit paracrine effects through the production of cell-protective and immune-regulatory factors [8; 9]. Consequently, there is great interest in tissue engineering and regenerative medicine grafting MSCs for bone or cartilage-destructive diseases and in MSC-based cell therapies targeting immune diseases. To reveal the biological properties of MSCs under 3D conditions and exert their optimal cellular function at the graft site, the spheroid culture method has been frequently employed [10; 11]. MSC spheroids, formed by using the hanging drop method or non-adhesive plates, primarily consist of cell-cell contact molecules, such as cadherins [12; 13]. Intriguingly, MSC spheroids, when compared to 2D cultured MSCs, exhibit a significantly increased production of cell-protective and immune-regulatory factors [14; 15]. Moreover, it is also well reported that MSC spheroid culture positively affects cell viability and cell differentiation capacities more than a conventional 2D cell culture system [16; 17]. On the other hand, we have originally developed a novel 3D culture technique, termed \"clumps of MSCs/ECM complexes\". Unlike MSC spheroids, which are derived from cell suspension cultures, C-MSCs are formed from ECM-rich cell sheets. When the cellular sheets are detached from the plastic culture plate and maintained in the floating condition, they naturally contract and aggregate to form C-MSCs [18]. C-MSCs, approximately 1 mm in diameter, can be directly transplanted into bone defect sites without artificial materials, inducing effective bone regeneration [18; 19]. Moreover, the bone regenerative capacities of C-MSCs can be regulated by osteogenic or chondrogenic inductions [20; 21]. Similarly to MSC spheroids, C-MSCs also show increased production of cell-protective factors compared to the cells on 2D plastic culture plates [22]. As described above, both 3D cell culture systems, MSC spheroids, and C-MSCs offer advantages over 2D cultures. However, given their contrasting manufacturing methods, it is anticipated that there are inherent biological differences between MSC spheroids, primarily formed through cell-cell contacts, and C-MSCs, shaped mainly by cell-ECM interactions. To deepen the understanding regarding cultured MSCs, it is imperative to clarify the distinctive characteristics of these artificial scaffold-free 3D cell aggregates.