What is Tissue Culture?

Today’s Objectives Give a brief overview of Tissue Culture Plant and Animal Give a brief history of Animal Tissue Culture & It’s uses Some 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

Definition A 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.

Advantages Many plants can be produced from a single plant in small space and short period of time. Diseases can be eliminated by quickly dividing cells. Produce plants with identical flower color for the cut flower industry. Promote the growth of genetically engineered plant cells.

Popular Plants to Culture African Violet Ferns Orchid Bamboo Plumeria Rose Carnivorous Hosta Banana Cactus Hibiscus PawPaw Palm/Cycad Arabidopsis Daylily

Some Vocabulary we Will Learn AGAR 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 Refer to Carolina’s “Plant Tissue Culture Definitions and Techniques” packet

Animal Cell Culture First developed in beginning of 20th century to study behavior of animal cells Used tissue fragments restricted growth to migration of cells from fragments Culture of cells from primary explants dominated field for >50 years Primary explants = harvest cells, culture them Most expansion in field was 2nd half of century Possible by using dispersed cell cultures

In 1950s trypsin (enzymatic) used more for subculture Rous first demonstrated disaggregation of explanted cells and subcultures from cells By surgical subdivision (rather than chemical – enzymatic) L929 first cloned cell strain (cells are identical) In 1950s trypsin (enzymatic) used more for subculture Dulbecco’s procedures obtain monolayer cultures Generate single cell suspension by trypsinization Facilitated further development of single cell cloning

Gey established first continuous human cell line Called HeLa Tissue culture became more popular because of antibiotic production Facilitated long-term cell line propagation 1950s also developed defined media Lead 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 culturing Generates “tissue equivalent”

Development of Cell Culture Relies on 2 major branches of research Production of antiviral vaccines Understanding of neoplasia (tumors) Standard conditions, cell lines & assay of viruses inspired development of modern tissue culture tech Particularly large amounts of cells for biochem analysis Tech 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 manipulation Somatic 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 transfer Added 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 cells Includes calcium phosphate coprecipitation, lipofection, electroporation & retroviral infection

“Tissue Engineering” Generation of tissue equivalents by organotypic culture, isolation &differentiation of human embryonic &adult totipotent SCs From techniques that implant normal cells from adult or fetal tissue Matched donors or implanting genetically reconstituted cells from same patient Gene transfer, materials science, bioreactors &transplanting tech SC= stem cell “organotypic”= recombining cells of different lineages with procedures similar to histotypic culturing

IVF Developed from early experiments in embryo culture Now widely used &accepted Ethical debate in generation of gametes in vitro from primordial germ cells Oocytes cultures from embryonic mouse ovary &implanted to generate normal mice Spermatids cultured from newborn bull testes &co-cultured w/ Sertoli cells Similar work w/ mouse testes to fertilize mouse eggs

Primary Culture Stage of culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they completely occupy the substrate Monolayer – Reach Confluence Then 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 type Homogeneous cell line emerges from heterogeneous primary culture Can now be propagated, characterized and stored

Cell Strain Subpopulation of a cell line gets positively selected from a culture by cloning Becomes a Cell Strain Often 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 proliferate Finite Determined by genetic event called Senescence Transformed Spontaneously or chemically or virally induced Tumors Can divide indefinitely Continuous Cell Line

Be Sterile! Use 70% ethanol Spray on surfaces and wipe Spray on containers (bags, glass jars, etc.) containing sterile material before bringing into sterile hoods Keep supplies, i.e. forceps, submerged in container filled with 70% ETOH when not in use Autoclave distilled water to yield sterile water Dip supplies in 70% ETOH in sterile water and swirl for a few seconds to sterilize before use Refer to Carolina’s “Plant Tissue Culture Definitions and Techniques” packet Wash your hands! Follow given directions!

Sterile Technique Wipe 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