**3. Morphological evaluation of two-dimensional stem cell culture**

**Figure 1** shows morphology of the stem cells cultured in an alpha-minimal essential medium (α-MEM, Gibco, Grand Island, NY, USA) containing 15% fetal bovine serum (Gibco), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Sigma-Aldrich Co., St. Louis, MO, USA), 200 mM of lglutamine (Sigma-Aldrich Co.), and 10 mM of ascorbic acid 2-phosphate (Sigma-Aldrich Co.) on Day 12. We plated stem cells at the seeding density of 1.3 × 104 cells/cm<sup>2</sup> . The media were changed every 2–3 days, and cells were incubated in an incubator with 5% CO<sup>2</sup> and 95% O<sup>2</sup> at 37°C. The cells were observed under an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany) (CKX41SF, Olympus Corporation, Tokyo, Japan), and the images were saved as JPGs.

**Figure 1.** Morphology of the stem cells cultured in growth media on Day 12. (a) The view shows the cells with higher confluence (original magnification 200×) and (b) higher magnification (original magnification 400×).

**Figure 2** shows morphology of the stem cells cultured in adipogenic media (STEMPRO® Adipogenesis Differentiation Kit, Gibco, Grand Island, NY, USA). The cells were supplied with adipogenic induction medium and adipogenic maintenance medium alternately. The cells' morphology was viewed under an inverted microscope (Leica DM IRM). **Figure 3** shows morphology of the stem cells cultured in adipogenic media for a longer period of 13 days.

colony stimulating factor, when compared with that from two-dimensional cultures [6]. The aim of this review was to describe the two- and three-dimensional cultures, and the morphological comparison will be performed between two-dimensional culture and spheroid culture.

Mesenchymal stem cells are characterized by the capability of osteogenic, adipogenic, and chondrogenic differentiation [7]. Previously, the stem cells derived from the periosteum and bone marrow of the jaw bone (mandible) and long bone (tibia) were compared in order to determine a suitable cell source [8]. A bone marrow-derived mesenchymal stem cell sheet with platelet-rich plasma could promote bone regeneration [9]. Bone marrow is an attractive source of stem cells, but gaining stem cells from bone marrow may produce greater pain and morbidity [10]. Stem cells can also be achieved intraorally, and gingiva may serve as a more feasible source for stem cells because obtaining gingival-derived stem cells can be done under

**3. Morphological evaluation of two-dimensional stem cell culture**

**Figure 1** shows morphology of the stem cells cultured in an alpha-minimal essential medium (α-MEM, Gibco, Grand Island, NY, USA) containing 15% fetal bovine serum (Gibco), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Sigma-Aldrich Co., St. Louis, MO, USA), 200 mM of lglutamine (Sigma-Aldrich Co.), and 10 mM of ascorbic acid 2-phosphate (Sigma-Aldrich Co.) on

cells were observed under an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany) (CKX41SF, Olympus Corporation, Tokyo, Japan), and the images were saved as JPGs.

**Figure 1.** Morphology of the stem cells cultured in growth media on Day 12. (a) The view shows the cells with higher

confluence (original magnification 200×) and (b) higher magnification (original magnification 400×).

cells/cm<sup>2</sup>

. The media were changed

at 37°C. The

and 95% O<sup>2</sup>

**2. Characteristics of stem cell research**

140 Cell Culture

local anesthesia with less pain and morbidity [11].

Day 12. We plated stem cells at the seeding density of 1.3 × 104

every 2–3 days, and cells were incubated in an incubator with 5% CO<sup>2</sup>

**Figure 2.** Morphology of the stem cells cultured in adipogenic media on Day 5. (a) The morphology of the cells at low magnification (original magnification 100×) and (b) higher magnification (original magnification 200×).

**Figure 3.** Morphology of the stem cells cultured in adipogenic media on Day 13. (a) The morphology of the cells at low magnification (original magnification 100×), (b) higher magnification shows that cells have a ghost-like feature (original magnification 100×) and (c) more distinct feature of abiogenesis is shown (original magnification 200×).

**Figure 4** shows that the morphology of stem cells treated with a chemotherapeutic agent of doxorubicin at 10 μg/mL on Days 1, 3, 5, and 7. A cell viability analysis of the stem cells was performed on Days 1, 3, 5, and 7. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H tetrazolium, monosodium salt] (CCK-8; Dojindo, Tokyo, Japan) was added to the cultures, and the spheres were incubated for 1 h at 37°C. Viable cells were identified by the assay, which relies on the ability of mitochondrial dehydrogenases to oxidize WST-8 into a formazan product. The spectrophotometric absorbance of the samples was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). No significant morphological change of the stem cells cultured in growth media was observed after the addition of the chemotherapeutic agent on Day 1. More significant changes in the morphology of the stem cells were seen in the chemotherapeutic agent-treated group with longer incubation. A decrease in cellular viability was noted with treatment of the chemotherapeutic agent.

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

**Figure 5** shows the morphology of the stem cell culture on modified titanium discs. Machined titanium discs measuring 10 mm in diameter and 2 mm in thickness were used. The stem cells

cultured. Each implant disc was fixed with 4% paraformaldehyde at room temperature for 30 min. Permeabilization was performed with 0.1% Triton X-100/Dulbecco's phosphate-buffered saline for 2 min and blocking solution consisting of 0.2 μm filtered 1% bovine serum albumin/ Dulbecco's phosphate-buffered saline for 30 min. Actin filaments were stained with rhodamineconjugated phalloidin (Molecular Probes, Eugene, OR), and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole. The cells were observed using a confocal laser microscope (LSM5 Pascal, Zeiss, Jena, Germany) at a magnification of 200×. The cells attached to the titanium discs showed well-organized actin cytoskeletons with blue nuclei with confocal microscopy.

In more recent years, three-dimensional cell culture methods have been widely applied and are regarded to have high importance in evaluating the biological processes [12]. Three-dimensional

**Figure 5.** The morphology of stem cells culture on modified titanium discs. (a) Disc with limited number of cells

cells/well on 24-well plates containing titanium discs and

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

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**4. Titanium research**

were plated at a density of 1.0 × 10<sup>5</sup>

**5. Three-dimensional culture**

(objective lens 20×) and (b) disc with higher number of cells (objective lens 20×).

**Figure 4.** Morphology of the stem cells in growth media: (a) untreated group on Day 1 (original magnification 200×); (b) chemotherapeutic group of doxorubicin at 10 μg/mL on Day 1 (original magnification 200×); (c) untreated group on Day 3 (original magnification 200×); (d) chemotherapeutic group of doxorubicin at 10 μg/mL on Day 3 (original magnification 200×); (e) untreated group on Day 5 (original magnification 200×); (f) chemotherapeutic group of doxorubicin at 10 μg/mL on Day 5 (original magnification 200×); (g) untreated group on Day 7 (original magnification 200×); (h) chemotherapeutic group of doxorubicin at 10 μg/mL on Day 7 (original magnification 200×); and (i) cellular viability of the stem cells on Days 1, 3, 5, and 7 using CCK-8. The bar indicates 200 μm.

WST-8 into a formazan product. The spectrophotometric absorbance of the samples was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). No significant morphological change of the stem cells cultured in growth media was observed after the addition of the chemotherapeutic agent on Day 1. More significant changes in the morphology of the stem cells were seen in the chemotherapeutic agent-treated group with longer incubation. A decrease in cellular viability was noted with treatment of the chemotherapeutic agent.
