development and maintenance of cell line

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Development and maintenance of cell line :

Development and maintenance of cell line N.STALIN M.PHIL ANIMAL BIOTECHNOLOGY


CELL CULTURE The term "cell culture" has come to refer to the culturing of cells derived from single cellular eukaryotes , especially animal cells. It is the technique in which the cells/tissues are taken from an animal’s body and grown, and maintained in a laboratory in a suitable medium These immortal cells are 'useful' for experimentation in labs as they are always available to researchers. Why ? Cellular and molecular studies. Protein over expression/cell structure and function studies Drug development Vaccine test/ production.


Cell Line The in vitro development of immortalized human tumor cells successfully cultured in the lab in 1952 for scientific research and Cell lines have been the backbone of cancer research. Cells that have undergone a mutation and becomes immortal (infinite life span ) i.e. Attain indefinite growth. Transformed cell line A cell line that has been transformed by a tumor inducing virus or chemical that has the ability to cause tumors if injected into animal. Hybrid cell line ( hybridoma ) Fused cells with two different characteristics CELL LINES


DEVELOPMENT OF CELL LINE The strategies to help increase understanding and improve the stability, quality and manufacturability of cell lines. You take cells from various sources (tumors) and grow them in various cell culture media, and asses their biological properties


CRITERIA FOR CELL LINE SELECTION Stability of cell. Product for biological activity Product expression: level, duration and indelibility Growth and productivity in large scale culture ease of selection of high producers Adaptation to protein free suspension culture apoptosis; proliferation rate; max cell number Safety issues


PRIMARY CELL CULTURE Primary culture : cell recently excised from specific organs of animals. When cells are surgically removed from the organism and placed into the environment, they will attach, divide and grow. Steps: Isolation of tissue Disintegration (separation)of cells Incubation and growth

Primary cell culture:

Primary cell culture + enzymes time


BASIC COMPONENTS IN THE CULTURE MEDIA The artificial environment created in the laboratory is generally known as media and its regulate the cell cycle Most animal cell culture media are generally having following 10 basic components and they are as follows: Energy sources: Glucose, Fructose, etc Nitrogène sources: Aminoacides Vitamins: Generally water soluble vitamins B & C Inorganic salts: Na+, K+, Ca2+, Mg2+ Fat and Fat soluble components: Fatty acids, cholesterols Nucleic acid precursors Antibiotics( ampicillin & tetracycline ) Growth factors and hormones pH and buffering systems Oxygen and CO2 concentration .


LAMINAR AIR FLOW When fresh air is passed in the laminar air flow it replaces the contaminated air inside and keeps it contamination free. PRINCIPAL- Laminar Air Flow is based on the flow of air current to create uniform velocity, along parallel lines, which helps in transforming microbial culture in aseptic conditions .


CO2 INCUBATOR Our blood contains CO2, usually around 40mmHg, which is close to 5%. The importance in mammalian cell culture is the same as in mammalian blood


INVERTED PHASE MICROSCOPE A phase contrast microscope with objectives below the specimen. A phase plate with an annulus will aid in exploiting differences in refractive indices in different areas of the cells and surrounding areas, creating contrast


VACUUM PUMP For permanent aspiration of liquids (media, PBS and TRED). Use unplugged glass pasteur pipettes, throw into sharps box when done.


CELL CULTURE FLASKS It stores stuff, and you can mix chemicals and make solutions easily without the use of a stirring rod or something like that. It could also be a tool for measuring milliliters


APPROACHES FOR CELL LINE ENGINEERING Identification of genes/proteins that are specifically up-regulated in bio processing conditions. Engineering of cells and selection of a new cell line Engineering of cells to over-express the gene(s) of interest (historical approach) Examine the effect and select new cell line Further understanding of genes/pathways directly regulated by the gene of interest


KEY GENES IN PROLIFERATION AND APOPTOSIS bcl-2 suppresses cell death p21 arrests the cell proliferation and enhance specific productivity c- myc enhances proliferation rate, reduces serum dependency and induces anchorage independence hTERT reduces apoptosis, enhances proliferation and increases attachment tendency in the absence of serum


THE ADVANTAGES Increases cell viability Prolongs culture duration Reduces serum dependency Improves nutrient metabolism Protects cells in stressful conditions Enhances adaptation in serum free media


Transformation Transformed cell lines divide more rapidly and do not require attachment to surface for growth, the loose contact inhibition(tumors), It occurs spontaneously or through interaction with viruses, oncogenes, radiation, or drugs/chemicals. Characteristics Infinite life span High growth potential Low growth factor dependence Suspension growth Aneuploid Methods Mutagens Viruses Oncogens Spontaneous Tumors *Not all transformed cell lines can form tumors, but all tumors contain transformed cells

Transformed cell lines:

Transformed cell lines These types of cells do not age in culture They are ‘immortal’ They often lose contact inhibition They often lose many normal characteristics They are not dependent on growth factors They may express ‘large T-antigen’ a p53 inhibitor


MAINTENANCE OF CELL LINE The process of maintaining: keeping cell line in good condition by using Provides the ideal environment of their growth.


MEDIA PREPARATION A growth medium or culture medium is a liquid or gel designe to support the growth of animal cells. There are different types of media for growing different types of cells. Such as: DMEM- Dulbecco's Modified Eagle Media RPMI - Roswell Park Memorial Institute medium


PASSAGING Passaging (also known as subculture or splitting cells) involves transferring a small number of cells into a new vessel. Suspension cultures are easily passaged with a small amount of culture containing fresh media- a few cell diluted in a large volume . Adherent cells first need to be detached- this is commonly done with a mixture of trypsin-EDTA. A small number of detached cells can then be used to seed a new culture.


CRYOPRESERVATION The process by which preserving the cells in frozen state for future use. Principle: freezing slowly to allow water not too slow thaw rapidly hydrophilic cryoprotectants Glycerol or DMSO - A cryoprotectant is a substance that is used to protect biological issue from freezing damage. Solid co2 at -79 degree celcius Refrozen at -80 degree celcius Vapour N2 at -150 degree celcius Liquid N2 at -196 degree celcius


ROLE AND USES OF CELL LINE Immortalized cell lines are widely used as a simple model for more complex biological systems, for example: The biochemistry and cell biology of mammalian (including human) cells. Immortalized cell lines can also be cloned giving rise to a colonel population (genetically identical cells). The testing toxicity of compounds or drugs to production of eukaryotic proteins.

. A: Performing all procedures in a laminar flow hood. :

25 BILAL . A: Performing all procedures in a laminar flow hood.

. B: Using flames to fix microorganisms on container necks :

26 BILAL . B: Using flames to fix microorganisms on container necks

C: Holding a bottle cap with the little finger. :

27 BILAL C: Holding a bottle cap with the little finger.

D: Avoid touching tops of open vessels while transferring their content.:

28 BILAL D: Avoid touching tops of open vessels while transferring their content.

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29 BILAL Filter sterilization Media that cannot be autoclaved must be sterilized through a 0.22 mm pore size membrane filter.

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4 Phases M Phase, mitosis occurs Chromatin condensation, sister chromatid separation Daughter cells G1 Phase Progression to DNA SYNTHESIS Alternatively Go OR differentiation Restriction Points S Phase DNA Synthesis Progression to G2 G2 Phase Integrity of DNA Checkpoints Apoptosis is an option DNA fragmentation, cell shrinkage, formation of small vesicles

Contact Inhibition:

Contact Inhibition When cells contact each other, they cease their growth. Cells arrest in G0 phase of the cell cycle Transformed cells will continue to proliferate and pile upon each other

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