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Introduction
How much medium should be used for cell culture? Some companies provide a recommended media volume for culture dishes, plates and flasks, however, there are few reports investigating the ideal media volume for individual wells, dishes and flasks. The optimal medium volume for the culture of embryos (1,2), renal epithelial cells (3) and chondrocytes (4–6) has been reported, and it has been recognized that cell proliferation and differentiation are largely influenced by culture conditions, such as cell density and autocrine and/or paracrine regulators. In a study investigating in vitro embryonic development, the volume of micro-drop culture medium and the density of embryos were important factors (2). Another study demonstrated that cell viability was influenced by the media glucose content, and with increasing volumes of culture medium there are increasing amounts of glucose (3,5). Furthermore, the rates of lactate reuptake appear to be highly dependent on the culture medium volume, indicating there may be a volume-induced stimulation of oxidative lactate metabolism (3). In addition, the concentration of oxygen in the medium has been demonstrated to decrease in line with increasing medium depth (6,7). Collectively, this indicates that the medium volume may impact a variety of cell culture factors.
Bone homeostasis is maintained via a balance between osteoblastic bone formation and osteoclastic resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. Osteoblasts undergo proliferation, matrix maturation and extracellular matrix mineralization (8). During this process, osteoblasts synthesize extracellular matrix components including type I collagen. Notably, the activity of osteoblast alkaline phosphatase (ALP) increases upon reaching confluence in culture (9,10). ALP hydrolyzes substrates, and the extracellular matrix is subsequently mineralized, which occurs by increasing the local calcium phosphate concentration. ALP activity has therefore been used as an important indicator of bone formation (11,12). Osteoblastic MC3T3-E1 cells undergo a process of proliferation and differentiation, and then produce mineralized nodules (13). Osteoclasts are derived from hematopoietic cells and are regulated by various cytokines and hormones including interleukin (IL)-1, IL-6 and parathyroid hormone (14). It was previously reported that macrophage colony-stimulating factor and receptor activator of nuclear factor-κB (RANK) ligand (RANKL) are necessary and sufficient for osteoclast differentiation (15,16). Osteoprotegerin (OPG) is the decoy receptor for RANKL and inhibits RANKL-RANK...