Plant and Mammalian Tissue Culture

1. Plant and Mammalian Tissue Culture Plant and Animal Cell Culture Medium

2. Animal Culture Medium Basics • Medium components • Buffer(s) to maintain pH • Salts for osmosis and cell needs • Amino acids – essential and other • Growth stimulants (hormones and agonists) • Serum (fetal calf/bovine) • Lipids including cholesterol • Vitamins • Food (typically glucose) • Trace Minerals for metabolism/enzyme function • Nucleic Acids (deoxy and NAD)

3. Animal Culture Medium Basics • “Base” medium • Just components without additives (DMEM, RPMI…) no serum or antibiotics • “Complete” medium • 5 or 10% serum • Basic medium • Antibiotics or antimycotics • “low serum or starving” medium • Same as complete but with no or low levels of serum (0.5%). • Used to synchronize cells (quiescence) in Go of cell cycle

4. Selecting Media (animal cells) The choice of cell culture medium is extremely important, and significantly affects the success of cell culture experiments. Different cell types have highly specific growth requirements, and the most suitable medium for each cell type must be determined experimentally.

5. Selecting Media (animal cells) There are no set culture conditions for growing a certain cell type. A culture grown in MEM, can probably be just as easily grown in DMEM or Medium 199.

6. Selecting Media (animal cells) In general a good place to start is MEM for adherent cells and RPMI-1640 for suspension cells.

7. RPMI Media

8. Eagles Minimum Essential Media

9. Culture Media These contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and are available either as a powder or as a liquid from various commercial suppliers.

10. Culture Media • Requires • Ions; Na+, K+, Ca++, Mg++, Cl-, PO4-, HCO3- • Trace elements; iron, zinc, selenium • Sugars such as glucose • Amino Acids

11. Media Selection

12. Culture buffers Some media includes HEPES or MOPS buffers – give additional buffer strength if CO2 levels are off. Bicarbonate system is critical – thus caps of culture flasks must be vented or open to allow exchange of gasses CO2 + H2O -> H2CO3 -> H+ + HCO3-

13. Serum Serum is a partially undefined material that contains growth and attachment factors, and may show considerable variation in the ability to support growth of particular cells.

14. Serum Fetal calf serum (FCS) is the most frequently used serum, but for some applications less expensive sera such as horse or calf serum can be used. Different serum batches should be tested to find the best one for each cell type.

15. FCS vs FBS • Fetal Calve / Bovine Serum – typically used interchangeably but are different. • Fetal Calf Serum – taken from newborn Calves • Fetal Bovine Serum – from fetus • Some variation in content of growth factors • Variability in lot to lot and location to location – often times tested for several viruses that my impact cells. • Either can be different if mother or calve has been nursing or grazing.

16. L-glutamine • L-glutamine is an unstable amino acid that, with time, converts to a form that cannot be used by cells, and should be added to medium just before use. • Provides nitrogen for NAD NADPH and nucleotides • Serves (like pyruvate) as secondary energy source for metabolism • Breaks down (40%) within 3 to 4 weeks, faster if in cultured cells. • In culture, glutamine breakdown generates ammonium • Some supplements (glutamax) are more stable and can replace glutamine for long term culturing of slow cells

17. Antibiotic & Antimycotic Antibiotics and fungicides can be used as a supplement to aseptic technique to prevent microbial contamination.

18. Antibiotic & Antimycotic

19. Media & Components Media, serum, and supplements should be tested for sterility before use by incubation of a small aliquot at 37°C for 48 hours. If microbial growth has occurred after this incubation, the medium or supplement should be discarded.

20. Making Media Making Media from Liquid Video Making Media from Powder Video From powder or liquid form Very “recipe” dependent.

21. Plant Culture Medium • Plant Culture Medium Requirements Varies: • Plant cell type (woody, fern, orchid…) • Maintenance of callus or shoot formation (stage II) • Stimulation of Root and de-differentiation • Protoplast, suspension or batch cultures • General Components: • Macronutrients, micronutrients, vitamins, amino acids, nitrogen, phosphorous, sugar, organic supplements and solidifying agents/support systems • AND growth regulators (hormones)

22. Plant Culture Medium • Macronutrients (macroelements) - Needed in media in large amounts and make up ~0.1% of dry weight of plant: • Nitrogen – supplied in form of ammonium ion (H4NO3+) and nitrate (KNO3) – best if both are present and together act to buffer pH. • Some amino acids can supplement N requirements or take place as the N is removed via TCA and transglutaminases • High ammonium causes a pale, glassy culture (vitrification) • Potassium – come as counter ion with NO3-and PO4-2 • Phosphorus – K2HPO4, H4NO3(HPO4)2, • High concentrations of phosphate will lead to ppt with Ca+2 and other cations

23. Plant Culture Medium • Micronutrients (Microelements): Trace amount elements and salts necessary for growth: • Fe (FeSO4) – Chelated to EDTA is most critical. The complex allows for a slow continuous release and avoids free metal generation of radical oxides after reaction with water. • Others include: Zn, Cu, B, and Mo. • Carbon and Energy Source – cultures do little if any photosynthesis (heterotrophs). Must supply carbon to metabolize ATP and other energy molecules. • Sucrose is usually used • Galactose, sorbitol and maltose also are used.

24. Plant Culture Medium • Organic Supplements – Wide range of various needs • Amino Acids– can provide nitrogen and support for metabolism as well as biosynthesis for new proteins, lipids and nucleotides • Casine (milk protein) hydrolysates typically are the source of amino acids • Vitamins: Vitamin B1 (thiamin) and Vitamin B6 (nicotinic acid pyridoxine), and myo-inositol. The latter is not a vitamin but used as one for plant culture media. • Activated Charcoal (AC) –Used for it’s ability to bind hydrophobic compounds which inhibit growth. The actual role isn’t always clear nor is it always included in medium. • Gelling Agents (support systems) – Solidified surface typically from the complex carbohydrates (non-digestible) extracted from seaweed (agar). • Lots of variation between batches and suppliers • Gums from plants, agarose can also be used

25. Plant Culture Medium • Naturally produced in apical and root meristems seeds and developing fruit • Alters proton pump and ATP production in target cells • Induces cell elongation • Suppresses lateral bud growth and stimulates adventitious roots • Synthetic form(s) include 2-4 dichorophenoxyacetic acid (2-4D) • Acts as a herbicide by inducing unsustainable growth in broad leaf (dicot) weeds – corn, rice and wheat all have one leaf (monocot). • Can be used for trees to hold fruit for development 2-4-D Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. • Auxins- Promote cell division and growth – most auxins are synthetic and not found in plants. Naturally produced 1H-indol-3-acetic acid, is unstable to both heat and light.

26. Plant Culture Medium • Used to stimulate cell division, induce shoot formation and auxiliary shoot proliferation while inhibiting root formation. Not good for stage III. • Delays cell aging and increases as some fruits bloom and grow • Used to induce bud growth in orchids and daylilies • Prevents browning in salads • When mixed with gibberellins – can increase the size of a fruit (30-50% in pears and mangos) Zeatin – first isolated from corn Kinetin– first isolated herring spirm Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. • Cytokinins – Promote cell division and are produce in young leaves fruits and seeds.

27. Plant Culture Medium Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. • Ratio of Auxin and cytokinin control root formation • Root initiation occurs when more auxin than cytokinin is in media and adventitious and shoot growth takes place when more cytokinin than auxin ratio

28. Plant Culture Medium Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. • Gibberellins & Abciscic acid- Regulate cell elongation and determine plant height. • Gibberellins increase growth of low-density cultures, enhance callus growth and elongate dwarf plants • Abscisic acid alters callus growth, enhance bud and shoot formation, and inhibit cell division. Commonly used in somatic embryogenesis

29. Plant Culture Medium H2C=CH2 Growth Regulators- Five main classes; auxin, cytokinin, gibberellins, abscisic acid and ethylene. • Ethylene- volatile gas produced during ripening, stress, mechanical damage or infection. • Produced from methyl group of methionine • Nearly all plant tissues can produce • Natural role is to encourage fruit ripening and flower blooming • Used commercially to initiate flowering and ripen tomatoes, citrus and bananas – why brown bags? • Specific protein receptors for ethylene have been found which act as transcription factors • Can be a problem in culture without proper air circulation