**6. Spheroid 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 regulations. In short, gingivae were de-epithelialized, minced into 1–2 mm<sup>2</sup> fragments, 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 a humidified incubator with 5% CO<sup>2</sup> . After 24 h, the non-adherent cells were washed with 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 stem cells and bone marrow-derived stem cells in the amount of 1 × 10<sup>6</sup> were seeded and subsequently 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 gingivaderived stem cells.

**7. Conclusions**

**Acknowledgements**

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 three-

**Figure 6.** The morphology of the stem cell spheroids on Day 5. (a) The morphology of the stem cell spheroids at low magnification (original magnification 100×), (b) higher magnification (original magnification 200×) and (c) the number of

Morphological Comparison of Stem Cells Using Two-Dimensional Culture and Spheroid Culture

http://dx.doi.org/10.5772/intechopen.81471

145

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, Information and

Communication Technology & Future Planning (NRF-2017R1A1A1A05001307).

dimensional cell culture methods to evaluate the biological processes.

stem cell spheroids in the well is more than one (original magnification 200×).

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

Morphological Comparison of Stem Cells Using Two-Dimensional Culture and Spheroid Culture http://dx.doi.org/10.5772/intechopen.81471 145

**Figure 6.** The morphology of the stem cell spheroids on Day 5. (a) The morphology of the stem cell spheroids at low magnification (original magnification 100×), (b) higher magnification (original magnification 200×) and (c) the number of stem cell spheroids in the well is more than one (original magnification 200×).
