**3. Cell culture practices**

Benefits of nanoscale materials are numerous and the market for nanotechnology is enormous owing to the large turnout for profits. The only ethical way forward to manage the harmful effects of nanoparticles is in educated design with innovations, applications and in ethical disposal of nanowastes [2]. Understanding the mechanisms of toxicity attributed by nanoparticles is crucial to this endeavor. Some tools to assess dose and time dependent toxicity have already been developed. Toxicity can either be directly- cell death or the impairment of normal functions in the cell. There are different ways to test toxicity as there are different

Conventionally 'acute toxicity testing' has been carried out on a model organism, with each candidate, being tested for a single dose and a single exposure time. Following the exposure, biochemical and histological changes observed from different tissue samples of the dead animal were documented for analysis. The determination of LD50; administration dose with 50% lethality thus required the sacrifice of many animals. LD50 has long been used the comparative standard in assessing the degree of toxicity. Several skin sensitivity tests and lymph analysis have also been performed to assess immunogenic potential of an exposing agent. There are other function-based toxicity tests to study effects on reproduction, mutagenic potential, neu-

The advantage of cell-based toxicity assays is that large numbers of experiments can be conducted to screen the exponential dose-time combinations of exposure [3]. It is greatly time and cost effective as compared to *in vivo* testing. And ethical concerns of animal sacrifice and need for elaborate and regulated laboratories are avoided. No doubt, *in vitro* and *in vivo* results tend to vary depending on the case study, but the wider use of *in vitro* studies allows for only a fraction of the most promising outcomes to be further evaluated with live animal testing. Again, this alleviates many concerns associated with the using animal testing as the

Cell culture broadly denotes maintaining cell population, enabling both growth and propagation of cells. There are different types of cell culture methods. The primary culture involves desegregation of cells from the mother tissue by application of enzymatic or shear processes. Cells are transferred to a sterile system with favorable media for growth. This is usually done in glass or plastic containments such as flasks, petri plates, dishes and so on. Primary cultures are often heterogeneous, that is they are a mixed collection of different cell types that are present in the source tissue. Depending on the application requirements, the mixed pool can directly be considered for a study or the different cell populations may be identified by examining biomarkers and further sorted to obtain a culture of a single type of cells. Primary cultures are often further classified into adherent and suspension cultures. Adherent cultures are anchorage dependent [4]. They require surface support for normal proliferation. Adherent cultures are grown in containers coated with a basal polymeric protein matrix such as lysine. When normal cells are isolated for a period, detached from the surrounding extracellular matrix, it leads to growth arrest and even the induction of anoikis. The cell-cell contact [5]

routes of exposure to the toxic substance.

98 Cell Culture

ral management and embryonic development.

first line of investigation.

**2. Types of cell culture**

All cultures face challenges of contamination through contact and lack of appropriate sterility techniques. Bacterial and fungal contaminations cloud the culture and shift the pH. pH imbalances also occur due to presence of incorrect salts, bicarbonate buffering and gaseous tension. pH changes may at times result in media precipitation, although this may also be the case for contamination with detergent phosphate used for cleaning the culture vessels and equipment. Contamination with magnesium and calcium ions can lead to cell clumping and lysis particularly in a suspension culture. Increased duration of enzyme treatment such as trypsin not only results in subsequent cell adherence issues but may adversely affect cell's survivability due impairment of membrane integrity. Cell death can ultimately be induced by several parameters such as fluctuation in the conditioning temperature, CO<sup>2</sup> , repeated freeze and thawing of cells, bad cryopreservation techniques, production of toxic metabolites in the culture media etc. [6]. Thus, proper handling and care are vital to cell culture.

are within the recommended shelf life. All equipment need to be well calibrated and safety cabinets need to be tested for efficacy. Water baths need to be routinely cleaned to avoid contamination. Always sterile water needs to be used in water baths. All work surfaces need to be free of clutter. There must be minimal cardboard packaging if at all required. A splash

*In Vitro* Toxicity Testing of Nanomaterials http://dx.doi.org/10.5772/intechopen.80818 101

Nanoparticle formulations to be administered in *in vitro* experiments need to be prepared with care. To avoid inhaling aerosolized nanoparticle, an appropriate pollution mask needs to be worn while handling nanoparticles. It is best to sonicate or vortex and add nanoparticles to allow for dispersion in the nanoscale. This process is called charging. If charging is not done properly, nanoparticle may aggregate and present themselves as micro range particles to the experimental set up. Another precaution to avoid aggregation is to use stock solutions with least possible nanoparticle concentration. Incubation with nanoparticles need to be followed with appropriate washing steps to remove as much particles as possible that have adhered to

proof apron and eye protection are to be utilized where necessary.

**nanoparticles**

**4. Cell culture-based assays to evaluate toxicity associated with** 

the cell surface. This prevents interference to the downstream processing of the cell.

counting slide, has been conventionally used to count cells.

corresponding to the number of live cells.

One of the first investigations in understanding the effects of any exposing agent is to conduct a cell proliferation assay. Cellular viability, a synonym term is the number of healthy cells in a sample. Viability monitored as a function of dose and time provides information on cell death and hence is also a measure of cytotoxicity [8]. Different parameters indicate the viability of the cell and thus can be used to quantify the cytotoxicity [9]. Some of the methods used to study nanoparticle exposure on cells are described as follows. The dye exclusion methods are a preliminary test based on permeation of dye in dead or dying cells owing to loss of membrane integrity. Trypan blue, eosin and propidium iodide can all be implemented in the dye exclusion test [10]. Dyes are added to cell suspensions and appropriate volumes are loaded onto a counter to aid determination of live cells. Neubauer hemocytometer, a manual

Another cell viability assessment is through the documenting the metabolic or enzymatic activity of viable cells. These cells convert the substrate to a colored or fluorescent product and as cell death increases, the degree of this conversion also lacks behind. Examples of this type of tests include protease activity assay and reduction of tetrazolium and resazurin salts [11]. The protease viability assay includes the use of glycylphenylalanyl-aminofluorocoumarin [12]. Abbreviated as GF-AFC, it's a recently developed marker. It permeated live cells and is acted upon by cytoplasmic aminopeptidase. This results in cleavage of glycine and phenylalanine amino acid, releasing AFC (aminofluorocoumarin). AFC generates fluorescent signals

MTT [13] (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) a positively charged tetrazolium dye readily penetrates the cell. It is converted to the colored formazan product by specific NADH dependent mitochondrial enzymes. Formazan crystals accumulate

Some common cell culture techniques are exercised to ensure maintaining an aseptic environment for culture [7]. These include routine cleaning of designated rooms and facilities for removal of dust and grease. Use of chlorine and xylenol-based disinfectants for open surfaces is another precaution commonly employed. Decontamination of culture premises by fumigation using potassium permanganate and formaldehyde is carried out as per requirement such as the instances of biological contamination. Steam sterilization of all glass and plastic ware, particularly those used for actual culturing within the biosafety cabinet is undertaken. 70% ethanol or isopropyl alcohol is commonly used to swab hands and wipe surfaces during handling. Use of single open glass flame and sterile tissue rolls also supplements this purpose. Double autoclaved and ion free water is to be used to prepare all sterile solutions. Filtration with at least a 0.22-micron pore sized filter is advised to remove biological contaminants in heat labile substances such as trypsin and antibiotic/antimycotic agents. Use of powder free and sterile oil-resistant gloves is recommended where necessary. The laminar air flow system should house a high efficiency particulate air flow system (HEPA filter). A good HEPA filter must be able to retain and remove at least 99% of particles of 0.3 micron in diameter, suspended in the penetrating air. The maximum speed of the filter should not exceed 0.025 m/s as low speed penetration achieves maximum filtration capacity. As a user dependent precaution, it is recommended, to avoid talking during handling, to prevent generation of contaminant carrying aerosols. Proper planning of experiments routes in better execution. Experiments need to be performed as quickly as possible avoiding all unnecessary steps especially those that involve physical contact with the culture.

Once the experiments are conducted, the right and efficient disposal of culture waste is also equally crucial for ethical reasons. Use of 70% alcohol, isopropyl alcohol, sodium hypochlorite (bleach) and autoclaving can all be employed depending upon the material.

Another crucial step to cell culture is the use of ideal media and storage conditions for preservation of cells. Cryopreservation either by storing vials containing cells in −80°C or liquid nitrogen is commonly followed. Components of freezing media are usually serum and dimethyl sulfoxide. Cryovials can be snap frozen by adding them directly to storage conditions or by gradual incubations with decreasing temperatures. The latter is particularly preferred for sensitive cells.

Some safety measures to be considered while dealing with unauthenticated source is to complete quarantine procedures. Particularly new samples need to be tested for mycoplasma, bacterial and fungal contamination. Unless absolutely required, use of antibiotics is not recommended as they may lead to development of resistant strains and may put stress the cultured cells. Sub culturing needs to be done around 80% confluency, to avoid effects of growth arrest by contact inhibition. Cells need to be routinely frozen and revived. Otherwise a continuous culture that runs for months especially for transformed cells has risks of picking up uncharacterized mutations. Care needs to be taken that all reagents used for cell culture are within the recommended shelf life. All equipment need to be well calibrated and safety cabinets need to be tested for efficacy. Water baths need to be routinely cleaned to avoid contamination. Always sterile water needs to be used in water baths. All work surfaces need to be free of clutter. There must be minimal cardboard packaging if at all required. A splash proof apron and eye protection are to be utilized where necessary.
