**7. Conclusions**

culture systems may simulate the intercellular interactions in regulation of stem cell self-renewal and differentiation [13]. It was shown that a three-dimensional culture enhanced the production of extracellular matrix-related genes when compared with two-dimensional monolayer culture

Spheroid cultures have an advantage of making three-dimensional cell aggregates without using exogenous materials [14]. Three-dimensional cell spheroids can be fabricated using various methods including silicon elastomer-based concave microwells and the hanging drop method [14]. **Figure 6** shows the morphology of cell spheroids cultured in growth media. Gingival tissues were collected from the healthy participants visiting the Department of Periodontics, Seoul St. Mary's Hospital. The Institutional Review Board of Seoul St. Mary's Hospital College of Medicine, Catholic University of Korea, Seoul, Republic of Korea, approved the study, and informed consent from the study participants was obtained. All the methods used in this study were performed in accordance with the relevant guidelines and

digested in an alpha-modified minimal essential medium (α-MEM, Gibco, Grand Island, NY, USA) containing collagenase IV (2 mg/mL, Sigma-Aldrich Co., St. Louis, MO, USA) and dispase (1 mg/mL, Sigma-Aldrich Co.). The cell suspension was filtered with a 70 μm cell strainer (Falcon, BD Biosciences, Franklin Lakes, NJ, USA), and the cells were incubated at 37°C in

phosphate-buffered saline (Welgene, Daegu, South Korea). Fresh media was replaced every 2–3 days. Stem cell spheroids were formed in the silicon elastomer-based concave microwells (H389600, StemFIT 3D; MicroFIT, Seongnam, Korea) with 600 μm diameters. Gingiva-derived

sequently cultured to investigate cellular behavior. Inverted microscopy (CKX41SF, Olympus Corporation, Tokyo, Japan) was used to evaluate the morphology of the tested stem cells. Spheroids were well formed in silicon elastomer-based concave microwells using gingiva-

Secretion of growth factors may differ between two-dimensional cultures and three-dimensional cell spheroids [6]. In a previous report, two- and three-dimensional systems were used for the determination of secreted human vascular endothelial growth factor using a commercially available kit (Quantikine® ELISA, R&D Systems, Inc., Minneapolis, MN, USA) [6]. The osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids were evaluated by comparing two- and three-dimensional cultures, and the results indicated that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture [11]. The co-culture of various cells including stem cells and primary cells can be done at various ratios [5]. Enhanced osteogenic differentiation may be

. After 24 h, the non-adherent cells were washed with

fragments, and

were seeded and sub-

regulations. In short, gingivae were de-epithelialized, minced into 1–2 mm<sup>2</sup>

stem cells and bone marrow-derived stem cells in the amount of 1 × 10<sup>6</sup>

achieved by applying the co-culture of stem cells and endothelial cells [15].

[12].

144 Cell Culture

**6. Spheroid culture**

a humidified incubator with 5% CO<sup>2</sup>

derived stem cells.

This report describes the two-dimensional culture and spheroid culture, and the morphological comparison will be performed between two-dimensional culture and spheroid culture. Spheroid cultures have an advantage of making three-dimensional cell aggregates without using exogenous materials, and this approach will be more widely applied as one of the threedimensional cell culture methods to evaluate the biological processes.
