Transcript Cell and Tissue Culture

Tissue Culture
Unit one
Cell and Molecular Biology
Advanced Higher Biology
Arrangements
Differentiation of cells into tissues and
organs.
• Cells undergo differentiation to
become specialised cells that are
organised into tissues and organs.
• Cellular differentiation depends on
changes in gene expression resulting
in genes being switched on and off
Arrangements
Cell and tissue culture
• The ability of stem cells to
differentiate, unlike specialised cells.
• The lac operon in E.coli.
– Repressor molecule,
– regulator gene,
– inducer,
– operator
– structural gene
Arrangements
Mammalian cell culture.
• Requirement of aseptic conditions,
solid surface, growth factors and
nutrients in complex growth media.
• Use case studies to illustrate the
applications of cell culture.
Arrangements
Mammalian cell culture.
• The addition of animal serum such as fetal
bovine serum (FBS) to promote cell
proliferation and antibiotics to prevent
bacterial growth.
– Use of proteolytic enzymes to release cells
from source tissue.
• Cells adhere to the surface, spread out and
divide until a monolayer is formed and the
cells are confluent.
Arrangements
Mammalian cell culture.
• Difficulty in maintaining cultures of
mammalian cells due to cells dying after a
finite number of divisions in culture.
• Cell lines prepared from cells which undergo
a genetic change that makes them
immortal or from cancer cells. A clone is the
result of cell cloning in which a single cell is
isolated and allowed to proliferate to form a
large colony.
Arrangements
Bacterial and fungal cultures
• The advantages of the simpler growth
media requirements and culture
conditions for bacteria and fungi
compared to mammalian cells.
Arrangements
Plant tissue culture.
• Techniques used (including
requirement for aseptic conditions and
suitable growth medium).
• Growth of explants on suitable media
to produce a callus.
• The use of growth regulators such as
auxins and cytokinins to cause tissue
differentiation.
Arrangements
Plant tissue culture.
• Production of pathogen-free
plantlets and plants, generation of
new varieties of plants and use in
plant propagation.
• Use of terms protoplast and
totipotent.
• Examine photographs of protoplasts
at different stages of culture and as
fusion products.
Differential gene
expression in
development
Needed in similar depth to that
taught at Higher Biology
Revision Questions
• Name the process in which DNA is
copied into RNA.
• Name the process in which RNA is read
into protein
• What is a transcription factor?
Revision Questions
• Name the process in which DNA is copied
into RNA.
– transcription
• Name the process in which RNA is read into
protein
– translation
• What is a transcription factor?
– A protein that interacts with RNA
polymerase, or other transcription factors,
to regulate gene expression.
Control of gene expression
• Temporal
• Spatial
• Drosophila melanogaster
– Fruit fly
– Used in studies
Development in Drosophila
Egg to
organism
Mouse
Cell and Tissue
Culture
Four major applications
•
•
•
•
agriculture
pharmaceuticals
food production
biodegradation
Conditions for growth
• a source of suitable cells;
• the growth medium;
• the type of growth container or
fermenter;
• temperature;
• pH;
• gas exchange
• aseptic conditions;
• a method for monitoring cell growth;
• safety measures
Data interpretation
• Assume that bacterial cells have a
doubling time of 30 minutes, and that
mammalian cells have a doubling time of
24 hours.
– Calculate the number of cells that would
exist after one day of growth if you start
with one cell in each culture.
– For the bacterial culture only, draw a
graph to show the pattern of growth for
the first 4.5 hours.
– Explain why the bacterial growth
achieved after one day is unlikely to be
achieved in practice.
Micro-organisms
• Categories of Micro-organisms
– bacteria;
– fungi;
– protozoa;
– algae.
• Microorganisms are relevant to
many aspects of human existence
– examples
Growth requirements
•
•
•
•
•
nutrient medium
temperature
pH
gaseous environment
light
Bacterial Culture
• Revision of bacterial growth
Questions
• In which phase are the bacteria
dividing at a constant rate?
• In which phase does the rate of cell
division equal the rate of cell death?
• In which phase are the bacteria
metabolically active but not
dividing?
• In which phase does bacterial cell
death exceed cell division?
Mammalian Cell Culture
• More carefully controlled growth conditions
• Anchorage dependent
– Monolayer
– Confluence  sub-culturing
• Non-anchorage dependent
– E.g. blood
– Grown in suspension
Growth medium
• Balanced salt solution with amino
acids, glucose and vitamins
• Serum e.g. fetal bovine serum
– Essential for animal cell proliferation
– Usually 5-10% FBS
Primary cell culture
• Tissues treated with a proteolytic
enzyme to separate cells from each
other.
• Normal cells – finite division
• Immortalised cell lines
– E.g. cells derived from tumours
– neoplastic
Uses of cell cloning
• Isolation of mutant cells
• Investigating cell growth
• Biotechnology
– Vaccines
– hormones
Plant Tissue Culture
• Nuclear totipotency
– Plant cells have ability to regenerate
complete plants under appropriate
conditions.
Plant tissue culture
explant
Grown in media containing
plant growth regulators
Cell proliferation
Callus
Sub-culture
Change growth regulators
Differentiation roots and shoots
Hybrid Plant cells
• Protoplast fusion
– Revise
• Formation of protoplasts
• Growth of protoplasts
Essay
• Discuss the requirements for
the growth of microbial cells
and describe, with the aid of a
labelled diagram, the pattern
of growth of a batch culture of
microbial cells.