Browsing by Author "Mohd Azmir bin Arifin"

Microcarrier cell culture offers many advantages over conventional cell culture systems and has been widely used for the production of many important biological products. Nevertheless, the price of commercial microcarriers is quite expensive and can significantly increase the cost of culturing when used on an industrial scale. The present study attempts to address this problem by developing low cost, yet, efficient gelatin coated polystyrene (PS) microcarriers using an ultraviolet/ozone (UVO3) system as the main surface modification method. In this study, polystyrene (PS) microspheres were prepared by using oil-in-water (O/W) emulsion solvent-evaporation method. In order to promote good cell attachment and proliferation on PS microspheres, their surfaces were first treated with UVO3 system to introduce carboxyl (COOH) functional groups and subsequently coated with bovine gelatin using zero length cross-linker reagents. One-factor-at-a-time (OFAT) method and face centred central composite design (FCCCD) were employed to optimize UVO3 treatment process conditions for maximal COOH concentration on the surface of PS microspheres. Factors affecting cross-linking conditions were also optimized using similar OFAT and FCCCD, to maximize the amount of immobilized gelatin on the surface of UVO3 treated PS microspheres. The cytocompatibility of gelatin coated PS microspheres and the intermediate product, UVO3 treated PS microspheres were evaluated by using them as attachment substrates in suspension cultures of Vero, CHO-K1 and AFSC in spinner vessel. Based on experiments, the highest amount of microspheres with appropriate mean size for microcarrier production could be generated at PS to CHCl3 ratio of 1:5 (w/v), agitation speed of 300 rpm, surfactant PVA concentration of 0.25%, oil to water phase ratio of 1:5 (v/v) and agitation temperature of 80°C. Statistical analyses showed that the optimum UVO3 process conditions; ozone concentration of ~64,603 ppm, exposure time of ~60 minutes and sample amount of 5.05 g resulted in the maximum COOH concentration of ~1,505 nmol/g. While at EDAC to COOH ratio of 2.5:1, NHS concentration of 0.5 mM and gelatin concentration of 40 mg/mL, the maximum gelatin immobilization at 2,524.74 µg/g was obtained. FTIR analysis revealed that UVO3 treatment has successfully introduced COOH functional groups on the surface of PS microspheres whereas gelatin immobilization was proven by the presence of several amide peaks. The surface wettability and the dispersion stability of PS microspheres in liquid medium were also found to be highly improved after UVO3 treatment and gelatin immobilization. Furthermore, UVO3 treated and gelatin coated PS microspheres were also revealed as able to withstand different sterilization procedures without losing much of their surface functionalities. By using UVO3 treated and gelatin coated PS microspheres, high cell density cultures of Vero and CHO-K1 cells were successfully achieved with results that were highly comparable to those obtained when using expensive commercial microcarriers. Additionally, EBs formation assay has confirmed that, AFSC cultured using both types of microspheres were able to maintain their ‘stemness’. In conclusion, by using the UVO3 system, this study has successfully developed two types of low cost and efficient microcarriers that have huge potentials for commercialization. Further studies involving the use of both types of microcarriers in the production of important biological products such as vaccines, recombinant proteins, gene therapy vector and etc. is highly encouraged.

The present work aims to prepare a model for the production of lentogenic Asplin F strain of Newcastle disease virus (NDV) using cell culture in bioreactor for live vaccine preparation. First NDV was adapted in four different cell lines namely DF-1, CEF, MSB-1 and Vero cells in T-flasks. DF-1 was found to be the most potential for NDV propagation in terms of high NDV production, rapid proliferation rate and several ethical values. Viruses produced in DF-1 cell culture were confirmed as lentogenic NDV strain by molecular diagnosis by using reverse transcriptase polymerase chain reaction (RT-PCR). In a screening experiment, high cell concentration during time of infection was found to be the most significant factor for NDV production. In another screening experiment, DF-1 was cultured in DMEM, DMEM-F12, RPMI 1640 and MEM. Culture of DF-1 cells in DMEM has the highest cell concentration of 1.240 × 106 cells/ml with specific growth rate and doubling time of 0.0180 h-1 and 38.4967 hours respectively. Composition of culture medium was later optimized and it was observed that culture has optimum cell concentration of 1.368 x 106 cells/ml when 2.1 g/L of NaHCO3 and 7.5% serum is included in the culture medium. Experiment was continued with culture of DF-1 cells in spinner vessel. In an optimization of microcarrier concentration study, culture with 3 g/L microcarrier achieved maximum cell concentration of 0.515 × 106 cells/ml compared to culture having 1 g/L, 2 g/L, 4 g/L and 5 g/L microcarriers. Based on this result and previous optimization studies, NDV was propagated in DF-1 cell culture in spinner vessel and the maximum virus titre achieved was 128 HA unit with infectivity titre of 3.08 x 107 TCID50/ml. In a separate experiment using 2-L stirred tank bioreactor, an optimization of process conditions study was conducted and it was analyzed that maximum cell concentration of 1.213 × 106 cells/ml will be obtained when the agitation rate is set at 71 rpm, microcarrier concentration at 2.9 g/L and pO2 pressure at 20%. NDV was also propagated in DF-1 cell culture in stirred tank bioreactor and the maximum virus titre achieved was 4 HA unit with infectivity titre of 1.03 x 103 TCID50/ml. Viruses produced from both types of bioreactors were later concentrated by using the high speed centrifugation method. An optimization study on process conditions of high speed centrifugation was conducted and it was revealed that optimum virus titre of 256 HA unit was achieved when the sample concentration was set at 50%, centrifugation speed was set at 12800 rpm and centrifugation time was set at 8 hours. The ability of cell culture in producing NDV in bioreactors has been shown in this study thus the model can be proposed for ND vaccine production in the future.