Despite advancements in therapeutic approaches, radiotherapy and cisplatin-based chemotherapy remain primary noninvasive treatments for patients with oral squamous cell carcinoma (OSCC). Moreover, the 5-year survival rate for patients with OSCC has remained almost unchanged for several decades, and many side effects of chemotherapy still exist. In this study, three-dimensional (3D) models of OSCC were established using fibroblasts, and the efficacy of various biological inhibitors was evaluated. A culture of epithelial cells with two types of fibroblasts (hTERT-hNOFs and cancer-associated fibroblasts) within a type I collagen matrix resulted in the formation of a continuous layer of tightly packed cells compared to models without fibroblasts. Furthermore, the effects of biological chemicals, including Y27632, latrunculin A, and verteporfin, on these models were investigated. The stratified formation of the epithelial layer and invasion in OSCC 3D-culture models were effectively inhibited by verteporfin, whereas invasion was weakly inhibited by Y27632 and latrunculin. Collectively, the developed OSCC 3D-culture models established with fibroblasts demonstrated the potential for drug screening, with verteporfin showing promising efficacy.

Mesenchymal stem cells in the dental pulp exhibit a tendency for differentiation into various dental lineages and hold great potential as a major conduit for regenerative treatment in dentistry. Although they can be readily isolated from teeth, the exact characteristics of these stem cells have not been fully understood so far. When compared to twodimensional (2D) cultures, three-dimensional (3D) cultures have the advantage of enriching the stem cell population. Hence, 3D-organoid culture and 3D-sphere culture were applied to dental pulp cells in the current study. Although the establishment of the organoid culture proved unsuccessful, the 3D-sphere culture readily initiated the stable generation of cell aggregates, which continued to grow and could be passaged to the second round. Interestingly, a significant increase in SOX2 expression was detected in the 3D-spheroid culture compared to the 2D culture. These results indicate the enrichment of the stemness-high population in the 3D-sphere culture. Thus, 3D-sphere culture may act as a link between the conventional and 3D-organoid cultures and aid in understanding the characteristics of dental pulp stem cells.

Three-dimensional (3D) culture system is useful technique for study of in vivo environment and it was used various experiments. This study was investigated to establish of embryo co-culture system and changes of PAs activity in 3D cultured endometrial cells of pigs. In results, growth of stromal cells into gel matrix were detected only with endometrial and myometrial cells. The most rapid growth of stromal cells were confirmed in 2.5x105cells/ml and gel matrix containing 15% FBS. Expression of urokinase-PA (uPA) after treatment of hCG (0.5, 1.0, 1.5 and 2.0 IU/ml) were higher than without hCG, but, there are not significant difference among the treatment. On the other hand, expression of uPA after treatment of IL-1β (0.1, 1, 10 and 100 ng/ml) were higher than without IL-1β, but, there are not significant difference. Expression of uPA after treatment of estrogen (0.2, 2, 20 and 200 ng/ml) were not difference, but PA activity was significantly decreased (p＜0.05). Blastocyst was producing in PZM-3 medium containing FBS and endometrial cells were grown in PZM-3 medium. When embryos development with cultured endometrial cells, cleavage rates were not significant difference and blastocyst were not produced in co-culture with stromal cells and 3D culture system. 3D culture system had similar activity to in vivo tissue and these features are very useful for study of in vivo physiology. Nevertheless 3D culture system was not proper in embryo co-culture system. Therefore, we suggest that 3D culture system with embryo co-culture need continuous research.

The aim of this study was to establish a three dimensional (3D) culture system of endometrial cells and to examine the plasminogen activators (PAs) activity in porcine uterine. The 3D culture system in porcine endometrial cells was composed to mixture 3D gel, stromal cells and epithelial cells. The 3D culture system was used to identify normal structure search as uterine tissue and PAs expression in this study. In results, porcine endometrium epithelial cells forming a top monolayer and endometrium stromal cells developed as fibroblast-like within 3D matrix scaffold. Expression of urokinase-type PA (uPA) and tissue-type PA (tPA) were observed during the 3D culture using immunofluorescence. PA activity in 3D-cultured endometrial cells was no significant difference between the tissue type, but 2D culture system were significantly lower than in 3D-cultured endometrial cells (P<0.05). Therefore, basic system and functional aspect of 3D culture could be established with similar system of endometrium tissue. We suggest that this study was assumed applicable as baseline data to investigate mechanism between porcine uterus cells in vitro.

Epithelial-mesenchymal interaction is well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular micro-environment which provide a epithelial-mesenchymal interaction. The aims of this study were to develop and evaluate the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted human normal oral kertinocyte(NHOK) and immortalized human oral keratinocytes(IHOK) by histological and immunohistochemical analysis. The results were as follows; 1. Best condition of three dimensionally reconstituted IHOK & HaCaT cells are 14 days air-exposure cultivation, 3 days of submerged state, and dermal equivalent consisting type I collagen and IGF cells. 2. In comparison to IHOK, there was better preservation of the overall epidermal strucutures in oral cracioma cells (HN30) & HaCaT cells by organotypic cultures. But orgnaotypic co-culture of the normal keratinocyte showed the thinnest epithelial layer formation. 3. PCNA was detected primarily in the basal layer of normal mucosa and NHOK, whereas was shown throught the epithellium except surface layer of IHOK cells, and it's expression was similar to that of CIS of biopsied patient's tissue 4. Involucrin is expressed in the upper layer of oral mucosa and NHOK raft, but staining for involucrin was induced in the IHOK rafts indicating differentiation is incomplete, and the staining pattern in the IHOK raft was not uniform. 5. Normal oral keratinocyte raft showed weak immunostaining for p53, and p53 expression of IHOK raft increased rahter than in NHOK. In organotypic cultures of normal cells and IHOK, p53 expression was restricted to the proliferative part of epithelium. This is consistent with expression pattern in biopsy specimens of the normal and CIS tissue. 6. In artifically reconstructed NHOK, the pattern of keratin staining showed both similarity and differences from that of intact normal mucosa. An obvious difference was increased expression of CK10 & CK19, and decreased expression of CK6 in a reconstructed NHOK rather than in normal mucosa, and similar expression was in CK4 and CK16. 7. CK19 & CK16 were strongly positive in HPV immortlaized keratinocyte rafts rahter than in NHOK, an indicator of premalignant or malignant changes, while CK10 & CK6 were decreased, and organotypic cultures of IHOK express was similar keratin expression as epithelial dysplasia or CIS tissue. These results suggest that three-dimensional organotypic co-culture of normal oral & immortalized keratinocytes with dermal equivalent consisting type I collagen and fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. Thus this organotypic system can be used for studing mechanism of epitehlial-mesenchymal interaction in particular regulating epidermal diffenentiation and morphogenes

We used three-dimensional Matrigel culture system to examine the morphognesis of normal and malignant salivary glands cell in vitro including acinar cells(AC), myoepithelial cell(MC), salivary gland adenocarcinoma cells(SGT), mucoepidermoid carcinoma cells(MEC), and immortalized human salivary gland cells(HSG). For this purpose, normal and salivary gland tumor cells cultured in 3-D Matrigel, and characterized histologically and immunohistochemically, compared with same cells grown on monolayer culture and patient tissue from biopsy. 1. In three-dimensional Matrigel culture, HSG cells form acinar structure, SGT cells shows duct like structure, and other AC, MC and MEC cells dont' form any structure , and their morphology was different from that of monolayer cells. 2. Matrigel involved cell proliferation at a similar pattern to cells on plastic monolayer cell cultures, and monolayer cell revealed higher cell viability than that of Matrigel cultured cells. 3. All salivary glands cells on Matrigel or monolayer showed strong PCNA expression, and there is no expression difference in these cells. But some cells including myoepithelial cells in normal and salivary gland tumor tissue showing PCNA lavelling, so there is PCNA expression difference among normal and tumor tissue cells. 4. Actin expression was noted in AC cells on Matrigel, were rare expressed in the other cells except in MEC cells, and was present in myoepithelial cell and ductal cells of normal gland tissue. There is actin expression difference between tissue and cultured cells . 5. S-100 immunoreaction was moderateively positive in MC cells of monolayer culture, myoepithelial cells of normal tissue and pleomorphic adenoma, all cancer cells of mucoepidermoid carcinoma tissue, but significantly decreased in all salivary cells on Matrigel. 6. TGase 2 expression was prominent in MC cells of monolayer and Matrigel cultured, in myoepithelial cells of normal gland and pleomorphic adenoma, epidermoid cells of mucoepidermopid carcioma, and strong reaction in MEC and AC cells of monolayer and Matrigel cultured. 7. Expression of CK in monolayer culture showed strong reaction to CK6 in all sailvary gland cells, and mild reaction to CK10 and CK16 for all salivary cells, CK16 and CK19 expression in monolayer culture was similar to that of Matrigel culture. 8. CK6 and CK10 expression was strongest in AC and MC cells on Matrigel, and CK 4 was negative reaction in AC, SGT, MEC cells, strong reaction in MC cells but mild in SGT cells on Matrigel. Expression of CK was rare in HSG cells compared with other salivary gland cells, CK16 was prominent in SGT cells, CK10 and CK16 showed strongest expression in MEC cells of Matrigel. 9. Monolayer culture of HSG cell shwoing strong reaction to CK6, moderate to CK19 and mild to the others CK, but 3D cultured HSG cells reveal mild expression to CK16, and rare to others CK, intercallated duct in normal gland tissue showing strong to CK19, and mild to the others Ck, so there are CK expression difference in tissue, monolayer and 3-D cultured cells. 10. Monolayer culture of MEC cells represent strong reaction to CK6, mild to other CK, 3-D cells showing increased CK expression including CK6, epidermoid cells and intermediate cells in mucoepidermoid carcinoma tissue reveal positive to CK6 and CK16, mucous cell positive to CK10 and CK19, so Matrigel showed similar CK pattern compared to mucoepidermoid carcinoma tissue rather than monolThese data indicate that the interaction of salivary gland cells with basement membrane is an important factor in salivary gland development and cytodifferentiation, so this model system will be useful to study acinar or ductal differentiation in vitro.ayer cultred.