# Comparison of 2- and 3-Dimensional Cultured Periodontal Ligament Stem Cells; a Pilot Study
## 4 Discussion  
A scaffold-free 3D culture method, Stemfit 3D[8], was used for the 3D culture of PDLSCs and compared to traditional 2D-cultured PDLSCs. The morphology, viability, proliferation ability, differentiation ability, and gene expression of the two groups were analyzed, and it was confirmed that they have different functions and characteristics depending on the culture method although the cells are of the same origin. The viability of the 3D-cultured PDLSCs was investigated by the 3D culture of PDLSCs and compared to traditional 2D-cultured PDLSCs. The morphology, viability, proliferation ability, differentiation ability, and gene expression of the two groups were analyzed, and it was confirmed that they have different functions and characteristics depending on the culture method although the cells are of the same origin. The viability of the 3D-cultured PDLSCs is also investigated by the 3D culture of PDLSCs and compared to traditional 2D-cultured PDLSCs. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Gene Name** & **Fold Change** & **Description** & **Related Function** \\ \hline EREG & 169.3 & epiregulin & Angiogenesis, cell cycle, cell differentiation, \\ AREG & 167.8 & amphiiregulin & Cell differentiation, \\ CSF3 & 159.6 & colony stimulating factor 3 & Cell differentiation, \\ TAC1 & 136.1 & tachycardia precursor 1 & Immune response, \\ IER3 & 125.0 & immediate early response 3 & Apoptotic process, cell death \\ COMP & 117.5 & cartilage oligomeric matrix & Apoptotic process, cell death, \\ protein & & extracellular matrix \\  & & & Apoptotic process, cell death, \\ S100A8 & 110.7 & S100 calcium binding protein & cell differentiation, cell \\ A8 & & migration, immune response, \\ inflammatory response & & & Cell migration, immune response, \\ CXCL3 & 87.3 & & ligand 3 \\ C3 & 86.3 & complement component 3 & Immune response, \\ LRP4 & 84.2 & LDL receptor related protein 4 & Cell differentiation \\ PTGS2 & 83.4 & prostaglandin-endoperoxide & Angiogenesis, cell differentiation, \\ SPON1 & 80.9 & spondin 1 & Extracellular matrix \\ COL5A3 & 73.4 & collagen type V alpha 3 & Extracellular matrix \\ PTPN22 & 69.5 & protein tyrosine phosphatase, & Cell differentiation \\ IL24 & 67.2 & interleukin 24 & Apoptotic process, cell death \\ TNFAIP6 & 65.9 & TNF alpha induced protein 6 & Immune response, \\ CYP7B1 & 64.1 & cytochrome P450 family 7 & inflammatory response \\ PRDM1 & 63.7 & PR domain 1 & Cell differentiation, cell \\ STC1 & 53.3 & stanniocalcin 1 & proliferation, \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._ \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Gene Name** & **Fold Change** & **Description** & **Related Function** \\ \hline RSPO2 & 152.7 & R-spondin 2 & Cell differentiation \\ DCLK1 & 86.8 & doublecortin such as kinase 1 & Cell migration \\ CXCL4 & 73.9 & C-X-C motif chemokine & Cell migration, \\ SEMA3D & 65.6 & semaphorin 3D & Cell differentiation \\  & & teneurin & \\ TENM2 & 64.3 & transmembrane protein & Cell differentiation \\  & & 2 & \\ ADRA1B & 58.2 & adrenoreceptor alpha 1B & Cell proliferation \\ \hline \hline \end{tabular}
\end{table} 
Table 2: List of genes downregulated more than fifty-fold in 3D-cultured PDLSCs compared to 2D-cultured PDLSCs. cells was decreased, but they showed superior osteogenic differentiation compared to 2D-cultured cells. Additionally, the function of PDLCs differed by the cell culture method. Stemfit 3D\({}^{\text{\#}}\) utilizes a non-adhesive plate with a 600 um diameter polydimethylsiloxane-based concave micromold. The reason for using concave micromolds is that they more rapidly aggregate the cells than other methods (plane and cylindrical), and the cells form spheroids that are highly uniform size and easily harvested [23]. Due to its economy and ease of use, Stemfit 3D is commonly used in stem cell cytology and various other cellular studies [24; 25; 26; 27; 28]. PDLSC spheroids from 3D cultures showed a rapid decrease (67.1%) in diameter over the first 5 days, followed by a moderate decrease after day 5 (Figure 1E-H).