ABSTRACT
@#Introduction: A crucial factor in cell culture technology is the use of appropriate culture medium which can promote cell growth and cellular functions. Development of serum free chemically defined medium enables the researchers to conduct the experiment in a more controlled manner. Myofibroblasts of the tumour microenvironment drive the colorectal carcinogenesis. In vitro study of the tumour-myofibroblast interaction using serum free medium may give a better insight into potential treatment for colorectal cancer (CRC) in the future. This study aims to establish serum free chemically defined medium to study the interplay between myofibroblast and CRC cells. Methods: A myofibroblast-specific serum free culture medium named as M-CIL, was developed to study the interactions between myofibroblasts and CRC cell lines in vitro. The influence of substrate (collagen type I) and subculturing of cells under incubation with M-CIL medium were also analysed. The effect of M-CIL medium on CRC cell growth also was studied. Gene expression analysis using quantitative real time polymerase chain reaction on amine oxidase, copper containing 3 (AOC3) was conducted to investigate the effect of individual components of the medium on myofibroblasts. Results: M-CIL medium supports the proliferation of myofibroblasts and produce minimal effect on CRC cells’ growth. Our data also shows the influence of M-CIL components on gene expression in myofibroblasts. Conclusion: M-CIL culture medium, which was designed with known and defined components, proved to be a suitable alternative to complete medium (DMEM + 10% FBS) for co-culture experiments of myofibroblasts and CRC cell lines.
ABSTRACT
Perivitelline fluid [PVF] of the horseshoe crab embryo has been reported to possess an important role during embryogenesis by promoting cell proliferation. This study aims to evaluate the effect of PVF on the proliferation, chromosome aberration [CA] and mutagenicity of the dental pulp stem cells [DPSCs]. This is an in vitro experimental study. PVF samples were collected from horseshoe crabs from beaches in Malaysia and the crude extract was prepared. DPSCs were treated with different concentrations of PVF crude extract in an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide [MTT] assay [cytotoxicity test]. We choose two inhibitory concentrations [IC[50] and IC[25]] and two PVF concentrations which produced more cell viability compared to a negative control [100%] for further tests. Quantitative analysis of the proliferation activity of PVF was studied using the AlamarBlue[Registered sign] assay for 10 days. Population doubling times [PDTs] of the treatment groups were calculated from this assay. Genotoxicity was evaluated based on the CA and Ames tests. Statistical analysis was carried out using independent t test to calculate significant differences in the PDT and mitotic indices in the CA test between the treatment and negative control groups. Significant differences in the data were P<0.05. A total of four PVF concentrations retrieved from the MTT assay were 26.887 mg/ml [IC[50]], 14.093 mg/ml [IC[25]], 0.278 mg/ml [102% cell viability] and 0.019 mg/ml [102.5% cell viability]. According to the AlamarBlue[Registered sign] assay, these PVF groups produced comparable proliferation activities compared to the negative [untreated] control. PDTs between PVF groups and the negative control were insignificantly different [P>0.05]. No significant aberrations in chromosomes were observed in the PVF groups and the Ames test on the PVF showed the absence of significant positive results. PVF from horseshoe crabs produced insignificant proliferative activity on treated DPSCs. The PVF was non-genotoxic based on the CA and Ames tests