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Cytotoxicity - A Crucial Toxicity Test for In Vitro ExperimentsCYTOTOXICITY: AN ESSENTIAL ASSAY FOR DRUGS, C...Edited by Pınar Erkekoglu
From the Edited Volume
Cytotoxicity - A Crucial Toxicity Test for In Vitro Experiments - CYTOTOXICITY: AN ESSENTIAL ASSAY FOR DRUGS, COSMETICS AND MEDICAL DEVICES [Working Title]
Cell lysis and disruption involve breaking down cells through natural processes or induced damage to the outer membrane. This process can occur during apoptosis, which helps detoxify and clear cells. However, when extracting valuable intracellular products from microbial or mammalian cells, controlled lysis techniques are essential. Lysis and cytotoxicity can lead to loss of desired products, toxin production, and complications from exopolysaccharides and endotoxins. While it may aid in detoxification when regulated, uncontrolled cytotoxicity complicates product extraction. To address these issues, researchers are developing novel extraction strategies tailored to the type of cells and the stability of the target products. Methods for cell disruption include physical, biological, and chemical approaches, with physical techniques often favoured in biological applications to enhance product recovery. The current chapter will provide an insight into the recent developments in the field of cell disruption, analysis of cell toxicity, and challenges associated with the phenomena. This chapter explores the various obstacles encountered in the process of cell lysis, a critical step in bioprocessing. This chapter delves into the underlying principles of cell lysis, examines the most common issues faced by researchers, and presents innovative strategies and cutting-edge technologies designed to overcome these challenges.
Centre for Biopharmaceutical Technology, Department of Chemical Engineering, Indian Institute of Technology, Delhi, India
Shoaib Haidar
Centre for Biopharmaceutical Technology, Department of Chemical Engineering, Indian Institute of Technology, Delhi, India
Hare Ram Singh*
Department of Biotechnology and Bio-Engineering, Birla Institute of Technology Mesra, Ranchi, India
*Address all correspondence to: hrsingh@bitmesra.ac.in
1. Introduction
Cell lysis and cell disruption refer to the breakdown of cells by self-induction or induced damage to the outer membrane. The cell lysis is carried out under various conditions, and with time multiple techniques have been developed for cell disruption. Naturally, cell lysis occurs with the cells entering apoptosis whenever there is a need for detoxification and clearance of cells. However, since a number of biological products are being produced using microbial or mammalian cells, cell lysis is performed to get the intracellular product in its crude form [1]. There are numerous challenges researchers face when moving with lysis methods and one of them is cytotoxicity. Cytotoxicity is the effect of internal or external stimuli/environment leading to cell death [2]. The result of cytotoxicity is loss of product, production of toxins, and availability of exopolysaccharides and endotoxins. The cytotoxicity leads to multiple complications because of cellular material on availability and stability of the desired product. While cytotoxicity leads to clearance of toxins and when in controlled form is good as a natural maintenance of cellular systems, its induction and uncontrolled nature may lead to serious complications while extracting a desired product [3].
Cytotoxicity is one issue related to cell lysis while the procedures performed for inducing cell lysis also creates many barriers to the extraction of the desired product. With the development of novel strategies for the extraction of intracellular products, the focus has been to develop strategies keeping in mind the recovery of the desired product. Depending on the type and class of cells and the sensitivity, and stability of the material, the strategy is decided. It can be physical methods, biological, and chemical methods. In terms of biological applications, the physical methods for cell disruption are usually used [4].
Cytotoxicity refers to the quality of being toxic to cells, which can result from various factors, including chemical exposure, radiation, and biological agents. The chapter highlights the significance of studying cytotoxicity, particularly in the context of cancer treatment and environmental toxicology. Understanding how different substances affect cell viability is crucial for developing effective therapeutic strategies and assessing the safety of chemicals [5].
Cytotoxicity is the adverse effects observed from reactions with internal or external factors such as metal ions, chemicals, phytotoxins or microbial infections. Once the cytotoxicity is observed the immune system responds to it by inducing apoptosis and clearance from the system. Depending upon how the cells are impulsive for cytotoxic agents, it results in a variety of prognoses. While it may induce apoptosis, i.e., programmed cell death, it may also induce necrosis, and as a result, cell may undergo lysis. As cytotoxicity is important in biological and specifically physiological processes, it becomes important to measure the cytotoxicity to determine its extent and further plan its control if needed. Cytotoxicity is measured in multiple ways and one of the common strategies is ATP measurement. The basic mechanism behind the estimation of cytotoxicity is that, due to altered cellular membranes, the cellular material leaks out of the cell and hence allows space for the labeling dyes to penetrate through. This mechanism of uptake of dyes by cells, however, can lead to inaccurate estimation of cytotoxicity. To overcome this, modern non-radiolabeled methods have been developed which act on the specific class of molecules present in both proliferating and non-proliferating cells to determine cytotoxicity.
2.2 Analysis of cytotoxicity
The analysis of cytotoxicity can be divided into several types. Some are traditional, while others are newly developed and focus on the evolving needs of the future.
2.2.1 Agar overlay test
The agar overlay test is a traditionally used method to determine the toxic effects of various materials on living cells. It is used to test toxicity toward bacterial cells or any particular bacterial strain. This involves careful preparation of an extract or purified material to be tested for cytotoxicity, as the purity of the material and external environmental conditions may lead to false results. Also, this method cannot be used to determine cytotoxicity in animal cell culture and hence has limited applications [6].
2.2.2 MTT assay
The MTT assay is the most common assay to study cytotoxicity in animal cell culture. The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) is a mono-tetrazolium salt that consists of a positively charged quaternary tetrazole ring core containing four nitrogen atoms surrounded by three aromatic rings including two phenyl moieties and one thiazolyl ring. The reaction leads to the reduction of MTT which results in disruption of the core tetrazole ring and the formation of a violet-blue water-insoluble molecule called formazan. The MTT assay has multiple applications in cancer research and drug development. It also helps in the development of new cell lines and is also used for the analysis of stable cell lines. Adapting to modern-day research this can also be used for high-throughput screening applications [7].
Despite the applications, this method has many limitations, and various factors contribute to its misinterpretation of results. One of the limitations is that the measurement of cellular toxicity is based on the metabolic activity and not on cell viability, which may lead to false interpretation as the low metabolic activity cell may be viable. Also, the non-viable cells can be metabolically active for some or longer duration of time [7]. Like the agar test the MTT test is also impacted by environmental factors such as temperature, pH, osmolarity, and other than that cell density.
Since the MTT assay is not always automated, hence, there are high chances of manual errors while handling the cell lines, storage and preparation of reagents, delay time, and mechanical errors. While these errors are tough to avoid, one of the common errors that happens is the designing of experiments and protocols. It is important to note here that for every cell line or treatment condition, the MTT assay protocol changes, and hence, it is difficult to avoid inaccuracy at certain levels. However, despite the limitations and vulnerability, MTT assay remains a popular test of cytotoxicity due to its replicability, ease of doing, and cost-effectiveness [8]. However, it is needed to put in controls so that the impact of external factors can be minimized. Also, the automation of the MTT assay could lead to a reduction in manual and mechanical errors.
2.2.3 Cytokinesis-block micronucleus-cytome assay
This is a comprehensive method which measures cytotoxicity based on DNA damage. Multiple events such as binucleated cells, presence of micronuclei, chromosome loss, and chromosome damage are counted as scores. A ratio of binucleated and multinucleated cells to the necrotic and/or apoptotic cells is taken into consideration. More information on the DNA damage is sought using centromere or telomere probes. The assay is applied for analysis of in-vivo genotoxin exposure and in-vitro genotoxicity test [9].
2.2.3.1 Methodology
Sample preparation: Whole blood is diluted and layered over Ficoll-Paque to separate leukocytes. After centrifugation, the leukocyte layer is collected and further processed to obtain a pure lymphocyte suspension.
Cell culture: The isolated lymphocytes are cultured in a suitable medium, typically supplemented with fetal bovine serum (FBS). The article corrects a previous error regarding the concentration of FBS required, emphasizing the need for a 100% solution.
Cytokinesis block: A key feature of the assay is the use of cytochalasin B, which inhibits cytokinesis, allowing for the formation of binucleated cells. This step is crucial for the accurate scoring of micronuclei.
Slide preparation: The article details the process of cytocentrifugation to prepare slides for microscopic examination. Proper slide preparation is critical for obtaining clear and interpretable results.
Staining and scoring: After air-drying and fixing the slides, cells are stained to visualize the micronuclei. The scoring of micronuclei is performed under a microscope, where the number of micronuclei per 1000 binucleated cells is counted. The article emphasizes the importance of maintaining consistent scoring criteria to ensure reproducibility.
2.2.3.2 Applications of the CBMN assay
The CBMN assay has a wide range of applications, including:
Genotoxicity testing: The assay is used to evaluate the genotoxic potential of various substances, including pharmaceuticals, environmental pollutants, and dietary components.
Cancer research: By assessing the frequency of micronuclei in lymphocytes, researchers can identify individuals at higher risk for cancer due to genetic predispositions or environmental exposures.
Radiation sensitivity: The assay can help predict individual responses to radiation therapy, allowing for personalized treatment plans based on a patient’s cellular response to DNA damage.
Nutrigenomics: The CBMN assay is also applied in studies examining the effects of dietary components on DNA integrity and repair mechanisms.
2.2.3.3 Advantages of the CBMN assay
Sensitivity: The assay is highly sensitive and can detect low levels of DNA damage, making it suitable for biomonitoring studies.
Relevance: Since lymphocytes are easily accessible and reflect systemic exposure to genotoxic agents, the results are relevant to human health.
Comprehensive assessment: The assay provides a comprehensive assessment of cellular responses, including cytotoxicity, cell proliferation, and DNA damage.
2.2.3.4 Challenges and considerations
Technical variability: The assay requires careful attention to detail in each step to minimize variability. Factors such as cell density, culture conditions, and staining protocols can influence results.
Interpretation of results: The presence of micronuclei can be influenced by various factors, including cell cycle stage and individual genetic differences. Therefore, results should be interpreted in the context of other biological data.
Ethical considerations: The use of human lymphocytes necessitates ethical considerations regarding sample collection and informed consent.
The cytokinesis-block micronucleus cytome assay is a valuable method for assessing DNA damage and cellular responses to genotoxic agents. The detailed protocol provided in the article serves as a comprehensive guide for researchers looking to implement this assay in their studies. By understanding the methodology, applications, and implications of the CBMN assay, scientists can contribute to advancing knowledge in fields such as cancer research, environmental health, and personalized medicine.
As research continues to evolve, the CBMN assay remains a critical tool for understanding the complexities of DNA damage and repair, ultimately aiding in the development of strategies for cancer prevention and treatment.
Apart from these tests, number of other traditional cytotoxicity assays have been discovered and been in use such as the chromium release test, model cavity test, neutral red uptake assay, and Millipore filter test (Table 1).
Method
Advantages
Limitations
Application
References
Development of oncolytic viruses
Can be used in immunotherapy for cancer treatment
Selection of oncolytic viruses, virus entry and selection
Recent developments in the innovative approaches for cell lysis.
2.3 Cytotoxicity and cell death
Cell death is an essential biological process that occurs in a multitude of contexts, ranging from the development of organisms to the maintenance of tissue homeostasis. Cells can cease to function in various ways, and understanding these mechanisms is crucial to grasping how organisms regulate life and death at the cellular level. Below, we explore the primary types of cell death and their implications.
2.3.1 Apoptosis (type I cell death)
Apoptosis can be likened to a graceful ballet of cellular demise, representing a highly regulated form of programmed cell death (PCD). This process occurs in a controlled manner and is independent of external stimuli, emphasizing the intrinsic mechanisms of cellular regulation. The initiation of apoptosis is dictated by biochemical signals encoded within the cell’s DNA, which orchestrate a well-defined pathway leading to self-destruction [19].
2.3.1.1 Triggers of apoptosis
The onset of apoptosis can be triggered by a variety of factors, both internal and external to the cell. Internal triggers may include mild cellular injury or stress, while external cues could involve signals related to developmental processes or the withdrawal of survival signals. These factors signal the cell to commence the apoptotic process, ensuring that cellular turnover is maintained [20].
2.3.1.2 The process of apoptosis
During apoptosis, cells undergo a series of morphological changes. Initially, the cell will shrink and lose its characteristic shape, which is a hallmark of this process. The cell’s internal structure begins to break down, leading to distinct features such as chromatin condensation and the fragmentation of the nucleus. Ultimately, these changes result in the formation of apoptotic bodies, which are then phagocytosed by neighboring cells or immune cells, ensuring that the remnants of the dying cell are efficiently removed without eliciting an inflammatory response.
2.3.1.3 Importance of apoptosis
Apoptosis plays a vital role in the development and maintenance of multicellular organisms. This process is crucial for controlled growth, proper development, and the ongoing turnover of cells within tissues. A classic example of apoptosis in action can be observed during embryonic development, where it facilitates the separation of digits in the developing hands and feet. By selectively eliminating cells, apoptosis contributes to the formation of functional structures and helps to sculpt the organism.
2.3.1.4 Mechanisms of apoptosis
To date, two primary pathways have been identified in the process of apoptosis: the extrinsic pathway and the intrinsic pathway. The extrinsic pathway is activated by the engagement of death receptors on the cell surface, while the intrinsic pathway is governed by mitochondrial enzymes that play a pivotal role in the apoptotic process. These pathways highlight the complexity and precision with which cells regulate their own demise [21].
2.3.2 Autophagy (type II cell death)
Autophagy can be viewed as a form of cellular spring cleaning, where cells actively degrade and recycle their own components. This process involves the engulfment of organelles and cytoplasmic contents, which are then directed to lysosomes for degradation. Depending on the context, autophagy can serve dual functions: promoting cell survival under nutrient-limited conditions or facilitating cell death when cellular damage is extensive.
2.3.2.1 The autophagic process
During autophagy, cells essentially take stock of their internal components, identifying worn-out organelles and misfolded proteins that require removal. This self-eating process not only cleans up cellular debris but also recycles essential components to support cellular metabolism and energy production. Thus, autophagy plays a critical role in maintaining cellular health and homeostasis [22].
2.3.3 Necrosis (type III cell death)
Necrosis has historically been regarded as the chaotic and uncontrolled cousin of cell death, often resulting from acute cellular injury. However, recent research has illuminated specific pathways of regulated necrosis that add complexity to this process. Unlike apoptosis, necrosis typically occurs in response to severe stress or injury, leading to cell swelling and eventual rupture [23].
2.3.3.1 Regulated necrosis pathways
Within the umbrella of necrosis, several distinct pathways have been identified:
Necroptosis: This is a programmed form of necrosis that occurs when cells experience certain signals, often when apoptosis is inhibited. It involves receptor-interacting protein kinases (RIPKs) that promote cell death in a regulated manner.
Pyroptosis: This is characterized by inflammatory cell death, primarily in response to infections. Pyroptosis leads to the release of pro-inflammatory cytokines, making it a crucial response in the immune system.
Ferroptosis: This pathway involves iron-dependent lipid peroxidation, leading to cell death that is distinct from other forms of necrosis. Ferroptosis has garnered attention for its implications in various diseases, including cancer.
NETosis: This is a unique form of cell death observed in neutrophils, where they release extracellular traps composed of DNA and antimicrobial proteins. This process helps to trap and eliminate pathogens but can also contribute to tissue damage if dysregulated.
3.1 Challenges in physical and chemical methods of cell lysis
Most of the bioproducts are needed to be extracted before proceeding toward purification. Since the products are mostly intracellular, the cell wall and/or cell membrane need to be ruptured for release of products in crude form. One of the basic methods for the rupturing of cells is physical and chemical-based methods. Depending upon the nature and properties of the product and its source of production, cell lysis methods are decided. Many processes involve single-step procedures while others may involve a cascade of multiple procedures for efficient extraction. However, these methods often come with several limitations which one has to deal with while implying or integrating in the bioprocessing platforms.
The bead mill method is one of the oldest mechanical methods of cell disruption. It heavily depends on the cell type be it bacterial, yeast or plant, and the nature of the cell wall. It involves mild to heavy grinding of the cells. The grinding process is done by using microbeads, and its size and weight depend on the cell type and its vulnerability to disruption. One of the limitations is the heat generation process. As the bead size increases, the heat generation also increases which may affect the stability of the desired product. The limitation is backed up by poor recovery and poor scale-up [24].
Ultrasonication is another cell disruption method which is used for cell lysis. There are two ways to perform a cell lysis using sonication. One is the water bath method, and the other is the dip probe method. While water bath sonication is a very mild method of sonication, the dip probe method is a more efficient method for the recovery of biological products. These are mostly used for bacterial and yeast-based products. The method is used in combination with any other physical or chemical methods [1]. However, it also has serious limitations such as poor scale-up and high energy usage, another major limitation to this is that the process requires a separate area as the ultrasonic waves pose a health hazard [1].
Thermolysis and osmotic shock are the methods which do not involve mechanical setup; however, these are also physical-based methods. Thermolysis involves the treatment of cells at high temperatures causing cell disruption. For effective disruption, a cold cycle may be integrated, but this leads to limitations of poor recovery. One of the important things to note here is that temperature variation may change the properties of the desired biological product, especially in the case of enzymes which become highly critical. One of the lysis methods is osmotic shock which involves the treatment of cells with high and low-salt solutions in a disruptive manner. The disruption causes the development of osmotic pressure across the membranes disrupting the cell membranes. This method is utilized in biopharmaceutical and biotechnological processes, but the yield and inefficiency serve as limitations of this method [25].
Chemical methods of cell lysis involve the use of detergents and enzymes for cellular disruption. Chemicals such as Sodium dodecyl sulfate (SDS), TritonX-100, and Tween- 80, etc. are also used. The detergents interact with the receptors on the cell membrane and lead to membrane damage. They work by solubilizing the membrane proteins and thereby creating space for the release of cellular components. While the use of chemicals leads to issues with the recovery and stability of the desired compounds, the enzymatic methods such as lysozymes and peptidases cause issues with the recovery of the biological products as their activity may lead to the inactivity of the biomolecules [26].
Another approach utilized for cell lysis is the use of solvents for extraction and cell disruption, while this approach is widely utilized in the case of natural products from plants as the cell wall has a significant resistance to the solvents. Organic solvents such as ether, methanol, chloroform, etc. are used. This method is accompanied by the limitations of product stability and additional downstream costs due to solvent removal and recovery steps [26].
4. Development of innovative cell lysis approaches
To overcome the limitations of the traditional cell lysis methods, we need to focus on developing new roads to the development of lysis techniques.
Nanotechnology: The use of nanoparticles for cell lysis is a promising area of research. For instance, Seo et al. [27] developed octahedral ZnFe2O4 nanoparticles that act as magnetic flocculants, enabling simultaneous harvesting and lysis of microalgal cells. These nanoparticles can be functionalized to enhance their lytic capabilities, allowing for targeted disruption of specific cell types.
Microfluidic devices: Advances in microfluidics have led to the development of devices that can achieve cell lysis through mechanical means. Yun et al. [28] created a microfluidic device with ultra-sharp nano-blades that apply shear forces to disrupt cell membranes. This method allows for precise control over the lysis process and can be scaled for high-throughput applications, making it suitable for various research and clinical settings.
Molecular dynamics simulations: Capozza et al. [29] conducted in-silico studies to understand the behavior of cell membranes under mechanical stress. Their findings suggest that creating curvature in the membrane can significantly reduce the force required for lysis, paving the way for more efficient mechanical lysis techniques. This approach can inform the design of new lysis devices that optimize the mechanical forces applied to cells.
Laser-based methods: Pulsed laser microbeam techniques have been explored for cell lysis, where focused laser beams induce localized heating and pressure changes, leading to cell disruption. This method allows for precise targeting of specific cells without affecting surrounding tissues, making it a valuable tool for applications in single-cell analysis and tissue engineering [30].
Acoustic lysis: This method utilizes ultrasonic waves to create cavitation bubbles in the surrounding medium, which collapse and generate shock waves that disrupt cell membranes. Acoustic lysis is effective for lysing various cell types, including bacteria, yeast, and mammalian cells.
Despite the advancements in cell lysis technologies, several challenges remain. The review identifies issues such as the need for methods that are gentle enough to preserve sensitive biomolecules while still being effective at lysing cells. Additionally, scalability and cost-effectiveness are critical factors that need to be addressed for widespread adoption of new technologies (Figure 1) [31].
Figure 1.
Incidents that induce cell death and some of the cellular factors associated with it.
Future research directions include:
Hybrid methods: The development of hybrid methods that combine physical, chemical, and biological approaches to achieve optimal lysis efficiency. For example, combining enzymatic lysis with mechanical disruption could enhance the overall lysis process while minimizing damage to sensitive biomolecules.
Artificial intelligence and machine learning: There is a growing interest in exploring the use of artificial intelligence and machine learning to optimize lysis protocols based on specific cell types and applications. These technologies can analyze large datasets to identify the most effective lysis conditions, leading to more efficient and tailored lysis strategies.
Nanostructured materials: Continued research into nanostructured materials for cell lysis could lead to the development of new lysis agents that are more effective and selective. For instance, nanoparticles that can be targeted to specific cell types or that respond to external stimuli (e.g., light or temperature) could enhance the precision of lysis methods.
Integration with other techniques: Integrating cell lysis methods with downstream applications, such as nucleic acid extraction and analysis, could streamline workflows and improve overall efficiency. For example, developing lysis methods that are compatible with microfluidic platforms could facilitate the automation of sample processing.
We acknowledge the use of ChatGPT for language polishing of the manuscript.
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Written By
Pragya Prakash, Shoaib Haidar and Hare Ram Singh
Submitted: 19 September 2024Reviewed: 07 November 2024Published: 07 January 2025
Open access peer-reviewed chapter - ONLINE FIRST
