Effects of Secretome from Dynamic 3D Cell Culture System onto Growth and Cytoprotection of Nasal Fibroblast
Effects of Secretome from Dynamic 3D Cell Culture
Abstract
Three 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.
Keywords :
airway epithelium,
Centella asiatica,
cytoprotection,
microsphere,
respiratory,
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). Diketahui juga, rembesan sel mempunyai kesan parakrin kepada pertumbuhan sel-sel lain. Dalam kajian ini, pengkulturan fibroblast hidung menggunakan system kultur sel 3D telah dioptimumkan dan kesan bahan rembesan (BR) daripada kultur 3D terhadap kadar pertumbuhan dan perlindungan sel telah dikaji. Fibroblas hidung dipencilkan daripada turbinate hidung manusia. Mikrosfera yang sesuai telah dipilih melalui pengkulturan fibroblast pemindahan ke-3 pada pelbagai jenis mikro sferapolisterin PolyGEM™. Kemudian, sel-sel telah dikulturkan pada mikrosfera yang terpilih menggunakan system kultur 3D dan media terkondisi (MT) telah dikumpulkan. Media terkondisi tiga dimensi (MT3D) telah ditambah kepada fibroblast untuk mengkaji kadar perlekatan sel, kadar proliferasi, dan perlindungan sel terhadap kesitotoksikan Centella asiatica. Asai protein asid bicinchonic dijalankan untuk mengetahui kuantiti protein di dalam BR. Elektroforesis gel poliakrilamida-Sodium Dodesil Sulfat (SDS-PAGE) telah dilakukan untuk memperoleh profil awal protein dan membandingkan profil MT3D dengan protein media terkondisi dua dimensi (MT2D). Kajian ini menunujukkan MT3D tidak menggalakkan perlekatan dan proliferasi sel secara signifikan. BR didapati memberikan perlindungan sel yang signifikan pada fibroblast hidung terhadap kesitotoksikan Centella asiatica. MT3D mempunyai kepekatan protein yang lebih tinggi berbanding MT2D. SDS-PAGE menunjukkan MT3D mempunyai 3 jalur ekslusif manakala MT2D mempunyai 4 jalur eksklusif. Kajian masa depan harus dijalankan keatas penggunaan BR fibroblast hidung untuk perlindungan sel terhadap agen-agen yang memudaratkan di alam sekitar dan produk herba yang sitotoksik.
Kata Kunci :
Centella asiatica,
epitelium salur pernafasan,
microsfera,
perlindungan sel,
respiratori,
Correspondance Address
Yogeswaran Lokanathan. Tissue Engineering Centre, 12th Floor, Clinical Block, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia. Tel: +603-91457704 Fax: +603-91457678 E-mail: lyoges@ppukm.ukm.edu.my
