Tissue Culture Biotechniques (BIOL 410)

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

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

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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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

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

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

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

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

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

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

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

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

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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

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

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

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

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 Fume HoodLaminar Flow Hood (Clean Bench)

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

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

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

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

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Biomedical Commercial Use 27

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

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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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

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

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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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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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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