**4.14 Cell harvest**

Knowledge of splitting, media, trypsinization, cell handling is essential to harvest cells. Firstly, remove and discard the media from CCF. For 75 cm<sup>2</sup> CCF, mild PBS wash is required before the application of warm trypsin or trypsinization process. Add trypsin (2–4 ml/75 cm2 ) then incubate as like trypsinization process. When detached cells appear then add 2–5 ml growth media (GM) to inactivate trypsin. Gently pipette to disperse the medium to ensure recovery of >95% of cells. Sometimes commercial trypsin inhibitor is added. Carefully centrifuge the collected cell suspension at 300–1000 X g for 5–10 min. Discard the supernatant and add GM at the required amount for the preparation of cell count. Split the cells after counting or go for further processes that need. There is a lot of problems that may be appeared like detachment difficulty of cells from culture flasks, cell adherence difficulty, insufficient attachment of cells, low viability of cells, clumping after detachment, damage of cell membrane, and cell death. The possible solutions to the above problems will be careful during the following such as a). Check the quality, date, and concentration of trypsin before use b). Be careful during antibiotic application if any c). Splitting, media replacement required before harvesting if cells are in stress d). Avoid vigorous pipetting and long centrifugation.

#### **4.15 Antibiotics**

Routine cell and tissue culture according to good cell culture practice (GCCP) [33] should not require the use of antibiotics as they can never be relied on as a substitute for effective aseptic techniques. However, its use is still widespread e.g., OECD TG 432 [34] due to established routine procedures in many laboratories. Antibiotics are agents that may arrest or disrupt fundamental aspects of cell biology, and, while they are effective against prokaryotic cells (i.e. Bacteria), they are also capable of causing toxic effects in animal cells. Not surprisingly, antifungal agents, being directed at higher order, eukaryotic microorganisms, are likely to be more toxic to animal cell cultures. In addition, antibiotics often make it more difficult to detect microbial contamination. These obvious contraindications, the use of antibiotics in cell and tissue culture should be focused in two areas: a) Protection of materials at high risk of contamination such as tissues, organs, and primary cultures in cases where sterility cannot be guaranteed, and b) The positive selection of recombinant cell clones based on the expression of antibiotic resistance genes [33]. If antibiotics are needed, a justification for the use of antibiotics in the procedure is suggested.

#### **4.16 Laboratory management**

Good laboratory setup is essential for cell culture as well as good laboratory practice is also essential for better and smooth work. The following discussion and points are very much important for a cell culture laboratory and are also supported by Maneesha et al. [35] and Coecke et al. [36].

Location and ideal layout with the purpose-built facility is the first priority. Room data sheet (RDS) in which available all the data of every facility, specification of the room define its location, a number of doors, windows, pass box, ventilator, light, air conditioner, fire alarm like everything present on that room permanently. Specification of instruments must be available at the working area that helps the laboratory personnel to operate it. Standard operating procedure (SOP) helps to do research/ laboratory work in a defined way to get a better outcome. Define the area of a room with a specific class and biosafety level by the standard of ISO, GMP. Define or specify works of laboratory personnel with defined working areas according to CDC, NIH-USA also beneficial for a good outcome. There are four biosafety levels based on hazard or pathogenicity or virulence of microorganism and toxicity of agents. The basic biosafety level known as biosafety level 1 (BSL-1). In this level generally very common research work done with easy protection. Normally in BSL-1 working with those organisms or agents which are not harmful to healthy person. In biosafety level 2 (BSL-2) working with those organisms or agents which is known as moderate-risk agents, and known as a potential threat to human resulting produce disease (by ingestion or through percutaneous or mucous membrane exposure) of varying severity. Generally, cell culture should be performed at BSL-2 laboratory. Sometimes the biosafety level depends on the type of cell line and working style. In biosafety level-3 (BSL-3) working with that agents which have the capability to transmit through air (aerosol transmission). BSL-3 agents may be indigenous or exotic, having potential threat to human, may cause serious health issue, and may be potentially lethal. Biosafety level-4 (BSL-4) deals with exotic agents that create life-threatening disease of an individual through infectious aerosols and for which no treatment is available. These organisms or exotic agents are restricted to high containment laboratories. The easily accessible facility of the emergency shower should be available in the laboratory area. Access

## *A Brief Concept of Cell Culture: Challenges, Prospects and Applications DOI: http://dx.doi.org/10.5772/intechopen.99387*

control in laboratories should be helpful to maintain unwanted occurrences. A separate logbook is very much helpful for liquid nitrogen and CO2 management. Ventilation and pressure control like negative and positive pressure controlled area must be defined, HEPA filtered providing positive pressure to clean areas, is recommended where space and resources allow. Electricity room (uninterrupted power supply (UPS) units should be provided for essential equipment (class II cabinets, incubators, air filtration) and to allow cell culture procedures to be completed. Accountability for all the staff will provide a smooth working environment. Documentation, Training (Fumigation, 5 s, SOP, etc.) and Monitoring of staffs, Emergency service provider contract with the third party are essential for good laboratory management. Before receive and entering all the reagents, chemicals, and supplier's documents must be checked by maintaining a logbook. For the management of inventory and documents should be established hard copy or electronic form that stored the information of materials, cells, suppliers, overall all the possible information. Staff safety is a vital issue. The primary concerns regarding safe management of liquid nitrogen storage are frostbite burns from skin contact with liquid nitrogen and asphyxiation due to exposure to low oxygen levels when nitrogen gas is released from vessels. Finally, all service personnel entering the laboratory should receive instruction in special laboratory hazards and any necessary procedures for working in clean areas e.g. gowning, hand disinfection. All the person needs to use a separate biohazard bag/bin to ensure safe hazard management. Like-Infectious non-sharp waste (incineration/deep burial)-Yellow bag; Plastics and sharps (chemical treatment/autoclaving/shredding/microwaving)- Blue bag; Infectious nonsharp waste (chemical treatment/autoclaving/microwaving)-Red bag; Incineration ash and solid chemical wastes (secure landfill)- Black bag.
