1.Advanced Aminal Cell Culture 2013 2nd Semester Department of Animal Science Chungbuk National University 2st Lecture

2. Syllabus Date Topics September 5, 2013 Introduction : What is Cell Culture? September 12, 2013 Cell Culture As Model System For Research September 26, 2013 Cell Culture For Antibody / Protein Production October 10, 2013 Protein Production/Purification October 24, 2013 Stem Cell I November 7, 2013 Stem Cell II November 21, 2013 TG/KO Animals December 5, 2013 Genome Engineering/NGS December 12, 2013 Final Exam 3. Date September 26, 2013 Cell Culture For Antibody / Protein Production , October 10, 2013 Protein Production/Purification , October 24, 2013 , November 7, 2013 Stem Cell I Jia Jia Lin, November 21, 2013 Stem Cell II Zhao MingHui, December 5, 2013 Transgenic Animals Lin Zili, December 12, 2013 Genome Engineering/NGS , 4. Animal Cell Culture for the Model System for the research By the way, what is the model system? - Sometime you should find the replacement for your subject of research to reduce difficulty in practicing research. -In many cases, human experiment is not feasible or unethical. We cannot test unprove Chemical/treatment to human directly without animal tests. - In those case, we need to find non-fuman species to understand particular biologica Phenomena, with the expectation that discoveries made in model system will provide Insight into working of other organisms. 5. Commonly Used Model Organism - E.coli - Drosophila - ZebraFish - Mouse - Pig - Chimpanzee.. 6. Cultured Cell as Model System For Research - You can avoid the interferences from other kinds of cell, tissue, organ and can focus on the specific kind of cell you want to study - You can observe and monitor during the treatment in vitro conditions - Much easier for the study using microscopic/biochemical/Molecular Biologic methodology.. - Cheaper and Faster than most animal experiments - Ethical Consideration : Reduce laboratory animal sacrification.. 7. Cultured Cell as Drug Discovery Platforms - Screening of chemical Library 8. Choice of Cell - Primary Cell or Cell Line? - Source of Organism? - Source of Tissues Many Cell Line Derived from various tissues can be used as model system for studies HUVEC (Human umbilical vein endothelial Cells) : model system for endothelial cells RAW264.7 (Murine Macrophage) Generally cell line is chacterized better than primary cell and easy to culture Limited life spanning of primary culture limits experiment In some cases (Neuron for example), Primary cell is the only source for cultured Cell Primary Cell is thought to be much closer than Cell Line 9. Commonly Used Cell Line 10. Experimental Facillities - Incubator with CO2 - Microscope - Cell Culture hood - Cryogenic Storage 11. Asceptic Techniques - Contamination is the major problem during animal cell Culture - Contamination can be divided by source * Bacteria * Fungi * Microplasma * Virus * Other animal cell (e.g. HeLa) - You have to do your best to avoid contaminations Sanitization / Sterillization (UV) Laminar Flow Hood Restricted/Designated Cell Culture Area 12. Contaminants (Bacteria) 13. Contaminants (Yeast) 14. Mycoplasma : simple bacteria that lack a cell wall (smallest self-replicating organism) Extremely small (less than one micrometer), difficult to detect Only way to detecting mycoplasma contamination : immunostaining, fluorescent staining (Hoechst 33258), PCR Contaminants (Mycoplasma) 15. Subculture - Removal of the medium and transfer of cells from a previous culture into fresh medium - When the cells in adherent cultures occupy all the available substrate and have no room left for expansion, or when the cells in suspension cultures exceed the capacity of the medium to support further growth, cell proliferation is greatly reduced or ceases entirely. 16. Cell Density - In adherent cell, cell will not grow when they cover the surface of culture dish (Contact inhibitions) Exhaustion of Medium - Exhaustion of Medium - pH change - Accumulation of toxic compounds (secreted from the cell) 17. Subculture of adherent Cell 1. Remove and discard the spent cell culture media from the culture vessel 2. Wash cells using a balanced salt solution without calcium and magnesium 3. Remove and discard the wash solution from the culture vessel 4. Add the pre-warmed dissociation reagent such as trypsin to the side of the flask 5. Incubate the culture vessel at room temperature for approximately 2 minutes. 6. Observe the cells under the microscope for detachment. 7. When 90% of the cells have detached, tilt the vessel for a minimal length of time to allow the cells to drain. Add the equivalent of 2 volumes (twice the volume used for the dissociation reagent) of pre-warmed complete growth medium. 8. Transfer the cells to a 15-mL conical tube and centrifuge then at 200 g for 5 to 10 minutes 9. Resuspend the cell pellet in a minimal volume of pre-warmed complete growth medium and remove a sample for counting 10. Determine the total number of cells and percent viability using a hemacytometer 18. Subculture of suspension cell 1. Remove the flask from the incubator and take a small sample from the culture flask using a sterile pipette. 2. determine the total number of cells and percent viability 3. Calculate the volume of media that you need to add to dilute the culture down to the recommended seeding density 4. Aseptically add the appropriate volume of pre-warmed growth medium into the culture flask 19. Cell should be stored as frozen as a seed stock Cell will be damaged by Freezing/Thawing - Protection against freezing : Cryoprotection Best Method for Cryopreserving cultured cell - store in liquid nitrogen in complete medium in the presence of cryoprotectant like DMSO DMSO : Dimethyl Sulfoxide Cell Freezing 20. Serum Based or Serum Free? Serum : Fetal Bovine Serum(FBS) Media 21. Research Methods using cultured Cell Microscopic Biochemical Molecular Biology 22. Optical Microscope : Main workhorse of Cell Research 23. Phase-Contrast Microscope Contrast-enhancing techniques 24. Differential-Interference-Contrast Microscope 25. Fluorescences Fluorescence : some molecules can absorb one color and emits different colors 26. Fluorescences Microscope 27. Filters: the key to successful fluorescence microscopy 28. Staining of different components of the cell Q : We want to localize the location of a specific protein in cell. How we can do that? A : Use Antibody! By labeling antibody with fluorescence, you can locate the desired protein in 29. Alpha-Tubulin Actin Mitochondria Synaptic Vesicle 30. Immunofluorescence - Direct Immunofluoresence * Antibody (or chemical) which can bind a desired protein is labeled with fluorochrome * Pros - Convienient - More Sensitive * Cons - You should have a primary antibody labeled with fluorochrome - If you dont have it, you should do it by yourself or use Indirect Immunofluoresence 31. Alexa-Phalloidin Phalloidin : Actin binding Chemical 32. - Indirect Immunofluoresence * Unlabeled Antibody is applied on the fixed tissue * Antibody was detected by secondary antibody conjugated with fluorochrome Primary Antibody Recognize Antigen Secondary Antibody recognize Primary Antibody It is labeled by fluorescence Pros You dont need to label primary antibody Based on the selection of secondary antibody, you can change wavelength of signal Cons * More complicated (Two step process) 33. Fixation and Section We need to stop the cellular process and preserve the component inside in cell. Crosslinking Fixation Commonly used for luoresence microscopy Generate covalent cross-links between intracellular components Most commonly used agent : aldehyde Formation of bond between amine grouop Glutaraldehyde formaldehyde - Precipitating Fixatives : Disctrupt hydrophobic interaction Denature proteins Methanol, Ethanol, Acetic Acid 34. Colocalization Using two different fluorophore with different wavelength, we can test cellular locations of Two protein simultaneously. A B 35. Choice of fluorophore * Choice of two closely distributed spectrum may cause bleeding 36. Fluorescence Protein as Reporter GFP Gene of Interest - Drawbacks of immunoflorescences You need to have (specific and high-quality) antibody against your protein of interest You need to fix a cell (i.e. Dead Cells), so you cannot observe live event in live cell - You need a probe which will work in In the Living cell (and even organism) GFP(RFP) Your Gene of Interest Transfection 37. Time-Lapse Imaging of Live Cell 38. Confocal Microscopy The main problem in the florescence microscopy is that strong illumination background from other focal planes 39. Biochemical Characterization of Cell 40. Immunoprecipitation 41. Molecular Biology techniques Loss-of-function Study - Knockdown of functions for GOI (Gene of Interest) - Find a Phenotype caused by the Ablation of Gene Function - Find a function of Gene/Protein - Can be classified as - RNAi - Morpholino - Dominant Negative Mutant Gain-In-Function Study - Introduction / overexpression of GOI - Find a Phenotype caused by the (over)expression of Gene 42. RNAi (RNA interferences) Temporal knockdown of desired gene Loss-of function Study 43. Transfection