Cell and Tissue Culture Essential if experimental work on cells is to be done e.g., manipulation of genes, gene analysis, cancer studies, new plant cells. Stem cells can differentiate Cell cultures involving animal, plant, bacterial and fungal cells.

Growth curve for cells

Basic requirements

Micro-organisms

Batch culture Grown until maximum strength reached No dilution Exponential growth but slows as waste products build up Continuous culture Add fresh media Remove equal volume of culture Dilution of culture occurs Growth phase is maintained

Mammalian cell culture Animal cells: fragile (need more carefully controlled conditions) basic requirements similar growth medium – balanced salt solution, amino acids, vitamins glucose, pH indicator/buffer, antibiotics Essential addition of Animal serum (foetal bovine serum FBS 5 - 10%)

Sequence of events when cells are cultured in vitro Adhesion Spreading Division Confluence

Obtaining cells Treat tissue with proteolytic enzymes (e.g. trypsin) Seperate cells to give primary culture Cells die after a number of divisions secondary culturing is necessary Longest cell lines come from TUMOURS or transformed cells which have been immortalised NEOPLASTIC and these will cause cancer if put in animals. Scholar examples in table.

Cell growth Mono-layers grown in plastic flasks Surface area covered (confluent) cells stop proliferating need to be sub-cultured. Advantages mutant cell lines help understanding of cell growth cell lines can be fused to give new HYBRID cells cloning and selection experimental work

Plant Tissue culture Similar to animals in many instances but requirements simpler and media easier to prepare. Under correct conditions plants can regenerate completely as many possess NUCLEAR TOTIPOTENCY. (Animals too complex to regenerate whole organism) Regeneration important for ornamental commercial use and to produce pathogen free plants.

Method of culture Explants (small pieces - sterilised) grown in media (hormones e.g. auxins/cytokinin) callus produced (undifferentiated mass of cells) subculture (alter balance of growth regulators to produce shoot/root) regeneration of whole plants

Hybrid Plants Hybrid plants containing genetic information from 2 species can be produced by PROTOPLAST FUSION. Remove cell walls (cellulase and pectinase) fuse protoplasts using electric current grow to callus (hormones) stimulate shoot/root growth with hormones Why? Overcomes reproductive incompatability Can produce new variety of plants Can produce plants with useful characteristics of 2 species.