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