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Tissue culture basics 2014
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TISSUE CULTURE: AN EXCELLENT TOOL
FOR VIRUS RESEARCH
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Cell culture
Explant culture
Organ
culture
Tissue culture is used as a generic term to include the in vitro cultivation of organs, tissues and cells
The ability to survive and grow tissues outside the body in an artificial environment
Tissue culture can be subdivided into three major categories
Tissue Culture
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Types of Tissue Culture
Organ culture: a spherical or three-dimensional shapespecific histological interaction
Primary explant culture:a fragment of tissueattachment and migration occurs in the plane of the solid substrate
Cell culture:adherent monolayer on a solid substrate (various cell types)suspension in the culture medium (few cell types)
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What is Cell Culture?
• Removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment
Tissue
Primary culture
Cell line Continuous cell line
Subculture
Stored Stored
Animal Plant
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Tissue
Primary culture
Cell line Continuous cell line
Subculture
Stored Stored
Animal Plant
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Primary Culture
Characteristics:Morphologically similar to the parent tissueLimited number of cell divisionsBest experimental models for in vivo situations
Cells removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation
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Primary Culture Preparation
Fig: Primary culture preparation
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• Difficult to obtain
• Relatively short life span in culture
• Very susceptible to contamination
• May not fully act like tissue due to complexity of media
Primary Culture: Limitations
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Sub-culturing /Passaging of Primary culture
Primary culture acts like ‘Seed’ for a particular type of cell
So, sub-culturing/passaging and subsequent treatment allows to establish a culturally ‘immortal’ progeny of cells
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• Cell lines derived from primary cultures have a limited life span
• After the first subculture, the primary culture becomes cell line
• When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line
Cell Line
Transformation can occur:Chemically Virally inducedSpontaneously
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Comprised of a single cell type that can be serially propagated in culture either for a limited number of cell divisions (approximately thirty) or indefinitely
Continuous Cultures
Characteristics :• Cell lines of a finite life are usually diploid • maintain some degree of differentiation• such cell lines senesce after approximately thirty cycles• Continuous cell lines that can be propagated indefinitely
by transforming into tumor cell
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• Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate
• Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches
• Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface
Morphology of Cells in Culture Based on shape and appearance: 3 types
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Anchorage-dependentMust be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture)Example: MDCK, Vero
Anchorage-Independent (suspension culture)Can be grown floating in the culture mediumExample: MNFS-60
Morphology of Cells in Culture
Depending on the adherence property : 2 types
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Adherent Cell Culture Suspension Cell Culture Attached to a solid or semi-solid substrate Grown floating in the culture medium
Easy visual inspection under inverted microscope
Requires daily cell counts and viability determination to follow growth patterns
Cells are dissociated enzymatically (e.g. trypsin) or mechanically
Does not require enzymatic or mechanical dissociation
Growth is limited by surface area Growth is limited by concentration of cells
No agitation required Requires agitation (i.e., shaking or stirring) for adequate gas exchange
Used for cytology, harvesting products continuously, and many research applications
Used for bulk protein production, batch harvesting, and many research applications
Adherent Cell vs Suspension Cell
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Cell Culture Environment
Physico-chemical environment • Growth media (pH, osmotic pressure, O2 and
CO2 tension)• Temperature
Physiological environment • Hormone and nutrient concentrations
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The culture medium is the most important component of the culture environment, because it provides the necessary nutrients, growth factors, and hormones for cell growth, as well as regulating the pH and the osmotic pressure of the culture.
The three basic classes of media are
1. Basal media 2. Reduced-serum media and 3. Serum-free media
Cell Culture Media
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Basal Media• Contains amino acids, vitamins, inorganic salts, and
a carbon source such as glucose• Basal media formulations must be further
supplemented with serum
Reduced-Serum Media• Basal media formulations enriched with nutrients and
animal-derived factors with reduced amount of serum
Cell Culture Media
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Serum-Free Media• Appropriate nutritional and hormonal formulations
replaces serum completely
• Serum-free medium in combination with growth factors has the ability to make the selective medium for primary cell culture
Cell Culture Media
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• Commonly used Medium: GMEM, EMEM,DMEM etc.
• Media is supplemented with Antibiotics (penicillin, streptomycin)BSANa-bicarbonate L-glutamateNa-pyruvateHEPESGrowth factors etc.
Cell Culture Media
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a) Serum:
• Vitally important source of growth factors, adhesion factors, hormones, lipids and minerals
• Regulates cell membrane permeability
• Serves as a carrier for lipids, enzymes, micronutrients, and trace elements into the cell
Factors affecting culture environment
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b) pH Level:
Factors affecting culture environment
Cell line Optimal pH
Mammalian cell lines 7.4
Transformed cell lines 7.0 – 7.4
Normal fibroblast cell lines 7.4 – 7.7
Insect cell lines 6.2
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The growth medium controls the pH of the culture and buffers the cells in culture against changes in the pH
Buffering is achieved by an organic (e.g., HEPES) or CO2-bicarbonate based buffer
4 – 10% CO2 is common for most cell culture experiments
C) CO2 Level
Factors affecting culture environment
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The optimal temperature for cell culture largely depends on the body temperature of the host from which the cells were isolated
Factors affecting culture environment
d) Temperature
Cell line Optimal Temperature
Human and mammals 36°C - 37°Insect cells 27°C Avian cell lines 38.5°C Cold-blooded animals (e.g., amphibians, cold-water fish)
15°C - 26°C
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e) In Vitro Age of a Cell Culture
The in vitro age of a cell culture is particularly useful to know for cell lines with a finite lifespan or unstable characteristics that change over time in continuous culture
Two terms are predominantly used to define the age of a cell culture:
(i) Passage number:Indicates the number of times the cell line has been sub-cultured(ii) Population doubling (pd) number:Indicates the number of cell generations the cell line has
undergone i.e. the number of times the cell population has doubled
Factors affecting culture environment
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Cell Culture Equipments
Basic Equipments
laminar-flow hood or biosafety cabinetIncubator (humid CO2 incubator recommended)Water bathCentrifugeRefrigerator and freezer (–20°C)Cell counterLiquid nitrogen freezer or cryo-storage containerSterilizer (i.e., autoclave)
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Expanded Equipment & Additional Supplies
Aspiration pump (peristaltic or vacuum)pH meterMicroscope (inverted and confocal)Flow cytometerCell culture vesselsPipettes and pipette-aidSyringes and needlesWaste containersMedia, Sera and reagentsCells
Cell Culture Equipments
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Cell Culture Contamination
Cell culture contaminants can be divided into two main categories:
Chemical contaminants such as impurities in media, sera, and water, endotoxins, plasticizers, and detergents
Biological contaminants such as bacteria, molds, yeasts, viruses, mycoplasma, as well as cross contamination by other cell lines
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Fig: adherent 293 cells contaminated with E. coli
Cell Culture Contamination
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Cell Culture Contamination
Fig: 293 cells contaminated with yeast
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Cells can be stored for future use by using appropriate protective agent such as: Dimethyl Sulfoxide (DMSO) Glycerol etc.
Storage temperature :Below –130°C (cryopreservation)
Cryopreservation
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• Excellent model systems for studying :-the normal physiology and biochemistry of cells-the effects of drugs and toxic compounds on the cells-mutagenesis and carcinogenesis
• Used in drug screening and development
• Large scale manufacturing of biological compounds (vaccines, insulin, interferon, other therapeutic protein)
Applications of Cell Culture
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• Monoclonal antibody production (immunology)
• Tissue engineering: -Homografting -Reconstructive surgery using individual’s own cells
• In vitro fertilization (embryo culture)
• Implanting normal fetal neurons into patients with Parkinson diseases
Applications of Cell Culture
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Sample (NPW, TS, NS)
Inoculation(Passage 1)
Inoculation(Passage 2)
Inoculation(Passage 3)
HA Titer: ≥ 32
HA Titer: 0 Discard
HAI
Characterization
Lab Activity
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• Samples (NPW, TS, NS) inoculated into the small flasks (80-90% cell confluency)
• Observation of CPE (consecutive 7 days)• 85-95% CPE reached• Hemagglutination assay (HA) done• Cells harvested• If the HA titre is ≥32• Hemagglutination Inhibition assay (HAI) done for virus
characterization
Lab Activity
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Thank You
