Introduction to Cell Culture PDF

INTRODUCTION TO CELL CULTURE What is Cell Culture?  Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.  The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means befor...

INTRODUCTION TO CELL CULTURE What is Cell Culture?  Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.  The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or  they may be derived from a cell line or cell strain that has already been already established. 1) Primary Culture:- Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence).  At this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth. 2) Cell Line :-  After the first subculture, the primary culture becomes known as a cell line or subclone.  Cell lines derived from primary cultures have a limited life span (i.e., they are finite), and as they are passaged, cells with the highe growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. 3) Cell Strain:-  If a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line becomes a cell strain.  A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line.  Finite vs Continuous Cell Line:-  Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is genetically determined event known as senescence; these cell lines are known as finite cell line.  some cell lines become immortal through a process called transformation, which can occur spontaneously or can be chemically or virally induced. When a finite cell line undergoes transformation an acquires the ability to divide indefinitely, it becomes a continuous cell line.  Culture Conditions:-  Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of 1) a suitable vessel containing a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), 2) growth factors, 3) hormones, 4) gases (O2, CO2), 5) regulates the physicochemical environment (pH, osmotic pressure, temperature). 6) Most cells are anchoragedependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), 7) others can be grown floating in the culture medium (suspension culture). Cryopreservation:- If a surplus of cells are available from subculturing, they should be treated with the appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below –130°C (cryopreservation) until they are needed  Morphology of Cells in Culture :-  three basic categories based on their shape and appearance (i.e., morphology). cells shape growth Fibroblastic  are bipolar or grow attached to (or fibroblast-like) multipolar, a substrate  have elongated shapes Epithelial-like polygonal in shape Grow attached to a cells with more regular substrate in discrete dimensions patches Lymphoblast- spherical in shape grown in suspension like cells without attaching to a surface Applications of Cell Culture:- 1) Cell culture is one of the major tools used in cellular and molecular biology, 2) providing excellent model systems for studying  the normal physiology and biochemistry of cells (e.g., metabolic studies, aging),  the effects of drugs and toxic compounds on the cells,  mutagenesis and carcinogenesis. 1) It is also used in drug screening and development, and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). 2) The major advantage of using cell culture for any of the these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells. Cell Culture Laboratory :- Safety:- a cell culture laboratory has a number of specific hazards associated with handling and manipulating human or animal cells and tissues, as well as toxic, corrosive, or mutagenic solvents and reagents. The most common of these hazards are  accidental punctures with syringe needles or other contaminated sharps,  spills and splashes onto skin and mucous membranes,  ingestion through mouth pipetting,  inhalation exposures to infectious aerosols. The fundamental objective of any biosafety program:- is to reduce or eliminate exposure of laboratory workers and the outside environment to potentially harmful biological agents The most important element of safety in a cell culture laboratory is the strict adherence to standard microbiological practices and techniques. Biosafety Levels :- Acc to Centers for Disease Control (CDC) and the National Institutes of Health (NIH), four ascending levels of containment, referred to as biosafety levels 1 through 4:- 1)Biosafety Level 1 (BSL-1)  BSL-1 is the basic level of protection common to most research and clinical laboratories, and is appropriate for agents that are not known to cause disease in normal, healthy humans. 2)Biosafety Level 2 (BSL-2) BSL-2 is appropriate for moderate-risk agents known to cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. Most cell culture labs should be at least BSL-2, but the exact requirements depend upon the cell line used and the type of work conducted. 3)Biosafety Level 3 (BSL-3) BSL-3 is appropriate for indigenous or exotic agents with a known potential for aerosol transmission, and for agents that may cause serious and potentially lethal infections. 4)Biosafety Level 4 (BSL-4) BSL-4 is appropriate for exotic agents that pose a high individual risk of life- threatening disease by infectious aerosols and for which no treatment is available. These agents are restricted to high containment laboratories.  Safety Data Sheet (SDS) :-  also referred to as Material Safety Data Sheet (MSDS), is a form containing information regarding the properties of a particular substance.  The SDS includes physical data such as melting point, boiling point, and flash point, information on the substance’s toxicity, reactivity, health effects, storage, and disposal, as well as recommended protective equipment and procedures for handlin Safety Equipment :- 1) primary barriers such as biosafety cabinets, enclosed containers, and other engineering controls designed to remove or minimize exposure to hazardous materials. 2) personal protective equipment(PPE):- that is often used in conjunction with the primary barriers. 3)The biosafety cabinet :- (i.e., cell culture hood) is the most important equipment to provide containment of infectious splashes or aerosols generated by many microbiological procedures as well as to prevent contamination of your own cell culture. Personal Protective Equipment (PPE):- form an immediate barrier between the personnel and the hazardous agent, they include items for personal protection such as gloves ,laboratory coats and gowns, shoes covers, boots, respirators, face shields, safety glasses, or goggles. They are often used in combination with biosafety cabinets and other devices that contain the agents or materials being handled. We recommend that you consult your institution’s guidelines for the appropriate use of PPE in your laboratory.  Safe Laboratory Practices:-  The following recommendations are simply guidelines for safe laboratory practices, and they should not be interpreted as a complete code of practice. Consult your institution’s safety committee and follow local rules and regulations pertaining to laboratory safety. 1)Always wear appropriate personal protective equipment. Change gloves when contaminated, and dispose of used gloves with other contaminated laboratory waste. 2)Wash your hands after working with potentially hazardous materials and before leaving the laboratory. 3) Do not eat, drink, smoke, handle contact lenses, apply cosmetics, or store food for human consumption in the laboratory. 4) Follow the institutional policies regarding safe handling of sharps (i.e., needles, scalpels, pipettes, and broken glassware). 5) Take care to minimize the creation of aerosols and/or splashes. 6) Decontaminate all work surfaces before and after your experiments, and immediately after any spill or splash of potentially infectious material with an appropriat disinfectant. Clean laboratory equipment routinely, even if it is not contaminated. 7) Decontaminate all potentially infectious materials before disposal. 8) Report any incidents that may result in exposure to infectious materials to appropriate personnel (e.g., laboratory supervisor, safety officer). Cell Culture Equipment :-  The specific requirements of a cell culture laboratory depend mainly on the type of research conducted; for example, the needs of mammalian cell culture laboratory specializing in cancer research is quite different from that of an insect cell culture laboratory that focuses on protein expression  All cell culture laboraties have the common requirement of being free from any microorganism 1) Basic Equipment :- Cell culture hood (i.e., laminar-flow hood or biosafety cabinet) Incubator (humid CO2 incubator recommended) Water bath Centrifuge Refrigerator and freezer (–20°C) Cell counter (e.g., Countess® Automated Cell Counter or hemacytometer) Inverted microscope Liquid nitrogen (N2) freezer or cryostorage container Sterilizer (i.e., autoclave) 2) Expanded Equipment :- Aspiration pump (peristaltic or vacuum) pH meter Confocal microscope Flow cytometer 3) Additional Supplies:- Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multi-well plates) Pipettes and pipettors Syringes and needles Waste containers Media, sera, and reagents Cells Aseptic Work Area :-  The major requirement of a cell culture laboratory is the need to maintain an aseptic work area that is restricted to cell culture work.  Although a separate tissue culture room is preferred, a designated cell culture area within a larger laboratory can still be used fort sterile handling, incubation, and storage of cell cultures, reagents, and media.  The simplest and most economical way to provide aseptic conditions is to use a cell culture hood (i.e., biosafety cabinet). Cell Culture Hood :-  The cell culture hood provides an aseptic work area while allowing the containment of infectious splashes or aerosols generated by many microbiological procedures.  Three kinds of cell culture hoods, designated as Class I, II and III, have been developed to meet varying research and clinical needs. Classes of Cell Culture Hoods :- Class I :-  cell culture hoods offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques, but they do not provide cultures protection from contamination.  They are similar in design and air flow characteristics to chemical fume hoods. Class II :-  cell culture hoods are designed for work involving BSL-1, 2, and 3 materials, and they also provide an aseptic environment necessary for cell culture experiments.  A Class II biosafety cabinet should be used for handling potentially hazardous materials (e.g., primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents). Class III :-  biosafety cabinets are gas-tight, and they provide the highest attainable level of protection to personnel and the environment.  A Class III biosafety cabinet is required for work involving known human pathogens and other BSL-4 materials.  Air-Flow Characteristics of Cell Culture Hoods :-  Cell culture hoods protect the working environment from dust and other airborn contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area. Depending on its design :- horizontal flow hood Vertical flow hoods blowing parallel to the work blowing from the top of the cabinet surface. onto the work surface. provides protection to the culture Provide significant protection to (if the air flowing towards the the user and the cell culture user) or to the user (if the air drawn in through the front of the cabinet by negative air pressure inside). Clean Benches:- Horizontal laminar flow or vertical laminar flow “clean benches” are not biosafety cabinets; these pieces of equipment discharge HEPA-filtered air from the back of the cabinet across the work surface toward the user, and they may expose the user to potentially hazardous materials. These devices only provide product protection. Clean benches can be used for certain clean activities, such as the dust-free assembly of sterile equipment or electronic devices, they should never be used when handling cell culture materials or drug formulations, or when manipulating potentially infectious materials.  Cell Culture Hood Layout:- A cell culture hood should be 1) large enough to be used by one person at a time, 2) be easily cleanable inside and outside, 3) have adequate lighting, 4) be comfortable to use without requiring awkward positions. 5) Keep the work space in the cell culture hood clean and uncluttered, 6) keep everything in direct line of sight. 7) Disinfect each item placed in the cell culture hood by spraying them with 70% ethanol and wiping clean. The arrangement of items within the cell culture hood usually adheres to the following right-handed convention :- 1)A wide, clear work space in the center with your cell culture vessels 2)Pipettor in the front right, where it can be reached easily 3)Reagents and media in the rear right to allow easy pipetting 4) Tube rack in the rear middle holding additional reagents 5) Small container in the rear left to hold liquid waste  The basic layout of a cell culture hood for right-handed workers. Left-handed workers may switch the positions of the items laid out on the work surface Incubator :- The purpose of the incubator:- is to provide the appropriate environment for cell growth. Desine:- 1)The incubator should be large enough for your laboratory needs, 2)have forcedair circulation, 3)should have temperature control to within ±0.2°C. 4)Stainless steel incubators allow easy cleaning and provide corrosion protection, especially if humid air is required for incubation.  Although the requirement for aseptic conditions in a cell culture incubator is not as stringent as that in a cell culture hood, frequent cleaning of the incubator is essential to avoid contamination of cell cultures Types of Incubators:- (There are two basic types of incubators) Dry incubators Humid CO2 incubators  are more economical.  are more expensive.  require the cell cultures to be  allow superior control of culture incubated in seale flasks to prevent conditions. evaporation. Placing a water dish  They can be used to incubate cells in a dry incubator can provide cultured in Petri dishes or multi-well somehumidity, plates which require a controlle  but they do not allow precise atmosphere of high humidity and control of atmospheric conditions increased CO2 tension. in the incubator. Storage :- A cell culture laboratory should have storage areas for liquids such as media and reagents, for chemicals such as drugs and antibiotics, for consumables such as disposable pipettes, culture vessels, and gloves, for glassware such as media bottles and glass pipettes, for specialized equipment, for tissues and cells.  Glassware, plastics, and specialized equipment can be stored at ambient temperature on shelves and in drawers; however, it is important to store all media, reagents, and chemicals according to the instructions on the label.  Some media, reagents, and chemicals are sensitive to light; while their normal laboratory use under lighted conditions is tolerated, they should be stored in the dark or wrapped in aluminum foil when not in use. Storage :- 1) Refrigerators :- For small cell culture laboratories: a domestic refrigerator (preferably one without a autodefrost freezer) is an adequate and inexpensive piece of equipment for storing reagents and media at 2–8°C. For larger laboratories:- a cold room restricted to cell culture is more appropriate.  Make sure that the refrigerator or the cold room is cleaned regularly to avoid contamination. 2) Freezers:- Most cell culture reagents can be stored at –5°C to –20°C; therefore an ultradeep freezer (i.e., a –80°C freezer) is optional for storing most reagents. A domestic freezer is a cheaper alternative to a laboratory freezer. While most reagents can withstand temperature oscillations in an autodefrost (i.e., self-thawing) freezer, some reagents such as antibiotics and enzymes should be stored in a freezer that does not autodefrost Storage :- 3) Cryogenic Storage :- Cell lines in continuous culture are likely to suffer from genetic instability as their passage number increases; therefore, it is essential to prepare working stocks of the cells and preserve them in cryogenic. Do not store cells in –20°C or –80°C freezers?? because their viability quickly decreases when they are stored at these temperatures.  There are two main types of liquid-nitrogen storage systems, Vapor phase systems liquid phase systems  minimize the risk of  have longer static explosion with cryostorage holding times, and are therefore tubes, and are required for more economical. storing biohazardous materials  which come as wide-necked or narrow-necked storage containers Narrow-necked containers wide-necked containers * a slower nitrogen evaporation rate * allow easier access * are more economical * larger storage capacity. Storage :- 4) Cell Counter :- is essential for quantitative growth kinetics, and a great advantage when more than two or three cells  The Countess® Automated Cell Counter :-  is a bench-top instrument designed to measure cell count and viability (live, dead, and total cells) accurately and precisely in less than a minute per sample,  using the standard Trypan Blue uptake technique. the same amount of sample that you currently use with the ,... hemacytometer,  takes less than a minute per sample for a typical cell count and is compatible with a wide variety of eukaryotic cells.ell lines are cultured in the laboratory. Aseptic Technique :-  designed to provide a barrier between the microrganisms in the environment and the sterile cell culture,  depends upon a set of procedures to reduce the probability of contamination from these sources.  The elements of aseptic technique are :- 1) sterile work area, 2) good personal hygiene, 3) sterile reagents and media, 4) sterile handling. Aseptic Technique :- 1) Sterile Work Area :-  The simplest and most economical way to reduce contamination from airborne particles and aerosols (e.g., dust, spores, shed skin, sneezing) is to use a cell culture hood. The cell culture hood should be properly set up and be located in an area that I restricted to cell culture that is free from drafts from doors, windows, and other equipment, and with no through traffic. The work surface should be uncluttered and contain only items required for a particular procedure; it should not be used as a storage area. Before and after use, the work surface should be disinfected thoroughly, and the surrounding areas and equipment should be cleaned routinely. For routine cleaning, wipe the work surface with 70% ethanol before and during work, especially after any spillage. You may use ultraviolet light to sterilize the air and exposed work surfaces in the cell culture hood between uses. Using a Bunsen burner for flaming is not necessary nor recommended in a cell culture hood. Leave the cell culture hood running at all times, turning them off only when they will not be used for extended periods of time. Aseptic Technique :- 2) Good Personal Hygiene :- 1)Wash your hands before and after working with cell cultures. 2) In addition to protecting you from hazardous materials, wearing personal protective equipment also reduces the probability of contamination from shed skin as well as dirt and dust from your clothes. 3) Sterile Reagents and Media:-  Commercial reagents and media undergo strict quality control to ensure their sterility, but they can become contaminated while handling.  Follow the guidelines below for sterile handling to avoid contaminating them. Always sterilize any reagents, media, or solutions prepared in the laboratory using the appropriate sterilization procedure (e.g., autoclave, sterile filter). Aseptic Technique :- 4) Sterile Handling :- Always wipe your hands and your work area with 70% ethanol. Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell culture hood. Avoid pouring media and reagents directly from bottles or flasks. Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids, and use each pipette only once to avoid cross contamination. Do not unwrap sterile pipettes until they are to be used. Keep your pipettes at your work area. Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in resealable bags to prevent microorganisms and airborn contaminants from gaining entry. Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to use it and never leave it open to the environment. Return the cover as soon as you are finished. If you remove a cap or cover, and have to put it down on the work surface, place the cap with opening facing down. Use only sterile glassware and other equipment. Be careful not to talk, sing, or whistle when you are performing sterile procedures. Perform your experiments as rapidly as possible to minimize contamination.  Biological Contamination:-  Cell culture contaminants can be divided into two main categories, 1)chemical contaminants:- -such as impurities in media, sera, and water, endotoxins, plasticizers, an detergents, and 2)biological contaminants:- -such as bacteria, molds, yeasts, viruses, mycoplasma, as well as cross contamination by other cell lines.  it is impossible to eliminate contamination entirely,  it is possible to reduce its frequency and seriousness by gaining a thorough understanding of their sources and. by following good aseptic technique