SIO2004 Animal Cell and Tissue Culture Lecture 3 PDF

SIO2004 Animal Cell and Tissue Culture Lecture 3 Biotechnology Program University of Malaya Instructor: Dr. Nuradilla Mohamad Fauzi  Now that you have cells … how do you maintain them in vitro? Culture Requirements Factors affecting cell behavior in the comple...

SIO2004 Animal Cell and Tissue Culture Lecture 3 Biotechnology Program University of Malaya Instructor: Dr. Nuradilla Mohamad Fauzi  Now that you have cells … how do you maintain them in vitro? Culture Requirements Factors affecting cell behavior in the complex in vivo environment: The local micro-environment: metabolites, local growth factors, ECM, architecture Cell-cell interactions Circulating proteins, cytokines, hormones Physicochemical parameters How to best mimic this in vitro? Requirements for cell maintenance a) Culture surface b) Gas phase c) Temperature and humidity d) Media: amino acids, vitamins, salts, energy source, etc. e) pH and buffering f) Osmotic balance g) Serum factors: growth factors, hormone, lipids, etc. h) Sterility Culture Surface Most adherent cells (anchorage dependent) require attachment to proliferate Plastics for cell culture are specially treated Increase negative charge to make the hydrophobic plastic more hydrophilic Also, some are coated with extracellular matrix, attachment and adhesion proteins Collagen, laminin, fibronectin For basic culturing: cells are cultured in disposable plastic flasks, dishes and plates  “T” flasks Named after surface area e.g. T-25, T-75, T-175 Dishes Named by diameter e.g. 10-cm dish Multi-well plates Named by number of wells e.g. 6-well plates, 24-well plates, 96-well plates The gas phase Oxygen Important for aerobic metabolism Standard culture conditions: ~20% (from ambient air) Some cell cultures prefer lower oxygen levels In vivo levels are lower Carbon dioxide Atmospheric 0.03% Standard culture conditions: 5% Buffering (pH) Temperature and humidity Normal body temperature in mammals: 37°C Humidity must be maintained at saturating levels as evaporation can lead to changes in Osmolarity Volume of media and additives Media formulation Initial studies used body fluids Plasma, lymph, serum, tissue extracts Early basal media Salts, amino acids, sugars, vitamins, supplemented with serum Today: more defined media Extremely complex media have been developed to meet the needs of specific cell types Plus serum (mostly) Also, serum-free media have been developed Basal Media A basic cell culture medium contains sugars, amino acids, vitamins, salts and other components Classical basal media are chemically defined formulations, each developed to support a particular cell line or culture condition. The main differences between the various classical basal media are the identity and quantity of buffers, salts, and growth supplements. Many were originally developed using mouse fibroblasts, HeLa, or CHO cell lines, and modifications over time have established basal media suitable for a wide range of cell types. Interestingly, the names of classical basal media represent the researcher or the institute who developed them. e.g. RPMI = Roosevelt Park Memorial Institute BME = Eagle’s Basal Medium (Eagle = developing researcher) MEM = Modified Eagle’s Medium (Eagle = developing researcher) DMEM = Dulbecco’s Modified Eagle’s Medium (Dulbecco = developing researcher) you can also see how basal media can be modified over time by multiple researchers! Dulbecco’s Modified Eagle Medium (DMEM) Media formulation Inorganic ions Osmotic balance – cell volume Trace Elements Co-factors for biochemical pathways (Zn, Cu) Amino Acids Protein synthesis Glutamine required at high concentrations Vitamins Metabolic co-enzymes for cell replication Energy sources Glucose  pH pH is a measure of hydrogen ion concentration Physiological pH is 7.4 for most mammalian cells There are exceptions for some cell types; e.g. blood monocytes (a type of white blood cells) require pH ~7.0 pH can affect Cell metabolism Growth rate Protein synthesis Availability of nutrients Buffering Maintain small pH range in media is critical Although salts and amino acids within the medium can provide some buffering capacity, additional buffering compounds are added to ensure that a proper physiological pH is maintained Sodium bicarbonate is normally used as a buffer CO2 acts as a buffering agent in combination with sodium bicarbonate in the media CO2 gas supplied into the incubator typically at 5% CO2 gas in the atmosphere dissolves into the cell culture medium and establishes equilibrium with bicarbonate ions (HCO3-) CO2 is acidic, lowers the pH of the medium. Sodium bicarbonate buffers this reaction Buffering (cont’d) HEPES is an organic buffer that is also used to maintain physiological pH of cell culture media HEPES is recommended when the cell culture system is very sensitive, increasing the buffering capacity and stabilizing the pH within the range of 7.2 to 7.6 not dependent on CO2levels: does not require elevated levels of CO2 disadvantage: can become toxic to the cells  Phenol Red: pH indicator In many cell culture media, phenol red is added Its color exhibits a gradual transition from yellow to red over the pH range 6.8 to 8.2 Above pH 8.2, phenol red turns a bright pink (fuschia) color Below pH 6.8, it turns bright yellow At physiological pH (~7.4), it is bright red A convenient way to rapidly check on the health of tissue cultures In the event of problems, waste products produced by dying cells or overgrowth of contaminants will cause a change in pH, leading to a change in indicator color. Sem II, 2021/2022 What cells look like when all is good! Sem II, 2021/2022 What cells look like when all is good! 5.0% CO2, all is well After 0.0% CO2, all is sad  The waste products produced by the mammalian cells themselves will slowly decrease the pH, gradually turning the solution orange and then yellow. This color change is an indication that even in the absence of contamination, the medium needs to be replaced (generally, this should be done before the medium has turned completely orange) A culture of relatively slowly dividing mammalian cells can be quickly overgrown by bacterial contamination. This usually results in an acidification of the medium, turning it yellow. Osmotic balance The cell membrane is permeable Thus, the fluid surrounding the cells must contain the same concentration of soluble molecules as in within the cells The osmotic pressure of the two fluid compartments are equal = no net water movement occurs. This is called iso-osmotic or isotonic Osmolality (“osmo”) is a measure of how "salty" the media is and significantly impacts the cellular environment of cell culture cells in a low osmo environment are bursting at the proverbial seams and conversely in high osmo environments are shriveled. High osmolality can cause delayed cell growth or accelerate cell death Media is designed to be somewhere between 270 - 330 mOsm/kg (mammals have interstitial osmo of 290 mOsm) for the typical media While there are no formal osmolality controls for the cell culture, there are typically osmolality specifications for the media and batch feed. Typically media preparations targets final osmolality near 290 to 300 mOsm/kg so that the cell culture has a fighting chance at staying within biological range for the cells  Serum Serum is the liquid component of clotted blood Serum contains: Basic nutrients Hormones and growth factors Attachment and spreading factors Binding proteins (albumin, vitronectin, transferrin), hormones, vitamins, minerals, lipids Protease inhibitors pH buffer Most mammalian cell cultures require the addition of animal or human serum to the media Some cell cultures depend on serum to provide not only nutrients, but also compounds that promote cell proliferation and attachment  Serum (cont’d) Fetal Bovine Serum (FBS) is the most widely used serum for cell culture due to being low in antibodies and containing more growth factors, allowing for versatility in many different cell culture applications. FBS comes from the blood drawn from a bovine fetus via a closed system vein puncture at the slaughterhouse. The globular protein, bovine serum albumin (BSA) is a major component of FBS. The rich variety of proteins in FBS maintains cultured cells in a medium in which they can survive, grow and divide. Media that includes serum commonly contains between 10 to 20% FBS Freshney (1992) Animal Cell Culture. Type of serums and serum replacements Fetal bovine serum / fetal calf serum Calf serum Goat serum Human serum Porcine serum Rabbit serum Bovine serum Horse serum Sheep serum Chick embryo extract Embryonic serum  Serum (cont’d) There are many serum components and most are with unknown functions Although serum used in cell culture media generally undergoes numerous modification and purification steps, the end product will always hold the same key disadvantages for cell culture applications Serums are not fully defined! Variability in quality and composition between serum batches Derived from an animal source Risk of contamination (mycoplasma, viruses, etc.) Can be expensive Can avoid this limitation by using serum-free media!  Serum-Free Media Serum-free media allow researchers to culture cells in the absence of serum. In general, serum-free media is considered defined media, containing albumin, insulin, selenium, and transferrin proteins in place of FBS. Defined serum-free media can include serum-derived proteins, however, such as human serum albumin (HSA). The main advantage of serum-free stem cell media is the consistent and reproducible experimental results from a more pure media formulation. Disadvantage: stem cells can be much more sensitive when cultured in a serum- free environment. Can be more sensitive to mechanical and chemical stresses, including dissociation enzymes and antibiotics Cell Culture Incubators The purpose of the incubator is to provide the appropriate environment for cell growth Provide Control of physical parameters (temperature, humidity, gas pressure) Protection against contamination CO2 incubators More expensive, but allows superior control of culture conditions Hooked on to a CO2 tank, which supplies CO2 at desired setting (typically 5%) Humidified: Has a tray filled with water to add humidity Temperature controlled: usually set at 37°C Used to incubate cells cultured in dishes, flasks or multi-well plates, which require a controlled atmosphere of high humidity and increased CO2 tension Humans shed particles of skin, bacteria, fungi, etc. all the time! “Sitting or standing with no movement, wearing cleanroom garments, an individual will shed approximately 100,000 particles of 0.3um and larger per minute. The same person with only simple arm movement will emit 500,000 particles. Average arm and body movements with some slight leg movement will produce over 1,000,000 particles per minute; average walking pace 7,500,000 particles per minute; and walking fast 10,000,000 particles per minute. Boisterous activity can result in the release of as many as 15x106 to 30x106 particles per minute into the cleanroom environment.” Cell Culture is a Fussy Discipline! In the tissue culture laboratory: Bench tops should be kept clear and clean Wear a long sleeve lab coat: minimizes contamination from street clothing, hair, etc. Wear gloves while doing tissue culture work: minimizes contamination from skin organisms Surfaces, gloves, solutions and plastic-ware sprayed with 70% ethanol (EtOH) before placed into the biological hood Solutions, reagents and glassware used in tissue culture work should not be shared with non-tissue culture work Sterility Contaminating organisms (such as bacteria, yeast, etc.) grow much faster than animal cells in culture If culture is contaminated, the contaminants will overrun the culture and your cells’ growth will be affected and they will eventually die! Aseptic technique “Non-dirty techniques” To avoid/ minimize chances of contamination Antimicrobials additives to the media Antibiotics: protect against bacteria Antimycotics/anti-fungals: protect against fungi May interfere with some cell types or experiments Aseptic Technique is the best prevention! Controlled environment Traffic, air flow Dedicated cell culture room Sterile media and reagents Avoid aerial contamination of solutions Avoid manual contamination of equipment Avoid repeated opening of bottles 70% ethanol swab UV irradiation of the cell culture hood before and after Only use disposable equipment once  Aseptic Technique Lab coat Gloves Pipette tip does not touch the tube Holding of the tube Placement of items in the hood Wide, clear work space in the center with your cell culture vessels Pipettor in the front right, where it can be reached easily Reagents and media in the rear right to allow easy pipetting Tube rack in the rear middle holding additional reagents Small container in the rear left to hold liquid waste Spray/wipe with 70% EtOH Hoods for Cell Culture Principle – to make available a space free of microorganisms and spores Types of hoods used for cell culture: Laminar Flow Hood Biological Safety Cabinet / Tissue Culture Hood Class II: most common Class III: provides maximum protection to the environment and the worker High Efficiency Particulate Air (HEPA) filter trap airborne pollutants including dust, allergens, and microorganisms Laminar Flow Hood Horizontal air flow OK for cells not hazardous to the user Not suitable for working with human cells and pathogens Less protection compared to biosafety cabinets Biosafety Cabinet (Class II) Top-down air flow Most commonly used for cell culture Biosafety Cabinet (Class III) Requirements of a cell culture hood Large enough space to work Easily cleanable inside and outside Adequate lighting, and be comfortable to use without requiring awkward positions UV lighting for disinfecting