**4. Cytotoxicity assessment**

Cytotoxicity assessment is an essential component of the evaluation process for pharmaceutical agents, medical devices, and other substances that may come into contact with living cells or tissues. It involves the study of the potential harmful effects of these substances on cells, including cell damage and cell death. Cytotoxicity is the degree to which a substance can cause damage to cells. Assessing cytotoxicity is crucial for ensuring the safety and effectiveness of medical devices and pharmaceutical agents, as well as other substances that may come into contact with living cells or tissues. Understanding the cytotoxic effects of these substances helps researchers identify potential hazards and optimize the design and formulation of products to minimize the risk of adverse effects on the human body.

Moreover, cytotoxicity assessment is a valuable tool in various other aspects of biomedical research, such as the investigation of the mechanisms of cell death and the identification of novel therapeutic targets. By studying the cytotoxic effects of specific compounds or treatments, researchers can gain insights into the biological processes involved in cell damage and death, which may contribute to the development of new therapeutic strategies for a wide range of diseases and conditions.

#### **4.1 Applications of cytotoxicity assessment**

Cytotoxicity assessment has a wide range of applications in biomedical research and drug development. Some key applications include:

*Evaluating the safety of medical devices and pharmaceutical agents:* Cytotoxicity assays can be used to determine the potential harmful effects of medical devices and pharmaceuticals on living cells, ensuring that these products do not cause unacceptable levels of cell damage or death.

*Screening for potential therapeutic agents:* High-throughput cytotoxicity assays can be used to screen large libraries of compounds for their ability to selectively kill cancer cells or other target cell populations, facilitating the identification of novel therapeutic agents.

*Investigating the mechanisms of cell death:* By studying the cytotoxic effects of specific compounds or treatments, researchers can gain insights into the biological processes and signaling pathways involved in cell damage and death. This knowledge can contribute to a deeper understanding of the mechanisms underlying various diseases and conditions, as well as the development of new therapeutic strategies.

*Evaluating the efficacy of therapeutic interventions:* Cytotoxicity assessment can be used to measure the effectiveness of therapeutic interventions, such as chemotherapy or radiation therapy, in inducing cell death in target cell populations. This information is crucial for optimizing treatment regimens and developing more effective therapeutic strategies.

*Assessing the potential toxicity of environmental contaminants:* Cytotoxicity assays can be employed to evaluate the potential harmful effects of environmental contaminants, such as pollutants, pesticides, and industrial chemicals, on living cells. This information is vital for understanding the risks associated with exposure to these substances and developing strategies to minimize their impact on human health and the environment.

Cytotoxicity assessment is an indispensable tool in the evaluation of medical devices, pharmaceutical agents, and other substances that may come into contact with living cells or tissues. The various methods available for measuring cytotoxicity offer researchers a range of options for assessing the potential harmful effects of these substances on cells and for investigating the mechanisms of cell damage and death.

#### **4.2 Methods for measuring cytotoxicity**

There are numerous methods available for measuring cytotoxicity, each with its advantages and limitations. Some of the most commonly used methods include:


#### **4.3 MTT assay**

The MTT assay, also known as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, is a widely used colorimetric method for assessing cell viability and cytotoxicity [12]. This assay measures cellular metabolic changes using colorimetric shifts. It is based on the conversion of the purple tetrazolium dye MTT into insoluble formazan by the nicotinamide adenine dinucleotide phosphate (NADPH) dependent cellular oxidoreductase enzymes. The reductive activity occurring in the mitochondria of living cells is employed to assess cell viability [6]. The MTT assay quantifies live cells by gauging mitochondrial activity, as it correlates with the number of formazan crystals [13]. Despite being the gold standard for cytotoxicity testing, the conversion to formazan crystals is influenced by various factors like metabolic rate and the number of mitochondria [14].

The MTT assay is based on the idea that proliferating cells exhibit a higher rate of MTT conversion, while nonviable or slow-growing cells have reduced metabolism and lower MTT reduction levels. After MTT application, formazan crystals are dissolved in a solution containing dimethyl sulfoxide or sodium dodecyl sulfate. Formazan concentrations can be measured using a spectrophotometer between 540 and 720 nm [15]. This method can provide an accurate dose-response curve for small cell numbers, test multiple parameters simultaneously, and is straightforward and highly replicable. The MTT assay is primarily used for in vitro testing of cytotoxic effects of various novel drugs at different concentrations and evaluating drug resistance in cell lines. It also assesses in vitro drug effects and their potential clinical applications. Due to its simplicity, the MTT assay is widely used to determine the toxicities of polymers, alloys, and ceramics [16]. However, the assay does not differentiate between cytostatic and cytotoxic effects, and results may be inaccurate if the cell population is low [17, 18]. Additionally, the MTT test is cell-specific and requires solubilization. Although highly sensitive, it works only with adherent cell targets. Since all cells must be killed during the protocol, this assay cannot be used for follow-up studies.

The MTT assay is relatively easy to perform, requiring only the addition of the MTT reagent to cell cultures, incubation, and subsequent solubilization and quantification of the formazan product. Non-destructive: As the MTT assay measures cell viability indirectly through the reduction of the MTT reagent, it does not require the destruction of cells or the use of invasive techniques.

#### **4.4 AlamarBlue assay**

The AlamarBlue assay offers a straightforward and dependable approach to measuring cell viability. It employs a fluorometric technique to detect cellular metabolic activity. Mitochondrial enzymes with diaphorase activity, such as NADPH dehydrogenase, reduce resazurin (oxidized form; 7-hydroxy-3H-phenoxazin-3-1-10-oxide) to resorufin (reduced form) [19]. The AlamarBlue assay has been utilized to examine trophoblast viability, migration, and invasion [20].

Cell viability can be assessed in 96-well plates after exposure to the biomaterial being studied. This test offers several advantages [19]. It is a straightforward method that uses a water-soluble substance, applicable to both suspended and attached cells, and features a fluorometric and colorimetric growth indicator [21]. Furthermore, the reagents are harmless to both cells and technicians. This test eliminates the necessity for washing and extraction steps, allowing for easy differentiation of endothelial cell viability and cell concentrations. It is a cost-effective test that enables continuous monitoring of endothelial cell metabolism and viability [22]. However, the reduction process may reverse with high cell numbers and extended culture times. One limitation of this assay is that it is not a direct cell counting technique. The assay relies on metabolic pathways that can be influenced by various factors, such as individual cell-reducing capacity and agents that affect mitochondrial activity or directly reduce resazurin [23].

#### **4.5 LDH (lactate dehydrogenase) release assay**

The LDH (lactate dehydrogenase) release assay is a widely used method for evaluating cell viability, cytotoxicity, and membrane integrity in various cell types. LDH is an intracellular enzyme that is released into the extracellular environment upon cell membrane damage or cell lysis. Lactate dehydrogenase release assay is a two-step rapid colorimetric test to assess the quantification of cell numbers in vitro [24]. The principle of the LDH release assay is based on the conversion of lactate to pyruvate by LDH in the presence of a cofactor, NAD+ (nicotinamide adenine dinucleotide). During this process, NAD+ is reduced to NADH, which then reacts with a specific tetrazolium salt, producing a colored formazan product. The absorbance of the formazan product can be measured using a spectrophotometer or microplate reader, with the intensity of the color directly proportional to the amount of LDH released and, consequently, the number of damaged or non-viable cells. Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme in every cell. Cytotoxicity is assessed by the activity of cytoplasmic enzymes released by damaged cells [25]. LDH is released into the cell culture when there is apoptosis, necrosis, or other cellular destruction in the membrane [26]. This test can detect the cytotoxic effects of various agents or environmental factors [27]. In the first stage, LDH catalyzes the conversion of lactate to pyruvate by reducing NAD+ to NADH. Following this, diasphorase enzymes reduce the tetrazolium salt to a red formazan in the presence of NADH.

Colorimetric lactate dehydrogenase (LDH) assay has also been used for the evaluation of antiviral activity against bovine viral diarrhea virus in vitro. Using the NADH produced during the conversion of lactate to pyruvate to reduce a second compound in a coupled reaction into a product with easily quantifiable properties makes it simple to quantify LDH activity. In this assay, the reduction of a yellow tetrazolium salt, INT, by NADH into a red, water-soluble dye of the formazan class is measured using absorbance at 492 nm. The amount of formazan is proportional to the amount

of LDH in the culture, which is proportional to the number of dead or damaged cells. The advantages are that LDH assay reflects the membrane integrity, and the reagent does not damage viable cells. The drawback is that it is not super sensitive. Despite these advantages, there are some limitations to the LDH release assay.

#### **4.6 MTS assay**

The MTS assay is utilized to evaluate cell proliferation, cell viability, and cytotoxicity. It can determine cell viability after exposure to various cytokines, growth hormones, cytotoxic drugs, and anticancer agents [28]. The MTS assay can also be used to assess the effects of chemical and physical treatments on the biocompatibility of human bone and tendon tissues for clinical applications [12]. The test's principle is that a colored formazan dye is generated when MTS tetrazolium molecules are reduced by live mammalian cells and other species' cells.

Living cells' mitochondrial reductase enzymes convert MTS to formazan crystals in the presence of phenazine methosulfate, an electron-coupling agent. These reduced formazan crystals are water-soluble, eliminating the need for an additional solution or washing step to dissolve them in the cell culture medium [29]. A spectrophotometer can measure these formazan crystals at 490–500 nm. The MTS reagent solution has better storage stability compared to MTT or XTT molecules. One advantage of tetrazolium assays that yield a water-soluble formazan is the ability to periodically measure absorbance from the test plates during the initial incubation stages. Multiple readings may be useful during assay development, but it is crucial to keep the plates in the incubator between readings to maintain a relatively constant environment [30].

#### **4.7 XTT assay**

The XTT (2,3-bis-(2- methoxy-4- nitro-5-sulfophenyl) -2H-tetrazolium −5-carboxanilide) assay is similar to the MTT assay but relies on the reduction of the XTT reagent to a soluble orange formazan product. This allows for a simpler and faster assay, as the formazan product can be directly quantified in the cell culture medium without the need for solubilization. The colorimetric change indicates cell viability, proliferation, and cytotoxicity through a nonradioactive test [31]. The biomaterial to be evaluated is placed in 96-well microplates and an adherent or suspension cell culture. Metabolically active cells reduce yellow tetrazolium salt (sodium 3′-[1- (phenylaminocarbonyl)- 3,4- tetrazolium]-bis (4-methoxy6-nitro) benzene sulfonic acid hydrate or XTT) into an orange formazan dye.

A scanning multiwell spectrophotometer (ELISA reader) measures formazan dye. XTT assay can assess cell proliferation when exposed to growth factors, cytokines, and nutrients. It can measure the increase in the overall activity of mitochondrial dehydrogenases that corresponds to the increase in the number of living cells and the amount of orange formazan formed. Cytotoxicity can also be measured using XTT assay by measuring the cytotoxic or growth-inhibiting agents such as inhibitory antibodies [32]. This assay is compatible with resorbable and nonresorbable guided tissue regeneration membranes in cultures of primary human periodontal ligament fibroblasts and human osteoblast-like cells [33]. Unlike MTT, where cells must be lysed to solubilize the formazan salt before absorbance measurement, XTT does not require cell lysis. This allows easier monitoring of the same samples at different time intervals [32].

#### **4.8 WST-1 assay**

The WST-1 assay (Water-Soluble Tetrazolium Salt-1) is a colorimetric method used to evaluate cell viability, cytotoxicity, and proliferation. It is based on the reduction of a tetrazolium salt, WST-1, to formazan by cellular dehydrogenase enzymes present in metabolically active cells. The formazan dye produced is directly proportional to the number of viable cells, and its absorbance can be measured using a spectrophotometer [34]. WST-1 assay is an improved version of the MTT assay and offers several advantages. Unlike the MTT assay, which requires solubilization of the formazan crystals in a separate step, the WST-1 assay generates a water-soluble formazan product. It is water-soluble, quick and sensitive [35]. This feature simplifies the experimental procedure and allows for the absorbance measurement without the need for additional steps or cell lysis. As a result, it is possible to measure cell viability more quickly and monitor the same cell samples at multiple time points.

The WST-1 assay is widely used in various applications, such as assessing the cytotoxic effects of drugs, chemicals, or nanoparticles on cell lines, screening for potential anticancer agents, and testing the biocompatibility of biomaterials. The assay is performed by adding the WST-1 reagent to cell cultures in a 96-well plate format, followed by incubation for a specific period, usually ranging from 1 to 4 hours. The absorbance of the formazan dye produced is then measured at a wavelength of around 450 nm using a microplate reader. One of the limitations of the WST-1 assay is its sensitivity to environmental factors and culture conditions, such as pH and serum concentration [35]. WST-1 assay is unsuitable for assessing the cell toxicity of Mn-containing materials in vitro [35]. It is essential to optimize the experimental conditions and maintain a consistent environment during the assay to obtain reliable and accurate results. Additionally, the WST-1 assay measures metabolic activity rather than directly counting the number of viable cells, so it may not always accurately reflect the true cell viability, especially in cases where metabolic activity is altered by the experimental treatments. Despite these limitations, the WST-1 assay remains a popular and convenient method for assessing cell viability, cytotoxicity, and proliferation due to its simplicity, speed, and compatibility with various cell types and experimental conditions.

#### **4.9 ATP assay**

The ATP assay is a sensitive and reliable method for evaluating cell viability, metabolic activity, and cytotoxicity in various fields of biomedical research. It is based on the quantification of adenosine triphosphate (ATP), the primary energy currency of living cells, which serves as an indicator of cell health and metabolic activity. The ATP assay is centered on the detection and quantification of intracellular ATP levels, which directly correlate with the number of viable, metabolically active cells in a given sample. The most common method of ATP quantification involves the use of a bioluminescent enzyme, luciferase, which catalyzes the oxidation of luciferin in the presence of ATP, producing light as a byproduct. The emitted light is then measured using a luminometer, with the intensity of the luminescent signal being proportional to the ATP concentration and, consequently, the number of viable cells [36].

The ATP assay is highly sensitive, capable of detecting even small changes in ATP levels and cell viability, making it suitable for assessing the effects of various substances on cellular metabolism and health. The ATP assay provides rapid results, often within minutes, allowing researchers to quickly assess cell viability and metabolic activity in response to various experimental conditions. The ATP assay is a powerful tool for
