Lecture 2 Animal Culture PDF

Dr Mohd Nazmi Bin Abd Manap Cell Culture Room Design Layout of cell culture room Depends on the type of scale of operations and user number Room must be designed so that it is easily to clean Furniture tightly fitted to the floor with space underneath for cleaning Floor material made of...

Dr Mohd Nazmi Bin Abd Manap Cell Culture Room Design Layout of cell culture room Depends on the type of scale of operations and user number Room must be designed so that it is easily to clean Furniture tightly fitted to the floor with space underneath for cleaning Floor material made of vinyl/acrylic coating (dustproof) Floor levelling – slight fall towards drain Preferably tissue culture lab separated from preparation, washup and sterilization area (same floor, adjacent room, no stairs for movement of cart/materials) Build requirement Number of users Space availability Location of preparation area Storage Accessibility Containment and sterility Small Cell Culture Lab Layout Self-contained lab suitable for 2- 3 persons. Cell Culture Lab with Adjacent Prep Room Medium size cell culture lab Attached to preparation area, microscope room and 37°c room. Advantages of separate adjacent room Offers protection against contamination Allows cell culture stock to be kept separately from regular laboratory reagents, glassware and apparatus (safety purpose) Services Requirement: water, electricity, gas methane/propane, carbon dioxide and compressed air Electricity must be sufficient to power equipment Water supply from sink with proper drainage including adequate drainage on floor especially in preparation/washup area Gas such as carbon dioxide must be piped into the facility Air Pressure Balance Ventilation Cell culture lab should be at positive pressure relative to surrounding work areas (avoid contaminated air from going in) Capability changing to negative pressure if containment is required. To fulfill both requirement lab must contain a buffer zone equip with HEPA filter to received air from outside the lab. Laminar flow hood must duct air outside and must be run continuously. Must have alternative air duct, if there’s a need to switch off the laminar flow. Layout Single room design must have ‘sterility gradient’. Preparation area adjacent to wash up & strerilisation area Storage and incubators accessible to sterile working area Clean area Storage & Wash up & for sterile Preparation Incubation Sterilization handling Service Bench Must be positioned and reachable to the sterile handling area. Bench for a cell counter, microscope, and other critical instruments. Provide storage of sterile glassware,plastics, pipettes, screw caps, and syringe. Additional equipment can be placed. Ex: Small Centrifuge. Horizontal Laminar flow There are two types of Laminar flow. Horizontal laminar flow chamber Airflow blows from the side facing users and the air is not circulated Aseptic environment due to stable airflow The downside is that the air flow is direct to the user and can harm the user Cheaper to obtain Vertical Laminar flow Vertical flow chamber Using the principle of vertical air flow The flow of sterile air is from the top to the bottom (working area), The air either recirculated or vented. The airflow at the front of the chamber is also drawn in to create a sterile working area Does not harm the user Suitable for techniques involving hazardous materials such as radioactivity, viruses and animal cells especially a Class II biosafety cabinet. Some have UV light to sterilize the working area. Usually switch on before commencing work. Exhaust air is passed through a certified HEPA filter and is particulate-free, which provides environmental protection. Biosafety Cabinets Containment devices with HEPA (high efficiency particulate air filter) and is designed to protect user, working environment and product from biohazardous materials. A place for aseptic technique manipulation The work area is prevented from contamination by a stable air flow. The airflow is sterile due to the presence of a HEPA (High Efficient Particulate Air) filter, which works to filter air particulate. Biosafety cabinets provide protection to environment, personnel and product while laminar flow only provide protection to environment and product, neglecting user. Type & Class of biosafety cabinets Class I Class II Class III Class I Biosafety Cabinet Description: Class I biosafety cabinets are basic cabinets that provide protection to the user and the environment. They have a HEPA filter that filters the air and a blower that creates negative pressure to prevent contaminants from escaping. Usage: They are suitable for handling low to moderate-risk materials, including aerosols, powders, and liquids. They are commonly used for tasks such as transferring liquids, preparing cultures, and handling animal tissues. Class II Biosafety Cabinet Description: Class II biosafety cabinets are more advanced than Class I cabinets and provide protection to the user, the environment, and the sample. They have HEPA filters for air filtration, and a blower system that creates negative pressure. Usage: Class II biosafety cabinets are suitable for handling low to high-risk materials, including infectious agents, toxic chemicals, and carcinogens. There are three subtypes of Class II biosafety cabinets, including Type A2, Type B1, and Type B2. Most cell culture laboratory are equip with Class II BSC which provide moderate protection. Class III Biosafety Cabinet Description: Class III biosafety cabinets are highly specialized and provide maximum protection to the user, the environment, and the sample. They are completely enclosed and have airtight seals. Usage: Class III biosafety cabinets are suitable for handling high- risk materials, including highly infectious agents and radioactive materials. Biosafety Cabinet Class I and II Biosafety cabinets are used for Biosafety levels I and II but, when used correctly in conjunction with useful microbiological techniques, these provide an effective containment system for safe manipulation of moderate and high-risk microorganisms. Class III BSCs are most suitable for work with hazardous agents that require Biosafety Level 3. Essential Equipment in Cell Culture Lab Laminar flow hood (BSc Type II) Pipette controller Inverted microscope (optional: equip with fluorescence detection) Water bath CO2 Incubators Refrigerator and freezer Centrifuge Liquid N2 storage Dewar Pipette controller Dispensing and withdrawing medium, reagents, and cells Transfer amount ranging from 1ml to 100ml. Motorized pipette which may be obtained with a separate or built-in pump and can be mains operated or rechargeable. Usually have a filter at the pipette insert to minimize the transfer of contaminants Serological pipettes Disposable one time use Sterilised, cotton plugged and packed individually. Made of plastic material. Could range from 1ml to 100ml Inverted Microscope For routine view of cultured cells, as well as to detect morphological changes and detect contamination. View the cell tissues in their original vessel, which are larger than microscopic slides, which makes it better than the upright microscope which only views specimens in small microscopic slides. Water Bath Used to heat or cool certain solutions or reagents that are used in cell culture preparation. For example, media and buffers used in cell culture are often stored at low temperatures to prevent bacterial growth and maintain their stability. A water bath can be used to warm these solutions to the desired temperature before they are added to the cell culture. CO2 incubator Essential for cell culture because they provide a controlled environment that mimics the conditions found invivo. Designed to maintain a stable temperature, humidity, and pH level, as well as a specific concentration of CO2 gas. Typically, mammalian cells require a CO2 concentration of 5%, which is the same as that found in human blood. It provides a closed system where the concentration of CO2 can be precisely controlled, ensuring the cells are able to grow and proliferate. Humidified environment provided by these incubators helps to prevent the cells from drying out, which can lead to cell death. The stable temperature also ensures that the cells remain at an optimal temperature for growth, while the filtered air prevents contamination from bacteria and other microbes. Centrifuge Usage: Cell washing: After cells have been cultured, they may need to be washed to remove any residual media, serum, or other contaminants. Centrifugation can be used to gently pellet the cells, allowing for easy removal of the supernatant without disturbing the cell pellet. Harvesting: When cells have reached a certain density or have undergone a specific treatment, they may need to be harvested for further analysis or experimentation. Centrifugation can be used to collect and concentrate the cells, making them easier to work with. Liquid N2 storage Dewar Commonly used in a culture lab to store and preserve biological samples such as cells, tissues, and microorganisms. This is because liquid nitrogen is an extremely cold and stable temperature (-196°C or -320.8°F), which can help to maintain the viability and integrity of these biological samples over extended periods of time. When samples are stored at ultra-low temperatures, their metabolic processes slow down, preventing cellular damage and degradation. Therefore, by using a liquid nitrogen dewar to store biological samples, researchers can ensure that the samples remain viable and functional for future experiments or analysis. In addition to its use in sample storage, liquid nitrogen can also be used to flash freeze samples for cryopreservation, which involves freezing the sample at an extremely rapid rate to minimize ice crystal formation and preserve the sample's integrity. Liquid N2 storage Dewar Consumables T25 and T75 Falcon tubes Multiwell plates Serological Pipettes T25 and T75 flask Provide a convenient way to grow and maintain cells in culture. Designations refer to the approximate surface area of the flask, which is important because it determines the maximum number of cells that can be grown in the flask. Example: T75 flasks have a surface area of approximately 75 cm² Made of polystyrene, which is a material that is compatible with a wide range of cell types. Convenient, versatile, and provide a reliable way to grow and maintain cells in culture. Falcon tubes Used to prepare and store cell culture media Used for centrifugation to separate cells based on their size and density Used to store cell samples in a refrigerator or freezer until they are needed for experiments or analysis Multiwell Plate Providing a sterile surface for cell growth Facilitating cell observation and manipulation Come in various sizes and shapes, including multi-well plates, which allow for multiple cell culture experiments to be performed simultaneously. The wells in these plates also make it easy to observe and manipulate cells under a microscope. Used to test the effect of different experimental conditions on cell growth and behavior. For example, researchers can test how different nutrients, growth factors, or drugs affect cells by culturing them under different conditions in different wells of the plate. Media Formulations for Cell Culture Dr Mohd Nazmi Bin Abd Manap Types of Cell Culture Media Basal Media Complete Media Serum Free Media (SFM) Basal Media Contain essential nutrients needed for cell growth and maintenance, such as amino acids, vitamins, glucose, minerals, and salts. Examples of basal media include Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), and RPMI-1640. Complete Media Contain all the essential nutrients needed for cell growth and maintenance, as well as serum or other supplements. Serum is a complex mixture of growth factors, hormones, and other biomolecules derived from animal blood. These media are commonly used for routine cell culture and can support a wide range of cell types. Examples of complete media include DMEM with 10% fetal bovine serum (FBS) and MEM with 10% calf serum. Complete Media Constituents of complete media Amino acids – essential amino acids such as cysteine, arginine, glutamine, and tyrosine Vitamins – B, choline, folic acid, inositol, nicotinamide. Some media such as M199 contains A,D,E,K. Salts - Na+, K+, Mg2+, Ca2+, Cl−, SO42−, PO43−, and HCO3− contribute to medium osmolality Glucose – Source of energy and will be metabolized by glycolysis Organic Supplement - proteins, peptides, nucleosides, citric acid cycle intermediates, pyruvate Serum - contains growth factors, which promote cell proliferation, and adhesion factors and antitrypsin activity, which promote cell attachment. Serum Free Media Do not contain serum, but instead, are supplemented with defined components, such as growth factors, hormones, and lipids, to support cell growth. These media are often used for specific applications, such as drug discovery and tissue engineering, and can minimize the variability associated with using serum- containing media. Examples of serum-free media include Chemically Defined Media (CDM) and Serum- free Media (SFM). Advantages of Serum Free Media Better defined composition: SFM have a well-defined composition, which makes them more reproducible and easier to standardize than serum-containing media. This allows for greater experimental control and reduces the risk of batch-to-batch variation. Reduced variability: Because serum is a complex mixture of biomolecules, it can introduce a lot of variability into cell culture experiments. SFM can reduce this variability and improve the consistency of experimental results. Advantages of Serum Free Media Lower risk of contamination: Serum is a potential source of contamination with viruses, bacteria, and other microorganisms, which can affect cell growth and function. SFM can reduce this risk and improve the safety of cell culture experiments. Better control of growth factors: SFM can be formulated with specific growth factors, cytokines, and other signaling molecules to support the growth and differentiation of specific cell types. This allows for greater experimental flexibility and control. Disadvantages of Serum Free Media Higher cost: SFM are often more expensive than serum-containing media, which can be a barrier to their use, especially for large-scale cell culture experiments. Lower cell growth: Some cell types may not grow as well in SFM as they do in serum-containing media, which can limit their usefulness for certain applications. More difficult to optimize: Because SFM have a defined composition, it can be more difficult to optimize their formulation for specific cell types and experimental conditions. This can require more extensive testing and optimization to achieve optimal results. Limited availability: SFM may not be available for all cell types and applications, which can limit their usefulness in certain experiments. Physicochemical Properties pH pH of cell culture media should be maintained within a narrow range, typically between 7.2 and 7.4, to support optimal cell growth and function. Changes in pH can affect enzyme activity, protein conformation, and membrane integrity, leading to cell death or dysfunction. Phenol red is commonly used as an indicator. pH 7.8 - purple pH 7.6 – pink ph 7.4 – red pH 7.0 – orange pH 6.5 – yellow pH