1. Tissue Culture Biotechniques (BIOL 410)

2. Tissue Culture The Growth of tissues or groups of cells outside the organism – Multicellular organisms – In vitro Can include the growth in separate cells in culture from a multicellular organism – Or groups of cells as a tissue mass 2

3. Tissue Culture vs. Cell Culture Cell Culture Cell colony lack structure – Disassociated cells Tissue Culture Cells from multi-cellular organism – Contains some of the original cells structure (e.g. adhesion) 3

4. Tissue Culture Rely on on other organ systems – Division of labor within the organism Tissue culture needs to compensate for lack of other systems – Provide resources 4 Much like organelles, tissue, organs and organ systems cannot survive outside of the body – ex vivo

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6. Tissue Culture Tissue – groups of cells that share a morphology – Specialized function Types of tissue culture can include any of the major organ systems 6

7. Tissue Culture Similar tissues can be found throughout the body – Epithelial – Striated muscle Some tissues are localized – Liver – Lung 7

8. Tissue Culture Some organ systems share similar tissue – Common morphology – E.g. epithelia tissue 8 Similar structure, and origin – Similar function Some specification/specia lization

9. Considerations 1.Normal cells have limited longevity in vitro – Cancer cells 2.Some cells are easier to grow and work with outside of the body – May be similar to target cells, but not the same 3.In vitro experimentation may not represent what happens in nature – Physiological Relevancy – Scientific manipulation easier than in vivo 9

10. Cancer Cells Cells that have lost growth inhibition Creating immortal cell lines – Making them easier to use in biomedical research Induced pluripotency 10

11. Density dependent inhibition When cell density gets high enough cells stop dividing Cancer cells loose density dependence inhibition – And Contact inhibition – Grow out of control 11

12. Stem cells (potency) 12 The ability to form multiple tissue types – Decreases with subsequent stages of development

13. Stem Cell Medicine Stem cells can be generated by: – Harvesting embryonic stem cells (ESC) or adult stem cells – Replacing DNA in a ESC with Autosomal DNA from an adult – Chemical manipulation of an adult cell to induce pluripotency (IPS) 13

14. Stem Cell Research Cultured in the lab – Using chemical signals cells are kept pluripotent. 14

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16. Requirements for Tissue Culture Stable environment (e.g. Human) – Temperature (37 o c) – pH (varies) – Tonicity (isotonic; 0.9%) Nutrient rich Oxygen rich – But not too rich! 16

17. Requirements for Tissue Culture Tissue culture media – Nutrient rich environment to promote cell health – Solid – Liquid Growth Factors – Chemical signals that stimulate in vitro growth Sterile environment for culture 17

18. Tissue culture media Mixture of nutrients and antimicrobial compounds – Provide materials for cells growth (carbohydrates, proteins, lipids, nucleotides, vitamins and ions) – Prevent contamination (bacteria, fungi, protozoans) Liquid or Solid – Depending on tissue types, and questions asked – Effects growth (structural) of cells 18

19. Growth Factors Protein signals instruct cells to divide – Signaling pathways – PDGF is a growth factor 19

20. Sterile Environment Prevent contamination Without other organ systems unable to fight infections – i.e. immune system Culture is easily contaminated – We will practice extreme caution to prevent this – Laminar Flow hood 20

21. 21 Fume HoodLaminar Flow Hood (Clean Bench)

22. Types of tissue culture Variation in media and growth chamber can affect patterns of growth – Depending on what you want to research 22

23. History of Tissue Culture 1860’s Sidney Ringer created a salt solution to observe frog embryos over several weeks Solution did not kill cells – Inhibited growth 23

24. History of Tissue Culture 1885 Wilhem Roux removed epiblast of a developing chicken embryo – Kept it alive for several days in warm saline This established the principles for tissue culture – Environment that mimicked natural conditions – Lacked nutrients, which is why embryo eventually died 24

25. Uses of tissue culture Bioreactors (molecule synthesis) Cell Engineering Cell Stability Therapeutics Diagnostics 25 Engineering Medicine Research (Biology)

26. 26 Commercial Use?

27. Biomedical Commercial Use 27

28. Similar technique to animal cell propagation – Hearty cultures – Form natural structures (patterning) Plant Tissue Culture (Micropropigation) Plant culture is also an important field of study Many fruits rely on micropropigation for survival – “Seedless” 28

29. Chicken Cell Cultures Red Jungle Fowl (Gallus gallus) – The common chicken Cells of different tissue lines can be cultured from embryonic cells – Like stem cells in humans Chickens are a model organism for studying development – Genetics 29

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31. Chicken Tissue Culture Experiment 10 days post-fertilization – Limb structures – Eyes – Organogenesis We will be culturing cells from chicken embryos to gain familiarity with the technique – Cell extraction – Culturing 31

32. Howard’s Ringer Solution Warm solution simulates pre-natal environment – Used in transfer to in vitro – Use Plenty to rinse and hold tissue during observations and transfer 32 Saline Solution used in extraction and culture – Isotonic – Similar to IV drip solution

33. Explantation 1.Gather all tools and sterilize with alcohol and heat – Wipe down microscope and place in the hood 2.Place egg on rubber stand inside a petri dish – This will keep the egg from rolling – Petri dish contains the mess 3.Gently use probe to crack the egg, and begin peeling back the shell – It is ok if you break the membrane – Do not break the yolk 33

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35. Explantation 4.Once you have created a large enough hole – Observe (using the dissection microscope and record observations 5.Set up a small beaker with warm ringer solution – You can use the microscope light to keep it warm 6.Begin to remove tissue from embryo using scissors and forceps – Place into a clean petri dish – Rinse with ringer solution 35

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37. Explantation 7.Transfer explant to the dish with culture media – Transfer as little ringers as possible 7.Make additional observations. – You will make observations over the next few days 8.Clean up when you are done. 9.Cleanliness is KEY!!! 37

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