Cell Culture Set 2 of 13
Cell Culture Set 2 of 13 A214
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This 7 page Class Notes was uploaded by Tarn Notetaker on Saturday November 7, 2015. The Class Notes belongs to A214 at Republic Polytechnic taught by Souvik Chatterji in Fall 2015. Since its upload, it has received 17 views. For similar materials see Cell Culture in Natural Sciences and Mathematics at Republic Polytechnic.
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Date Created: 11/07/15
ATCC: American Type Cell Culture A private nonprofit organization in the biotechnology field Mission focuses on the acquisition, authentication, production, preservation, development and distribution of standard reference microorganisms, cell lines and materials for research in life sciences Subculture ratio: Split ratio Can be used to ensure cells are ready for an experiment Can keep the cell culture running for future use or as a backup Suspension cell lines Have a recommended subculture seeding density Check guidelines Growth Some slow growing cells may not grow if a high split ratio is used Some fast growing cells may require a high split ratio to make sure they do not overgrow Most cells must not be split more than 1:10 as the seeding density will be too low for the cells to survive Types of cells: Cell line difference Origin tissue Cell morphologies and growth characteristics Life span (finite or continuous) Specific requirements for growth Genetic makeup Types Adherent or suspension cells Primary or transformed cells Stem cells or differentiated cells DMEM: Widely used basal medium for supporting growth of many different mammalian cells Cells successfully cultured in DMEM include primary fibroblast, neurons, glial cells, HUVECS and smooth muscle cells Unique media as it contains 4 times the concentration of amino acids and vitamins than the original Eagle’s Minimal Essential Medium Originally formulated with low glucose and sodium pyruvate but often used with higher glucose levels, with or without sodium pyruvate Does not contain no proteins, lipids, or growth factors Requires supplementation Sterilizing reagents: Autoclave Dry Solid consumables Wet Liquids Filter sterilization Liquids with protein, enzymes or serum Buffer Solution: A balanced salt solution Used for washing cells before dissociation, transporting cells or tissue, diluting cells for counting and preparing reagents Basal media: Basal medium for supporting the growth of many different mammalian cells Those that may be used for growth culture of bacteria that do not need enrichment of the media Contains ten essential amino acids, exclusive of cystine, tyrosine and glutamine Modification DMEM → Dulbecco Modified Eagle Medium RPMI → Rosewell Park Memorial Institute medium Used in cell culture and tissue culture. Traditionally used for growth of human lymphoid cell Lglutamine An amino acid required for cell culture Participates in the formation of purine and pyrimidine nucleotides, amino sugars, glutathione, Lglutamate, other amino acids and is used in protein synthesis and glucose production Unlike most other amino acids, it is not stable in solution The rate at which this degradation proceeds is a function of time, temperature and pH. Trypsin: Member of the serine protease family Cleaves peptides on the Cterminal side of lysine and arginine Most commonly used enzyme in tissue culture to release the adherent cells from culture vessel surface and/or to disaggregate tissue into single cell suspension Spore test: Way of ensuring that an autoclave is working Works by running a package of living spores through an autoclave If anything is still alive, then the autoclave did not achieve sterility Melanoma: This cancerous growths develop when unrepaired DNA damage to the skin cells triggers mutations that lead the skin cells to multiply rapidly and form malignant tumors. Skin cancer on the skin pigment Melanin cancer Cell morphologies: Epithelial Polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches Lymphoblast Spherical in shape and usually grown in suspension without attaching to a surface Fibroblast Bipolar or multipolar, have elongated shapes, and grow attached to a substrate Neuronal cells Exist in different shapes and sizes STR profile (short tandem repeats): STR are repeated segments of the DNA that are typically 26 base pairs in length STR are scattered throughout the genome Helps ensure the quality and integrity of human cell lines in the scientific community STR analysis measures the exact number of repeating units Cord blood banking (umbilical cord blood): Blood that circulates through the umbilical cord from the foetus to the placenta Blood left in the umbilical cord and placenta after the baby and the cord is cut. Umbilical cord, placenta and residual blood in it is usually discarded after the baby has been delivered. The umbilical cord would be clamped and is noninvasively cut. Pained for the mother and child. Donation of cord blood after a baby’s safe delivery is encouraged as it has the potential to save the lives of patients searching for matching stem cell unit to survive Stem cells found in umbilical cord blood can help to cure patients of diseases such as leukaemia, thalassemia’s sickle cell anaemia, other blood cancers and disorders, as well as immune disorders The cord blood is a rich source of HSCs or haematopoietic stem cells. It is well recognised to be useful to treat long bloodrelated cancer, immune and some genetic disorders Dental pulp banking: Different types of stem cells exist in different body tissues and in varying concentrations Mesenchymal stem cell MCS can form tissues such as bone, nerve muscle and blood vessels Help body to repair itself and heals by suppressing inflammation Located in places that have high concentrations in the healthy dental pulp of teeth Involves regenerative applications like bond and cartilage repair and cardiac muscle repair after myocardial infraction. Formula 1: Concentration can be in any units (M, g, g/ml) Same for volume M1V1 = MV2 Units must be same on both sides Adherent Vs Suspension cells: Adherent cells Suspension cells Requires tissue culture treated surface, Tissue culture treated surface is not usually horizontal, to adhere to required. They require agitation or shaking in vertical spinner flasks Not easy to scaleup Easy to scaleup Periodic passaging by enzymatic or Mechanical or enzymatic passaging is not mechanical dissociation is required required. Easier to passage by dilution Daily cell counts are not required Daily cell counts required to monitor cell concentration Growth is limited to the surface of the Growth is limited by the concentration of culture dish cells in medium Used in cytology and research applications Used in bulk protein harvesting and (example, skin cells) research applications (example, blood cells) Passage: A technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90% to 100% confluent Cell line: A propagated culture of specific cells after the initial subculture, representing generations of a primary culture Primary culture (heterogeneous) after the 1 st subculture (passage) Done when Pculture occupies all the available area for growth Two types Continuous Finite Go through some (couple of) of divisions than die Formula 2: Molarity (M – 1000ml) % (Mass – 100ml) (v/v) (m/v) 1M = 1000mm 1M = 1mole/ 1L Mole = mass/ mr X% means xg/(100ml of H20) Other important terms: Serum is an animal origin Cell micrograph is an image of the cell Collagenase Enzymes that break the peptide bonds in collagen Assist in destroying extracellular structures
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