Función interaccional

2.3.1.1 Cell lines

Three cell lines were used for the anti-cancer test of the crude extracts; human acute T lymphoblastic leukaemia (MOLT-4), human Caucasian chronic myelogenous leukaemia (K562), and human Caucasian lung cell carcinoma (COR-L23). For the cytotoxicity test, mouse embryonic fibroblast cells (3T3) were used. All cell lines used were grouped into

67 adherent cells (COR-L23 and 3T3 cell lines) and suspension cells (MOLT-4 and K562). All cell lines were cultured in culture medium containing 90% (v/v) RPMI 1640, 2mM L-glutamine, and 10% (v/v) Fetal Bovine Serum (FBS). To prevent bacterial infection during cell culturing, 50 IU/ml penicillin and 50µg/ml streptomycin were added into the media. Additional reagents or materials needed for performing cell culture or anti-cancer test of Phyllanthus niruri L extracts were sterilized by autoclaving, or filtered through a sterile 0.2µm filter. The cells were cultured in 25 cm2 sterile flasks and incubated at 37 ˚C, in an atmosphere of 95 % air and 5% CO2. All procedures during cell culture were performed in a class II biological

safety cabinet and, in addition, all precautions were carefully taken to prevent the occurrence of any aspects of contamination (Markovic and Markovic, 1998).

2.3.1.2 Recovery of frozen cell lines

All cell stocks were kept in a 0.5 ml aliquot of cell suspension in a medium containing 10% (v/v) concentration of DMSO at a -80 ˚C environment or in liquid nitrogen (-196 ˚C), which is suitable for a long term storage. Prior to the regular culturing procedure, the cells were recovered from the frozen condition and prevented from the toxic effect of DMSO. At a concentration of more than 4%, DMSO induced monolayer disruption and an alteration of cell-to-cell tight junctional complexes, whereas at higher concentration (more than 10%), DMSO will disturb cell membrane permeability (Da Violante et al., 2002). Accordingly, cell aliquots were quickly thawed in a 30 ˚C waterbath after removal from the -80 ˚C condition. Immediately after the ice crystal had melted, the cell suspension was transferred into a flask containing pre-warmed medium. By doing so, the containing DMSO was diluted to a lower concentration that is tolerable for the cells and without giving any deleterious effects. Subsequently, the cell suspension was incubated at a 37 ˚C incubator with 5% CO2

68 environment for a minimum of one passage to allow the cells to recover and achieve the optimum conditions for the anti-cancer test by MTT assay and protein extraction.

2.3.1.3 Maintaining the optimal condition of cell culture

The cells were monitored regularly to ensure the cell viability as well as to observe if there were any sign of contaminants. The colour and turbidity of media were monitored, as well as cell morphology and cell density. In conditions where the cell population was too dense, the media environment became acidic, and nutrient was depleted, cell culture grew which led to cell detachment and death. Therefore, when the cell suspension had reached sub- confluent condition or 70-80% confluency, the medium was changed to replenish the nutrient supply, restore the optimum conditions for cell growth, as well as to restore the cell density back to optimum condition for cell growth (Macleod and Landon, 2004).

To maintain the healthy growth of cells, the culture required regular subculture, the procedure in which the sub-confluent culture was split into a fresh medium. Generally, the subculture of the cells was performed every three days to ensure that the cells did not overgrow. Subculturing was also referred as harvesting because at this stage, cells are ready for further biological assays.

2.3.1.4 Subculturing adherent cell lines

Adherent cell lines grow by adhering to a plastic wall of the culture flask, which acts as the substrate, and proliferate as a monolayer of cells. As the first step of the subculture, the old medium was removed from the flask, followed by subsequent washing of the interior surface of the flask to remove the remaining medium and non-viable cells. Sterile phosphate buffer saline (PBS) was used in this step. A sterile 0.5 ml trypsin solution at 0.25% (w/v) concentration was added to the flask, which then was incubated in a 37 ˚C incubator for 2-3

69 minutes until the cells have detached. Trypsin is a proteolytic enzyme that breaks the cell- substrate and cell-cell bonds, which then results in cell detachment and creates a single-cell suspension. Subsequently, 4.5 ml of fresh medium was added into the flask, which eventually diluted the enzyme and halted the proteolytic activity. At this stage, healthy cells were ready for harvesting for further MTT and protein extraction (see section 2.3.2 and 2.3.3). Although the cell lines used in this study had different rate of cell growth, as a general rule, a split ratio of 1:5 was used for routine culture COR-L23 and, due to the fast rate of cell growth, split ratio used for 3T3 cell line was 1:50.

2.3.1.5 Subculturing suspension cell lines

Suspension cell lines grow as suspended cells in the medium, therefore subculturing does not require trypsinization step. The whole volume of cell suspension was transferred into a sterile universal bottle for subsequent centrifugation at 1500 rpm for 5 minutes to spin down all cultured cells. The spent medium was carefully pipetted and discarded, and a fresh medium was added into the cell pellet. At this stage, healthy cells were ready for harvesting for further MTT and protein extraction (see section 2.3.2 and 2.3.3). After homogenization the cell pellet into the medium by several times pipetting, the cell suspension was then split at a ratio of 1:10.

2.3.1.6 Cryopreservation of cell stocks

As mentioned in section 2.3.1.2, the cell stocks were stored at an extreme low temperature (below -80°C) with several conditions to maintain the cell viability. The important feature is to ensure that cells are in optimum condition without any feasible contaminants. For adherent or monolayer cells, the cultured cells were detached by trypsinization, followed by addition of fresh media to inactivate trypsin activity. Subsequently, for adherent and

70 suspension cells, the suspension was centrifuged at 1500 rpm for 5 minutes to obtain cell pellet, then a freeze-cold medium containing 10% (v/v) DMSO was added to re-suspend the cells at a concentration of approximately 107 cells/ml. DMSO is another crucial element in the cell cryopreservation, that acts as a cryoprotective agent which prevents the formation of ice crystal and fragmentation of the cell membrane. The final suspension of cells stock was aliquoted in several cryovials. To prevent the formation of intracellular ice crystal which causes cell lysis upon cell thawing, the cryovials were placed overnight in a cryocontainer containing isopropyl alcohol, which slowly reduced the cell temperature at a cooling rate of 1˚C/min when it was stored in a -80 ˚C freezer. This treatment is necessary to allow water from the intracellular environment to diffuse out of the cells thus inhibit the ice crystal formation. When the final temperature of -70 ˚C to 80 ˚C was achieved, cell were transferred to -80 ˚C freezer or liquid nitrogen storage tank.