**2. 2D and 3D cell culture systems**

Cell culture is used in vitro in cell biology, tissue morphology, and mechanisms of diseases, drug action, protein production, and the development of tissue engineering. The stage of the culture after the cells can be isolated from the tissue and proliferates under favorable conditions until they reach confluency. In this stage, the cells have to be passaged or subcultured by transferring them to a new vessel with fresh growth medium to provide more room for continued growth. Various researches in cancer biology are based on experiments using two-dimensional cell culture by growing cells in flat dishes, made of plastic. The dish culture system is mainly used for developing adherent two-dimensional cell monolayers. 3D cell culture applications are usually beneficial in tissue engineering and regenerative

medicine. 3D cell culture is an artificially created environment in which biological cells are allowed to grow and interact with their surroundings in all three dimensions. These three-dimensional cultures are grown in bioreactors, small capsules in which the cells can grow into spheroids, otherwise 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor 3D cell culture which allows cells in vitro to grow in all directions, similar to how they would in vivo [15]. An increasing shift in research is occurring, where 3D cell culture systems are replacing 2D cell culture systems and translating 2D in vitro research to 3D before or as an alternative to testing using in vivo animal models [16, 17].

### **2.1 Preparation of culture media**

Culture media provide artificial environment to grow the cancer cells in vitro. Culture media can be prepared by mixing DMEM powder (glucose, l-glutamine, pyridoxine, HCl, without pyruvate) in autoclaved triple distilled water. To this 1.95 g of HEPES buffer and 3.75 g sodium bicarbonate can be added. Antibiotics such as penicillin (500 μl) and streptomycin (500 μl) and fungicide-amphotericin-B (750 μl) can also be added. The volume can then be made up to 1000 ml, and the pH will adjust to 7.2–7.4. The medium will then filter under negative pressure using 0.22 μm cellulose filter. Sterility of the medium can be tested before use. Ten percent FCS can be mixed with the medium prior to culture.

#### **2.2 Maintenance of adherent cancer cell lines**

Adherent cell lines will grow in vitro until they form a monolayer over surface area available or medium depleted of nutrients. Adherent cells human oral cancer cells (KB), lung adenocarcinoma (A-549), and breast cancer (MCF-7) can be cultured in tissue culture flasks. The cells were disaggregated by trypsinization and subcultured when the monolayer reaches about 70% confluency. The cells will be cryopreserved at −80°C. With an inverted microscope, the degree of confluency of the cell monolayer can be assessed, and the absence of bacterial and fungal contaminants can be confirmed. Spent medium can be removed. Cells can be washed with PBS-EDTA for removing the traces of serum. Trypsin/EDTA (500 μl) will be applied onto the cell monolayer, and the flask is swirled to cover the monolayer with trypsin. The flask will be incubated at 37°C for 2–3 min. The flask can be examined under the inverted microscope to ensure uniform detachment of the cells. 1–2 ml of medium can be added to the flask as fast as possible to lessen the trypsin-induced stress, and the contents of the flask can be transferred to a centrifuge tube. Cells should be centrifuged at 1500 rpm, for 10 min. The supernatant will be discarded, and the cells were resuspended in minimum volume of medium. Cells can be counted using a hemocytometer, and the required numbers of cells can be subcultured in a new flask containing fresh DMEM with 10% FCS. This process can be repeated as demanded by the growth characteristics of the cell line.

#### **2.3 Maintenance of cancer suspension cell lines**

In general terms cultures derived from blood (e.g., lymphocytes grow in suspension) cells may be seen as single cells or clumps. For these types of cell lines, subculturing is done by dilution in small volume of media before counting. The culture can be viewed using an inverted phase contrast microscope; cells growing in exponential phase should be bright, round, and refractive. Cell suspension can be mixed well and dispersed uniformly by repeated pipetting in order to make singlecell suspension. The cells can be counted, and 1 × 106 cells can be seeded to a fresh

**105**

cell suspension.

*Cell-Based Assays in Cancer Research*

and antibiotics.

*DOI: http://dx.doi.org/10.5772/intechopen.90226*

**2.4 Establishing cell cultures from frozen cells**

medium. Add 10 ml of growth medium to a 75-cm2

bottle containing 10 ml of DMEM medium with 10% FBS antibiotics and incubated at 37°C and subcultured every third day. On the day of the experiment, single-cell suspension will be prepared. The cells will be counted and the viability can be checked. The concentration can be adjusted with the medium containing 10% FBS

About 10 ml of growth medium placed in a 15-ml conical tube. Thaw the frozen cryovial of cells for 40–60 s by gentle agitation in a 37°C water bath. Remove the cryovial from the water bath, and decontaminate the cryovial by immersing it in 70% (v/v) ethanol at room temperature (RT). Transfer the thawed cell suspension to the conical tube containing 10 ml of growth medium. Cells can be collected by centrifugation at 2000 rpm for 5 min at RT. Remove the growth medium by aspiration, and then resuspend the cells in the conical tube in 5 ml of fresh growth

5 ml of cell suspension to the same. Place the cells in a 37°C incubator at 5% CO2. Monitor cell density daily. Cells can be passaged when it will attain 50% confluency.

Trypan blue is a dye used to determine the viability of a cell. Living cells exclude the dye, and dead cells will take up the blue dye. The blue stain is easily visible, and cells can be counted using a light microscope. The reactivity is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the cells that exclude the dye are viable. When the cells are confluent, remove the cell media through aspiration, and add 5 ml of PBS swirl and aspirate. Then add 2 ml of trypsin/EDTA, and swirl to cover the monolayer of cells. Incubate for few minutes at 37°C. To remove the cells, strike the side of the plate or the flask with the palm. Check under a microscope to ensure that all the cells are dislodged. Add 8 ml of cell media containing fetal calf serum (FCS) to the cells containing the culture flask. The FCS will neutralize the action of trypsin. Transfer the cell suspension to a sterile centrifuge tube, and centrifuge the cell suspension at 1000*g* to pellet the cells. Wash the cell pellet twice with PBS. Resuspend the cell pellet in appropriate volume of PBS or cell media. Dilute 10 μl of cell suspension, and place 10 μl on a hemocytometer, and count the cells under a microscope. There are grid markings on the hemocytometer that can be seen under magnification. Count the cells in all four other quadrants of the grid. Divide this number by four to determine the average number of cells in one quadrant. To calculate the number of cells, multiply the average number of cells per quadrant by dilution factor. Multiply this number by 10,000 to calculate the number of cells in 1 ml of suspension. The equation is as follows: average number of cells per quadrant C dilution factor C 10,000 = number of cells/ml. To calculate the total number of cells, multiply the number of cells per ml by the volume (ml) of the

Calculating the % of viable cells: The cells (10,000) are suspended in 500 μl media and treated with varying concentrations of drug and incubate for 24 h. Centrifuge at 1500 rpm for 10 min. Discard 400 μl medium. Resuspended the pellet in the remaining medium. Mix 0.5 mg of trypan blue in 1 ml PBS. Take 10 μl of cell suspension and mix with trypan blue solution. Incubate for 5 min at room temperature. Count the numbers of unstained cells on the hemocytometer under a microscope. As mentioned above the dead cells will take up the trypan blue stain. First count the blue cells in the field and then white cells. Count the total number of

**3. Determination of cell viability by trypan blue exclusion method**

tissue culture flask, and transfer

#### *Cell-Based Assays in Cancer Research DOI: http://dx.doi.org/10.5772/intechopen.90226*

*Cell Growth*

medicine. 3D cell culture is an artificially created environment in which biological cells are allowed to grow and interact with their surroundings in all three dimensions. These three-dimensional cultures are grown in bioreactors, small capsules in which the cells can grow into spheroids, otherwise 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor 3D cell culture which allows cells in vitro to grow in all directions, similar to how they would in vivo [15]. An increasing shift in research is occurring, where 3D cell culture systems are replacing 2D cell culture systems and translating 2D in vitro research to 3D before or as an alternative

Culture media provide artificial environment to grow the cancer cells in vitro. Culture media can be prepared by mixing DMEM powder (glucose, l-glutamine, pyridoxine, HCl, without pyruvate) in autoclaved triple distilled water. To this 1.95 g of HEPES buffer and 3.75 g sodium bicarbonate can be added. Antibiotics such as penicillin (500 μl) and streptomycin (500 μl) and fungicide-amphotericin-B (750 μl) can also be added. The volume can then be made up to 1000 ml, and the pH will adjust to 7.2–7.4. The medium will then filter under negative pressure using 0.22 μm cellulose filter. Sterility of the medium can be tested before use. Ten

Adherent cell lines will grow in vitro until they form a monolayer over surface area available or medium depleted of nutrients. Adherent cells human oral cancer cells (KB), lung adenocarcinoma (A-549), and breast cancer (MCF-7) can be cultured in tissue culture flasks. The cells were disaggregated by trypsinization and subcultured when the monolayer reaches about 70% confluency. The cells will be cryopreserved at −80°C. With an inverted microscope, the degree of confluency of the cell monolayer can be assessed, and the absence of bacterial and fungal contaminants can be confirmed. Spent medium can be removed. Cells can be washed with PBS-EDTA for removing the traces of serum. Trypsin/EDTA (500 μl) will be applied onto the cell monolayer, and the flask is swirled to cover the monolayer with trypsin. The flask will be incubated at 37°C for 2–3 min. The flask can be examined under the inverted microscope to ensure uniform detachment of the cells. 1–2 ml of medium can be added to the flask as fast as possible to lessen the trypsin-induced stress, and the contents of the flask can be transferred to a centrifuge tube. Cells should be centrifuged at 1500 rpm, for 10 min. The supernatant will be discarded, and the cells were resuspended in minimum volume of medium. Cells can be counted using a hemocytometer, and the required numbers of cells can be subcultured in a new flask containing fresh DMEM with 10% FCS. This process can be

to testing using in vivo animal models [16, 17].

percent FCS can be mixed with the medium prior to culture.

repeated as demanded by the growth characteristics of the cell line.

In general terms cultures derived from blood (e.g., lymphocytes grow in suspension) cells may be seen as single cells or clumps. For these types of cell lines, subculturing is done by dilution in small volume of media before counting. The culture can be viewed using an inverted phase contrast microscope; cells growing in exponential phase should be bright, round, and refractive. Cell suspension can be mixed well and dispersed uniformly by repeated pipetting in order to make single-

cells can be seeded to a fresh

**2.3 Maintenance of cancer suspension cell lines**

cell suspension. The cells can be counted, and 1 × 106

**2.2 Maintenance of adherent cancer cell lines**

**2.1 Preparation of culture media**

**104**

bottle containing 10 ml of DMEM medium with 10% FBS antibiotics and incubated at 37°C and subcultured every third day. On the day of the experiment, single-cell suspension will be prepared. The cells will be counted and the viability can be checked. The concentration can be adjusted with the medium containing 10% FBS and antibiotics.

## **2.4 Establishing cell cultures from frozen cells**

About 10 ml of growth medium placed in a 15-ml conical tube. Thaw the frozen cryovial of cells for 40–60 s by gentle agitation in a 37°C water bath. Remove the cryovial from the water bath, and decontaminate the cryovial by immersing it in 70% (v/v) ethanol at room temperature (RT). Transfer the thawed cell suspension to the conical tube containing 10 ml of growth medium. Cells can be collected by centrifugation at 2000 rpm for 5 min at RT. Remove the growth medium by aspiration, and then resuspend the cells in the conical tube in 5 ml of fresh growth medium. Add 10 ml of growth medium to a 75-cm2 tissue culture flask, and transfer 5 ml of cell suspension to the same. Place the cells in a 37°C incubator at 5% CO2. Monitor cell density daily. Cells can be passaged when it will attain 50% confluency.
