Tissue Culture Unit one Cell and Molecular Biology Advanced Higher Biology.

Tissue Culture

Unit oneCell 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.

ArrangementsPlant 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.

ArrangementsPlant 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 developmentNeeded 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 cultureexplant

Grown in media containing plant growth regulators

Cell proliferation

Callus

Sub-cultureChange 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.