Cell and Tissue Culture

Cell cultures involving animal, plant, bacterial and fungal cells.

Stem cells can differentiate

Essential if experimental work on cells is to be done e.g., manipulation of genes, gene analysis, cancer studies, new plant cells.

Growth curve for cells

Basic requirements

Micro-organisms

Batch culture

Continuous culture

•Exponential growth but slows as waste products build up

•No dilution

•Grown until maximum strength reached

•Growth phase is maintained

•Dilution of culture occurs

•Remove equal volume of culture

•Add fresh media

Mammalian cell culture

Animal cells:

•Essential addition of Animal serum

(foetal bovine serum FBS 5 - 10%)

•growth medium –

balanced salt solution, amino acids, vitamins

glucose, pH indicator/buffer, antibiotics

•basic requirements similar

•fragile (need more carefully controlled conditions)

Sequence of events when cells are cultured in vitro

Adhesion

Spreading

Division

Confluence

Obtaining cells

•Scholar examples in table.

•Longest cell lines come from TUMOURS or transformed cells which have been immortalised NEOPLASTIC and these will cause cancer if put in animals.

•Cells die after a number of divisions secondary culturing is necessary

•Seperate cells to give primary culture

•Treat tissue with proteolytic enzymes (e.g. trypsin)

Cell growth

•cloning and selection experimental work

•cell lines can be fused to give new HYBRID cells

•mutant cell lines help understanding of cell growth

Advantages

•Surface area covered (confluent) cells stop proliferating need to be sub-cultured.

•Mono-layers grown in plastic flasks

Plant Tissue culture

Regeneration important for ornamental commercial use and to produce pathogen free plants.

Under correct conditions plants can regenerate completely as many possess NUCLEAR TOTIPOTENCY.

(Animals too complex to regenerate whole organism)

Similar to animals in many instances but requirements simpler and media easier to prepare.

Method of culture

•regeneration of whole plants

•subculture (alter balance of growth regulators to produce shoot/root)

•callus produced (undifferentiated mass of cells)

•grown in media (hormones e.g. auxins/cytokinin)

•Explants (small pieces - sterilised)

Hybrid Plants

•Can produce plants with useful characteristics of 2 species.

•Can produce new variety of plants

•Overcomes reproductive incompatability

Why?

•stimulate shoot/root growth with hormones

•grow to callus (hormones)

•fuse protoplasts using electric current

•Remove cell walls (cellulase and pectinase)

Hybrid plants containing genetic information from 2 species can be produced by PROTOPLAST FUSION.