Laboratory Safety: Hazards and Precautions

Laboratory Safety

• Laboratory safety reduces workplace injuries and illnesses
• It includes recognizing, minimizing, assessing, and preparing for hazards to create a safe environment
• The acronym RAMP guides safety:
  • Recognize hazards
  • Assess hazard risks
  • Minimize hazard risks
  • Prepare for emergencies

Historical Background of Laboratory Hazards

• A laboratory hazard is anything that can harm a vulnerable target
• Past incidents emphasize the importance of lab safety
• Marburg virus outbreak (1967): In Germany, lab workers experienced fever, diarrhea, vomiting, and internal bleeding
  • Seven died
  • The outbreak came from a previously unknown virus in monkeys imported from Uganda for polio research
  • The virus was named after the city where it was discovered
• Chernobyl disaster (1986): A safety test at the Chernobyl nuclear power plant in Ukraine caused an explosion and meltdown
  • Massive radioactive material was released
  • This caused immediate deaths, long-term health effects, and environmental contamination
  • The disaster made it the worst nuclear accident in history
• Karen Wetterhahn mercury poisoning (1997): Karen Wetterhahn at Dartmouth College in the USA was exposed to dimethylmercury during an experiment
  • A few drops went through her latex gloves and onto her skin
  • This resulted in severe mercury poisoning, neurological damage, and death within months
  • This indicated that latex gloves provides limited protection against penetrating toxic chemicals
• University of Minnesota glass vessel explosion (2008): A researcher lost an eye when a glass vessel exploded under pressure
  • The cause was a lack of proper shielding and safety protocols for pressurized experiments
• University of California electrical arc flash (2012): A graduate student suffered severe burns from an electrical arc flash while working on high-voltage equipment
  • The incident resulted from lacking training and safety protocols for electrical work
• Gold King Mine wastewater spill (2015): A cleanup operation at the Gold King Mine in the USA accidentally released 3 million gallons of toxic wastewater into a river
  • The environmental disaster resulted from poor planning and a lack of environmental safety protocols

Hazards in Cell Culture Laboratories

• Cell culture labs involve hazards when handling human or animal cells, tissues, and toxic/mutagenic reagents
• Common hazards:
  • Accidental punctures from contaminated sharps like syringe needles
  • Spills and splashes onto skin or mucous membranes
  • Ingestion through mouth pipetting
  • Inhalation of infectious aerosols

Types of Hazards

• Chemical hazards include toxic, corrosive, or irritating substances like medications, solutions, gases, vapors, aerosols, and particulate matter
  • Some chemical reactions generate heat, leading to thermal burns
  • Significant risks are Inhalation of toxic solvents and ingestion of chemicals (e.g., through contaminated hands or food)
  • It is critical to keep safe storage of food and drinks away from chemical exposure
• Physical hazards involve risks that cause physical harm due to improper handling of equipment, exposure to extreme temperatures, noise, or vibration
  • Common injuries include cuts from glassware or sharp objects and burns from hot apparatus
• Safe handling practices prevent injuries
• Electrical hazards arise from faulty wiring or equipment near liquids
  • Electrical fires are possible of due to unsafe cords or plugs
  • Ground-fault circuit interrupters prevent electrical shocks
• Radiological hazards involve the uncontrolled release of radioactive materials, which can harm people or the environment
  • Proper shielding and handling protocols prevent exposure.
• Biological hazards include bacteria, viruses, blood, tissues, and bodily fluids that can carry diseases or allergens
  • These hazards can be transmitted to lab workers, who may then become carriers, posing risks to others

Importance of Laboratory Safety

• Laboratory safety is crucial for reducing injuries and illnesses
• By minimizing or eliminating exposure to hazards, lab workers protect their health and lives
• Taking simple precautions, such as wearing seat belts to prevent car accident injuries, demonstrates proactive safety measures

Biosafety in Cell Culture Laboratories

• The primary goal of biosafety programs is to minimize or eliminate exposure to harmful biological agents, protecting workers and the environment
• Adhering to standard microbiological practices and techniques are most critical for safety in cell culture labs
• Safety equipment includes primary barriers (e.g., biosafety cabinets) and personal protective equipment (PPE), along with Material Safety Data Sheets (MSDS) for hazard communication

Biosafety Cabinets

• Biosafety cabinets reduce exposure to hazardous materials by providing an aseptic work area
• Biosafety cabinets contain infectious splashes or aerosols during microbiological procedures
• There are three classes of biosafety cabinets (Class I, II, and III) designed to meet varying research and clinical needs
• Class I biosafety cabinets protect lab personnel and the environment but do not protect cultures from contamination
  • In design and airflow characteristics, they are similar to chemical fume hoods
• Class II biosafety cabinets protects the safety of personnel, environment and samples
  • They are for work involving BSL-1, 2, and 3
  • Class II biosafety cabinets provide the aseptic work area in cell culture experiments
  • Class II biosafety cabinets help handle potentially hazardous materials (e.g., primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents)
  • Class II cabinets are most popular in cell culture laboratories because they protect the user and the samples
• Class III biosafety cabinets are gas-tight providing the highest level of protection for personnel and the environment
  • Class III biosafety cabinets are needed for work involving known human pathogens and BSL-4 materials
  • Class III biosafety cabinets are used in highcontainment laboratories where the risk of exposure to life-threatening agents is significant

Air-Flow Characteristics of Cell Culture Hoods

• Biosafety cabinets protect the working environment from dust and other airborne contaminants
• This is achieved by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area
• HEPA (High-Efficiency Particulate Air) filters can remove at least 99.97% of airborne particles (as small as 0.3 microns), including dust, pollen, mold, and bacteria
• The airflow can be horizontal (parallel to the work surface) or vertical (from the top of the cabinet onto the work surface)

Personal Protective Equipment (PPE)

• PPE forms an immediate protective barrier between personnel and hazardous agents
• PPE includes items such as gloves, laboratory coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, and goggles
• PPE is often used in combination with biosafety cabinets and other containment devices

Material Safety Data Sheets (MSDS)

• MSDS documents include and lists information about properties of a substance:
  • Physical data (melting point, boiling point, and flash point)
  • hazard information (toxicity, reactivity, and health effects)
  • safety guidelines (storage, disposal, recommended protective equipment, and spill-handling procedures)

Biosafety Levels (BSL)

• Biosafety Levels (BSL) are biocontainment precautions designed to protect lab personnel, the environment, and the community from exposure to infectious agents
• BSLs range from BSL-1 (minimal risk) to BSL-4 (highest risk)
• Biosafety Level 1 (BSL-1) has minimal risk to personnel and the environment, representing the basic level of protection
  • BSL-1 is appropriate for agents that are not known to cause disease (non-pathogenic, such as E. coli) in normal, healthy humans
  • This level requires standard microbiological practices, like handwashing and prohibiting eating or drinking in the lab
  • Work can be performed on open bench tops
  • Required safety equipment includes basic PPE, such as lab coats, gloves, and safety glasses
  • BSL-1 is commonly found in teaching laboratories for undergraduate students
• Biosafety Level 2 (BSL-2) involves moderate risk where agents pose a hazard to personnel but are unlikely to spread to the community
  • Examples include pathogens that cause mild to moderate disease, such as Salmonella spp. and hepatitis A/B/C viruses
  • In this biosafety level, access to the lab is restricted
  • Procedures that may create aerosols are conducted in biosafety cabinets (BSCs)
  • Required safety equipment includes PPE (lab coats, gloves, and face shields) and Class I or II biosafety cabinets for aerosol-generating procedures
  • An autoclave for waste decontamination and self-closing doors are required
  • BSL-2 is typically found in clinical laboratories handling human blood or tissues
• Biosafety Level 3 (BSL-3) involves high-risk agents that can cause serious or potentially lethal disease through inhalation
  • Examples include pathogens that cause severe disease, such as Mycobacterium tuberculosis and SARS-CoV-1
  • Strict access control and medical surveillance for personnel are mandatory
  • All work is conducted in biosafety cabinets or other physical containment devices
  • Required safety equipment includes enhanced PPE, such as respirators and fullbody suits, and Class II or III biosafety cabinets
  • Facility requirements include double-door entry (airlock) and HEPA filtration for exhaust air
  • BSL-3 is commonly found in research laboratories studying tuberculosis or SARS-CoV-1
• Biosafety Level 4 (BSL-4) involves maximum-risk agents that are life-threatening, with no available vaccines or treatments
  • Examples include exotic pathogens that cause severe or fatal disease, such as the Ebola and Marburg viruses
  • Strict access control and extensive personnel training are required
  • Full-body, air-supplied positive pressure suits are worn
  • All work is conducted in Class III biosafety cabinets or isolated rooms
  • Required safety equipment includes full-body, air-supplied suits with independent breathing apparatus, Class III biosafety cabinets, and an isolated building or zone within a building
  • Double-HEPA filtration for exhaust air is also required
  • BSL-4 is found in laboratories studying Ebola virus or other hemorrhagic fever viruses

Introduction to Cell Culture

• Cell culture is the process in which cells in vivo are grown outside the body under controlled conditions
• The term, “cell culture,” applies to all types of cultures including plant cells, animal cells, microorganisms, and fungi
• However, the development of animal and human cell culture techniques has driven breakthroughs in medical biotechnology
• Since the mid-20th century, in vitro cell culture methods have become more robust and reliable
• Defined broadly, cell culture refers to the maintenance and propagation of isolated cells, tissues, or organs under optimal conditions outside the body (in vitro)

History of Animal Cell Culture

• Cell culture began in the nineteenth century
• Pioneering scientists showed that cells could remain viable outside the body during this time
• In 1907, Ross G. Harrison became the first pioneer researcher to develop cell cultivation for in vitro in the early twentieth century
  • He isolated living embryonic nervous tissue from frog embryos and grew these nerve cells in a lymph solution
  • Harrison conducted extensive studies on maintaining nerve cells and monitoring fiber development
• Ross Granville Harrison encountered challenges due to bacterial contamination
  • He introduced the aseptic technique
  • This innovation enabled creating sterile preparations that could be maintained in vitro for over five weeks
• In 1912, Alexis Carrel and Montrose Burrows established in vitro cell cultures of adult tissues
  • Tissue included Connective tissue, cartilage, bone, skin, kidneys
  • They sourced from various species such as dogs, cats, chickens, guinea pigs, and rats
  • They experimented with cancerous human cells
  • The success was due to using chicken plasma as a more accessible and consistent growth medium
• Seeking advancements, Carrel explored additional media, such as diluted plasma with varying salt solution concentrations and serum
  • These innovations enabled cells to be maintained in vitro for several months, paving the way for the establishment of long-term cell lines.
• In 1916, Rous (an American pathologist) and Jones (an American scientist) introduced trypsin to detach adherent cells in culture, enabling passage and subculturing
  • Trypsin cleaves peptide bonds of proteins responsible for cell adhesion
• In 1923, Carrel and Baker identified essential nutritional requirements for cells, significantly improving culture media properties
  • They added different components to the culture media, such as glucose, amino acids, hormones, and vitamins
• Carrel also developed 'Carrel' or T-flask as the first specifically designed cell culture vessel
• In the 1940s, antibiotics (penicillin/streptomycin) were introduced to cell cultivation
  • This helped avoid contamination of the culture cells by bacteria and fungi
• In 1951, the first continuous cell line, HeLa cells, was established by George O. Gey
  • These cells were derived from a cervical carcinoma of Henrietta Lacks, after whom the cell line is named
  • Henrietta Lacks passed away from cancer in the same year
  • The immortal HeLa cell line was isolated from her cervical cancer tissue and developed at Johns Hopkins Hospital in Baltimore, Maryland
• In 1955, Harry Eagle determined the nutrient requirements for human cell culture and developed the first defined medium known as Minimum Essential Medium (MEM)
  • MEM contained essential components for cell survival and growth
• Cultures were established to investigate the development of cells and normal physiological processes like nerve development
  • Ross Harrison, in 1907, demonstrated in vitro growth of nerve fibers
• Animal cell culture was performed at an industrial scale in the 1950s and was brought to light by significant polio outbreaks
  • One of the first commercial products from cultured animal cells was the polio vaccine, which was made from a deactivated virus

Types of Animal Cell Culture

• Cell culture is the process of isolation and cultivation of human, animal, or insect cells in a favorable artificial environment outside the body to allow them to grow and divide in conditions similar to that of the body (in vitro)
• Cells may be obtained from tissues and organs by enzymatic or mechanical disintegration, or from a cell line or cell strain that has already been established.
• Two types of animal cell culture: primary cell culture and secondary cell culture
• Primary cell culture refers to cells directly isolated from human or animal tissues and organs using enzymatic or mechanical methods
  • They are placed in an artificial environment in plastic or glass containers
  • They're supported by a specialized medium containing essential nutrients and growth factors to support proliferation until they achieve confluence
  • At this stage, the cells sub-cultured by transferring them to a new vessel with fresh growth medium
• Primary cell culture can be divided into anchorage-dependent cells or anchorage-independent cells
  • Anchorage-dependent (adherent) cells are types that require a stable, nontoxic, and biologically inert surface for attachment and growth as a monolayer
  • They stop proliferating once they become confluent
  • They need periodic passaging to stimulate growth
  • Majority of vertebrate cells, a few others, are anchorage-dependent
• Anchorage-independent (suspension) cells do not require a solid surface for attachment or growth
  • They can be grown and proliferate continuously in liquid media (suspension)
  • Growth is limited by cell concentration in the medium in rotating spinner flasks, and the culture can be diluted to stimulate growth
  • Blood cells are vascular in nature and are suspended in plasma, making them easily established in suspension cultures
• Secondary cell cultures are primary cell cultures passaged or subcultured to grow long periods of time in fresh medium
  • Long-lasting of secondary cells compared to primary cell cultures is due to having fresh nutrients at regular intervals
  • Passaging or subculturing involves enzymatic digestion of adherent cells, followed by washing and re-suspending the cells in appropriate volumes of growth media
  • Secondary cell cultures are popular because they are easy to grow and readily available

Cell Line

• The primary culture becomes a cell line or sub-clone after the first subculture
• Cell lines are finite or continuous, depending on lifespan in culture
• Finite cell lines have a limited lifespan due to their restricted number of cell divisions before entering a senescence stage
  • The cells slow growth (24–96 hours) and anchorage dependence and density limitation
  • Normal adult cells have finite growth
• Indefinite cell lines have the potential to be subcultured indefinitely (immortal cell lines)
  • The cells divide rapidly, a generation time of 12–14 hours and are grown in monolayer or suspension form
  • Are anchorage-dependent and anchorageindependent cells
  • Derived from in vitro transformed or cancerous cells and can exhibit indefinite proliferation and are called continuous cell lines
• A cell strain is a subpopulation of a cell line that has been positively selected from the culture by cloning or other methods
  • Acquires additional genetic changes subsequent to initiation of the parent line

Commonly Used Cell Lines and their Origins

• HeLa - Cervix carcinoma - Human
• MCF-7 - Breast carcinoma - Human
• A549 - Lung carcinoma - Human
• HepG-2 - Hepatocellular carcinoma - Human
• HCT-116 - Colorectal carcinoma - Human
• PC-3 - Prostate carcinoma - Human
• A498 - Renal cell carcinoma - Human
• A-431 - Epidermoid carcinoma - Human
• HL-60 - Leukemia cells - Human
• C-26 - Colon carcinoma - Mouse
• CHO - Ovary - Chinese hamster
• DT40 - Lymphoma - Chick

Morphology of Cells in Culture

• The structure of cells in culture can vary and includes various types
• Epithelial-like cells: polygonal in shape and appear flattened as they attach to a substrate, forming a continuous monolayer
• Fibroblast-like cells: bipolar or multipolar cells with an elongated shape, forming an open network instead of tightly packed ones, and they attach to the substrate
• Lymphoblast-like cells: spherical in shape and typically grow in suspension without attaching to a substrate

Environmental Requirements for Animal Cell Cultivation

• The artificial environment for culturing cells consists of a vessel containing a medium that supplies nutrients (amino acids, carbohydrates, vitamins, minerals)
  • Growth factors, hormones, and regulation of the physicochemical environment (e.g., temperature, pH, osmotic pressure, O2 and CO2 tension)
  • The culture environment is controlled by the growth medium, except for temperature
• Culture media is a most important factors in animal cell culture that affects in vitro growth of animal cells require appropriate nutritional, hormonal, and growth factors
  • Environmental conditions closely resemble their in vivo milieu
• Basic components in culture media:
  • Energy sources: Every growth medium requires a carbon source that cells can metabolize
    • Glucose is considered the primary fuel source in cell culture and is used to synthesize adenosine triphosphate (ATP)
    • Commonly added at 5.5 mM, approximately equal to the glucose levels found in human blood
    • Media contains a wide range of glucose concentrations to suit needs
    • Sugars/carbon sources other than glucose, such as fructose, sucrose, pyruvate and glutamine, can be used
  • Vitamins: Vitamins play roles in cell growth and productivity, such as thiamine (B1), riboflavin (B2), biotin (B7), and cobalamin (B12)
  • Inorganic salts: Inorganic salts provide ions such as Ca2+, Mg2+, Na+, and K+, for cell growth and sustenance
    • Salts maintain osmotic balance of the cell culture medium and structural integrity
  • Amino acids: Glutamine is an essential amino acid for almost all cultures and serves as the major energy source for cell proliferation, even in preference to glucose
    • Glutamine is not stable in solution, even at +4°C, so it must be added just before use.
  • Fat and fat soluble components: Fat and fat-soluble components, such as fatty acids/cholesterol serve as energy stores
  • Growth factors and hormones: Growth factors, such as epidermal growth factor (EGF) and fibroblast growth factor (FGF), play roles in proliferation and differentiation
    • Hormones like insulin and transferrin are also crucial
    • Insulin regulates glucose and protein metabolism
    • Transferrin incorporates iron into cells
  • Antibiotics: Typically added to minimize the risk of microbial contamination, even aseptic techniques are used
    • The most common antibacterial supplement is a combination of penicillin and streptomycin (pen/strep), used at 50–100 IU/mL penicillin and 50–100 µg/mL streptomycin in a complete cell culture medium
  • pH and buffering systems: Biological processes are generally only possible when the pH is within a specific range
    • Changes in pH within the cell culture environment can affect cellular process, such as metabolism and cell growth
    • Extracellular pH is slightly alkaline, from 7.3 to 7.4, while intracellular pH is slightly lower, at approximately 7.2
• pH buffers & how they works
  • A buffer is a mixture of a weak acid or weak base and its conjugate base or acid
    • Buffers neutralize acid (H⁺ ions) or base (OH⁻ ions) for optimal pH
    • A pH buffer acts as either a weak acid or a weak base, ensuring that the medium remains somewhat resistant to changes in pH
    • This occurs because the buffer can donate or accept hydrogen ions, for establishing pH
    • Acid + Base ⇌ Conjugate Base + Conjugate Acid
  • A 5% CO₂ level is needed to buffer the system and maintain normal physiological pH for optimal cell growth
    • In incubators with high carbon dioxide levels, CO₂ reacts with water to produce hydrogen ions, making the medium acidic
    • Most media uses a chemical buffering system to counteract this acidity and maintain the optimal pH in culture
    • The most common buffer used in mammalian cell culture is sodium bicarbonate
    • Sodium bicarbonate-buffered media are sensitive to carbonic acid, due to CO₂ concentration
    • The medium pH is maintained when CO₂ level is controlled
    • HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), a stronger buffer, may also be used
      • Higher concentrations of HEPES than bicarbonate are required when used in a CO₂ atmosphere
      • Density of the culture may affect CO₂ requirement
      • Phenol red in the medium indicates the pH state at any given time
  • Phenol Red
    • Many media formulations include a pH indicator called phenol red for maintain pH
    • Phenol red turns yellow in acidic conditions (<6.8) and pink in basic conditions (>8.2)
    • A drop in medium pH typically indicates buildup of lactic acid, a byproduct of cellular metabolism or contamination
    • Conversely, a rise in pH values suggests insufficient CO₂ in the incubator
• Serum: The clear. yellowish fluid is obtained after fibrin and cells are removed from blood
  • Animal-derived serum (5–10%), serves as a source of amino acids, hormones, growth factors, vitamins, proteins, inorganic salts, carbohydrates, and lipids for cellular metabolism and growth
  • Initially, fetal bovine serum (FBS) only supported all growth, but FBS has essential components for cell attachment, proliferation, and maintenance
    • Albumin, contains lower amounts of y-globulin (a cell growth inhibitor)
    • Regulates cell membrane permeability and acts as a carrier for lipids, enzymes, micronutrients, and trace elements into the cell
  • Three basic classes of media can differ in their need for serum supplementation
    • Basal media: Most cell lines grow best in basal media (amino acids, vitamins, inorganic salts, and a carbon source like glucose) - These basal media formulations are supplemented with serum - Dulbecco’s Modified Eagle’s Medium (DMEM) contains amino acids, salts, vitamins, and glucose
    • Reduced-serum media: used for cell culture experiments to minimize the serum - Reduced-serum media is enriched with nutrients and animal factors, which reduce the amount of serum - Reduced-serum media is an improved version of Minimal Essential Medium (MEM) that allows for at least a 50% reduction in FBS supplementation without affecting growth rate or morphology - Useful for synchronizing the cell cycle and reduce serum protein interference during the transport of genetic material cells in transfection experiments
    • Serum-free media: Eliminate the issues associated with animal sera, such as contamination - serum-free media are often replaced with appropriate nutritional and hormonal formulations - The ability to make the medium selective for specific cell types by choosing the right combination of growth factors - Low growth rates and saturation densities are commonly achieved using supplement media - SFM tends to be expensive and supplemented with hormones. - Ham’s medium exists as an option

Temperature

• Temperature affects growth and production processes
• The optimal temperature for cell culture depends on the body temperature of the host that the cells were
• Mammalian cells should be at 36.5 °C to 37.5 °C
• While cell lines derived from cold-blooded animals are at 15 °C and 30 °C

Growth Curve of Cells

• A typical growth curve displays a sigmoid pattern of proliferation, defining Lag phase, exponential (log) phase, and plateau (stationary) phase
  • Lag phase
    • This follows subculture and reseeding that is the cell population takes time.
    • The cell number is constant before the rapid growth begins
    • Phase: cells replace the glycocalyx elements and attach to the substrate, and spread out
  • Logarithmic (Log) phase increases rapidly due to cell division
    • Length depends on seeding density, growth rate, and which the inhibited
    • Provides a high grow rate cells are most reproducible
    • Passaging cells is popular
  • Plateau phase is most cells being inhibited and exhausted to grow
    • Growth is 0% to 10% if the injury limited to a low culture

Subculture (Cell Passaging) and Use of Trypsin

• Passaging subcultures cells to harvest more cells
  • The homogeneity must be high
  • Reduces a large transfer to new cell
  • Then they will continue to transfer on
• Attached cells will be attached cells on media
  • EDTA and trypsin will make sure cells stick properly

The growth of cells

• In culture progresses goes from seeding
• Cells will occupy where need to have space
• Maintenance must come from original

Quantitation of Cells (Cell Counting)

• Determine when viable before use
  • Its known for steps
  • Its to improve quality of culture
• Hemocytometer
  • Cell need to be know
  • The tool is in most facilities.
  • The hemocytometer is two devices
  • To make the cell right count
  • The amount of cells right is need to use optics
  • Trypan blue is useful to find viable
  • Then you known what you using

Contamination

• One of the most frequent the cells
  • Is two main
  • Chemical are media or anything with water
  • Also biology like Bactria
  • Reduce bacteria to the best use

Microorganisms

• Is useful when need aseptic tool