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Overview • Introduction to Cell Culture. – History and definitions of cultured cells • General Modern Cell culture techniques – Aseptic technique – Media – Substrates – Passage methods • Culturing and Human Pluripotent Stem Cells – Morphology – Media – Passaging References Lecture notes (hyperlink) Activity notes (hyperlink) More links… History of Cell Culture References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Sydney Ringer (1882): Ca2+ containing salt solution that could keep a frog heart beating in a dish – Ringer’s Solution still used to rapidly restore blood volume in trauma and burn victims • Wilhelm Roux (1885): Successfully maintained chick embryonic neural tissue in a dish • Ross Granville Harrison (1907): Grew frog embryonic neural tissues in hanging-drops of clotted lymph – Proved that nerve fibers form as extensions from single nerve cells • Steinhardt, C. Israeli, and R. A. Lambert (1913): grew vaccinia virus in guinea pig corneal tissue culture [should we include more history, including use of human fetal cells for vaccine production - I think there is little awareness of this important fact…] The “Immortal Cell” Dogma Lasted 50 years References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Alexis Carrel’s Immortal Cells – 1906: immortal cells- chick heart fibroblasts, • Explant of chicken heart placed on glass slide, fed with clotted serum and chicken embryo extract • Claimed it continuously cultured for 34 years (long after the lifespan of a chicken) with no changes in appearance or growth rate • Hypothesized that all cells were immortal in that they could grow indefinitely without changing. Therefore the mechanisms of aging at the organism level could not have root mechanisms inside the cell. – Other labs couldn’t reproduce Carrel’s results –explained by their poor technique HeLa as a pioneer References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • HeLa is the first successful human cell culture • Obtained from Mrs. Henrietta Lacks in 1951 • Advanced stage of cervical adenocarcinoma Photo source: http://en.wikipedia.org/wiki/File:Henrietta_Lacks_(1920-1951).jpg http://en.wikipedia.org/wiki/File:Hela_Cells_Image_3709-PH.jpg Doubting the Dogma References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Hayflick Limit: Normal cells have a limited lifespan • Human fibroblast had a reproducibly limited number of times that they will divide before replicative senescence: – • the Hayflick limit ~50 population doublings Senescence – Exceptions germ cells, stem cells and transformed cells – 50 generations –crisis =stop dividing but may be viable for up to 18 months – Somehow cells have a “replicometer,” a method for keeping track of the number of times the cell has divided. Telomeres References Lecture Lecturenotes notes(hyperlink) (hyperlink) Activity Activitynotes notes(hyperlink) (hyperlink) MoreMore links…links… Telomeres A region of repetitive DNA at the end of a chromosome protecting the end of the chromosome from deterioration. In normal somatic cells, each time the cell divides, the length of the telomere shortens. When the telomere length gets too short, the cell dies. Culturing Stem Cells: Embedded Assessment 1 References Lecture notes (hyperlink) Activity notes (hyperlink) More links… What’s the Big Deal? • Discuss with a person next to you: Why is are immortalized cells so important to advancing scientific research? What are the advantages? disadvantages? Culturing Stem Cells: Embedded Assessment 2 References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Compare and Contrast Carrel’s “immortal” cells Genetics Potency Lifespan HeLa Cells Embryonic Stem Cells Aneuploidy References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Aneuploidy refers to any number of chromosomes other than the normal count • Gains of chromosomes or translocations contribute to abnormal cell growth An aneuploid karyotype in a cell taken from an individual with advanced stage leukemia. There are chromosome losses (Chr. 7 and 11), missing pieces of chromosomes (Chr. 9 and 16), completely lost chromosome pairs (Chr. 19), unidentified marker chromosomes (at bottom), and multiple translocations among other problems. Cell lines: Definitions • • A "primary cell line" is a population of cells derived by direct isolation from an animal. It may not necessarily divide indefinitely. An “established cell line” refers to a population of cells which has been expanded through many cell doublings – – • • • • • often beyond the Hayflick limit no apparent changes in growth behavior or phenotype Cell doubling = the number of times the culture has grown as one cell divides to make two, two to make four, four to make eight and so on… Population doubling time = the time it takes for the number of cells in a culture to double – • References Lecture notes (hyperlink) Activity notes (hyperlink) More links… may be different from the time it takes for one cell to complete a cell cycle, e.g. if some of the cells are lost due to cell death Cell passaging = expanding or "splitting" a cell culture into a new flask or series of flasks as the population grows Immortalization = dividing endlessly Transformation = alteration in certain growth characteristics. Often associated with cancer. Aneuploid = an abnormal number of chromosomes Overview • Introduction to Cell Culture. – History and definitions of cultured cells • General Modern Cell culture techniques – Aseptic technique – Media – Substrates – Passage methods • Culturing and Human Pluripotent Stem Cells – Morphology – Media – Passaging References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Aseptic technique Laminar hood Airflow = recycled air = use toxic compounds with care References Lecture Lecturenotes notes(hyperlink) (hyperlink) Activity Activitynotes notes(hyperlink) (hyperlink) MoreMore links…links… Tissue Culture Incubators References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Maintain – Temperature (37C) – Humidity (close to 100%) – Gas concentrations • CO2 - typically ~5-7% (important for CO32--buffered media) • O2 - optional (some incubators inject N2 to displace O2 down to O2 concentrations of as low as 2%) • Vibration-free • Air inside is not sterile! • Must be cleaned regularly to reduce risk of mold and bacterial growth Media: Food for cells • Early used crude cell and tissue extracts. • Chemically defined media based on body fluids and nutritional biochemistry • Harry Eagle: Eagle’s Minimal Essential Media (MEM) – 13 amino acids, six ionic species, 8 vitamins, and glucose. References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Cell Culture Media • pH Buffer – e.g., Bicarbonate, HEPES • Inorganic salts – Ca2+, Mg2+, K+, Na+, Cl-, SO42-, PO43- • Small organic molecules – Energy substrates (glucose, pyruvate) – Amino Acids – Vitamins • Anti-oxidants – 2-Mercaptoethanol, ascorbic acid, monothioglycerol, glutathione • Serum / Serum Replacement – Proteins (serum albumin) – Growth factors and hormones (insulin etc) – Lipids • Antibiotics – Penicillin, Streptomycin – Phenol Red References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Chemically Defined Media Example: mTeSR hESC Media References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Serum and growth factors References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Most medias still rely on serum. • Serum: Protein-rich fluid that separates out when blood coagulates • Contains • Commonly used serum – Fetal bovine serum (FBS) – Horse serum NGF NGF Y Y 17 Surfaces References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • TC Plates: Gas plasma treated polystyrene – Modifies the hydrophobic plastic surface to make it negatively charged and hydrophilic • Gelatin= hydrolyzed collagen • Matrigel – From Engelbreth Holm Swarm (EHS) sarcoma= laminin, fibronectin and proteoglycans • Ultra Low Attachment Plates – Polystyrene treated with a hydrophilic, neutral coating – Only cell-cell adhesions remain, resulting in formation of cell aggregates (e.g. embryoid bodies from embryonic stem cells) – Also useful for culture of non-adherent cell (e.g., hematopoietic cells) Overview • Introduction to Cell Culture. – History and definitions of cultured cells • General Modern Cell culture techniques – Aseptic technique – Media – Substrates – Passage methods • Culturing and Human Pluripotent Stem Cells – Morphology – Cell dissociation techniques – Passaging References Lecture notes (hyperlink) Activity notes (hyperlink) More links… References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Two Types of ESCs! mESCs Inner Cell Mass (epiblast+hypoblast) hESCs mEpiSCs Epiblast Amnion (epithelialized) posterior anterior Trophoblast Blastocoele Primary Yolk Sac (human embryo) Hypoblast (primitive endoderm) 20 Mouse and Human ES cells are typically grown on Feeders mESCs Phase-Contrast Microscopy References Lecture notes (hyperlink) Activity notes (hyperlink) More links… hESCs grown on mouse embryonic fibroblasts (MEFs) Mouse Embryonic Fibroblasts (a.k.a. MEFs or feeders) References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Derived from ~13.5 days old mouse embryos – Culture of adherent cells from whole embryo excluding visceral organs (and head) • Grown on gelatin-coated dishes in DMEM/FCS media • Produce Proteins (e.g., ECM and growth factors) that: – facilitate adhesion, – promote self-renewal, – and inhibit differentiation of pluripotent stem cells in culture • MEFs used as stem cell feeder cells are treated to prevent proliferation – irradiated or treated with the cell cycle drug mitomycin C – lack of MEF cell proliferation helps maintain an optimal feeder cell density • Feeder cell density – optimal MEF feeder cell density differs between mESCs and mEpiSCs or hESCs 22 Stem Cell Microscopy References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Inverted Microscope Dissecting- or StereoMicroscope 23 Stem Cell Microscopy References Lecture notes (hyperlink) Activity notes (hyperlink) More links… hESC Colony Phase Microscopy Dark-field Microscopy 24 Stem Cell Culture Quality References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Watch for changes in morphology, growth and plating rate, as well as ability to differentiate • Aneuploidy can occur even in low-passage cultures (particularly when sub-optimal culture methods are used) • Regularly check karyotype (~every 10-20 passages, and before/after experimental manipulations) • Certain contaminations can be difficult to detect without special testing (mycoplasma, viruses) • Take extreme care to prevent cross-contamination of cell lines (contaminations can be detected by DNA fingerprinting Stem Cell Homeostasis (1) References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Tissue-restricted adult stem cells reside in their niche as quiescent or slow-cycling cells • Homeostasis: adult stem cells proliferate when signaled to do so – for example, when cells need to be replaced after an injury • There is no culture system for keeping ESCs in a stable, quiescent state (like Alice in Wonderland’s the “Red Queen”, they must run to stay in place) Stem Cell Homeostasis (2) References Lecture notes (hyperlink) Activity notes (hyperlink) More links… The decision to differentiate occurs during the G1 phase of the cell cycle (G1 phase is extremely short in fast-cycling ESCs) • When cultures of ESCs get very dense, metabolic stresses and their cell cycle time can increase • ESCs differentiate in an uncontrolled fashion • IMPORTANT: regularly feed, monitor, and split (passage) embryonic stem cells in order to maintain optimal growth conditions and cell densities! Cell dissociation techniques References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Enzymatic: protease digestion of the ECM, cell-cell-, and cell-ECM interaction proteins (receptors) – Trypsin – Collagenase – Dispase • Chemical: many cell-cell interactions are dependent on bivalent ions (Ca2+) – EDTA • Mechanical – Scraping – Mechanical dissection – Triturating Culturing Stem Cells: Concept Mapping Terms References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Add the key terms/concepts from today’s lecture to your previous concept map. You should include (but are not limited to) the following terms/concepts: • Confluence • Senescence • Transformed cells • Immortalized cell line • Ploidy • Aneuploid • Serum • Telemere Due by _________. 29