**2. Primary cell culture**

Primary cell culture is a gold standard testing platform for *in-vitro* research in oncology as they reflect the tumor state more accurately compare to most commonly employed cell lines [20]. It is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment [21]. Cell culture studies have made positive contributions to the initialdevelopment of drugs. Contrary to animal studies *in vitro* method requires low drug dose and short response time, which is characteristic feature of in vitro cell culture methods [22]. Primary cell culture is also called as *ex-vivo*. Because primary cells are directly taken from tissue origin and cultured under favorable conditions hence it is more similar to the *in-vivo* state and exhibit normal physiology. It maintains the cross talk between malignant and healthy components [23]. This is the main reason why primary cell culture is called as excellent model system to carry studies in metabolic, aging, signaling studies and effects of drugs, toxic compounds in the cell.

Primary cells are non-immortalized and non-transformable as they imitate the appearance of living model and hence these can help to model 3D tissues. In this culture, cells will grow in 3D aggregates and presents interesting application [24]. It helps for detection, isolation, growth and developmental stages of viruses and helps to analyze the mode of infection. Drug candidate and its toxicity screening rely on results of early-stage *in vitro* cell based assays. Particularly in pharmaceutical industry primary cell culture is used to synthesize verity of biomolecules in high scale, various research project on cell-based product are developed. It is alternative for animal model to test effect of drugs and cosmetics [25]. There are few technical hurdles associate with primary cell culture. For instance, culturing might be difficult if the quality of the surgical material is poor. Also, due to early onset of cell senescence, difficulty arises to maintain sufficient number of cell passages but researchers have made many attempts to resolve this problem [26, 27].

Primary cell culture has been subdivided in to adherent cell culture and suspension cell culture. In the adherent cell culture, cells are arranged in monolayer and attach to the surface of the culture flask. Adherent cells are usually derived from tissues of organs. Growth is limited to surface area and it needs to be detached from the surface before cells get sub-cultured. Viral vaccine, gene therapy and cell therapies depend only on adherent cell culture. Suspension cells are derived from the peripheral blood and do not require any attachment for growth. They do not get attached the surface area of the cultural flask. The cells are free floating and growth is limited to the concentration of cells. The steps involved in the primary cell culture are represented in (**Figure 1**).

**Figure 1.** *Flowchart showing the process of primary cell culture technique.*
