Animal cell culture is a common laboratory technique for maintaining live cells separated from their original tissue (primary cells) or immortal cells (cell lines). The technique of cell culture was discovered in the 19th century after the discovery of the microscope. Presently, the culture of cells is one of the most important tools of life sciences research and is being used by investigators to understand advanced understanding of cell growth and differentiation and mechanisms of normal/ abnormal functioning of various cells. In addition to that, cell culture is employed in the generation of vaccines, the production of pharmaceutical proteins, and the safety testing of drugs. The Faculty of the College of Animal Biotechnology authored or adapted the protocol contained in this manual for the purpose of creating basic information resource for students beginning studies in the field of Biotechnology to the course “Principles and Procedures of Animal Cell Culture (Biotech 421). This manual may be useful for some other undergraduate program courses in animal biotechnology. Starting with Experiment No. 1, you will delve into the fundamental principles of laboratory layout, safety protocols, and guidelines crucial for establishing and maintaining cell culture facilities. Subsequent experiments cover various topics, including basic equipment setup, sterilization techniques, media preparation, and isolation of primary cell cultures. Moreover, this manual incorporates emerging trends in cell culture, including cryopreservation methods, virus-mediated cell line development, and virus-neutralization assays, reflecting the evolving landscape of biomedical research.

The tissue/cell culture lab has the requirement of maintaining an aseptic environment. There are several aspects to the design of good tissue culture facilities. Ideally, cell culture work should be conducted in a single-use facility. Therefore, cell culture lab unit should be separated from the rest of the lab area. It can be either separate lab or a closed partition in a big lab. All new material should be handled as ‘quarantine’ material in a separate area, and materials known to be free of contaminants should only be allowed inside the culture room. In addition, the work surfaces should be thoroughly cleaned and disinfected between activities. For most of the work involving non-infectious primary culture and cell lines, the laboratory should be designated to at least BSL2 facility. The location of the Biosafety cabinet is the most important point to consider. When operated correctly, the cabinet will provide a clean working environment, whilst protecting the operator from aerosols. Environmental monitoring with Tryptone Soy Broth agar settle plates inside the biosafety cabinet is a good indicator of the cleanliness of a cabinet. There should be no growth of bacteria and fungi after 3-5 days of incubation.

Things required for cell culture 1. Lab – having negative pressure (HEPA filter) 2. Safety cabinet (Hood/laminar flow) a) Biosafety level 1 (BSL-1) – Basic level of protection. Agents handled under this cabinet are NOT infectious. b) BSL-2 – Appropriate for moderate risk agents. Most of the non infectious cell cultures are done under this facility.  

Working Principle: An autoclave is a sealed device that creates a different environment from the outside world under extraordinarily high pressure (Auto + clave = automatic locking). It uses moist heat or saturated steam under pressure to kill microorganisms and heat-resistant endospores. Its function is compared to that of a pressure cooker, i.e., steam is used to create a high pressure environment. However temperature of autoclave or steam sterilizer is generally maintained at 121°C for 15-20 minutes that is achieved by raising the pressure inside the device. This high pressure increases the boiling point of water and therefore raises the temperature that is too high for the microbes to survive, so used for sterilization. Types of Autoclave cycle 1. Liquid cycle: Liquids take time to sterilize as they have high heat capacity. It is based on the principle of pressurized steam at 15 psi, 121°C temperature for 20 minutes. It has latent heat, which is 7× heat 100°C. Liquids and biological waste require slow exhaust to prevent the boiling over of super-heated liquids. This excess temperature is enough to hydrolyze the proteins of bacteria, viruses, or any other contaminant and therefore kill it. However, wet heat is seven times more intense than dry heat, so it requires less temperature than the dry heat cycle of the autoclave. 2. Dry Cycle: Here, steam is the sterilization medium, which penetrates inside the load and makes contact with all the surfaces, so a fast exhaust cycle is used. It has a high temperature of 160°C for 60 minutes.

In the realm of cell culture, the quality and composition of the growth media and reagents play a pivotal role in growth and sustaining cells in vitro. The preparation of these essential components requires precision, attention to detail, and adherence to the strict protocols to ensure optimal cellular growth, viability, and experimental reproducibility. This introduction serves as a foundational overview of the critical aspects involved in the preparation of media and reagents for cell culture experiments. Furthermore, the preparation of media and reagents for cell culture necessitates adherence to stringent quality control measures to ensure consistency and reproducibility across experiments. Validation of media batches, sterility testing, and periodic quality assessments are essential practic

What is primary cell culture? In vitro culture of cells freshly obtained from the tissue is called primary cell culture. Unlike that of cell line, primary cell culture grows for a limited period of time. Examples, are primary mammary epithelial cells, primary hepatocyte culture. Aim: To prepare the primary cell culture from chicken embryos. Materials required for the primary cell culture • 70% alcohol • Cotton swabs • Small beaker 20-50 ml • Forceps • Straight and required curved, • 9 cm Petri dishes • 11 day embryonated eggs • Balanced Salt solution (BSS) • 250 ml conical flask • Magnetic centrifuge tubes • Haemocytometer • Growth medium with serum • Culture flasks

What is keratinocyte? The epidermal layer consists of keratinocytes, melanocytes, Langerhans cells, and Merkel cells. Their major function is to produce Keratinocytes which are the most abundant cells in the epidermis, keratin protein. Keratinocyte synthesizes major structural components of the epidermal barrier through a programmed process of differentiation. They are present in the innermost layer of the epidermis (Eckert et al?, 1989). The epidermis is composed of several layers, stratum corneum (horny layer), stratum lucidum (clear layer), stratum granulosum (granular layer), stratum spinosum (prickle cell layer), and stratum basale (basal layer), from the outermost to the deepest layer respectively. The process of maturation and movement of the keratinocytes from the stratum basale to the stratum corneum is what we call epidermal differentiation. Procedures for isolation of canine karatinocytes When studying skin cells, isolating keratinocytes from tissue samples, such as skin biopsies, is a critical initial step. Here is a general guide on how to isolate keratinocytes:

What is dermal fibroblast? Skin fibroblasts are a type of cell found in the dermis, the middle layer of the skin. They are the most common cells in connective tissue and play a crucial role in maintaining the structural integrity of the skin. Fibroblasts are responsible for synthesizing and secreting extracellular matrix proteins such as collagen, elastin, and fibronectin, which provide support and elasticity to the skin. Additionally, fibroblasts play a role in wound healing by proliferating and migrating to the site of injury, where they aid in tissue repair and scar formation. Aim: To prepare the primary cell culture of fibroblasts from canine skin. Tools and instrument required for the procedure 1. Tissue collection box with ice cubes - To prevent contamination and maintain the stable temperature 2. Surgical Gloves - To prevent contamination 3. Tissue collection tube with 1X PBS - To collect sample 4. Laminar flow hood - To work in a sterile environment and prevent contamination 5. Cell culture dish - To digest the skin 6. Sterile 1000 μL and 200 μL pipette and tips-For accurate measurement and transfer of cell suspension and culture media. 7. Forceps - Fine-tipped forceps for handling and manipulating tissue. 8. Scalpel and blade holder - Scalpel for the dissection of skin tissue. 9. Surgical scissors - Chopping of the skin tissue 10. Falcon 70 μm cell strainer - To filter out undigested tissue debris and obtain a homogeneous cell suspension. 11. 15 ml and 50 ml Centrifuge tubes - To collect and centrifuge the digested cell suspension.

What is subculture? In cell culture, a process called subculturing, or passaging, keeps cells alive and growing. It involves taking cells from an existing container, removing the used medium, and placing them in a new container with fresh nutrients. This allows the cells to continue multiplying and creates more cells for research. Adherent subculture protocol Cells nearing 80% coverage need splitting! 1. Warm things up: Get your media, detachment solution, and incubator ready at 98.6°F (37°C). 2. Safety first! Disinfect your work area and supplies inside a biosafety cabinet. 3. Gently rinse: Remove the old media and wash the cells with a salt solution at room temperature. Be careful not to dislodge the cells. 4. Detach the cells: Add a pre-warmed enzyme solution to detach the cells from the flask surface. Keep an eye on them to make sure it works (usually 2 minutes). Note: Not all cells need this enzyme, and it can be harmful to some. 5. Stop the enzyme: Once all the cells are detached, add a culture medium with serum to neutralize the enzyme. 6. Collect the cells: Transfer the cell suspension to a tube, rinse the flask with fresh medium to get all the cells, and add it to the same tube. 7. Spin it down: Centrifuge the cell suspension for 5 minutes at room temperature (1000 rpm). 8. Get ready for counting: Remove the liquid, loosen the cell clump, and resuspend the cells in a fresh medium at a good volume for counting. 9. Count your cells: Use a hemocytometer to count the cells and determine their health.

Cell counting is the fundamental techniques in various fields of biology, medicine and biotechnology. Importance of cell counting 1. Research: Cell counting is a critical component of biological and medical research. It helps scientists understand the behavior of cells in various contexts, such as studying cell proliferation, differentiation, and response to stimuli. 2. Disease diagnosis: Monitoring various medical conditions, including infections, autoimmune diseases, cancer and blood disorders. Complete blood counts (CBCs) are routinely used to assess the levels of different blood cell types and assess disease conditions. 3. Stem Cell Research: Cell counting plays a significant role in stem cell research, where precise quantification of stem cells is necessary for various applications, including regenerative medicine and tissue engineering. 4. Cell Culture: Cell counting is integral part to determine seeding density. Seeding of cells to available surface area for growth is important consideration for successful culture. 5. Cell-Based Assay: The cell-based assays are used for drug screening, toxicity testing, and evaluating the efficacy of new compounds. Accurate cell counts are essential to ensure the reliability of assay results.

Introduction Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitization in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro. Background information Blood is a liquid mixture comprised of plasma, cells and proteins. The liquid portion, plasma, makes up half of the blood volume, and cells the other half. Isolation of lymphocytes is a common procedure in immunology and cell biology that involves separating and collecting lymphocytes.

Introduction The protocol below describes the use of cell freezing methods involving an electric -80°C freezer for the cryopreservation of cell lines. ECACC routinely use a programmable rate-controlled freezer. This is the most reliable and reproducible way to freeze cells but as the cost of such equipment is beyond the majority of research laboratories the methods below are described in detail. If large numbers of cell cultures are regularly being frozen then a programmable rate-controlled freezer is recommended. Materials • Freeze medium (commonly 90% FBS, 10% DMSO or glycerol • 70% (v/v) alcohol in sterile water • PBS without Ca2+/Mg2+ • 0.05% trypsin/EDTA in HBSS, without Ca2+/Mg2+ • DMSO

Introduction Structural changes in host cells by the viral infection is referred as cytopathic effect (CPE) and the responsible virus is called cytopathogenic virus. As a virus hijacks a cell to make copies of itself, the cell goes through distinct changes in its chemistry and structure. These effects are easiest to see in lab grown cell cultures. The severity of the damage caused by the virus depends on the specific virus itself, the type of cell it infects, how many viruses infect each cell (MOI), and other factors. The early stages of this damage, called cytopathic effects (CPE), can be spotted directly under a microscope without any dyes or special treatments. While various types of CPE exist in living cells, some signs of viral infection, like inclusion bodies, require staining the cells to be visible. We can classify viruses based on how quickly they cause visible damage (cytopathic effect) in cell cultures. Viruses are typically considered slow if it takes 4-5 days to see this effect in cultures with a low starting dose of virus (MOI). On the other hand, viruses are considered fast if the damage shows up in just 1-2 days under the same conditions. It is important to note that at a high MOI all CPE can occur rapidly. So all decisions about rate of CPE appearance should be based on the lowest MOI that produces CPE

Principle Isolating the virus in a laboratory is the gold standard for diagnosing many viral infections. Virus isolation is a highly specialised technique used to diagnose viral infections and produce a stock of viruses for research purposes. The isolation of the virus relies on the suitability of the sample, the ability of the cells to allow entry, the successful replication of the virus, and the release of viral particles from the cells. The choice of cell lines is dependent upon the suspected viral disease and the availability of such cell lines in the laboratory. Materials required Equipment a) Biosafety cabinet level 2 (B2) b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere) c) Refrigerated centrifuge d) Refrigerator e) Inverted Microscope f) Water bath g) -86°C/-80°C deep freezer h) Autoclave i) Hot air oven j) Filtration Assembly

Principle The one-step growth experiment refers to a scientific methodology. A study aimed at observing the molecular events that take place during viral reproduction. This elucidates the fundamental features of the virus replication process and determines the optimal multiplicity of infection and timing for virus harvest. The initiation of modern bacteriophage research can be traced back to the development of the one-step growth curve by Max Delbrück and Emory Ellis in 1939, which utilised the Escherichia coli-T4 bacteriophage system. One step growth curve consists of three clearly defined phases 1. Latent period refers to the initial phase of a process where no significant changes or activities are observed. 2. Burst or Rise period is the subsequent phase characterised by a sudden increase virus particle. 3. Plateau period is the following phase where a stable and constant level virus replication. Materials required

Principle Unlike bacteria, which can be cultivated on an artificial nutrient medium, viruses rely on a living host cell for replication. Eukaryotic or prokaryotic host cells that have been infected with viruses can be used to produce viruses. Subsequently, the medium and cells can be collected and used as a virus source. The production of viruses usually consists of a two-step procedure: f irst, the growth of host cells, and then the production phase. Understanding the complex relationship between the host cell and virus is essential. Various crucial factors have been recognised, such as the concentration of host cells during infection and the multiplicity of infection (MOI). Materials required Equipment a) Biosafety cabinet level 2 (B2) b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere) c) Refrigerated centrifuge d) Refrigerator e) Inverted Microscope f) Micro pipettes(1-10 μl, 10-200 μl, 100-1000 μl) g) -86°C/-80°C deep freezer h) Autoclave i) Hot air oven j) Filtration Assembly

Principle The quantification of infectious virus particles is commonly achieved through the utilisation of the Median Tissue Culture Infectious Dose (TCID50) assay. The assay functions through the introduction of a serial dilution of the virus sample into cells in a 96 well plate format. The chosen cell type is specifically intended to exhibit a cytopathic effect (CPE), which refers to observable changes in morphology or cell death resulting from viral infection. Following a period of incubation, the cells are examined for cytopathic effects or cell death, and each well is categorised as either infected or uninfected. The concentration at which 50% of the wells exhibit a cytopathic effect (CPE) is employed to determine the 50% tissue culture infectious dose (TCID50 ) of the viral sample. The end point is the dose or dilution that infects or kills or shows CPE of the cells. The calculation can be performed using various mathematical techniques, such as the Spearman-Karber method or the Reed Muench method. The virus titer is quantified as the number of tissue culture infectious dose 50 (TCID50 ) per millilitre (ml). The non-cytopathic virus can be quantified by employing the fluorescent antibody test, immune-peroxidase assay, or other appropriate methodologies.

Principle Red blood cell sticking (hemadsorption) can reveal the presence of certain viruses. This happens because these viruses, like influenza or measles, sprout proteins (hemagglutinin) on their infected host cells. These proteins act like Velcro, attracting red blood cells and causing them to cling to the surface. A hemadsorption assay uses this natural process to check for viruses. It's faster than waiting for the cells to show damage (cytopathic effect), allowing for earlier detection. Haemadsorbing viruses can be detected several days prior to the appearance of a cytopathic effect. Equipment a) Biosafety cabinet level 2 (B2) b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere) c) Refrigerated centrifuge d) Refrigerator e) Inverted Microscope f) Micro pipettes (1-10 μl, 10-200 μl, 100-1000 μl) g) -86°C/-80°C deep freezer h) Autoclave i) Hot air oven j) Filtration Assembly

Principle The plaque assay was developed in order to enumerate and quantify the infectivity of bacteriophages. This technique has been subsequently adapted for application in animal virology and has proven to be a dependable method for quantifying the concentration of various viruses that have been adapted for growth in cell cultures. This assay is designed to quantify the capacity of a solitary infectious virus to create a visible area of cell death on a layer of cultured cells. A plaque forms during the viral infection cycle, in which the host cell dies after viral replication. Some viruses cause cell lysis during replication, while others cause minimal damage to the cells. The plaque assay is a technique used to measure the quantity of viruses that cause the destruction of their host cells, resulting in cytopathic effects (CPE). During a plaque assay, the host cells and virus are briefly co-cultured to enhance the virus's attachment and entry into the host cell. Subsequently, the concoction is introduced into a partially solidified agar medium. The thick agar is poured onto a layer of "bottom agar" which supplies ample nutrients for the host cell. After one round of virus replication, an infected cell will undergo lysis, leading to the release of multiple new viruses. Within the gelatinous substance, these newly released viruses can only infect neighbouring cells. After a subsequent round of replication, these neighbouring cells will undergo lysis. The cells that manage to avoid infection will continue to proliferate. After a period of 24 to 48 hours, plates that were not contaminated with a virus will display a consistent layer of cells. The plates infected with a multitude of viruses exhibit plaques. Quantifying viral titer is essential for assessing viral infectivity. The determination can be made either by conducting a plaque assay or by calculating the infectious dose.

Principle Neutralization refers to the process of reducing or nullifying the effect of a specific component. Neutralization is a process in which the effects of viral antigens or viruses reduce through Ag-Ab reactions. Neutralizing antibodies are a specific type of antibodies that play a role in neutralization. Not all antibodies generated within our body possess the capability to impede the effects of antigens. Only antibodies with the ability to neutralize can prevent the uptake of viral particles by cells and counteract their effects. The virus neutralization assay is a serological test utilized to identify the existence and extent of functional systemic antibodies that hinder the ability of a virus to cause infection. Media and Materials Equipment a) Biosafety cabinet level 2 (B2) b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere) c) Refrigerated centrifuge d) Refrigerator e) Inverted Microscope f) Micro pipettes (1-10 μl, 10-200 μl, 100-1000 μl) g) -86°C/-80°C deep freezer h) Autoclave i) Hot air oven j) Filtration Assembly

Principle The majority of cell lines are cultivated as monolayer adherent cells, meaning they only grow on the surfaces of culture vessels. However, there are particular cells that are non-adhesive and do not rely on external support for their growth, referred to as suspension cells. A suspension culture, is a cell culture where individual cells or small cell clusters grow and multiply in a stirred growth medium, forming a suspension. Subculturing, also known as passaging cells, involves transferring cells from a previous culture into fresh growth medium to allow the cell line to continue growing. Suspension cultures involve the suspension of cells in a liquid medium and agitation at a specific speed to ensure that the cells are uniformly exposed to nutrient media from every angle. This method prevents cell aggregation and enhances nutrient absorption, thereby promoting cell growth. The maintenance of these cultures is achieved by transferring the cells from the early stationary phase to a fresh medium through subculturing. During the incubation period, there is an increase in both cell division and cell enlargement, leading to rapid cellular growth. The viability of cells in the suspension decreases after the stationary phase due to the exhaustion of certain factors or the accumulation of toxic substances in the medium. This decrease in the viability of the cells further reduces the growth rate of the entire culture. Next, a portion of the cell suspension is added to the freshly prepared medium with the same composition.

Principle Adenoviral, oncoretroviral, and lentiviral vectors are commonly utilised for gene delivery in mammalian cell culture and in vivo. Additional important examples of viral gene transfer involve baculovirus and vaccinia virus vectors. Stable cell lines produced with lentivirus technology provide consistent, long-term protein expression compared to the short-term expression achieved through transient transfection. This chapter focuses on cell transformation by lentiviruses. Equipment a) Biosafety cabinet level 2 (B2) b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere) c) Refrigerated centrifuge d) Refrigerator e) Inverted Microscope f) Micro pipettes (1-10 μl, 10-200 μl, 100-1000 μl) g) -86°C/-80°C deep freezer h) Autoclave i) Hot air oven j) Filtration Assembly

Principle Cell viability refers to the quantity of healthy cells in a sample, while cell proliferation is a crucial indicator for comprehending the mechanisms and pathways involved in the survival of cells or death following exposure to toxic substances. The methods used to assess viability are typically the same as those used to detect cell proliferation. Cell cytotoxicity and proliferation assays are commonly employed in drug evaluations to determine if the test compounds impact cell proliferation or exhibit direct cytotoxic effects. Cell viability can be determined by calculating the ratio of total live cells to the total number of cells (live and dead). Staining also helps to observe the general cell structure. Cell proliferation assay by MTT. Metabolically active cells reduce the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) through dehydrogenase enzymes to produce NADH and NADPH. The purple formazan produced inside the cell can be dissolved and measured using spectrophotometry. The MTT Cell Proliferation Assay quantifies cell proliferation rate and detects decreases in cell viability caused by metabolic events leading to apoptosis or necrosis. The MTT Reagent produces minimal absorbance values when no cells are present.

293T (HEK 293T) is a human cell line. It is frequently utilised in biological research to synthesise proteins and generate recombinant retroviruses. Antigen recall assay: It is an antigen specific system assesses the activity of potential immune checkpoint inhibitors (ICI) candidates, in a relatively physiologically relevant manner. Apoptosis: is the process of programmed cell death. It is opposite to the cell proliferation. BSS: BSS, which stands for Balanced Salt Solution, plays a critical role in keeping cells happy and healthy in cell culture experiments. BSS is formulated to closely resemble the natural fluids surrounding cells within the body. This includes having the right balance of salts (like sodium, potassium, calcium, and magnesium) at a concentration similar to what cells are used to. Cell line is a specific group of cells that can be cultured for a long time while maintaining consistent characteristics and functions. Cell proliferation is the cellular process of growth and division resulting in the production of two daughter cells. Confluency is the percentage of the culture flask's or plate's surface area that is occupied by cells adhering to it.