Effects of Secretome from Dynamic 3D Cell Culture System onto Growth and Cytoprotection of Nasal Fibroblast

Abstract

ABSTRAK Model tiga dimensi (3D) menyerupai ciri-ciri persekitaran tisu asli, justeru morfologi dan isyarat-isyarat sel daripada kultur 3D selalunya lebih menyerupai fisiologi asal berbanding sel kultur dua dimensi (2D ABSTRACTThree dimensional (3D) models mimic the features of native tissue environment. Thus, morphology and signalling of cells from 3D culture are often more physiological than routine two dimensional (2D) cell culture. It is also known that the cell-secreted products have paracrine effect on other cells growth. In this experimental study, we optimised the nasal fibroblast culture on a 3D cell culture system and studied the effects of secretome from the 3D culture (3DCM) onto fibroblast growth and cytoprotection. Nasal fibroblast was isolated from human nasal turbinates. The suitable microcarrier was selected by culturing the fibroblasts in passage 3 on various types PolyGEM™ polystyrene microcarriers. Then, the cells were cultured on selected microcarrier using a 3D culture system and the conditioned medium (CM) was collected. 3DCM were supplemented to fibroblasts to study for attachment, proliferation, and cytoprotective effects against cytotoxicity of Centella asiatica. Bicinchonic Acid Assay (BCA) was performed to quantify protein amount in CMs. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for preliminary profiling and comparison of 2DCM and 3DCM protein profile. Our study showed that the 3DCM did not significantly enhance cell attachment and proliferation. The secretome of both 2DCM and 3DCM found to have significant cytoprotective effect onto nasal fibroblast against cytotoxicity of C.asiatica extract. 3DCM had higher protein concentration than 2DCM. SDS-PAGE showed three exclusive proteins in 3DCM and four exclusive proteins in 2DCM. Future study should be conducted on utility of nasal fibroblast secretome on cytoprotection against harmful agents in environment and cytotoxicity of natural products.

Application of Conditioned Medium for In Vitro Modeling and Repair of Respiratory Tissue

“…In vitro research has investigated the effects of CM from differentiated cells on respiratory epithelium tissues. The findings suggested that CM may be used successfully to improve the functioning of both healthy and diseased cells [42,46,50]. CM derived from primary cells such as nasal fibroblasts enhance cell proliferation, attachment, and migration [42,46] and also exert a cytoprotective effect on REC [42].…”

“…The findings suggested that CM may be used successfully to improve the functioning of both healthy and diseased cells [42,46,50]. CM derived from primary cells such as nasal fibroblasts enhance cell proliferation, attachment, and migration [42,46] and also exert a cytoprotective effect on REC [42]. The CM from cell lines such as A549, when cocultured with THP-1, aids in the reduction of cancer cell metastasis [50].…”

“…The result metrics used included cell viability assays, proliferation assays, scratch wound migration assays, immunofluorescence, Western blotting, and enzyme-linked immunosorbent assays (ELISA). While most studies examined both proliferation and migration assays, some found that a faster migration rate was often associated with a greater impact of tissue-conditioned media on cell proliferation [37,42,43]. Four studies examined cell viability assays [41,[44][45][46], where a higher proportion of viable cells was accompanied by a larger estimation of the number of live cells.…”

“…The 10 studies also conducted specific marker expression experiments, namely immunofluorescence assays, PCR, ELISA, and Western blotting. CK18, KI67, and MUC5B were among the genes expressed [42]. Table 1 summarizes where cell-released factors from respiratory disorder-related tissue, such as LAM-associated fibroblasts and idiopathic pulmonary fibrosis fibroblasts, demonstrated positive effects on respiratory regeneration in vitro, including increased proliferation, migration, and protection against apoptosis.…”