What is Tissue Culture?

1. What is Tissue Culture?

2. 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

3. 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

4. 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.

5. 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.

6. 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.

7. Popular Plants to Culture

8. Some Vocabulary we Will Learn • AGAR • CALLUS • CYTOKININ • EXPLANTS • HORMONES • LAMINAR FLOW HOOD • PARENT PLANT • PLANTLETS • STERILE TECHNIQUE

9. 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

10. 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

11. 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

12. 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

13. 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”

14. 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

15. 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

16. 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

17. 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

18. “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

19. 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

20. 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)

21. 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

22. 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

23. 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

24. 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 • Wash your hands! • Follow given directions!

25. 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

26. 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/

27. Experiments • African violet tissue culture • Carrot tissue culture • Tobacco hormone tissue culture • Chick embryo culture, propagation, fixation and staining