Download What is Cell culture

Lec # 15
Animal cell lines and culturing
Shah Rukh Abbas, PhD
26.2. 2015
What is Cell culture ?
• Cell culture refers to the removal of cells from
an animal or plant and their subsequent
growth in a favorable artificial environment.
• The process by which prokaryotic,
eukaryotic or plant cells are grown under
controlled conditions
• But in practice it refers to the culturing of
cells derived from animal cells/tissue
Why we need to culture animal cells?
- Cell is the Basic Unit for the Life
- Understanding functions and roles of various cell are crucial
approach for modern biology
• With isolated cell culture, we can study/investigate desired cell using
various techniques without interference of other cells/tissues/organ
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Microscopic
Biochemical
Molecular Biology
Toxicology…
• Cost effective than animal experiments
• Avoid ethical problem in animal/human experiments
• Production of Protein/Antibody/Virus/Vaccine using cultured animal cell
culture
History
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1885 : medullary plate of an embryonic chickens (Wilhelm Roux)
1907 : Grow Frog nerve fiber in using hanging drop culture
1912 : Alexis Carrel culture chicken heart using chick embryo extract
1916 : Trypsinization and subculture of explants
1923 : Development of first cell culture flask
1925 : Subculture of fibroblastic cell lines
1940s : Discovery of Antibiotics -: The use of the antibiotics penicillin and
streptomycin in culture medium decreased the problem of contamination in cell
culture.
1952: Cloned Tadpoles (Briggs and King)
1954 : Discovery of Contact Inhibition (Abercrombie)
1955: nutrient requirements of selected cells in culture and established the first
widely used chemically defined medium.(Eagle) DMEM Dulbecco’s Modified
Eagle’s Medium
1961: isolated human fibroblasts (WI-38) and showed that they have a finite
lifespan in culture.
History
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1965: first serum-free medium which was able to support the growth of some
cells. (Ham)
1975: First hybridoma capable of secreting a monoclonal antibody (Milstein)
1978: development of serum-free media from cocktails of hormones and growth
factors. (Sato)
1981 : Mouse Embryonic Stem Cell
1982: Human insulin : the first recombinant protein to be Licensed as a
therapeutic agent.
1985: Human growth hormone produced from recombinant bacteria
1989 : Knockout mouse using Mouse ES Cell (Capecchi, Evans, Smithies)
1996 : The First mammal cloned from adult cells (Dolly, the sheep)
1998 : Human Embryonic Stem Cell (Thomson)
2006-2007 : First Induced Pluripotent Stem Cell (iPSC : Yamanaka and others)
2013 : First Human stem Cell generated from SCNT
•Mouse, mammals,
•Embryo
•Embryonated Eggs
because stage of differentiation)
Cell culture
Finely cut
Finely cut
tissue or explant
explant
•Sterilize the site with 70% alcohol.
•Remove tissue aseptically.
•Transfer to the laboratory in
transport medium
•If delay in transporting to lab,
Enzymic digestion
keep at 4°C for up to 72hour.
Grow in media
-monolayer
-suspension cells
STAGES OF CULTURE
Isolated tissue
(disaggregation)
Primary cell culture(limited lifespan after certain
proliferations undergo senescence)
Finite cell cultures
Continous cell lines(immortalized cell line acquires ability to proliferate
indefinitely by transformation)
Primary Cell Culture
• Cells taken directly from living tissue (e.g. biopsy
material) and established for growth in vitro
• Undergone very few population doublings
• Proteolytic enzymes (trypsin and Collagenase)
are commonly used to break the protein
Primary Cell Culture
Pros
• more representative of the main functional
component of the tissue
Cons
• They are not well characterized,
• Have limited life span,
• Slow in proliferation
Established Cell Line
• After the first subculture, primary culture may be called secondary
cultures
• Thereafter, if continued passage is possible, a cell line.
• Established or immortalised cell line : ability to prolierate indefinetely
by
• Random Mutation
• Artificial Modification : expression of telomerase, insertion of
cancer antigen
Cell Line
Organism
Origin Tissue
HeLa
Human
Cervical Cancer
293-T
Human
Embryonic Kidney
A-549
Human
Lung carcinoma
ALC
Murine
Bone Marrow
CHO
Hamster
Ovary
HB54
Hybridoma
Hybridoma
Growth Curve
‘Normal’ mammalian cells have the following properties:
• a diploid chromosome number (46 chromosomes for human
cells)
• anchorage dependence
• a finite lifespan
• nonmalignant (non-cancerous)
• density inhibition
Transformed cell characteristics
• infinite growth potential
• loss of anchorage-dependence
• aneuploidy (chromosome fragmentation)
• high capacity for growth in simple growth medium, without
the need for growth factors
• called an “established” or “continuous” cell line
Example of Anchorage Dependence
Passaging - establishing Secondary → Tertiary
Cultures
• growth of cells prolonged by inoculating some of the cells into fresh
medium
• ‘cell line’ refers to cell population that continues to grow through
passaging or subculturing
• genetic alteration may occur during the first few passages as cells adapt to
a new chemical environment
• subculture within a day or two of maximum cell density
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must detach anchorage-dependent from growth surface
→ trypsinization
→ EDTA in Ca 2+ - and Mg 2+ -free solution
Contamination
• bacteria and fungi are main sources of contamination
• culture contamination observed by:
→ drop in pH
→ turbidity of medium
→ may observe granules between mammalian cells
HeLa
• The oldest and most commonly used human cell line
• 1951 : Derived from cervical cancer cell taken from
Henrietta Lacks (1920-1951)
Henrietta Lacks (circa 1945)
HeLa is famous for…
Immotallised Cell Line : Due to mutation, it can evades
normal cellular senescence and can keep undergo division
Used to test the first polio vaccine / virus Culture
More than 60,000 scientific articles has been published
using HeLa
Extensively Used for the Cancer Studies
It has abnormal chromosome number : 82
4 copies of chromosome 12
3 copies of chromosome 6,8,7
How to grow/select specific cells?
• selective overgrowth of a particular cell type
• controlling media composition
• gradient centrifugation
Type of Cell by Morphology
Fibroblast like
• spindle-shaped, often striated, form
parallel lines as they attach to
substratum/substrate
→ in vivo – wrap around collagen
(fibrous protein)
→ in vitro – glass
Lymphoblast-like
• large nuclei
• found in vivo in blood (liquid
suspension)
• can grow in suspension in liquid
medium in lab
Epithelial-like
• cover organs and line cavities (i.e.
skin)
• cobblestone morphology, form
monolayer
• anchorage dependent, need solid
substratum
Type of Cells
• Anchorage-independent cells: which can propagate in the
suspension culture
- Blood Cells
- Cancer Cells
- Hybridoma
• Anchorage-dependent cells which can propagate as a
monolayer attached to the cell culture vessel
- Will cease proliferating once they become confluent
(completely cover the surface of cell culture vessel)
Suspension Culture
• Appropriate for cells adapted to suspension culture and a few
other cell lines that are non-adhesive (e.g., hematopoietic)
• Used for bulk protein production, batch harvesting, and many
research applications
• Can be maintained in culture vessels that are not tissueculture treated, but requires agitation (i.e., shaking or stirring)
for adequate gas exchange
• Easier to passage, but requires daily cell counts and viability
determination to follow growth patterns;
• culture can be diluted to stimulate growth
Spinner Flask
Adherent Cell Culture
• Adherent cell require surface to attach to grow
• Appropriate for most of cell types (including primary
cultures)
• The majority of the cells derived from vertebrates are
anchorage-dependent and have to be cultured on a suitable
substrate that is specifically treated to allow cell adhesion
and spreading
• Growth is limited by surface area
• Cells are dissociated enzymatically or mechanically from
surface
Utilization of Cell Culture
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Model System for Basic Science
In Vitro Cell Toxicity / Screening
Animal Cell Culture for Protein Productions
Tissue/embryo Engineering