Transcript Research Focused Undergraduate Education

Plant and Mammalian Tissue Culture
Plant and Animal Cell Culture Medium
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)
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
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.
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.
Selecting Media (animal cells)
In general a good place to start is MEM
for adherent cells and RPMI-1640 for
suspension cells.
RPMI Media
Eagles Minimum Essential
Media
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.
Culture Media
 Requires
Ions; Na+, K+, Ca++, Mg++, Cl-, PO4-, HCO3Trace elements; iron, zinc, selenium
Sugars such as glucose
Amino Acids
Media Selection
Cell Line
Cell Type
Species
Tissue
Medium
293
Fibroblast
Human
Embryonic
Kidney
MEM
10% HI
Horse Serum
3T6
Fibroblast
Mouse
Embryo
DMEM
10% FBS
A549
Epithelial
Human
Lung
Carcinoma
F-12K
10% FBS
H9
Lyphoblast
Human
T-Cell
Lymphoma
RPMI-1640
10% FBS
HeLa
Epithelial
Human
Cervix
Carcinoma
MEM
10% FBS
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-
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.
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.
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.
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
Antibiotic & Antimycotic
Antibiotics and fungicides can be used
as a supplement to aseptic technique to
prevent microbial contamination.
Antibiotic & Antimycotic
Antibiotic / Antimycotic
Working
Concentration
Stability at 37°C
Amphotericin B
2.5 µg / ml
3 days
Ampicillin
100 µg / ml
3 days
Penicillin
100 U / ml
3 days
Streptomycin
100 µg / ml
3days
Puromycin
20 µg / ml
Unknown
Kanamycin
100 µg / ml
5 days
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.
Making Media
From powder or liquid form
Very “recipe” dependent.
Making Media
from Powder
Video
Making Media
from Liquid
Video
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)
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
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.
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
Plant Culture Medium
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.
• 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
Plant Culture Medium
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.
• 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
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
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
Plant Culture Medium
Growth Regulators- Five main classes; auxin, cytokinin,
gibberellins, abscisic acid and ethylene.
 Ethylene- volatile gas produced during ripening, stress,
mechanical damage or infection.
H2C=CH2
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
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• Can be a problem in culture without proper air circulation