**5. Culture media**

Cell pellets collected from centrifugation has to be placed in micro well plate or flask that contains culture media. It provides artificial environment for cell to grow. Basic requirement of culture media are controlled temperature, substrate to attach cell, growth medium and incubator to maintain pH [45]. Main step in culture is to choose culture media. It generally composed of amino acid, vitamins, inorganic salts, glucose, hormones, growth factor, and attachment factor which provides energy and helps to complete the cell cycle. Commercially available cell media for primary epithelialcancer cells are less effective compare to tissue specific primary cell media prepared in lab (**Table 6**) [46–49].

Choice of culture media is very important to get significant result in experiment. Selection of media completely depends upon type of cell, purpose and resource [62].


#### **Table 6.**

*Components of media from different studies.*

*Overview of Primary Cell Culture Models in Preclinical Research of Prostate and Bladder Cancer DOI: http://dx.doi.org/10.5772/intechopen.99493*


#### **Table 7.**

*Commercially available media for epithelial cells [44, 63].*

As primary culture provides valuable research data, preparation of quality culture media is required, or to avoid limitation (cell number) of primary cell culture, commercially produced medias are available (**Table 7**) [58–61].

It is very important to maintain cell viability after isolation process which is totally depends on skillful handling and culture conditions. The culture condition will differ depending on the cell type. Cell growth has to be observed till 11 or 12 days. Additional extra media, Fetal Bovine Serum (FBS) and antibiotics need to be provided to avoid contamination. Culture media has to be changed between 2 and 3 days [60]. Initially apoptosis is 5% from 0 to 1 day but as days will pass apoptosis rate will increases from 7 to 14 days. But functional validity of benign and prostate cancer cells was 5 days after confirming it with histochemically, biochemical and by immunohistochemical assay [63]. Use of serum free culture media with low calcium condition increases the longevity of the cell. Cryopreservation (Preservation of structurally intact cells) can be achieved by adding 10% FBS (Fetal bovine serum) and 10% DMSO (Dimethyl sulfoxide) in 80% confluence primary cell culture [64].

Each day morphological changes have to benoted. Normal cells get counted every day and cancer cells get counted every 2 days [65]. Cell viability is determined by trypsin blue dye, equal volume of PBS and trypsin blue dye allowed to sit on cells for few minutes then to count the cells samples are loaded on hemocytometer, cells scored as leaving or dead based on uptake of tryptophan blue dye [66]. Once confluence reaches to 80 to 90% it has to get counted by Neubauer camera at 1:2 dilution with tryptophan dye exclusion, MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) can be used to determine cell viability [67]. Cell growth curve can be plotted from the graph to check the time when the cell viability increases in the culture. Once cell get cultured properly depending upon need of investigator, cells can be passaged and characterization of the cells can be done.
