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

survivability due impairment of membrane integrity. Cell death can ultimately be induced by

and thawing of cells, bad cryopreservation techniques, production of toxic metabolites in the

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

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 hypochlo-

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

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

rite (bleach) and autoclaving can all be employed depending upon the material.

, repeated freeze

several parameters such as fluctuation in the conditioning temperature, CO<sup>2</sup>

culture media etc. [6]. Thus, proper handling and care are vital to cell culture.

that involve physical contact with the culture.

preferred for sensitive cells.

100 Cell Culture

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

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 counting slide, has been conventionally used to count cells.

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 corresponding to the number of live cells.

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 as insoluble precipitates inside the cell. Cells are lysed, and these crystals are solubilized through combinations of various reagents such as detergent, DMSO, SDS, acidified isopropanol, dimethylformamide, etc. Absorbance readings are then documented, where maximum absorbance is proportional to higher survival. There are some negatively charges tetrazolium dyes such as MTS, XTT, and WST-1. These do not readily penetrate the cell. However, they can still be used for the assay by incorporating an intermediate electron acceptor.

morphology. This blue fluorescent dye is membrane permeable and binds to DNA. Hoechst staining has also been successfully used to track changes in the nuclear integrity, thus documenting the progress of cell fate. Necrosis, apoptosis and cell enlargement have been successfully shown as consequences of different nanoparticle exposure in various cell-based studies. Phalloidin, a mushroom toxin, with a high affinity for F-actin can be used to track changes in actin dynamics [21]. Fluorescent probes are bound to phalloidin and thus help visualize the actin networks. Gap junctions in neuronal cells can be imaged by using biotinylated dextrans. Biotinylated dextran amines can be introduced in neural cell cultures by pressure injections [22]. Further they can be visualized by avidin conjugated horse radish peroxidase with a

*In Vitro* Toxicity Testing of Nanomaterials http://dx.doi.org/10.5772/intechopen.80818 103

Biocytin hydrazide, an aldehyde-based fixative, is another transneuronal tracer. It detects glycoconjugates and can be used to trace neuronal projections and visualize gap junctions. Biocytin hydrazide [23] in turn is detected by a fluorescent dye conjugated to streptavidin. Cholera toxin subunit B is a protein commonly used to retrograde tracer [24]. It binds to glycosphingolipids in axonal membranes [25]. It can also be used to visualize retinofugal projections. Subunit B is non-toxic and aids internalization and transport of conjugates. Wheat germ agglutinin (WGA) is a lectin protein that binds to N-acetyl-D-glucosamine and sialic acid [26]. Neurons can endocytose WGA-HRP, thus they can be used as tracers as they cross through synapses. Isolectins from legumes *Griffonia simplicifolia* can be used to differentiate between neuronal subtypes [27]. The A subunit prefers N-acetyl-D-galactosamine end groups while the B subunit is selective for terminal a-D-galactosyl residues. Its therefore used as a vascular stain for study of adult neurogenesis. Carbocyanine is a lipophilic dye used to stain plasma membranes [28]. They are highly fluorescent in lipid bilayers while being weakly fluorescent

metal enhanced diaminobenzidine reaction.

in aqueous phase providing a strong contrast for visualization.

illuminator using ethidium bromide staining.

**6. Evaluation of key signaling pathways in studying cell fate**

Simple but conclusive evaluation of changes in expression of molecules is to conduct a reverse transcriptase polymerase chain reaction (RT-PCR) and carry out a western blot analysis. RT-PCR documents the changes in mRNA (transcriptional) levels, while western blot records changes at the protein (translational) level. Cells exposed to nanoparticles are harvested and thoroughly washed before downstream processing. RNA extracted is converted to cDNA and used as a template for PCR. Appropriate primers are designed for target amplification. Amplicons from the PCR are resolved using agarose gel electrophoresis and visualize by a UV

For western blot analysis, protein is carefully extracted. Whole cell lysate (at least 40 μg) is resolved by SDS-PAGE [29]. Western blot is made by electrophoretic transfer on to membranes. Following transfer membranes are blocked with either non-fat dried milk or BSA. Overnight incubation with primary antibody is usually allowed. Post this, appropriate incubation is carried out with secondary antibody. Number of washes after each step, antibody dilution and incubation time all need to be optimized. Horse radish peroxidase conjugates are commonly

Resazurin (7-hydroxy-10-oxidophenoxazin-10-ium3-one) is particularly used to assay for mitochondrial dysfunction. Resazurin, a deep blue colored complex is reduced to resorufin, a pink colored complex by the enzymes in the inner mitochondrial membrane. Use of resazurin is inexpensive and more sensitive than using tetrazolium dyes.

ATP cell assay is yet another type of evaluation for cell viability. It overrides any incubation with live cell population. It is quicker, more sensitive and less prone to artifacts. Cells with damaged membranes cannot synthesize ATP and endogenous ATPases rapidly depletes cellular ATP concentration. Cells are lysed through detergent activity for this assay, in the presence of ATPase inhibitors to stabilize the total ATP content. Firefly or shrimp derived luciferase acts on substrate luciferin to generate light in the presence of ATP. Higher intensity of light is proportional to the number of live cells.

Sulforhodamine B (SRB) is a fluorescent dye [14]. It is bright pink in color. This aminoxanthene dye binds to cellular protein under mildly acidic conditions. Extraction in a basic environment is proportional to cell mass and thus an indicator of cell viability. Clonogenic cell survival assay investigates the cell's capability for propagation [15]. Treated sample of cells are plated and colonies stained and counted. DNA synthesis cell proliferation assay involves incubating cells with 3H-thymidine. Proliferating cells incorporates this radioactive tracker. Proliferation and thus cell viability can be measured by a scintillation counter. A non-radioactive alternative such as 5-bromo-2′-deoxyuridine (BrdU) can also be implemented. Although this includes an additional step of binding with BrdU-specific antibody and probably a secondary antibody before the colorimetric estimation [16]. A developed 5-ethynyl-2′-deoxyuridine (EdU) can be detected by a fluorescent azide through a Cu(I)-catalyzed cycloaddition reaction. This fast and sensitive method has additional advantages over BrdU assay [17], which are lack of sample fixation and preservation of DNA structure [18]. Raman micro spectroscopy detects variations in Raman bands associated with O–H stretching in water [19]. These variations can non-destructively correspond with ionic concentrations in the intracellular and extracellular fluids. This can be useful in determining the rate and direction of ionic transport. Loss of membrane integrity is dead and dying cells are often associated with leaching of ions, which can be detected and quantified by this technique.
