References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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 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…]

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… The “Immortal Cell” Dogma Lasted 50 years 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… HeLa as a pioneer HeLa is the first successful human cell culture Obtained from Mrs. Henrietta Lacks in 1951 Advanced stage of cervical adenocarcinoma Photo source:

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Doubting the Dogma 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.

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Telomeres References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More 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.

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Culturing Stem Cells: Embedded Assessment 1 Discuss with a person next to you: Why is are immortalized cells so important to advancing scientific research? What are the advantages? disadvantages? What’s the Big Deal?

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Culturing Stem Cells: Embedded Assessment 2 Compare and Contrast Carrel’s “immortal” cells HeLa CellsEmbryonic Stem Cells Genetics Potency Lifespan

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Aneuploidy 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.

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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 – 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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 References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Laminar hood Airflow = air flow duct recycled air = use toxic compounds with care

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Tissue Culture Incubators Maintain – Temperature (37C) – Humidity (close to 100%) – Gas concentrations CO 2 - typically ~5-7% (important for CO buffered media) O 2 - optional (some incubators inject N 2 to displace O 2 down to O 2 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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 – Ca 2+, Mg 2+, K +, Na +, Cl -, SO 4 2-, PO 4 3- 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 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 17 Y Y NGF

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Surfaces 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)

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… 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… Two Types of ESCs! 20 Inner Cell Mass (epiblast+hypoblast) Trophoblast Blastocoele Epiblast (epithelialized) Hypoblast (primitive endoderm) Primary Yolk Sac (human embryo) anterior posterior mESCs hESCs mEpiSCs Amnion

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Mouse and Human ES cells are typically grown on Feeders Phase-Contrast Microscopy mESCshESCs grown on mouse embryonic fibroblasts (MEFs)

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Mouse Embryonic Fibroblasts (a.k.a. MEFs or feeders) 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Stem Cell Microscopy 23 Inverted Microscope Dissecting- or Stereo- Microscope

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Stem Cell Microscopy 24 hESC Colony Phase Microscopy Dark-field Microscopy

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Stem Cell Culture Quality 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 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

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Stem Cell Homeostasis (1) 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)

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Stem Cell Homeostasis (2) 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! The decision to differentiate occurs during the G1 phase of the cell cycle (G1 phase is extremely short in fast-cycling ESCs)

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Cell dissociation techniques 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 (Ca 2+ ) – EDTA Mechanical – Scraping – Mechanical dissection – Triturating

References  Lecture notes (hyperlink)  Activity notes (hyperlink)  More links… Culturing Stem Cells: Concept Mapping Terms 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