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Cell Culture Set 2 of 13

by: Tarn Notetaker

Cell Culture Set 2 of 13 A214

Tarn Notetaker

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About this Document

Contains quite abit of information
Cell Culture
Souvik Chatterji
Class Notes
25 ?




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Popular in Natural Sciences and Mathematics

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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