Tc ppt v7

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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