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www.icddrb.org
TISSUE CULTURE:
AN EXCELLENT TOOL
FOR VIRUS RESEARCH
www.icddrb.org
Cell culture
Explant
culture
Organ
culture
Tissue culture is used as a
generic term to include the
in vitro...
www.icddrb.org
Types of Tissue Culture
Organ culture:
a spherical or three-dimensional shape
specific histological interac...
www.icddrb.org
What is Cell Culture?
• Removal of cells from an animal or plant and their
subsequent growth in a favorable...
www.icddrb.org
Tissue
Primary culture
Cell line Continuous cell line
Subculture
Stored Stored
Animal Plant
www.icddrb.org
Primary Culture
Characteristics:
Morphologically similar to the parent tissue
Limited number of cell divisi...
www.icddrb.org
Primary Culture Preparation
Fig: Primary culture preparation
www.icddrb.org
• Difficult to obtain
• Relatively short life span in culture
• Very susceptible to contamination
• May not...
www.icddrb.org
Sub-culturing /Passaging of
Primary culture
Primary culture acts like ‘Seed’ for a particular type of
cell
...
www.icddrb.org
• Cell lines derived from primary cultures have a limited life
span
• After the first subculture, the prima...
www.icddrb.org
Comprised of a single cell type that can be serially propagated
in culture either for a limited number of c...
www.icddrb.org
• Fibroblastic (or fibroblast-like)
cells are bipolar or multipolar,
have elongated shapes, and grow
attach...
www.icddrb.org
Anchorage-dependent
Must be cultured while attached to a solid or
semi-solid substrate (adherent or monolay...
www.icddrb.org
Adherent Cell Culture Suspension Cell Culture
Attached to a solid or semi-solid substrate Grown floating in...
www.icddrb.org
Cell Culture Environment
Physico-chemical environment
• Growth media (pH, osmotic pressure, O2 and
CO2 tens...
www.icddrb.org
The culture medium is the most important component of the
culture environment, because it provides the nece...
www.icddrb.org
Basal Media
• Contains amino acids, vitamins, inorganic salts, and a
carbon source such as glucose
• Basal ...
www.icddrb.org
Serum-Free Media
• Appropriate nutritional and hormonal formulations
replaces serum completely
• Serum-free...
www.icddrb.org
• Commonly used Medium:
GMEM, EMEM,DMEM etc.
• Media is supplemented with
Antibiotics (penicillin, streptom...
www.icddrb.org
a) Serum:
• Vitally important source of growth factors, adhesion
factors, hormones, lipids and minerals
• R...
www.icddrb.org
b) pH Level:
Factors affecting culture environm
Cell line Optimal pH
Mammalian cell lines 7.4
Transformed c...
www.icddrb.org
The growth medium controls the pH of the culture and
buffers the cells in culture against changes in the pH...
www.icddrb.org
The optimal temperature for cell culture largely depends on
the body temperature of the host from which the...
www.icddrb.org
e) In Vitro Age of a Cell Culture
The in vitro age of a cell culture is particularly useful to know for
cel...
www.icddrb.org
Cell Culture Equipments
Basic Equipments
laminar-flow hood or biosafety cabinet
Incubator (humid CO2 incu...
www.icddrb.org
Expanded Equipment & Additional Supplies
Aspiration pump (peristaltic or vacuum)
pH meter
Microscope (in...
www.icddrb.org
Cell Culture Contamination
Cell culture contaminants can be divided into
two main categories:
Chemical cont...
www.icddrb.org
Fig: adherent 293 cells contaminated with E. coli
Cell Culture Contamination
www.icddrb.org
Cell Culture Contamination
Fig: 293 cells contaminated with yeast
www.icddrb.org
Cells can be stored for future use by using appropriate
protective agent such as:
Dimethyl Sulfoxide (DMSO)...
www.icddrb.org
• Excellent model systems for studying :
-the normal physiology and biochemistry of cells
-the effects of d...
www.icddrb.org
• Monoclonal antibody production (immunology)
• Tissue engineering:
-Homografting
-Reconstructive surgery u...
www.icddrb.org
Sample
(NPW, TS, NS)
Inoculation
(Passage 1)
Inoculation
(Passage 2)
Inoculation
(Passage 3)
HA Titer: ≥ 32...
www.icddrb.org
• Samples (NPW, TS, NS) inoculated into the small flasks
(80-90% cell confluency)
• Observation of CPE (con...
www.icddrb.org
Thank You
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Tissue culture basics 2014

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Tc ppt v7

  1. 1. www.icddrb.org TISSUE CULTURE: AN EXCELLENT TOOL FOR VIRUS RESEARCH
  2. 2. www.icddrb.org 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
  3. 3. www.icddrb.org Types of Tissue Culture Organ culture: a spherical or three-dimensional shape specific histological interaction Primary explant culture: a fragment of tissue attachment 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)
  4. 4. www.icddrb.org 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
  5. 5. www.icddrb.org Tissue Primary culture Cell line Continuous cell line Subculture Stored Stored Animal Plant
  6. 6. www.icddrb.org Primary Culture Characteristics: Morphologically similar to the parent tissue Limited number of cell divisions Best experimental models for in vivo situations Cells removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation
  7. 7. www.icddrb.org Primary Culture Preparation Fig: Primary culture preparation
  8. 8. www.icddrb.org • 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
  9. 9. www.icddrb.org 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
  10. 10. www.icddrb.org • 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 induced Spontaneously
  11. 11. www.icddrb.org 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
  12. 12. www.icddrb.org • 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
  13. 13. www.icddrb.org Anchorage-dependent Must 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 medium Example: MNFS-60 Morphology of Cells in Culture Depending on the adherence property : 2 types
  14. 14. www.icddrb.org 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 Cel
  15. 15. www.icddrb.org Cell Culture Environment Physico-chemical environment • Growth media (pH, osmotic pressure, O2 and CO2 tension) • Temperature Physiological environment • Hormone and nutrient concentrations
  16. 16. www.icddrb.org 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
  17. 17. www.icddrb.org 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
  18. 18. www.icddrb.org 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
  19. 19. www.icddrb.org • Commonly used Medium: GMEM, EMEM,DMEM etc. • Media is supplemented with Antibiotics (penicillin, streptomycin) BSA Na-bicarbonate L-glutamate Na-pyruvate HEPES Growth factors etc. Cell Culture Media
  20. 20. www.icddrb.org 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 environm
  21. 21. www.icddrb.org b) pH Level: Factors affecting culture environm 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
  22. 22. www.icddrb.org 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 environm
  23. 23. www.icddrb.org The optimal temperature for cell culture largely depends on the body temperature of the host from which the cells were isolated Factors affecting culture environm 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
  24. 24. www.icddrb.org 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 environm
  25. 25. www.icddrb.org Cell Culture Equipments Basic Equipments laminar-flow hood or biosafety cabinet Incubator (humid CO2 incubator recommended) Water bath Centrifuge Refrigerator and freezer (–20°C) Cell counter Liquid nitrogen freezer or cryo-storage container Sterilizer (i.e., autoclave)
  26. 26. www.icddrb.org Expanded Equipment & Additional Supplies Aspiration pump (peristaltic or vacuum) pH meter Microscope (inverted and confocal) Flow cytometer Cell culture vessels Pipettes and pipette-aid Syringes and needles Waste containers Media, Sera and reagents Cells Cell Culture Equipments
  27. 27. www.icddrb.org 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
  28. 28. www.icddrb.org Fig: adherent 293 cells contaminated with E. coli Cell Culture Contamination
  29. 29. www.icddrb.org Cell Culture Contamination Fig: 293 cells contaminated with yeast
  30. 30. www.icddrb.org 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) Cryopreservati on
  31. 31. www.icddrb.org • 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
  32. 32. www.icddrb.org • 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
  33. 33. www.icddrb.org Sample (NPW, TS, NS) Inoculation (Passage 1) Inoculation (Passage 2) Inoculation (Passage 3) HA Titer: ≥ 32 HA Titer: 0 Discard HAI Characterization Lab Activity
  34. 34. www.icddrb.org • 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
  35. 35. www.icddrb.org Thank You

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