SIO2004 Animal Cell and Tissue Culture Lecture 5 PDF

SIO2004 Animal Cell and Tissue Culture Lecture 5 Biotechnology Program Universiti Malaya Instructor: Dr. Nuradilla Mohamad Fauzi How do you maintain a cell culture? You: Keeping your cultured cells alive Your cell...

SIO2004 Animal Cell and Tissue Culture Lecture 5 Biotechnology Program Universiti Malaya Instructor: Dr. Nuradilla Mohamad Fauzi How do you maintain a cell culture? You: Keeping your cultured cells alive Your cells: Media changes The purpose of media changes is to replenish nutrients and avoid the build up of potentially harmful metabolic by products and dead cells. In the case of adherent cultures the media can be removed directly by aspiration and replaced. In the case of suspension cultures, cells can be separated from the media by centrifugation and resuspended in fresh media.  Check the morphology of cells in culture! Regularly examining the morphology of the cells in culture (i.e., their shape and appearance) under the microscope is essential for successful cell culture experiments Confirm the healthy status of your cells! Signs of deterioration of cells include granularity around the nucleus, detachment of the cells from the substrate, and cytoplasmic vacuolation. May be caused by a variety of reasons, including contamination of the culture, senescence of the cell line, or the presence of toxic substances in the medium, or they may simply imply that the culture needs a medium change. Allowing the deterioration to progress too far will make it irreversible. Now that you have isolated cells, what kind of cells do you actually get? Primary culture freshly isolated from tissue source Cell line Finite cell line: has a finite lifespan; dies after several sub-cultures Continuous cell line: ‘immortal’ cancer cells, ‘transformed’ cells Generally, cells that can be passaged and maintained in culture are cells that can divide! (not all cells can)  Many cell types in culture move to avoid each other and proliferate. This is known as contact inhibition. Proliferation ceases when the dish is covered with a single layer of cells (monolayer). Once the cells form a continuous sheet they stop dividing. This is known as confluence.  Contact inhibition and confluence Confluence is also commonly used as an estimate of the number of adherent cells in a culture dish or a flask, referring to the proportion of the surface which is covered by cells e.g. 50% confluence means roughly half of the surface is covered and there is still room for cells to grow 100% confluence means the surface is completely covered by the cells, and no more room is left for the cells to grow as a monolayer The cessation of movement at confluence is known as contact inhibition and cells eventually stop dividing (withdrawal from the cell division cycle). Cells may differentiate (change their characteristics) when they reach confluence, depending on the microenvironment. ? Subculturing -> Passaging At high confluency, cells become too crowded and nutrients become limiting. To avoid differentiation or cell death and to maintain proliferation, cells need to be passaged. Removal of the medium and transfer of cells from a previous culture into fresh medium For adherent cells, detach cells by enzymatic dissociation Trypsin, Trypsin-EDTA Trypsin+collagenase Dispase (for detaching confluent, intact sheets from the surface of culture dishes without dissociating the cells) OR by mechanical dissociation, via scraping Cell scraper For cells sensitive to proteases For cells in suspension, simply take a small volume of the parent culture and diluting it in fresh growth medium  To move adherent cells, adhesion molecules such as integrins and cadherins have to be broken. Those proteins anchor cells to one another as well as to the extracellular matrix (ECM). Trypsin which cleaves peptides at lysines and arginines if not followed by a proline. EDTA is used as a chelator for Ca2+and Mg2+ ions which are needed by adhesion molecules. https://doodlesindishes.wordpress.com/tag/comic/ After an individual incubation time of 5 – 10 min all cells should have detached and ’rounded up’. You can keep growing cells and passaging them … but what if you have to press pause?  Cryopreservation of cells Why freeze cells? Maintaining cells when you don’t need them can be expensive! Cell lines in continuous culture are bound to change characteristics Finite cell lines have a limited lifespan (not immortal; will stop dividing) Preserve stocks for long-term storage Dimethyl sulfoxide (DMSO) is widely used for the freezing of cells DMSO is a cryoprotectant added to prevent the formation of ice crystals during the freezing process, otherwise cells would be destroyed Freeze at the rate of 1°C per min Store in liquid nitrogen Interstellar (2014) “Plan B” thaw Now that you have isolated cells, what kind of cells do you actually get? Primary culture freshly isolated from tissue source Cell line Finite cell line: has a finite lifespan; dies after several sub-cultures Continuous cell line: ‘immortal’ cancer cells, ‘transformed’ cells Generally, cells that can be passaged and maintained in culture are cells that can divide! (not all cells can) Which cells actually divide? Stem cells Precursor cells (unipotent stem cells; “-blasts”) e.g. fibroblasts Germ cells (makes gametes) … and cancer cells! Most differentiated (specialized) cells normally do not divide. Cultured cell lines are more representative of precursor cell compartments in vivo than of fully differentiated (specialized) cells. Commonly Used Cell Lines Not all cells divide at the same rate, or at all! Other than stem cells and cancer cells, cells can generally be categorized into cells that do not divide e.g. nerve cells, heart muscle cells (why spinal injuries, heart attacks, and strokes are that much more scary and difficult to treat in the hospital!) Their functions are so specialized that they've lost the ability to replicate. Muscles, for example, are composed of many cells fused end-to-end (each fiber is, in effect, one giant multinucleated cell). retain but do not normally utilize their capacity for division, unless stimulated e.g. liver, kidney cells (that’s why we can do liver transplants) e.g. fibroblasts – wound recovery Not all cells divide at the same rate, or at all! cells that are constantly being renewed at various rates Cells makes up tissues where brand new cells are always being made to replace “old” cells. e.g. skin cells, the cells in your gut lining, and blood cells e.g. blood cells for example are produced from hematopoietic stem cells in the bone marrow and the average adult produces 3-10 billion cells per hour Some cells divide rapidly (beans, for example take 19 hours for the complete cycle; red blood cells must divide at a rate of 2.5 million per second) Environmental factors such as changes in temperature and pH, and declining nutrient levels lead to declining cell division rates. When cells stop dividing, they stop usually at a point late in the G1 phase, the R point (for restriction), in the cell cycle. Cell cycle Cells are regenerated from cells, and the only way to make more cells is by division of those that already exist! A cell reproduces by performing an orderly sequence of events in which it duplicates its contents and divides in two Eukaryotic cells have evolved a complex network of regulatory proteins, known as the cell-cycle control system Governs progression through the cell cycle Can respond to various signals from both inside and outside the cell e.g. respond to signals from other cells, stimulating cell division when more cells are needed and blocking it when they are not Has central role in regulating cell numbers in the tissues of the body Remember the cell cycle??? G1? G2? M? S??? The cell checks newly The cell scans the internal replicated DNA for errors and external environment to and repairs them assess whether conditions If not successful, are favorable for division apoptosis If yes, proceed to S BRCA1, BRCA2 are If no, exit to G0 involved in DNA repair p53 = master regulator pathways Rb R G0 Senescence and/or apoptosis Or go back to G1 (quiescence) https://www.youtube.com/watch?v=aDAw2Zg4IgE “Actual Footage of Cell Division (Kidney Cells)” by Hashem Al-Ghaili Can normal cells divide forever and ever? (in culture) Nope, most normal cells have a finite lifespan! (most of them will eventually stop dividing)  In Vitro “Age” of a Cell Culture The in vitro age of a cell culture is particularly useful to know for cell lines with a finite lifespan or unstable characteristics that change over time in continuous culture. Two terms are predominantly used to define the age of a cell culture: (i) Passage number: Indicates the number of times the cell line has been sub-cultured (ii) Population doubling (pd) number: Indicates the number of cell generations the cell line has undergone i.e. the number of times the cell population has doubled Replicative cell senescence Multicellular organisms replace worn-out cells through cell division. However, in many cells, proliferation slows down until cell division eventually halts. Cells enter a non-dividing state, known as senescence. In humans this occurs, on average, after 52 divisions, known as the “Hayflick limit”. e.g. Human fibroblasts permanently cease dividing after 25-40 divisions (population doublings) In culture, primary cells have a finite lifespan. Cells stop dividing because the telomeres, protective bits of DNA on the end of chromosomes, shorten with each division, eventually being consumed.  Telomeres A telomere is a region of repetitive nucleotide sequences at each end of a chromosome. Protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. Disposable buffers at the ends of By U.S. Department of Energy Human Genome Program - chromosomes which are truncated during http://science.nasa.gov/media/medialibrary/2006/03/16/22mar_telomeres_resources/caps.gif, Public Domain, https://commons.wikimedia.org/w/index.php?curid=5234303 cell division; their presence protects the genes before them on the chromosome from being truncated instead. For vertebrates, the sequence of nucleotides in telomeres is TTAGGG.  Telomeres and cellular ageing Each time a cell divides, some of the telomere is lost (usually 25-200 base pairs per division) In humans, average telomere length declines from about 11 kilobases at birth to less than four kilobases in old age During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, so in each duplication the end of the chromosome is shortened When the telomere becomes too short, the chromosome reaches a "critical length" and can no longer replicate (the cell has become “old”) Cells undergo senescence or apoptosis  Telomerase Telomerase is a reverse transcriptase enzyme that lengthens telomeres in DNA strands Adds telomere repeats Prevents degradation of the chromosomal ends following multiple rounds of replication Telomerase activity is regulated during development. Post-natal somatic cells are deficient in telomerase.  Cells that express telomerase Which cells express telomerase? Fetal tissues Adult germ cells Stem cells Cancer and transformed cells Telomerase expression can allow senescent cells that would otherwise become post-mitotic and undergo apoptosis to exceed the Hayflick limit, extend the lifespan of the cells, and become potentially immortal (transformation!)