Upload
lesley-warner
View
267
Download
0
Tags:
Embed Size (px)
Citation preview
What is Tissue Culture?
Today’s ObjectivesGive a brief overview of Tissue Culture
Plant and Animal
Give a brief history of Animal Tissue Culture & It’s usesSome common Terminologies
We will look at Plant & Animal Cell culture in more detail before we try them.
Take a look around in the Tissue Culture Lab
DefinitionA method for studying the behavior of cells removed
from a plant or animal and the subsequent growth in favorable artificial conditions.
Use of solid, semi-solid or liquid growth medium
Brief History
First developed at beginning of 20th century
2nd half of 20th century began dispersing cell cultures, expanding on tissue culture
Widely used in research and commercial applications, including:antibiotics, transplants, various research, developing cell
lines, vaccine development, regulation of cell functions, growth factors, reconstituting tissues, etc.
Plant Tissue Culture• A very technical method of Asexual propagation
• The growing of plantlets from small pieces of plant tissue from a parent plant.
• Uses an artificial medium under sterile conditions.
• There are several advantages to
• tissue culture.
Advantages1. Many plants can be produced from a single
plant in small space and short period of time.
2. Diseases can be eliminated by quickly dividing cells.
3. Produce plants with identical flower color for the cut flower industry.
4. Promote the growth of genetically engineered plant cells.
Popular Plants to CultureAfrican Violet Ferns Orchid
Bamboo Plumeria Rose
Carnivorous Hosta Banana
Cactus Hibiscus PawPaw
Palm/Cycad Arabidopsis Daylily
Some Vocabulary we Will LearnAGAR
CALLUS
CYTOKININ
EXPLANTS
HORMONES
LAMINAR FLOW HOOD
PARENT PLANT
PLANTLETS
STERILE TECHNIQUE
Vocab You May Encounter
Aseptic- free of microorganisms
Auxin- group of plant growth regulators Endogenous auxins occur naturally Exogenous auxins are synthetic
Callus- unorganized cell mass
Cytokinin- group of growth regulators that enhances growth, morphogenesis, and cell division
Explant- source used to initiate cell culture
Animal Cell CultureFirst developed in beginning of 20th century to
study behavior of animal cellsUsed tissue fragments restricted growth to
migration of cells from fragmentsCulture of cells from primary explants dominated
field for >50 yearsPrimary explants = harvest cells, culture them
Most expansion in field was 2nd half of centuryPossible by using dispersed cell cultures
Rous first demonstrated disaggregation of explanted cells and subcultures from cellsBy surgical subdivision (rather than chemical –
enzymatic)L929 first cloned cell strain (cells are identical)
In 1950s trypsin (enzymatic) used more for subcultureDulbecco’s procedures obtain monolayer culturesGenerate single cell suspension by trypsinization
Facilitated further development of single cell cloning
Gey established first continuous human cell lineCalled HeLaTissue culture became more popular because of
antibiotic productionFacilitated long-term cell line propagation
1950s also developed defined mediaLead to serum-free media
Tissue Culture Terms“tissue culture”= general statement including organ
and cell culture“organ culture”= 3D culture of tissue retaining some or all
histological features in vivo“cell culture”= culture derived from dispersed cells taken
from another source“histotypic culture”= cells grown to recreate 3D structure
with tissue-like density“organotypic”= recombining cells of different lineages
with procedures similar to histotypic culturingGenerates “tissue equivalent”
Development of Cell Culture
Relies on 2 major branches of researchProduction of antiviral vaccinesUnderstanding of neoplasia (tumors)
Standard conditions, cell lines & assay of viruses inspired development of modern tissue culture techParticularly large amounts of cells for biochem analysisTech improvements possible by variety of media, sera
&sterile control
Increase in ethical concern promoted in vitro assays
Routine Applications in Medicine & Industry
Chromosomal analysis of cells derived by amniocentesis reveals genetic disorders
Determines quality of drinking water
Toxic effects of pharmaceutical compounds &potential environmental pollutants measured in in vitro assays
Areas Relying on Tissue Culture Techniques
Cancer research &virology
Introduction of cell fusion & genetic manipulationSomatic cell genetics became major component in
genetic analysis of higher animals
Study of cell interactions & intracellular control mechanisms in cell differentiation and development
Areas Relying on Tissue Culture Technique
Wide range of genetic recombination techniques including DNA transfer, monochromsomal transfer & nuclear transferAdded to somatic hybridization as tools for genetic
analysis &gene manipulation (In Situ Hybridization, KO Mouse)
DNA transfer spawned many techniques for transferring DNA to cultured cellsIncludes calcium phosphate coprecipitation, lipofection,
electroporation & retroviral infection
“Tissue Engineering”Generation of tissue equivalents by organotypic
culture, isolation &differentiation of human embryonic &adult totipotent SCsFrom techniques that implant normal cells from
adult or fetal tissueMatched donors or implanting genetically
reconstituted cells from same patientGene transfer, materials science, bioreactors
&transplanting tech
IVFDeveloped from early experiments in embryo
cultureNow widely used &acceptedEthical debate in generation of gametes in vitro
from primordial germ cellsOocytes cultures from embryonic mouse ovary
&implanted to generate normal miceSpermatids cultured from newborn bull testes &co-
cultured w/ Sertoli cells Similar work w/ mouse testes to fertilize mouse eggs
Primary CultureStage of culture after the cells are isolated from
the tissue and proliferated under the appropriate conditions until they completely occupy the substrateMonolayer –
Reach ConfluenceThen need to be
subcultured (Passaged)Split & Move to fresh
medium, new vessels
Chinese Hamster Ovary Cells (CHO)
Subcultures
Subcultures are needed when primary culture grows to capacity Indicates cell proliferation as important feature
Amount of growth depends on cell typeHomogeneous cell line emerges from heterogeneous
primary cultureCan now be propagated, characterized and stored
Cell StrainSubpopulation of a cell line gets positively
selected from a culture by cloningBecomes a Cell StrainOften acquires additional genetic changes from
the parent strain
Finite Vs. Continuous Cell Lines
Normal cells divide a finite number of times before losing their ability to proliferateFiniteDetermined by genetic event called Senescence
Transformed Spontaneously or chemically or virally inducedTumorsCan divide indefinitely Continuous Cell Line
Be Sterile!Use 70% ethanol
Spray on surfaces and wipeSpray on containers (bags, glass jars, etc.) containing sterile
material before bringing into sterile hoodsKeep supplies, i.e. forceps, submerged in container filled with
70% ETOH when not in use
Autoclave distilled water to yield sterile waterDip supplies in 70% ETOH in sterile water and swirl for a few
seconds to sterilize before use
• Follow given directions!• Wash your hands!
Sterile TechniqueWipe down equipment and lab areas with 70% ethanol
Minimize airflow- keep door closed
Turn on sterile hoods about 20 minutes before experiment and clean with 70% ETOH
Wash hands and arms, but don’t scrub too hard- can promote flaking
Spray outside of gloves and packages to enter the sterile hoods with 70% ETOH
• Keep equipment such as forceps and scalpels in 95% ETOH then dip in sterile water- makes equipment sterile for use
• Each time equipment is used, it must be sterilized before the next step/use
http://media.invitrogen.com.edgesuite.net/Cell-Culture/videos/CellCultureBasics.html?CID=ccbvid1
http://media.invitrogen.com.edgesuite.net/Cell-Culture/videos/SterileTechnique.html?CID=ccbvid2
http://www.benchfly.com/video/33/working-with-sterile-technique/
Experiments African violet tissue culture
Carrot tissue culture
Tobacco hormone tissue culture
Chick embryo culture, propagation, fixation and staining